Adrenal Glands From Pathophysiology To Clinical Evidence PDF

ENDOCRINOLOGY RESEARCH AND CLINICAL DEVELOPMENTS
ADRENAL GLANDS
FROM PATHOPHYSIOLOGY
TO CLINICAL EVIDENCE
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ENDOCRINOLOGY RESEARCH AND
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ENDOCRINOLOGY RESEARCH AND CLINICAL DEVELOPMENTS
ADRENAL GLANDS
FROM PATHOPHYSIOLOGY
TO CLINICAL EVIDENCE
GAETANO SANTULLI, MD, PHD

EDITOR
New York
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CONTENTS
Preface vii
Chapter 1 Imaging of Adrenal Gland 1
Michelle Tsang Mui Chung and Julie H. Song
Chapter 2 Mechanisms Integrating Endoplasmic Reticulum Stress, Cholesterol
Metabolism and Steroid Hormone Biosynthesis in the Adrenal Cortex 19
Zhi-qiang Pan, Yi-yi Zhang, Vivek Choudhary
and Wendy B. Bollag
Chapter 3 Regulation of Catecholamine Production from the Adrenal Medulla 53
Anastasios Lymperopoulos, Smit Chowdhary,
Kamarena Sankar and Isis Simon
Chapter 4 Interactions of the Adrenal Glands with Adipose Tissue 71
Janella León and Atil Y. Kargi
Chapter 5 Hypoadrenalism: Primary and Secondary Adrenal Failure 91
Marianna Minnetti and Ashley B. Grossman
Chapter 6 Clinical Management of Hyperaldosteronism 115
Jun Yang and Peter J. Fuller
Chapter 7 Cushing‘s Syndrome 143
Georgiana Alina Dobri, Divya Yogi-Morren and
Betul A. Hatipoglu
Chapter 8 Pheocromocytomas and Paragangliomas 171
Anna Heeney, Aoife J. Lowry,
Rachel K. Crowley and Ruth S. Prichard
Chapter 9 New Insights of Glucocorticoids Actions on the Homeostatic
Control of Energy Balance and Stress-Related Responses 207
Ernane Torres Uchoa, Silvia Graciela Ruginsk,
Rodrigo Cesar Rorato, Beatriz de Carvalho Borges,
Jose Antunes-Rodrigues and Lucila Leico K. Elias
vi Contents
Chapter 10 Role of Adrenal Hormones in the Fetal Programming of Hypertension 235

Sandhya Khurana, Collin J. Byrne, Stephanie Mercier,
Jeremy Lamothe, Chad R. Williamson, Julie Grandbois
and T. C. Tai
Chapter 11 Adrenal Cortical Neoplasms: Perspectives in Pediatric Patients 271
Shahrazad T. Saab and Gregory T. MacLennan
Chapter 12 Premature Adrenarche 281
Alexander K. C. Leung, Kam Lun Hon
and Benjamin Barankin
Chapter 13 Hyperandrogenism of Adrenal Origin 291
Dominik Rachoń
Chapter 14 Functional Roles of Corticosterone and Stress in Penile Morphology 299
Diogo B. De Souza, Dilson Silva, Célia M. Cortez,
Waldemar S. Costa and Francisco J. B. Sampaio
Chapter 15 Glucocorticoids and Neurodegeneration 307
Sheela Vyas, Ana Joao Rodrigues,
Joana Margarida Silva,
Francois Tronche, Nuno Sousa
and Ioannis Sotiropoulos
Chapter 16 Disorders of the Adrenal Glands: The Neurologists‘ Point of View 333
Tulio Bertorini and Lihong Shen
Chapter 17 Adrenocortical Cancer 361
Amudhan Pugalenthi and Eren Berber
Chapter 18 Adrenal Leiomyomas 377
Prashant B. Joshi
Chapter 19 Rare Tumors of the Adrenal Gland 385
Shahrazad T. Saab, Liang Chengand Gregory T. MacLennan
Editor Contact Information 411
Index 413
PREFACE
The present book provides a state-of-the-art overview of the adrenal gland

pathophysiology. It covers both up-to-date basic and clinical notions on adrenal glands.
Amidst the molecular aspects, discussed by leading scientists in the field, there are ER stress,
microRNA, hormone biosynthesis, and interactions with other organs. Clinical insights for
students and physicians are also provided, from the imaging to the therapeutic management of
common disorders of the gland, including adrenal failure, hyperaldosteronism, Cushing‘s
syndrome, neurologic disease, and cancer both in adults and pediatric patients. As mentioned
above, chapters are contributed by worldwide renowned expert scientists and physicians,
working in prestigious hospitals and universities including Brown, Oxford, Case Western
Reserve University, Cleveland Clinic, Mumbai, Shanghai, Chinese University of Honk Kong,
University of Tennessee, Indiana University School of Medicine, Ft. Lauderdale, Miami,
Monash Health in Clayton, Australia, University of Calgary, Northern Ontario School of
Medicine and Laurentian University, Canada, Medical University of Gdańsk, Université
Pierre et Marie Curie in Paris, France, University of Minho, Braga, Portugal, St. Vincent‘s
University Hospital in Dublin, Universities of Londrina, Alfenas, Sao Paulo, and Rio de
Janeiro, Brazil. Throughout these chapters, the authors spotlight future avenues for research
in basic pathophysiology and in therapy/prevention, in addition to thorough overviews of the
current literature pertaining to the adrenal gland and its functional roles. The book also
includes several color photographs, schemes and diagrams of molecular relationships, and
tables that support and complement the text. The comprehensive and systematic overview
provided within this book is expected to assist the reader in comprehending the importance of
taking into account the functional roles of the adrenal gland and also to address questions and
unresolved issues regarding the treatment of its disorders. Finally, the editor would like to
express his sincere appreciation to all the contributors for their dedicated collaboration in this
project. I also wish to thank Ms. Carra Feagaiga for her patient support. I sincerely hope this
book will enable readers to connect basic research principles with up-to-date clinical
knowledge, thereby encouraging future discoveries and developments of new therapeutic
strategies to treat disorders of the adrenal gland.
Gaetano Santulli, MD, PhD

New York, NY, USA
In: Adrenal Glands ISBN: 978-1-63483-550-3
Editor: Gaetano Santulli, MD, PhD © 2015 Nova Science Publishers, Inc.
Chapter 1
IMAGING OF ADRENAL GLAND
Michelle Tsang Mui Chung, MD,

and Julie H. Song, MD
Department of Diagnostic Imaging, Rhode Island Hospital,
Alpert School of Medicine, Brown University, Providence, RI, US
ABSTRACT
A variety of pathologies afflict the adrenal gland, including inert and biochemically-
active tumors and primary and metastatic malignancies. Of additional concern are the
adrenal masses that are detected incidentally because of expanding clinical indications of
cross sectional imaging and the high spatial resolution of current imaging tools.
Contemporary imaging techniques utilizing computed tomography (CT), magnetic
resonance imaging (MRI), and positron emission tomography (PET) play a key role in
the evaluation of the adrenal glands as they provide an accurate means to both detect and
characterize adrenal masses and ultimately to stratify these lesions into those that warrant
intervention and those which are of no consequence. In this chapter, we discuss the
imaging characteristics of adrenal masses in the contexts of hormonal abnormalities, a
positive oncologic history, and an incidentally discovered mass.
Keywords: adrenal gland, adrenal mass, adrenal tumor, hyperfunctioning adrenal tumor,
adrenal incidentaloma
INTRODUCTION
The adrenal glands are readily visualized on cross-sectional imaging studies. A wide
range of pathologies can affect the adrenal glands, and some adrenal abnormalities exhibit
distinguishing imaging characteristics. In others, the imaging findings must be considered in
the context of pertinent laboratory findings and past medical history.

Corresponding author; Dept. of Diagnostic Imaging, Rhode Island Hospital, Alpert School of Medicine, Brown
University, 593 Eddy Street, Providence, RI 02903, USA. E-mail: jsong2@lifespan.org.
2 Michelle Tsang Mui Chung and Julie H. Song
In this chapter we provide an overview of the contemporary techniques utilized in adrenal

imaging, discuss the characteristic imaging findings of a variety of adrenal lesions, and
present an imaging strategy applicable to the commonly encountered clinical scenarios of:
hormonal abnormalities, a positive oncologic history, and an incidentally discovered mass.
IMAGING MODALITY SELECTION

Indications for imaging the adrenal glands can be grossly stratified into two categories:
detection of a suspected adrenal mass and characterization of a known adrenal mass.
In the case of a suspected adrenal mass, selection of the most appropriate imaging
modality is guided by the patient‘s history, and one of the most common clinical scenarios is
metastatic work up in established extra-adrenal malignancy. In this setting, routine contrast
enhanced CT is the test of choice with PET-CT increasingly used in certain malignancies,
such as lung cancer.
Another important indication of lesion detection is localizing a hyperfunctioning tumor in
a patient with confirmed serum hormonal excess. In these cases, either CT or MRI is used for
lesion detection. Metaiodobenzylguanidine (MIBG) scintigraphy is occasionally utilized to
localize a suspected pheochromocytoma.
Characterization of an adrenal mass is determining its histology noninvasively by
imaging. Certain adrenal masses have characteristic appearance at detection. However, many
adrenal masses have non-specific appearance, especially when initially detected at routine
contrast-enhanced CT.
The main goal of characterizing an adrenal mass is to differentiate the benign,
inconsequential lesions from those that warrant treatment. CT and MRI are the workhorses in
lesion characterization, while PET-CT is generally reserved for patients with a history of an
extra-adrenal malignancy. Additional considerations for imaging modality selection include
contrast allergy, renal insufficiency, and radiation exposure in young patients.
IMAGING TECHNIQUES
CT
Most adrenal masses are readily identified by standard abdominal CT imaging, typically
reconstructed at 5 mm thickness. Dedicated adrenal CT is performed for the characterization
of a known adrenal mass. First, images are acquired through the adrenal glands and
reconstructed in axial and coronal planes at 2-3 mm slices prior to the injection of intravenous
contrast. A region of interest (ROI) is drawn over the adrenal mass to determine its density,
which is measured in Hounsfield Units (HU).
A density of less than or equal to 10 HU on the non-contrast enhanced series is diagnostic
of a benign, lipid-rich adenoma and hence the study is complete. If the density of the mass
measures greater than 10 HU, IV contrast is administered and subsequent images are obtained
using the same acquisition parameters at 60 seconds and at 15 minutes following contrast
injection. The density of the mass is measured at each additional time point and these values
Imaging of Adrenal Gland 3
are utilized to determine the percentage of contrast that is washed out of the mass. The
formula utilized to calculate percentage of washout and the interpretation of their values are
discussed later in this chapter.
MRI
The fundamental MR technique used in adrenal mass evaluation is chemical shift (CS-
MR) imaging which exploits the differing resonance frequencies of protons in water
molecules vs. protons in lipid molecules. CS-MR applies to microscopic fat and water protons
that are contained in the same voxel (a microscopic 3-dimensional imaging unit), which for
the purposes of this discussion is equivalent to the intracellular compartment.
When these protons are made to precess at their differing resonance frequencies, there are
specific points in time at which they precess in-phase and out-of-phase.
By choosing a pre-determined time point when these protons are out-of-phase, there is a
net cancellation of signal, which is observed as a drop in signal in the final image. Therefore,
most adenomas containing a sufficient amount of intracellular lipid will appear darker on out-
of-phase imaging than on the in-phase imaging.
In the case of a lipid-poor lesion, additional sequences including T2-weighted and
gadolinium-based, contrast-enhanced series are utilized for further characterization.
PET
The basis of PET imaging is that malignant tumors are more metabolically active and
consume more glucose. PET utilizes the radiopharmaceutical fluorine-18-2fluoro-2-deoxy-D-
glucose (FDG), which emits gamma rays when the glucose portion of the radiopharma-
ceutical is metabolized.
The patient is first injected with the radiopharmaceutical. After a period of approximately
75 minutes, a total-body non-contrast enhanced CT is obtained. This is immediately followed
by the PET acquisition, in which the emitted gamma rays are measured and a map is created
indicating the varying degrees of FDG radiopharmaceutical metabolism throughout the body.
The PET images are fused with CT images in order to provide the necessary spatial resolution
to correlate lesions identified on CT with areas of intense FDG activity.
NORMAL ADRENAL GLAND

The adrenal glands are located anterosuperior to the kidneys, contained within the
perirenal fascia. They are composed of two limbs that collectively range from 2-6 mm in
thickness and 2-4 cm in length (Figure 1) [1].
Figure 1. Normal adrenal glands in 45-year-old man. Axial contrast-enhanced CT image shows normal
adrenal glands (arrow).
They are highly vascular with arterial blood flow supplied by the superior, middle and
inferior suprarenal arteries, which arise from the inferior phrenic artery, aorta, and renal
artery, respectively. Venous outflow is via the suprarenal veins, which drain into the inferior
vena cava on the right and renal vein on the left.
The adrenal gland is composed of an outer cortex and inner medulla. The cortex and
medulla are indistinguishable by imaging, but functionally distinct. The adrenal cortex
produces cortisol, aldosterone, and androgen, and the adrenal medulla produces epinephrine
and norepinephrine.
HYPERFUNCTIONING ADRENAL MASSES

Hyperfunctioning adrenal masses are usually suspected when a patient presents with
clinical manifestations of hormonal excess, which is confirmed by biochemical analysis. The
suspected mass is then localized by imaging.
Cortisol-Producing Adenoma
Cortisol-producing adrenal adenomas are responsible for 20% of Cushing‘s syndrome,

and are the most common cause of adrenocorticotropic hormone (ACTH)-independent
Cushing‘s syndrome which is caused by primary adrenal disease. At CT, these masses are
typically larger than 2 cm and are low in density due to abundant intracellular lipid content
[2, 3]. At MR, these masses demonstrate signal loss on out-of-phase chemical shift imaging.
The overall appearance is similar to that of a nonfunctioning adrenal adenoma. In ACTH-
dependent Cushing‘s syndrome, there is overstimulation of the adrenal glands, usually by a
pituitary adenoma, and rarely by an ectopic source in the chest or the abdomen. In these
cases, the adrenal glands are often hyperplastic on CT or MR, but can also have normal
appearance.
Aldosterone-Producing Adenoma
Aldosterone-producing adrenal adenomas account for approximately one third of Conn‘s

syndrome. These masses are typically less than 2 cm in size, which can make differentiation
from adrenal hyperplasia challenging (Figure 2) [4, 5].
Figure 2. Aldosteronoma in 60-year-old man with hypertension. Axial contrast-enhanced CT image

shows a 1.0 cm mass (arrow) arising from the medial limb of left adrenal gland.
Hyperfunctioning adrenal adenoma is favored over adrenal hyperplasia in the setting of

unilateral gland enlargement and patient age less than 40 years [6]. Adrenal vein sampling
may be necessary for lesion localization when CT and MRI findings are inconclusive.
Pheochromocytoma
Pheochromocytoma is an uncommon catecholamine-producing tumor of adrenal medulla.

It is solitary in 90% of cases and multiple in 10%. Pheochromocytomas have a variable
imaging appearances [7]. On CT, smaller masses tend to be round and of uniform soft tissue
density; however, larger masses can appear heterogeneous due to internal hemorrhage or
necrosis.
On MRI, pheochromocytomas classically have been described to demonstrate ―light-bulb
bright‖ intensity on T2-weighted images, however recent reports have demonstrated a more
variable T2 appearance with some lesions appearing moderately hyperintense (Figure 3) or
even hypointense [2, 8].
Figure 3. Pheochromocytoma in 35-year-old woman. Coronal T2 weighted MR image shows a 4.1 cm

right adrenal mass with moderate hyperintensity.
Pheochromocytomas are highly vascular and avidly enhance following contrast

administration (Figure 4) [1]. For the exceptional cases where CT and MRI fail to identify a
mass, MIBG scintigraphy can be a problem-solving tool. MIBG is also helpful in patients at
increased risk for multiple pheochromocytomas or metastatic disease [9].
Aside from local invasion and metastases, imaging cannot differentiate between benign
and malignant lesions.
Figure 4. Pheochromocytoma in 19-year-old woman. Axial contrast-enhanced CT image shows a

mildly heterogeneous 2.9 cm left adrenal mass with avid enhancement.
Figure 5. Adrenocortical carcinoma in 86-year-old woman. Contrast-enhanced axial T1 weighted MR

image shows a 11 cm heterogeneous right adrenal mass with central necrosis. Note direct tumor
extension into intrahepatic IVC (arrow).
Adrenocortical Carcinoma
Adrenocortical carcinoma is a rare entity with a prevalence of 1–2 per million population
[10]. Approximately 50% are hormonally active, most commonly producing cortisol and
presenting clinically as Cushing‘s syndrome. On CT and MRI, these primary malignancies
are typically large (usually greater than 6 cm in diameter), heterogeneously enhancing and
frequently invade the inferior vena cava (Figure 5) [11]. They are often associated with
calcifications, central necrosis and hemorrhage, which contribute to their heterogeneous
appearance.
ADRENAL MASSES IN PATIENTS WITH AN ONCOLOGIC HISTORY:

ADENOMA VS. METASTASIS
While the majority of adrenal masses are benign, the adrenal gland is also a common site
of metastatic disease. Distinguishing a benign adenoma from a metastasis is especially critical
in an oncology patient with no other potential sites of metastatic disease, as this diagnosis can
mean the difference between a stage I and a stage IV designation. The most common
malignancies that metastasize to the adrenal gland are of lung, breast, pancreas and
gastrointestinal tract origin [12], but virtually any primary malignancy can metastasize to the
adrenal glands. In general, any adrenal mass that is increasing in size in a patient with a
known extra-adrenal malignancy should be considered malignant until proven otherwise.
Hence, comparison to prior imaging is critical.
In the absence of a prior study for comparison, differentiating between a benign adenoma
and a metastatic lesion can be difficult at routine contrast-enhanced CT as the findings can be
variable and nonspecific. Certain imaging features should be viewed as suspicious for
malignancy, such as central necrosis and irregular or infiltrative margins. In general, benign
adrenal masses are of homogenous density with smooth margins.
However the converse is not always true and a small metastatic lesion can appear
innocuous [13, 14]. The imaging features used most reliably to distinguish benign adenomas
from metastatic disease are as follow: the presence or absence of lipid content, contrast
washout pattern, and degree of metabolic activity.
Imaging of Intracellular Lipid: CT and CS-MR
The principle of determining the lipid content of an adrenal mass is predicated on the
finding that adrenal adenomas contain variable amounts of intracellular lipid, whereas
metastatic lesions generally do not [15]. At non-contrast enhanced CT, a density of 10 HU or
less has been shown to diagnose a lipid-rich adenoma with a sensitivity of 71% and
specificity of 98% (Figure 6) [16].
Figure 6. Lipid-rich adenoma in 60-year-old woman. Axial unenhanced CT image shows a well-defined
3.2 cm mass with attenuation of -5HU.
However up to 30% of adenomas do not contain an adequate amount of lipid and have
density values greater than 10 HU, making them indeterminate at CT. CS-MR is another tool
that can be utilized in the detection of intracellular lipid, as previously described.
When comparing the in-phase and out-of-phase images, adenomas with intracellular lipid
will become dark on the out-of-phase images, using spleen or muscle as the reference
standard (Figure 7), unlike most metastatic adrenal masses which will retain their signal [17,
18].
Figure 7. Lipid-rich adenoma in 71-year-old woman. Top: T1-weighted in-phase MR image shows a 3
cm left adrenal mass (arrow) that is mildly hyperintense relative to spleen. Bottom: On T1-weighted
opposed-phase MR image, the mass (arrow) shows marked signal intensity loss relative to spleen,
diagnostic of adenoma.
Perfusion Imaging: CT Washout
After enhancement with intravenous contrast, adenomas lose contrast more rapidly than
non-adenomas [19, 20]. Percentage of washout is calculated utilizing CT delayed phase
imaging, as previously described.
The absolute percentage washout (APW) is calculated when both unenhanced and
contrast enhanced images are available, as follows:
(E–D)/(E–U) x 100%
Where E is the attenuation value of the lesion on enhanced CT, D is the attenuation value of
the lesion on 15-minute delayed CT, and U is the attenuation value of the lesion on
unenhanced CT.
A value of 60% or greater is considered diagnostic of an adenoma (Figure 8) [19].
Figure 8. Lipid-poor adenoma in 51-year-old woman. Top: Axial unenhanced CT images shows a 1.5
cm left adrenal mass (arrow) with attenuation of 18 HU. Middle: On dynamic contrast-enhanced CT,
the mass enhances to 70 HU. Bottom: On 15-minute delayed CT image, attenuation of adrenal mass is
31HU. APW and RPW are 75% and 56%, respectively, diagnostic of adenoma.
If unenhanced CT images are not available, the relative percentage washout (RPW) can
be calculated, as follows:
(E–D)/E x 100%
A value of 40% or greater is considered diagnostic of an adenoma. As contrast washout is

independent of lipid content, this technique can be used to diagnose both lipid-rich and lipid-
poor adenomas [21, 22]. Perfusion imaging is a robust tool in separating adenomas from
metastasis [21-24].
Functional Imaging: PET and PET/CT

Malignant adrenal masses tend to be more metabolically active than benign adenomas
and will therefore demonstrate increased FDG uptake (Figure 9).
Figure 9. Adrenal metastasis in 68-year-old man with rectal cancer. Top: Axial unenhanced CT image
shows a 2.6 cm right adrenal mass with attenuation of 30HU. Bottom: Axial co-registered PET/CT
image shows markedly increased FDG uptake in right adrenal metastasis.
In practice, the liver is used as an internal standard of reference and adrenal masses that
appear qualitatively higher in intensity are considered suspicious for malignancy. PET and
PET/CT have been shown to detect malignant adrenal lesions with sensitivity ranging from
93-100% and specificity ranging from 80-100% [25-27].
Important pitfalls to consider are lesions less than 1 cm in size, which are below the
resolution of PET and can result in a false-negative interpretation [25]. Additionally, false-
positive interpretations can result from infectious or inflammatory processes, and rarely by
hypermetabolic adenomas [13].
Image-Guided Biopsy
The advent of more sophisticated imaging techniques has allowed adrenal masses to be
accurately characterized by noninvasive means [28]. However, there are several scenarios in
which imaging-guided biopsy is still required including: in oncology patients with
inconclusive imaging findings; a solitary adrenal metastasis requiring histologic correlation
for staging purposes; as well as in the case of an enlarging adrenal mass in a patient without a
known malignancy. Adrenal biopsies are usually performed under CT guidance in order to
ensure accurate and safe placement of the biopsy needle.
ADRENAL INCIDENTALOMAS
Adrenal ―incidentalomas‖ are adrenal masses identified on imaging studies performed for
unrelated indications. Their prevalence at CT is approximately 4-5%, which approaches the
estimated prevalence in the general population of 3-7% [29-33]. The majority of adrenal
masses incidentally discovered in patients without an oncologic history are benign and
extensive work up is not indicated for most small asymptomatic masses.
However in patients with a known extra-adrenal malignancy, a metastatic disease must
also be considered. In the case of a large adrenal mass, the possibility of primary malignancy
is an additional issue.
There are certain imaging features that are diagnostic of benignity. In the absence of
these features, several factors are used to determine the pre-test probability of malignancy for
an adrenal incidentaloma, as discussed below. Of note, imaging findings do not reveal the
functional status of an incidental adrenal mass. Subclinical hyperfunction of an incidental
adrenal mass is a well-recognized phenomenon, but its exact prevalence and management are
somewhat controversial. While most endocrinologists recommend biochemical assessment
for incidental adrenal masses, optimal endocrine evaluation of these masses are still debated.
Masses with Specific Imaging Features
Myelolipomas
Adrenal myelolipomas are benign, nonfunctional tumors composed of mature fat and
hematopoietic tissue.
They are usually asymptomatic, but when large, may rarely spontaneously hemorrhage
and cause pain. Identification of macroscopic fat is diagnostic of myelolioma at CT and MRI
with a varying amount of fat and soft tissue components (Figure 10).
Pseudocapsules and calcifications are additional common findings [34].
Figure 10. Myelolipoma in 74-year-old woman. Coronal reformatted contrast-enhanced CT image

shows a 10 cm encapsulated right adrenal mass predominantly containing macroscopic fat.
Figure 11. Adrenal cyst in 61-year-old woman. Axial T2-weighted MR image shows a well-defined 2
cm left adrenal mass with thin wall and fluid signal intensity.
Cyst and Pseudocyst

Adrenal cysts and pseudocysts are uncommon benign lesions. Most are asymptomatic,
with the exception of those that produce mass effect on adjacent structures and those that
become secondarily infected or develop internal hemorrhage.
Adrenal cysts appear as well circumscribed, homogeneous masses with density
measurements similar to that of water (0 HU).
At MRI, they follow the signal of water, appearing dark on the T1-weighted sequence
and bright on the T2-weighted sequence (Figure 11). Adrenal cysts do not internally enhance
following IV contrast administration, however the thin walls of the cyst may enhance or
contain thin calcifications [35].
Adrenal pseudocysts are the sequela of prior adrenal hemorrhage. They can appear more
heterogeneous than simple cysts with thicker walls, internal septations, and higher internal
density [35].
Hemorrhage
Adrenal hemorrhage can result from a wide range of clinical settings: trauma,
anticoagulation, bleeding disorder, sepsis, hypotension, renal vein thrombosis, and severe
stress. Rarely adrenal insufficiency can result when there is bilateral adrenal involvement.
On non-contrast enhanced CT, acute adrenal hemorrhage appears as a mass of higher
density (50-90 HU) than that of normal adrenal tissue (Figure 12). As the hematoma evolves,
the mass tends to decrease in size and can ultimately calcify or liquefy and persist as a
pseudocyst.
Adrenal hemorrhage has variable appearance at MRI, depending on the age of the blood
product.
Figure 12. Adrenal hemorrhage in 55-year-old man. Axial unenhanced CT image shows a 3.5 cm right
adrenal mss with attenuation of 60 HU, consistent with acute hemorrhage.
Masses without Diagnostic Imaging Features
In the absence of diagnostic imaging features, certain factors determine the pre-test
probability for malignancy of an adrenal incidentaloma. These include lesion stability, lesion
size, and patient history of malignancy.
Comparison to prior imaging is essential when assessing an adrenal incidentaloma. An
adrenal mass that has not increased in size over the course of at least one year is highly
unlikely to represent metastatic or primary malignancy [36]. As a general rule, a mass that
increases in size warrants further work up to exclude malignancy. There is no specific size
below which a mass can be definitively characterized as benign; however, a direct correlation
has been shown to exist between mass size and malignancy risk. Indeterminate adrenal
masses greater than 4 cm are generally resected in patients without a history of malignancy
because of the risk of adrenocortical carcinoma [36, 37].
A history of malignancy is one of the most important factors in determining the
malignant potential of an adrenal incidentaloma. It is extremely rare for an occult cancer to
present as an isolated adrenal metastasis [38, 39]. In a patient with known extraadrenal
malignancy, the work up of an incidental adrenal mass will be largely based on whether the
adrenal mass is the only potential site of metastasis vs. widespread disease, and may also
depend on the patient‘s co-morbidities.
Imaging Algorithm
A flow chart detailing the recommended imaging work up of an incidental adrenal mass
was published in the 2010 white paper of the American College of Radiology Committee on
Incidental Findings [36]. The recommendations are summarized as follows.
An adrenal mass with imaging features diagnostic of a myelolioma, lipid-rich adenoma or
cyst is benign and requires no further work up, regardless of size. In the absence of diagnostic
imaging features, an adrenal mass less than 4 cm in size is likely benign if it has been stable
for at least one year and no follow up is necessary. However if the lesion has demonstrated
interval growth, it should be considered suspicious for malignancy and biopsy or resection is
warranted.
If no prior imaging is available for an indeterminate lesion less than 4 cm in size, follow
up non-contrast CT or CS-MR can be obtained in 12 months if characteristics suggestive of
benignity are present (e.g., homogeneous density, low density, smooth margins) and there is
no cancer history. If suspicious characteristics are identified (e.g., heterogeneous density,
necrosis, irregular margins) or if the patient has a prior history of cancer, the lesion can be
further characterized using non-contrast CT or CS-MR, and if needed, adrenal CT with
washout analysis. If these imaging studies fail to lead to a diagnosis, then biopsy should be
considered. PET may be considered prior to biopsy in patients with known extra-adrenal
malignancies.
In the absence of an oncologic history, masses greater than 4 cm in size are typically
resected due to the risk of adrenocortical carcinoma. In patients with a history of extra-
adrenal malignancy, PET or biopsy should be performed to exclude metastatic disease.
CONCLUSION
The adrenal glands are a host to a variety of pathologies including inert and
biochemically active tumors and primary and metastatic malignancies. CT, MR, and PET are
excellent imaging tools, allowing accurate diagnosis of most adrenal masses. The diagnosis of
hyperfunctioning tumors is made in the context of pertinent biochemical analysis. With the
appropriate imaging strategy, most adrenal masses can be noninvasively stratified into those
that warrant intervention and those that are of no consequence.
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Chapter 2
MECHANISMS INTEGRATING ENDOPLASMIC

RETICULUM STRESS, CHOLESTEROL METABOLISM
AND STEROID HORMONE BIOSYNTHESIS
IN THE ADRENAL CORTEX
Zhi-qiang Pan2,3, Yi-yi Zhang4, Vivek Choudhary1,2

and Wendy B. Bollag1,2,
1
Charlie Norwood VA Medical CenterAugusta, GA, US
2
Department of Physiology, Medical College of Georgia
at Georgia Regents University, Augusta, GA, US
3
School of Basic Medical Science,
Shanghai University of Traditional Chinese Medicine, Shanghai, China
4
Longhua Hospital, Shanghai University of Traditional Chinese Medicine,
Shanghai, China
ABSTRACT
Cholesterol is an important component of mammalian cell membranes, and
intracellular cholesterol homeostasis plays a crucial role in physiological cellular
processes. When cells encounter adverse conditions, endoplasmic reticulum (ER) stress
may activate the unfolded protein response (UPR), which protects normal cell function
by stimulating a survival pathway; however, if the adverse conditions are severe and/or
persistent, the death pathway is activated. Accumulating evidence shows that ER
stress/UPR activation plays a critical role in cholesterol homeostasis and vice versa.
Excessive or insufficient cellular cholesterol results in cholesterol-associated diseases,
such as atherosclerosis, hypercholesterolemia, Niemann–Pick disease type C, and
Alzheimer‘s disease. In this review, we provide an overview of the various mechanisms
by which cellular cholesterol metabolism and ER stress signaling pathways are regulated.
We also discuss cholesterol absorption, synthesis and metabolism within various cells,

Address all correspondence to Wendy B. Bollag, Ph.D., Department of Physiology, Medical College of Georgia at
Georgia Regents University, 1120 15th Street, Augusta, Georgia 30912 (E-mail: wbollag@gru.edu).
20 Zhi-qiang Pan, Yi-yi Zhang, Vivek Choudhary et al.
and relate these processes with ER stress. Finally, we discuss the possible relevance of
ER stress to the synthesis of steroid hormones from precursor cholesterol in the adrenal
cortex.
Keywords: cholesterol, endoplasmic reticulum (ER), ER stress, steroidogenesis, unfolded

protein response (UPR)
INTRODUCTION
The endoplasmic reticulum (ER), an organelle found in all eukaryotic cells, forms a
membranous network of branching tubules and flattened sacs. This organelle is responsible
for protein synthesis, folding, maturation, quality control, and trafficking, and it is also the
major site of phospholipid synthesis and of intracellular calcium (Ca2+) storage [1-3].
The ER includes the rough endoplasmic reticulum (RER) and the smooth endoplasmic
reticulum (SER). The RER is studded with ribosomes on its cytosolic face and is the site of
protein synthesis; the SER is a smooth network lacking ribosomes. The SER is concerned
with lipid and carbohydrate metabolism and detoxification of organic molecules/metabolites.
In recent years, the study of ER function has become an important field in various
disciplines. If physiological conditions change or pathological factors impinge upon the ER to
cause ER stress (ERS), a set of signaling pathways called the unfolded protein response
(UPR), is activated [4, 5]. Such stressful conditions can include, for example, DNA damage
as a result of X-ray or UV irradiation [6-8], nutrient deprivation [9-12], elevated temperature,
induction of growth arrest [13], exposure to calcium ionophores [14], exposure to toxins or
treatment with anticancer agents [15, 16], hypoxia [17] and activation of the acute systemic
stress response [18].
In mammalian cells, the glucose-regulated protein of molecular weight 78,000 (GRP78)
is a pivotal regulator of the UPR [19], and inositol-requiring enzyme 1α (IRE1α), double-
stranded RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription
factor 6 (ATF6) are three key ER transmembrane protein sensors that initiate UPR signaling
cascades [4, 20].
Cholesterol is an important component of mammalian cell membranes and is required to
establish proper membrane permeability and fluidity; cholesterol also serves as a precursor
for the biosynthesis of steroid hormones [21], bile acids [22], and vitamin D [23]. Cholesterol
can be synthesized within the cytosol and endoplasmic reticulum in cells comprising organs
such as the liver, intestines, adrenal glands and reproductive organs.
The synthesis of cholesterol has been studied in considerable detail for several decades;
however, the physiological and molecular mechanisms mediating intracellular cholesterol
homeostasis are not fully elucidated. In general, intracellular cholesterol concentrations are
maintained within a narrow range and a variety of mechanisms are involved in this process
[24]. For example, when cholesterol levels are high, cells initiate an enzyme mechanism in
the ER to esterify excess cholesterol for storage in cytoplasmic lipid droplets; when
cholesterol is low, cholesterol esters are taken from the lipid droplets and de-esterified.
Under abnormal conditions, intracellular cholesterol metabolism may become disordered,
and an ER stress mechanism may be involved in cholesterol regulation. In this review, we
Mechanisms Integrating Endoplasmic Reticulum Stress, Cholesterol Metabolism … 21
will discuss the latest progress in terms of unraveling a potential connection between ER
stress and intracellular cholesterol homeostasis and steroidogenesis.
The UPR and Some Key Signaling Molecules
GRP78 Is a Pivotal Regulator of the Unfolded Protein Response

GRP78, also known as BiP (immunoglobulin heavy-chain binding protein) or HSPA5
(heat shock 70kDa protein 5), is an important ER chaperone and is essential for the normal
function of the ER (Figure 1). A study in which GRP78 was purified from a membrane
fraction isolated from virus-transformed chick embryo fibroblasts in 1977 identified this
protein to be localized in the endoplasmic reticulum [25]. Generally, GRP78 expression is
maintained at low basal levels in most adult organs including the heart, lung, and brain, but it
is strongly induced in many tumors such as breast, lung, prostate, liver, colon, and gastric
cancers [26-30]. In resting cells, GRP78 binds to the ER transmembrane sensor proteins
IRE1, PERK, and ATF6, and maintains them in an inactive form. However, upon the
development of ER stress, GRP78 is required for binding to the unfolded or misfolded
polypeptide chains and/or unassembled multisubunit proteins that characterize this condition.
As a result, GRP78 dissociates from the transmembrane sensors, leading to the activation of
the UPR signaling pathways [31, 32]. Therefore, GRP78 is a pivotal regulator of ER stress
due to its role as a major ER chaperone with antiapoptotic properties, as well as its ability to
control the activation of UPR signaling.
Figure 1. ER stress and the UPR signaling pathway. Once cells experience ER stress conditions, for
example, upon exposure to excess cholesterol or pharmacological agents such as thapsigargin or
tunicamycin, accumulation of misfolded or unfolded protein aggregates in the ER lumen activates three
ER transmembrane proteins: double-stranded RNA-dependent protein kinase-like ER kinase (PERK),
inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6). Normally, the ER
chaperone GRP78 binds to the luminal domains of these ER stress sensors in order to maintain these
proteins in an inactive state.
During ER stress, GRP78 preferentially binds to unfolded or misfolded proteins, with its
dissociation from the ER stress sensors leading to activation of the three UPR pathways. (1)
PERK phosphorylates the initiation factor eukaryotic translation initiator factor 2α (eIF2α) to
result in the attenuation of global protein synthesis, which reduces the protein load in the ER.
However, phosphorylated eIF2αstill allows ATF4 mRNA translation; the ATF4 protein then
translocates to the nucleus to promote the transcription of UPR target genes such as CHOP
and GADD34. (2) IRE1α dimerization, followed by activation and autophosphorylation,
triggers its RNase activity, such that unspliced X box-binding protein 1 (XBP1u) mRNA is
spliced to produce spliced XBP1 (XBP1s), which encodes a transcription factor that controls
the transcription of ER chaperones, other UPR target genes and ER-associated degradation
(ERAD). (3)
After ATF6 activation, it is transported to the Golgi apparatus through interaction with
the coat protein II (COPII) complex, where it is sequentially cleaved by site 1 protease (S1P)
and S2P, yielding an active cytosolic N-terminal ATF6 fragment (ATF6 p50) which migrates
to the nucleus, activating the transcription of UPR target genes.
Three Key Regulators Involved in the Activation of UPR Signaling Pathways

IRE1, PERK, and ATF6 are three key ER transmembrane sensor proteins, containing
three domains: an ER-luminal domain that senses unfolded proteins, a transmembrane domain
through which each protein is targeted to the ER membrane, and a cytosolic domain that
transmits signals to the transcriptional or translational component of the pathway. In resting
cells, all three ER stress sensors are maintained in an inactive state through association with
the abundant ER chaperone GRP78 [31, 32].
IRE1
IRE1 is a type 1 transmembrane serine/threonine protein kinase, which has two isoforms,
both of which participate in the ER stress response or UPR [33]. IRE1α is ubiquitously
expressed while IRE1β is tissue-specific. Upon ER stress, GRP78 dissociates from IRE1,
which allows its activation and autophosphorylation, as well as the appearance of its
endoribonuclease activity [34].
Additionally, unfolded or misfolded proteins may directly bind to IRE1 promoting its
homodimerization and autophosphorylation [35]. Upon activation, IRE1 splices a 26-
nucleotide sequence intron from the mRNA encoding the X-box binding protein 1 (XBP-1),
to produce XBP1s, which encodes a basic leucine zipper (bZIP) transcription factor with a
potent transactivation domain. Then, XBP1s translocates to the nucleus where it leads to
upregulation of ER chaperones and other UPR target genes, including molecular chaperone
DnaJ, endoplasmic reticulum DnaJ homolog 4 (ERdj4), ER degradation enhancer,
mannosidase alpha-like 1 (EDEM1), and protein disulfide-isomerase (PDI), all of which are
involved in protein folding and the ER-associated protein degradation (ERAD) response [36,
37].
PERK
Once ER stress commences, PERK dissociates from GRP78. Then, PERK
homodimerization, activation and autophosphorylation results in eukaryotic initiation factor
2α (eIF2α) phosphorylation. Phosphorylated eIF2α subsequently attenuates the rate of global
translation initiation to prevent further protein synthesis, in order to reduce the ER protein
load [38]. However, phosphorylated eIF2α can selectively increase the translation of certain
mRNAs with upstream open reading frames (uORFs) in the 5‘ untranslated region (5‘-UTR);
thus, these genes can escape from eIF2α-mediated translational attenuation. A representative
example of these selectively increased proteins is activating transcription factor 4 (ATF4).
Thus, phosphorylated eIF2α can lead to increased ATF4 protein expression, and ATF4 then
translocates to the nucleus to allow for transcription of UPR target genes by binding to the
UPR response element, for example, of C/EBP homologous protein (CHOP), a proapoptotic
transcription factor that results in cell death if ER stress persists [39]. Another such gene is
growth-arrest and DNA-damage-inducible protein 34 (GADD34), which acts as a negative
feedback mechanism for the PERK pathway by eventually dephosphorylating eIF2α, to
relieve the translational repression occurring during prolonged ER stress [40].
ATF6
ATF6 represents a group of ER stress transducers that encode basic leucine zipper (bZIP)
transcription factors, including ATF6α, ATF6β and cAMP responsive element binding
protein 3 family (e.g., CREB3, CREB3L1, CREB3L2, CREB3L3 and CREB3L4) [41].
Similar to other ER stress sensors, once ER stress occurs, the dissociation of GRP78 from
ATF6 results in its translocation from the ER to the Golgi, where it is cleaved by site 1 and
site 2 proteases. The sequential proteolysis by these proteases leads to the release of the N-
terminal ATF6 fragment, which translocates to the nucleus and binds to the ER stress
response element, thereby activating UPR target genes. Finally, these gene products, for
example, GRP78 and GRP94, as well as folding enzymes such as PDI, improve the ER‘s
protein folding capacity [42].
During ER stress, the activated UPR is essential in order to maintain (or restore) ER
homeostasis and cell survival [43]. The beneficial consequences of this response will result in
molecular signaling changes in downstream pathways. For example, enhancement of ER
protein-folding capacity through expansion of the ER and increased expression of chaperones
and foldases, inhibition of protein translation and reduced cell protein levels and ER protein-
folding load, and degradation of the misfolded or unfolded proteins via ERAD can all be
stimulated. However, if ER stress factors are not relieved, ER stress-induced cell death may
occur, generally through caspase activation [44].
The Consequences of Severe or Persistent ER Stress

Cell death is inevitable upon severe and/or persistent ER stress, and CHOP and apoptotic
caspases play a crucial role during this process [45].
CHOP Is a Key Regulator of ER Stress-Induced Apoptosis

CHOP, also referred to as growth arrest and DNA damage-inducible gene 153
(GADD153) or DNA-damage-inducible transcript 3 (DDIT3), is a member of the C/EBP
gene family of transcription factors [46]. First isolated from a hamster library by subtractive
hybridization, following induction of its expression by UV irradiation [47], CHOP is a
ubiquitously expressed mammalian gene encoding a basic region-leucine zipper (bZIP)
domain-containing transcription factor [48, 49].
CHOP has been considered the key UPR pro-apoptotic player [50]. It is not only a target
gene of the
IRE1 [51]. However, although all three branches of the UPR regulate the activation of CHOP,
ATF4 is considered the major inducer of CHOP expression [52]. Generally, CHOP is
expressed at very low levels under physiological conditions, but after severe or persistent ER
stress, its expression level increases significantly. As a transcription factor, CHOP has been
shown to regulate numerous proapoptotic and antiapoptotic genes, including Bcl-2, GADD34,
and tribbles homolog 3 (TRB3) [53]. Many factors that cause stress or malfunction of the ER
can increase CHOP gene expression [54], and CHOP can also negatively regulate C/EBP
transcription factors [55]. In addition, CHOP triggers critical early events that have been
reported to lead to the initiation of apoptosis [15, 56]. Thus, it is worth noting that CHOP may
be not only a marker of apoptosis but also the cause of apoptosis.
Caspases As Crucial Regulators of ER Stress-Induced Apoptosis

When cells undergo severe or prolonged ER stress, the UPR can trigger apoptosis [57].
Generally, there are two major pathways for the initiation of apoptosis: one is an
ER/mitochondria-independent cell death pathway, while the other is dependent upon the ER
and mitochondria [58].
(1) The ER/Mitochondria-Independent Cell Death Pathway

The ER/mitochondria-independent cell death pathway is also named the extrinsic
pathway, and is mediated by cell membrane death receptors. Caspase-12, one caspase isoform
activated by death receptor pathways, is localized to the ER in rodents and can be specifically
activated by ER stress [49, 59]. Mice that are deficient in caspase-12 are resistant to ER
stress-induced apoptosis, suggesting that caspase-12 plays a key role in the proapoptotic
process in rodents [49, 59]. Also likely involved is caspase-9: procaspase-9 is cleaved into
active caspase-9 by activated caspase-12, then activates caspase-3 and leads to apoptosis.
However, cytochrome c is not released from mitochondria during ER stress-induced
apoptosis, which suggests that cytochrome c is not involved in this caspase-12-dependent cell
death [60]. In addition, caspase-12 can also be activated by its downstream protein caspase-7,
which suggests that there may arise another loop pathway in the apoptotic cascade though
caspase-12 [61]. In humans, the caspase-12 homologue has been verified to be inactive as a
result of several mutations during evolution; however, caspase-4 has been proposed to fulfill
the function of caspase-12 in humans. Cleavage of caspase-4 is not affected by
overexpression of Bcl-2, which is involved in preventing mitochondrially mediated cell death,
suggesting that caspase-4 is not downstream of other effector caspases and is likely primarily
activated for ER stress-induced apoptosis [62]. It is noteworthy that the key caspase in ER-
induced apoptosis depends on the cell type and the cause of the ER stress; therefore, some
controversy has arisen. For example, it has been reported that caspase-12 and caspase-4 are
not required for the induction of caspase-dependent ER stress-related apoptosis in IL-3-
dependent murine pro-B cells (FL5.12) and human multiple myeloma cells (U266) [63]. In
another study caspase-8 deficiency reduced ER stress-induced apoptosis in P19 embryonal
carcinoma (EC) cell death [64].
(2) The ER/Mitochondria-Dependent Cell Death Pathway

The ER/mitochondria-dependent cell death pathway is also known as the intrinsic
pathway and is closely related to factors found in the mitochondria. With the insertion of
these proapoptotic proteins into the mitochondrial membrane, mitochondrial membrane
permeability increases, resulting in the release of cytochrome c into the cytosol. Released
cytochrome c binds to Apaf-1 and activates caspase-9 and then caspase-3, leading to cell
death [61]. Upon persistent ER stress, the expression of Bcl-2 family members, including
antiapoptotic (e.g., Bcl-2, Bcl-xL and MCL-1) and proapoptotic proteins (such as Bax, Bak,
and Bik) can be regulated by the UPR [65]. BH3-only proteins, such as Bim, Bad and Bax
play an important role in the triggering of programmed cell death and ER stress-induced
apoptosis [66]. Indeed, cells overexpressing Bcl-2 or deficient in Bax and Bak are resistant to
ER stress-induced apoptosis [67]. On the contrary, overexpressing Bax promotes cytochrome
c release and activates apoptotic enzymes, leading to cell death [68].
Crosstalk of Various Apoptotic Pathways

As cell communication is very complex, crosstalk among different pathways is prevalent.
For example, during ER stress, CHOP inhibits Bcl-2 and activates growth arrest as well as
GADD34 and ER oxidase 1α expression, thereby promoting apoptosis [40, 69]. Bim and Bax
can also be regulated by CHOP during ER stress [70]. However, the role of CHOP in cell
death and survival may be context-dependent because GADD34 up-regulation by CHOP
results in feedback inhibition of eIF2α phosphorylation. This can lead to recovery of
translation, which may be beneficial if the factors causing ER stress are no longer
problematic; conversely, when translation persists or is re-initiated under ER-stress
conditions, accumulation of abnormal proteins may further erode the capacity of the ER for
protein folding, leading to cell death.
During ER stress, p53-upregulated modulator of apoptosis (PUMA) can be induced by
p53, and PUMA-deficient cells are resistant to ER stress-induced apoptosis, indicating an
important role of p53 and PUMA in ER stress-associated cell death [71]. In addition, the
activation of c-Jun N-terminal kinase (JNK) is also involved in ER stress-initiated apoptotic
cascades [4]. For example, tumor necrosis factor receptor-associated factor 2 (TRAF2) is
recruited to the JNK-IRE1α complex; TRAF2 then interacts with procaspase-12 and promotes
its clustering and activation by cleavage in response to ER stress [72, 73]. Furthermore,
accumulating evidence suggests that depletion of ER calcium, which induces ER stress,
activates m-calpain, which subsequently cleaves and activates procaspase-12 to initiate the
caspase cascade [74, 75].
Bcl-2 family members have also been shown to influence ER calcium homeostasis, as
well as ER-mitochondrial crosstalk, and by these means might indirectly affect ER stress-
induced cell death [76]. However, proapoptotic Bax and Bak can also interact directly with
IRE1, modulating its functioning during ER stress, and thereby regulating ER stress-induced
cell death at the level of the signaling of this protein [77]. Finally, these apoptotic pathways
converge on caspase-3, resulting in its activation and the cleavage of other proteins and
leading to apoptosis.
Common Pharmacological Agents Capable of Inducing ER Stress
Experimentally, to study the mechanism of ER stress responses in different cells, a

variety of different pharmacological agents are often used to induce ER stress. For example,
tunicamycin and thapsigargin are the most commonly used pharmacologic agents to
experimentally induce ER stress [78]. These agents effect ER stress by the mechanisms
described below.
Thapsigargin
Thapsigargin was originally isolated from the Mediterranean plant Thapsia garganica L.
(Linnaeus) and belongs to a group of related, naturally occurring 6,12-guaianolides. The high
lipid solubility of this compound accounts for its excellent penetration of biological
membranes [79]. Thapsigargin can disrupt ER stores of calcium, which is required for proper
ER function, as this drug is a specific inhibitor of the sarcoplasmic/endoplasmic reticulum
calcium-ATPase (SERCA) pumps and causes a discharge of the intracellular calcium store by
preventing reuptake of calcium that leaks from storage. Thus, it was first shown that
thapsigargin can increase free cytosolic calcium in platelets in 1985 [49, 80]. In the past 20
years, there are increasing reports using thapsigargin as a tool to induce ER stress [81, 82].
Thapsigargin is also able to induce the transcription of the GRP78 and GRP94 genes [83].
Tunicamycin
Tunicamycin is a bacterial toxin that inhibits the first step in the biosynthesis of N-linked
oligosaccharides in cells; by inhibiting glycosylation, tunicamycin prevents protein
maturation leading to an accumulation of immature proteins in the ER to induce ER stress
[84, 85]. ER-associated proteins, members of the Sec61 complex, and several aminoacyl-
tRNA synthetases are affected by tunicamycin treatment [49, 86]. In addition, it has been
reported that tunicamycin selectively up-regulates the cell surface expression of tumor
necrosis factor-α-related apoptosis-inducing ligand (TRAIL)-receptor-2 and enhances
TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates [87].
Brefeldin A
Brefeldin A (BFA), a fungal fatty acid metabolite, induces ER and Golgi stress by
inhibiting the action of ADP ribosylation factor (ARF), involved in ER to Golgi vesicle
trafficking [88]. Thus, brefeldin A is used widely as an inhibitor of vesicle transport between
the ER and the Golgi. After cells are treated with brefeldin A, Golgi membranes fuse with
those of the ER, resulting in accumulation of proteins in the ER, ER stress, and ultimately
apoptosis [89, 90].
Calcium Ionophores (A23187 and Ionomycin)

Calcium ionophores are used widely to study the role of calcium in the regulation of gene
expression in mammalian cells. A23187, also known as calcimycin, is a calcium ionophore
that induces ER stress by disrupting intracellular calcium homeostasis to induce caspase-12
cleavage and GRP78 and GRP94 protein expression in PC12 cells [59, 91]. A23187 also
causes accumulation of the mRNA for GRP78 and other ER luminal proteins [92-94]. In
addition, A23187 and ionomycin block the movement of secretory proteins from the rough
ER to the Golgi in human hepatoma HepG2 cells.
Dithiothreitol
Dithiothreitol (DTT), as a reducing agent, blocks the ER to Golgi transport of newly
synthesized proteins, such as immunoglobulin molecules, via preventing the formation of or
breaking disulfide bonds. For example, CHO-ATF6 cells treated with DTT exhibit reduced
disulfide bond formation resulting in unfolding of proteins. DTT can act on folded proteins
directly by reducing disulfide bonds as they are synthesized, and DTT mobilizes ATF6 into
COPII vesicles from the ER in the absence of protein synthesis [95]. In HepG2 cells
following activation of the UPR by DTT and homocysteine, GRP94, Bip and CHOP mRNA
and protein are induced in a dose-dependent manner [49, 96].
All of the pharmacological agents mentioned above are generally used to experimentally
induce ER stress, because their effect is to interfere with ER functions and thereby lead to ER
protein misfolding and/or accumulation. Generally, cells must be exposed to these agents for
4-48 hours, but the concentration and time of treatment depends on the cell type being studied
and is determined individually for each system.
The Regulation of Cholesterol Homeostasis
Cholesterol is an important component of mammalian cell membranes and is required for

the maintenance of membrane fluidity and permeability, regulation of integral membrane
protein function and formation of membrane microdomains or lipid rafts [97, 98]. Because
cholesterol is synthesized or absorbed and distributed in different cells, such as those of the
liver, blood, intestine, skin, brain, vasculature and adrenal [49, 99], this important lipid has
been the focus of research for several decades.
Although cholesterol‘s biosynthetic pathway has been investigated extensively, with
considerable knowledge obtained [100], there is a need to determine molecular mechanisms
of cholesterol absorption, metabolite regulation, and intracellular homeostasis (Figure 2). As
is well known, the normal function of cells requires intracellular cholesterol homeostasis, and
when cellular cholesterol is in excess or depleted, cell dysfunction results. Thus, cells have
developed complex mechanisms to regulate sterols. On the one hand, cells acquire cholesterol
exogenously from the circulation in the form of apolipoprotein B-containing lipoproteins,
such as low-density lipoprotein (LDL) [101-103]; on the other hand, cholesterol is
synthesized de novo from acetyl-coenzyme A (acetyl-CoA) via a complex metabolic pathway
[100]. Overall, it is important for cells to control the synthesis, uptake, efflux and
intracellullar transport of cholesterol.
Cholesterol Synthesis
It is generally considered that in mammalian cells cholesterol originates from both the
diet and endogenous biosynthesis. The dietary intake of cholesterol is limited, and therefore
the physiological requirements for cholesterol are supplied also through de novo synthesis.
Since cholesterol synthetic pathways have been investigated thoroughly for nearly 50 years
[100], this review will not describe in detail the biosynthesis of cholesterol. In brief,
mammalian cells synthesize cholesterol from acetyl-CoA through the mevalonate pathway in
the ER. The newly synthesized cholesterol is transported to subcellular membranes by the
biosynthetic secretory pathway via the Golgi or by non-vesicular pathways with the help of
cholesterol transfer proteins, such as sterol carrier protein-2 (SCP-2), steroidogenic acute
regulatory (StAR) and related StAR domain-containing proteins and lipid rafts/caveolae
[104]. In order to prevent excess accumulation of free cholesterol in the cells, surplus
cholesterol in the ER is esterified by acetyl-CoA cholesterol acetyltransferase (ACAT) to be
stored in cytosolic lipid droplets. Cholesterol esters can be rapidly released as free cholesterol
as needed by enzymes such as cholesterol ester hydrolase, also known as hormone sensitive
lipase (HSL) [105, 106].
Figure 2. Schematic presentation of cellular cholesterol influx, synthesis, transport and efflux. Low-density
lipoproteins (LDL) carrying cholesterol bind to LDL receptors in the plasma membrane. These receptors are then
transported into the cell by endocytosis, going first to sorting endosomes, then to late endosomes and finally to
lysosomes, where cholesterol is released and can traffic to the endoplasmic reticulum (ER) or plasma membrane.
Moreover, in late endosomes and lysosomes cholesterol esters are cleaved by lysosomal acid lipase (LAL) to
produce free cholesterol, which can be bound by Niemann–Pick disease, type C2 (NPC2) protein, transferred to
NPC1, and finally transported to other subcellular organelles. In addition, high-density lipoproteins (HDL) can also
carry cholesterol into the cell via scavenger receptor B1 (SRB1). Cholesterol is also synthesized de novo in the ER.
Cholesterol can be reesterified by acyl-CoA:cholesterol transferase (ACAT) and stored in lipid droplets; cholesterol
esters can be cleaved to free cholesterol by hormone-sensitive lipase (HSL). Free cholesterol can also be transported
into mitochondria by various transport proteins, in particular the steroidogenic acute regulatory protein, and used as
a precursor to synthesize steroid hormones in steroidogenic cells, such as cells of the adrenal gland. Additionally,
newly synthesized free cholesterol in the ER is mostly transported to the plasma membrane by a Golgi-bypass
route, but a portion of the cholesterol is transported via the Golgi complex and the trans-Golgi network to the
plasma membrane, where it is distributed either to raft or nonraft microdomains.
Cholesterol Intake
As cholesterol is a hydrophobic molecule, dietary cholesterol absorption by enterocytes
requires hydrolysis of any dietary cholesterol esters by pancreatic carboxyl ester lipase as
well as emulsification, micellar solubilization and transport through the circulation in the
form of lipoproteins [107, 108]. Cells acquire cholesterol from the circulation in the form of
LDL and other ApoE/ApoB-containing lipoproteins via the low-density lipoprotein receptor
(LDLR), through classical receptor-mediated endocytosis [49, 109].
Thus, the circulating LDL particles are internalized through the LDLR and transported to
early endosomes. Then, the cholesterol is subsequently transported to late endosomes and
lysosomes where cholesterol esters are hydrolyzed by acid lipase [110]. Because of the lower
pH of the endosome, in early endosomes LDL dissociates from the LDLR, which is recycled
back to the cell membrane by vesicular mechanisms [111, 112]. Scavenger receptor class B
type I (SR-B1) is a cell surface receptor for high density lipoprotein (HDL), and a critical
player in cholesterol uptake by the liver and steroidogenic tissues [113-116].
Cholesterol Transport in Cells

Because of the presence of different subcellular organelles, the mechanisms of
intracellular cholesterol transport are complex. Currently, two types of transport pathways
have been revealed: one is vesicular transport, which plays an important role in some
cholesterol transport processes, and the other is non-vesicular, which is the major mode of
intracellular cholesterol transport [117-119]. Several proteins have also been implicated in
transporting cholesterol throughout the cell. For example, SCP-2 is a small soluble lipid
transfer protein that is capable of transporting sterols and a variety of other lipids between
membranes in vitro [120]. Other cholesterol transport proteins include the StAR-related lipid
transport (START) domain proteins, which can bind cholesterol and play an important role in
the intracellular trafficking of cholesterol.
These proteins are located in the cytoplasm, bound to membranes or within the nucleus
[121]. For example, in steroidogenic tissues StAR is required for the transfer of cholesterol
from the outer to the inner mitochondrial membrane and the initiation of steroidogenesis via
the activity of the side-chain cleavage enzyme complex (p450scc or CYP11A1). On the other
hand, MLN64/StarD3 is involved in transferring cholesterol from the endolysosomal
compartment to the mitochondria [122, 123].
Niemann–Pick disease, type C1 and 2 (NPC1 and NPC2) proteins are located in the late
endosome, where they participate in cholesterol exit from this organelle. A deficiency of
either protein results in the accumulation of free cholesterol in late endocytic organelles, to
result in the disease Niemann–Pick type C (NPC) [124], characterized by progressive
neurological decline. NPC1 is a lysosomal–endosomal transmembrane protein and regulates
the transport of cholesterol from late endosomes/lysosomes to other subcellular organelles
[49]. NPC2 is a soluble lysosomal protein and seems to be responsible for rapidly
transporting cholesterol to phospholipid vesicles [125-127].
Niemann-Pick C1 like 1 (NPC1L1) protein is present in the brush border membrane of
enterocytes in the small intestine and is a major executor of intestinal cholesterol absorption
[128, 129]. Several members of the oxysterol-binding protein-related protein (ORP) family,
including ORP-9, ORP4-S, ORP-1L, and ORP5, which were first identified as cytosolic
receptors for 25-hydroxycholesterol [130], have been shown to mediate cholesterol
trafficking in the late endosomal system. For example, ORP9 may play a role in cholesterol
transport between the ER and the Golgi [131].
Finally, since Rab GTPases are thought of as the crucial moderator of the recruitment of
membrane-tethering and docking factors that facilitate membrane traffic, these proteins are
involved in cholesterol transport within cells. For example, Rab1 is expressed on the ER,
Rab6 on the Golgi, Rab5 on early endosomes and Rab7 on late endosomes [132], at which
locations these small GTPases act to promote vesicular trafficking. LDL-cholesterol can be
transported out of the late endosomal system by vesicular mechanisms mediated by Rab7 and
Rab9 [133, 134]. Although there is increasing evidence confirming that many proteins play
key roles in intracellular cholesterol transport, their exact roles are not always well
understood.
Cholesterol Efflux
Because cholesterol cannot be degraded in cells, excess cholesterol must be removed to
maintain cholesterol homeostasis. Reverse cholesterol transport (RCT) is a very important
pathway for removing surplus cholesterol from extrahepatic cells and tissues and transporting
it to the liver. RCT is mediated mainly by HDL particles [135].
Members of the ATP-binding cassette (ABC) superfamily play an important role in
promoting intracellular cholesterol efflux; for example, ABCA1 mediates the transport of
cholesterol, phospholipids, and other lipid metabolites from cells to lipid-depleted HDL
apolipoproteins [136]. ABCG1 exports excess cellular cholesterol to HDL and reduces
cholesterol accumulation in macrophages [49, 137]. ABCG5 and ABCG8 together form
heterodimers, which are involved in preventing the absorption of excess dietary cholesterol
from the intestine and promoting cholesterol efflux from hepatocytes into bile [138-140].
LXRs also play a key role in regulating cellular cholesterol metabolism since they can induce
the expression of the cholesterol transporters ABCA1 and ABCG1, and later ABCG5 and
ABCG8 [141]. Another mechanism for cholesterol removal is mediated mostly by ApoA-I
(the major apolipoprotein of HDL) and leads to the assembly of HDL particles containing
cholesterol and phospholipid [142].
Key Mediators of Intracellular Cholesterol Homeostasis

With studies investigating microRNAs, data are accumulating to show that the expression
of gene products controlling intracellular cholesterol levels is tightly regulated not only at the
transcriptional, but also at the post-transcriptional level. In addition, several bio-molecules
also are involved in maintaining intracellular cholesterol balance, such as the sterol regulatory
element-binding proteins (SREBPs), the liver X receptor (LXR) and the LDLR, as well as the
microRNAs that regulate the levels of these and other proteins (miR-33 and -122) [111, 143].
SREBPs are members of the basic helix-loop-helix leucine zipper (bHLH-Zip) family.
SREBP-2 activates genes involved in cholesterol biosynthesis [111, 144]; moreover, SREBP-
2 is controlled by a highly regulated negative feedback mechanism via downstream products
of the cholesterol biosynthetic pathway [145]. Thus, when intracellular cholesterol levels are
high, SREBP-2 is retained in the ER by the cleavage–activating protein (SCAP)/insulin-
induced gene (INSIG) complex [146], and cannot be processed by proteases in the Golgi to
active SREBP-2. This inactive SREBP-2 is unable to enter the nucleus to modulate the
expression of genes involved in cholesterol uptake and synthesis [147, 148]. When
intracellular cholesterol levels are low or depleted, the SCAP–INSIG interaction is disrupted
[149, 150], allowing SCAP to interact with the COPII trafficking complex. SCAP then
escorts SREBP-2 from the ER to the Golgi where it is cleaved by two membrane-bound
proteases, site-1 protease (S1P) and site-2 protease (S2P) [151].
Active proteolyzed SREBP-2 can then enter the nucleus, bind to a sterol response
element in the enhancer/promoter region of many target genes involved in cholesterol
synthesis and finally, activate their transcription. One such gene is 3-hydroxy-3-
methylglutaryl coenzyme A reductase (HMGCR), which, as the rate-limiting enzyme in
cholesterol biosynthesis, increases de novo cholesterol production, and the LDLR, which
allows increased cellular cholesterol uptake [152-154].
The LXR nuclear receptor subfamily 1, group H, includes member 3 (NR1H3, LXRα)
and member 2 (NR1H2, LXR). LXRs are important transcriptional regulators of cholesterol
homeostasis. Compared to the ubiquitous expression of LXR, LXRα is primarily expressed
in the liver, adipose tissue and macrophages and plays an important role in lipid metabolism
[155, 156]. Generally, LXRs form heterodimers with retinoid X receptors (RXRs) and are
activated in response to cellular cholesterol excess [157]. LXRs activate the transcription of
genes involved in cholesterol efflux, for example, ABCA1 and ABCG1, which promote
cellular cholesterol efflux to HDL and ApoA-I [158, 159]. On the other hand, ABCG5 and
ABCG8 promote cholesterol excretion into bile [140]. When cellular cholesterol levels
exceed the biosynthetic rate, a feed-forward pathway is initiated which promotes the efflux of
cellular cholesterol and helps to maintain cholesterol homeostasis [160, 161].
MicroRNAs (miRNAs) are small (22-nucleotide) endogenous double-stranded noncoding
RNAs that have emerged as post-transcriptional regulators of physiological processes [162].
miRNAs repress gene expression via binding to complementary target sites in the 3‘-
untranslated regions (3‘UTRs) of messenger RNA (mRNA) to promote mRNA degradation
or prevent mRNA translation [163]. Recently, several miRNAs, such as miR-33, miR-122,
miR-370, miR-378/378*, miR-143, miR-125a, miR-27 and miR-335, have been considered to
regulate cholesterol homeostasis [143, 164-168]. For example, miR-33 plays a key role in
maintaining cholesterol homeostasis via regulating target genes involved in cholesterol
export, including ABCA1 and ABCG1 and the endolysosomal transport protein NPC1 [143,
164, 169, 170].
In humans miR-33a is located in intron 16 of the SREBP-2 gene, and miR-33b is present
in intron 17 of the SREBP-1 gene. However, in mice there is only one miR-33 isoform
(which is homologous to human miR-33a) [143, 164]. In addition, during low-sterol
conditions, miR-33a overexpression in human hepatocytes and macrophages strongly
represses ABCA1 expression and reduces cellular cholesterol efflux to ApoA-I, which leads
to increased cellular cholesterol levels. However, inhibition of endogenous miR-33 leads to
increased ABCA1 expression and promotes cholesterol efflux to ApoA-I, suggesting that
miR-33 can regulate ABCA1 physiologically [143, 164, 169, 170]. On the other hand, as
human ABCG1 lacks miR-33 binding sites in its 3‘UTR, miR-33 repression of ABCG1 is
clearly not conserved across species. Additionally, miR-33 strongly inhibits NPC1 protein
expression in human cells, whereas in mouse cells, miR-33 suppresses NPC1 protein
expression only modestly and has no effect on NPC1 mRNA levels [164].
A recent report showed miRNA-128-2 to be a new regulator of cellular cholesterol
homeostasis, with the administration of miR-128-2 leading to a decline in the protein and
mRNA levels of ABCA1, ABCG1 and RXRα. Conversely, anti-miRNA treatment results in
increased ABCA1, ABCG1 and RXRα expression. Furthermore, miR-128-2 increases the
expression of SREBP2 and decreases the expression of SREBP1 in HepG2, MCF7 and
HEK293T cells [171], indicating the importance of this microRNA in cholesterol
homeostasis.
Crosstalk Exists between Cholesterol Levels and ER Stress

in Different Cell Types
In mammals, almost all cells can synthesize cholesterol; however, the liver has been
considered the major organ controlling the maintenance of cholesterol homeostasis [102].
Other organs, such as the aorta, brain and adrenal gland, have their own mechanisms of
cholesterol homeostasis [172, 173].
Excess cholesterol in cells causes different effects in various subcellular organelles. In
particular, endoplasmic reticulum membranes, which normally contain low levels of
cholesterol [174], are likely to be particularly sensitive to abnormal free cholesterol
enrichment and dysregulation of cholesterol homeostasis. Indeed, evidence suggests that
elevated cholesterol levels and dysregulated cholesterol metabolism can induce ER stress. For
example, in macrophages cholesterol accumulation in the ER, rather than the plasma
membrane, triggers ER stress, induces CHOP and results in apoptosis [175]. On the other
hand, ER stress also appears to be able to alter cholesterol homeostasis.
Thus, when mouse embryo fibroblasts are incubated in a lipoprotein-deficient FBS
(LPDS)-containing medium, the expression of Tm7sf2, a gene product involved in the
conversion of lanosterol to cholesterol, is up-regulated, and cellular cholesterol levels are
increased. Cholesterol levels are also increased by exposure to ER stress-inducing
thapsigargin. This result suggests that Tm7sf2 is involved in cholesterol biosynthesis under
ER stress conditions; further, the stimulation of cholesterol synthesis upon induction of ER
stress implies that cholesterol may participate in decreasing vulnerability to ER stressors
[176]. Additional examples of this crosstalk between cholesterol levels and ER stress in
specific tissues and cell types are discussed below.
The Liver
Excess cholesterol in the liver may trigger ER stress, which can harm liver function. Free
cholesterol accumulation appears to induce ER stress by altering the critical free cholesterol-
to-phospholipid ratio of the ER membrane. The importance of free cholesterol accumulation
for triggering ER stress has been recently documented in hepatocytes where ER membrane
cholesterol accumulation, rather than total cellular cholesterol overload, induces hepatic ER
stress, while hepatocyte ER cholesterol lowering by two independent approaches resolves ER
stress [177]. On the other hand, ER stress can also modulate hepatic cholesterol metabolism.
For instance, overexpression of Ildr2 (immunoglobulin-like domain containing receptor 2), an
ER membrane-localized protein, increases the expression of the key ER stress molecules
PERK, ATF6 and IRE1 and concomitantly reduces hepatic triglyceride and total cholesterol
levels. On the other hand, Ildr2 knockout mice, which show decreased PERK, ATF6 and
IRE1 expression, exhibit hepatic lipid accumulation, indicating that manipulation of Ildr2
expression in liver affects both lipid homeostasis and ER stress pathways [178].
In addition, in transgenic mice with an eIF2α phosphorylation defect, the expression of

PPARγ and its lipogenic target genes is reduced under the stress of a high-fat diet. Repressed
expression of C/EBPα and C/EBPβ proteins is also observed in the livers of these transgenic
mice [179], again suggesting an association between ER stress and lipid metabolism. When
ER stress is induced in human hepatocarcinoma HepG2 cells, ABCA1 expression is
decreased thereby reducing cholesterol efflux to apolipoprotein A-1 (ApoA-I) by 80% in a
liver X receptor-independent manner [180]. Despite the reduced cholesterol efflux, cellular
cholesterol levels remain unchanged during the induced ER stress, due to impaired de novo
cholesterol synthesis upon reduction of HMG-CoA reductase activity by 70% [180].
This result may also explain the finding that plasma cholesterol and triglyceride levels are
markedly decreased in tunicamycin-injected mice [181]. In addition, tunicamycin challenge
provokes steatosis in C57BL/6 mice in vivo; in these mice alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), markers of liver injury, are increased, and the Bax/Bcl-
2 expression ratio as well as caspase-3 mRNA levels are significantly enhanced in the liver
upon tunicamycin treatment [182]. Moreover, livers from peroxisome-deficient PEX2-/- mice
exhibit activated SREBP-2 and ER stress pathways, especially the integrated stress response
mediated by PERK and ATF4 signaling, which perturbs the flux of mevalonate metabolites,
alters bile acid homeostasis and changes fatty acid levels and composition, and increases
oxidative stress, to result in an inability to maintain normal cholesterol homeostasis [183].
Finally, the ability of the liver to upregulate ER stress pathways appears to be important
in maintaining lipid homeostasis. Thus, homocysteine, dithiothreitol (DTT), and tunicamycin
all cause ER stress and significantly increase the steady-state mRNA levels of GRP78 and
CHOP in HepG2 (hepatocarcinoma) cells; however, overexpression of GRP78, which
attenuates ER stress and UPR activation, has been shown to decrease hepatic steatosis by
reducing SREBP-1c activity [184, 185]. This result thus demonstrates a direct role of ER
stress in the activation of cholesterol/triglyceride biosynthesis, leading to increased hepatic
biosynthesis and uptake of cholesterol and triglycerides [184, 185]. ER stress stimulated by
acetaldehyde also increases SREBP-1 in HepG2 cells [186].
The injection of tunicamycin into ATF6α-knockout mice induces the accumulation of
triacylglycerol and cholesterol in the liver as well as liver dysfunction and steatosis. Mouse
embryonic fibroblasts deficient in ATF6α are sensitive to ER stress, indicating the importance
of ATF6α-mediated upregulation of the levels of ER quality control proteins in protecting the
liver from ER stress-induced damage [187]. Thus, there is clear evidence linking hepatic
cholesterol metabolism and ER stress/UPR pathways.
Atherosclerosis and Macrophages

Atherosclerosis is a progressive and chronic disease characterized by accumulation of
lipids and fibrous elements in large arteries. Again, elevations in free cholesterol levels can
induce ER stress in macrophages and other cells associated with atherosclerotic lesions. For
example, THP-1 macrophages loaded with lipids by incubation with oxidized LDL (oxLDL)
show increased ER stress upon treatment with oxLDL, 9-hydroxy-octadecadienoic acid (9-
HODE) or 4-hydroxynonenal (4-HNE) (oxidation metabolites of the polyunsaturated fatty
acids in LDL). This induction of ER stress by oxLDL, 9-HODE and 4-HNE occurs by
activation of eIF-2α and stress-activated protein kinase (SAPK)/JNK (p54/p46) signaling
pathways. In these macrophages HDL3 diminishes ER stress by stimulating cholesterol efflux
from the lipid-loaded cells [188], suggesting the importance of cholesterol in inducing ER
stress in macrophages. Similarly, upon treatment with acetylated LDL (AcLDL) THP-1
macrophages demonstrate an 8-fold rise in ER free cholesterol levels. Concomitantly, a
significant increase in CHOP mRNA expression and a decrease in Bcl-2 mRNA level are
observed, suggesting that ER stress is involved in AcLDL-induced apoptosis upon
accumulation of free cholesterol in the ER [189].
In cultured peritoneal macrophages as well, the accumulation of free cholesterol in the
ER induces ER stress, and various components of the ER stress response are activated, to lead
to cell death [175, 190]. Transport to the ER appears to be required for this cholesterol-
induced apoptosis, as treatment of cholesterol-loaded macrophages with U18666A, which
selectively blocks cholesterol trafficking to the ER, results in a reduction in apoptosis and a
block of cholesterol-induced IκB kinase (IKK) activation and p65 NF-κB translocation [175,
190], indicating that NF-κB activation depends on cholesterol trafficking to the ER.
Furthermore, inhibition of cholesterol trafficking to the ER markedly and selectively
diminishes macrophage apoptosis and lesional necrosis in advanced atherosclerotic plaques
[190]. ER stress is likely involved in this process, as cholesterol-induced apoptotic cell death
is attenuated in CHOP-deficient macrophages [175].
IRE1 is also an important contributor to cholesterol-induced macrophage apoptosis since
in IRE1-deficient macrophages apoptosis induced by ER stress is inhibited [191].
Additionally, p38 mitogen-activated protein kinase signaling is necessary for CHOP
induction as well as apoptosis in macrophages, suggesting an involvement of this pathway in
destabilization of plaques in advanced atherosclerotic lesions [192].
ER stress can also regulate cholesterol metabolism in macrophages. Thus, with
atherosclerosis, apolipoprotein E (ApoE)-deficient, apolipoprotein B48 (ApoB48)-containing
(E-/B48) lipoproteins, by activating the PERK-eIF2α signaling cascade in cell types within
the developing atherosclerotic lesion, down-regulate lysosomal hydrolase synthesis, inhibit
lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester
accumulation, resulting in foam cell formation [193]. Indeed, ER stress is a key regulator of
macrophage differentiation and cholesterol deposition, and suppression of ER stress shifts
differentiated M2 macrophages toward an M1 phenotype and subsequently suppresses foam
cell formation by increasing HDL- and apoA-1-induced cholesterol efflux [194]. In addition,
exposure of the macrophage cell line RAW264.7 cells to mildly oxidized LDL induces
GRP78 expression, followed by CHOP up-regulation and ATF6 activation in a concentration-
and time-dependent manner.
Knockdown (via siRNA) of ATF6, which attenuates oxLDL-induced upregulation of
CHOP, also inhibits cholesterol accumulation and apoptosis in macrophages [195]. Also
suggesting a likely link between cholesterol homeostasis and ER stress in macrophages is the
fact that the expression of StarD5 increases 3-fold in free cholesterol-loaded macrophages in
which the ER stress response is activated [196]. StarD5 is a cholesterol transfer protein with
homology to the steroidogenic acute regulatory (StAR) protein critical for steroid hormone
production. Indeed, StarD5 can stimulate steroidogenesis in an in vitro overexpression assay,
indicating StAR-like cholesterol transfer activity [196]. Similarly, when NIH-3T3 fibroblasts
are treated with tunicamycin to induce the ER stress response, GRP78 increases 10-fold as
expected. However, in addition, StarD5 expression is increased 6-8-fold, again suggesting
that StarD5 expression is activated by ER stress and suggesting key interactions between
cholesterol metabolism and ER stress [196].
Macrophages from atherosclerosis-susceptible DBA/2 ApoE knockout (-/-) mice show

significantly higher levels of total and esterified cholesterol compared to atherosclerosis-
resistant AKR ApoE(-/-) mouse macrophages, while free cholesterol levels are higher in AKR
cells. In addition, the expression of DDIT3, TRIB3, and ATF4 is highly up-regulated by
cholesterol in AKR, and either down-regulated or unchanged in loaded DBA/2 macrophages,
thus associating a robust ER stress response with atherosclerosis resistance [197]. On the
other hand, a comparison of male ApoE knockout (Chop+/+/ApoE-/-) mice and Chop and
ApoE double-knockout (Chop-/-/ApoE-/-) mice fed a high-cholesterol diet indicated that
CHOP exacerbated apoptosis and plaque rupture. Thus, apoptosis is observed in a large
number of CHOP-expressing macrophages in ruptured advanced atherosclerotic lesions in
Chop+/+/ApoE-/- mice, whereas Chop-/-/ApoE-/- mice exhibit few apoptotic cells. In addition,
atherosclerotic plaque rupture is significantly reduced in high cholesterol–fed Chop-/-/ApoE-/-
mice compared with Chop+/+/ApoE-/- mice, suggesting in this case that ER stress may
contribute to atherosclerotic plaque rupture [198]. Studies in mice with bone marrow
transplantation indicate the importance of macrophage apoptosis to this process, and further
investigation demonstrates that unesterified free cholesterol accumulation in the ER induces
ER stress-mediated apoptosis in a CHOP-Bcl2-associated X protein (Bax) pathway-
dependent manner [198].
UPR activation occurs at all stages of atherosclerotic lesion development, including early
lesions in 9-week-of-age ApoE-/- mice and advanced lesions in 23-week-of-age ApoE-/- mice.
Nevertheless, despite the fact that accumulation of free cholesterol can be detected in
macrophage foam cells in early lesions, these cells exhibit no evidence of apoptotic cell
death; on the other hand, apoptotic cell death can be observed in a small percentage of
macrophage foam cells in advanced lesions, suggesting the possibility that activation of other
cellular mediators and/or pathways is required for apoptotic cell death in response to
cholesterol accumulation [199]. In sum, then, although there is clearly an association between
ER stress, apoptosis, cholesterol homeostasis and atherosclerosis, the exact relationship is
unclear at present and may differ according to the stage of the process.
Similarly to its effects on macrophages, oxLDL induces a time-dependent activation of
ER stress transducers in human microvascular endothelial cells, for example, increasing the
phosphorylation of PERK and IRE1α, which leads to CHOP activation and the expression of
protective ER chaperones. In these cells it has been suggested that oxysterols and lipid
peroxidation derivatives of polyunsaturated fatty acids participate in the activation of ER
stress by oxLDLs, and that ER stress plays a role in oxLDL-induced apoptosis through a
pathway involving IRE1α and JNK [200]. Likewise, in human umbilical artery endothelial
cells, silencing of endothelial ABCG1 (a cholesterol efflux transporter) increases GRP78 and
CHOP expression and promotes endothelial cell apoptosis. The apoptotic response and
expression of GRP78 and CHOP are suppressed by depletion of cellular cholesterol in
ABCG1-deficient endothelial cells, again suggesting the importance of intracellular free
cholesterol accumulation to subsequent ER stress and apoptosis [201].
Finally, cholesterol seems to induce ER stress in vascular smooth muscle cells as well.
Thus, incubation of rat aortic smooth muscle cells with "water soluble cholesterol" and an
acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor results in the development of free
cholesterol overload. In this model, dose-dependent expression of CHOP, as well as
upregulation of GRP78/GRP94 in cholesterol-overloaded smooth muscle cells, is observed,
suggesting that free cholesterol-induced death of smooth muscle cells can be mediated by ER
stress-related pathways [202].
The Brain
Excess cholesterol deposits and altered cholesterol metabolism appear to contribute to the
pathogenesis of Alzheimer's disease (AD), which is characterized in part by accumulation in
the brain of amyloid beta (Aβ) protein. For example, ApoE, a particular allele of which is a
known risk factor for the development of late-onset AD, is a crucial player in cholesterol
homeostasis in the brain and modulates Aβ clearance [203]. In addition, the oxysterol 27-
hydroxycholesterol, a cholesterol metabolite that is elevated with hypercholesterolemia,
induces Aβ production in rabbit hippocampus, and siRNA to CHOP reduces this response by
mechanisms involving a reduction in the levels of the β-amyloid precursor protein and the
protease that cleaves it, β-secretase. This result implies that ER stress-mediated CHOP
activation may play a central role in the triggering of the pathological hallmarks of AD [204].
In human retinal pigment epithelial cells treatment with 27-hydroxycholesterol also
increases Aβ peptide production, as well as caspase 12 and CHOP levels, and triggers Ca2+
dyshomeostasis and ER stress, suggesting that ER stress induced by high levels of 27-
hydroxycholesterol may represent a common pathogenic factor for both Alzheimer's disease
and age-related macular degeneration [203].
Aβ itself also promotes ER stress and can increase cholesterol synthesis and
mitochondrial cholesterol trafficking; Aβ–mediated ER stress also results in pathologic
progression in aging amyloid precursor protein/presenilin-1 (APP/PS1) mice as a result of
mitochondrial glutathione depletion. Treatment with the ER stress inhibitor 4-phenylbutyric
acid, which prevents mitochondrial cholesterol loading and glutathione depletion, protects
APP/PS1 mice from Aβ-induced neurotoxicity [205]. Finally, prolonged activation of the
UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis
models [206].
In contrast, other evidence suggests that cholesterol can also be protective in AD. Thus,
overexpression of 3β-hydroxysteroid-Δ24 reductase (DHCR24), which catalyzes the
conversion of desmosterol to cholesterol (the final step in cholesterol biosynthesis), protects
neuroblastoma N2A cells from apoptosis induced by tunicamycin-elicited ER stress.
DHCR24-overexpressing cells exhibit reduced Bip and CHOP protein expression, and
elevated intracellular cholesterol levels [207]. Thus, although the two are clearly related, the
exact interactions between cholesterol and ER stress in AD requires further investigation.
The Adrenal Gland

At present, there are few reports about ER stress and steroid hormone production in the
adrenal gland. However, since cholesterol serves as the precursor for the synthesis of all
steroid hormones, and ER stress and cholesterol metabolism are linked in other systems, it
seems possible that ER stress could affect steroidogenesis in the adrenal cortex. For example,
during the process of cholesterol uptake and transfer to the mitochondria as well as the
maintenance of cholesterol homoeostasis, LXRα plays a crucial role in regulating multiple
processes, including cholesterol uptake through lipoprotein receptors, cholesterol ester
hydrolysis, and endosomal transport [208]. LXRα is highly expressed in the adrenal gland;
therefore, similar cholesterol homeostatic mechanisms may operate in the adrenal gland as in
other cells. In addition, a stimulator of steroid hormone production, steroidogenesis activator
peptide (SAP) appears to be derived from GRP78 by proteolysis [209]. Thus, GRP78 is
cleaved to the approximately 30 kDa SAP, which acts synergistically with GTP to enhance
steroidogenesis [210]. We can therefore speculate that ER stress may be involved in steroid
hormone synthesis.
Further interaction between steroid hormone-producing cells and ER stress likely arises
through an ability of one steroid hormone, cortisol, to regulate metabolism. Thus, cortisol,
produced and released from zona fasciculata cells of the adrenal cortex during stress, is a
hormone that regulates glucose and lipid metabolism [211]. During periods of hypoglycemia,
such as occurs with fasting, the release of cortisol promotes the use of fatty acids as an energy
source in multiple tissues and increases gluconeogenesis in the liver, thereby ameliorating the
glucose deficiency [212].
Pioglitazone is a drug used to treat diabetes, a disease characterized by hyperglycemia
and dyslipidemia. It (as well as other thiazolidinediones) is thought to act through peroxisome
proliferation activator receptors (PPARs) [213-216], nuclear hormone receptors that are
activated by lipid metabolites and affect lipid metabolism [217, 218]. In diabetes pioglitazone
decreases serum glucose and lipid levels; however, it has the unwanted side effect of
promoting edema in some individuals [219]. We hypothesized that pioglitazone might
increase the production of aldosterone, which would then promote sodium retention thereby
contributing to edema.
To investigate this idea we used a model of adrenocortical cells, the HAC15 human
adrenocortical carcinoma cell line. HAC15 cells can produce all of the steroid hormones
synthesized by the adrenal cortex, including aldosterone and cortisol [220]. Treatment of the
HAC15 cells with pioglitazone resulted in the upregulation of the expression of a number of
genes, including steroidogenic acute regulatory protein (StAR), the early rate-limiting step in
the biosynthesis of all steroid hormones, and CYP11B2 encoding aldosterone synthase, the
late rate-limiting step in aldosterone production [221]. Somewhat unexpectedly, despite
increasing CYP11B2 mRNA levels dramatically, pioglitazone actually decreased CYP11B2
protein expression and aldosterone production and inhibited the ability of angiotensin II, the
primary physiologic regulator of aldosterone secretion, to enhance these parameters.
However, we found that pioglitazone also increased the expression of DDIT3, otherwise
known as CHOP, suggesting that pioglitazone might induce ER stress. Indeed, pioglitazone
increased CHOP protein levels as well as eIF2α phosphorylation, and our data suggest that
pioglitazone is able to inhibit CYP11B2 protein expression and thus aldosterone production
by blocking global protein translation initiation through ER stress and phospho-eIF2α. On the
other hand, pioglitazone increased the expression of CYP11B1, a key enzyme for the
synthesis of cortisol, and enhanced angiotensin II-induced cortisol production [221].
Therefore, it appears that ER stress induced by PPAR activation can modify the steroid
hormones produced by the adrenal cortex, promoting the secretion of cortisol, a hormone that
helps the body to cope with stress and regulate metabolism. However, under certain
conditions cortisol can also activate the mineralocorticoid receptor normally stimulated by
aldosterone to increase sodium retention. Indeed, cortisol has been proposed to play a role in
the metabolic syndrome, which is usually characterized in part by hypertension [222]. Thus,
the ability of pioglitazone to induce ER stress to promote the production of cortisol, rather
than aldosterone, in the adrenal gland may underlie its unwanted side effect of edema.
Cells derived from another steroidogenic tissue, Chinese hamster ovary (CHO) cells, also
exhibit changes in ER stress following an elevation of cholesterol levels. Thus, CHO cells
expressing human ApoE under a cholesterol-independent promoter and incubated with

cholesterol-cyclodextrin complexes show increased levels of cellular free and esterified
cholesterol, inhibition of SREBP-2 processing, and a mild induction of ER stress. Secretion
of ApoE from these cells is markedly inhibited by cholesterol accumulation, indicating that
intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and
secretion [223]. However, steroid hormone production was not investigated.
In summary, then, it appears that the relationship between ER stress and lipid metabolism
is bidirectional. Thus, while activation of ER stress pathways can result in lipogenesis and
altered lipid homeostasis, lipids and aberrant lipid metabolism can also cause ER stress in
many tissues (see above and [224]). Nevertheless, few studies have investigated the
relationship between ER stress and steroid hormone biosynthesis from cholesterol, and further
research is needed to fully understand the complex relationship between the two cell
responses.
CONCLUSION
In this review, we have described the ER stress/UPR activation pathways, and
intracellular cholesterol homeostasis regulatory mechanisms. There has been enhanced
interest in the connection between intracellular cholesterol and ER stress. The general
mechanism of both are fairly well established; however, some important properties remain
poorly characterized, and much work remains to be performed to better understand
cholesterol homeostasis in different tissues and cells, particularly steroidogenic cells, as well
as how this process is regulated by ER stress under abnormal conditions. It is likely that
important new findings concerning crosstalk among the signaling molecules remain to be
discovered.
The role of cholesterol in cells has been studied for decades, and many of the cellular and
molecular mechanisms of its action have been revealed in considerable detail. However, even
in this case, there are important unresolved questions that require probing: for example, how
does cholesterol move among organelles such as the ER, endosomes, lysosome and the Golgi
apparatus, and which proteins are key players in this intraorganellar transport? How are free
cholesterol and esterified cholesterol transferred between the ER and cytoplasmic lipid
droplets? How is cholesterol transferred and utilized under cell stress? How is cholesterol
exported to extracellular acceptors? And how is cholesterol deposited ectopically in vascular
endothelial and other cells to induce atherosclerosis and other diseases?
Moreover, since cholesterol is a major regulator of lipid organization, its cellular
concentration must be maintained within a narrow range, and cells have developed precise
mechanisms for accomplishing this. However, it is still not entirely clear what the normal
range of cholesterol concentration in each organelle is. When intracellular cholesterol is in
excess or deficient, ER stress is likely involved in regulating a return to homeostasis, but the
exact mechanisms by which ER stress signaling pathways effect cholesterol homeostasis are
incompletely understood. In addition, the severity and persistence of ER stress are difficult to
determine quantitatively among different cell types.
Therefore, it is necessary to study the connection between ER stress and cholesterol-
related diseases at different stages of the disease process, such as the early and later periods,
in order to understand the dynamic process of disease development. Since cholesterol is also a
precursor for steroid hormone synthesis, the ability of ER stress to modulate cholesterol
homeostasis suggests the possible involvement of this process in steroidogenesis, yet few
studies have examined the connection of these two processes. Thus, the response of
steroidogenic cells to ER stress and their involvement in cholesterol homeostasis requires
further investigation as well.
ACKNOWLEDGMENTS
Dr. Bollag was supported by a VA Research Career Scientist Award. The contents of this
article do not represent the views of the Department of Veterans Affairs or the United States
Government. Zhi-qiang Pan was a Visiting Scholar at Georgia Regents University.
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Chapter 3
REGULATION OF CATECHOLAMINE PRODUCTION

FROM THE ADRENAL MEDULLA
Anastasios Lymperopoulos, Smit Chowdhary,

Kamarena Sankar and Isis Simon
From the Laboratory for the Study of Neurohormonal Control of the Circulation,
Dept. of Pharmaceutical Sciences,
Nova Southeastern University College of Pharmacy,
Ft. Lauderdale, FL, US
ABSTRACT
The circulating catecholamines epinephrine (adrenaline) and norepinephrine
(noradrenaline) derive from two major sources in the whole organism: the sympathetic
nerve endings, which release norepinephrine on effector organs, and the chromaffin cells
of the adrenal medulla, which are cells that synthesize, store and release epinephrine
(mainly) and norepinephrine upon acetylcholine stimulation of the nicotinic cholinergic
receptors (nAChRs) present on their membranes. Thus, the chromaffin cells of the
adrenal medulla function, in essence, as postganglionic sympathetic neurons and
constitute the major source of circulating epinephrine in the body. All of the epinephrine
in the body and a significant amount of circulating norepinephrine derive from the
adrenal medulla. The secretion of catecholamines from adrenal chromaffin cells is
regulated in a complex way by a variety of membrane receptors present in these cells.
Most of these receptors are G protein-coupled receptors (GPCRs), including adrenergic
receptors (ARs), which act as ―presynaptic autoreceptors‖ in this regard. The majority of
hormones and of the receptors they activate on chromaffin cell membranes stimulate
catecholamine secretion, with the notable exception of the α2ARs, which inhibit
catecholamine secretion (inhibitory presynaptic autoreceptors). The present chapter will
discuss the molecular mechanisms by which all of these receptors regulate catecholamine

Corresponding author: Anastasios Lymperopoulos, PhD, FAHA, Associate Professor, Department of
Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, 3200 S. University Dr., HPD
(Terry) Bldg/Room 1338, Fort Lauderdale, FL 33328, USA, Tel.: 954-262-1338, FAX: 954-262-2278, E-mail:
al806@nova.edu.
54 Anastasios Lymperopoulos, Smit Chowdhary, Kamarena Sankar et al.
synthesis and secretion by the chromaffin cells of the adrenal medulla, highlighting
significant gaps in the knowledge requiring future research to fill them.
Keywords: adrenal medulla, chromaffin cells, catecholamines, sympathetic nervous system,

regulation of hormone secretion
INTRODUCTION
Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal
gland. Most existing knowledge of these cells has been the outcome of extensive research
performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the
physiology of this neuroendocrine cell in humans has been gained.
Precursor cells originating in the neural crest migrate from primitive spinal ganglia (6th
thoracic to first lumbar) to form the primitive sympathetic nervous system located dorsally to
the aorta. Some sympathetic cells (sympathogonia) migrate further in nerves that sprout from
the sympathetic chain, and alongside large blood vessels that penetrate into the (as yet) non-
encapsulated fetal adrenal cortex, primarily its caudal pole (head). The neural cells enter the
adrenal primordium as fingerlike processes and pass among the fetal cortical cells [1]. These
primitive sympathetic cells in the human fetal adrenal medulla can give rise to a neural or
endocrine–catecholamine-storing phenotype [2, 3]. The central areas of the primitive
elements do not undergo chromaffin differentiation and instead assume characteristics of
sympathetic neuroblasts.
Most of the medulla of the human adrenal gland occurs in the head of the gland, some
occurs in the body, and there is usually none in the tail [4]. The medulla extends in variable
measure into the crest of the gland (the ridge on the posterior surface) and into one or both of
the alae. Some areas of the medulla in the alae are not necessarily in direct continuity with the
main mass of the medulla around the central veins. The medulla sometimes extends into the
tail of the gland. Very occasionally, a narrow tongue of medulla accompanied by a vessel or
nerve, or unaccompanied, extends through the cortex to contact the capsule of the gland [1].
Splanchnic nerve activity or chemicals that reach the adrenal medulla via the bloodstream
may trigger catecholamine release from adrenomedullary chromaffin cells in the human body.
Catecholamine secretion induced by splanchnic nerve stimulation takes place in situations of
fear, anxiety, or organic stress. Allergic reactions or hypotension produce endogenous
compounds such as histamine, bradykinin, or angiotensin II that also stimulate catecholamine
release. One of the main roles of catecholamines is to ensure an adequate blood flow and
energy supply to vital organs to cope with stressful situations. Several nanograms of
catecholamines per minute are released under basal conditions. However, during a fight-or-
flight reaction there is massive adrenaline (70%) and noradrenaline (30%) input into the
circulatory system, increasing their plasma concentrations up to 60 times [5, 6].
Catecholamines also carry out important regulatory functions at several dopaminergic,
noradrenergic and adrenergic synapses. In addition, many other compounds such as opioids,
ATP, ascorbate, chromogranins and catestatin, co-stored with catecholamines and co-released
from the same vesicles, are able to regulate catecholaminergic effects in an
autocrine/paracrine manner.
Regulation of Catecholamine Production from the Adrenal Medulla 55
The present chapter will give a brief overview of the physiology of adrenal
catecholamine synthesis and secretion and then will outline the plethora of regulatory
mechanisms (hormones and their receptors) that tightly regulate these very important
biological processes in adrenal chromaffin cells.
PHYSIOLOGY OF CATECHOLAMINE SYNTHESIS, STORAGE, AND

SECRETION IN CHROMAFFIN CELLS
The fast secretory response evoked by short pulses of the physiological agonist
acetylcholine is mediated through the nicotinic current, partially composed of calcium ions
flowing across the ionophore receptor, and by the elicited depolarization that opens voltage-
dependent calcium channels (VDCC), with the subsequent calcium entry that triggers
neurotransmitter secretion. A more sustained response, yielded by long ACh pulses, is
achieved by activation of nAChRs, which recruit VDCC via prolonged depolarization, and
muscarinic receptors that mobilize calcium from the endoplasmic reticulum [7].
Catecholamines are co-stored in chromaffin vesicles together with soluble peptides,
including enkephalins [8], chromogranin A (CgA) [9], and neuropeptide Y (NPY) [6], which
are subject to exocytotic co-release with catecholamines [10, 11]. The main catecholamine
present in adult human adrenal gland is adrenaline [12]. Consequently, chromaffin cells
exhibit an adrenergic phenotype [13]. However, short-term, high-intensity dynamic exercise,
prolonged low-intensity dynamic exercise, and adoption of an upright posture, in decreasing
order of potency, predominantly stimulated noradrenaline release from sympathetic nerve
endings [6].
Thus, under different stress conditions, adrenaline is preferentially released from
chromaffin cells over noradrenaline. Catecholamine secretion in human chromaffin cells is
regulated by NPY, presumably acting via the putative y3 receptor [10], though factors such as
angiotensin II [14] or interleukin-1 [15] have also been found to regulate the release of
catecholamines and NPY (see also below).
As far as catecholamine biosynthesis is concerned, the enzyme tyrosine hydroxylase (TH)
catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA), a substrate for
the aromatic amino acid decarboxylase (LAAD), which converts L-DOPA to dopamine. This
initial step of the synthetic pathway is rate limiting and controls the rate of synthesis through
the entire pathway.
Several isoforms of TH can arise from alternative splicing of a single primary transcript.
Dopamine is converted to noradrenaline by dopamine beta-hydroxylase (DBH). Finally,
noradrenaline is converted to adrenaline by phenylethanolamine-N-methyl-transferase
(PNMT). In addition, vesicular monoamine transporter (VMAT) has been identified as a key
transporter in catecholamine biosynthesis in the human adrenal medulla. This protein is
integrated into the membrane of intracellular vesicles and transports cytosolic monoamines
into synaptic vesicles, using the proton gradient maintained across the synaptic vesicular
membrane.
Two VMAT isoforms are co-expressed in human chromaffin cells, VMAT1 and VMAT2
[16].
VMAT2, also known as SLC18A2, mostly transports dopamine, norepinephrine,

serotonin, and histamine. In humans, the VMAT2 protein is encoded by the SLC18A2 gene
[17].
Its correct function is key to the normal activity of monoaminergic systems that have
been implicated in a number of human neuropsychiatric disorders.
Catecholamines are co-stored and co-released with a group of acidic secretory proteins
(chromogranins). The main member of the chromogranin family is CgA, but human
chromaffin cells additionally possess B and C subtypes [18].
The adrenal medulla is the greatest normal neuroendocrine tissue source of CgA [19].
Elevated CgA is greater after intense adrenal medullary stimulation than after robust
sympathetic neuronal stimulation [6].
Chromaffin cells, like neurons, release transmitters by Ca2+-regulated exocytosis.
Catecholamines released from the cell are rapidly oxidized generating an electrical current
that is directly proportional to the number of catecholamine molecules. With suitable
stimulation protocols transient current spikes reflecting catecholamine release from individual
vesicular fusion events can be detected and analyzed. In situ, chromaffin cells are innervated
by splanchnic nerve terminals that release acetylcholine (ACh) and other transmitters,
including pituitary adenylate cyclase-activating polypeptide (PACAP).
Activation of nAChRs on the chromaffin cells causes membrane depolarization/action
potential firing, opening of voltage-gated Ca2+ channels, and Ca2+ influx that triggers fusion
of the vesicles with the plasma membrane [20]. Consequently, Ca2+ channels play pivotal
roles in stimulus-secretion coupling and are important targets for mechanisms that control
transmitter release. Significant advances have also been made toward identifying the protein–
protein interactions involved in exocytosis and it is now generally accepted that SNARE
(―Soluble NSF Attachment Protein Receptor‖) proteins constitute the core fusion machinery.
Several members of the synaptotagmin family serve as Ca2+ sensors for exocytosis [21],
and, in particular, synaptotagmins 1 and 7 are involved in chromaffin cells [22].
The SNAREs are also involved in other aspects of the secretory vesicle cycle and
recently it was proposed that docking of vesicles in mouse chromaffin cells is mediated by
binding of synaptotagmin-1 to binary t-SNARE complexes [23].
REGULATION OF CATECHOLAMINE SECRETION

FROM CHROMAFFIN CELLS
G protein-Coupled Receptor (GPCR) Signaling in Chromaffin Cells
GPCRs play central roles in orchestrating the dynamic modulation of transmitter release.
Heterotrimeric G proteins act as molecular switches to transduce extracellular ligand binding
to the GPCR into intracellular signaling cascades. Agonist binding to the GPCR catalyzes the
exchange of GDP to GTP on the G protein α-subunit (Gα) and both Gα and the free Gβγ
subunits signal to downstream effectors [24].
Chromaffin cells express a wide variety of GPCRs that sense and respond to changes in
the local environment and perhaps the overall physiological ―status‖ of the animal through
hormones and other blood borne signals. A common theme at chromaffin cells and synapses
is feedback modulation, whereby the released transmitters not only convey information to
downstream targets but also act in an autocrine manner to modulate subsequent secretory
activity.
In general, GPCRs that couple to Gi-type G proteins inhibit catecholamine release,
whereas Gq-coupled receptors and Gs-coupled receptors potentiate catecholamine release.
Autoreceptors for ATP (P2Y receptors), catecholamines (2ARs, see below), and enkephalin
(μ-opioid receptors) all couple to Gi-type G proteins and inhibit Ca2+ channels and,
consequently, catecholamine release [25-30]. Conversely, elevation of cAMP by Gs-coupled
GPCRs (e.g., D1 dopaminergic, or β-adrenergic) can augment electrically evoked
catecholamine release by increasing Ca2+ influx through L-type Ca2+ channels (LTCCs)
and/or direct protein kinase A (PKA)-mediated phosphorylation of the exocytotic machinery
[31-34]. Gq-coupled GPCRs (e.g., H1 histaminergic receptors) can release Ca2+ from
intracellular stores and promote influx of extracellular Ca2+ to evoke catecholamine release.
H1 receptors can also potentiate catecholamine release through generation of diacylglycerol
which binds munc13 to increase the size of the readily releasable pool of vesicles [35].
Acute activation of P2Y or μ-opioid receptors or direct application/transfection with Gβγ
can inhibit catecholamine release via direct inhibitory effects of the free Gβγ subunits on the
LTCCs but also independently of Ca2+ channel modulation [36, 37]. It is also interesting to
note that concomitant activation of PKC seems to prevent the effects of Gβγ on catecholamine
secretion [36]. Therefore, it is possible that there are opposing actions of Gβγ and PKC on the
exocytotic machinery to precisely control fusion pore kinetics. The molecular targets that
underlie these novel effects on catecholamine release remain unclear, but one plausible target
is the core fusion machinery.
Gβγ can bind to syntaxin-1A, synaptobrevin, SNAP25 and the ternary SNARE complex
in vitro [38, 39]. Moreover, Gβγ and Ca2+-bound synaptotagmin-1 compete for binding to the
SNARE complex in vitro [40]. Therefore, it is conceivable that Gβγ can modulate multiple
facets of exocytosis through interactions with SNARE proteins. Of course, Gβγ is known to
interact with an increasing number of downstream effectors.
Specific Receptors Regulating Catecholamine Secretion in Chromaffin Cells
NPY is a 36-amino acid peptide originally isolated from porcine brain [41]. This
neuropeptide acts as a co-transmitter, a neuromodulator and a neurohormone, and plays an
important role in numerous physiological processes such as food intake, hormone secretion or
regulation of the immune system [42]. NPY is also considered as a growth factor for several
cell types such as neuronal cells or smooth muscle cells [43, 44]. In human and other
mammalian species, high concentrations of NPY have been found in the brain and the
sympathetic nerve system, including adrenal medulla [45].
In the latter tissue, NPY concentrations are higher than those measured in the adrenal
cortex in all studied species [45, 46]. As mentioned above, NPY is co-secreted with
norepinephrine and exerts a strong vasoconstrictor effect on cardiovascular system vessels,
making this peptide an actor of the stress response [47]. In the human adrenal medulla, the
receptors Y1, Y2, Y4, and Y5 are expressed and functional, indicating that NPY exerts
autocrine effects in this tissue [10, 48, 49]. The expression of the CXCR4 in this tissue has
not been shown.
Few studies have reported on the effect of NPY on chromaffin cells. In human and
murine chromaffin cells in primary culture, NPY is able to stimulate catecholamine secretion
[10, 50]. It has also been shown that NPY treatment of rat or bovine chromaffin cells in
primary culture is able to inhibit cholinergic agonist-induced catecholamine secretion [51,
52]. Strikingly, the opposite effect has been observed when the adrenal gland was perfused
[51]. In addition to its role in the regulation of catecholamine secretion, NPY is also able to
act upstream, at the level of catecholamine biosynthesis, as shown by TH overexpression
observed in rat adrenal medulla in which NPY has been injected [53].
Moreover, a simultaneous treatment by ATP and NPY enhanced TH Ser31-
phosphorylation, which would stabilize this enzyme [54]. NPY levels are high but variable in
pheochromocytoma and this peptide could be differentially produced by different tumor
subtypes [55]. NPY may act in an autocrine manner on catecholamine production and
secretion and therefore participates in pathophysiological mechanisms involved in
pheochromocytoma.
PACAP is a ubiquitous neuropeptide of 27 or 38-amino acids involved in numerous
physiological functions [56]. In rat, several studies showed the expression of the PACAP
gene and the occurrence of peptide immunoreactivity in fibers innerving the adrenal medulla
[57]. Other studies showed the presence of PACAP in chromaffin cells of several mammalian
species and in human fetal chromaffin cells [58]. Numerous in vitro and in vivo studies
showed that PACAP acts as a neurotransmitter in order to regulate catecholamine secretion
by chromaffin cells in physiological and pathophysiological conditions [59-61].
These effects of PACAP on catecholamine secretion are associated with increased
expression of TH, DBH, and PNMT genes, concomitantly with increased activity of these
enzymes [62, 63]. However, immune labeling of TH and PNMT enzymes is similar in
PACAP knockout and wild type mice, suggesting that the peptide does not exert an important
role in the maintenance of the catecholaminergic phenotype of chromaffin cells and in the
development of the adrenal medulla [64]. In the rat pheochromocytoma PC12 cell line,
PACAP is able to stimulate catecholamine secretion, to inhibit cell proliferation and to induce
differentiation toward a sympathetic phenotype through molecular pathways similar to those
activated by nerve growth factor [65]. In addition, PACAP stimulates gene transcription of
TH and PNMT enzymes, VMAT1, the vesicular acetylcholine transporter and chromogranin
A and B, but inhibits the expression of secretogranin II [65].
In human intra- and extra-adrenal pheochromocytomas, a PACAP-like immunoreactivity
has been found in all studied tumors [66]. PACAP exerts its effects by binding to
VIP/PACAP receptors (VPAC1-R and VPAC2-R) and the PACAP-preferring receptor
(PAC1-R). In chromaffin cells, PAC1-R is the predominant receptor but VPAC2-R is also
present [67]. PAC1-R has also been detected in human fetal chromaffin cells [68]. PAC1-R is
involved in the effects of PACAP on peptide and catecholamine biosynthesis in the adrenal
medulla [69]. PACAP, through its PAC1 receptor, might play an important role into the
pathophysiology of pheochromocytomas. PACAP may also exert trophic and anti-apoptotic
effects on tumor cells and increase the biosynthesis and secretion of catecholamines and other
trophic peptides in these tumors.
Adrenomedullin (AM) is a 52-amino acid peptide originally isolated from a human
pheochromocytoma, which exhibits a high sequence homology with calcitonin gene-related
peptide (CGRP) [70]. AM is also present at high concentrations in the adrenal medulla (hence
its name), is secreted in the bloodstream and exerts hypotensive effects, acting on
vasodilatation and increasing diuresis and sodium secretion in urine [70].
In primary cultures of bovine chromaffin cells, it has been shown that AM is stored in
dense core vesicles and released along with catecholamines upon stimulation [71].
Moreover, there is an increase in AM and concomitant catecholamine secretion when
these cells are under hypoxia conditions [72]. So far, three AM receptors, also exhibiting
affinity for the CGRP, have been found: the adrenomedullin receptor (ADMR), the receptor
dog cDNA 1 (RDC1), and the calcitonin receptor-like receptor (CRLR) linked to the receptor
activity-modifying proteins 1, 2 or 3 (RAMP 1-3).
CRLR association with RAMP2 or 3 allows formation of a receptor displaying higher
affinity for AM than for CGRP, while association with RAMP1 results in the opposite effect
[73]. In rat adrenal medulla, CRLR and ADMR receptors are exclusively detected in
noradrenergic cells, while AM is mainly detected in adrenergic cells, suggesting a paracrine
role for this peptide [74].
Treatment of rat or human chromaffin cells with AM elicits catecholamine secretion [75].
However, this effect has not been observed in primary cultures of bovine chromaffin cells
[73]. In addition, in dog, injection of AM in the adrenal gland does not influence the secretion
of catecholamines, even if a stimulation of the splanchnic nerve or an injection of
acetylcholine is performed at the same time [76]. These results indicate that AM could have a
species-dependent role in the exocytosis of catecholamines from chromaffin cells. Significant
evidence exists for AM and its RDC1 receptor participating in the tumorigenesis of
chromaffin cells, supporting neoangiogenesis or allowing tumor cell survival [77]. Moreover,
overexpression of RDC1 in malignant pheochromocytomas suggests a role for this receptor
into tumor metastasis [77].
CHROMAFFIN CELL ADRENOCEPTORS, GRK2,

AND CATECHOLAMINE SECRETION: CONNECTION
WITH HEART FAILURE
The main source of circulating catecholamines in the body is the adrenal medulla [78].
The existing link between sympathetic overdrive and heart pathophysiology is well
established; since 1984, it has been clear that plasma concentration of noradrenaline is
negatively associated with survival in heart failure (HF) patients and the augmented plasma
concentrations lead to higher mortality [79]. Furthermore, sympathetic overdrive in HF
determines higher risk of arrhythmias and left ventricular dysfunction contributing to worse
prognosis [80]. In addition, this link is more evident when evaluating cardiac consequences in
pheochromocytoma. Nine mammalian AR subtypes are known: three α1ARs, three α2ARs,
and three βARs [81]. All ARs are GPCRs and, upon activation, are phosphorylated by the
family of GPCR-kinases (GRKs) that regulate their signaling and function [82-86]. It is now
widely recognized that cardiac GRK2 upregulation contributes significantly to the cardiac
dysfunction in HF [81-86].
The α2ARs are inhibitory autoreceptors that inhibit further release of catecholamines in
adrenergic nerves in the central sympathetic nervous system (SNS), and in the adrenal
medulla. The predominant inhibitory role of α2ARs in the adrenal gland becomes clear when
considering that PC12, a rat pheochromocytoma (chromaffin) cell line does not express these
receptors and secretes abnormal catecholamine quantities [87]. However, the mouse and rat
adrenal glands, as well as the human adrenal gland [88, 89], normally express various α2AR
subtypes endogenously [30], mainly α2A- and α2CARs (in the murine adrenal gland) [30]. As
far as the α2BAR subtype is concerned, this subtype is mainly expressed in the central SNS
and in vascular smooth muscle cells, where it mediates vasoconstriction [90].
Nevertheless, the α2BAR, like the other two α2AR subtypes, when expressed (separately)
in PC12 cells is capable of inducing neuronal differentiation into sympathetic-like neurons in
response to adrenaline, similarly to what the nerve growth factor (NGF) does in these cells
[87]. Of note however, this epinephrine-induced α2AR-mediated neuronal differentiation of
PC12 cells occurs to a different extent for each individual α2AR subtype, in part due to
subtype-dependent level of activation of the transcription factor NF-B (nuclear factor-
kappaB) and to subtype-specific activation of other signaling pathways, as well [91-93].
Finally, a human polymorphic α2BAR (Del301–303 α2BAR), which contains a three
glutamic acid deletion in its third intracellular loop important for its GRK2-dependent
phosphorylation and desensitization [94], is capable of enhanced inhibition of nAChR-
dependent catecholamine secretion compared to its wild type counterpart, when exogenously
expressed in PC12 cells [95]. This appears due to impaired phosphorylation and
desensitization of the Del301–303 α2BAR by GRK2, which is endogenously expressed in
PC12 cells [95].
In addition to the inhibitory α2AR autoreceptors, chromaffin cells also express
stimulatory βAR autoreceptors (primarily of the β2 subtype), which actually promote further
catecholamine secretion (facilitatory autoreceptors) [96]. Human chromaffin cells in
particular appear to express all three βAR subtypes, with β2- and β3ARs stimulating
catecholamine release, which is blocked by β2- or β3AR antagonists [97]. Similar to βARs,
mAChRs (muscarinic receptors), angiotensin II receptors, and histaminergic receptors also
seem to promote catecholamine secretion from chromaffin cells [98-100]. In contrast,
adenosine receptors may be present in chromaffin cells inhibiting catecholamine secretion
(like α2ARs), although their precise role and expression are not completely elucidated [101].
As mentioned above, upon agonist binding, ARs undergo phosphorylation by the GRKs,
leading to desensitization and/or downregulation [81-86]. GRK2, GRK3, and GRK5 are the
most important GRKs physiologically, because they are ubiquitous, phosphorylate most
GPCRs and are the most abundant. GRK2 is abundantly expressed in the adrenal gland
(including the medulla and, specifically, the chromaffin cells) and, by desensitizing the α2ARs
of the chromaffin cells, it exerts a tonic stimulation of catecholamine secretion [102], which is
particularly accentuated in chronic HF, when GRK2 expression in the adrenals (like in the
heart) is elevated [88].
Thus, hyperactivity of GRK2 towards chromaffin cell α2ARs underlies the elevated
sympathetic tone that accompanies and aggravates chronic HF [81]. More specifically,
increased catecholamine production, as evidenced by enhanced TH levels, and secretion of
both adrenaline and noradrenaline, as well as adrenal gland hypertrophy were evident during
chronic HF in two different experimental animal models [88]. In both of these models,
adrenal GRK2-dependent α2AR desensitization and downregulation led to enhanced
circulating catecholamines.
Importantly, this adrenal GRK2 elevation appears independent of the HF cause, since it
occurs also in HF induced chronic pressure overload of the heart (TAC model) [103]. Of note,
it also seems to be a fundamental process for the sympathetic overdrive of the failing heart
rather than the other way around, i.e., just an epiphenomenon or a consequence of the
declining cardiac function, since genetic deletion of chromaffin cell GRK2 in transgenic mice
prior to HF onset (i.e., from birth) significantly ameliorated both cardiac function and
sympathetic tone (circulating catecholamine levels) in these mice, after they were subjected to
myocardial infarction to develop HF [104]. Adrenal hypertrophy was also attenuated, as well
as cardiac GRK2 levels and AR dysfunction. Thus, adrenal GRK2 inhibition might be an
attractive sympatholytic strategy for HF therapy, which can also work in synergy with β-
blocker therapy or exercise training to improve HF symptoms [105, 106].
Another possible adrenal-targeted therapeutic strategy for HF, albeit significantly more
invasive, is of course bilateral or even unilateral adrenal denervation from the preganglionic
cholinergic nerves, which has been shown to ameliorate cardiac function and adrenal
hypertrophy in pressure overload-induced HF of experimental animals [103, 107].
CONCLUSION AND FUTURE PERSPECTIVES

A growing number of studies suggest that several GPCRs, as well as other types of
chromaffin cell membrane receptors are involved in regulation of the important and complex
physiological function of the adrenal medulla, catecholamine secretion. Several peptides play
important roles in this process, such as NPY and PACAP, adrenergic autoreceptors and
heteroreceptors for other important endogenous hormones, as well. The list seems to be
endless, especially with regards to receptors that stimulate this process; receptors that inhibit
it seem to be few and far between, with the most prominent ones being the α2ARs, and, to a
lesser extent, adenosine receptors. Full delineation of the roles of all of the receptors involved
in physiological and pathophysiological catecholamine secretion is absolutely warranted in
order to discover new, and improve upon existing therapies for a number of devastating
adrenal diseases (e.g., the adrenomedullary tumor pheochromocytoma, etc.). In addition,
known polymorphisms in the human genes for several of these receptors may affect (directly
or indirectly) catecholamine secretion in the carriers, which will have enormous implications
for the personalization of their adrenal disease treatment.
Importantly however, the therapeutic implications of studying adrenal GPCRs and their
related proteins extend far beyond endocrinologic conditions of the adrenal gland; as we and
others have uncovered over the past several years, adrenal GRK2 and arrestins, molecules
that regulate GPCR function in the adrenal gland, are directly implicated in cardiovascular
diseases, such as chronic heart failure, via elevation of catecholamine secretion from the
adrenal medulla but also of aldosterone secretion (and perhaps of secretion of other adrenal
steroids, as well) from the adrenal cortex in the context of heart failure [82, 108-110].
Therefore, targeting not only of chromaffin cell GPCRs per se but also of intracellular
proteins that regulate the function of these receptors in chromaffin cells (e.g., GRK2,
arrestin1, etc.), thereby modulating the multiple feedback loops that govern adrenal
catecholamine secretion, has recently begun to pose as an attractive therapeutic strategy to
combat both adrenal (e.g., pheochromocytoma) and cardiovascular (e.g., heart failure)
diseases. It seems more than certain that this particular area of chromaffin cell
physiology/biology, i.e., GPCR-dependent regulation of catecholamine secretion, will
continue to be the focus of intense research aiming to enrich the therapeutic armamentarium
of the future clinician, not only that of the future endocrinologist but perhaps of the future
cardiologist, as well.
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Chapter 4
INTERACTIONS OF THE ADRENAL GLANDS WITH

ADIPOSE TISSUE
Janella León, DO, and Atil Y. Kargi, MD

Division of Endocrinology, Diabetes and Metabolism,
Department of Medicine, University of Miami Miller School of Medicine,
Miami, FL, US
ABSTRACT
Adipose tissue and the adrenal glands both play fundamental roles in the regulation
of normal physiology and in the development of metabolic pathology. There has been an
increasing awareness of the complex interactions between adipose tissue and the adrenal
glands. Adipose tissue-derived efferent signals are implicated in a variety of alterations in
adrenal function. Adipocyte-derived secretory products such as adiponectin and leptin
have direct effects on adrenfal steroidogenesis, glucocorticoid metabolism, adrenal
medullary catecholamine output and hypothalamic-pituitary-adrenal (HPA) axis function.
Moreover, glucocorticoids and mineralocorticoids likely play a crucial role in the
development of obesity and insulin resistance through elaborate mechanisms.
Augmenting the effects of adrenal steroid hormones on adipose tissue is the enhanced
activity of the mineralocorticoid receptor in adipocytes. Elevated adrenal steroid levels
have been associated with weight gain, changes in body fat distribution and alterations in
circulating levels of several adipose tissue hormones, or ―adipokines.‖ In this chapter, we
summarize the current literature regarding the two-way communication between the
adrenal glands and adipose tissue; emphasizing the possible mechanisms of interactions
between these two endocrine organs and its relevance to the pathogenesis of obesity and
associated comorbidities, metabolic syndrome, and in primary adrenal disorders.
Keywords: adrenal glands, adipose tissue, adipokines

E-mail: akargi@med.miami.edu.
72 Janella León and Atil Y. Kargi
INTRODUCTION
While adrenal glands have long been known as important components of the endocrine
system and their contribution in the maintenance of homeostasis and hormonal influences on
physiology and disease have been well documented for over a century [1, 2], adipose tissue
has traditionally been perceived to serve principally as a reservoir for energy in the form of
triglycerides. Only recently it has come to light that the role of adipose tissue in human
physiology encompasses much more than the simple function of fat storage. It is now widely
acknowledged that adipose tissue operates as a dynamic, multifaceted endocrine organ,
pivotal in energy homeostasis, regulation of metabolism and predisposition to or protection
from disease [3-5]. Adipose dysfunction has been implicated in several disorders including
diabetes and insulin resistance, hypertension, dyslipidemia, reproductive disease and even
cancer.
The adrenal gland is composed of two endocrine organs: an outer cortex and an inner
medulla, each arising from distinct embryologic origins. The adrenal cortex, derived from
mesodermal tissue, synthesizes and secretes steroid hormones: mineralocorticoids,
glucocorticoids, and androgens. Regulation of these hormones is mostly determined by
hypothalamic – pituitary - adrenal hormone stimulation; however, importantly,
mineralocorticoid secretion is also influenced by the peptide angiotensin II. The adrenal
medulla, composed principally of cells derived from a subpopulation of neural crest cells,
secretes the catecholamines epinephrine, norepinephrine, and dopamine (see chapter 3). Each
of the adrenal hormones exert important effects on adipose tissue, with perhaps the most
striking example being the glucocorticoid induction of a remarkable degree of truncal obesity
characteristic of Cushing‘s syndrome (see chapter 7).
Adipose tissue is a multifaceted organ whose function is determined by histology and
location. The more prevalent white adipose tissue (WAT) serves mainly as energy storage in
the form of fat, whereas the mitochondria-rich brown adipose tissue (BAT) generates heat via
thermogenesis. Rodents possess both WAT and BAT during their entire lives, however in
human newborns, BAT constitutes 2%-5% of body weight, located mainly in the back, neck
and shoulders but is greatly reduced later in life, mostly scattered around the kidneys, adrenal
glands, aorta, and mediastinum [6]. Adipose tissue can be classified further as subcutaneous
and visceral adipose tissue (SAT and VAT, respectively), with VAT conferring a greater risk
of metabolic and cardiovascular disorders [6]. Adipose tissues secrete a variety of circulating
factors, termed ―adipokines‖ or ―adipocytokines.‖ These include hormones such as leptin,
involved in energy homeostasis, adiponectin, which is decreased in obesity and enhances
insulin sensitivity, and resistin, the levels of which are raised in obesity and may generate
insulin resistance [7]. In addition, there are a variety of other bioactive molecules released by
adipocytes. These include enzymes such as aromatase and 11-beta hydroxysteroid
dehydrogenase-1 (11HSD1), prothrombotic agents such as plasminogen activator inhibitor I,
and the cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), C-reactive
protein (CRP), and serum amyloid A (SAA), which are also secreted from cells of myeloid
origin, such as macrophages, found in adipose tissue stroma. Adipokines have regulatory
roles in many realms of human physiology including lipid homeostasis, blood pressure
control and vascular function, body weight regulation, and insulin sensitivity. The effects of
these adipokines on the adrenal glands is an emerging area of scientific inquiry [8, 9].
Interactions of the Adrenal Glands with Adipose Tissue 73
The investigation into the cross-talk between adrenal hormones and adipocytes has led to
the concept of an ―adipose-adrenal axis‖ [10, 11]. In this chapter, we explore this association
between the adrenals and adipose tissue as represented in the contemporary literature. We
focus in particular on emerging information supporting the impact of this relationship on the
development of obesity and on primary adrenal pathophysiology.
Mineralocorticoid-Adipose Interactions
Aldosterone is the chief mineralocorticoid hormone secreted by the adrenal zona

glomerulasa. Its cellular actions are mediated by activation of the mineralocorticoid receptor
(MR). The effects of aldosterone in regulating sodium transport in renal tubular cells,
resulting in net increase in Na reabsorption and K secretion, are widely known. However, in
addition to the kidney, a variety of other cell types, including adipocytes, have been described
as expressing MR [12].
Activation of MR in adipose tissue promotes preadipocyte differentiation and adipocyte
lipid accrual [13]. Treatment with the mineralocorticoid induces reactive oxygen species
(ROS) and promotes inflammation via increased production of various cytokines including
TNF-α, monocyte chemotactic protein-1 (MCP-1), and IL-6 in WAT [14]. In BAT, activation
of MR results in decreased thermogenesis and reduced transcription of uncoupling protein 1
(UCP1) [14]. Significantly higher MR mRNA levels have been observed in in WAT from
genetically obese mice when compared with lean control mice [15]. In humans, adipose tissue
MR gene expression correlates positively with body mass index (BMI) [14].
Mineralocorticoid Receptor Effects in Adipocytes Are Regulated Primarily

by Glucocorticoids
Glucocorticoids (cortisol in humans and corticosterone in rodents) circulate at 10-100

fold higher concentrations than those of aldosterone. MR has equal affinity for aldosterone
and glucocorticoids. Specifically, its affinity for cortisol is >10-fold higher than that of the
glucocorticoid receptor (GR) itself. Thus, the specificity of aldosterone-MR activation in
target epithelial cells relies on the intracellular enzymatic conversion of cortisol to MR-
inactive cortisone by the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (11HSD2).
In adipose tissue, however, MR does not exhibit selectivity for aldosterone and in fact
glucocorticoids constitute the main ligand for MR, given the relative lack of 11HSD2 activity
in adipose tissue. Moreover, there is greater regeneration of active glucocorticoid (conversion
of cortisone to cortisol) in adipocytes, as these cells exhibit enhanced 11-beta-hydroxysteroid
dehydrogenase type 1 (11HSD1) activity (Figure 1). This equilibrium between the
preferential activation and inhibition of these enzymes contributes to the increased activation
of MR by endogenous glucocorticoids [16].
Further proof that MR, rather than GR, mediates glucocorticoid effects in adipocytes, are
reports that treatment with the MR antagonist eplerenone mitigate local inflammation and
generation of ROS by glucocorticoid treatment of cultured adipocytes; yet, antagonism of the
glucocorticoid receptor (GR) with mifepristone does not result in a similar reversal of
alterations in adipocyte function [17].
Figure 1. 11-beta hydroxysteroid dehydrogenase (HSD) 2 activity is not significantly exhibited in

adipose tissue. However, adipocytes have considerable HSD1 activity, which results in higher levels of
cortisol, the active hormone, compared to its inactive counterpart, cortisone. Cortisol mediates its
actions on adipose cells via activation of both glucocorticoid receptor (GR) and mineralocorticoid
receptor (MR), for which cortisol has almost equal affinity. A positive correlation exists between
obesity and adipocyte HSD-1 activity, further boosting cortisol levels in adipose tissue.
Taken together, these findings suggest that many of the effects of glucocorticoids on
adipocyte function occur via activation of MR rather than GR.
Aldosterone in Obesity
Elevation in circulating plasma aldosterone levels has been detected in both obesity-
related hypertension and the metabolic syndrome. There are many possible explanations for
this finding, and one of the most important mechanisms by which this occurs likely involves
increased activity of the renin-angiotensin-aldosterone system (RAAS) in obesity [18].
This phenomenon, which represents an integral association between cardiovascular
disease and obesity, can be reversed with weight loss [18].
Another interesting possible etiology for the increased serum aldosterone concentration
observed in obesity is a direct influence on adrenal aldosterone secretion by adipose tissue
itself. It has been demonstrated that adipocyte secretory products induce adrenocortical
steroidogenesis, predominantly increasing aldosterone synthesis, illustrating the notion of
adrenal-adipose ―cross talk‖ [10].
This finding suggests the possibility of a ―vicious cycle‖ between adipose tissue and the
adrenal zona glomerulosa, with adipocytes secreting mineralocorticoid releasing factors
contributing to hypertension, fat cell mass, inflammation and derangements of endothelial
function. The stimulation of MR activation that would ensue would subsequently stimulate
differentiation of adipocytes and adipose tissue inflammation, thus promoting and
perpetuating the cycle. Although the precise adipocyte secretory factors acting on the adrenal
gland are unknown, oxidation products of polyunsaturated fatty acids, notably those derived
from linoleic acid, may be involved in this process [19].
A further possible explanation for the positive correlation found between serum
aldosterone concentrations and BMI is the demonstration of aldosterone synthase activity in
adipose tissue, making the adipocyte a possible secondary source of circulating aldosterone
[20]. Such locally generated aldosterone could modulate adipogenesis in an autocrine and
paracrine fashion, whereas adipocyte-derived circulating aldosterone might have effects on
distant target cells.
These observations regarding the role of MR activation in adipose tissue pathophysiology
introduce the prospect of inhibition of MR as an important therapeutic option in the
management of obesity and related metabolic disorders, including diabetes and hypertension.
Inhibition of MR in obese mice results in increased insulin sensitivity, a reduction in the
amount of hypertrophic adipocytes, decreased adipose tissue inflammation and a rise in
adiponectin [16, 21].
Clinical trials of MR blockade in humans with primary endpoints focused on effects on
adipose tissue biology or obesity have not been conducted.
Many clinical trials in patients with hypertension have shown a decrease in incident
diabetes with RAAS blockade [22]. It has been suggested that this finding may be mediated
by a direct effect of MR blockade on adipocyte differentiation, inflammation and regulation
of cytokines [16]. Antagonism of MR has been associated with reduction in morbidity and
mortality among patients with heart failure [23, 24], however no definite improvements in
BMI or metabolic syndrome have been demonstrated.
Adipose Tissue Dysfunction in Primary Hyperaldosteronism
Primary aldosteronism (PA), also known as Conn syndrome, is a condition in which there
is renin-independent hypersecretion of aldosterone that cannot be completely suppressed. The
most common etiologies include bilateral idiopathic hyperaldosteronism due to zona
glomerulosa hyperplasia, and unilateral aldosterone-producing adenomas. In investigating the
natural history of this disorder, we can achieve insight into the effects of hyperaldosteronism
on adipose tissue and on metabolism in general.
In a study of adrenalectomized rats, continuous administration of aldosterone for a
duration of twelve days resulted in weight gain [25]. However, a review of multiple cross-
sectional and longitudinal studies of patients with PA failed to show any consistent
correlation between aldosterone levels and body weight [15]. Previously researchers have
reported an association between PA and decreased insulin sensitivity and hyperglycemia [26],
yet, more recent investigations have not yielded similar results [14]. In some reports on PA,
investigators have described increases in serum leptin levels in patients following treatment of
PA [27] as well as lower serum adiponectin levels in patients with PA [28]. Patients with PA
have significantly elevated concentrations of the proinflammatory adipokine resistin in
comparison to hypertensive controls, and resistin levels were directly related to the existence
and severity of metabolic syndrome in the group with PA [29].
Glucocorticoid-Adipose Interactions
Glucocorticoids strongly influence adipose tissue development, metabolism and

adipocyte secretory actions. While a considerable amount of evidence substantiates the notion
that GR activation results in anti-inflammatory effects in adipose tissue, research regarding
the effects of glucocorticoids on adipogenesis and adipose tissue metabolism has yielded
differing conclusions.
Glucocorticoids increase the circulating fatty acids via enhanced food intake, increase
lipoprotein lipase activity leading to greater hydrolysis of circulating triglycerides which can
be deposited ectopically, and increase hepatic de novo lipid production [30]. The impact of
glucocorticoids on fat storage is likely determined by the physiologic context and differs by
location or type of adipose depot, generally being associated with central (particularly
visceral) fat deposition via lipogenic effects and depletion of peripheral subcutaneous fat
stores by predominantly lipolytic effects [31].
Glucocorticoids facilitate preadipocyte differentiation to mature adipocytes, and thus
stimulate adipogenesis [32]. Glucocorticoids reduce non-shivering thermogenesis by
decreasing UCP1 content and augment lipid storage by increasing the amount and size of
lipid droplets in BAT, essentially resulting in a phenotypic conversion of BAT to WAT [33].
Furthermore, glucocorticoids seem to exert both lipolytic and anti-lipolytic effects which may
be largely determined by the dose and duration of exposure (30). Though it is important to
point out that glucocorticoids influence lipid synthesis and lipogenesis variably depending
upon nutritional and hormonal conditions. For example, glucocorticoids downregulate
lipogenesis and free fatty acid (FFA) uptake in catabolic conditions, such as during fasting,
which allows for mobilization of crucial energy stores.
Conversely, in circumstances of increased nutritional supply and abundant circulating
insulin, as is observed with Cushing‘s syndrome, glucocorticoids behave additively or
synergistically with insulin to promote adipocyte expansion [31]. These concepts help to
clarify the finding that in Cushing‘s syndrome, excess cortisol is linked to increases in central,
particularly visceral adiposity while in undernourished, catabolic states such as anorexia and
acute illness, hypercortisolism is associated with decreased adiposity.
Abnormalities in circulating cortisol dynamics, resulting from activation of the
hypothalamic-pituitary-adrenal (HPA) axis, as well as altered metabolism of cortisol
metabolism in adipose tissue, have been associated with obesity and the metabolic syndrome.
Adipocyte Cortisol Metabolism in Obesity
Given the striking alterations in fat distribution typified by central obesity and peripheral
subcutaneous fat atrophy classically seen in Cushing‘s syndrome, it has been proposed that
changes in glucocorticoid activity may be involved in more common forms of visceral
obesity. Serum cortisol concentrations and dexamethasone suppression of cortisol are usually
normal in obesity, however, increased cortisol production occurs locally in adipose tissue.
This transpires because of increased local production of cortisol from cortisone as a result of
the increase in adipocyte HSD1 activity seen in obesity. Consequently, obesity can be thought
of as ―Cushing‘s syndrome of the omentum‖ [34]. A valuable point to note when considering
glucocorticoid effects on adipose tissue is the essential role that the MR receptor plays in
mediating corticosteroid-induced adipose conversion. Through the high activity of 11HSD1,

which generates active cortisol from inactive cortisone, and the lack of 11HSD2 expression in
adipose tissue (which inactivates glucocorticoid in mineralocorticoid target tissues), the
influence of glucocorticoid on adipocytes is in large part secondary to the effect of
glucocorticoid on MR rather than GR [35]. Moreover, it has been found that glucocorticoid
considerably increases 11HSD1 expression and activity, essentially acting in an autocrine
fashion in stromal cells and in a paracrine manner on neighboring adipose tissue, providing a
―fast-forward‖ feedback mechanism contributing to development of abdominal obesity [34].
11HSD1 activity is increased in obesity and metabolic syndrome [36, 37]. Transgenic
mice overexpressing 11HSD demonstrate elevated intra-adipose corticosterone
concentrations, abdominal obesity, hyperglycemia, dyslipidemia, hypertension, and insulin
resistance [38]. Contrarily, rodents with decreased exposure to 11HSD1 either by
pharmacologic inhibitors or via gene-knockout mechanisms seem to have cardiovascular
protection [39]. In humans, increased expression of 11HSD1 correlates with increased fat cell
volume, percentage of body fat, visceral and subcutaneous fat and with decreased insulin
sensitivity [36]. Numerous studies have shown increased 11HSD1 activity, transcription and
expression in SAT and VAT in obese individuals [37]. The impact on obesity and other
related metabolic derangements of 11HSD1 inhibition via naturally occurring and synthetic
inhibitors has been studied recently [40, 41]. A potent 11HSD1 inhibitor, INCB13739, was
found to significantly reduce HbA1c and fasting glucose in patients with type 2 diabetes, and
to reduce cholesterol and triglyceride levels in patients with hyperlipidemia and/or
hypertriglyceridemia [42]. These findings underscore the relevance of the HSD enzymes in
mediating the influence of glucocorticoids in adipose tissue and raise the prospect of HSD
modification as a potential novel approach for pharmacological intervention in the treatment
of obesity and its metabolic sequelae.
Glucocorticoids have been consistently found to suppress adipocyte inflammatory
Dexamethasone can be used to selectively stimulate GR in adipocytes and causes down-
regulation of adipocyte immune and inflammatory responses; while GR knockdown with
siRNA has the opposite pro-inflammatory effect [43]. Studies analyzing glucocorticoid
effects on adipocytokine production have demonstrated increased leptin mRNA levels and
increased serum leptin levels [44], with conflicting findings concerning glucocorticoid
influence on adiponectin [45].
Obesity and the HPA Axis
Obesity and the metabolic syndrome have been associated with dysregulation of the HPA
axis as manifested by alterations in adrenal cortisol production, peripheral cortisol
metabolism and dynamic tests of the HPA axis. It is unclear whether these changes are
directly responsible for the development and pathophysiology of obesity and related
comorbidities or simply represent a response, even perhaps an adaptation, to the obese state.
Basal serum concentrations of ACTH and cortisol and 24-hour urine free cortisol are
generally not increased in obesity. However, nighttime (7 pm-7 am) urine free cortisol levels
are elevated in females with abdominal obesity [46]. The most compelling proof of aberrant
HPA axis activity in obesity is derived from investigation into stimulation or suppression of
the axis. Several studies have demonstrated that obese individuals express higher ACTH and
cortisol responses to AVP and CRH, in effect exhibiting hypersensitivity of the HPA axis to
neuroendocrine stimuli [47, 48].
Obese women, in particular those with increased abdominal obesity, have elevated
responses of serum cortisol to both low dose and high dose ACTH stimulation tests, possibly
as a result of hyperactivity of the HPA axis [49, 50]. Although in general there is normal
cortisol suppression after 1 mg overnight dexamethasone suppression in obese subjects
compared to lean controls, one study demonstrated increased post-dexamethasone serum
cortisol concentrations in obese females with higher waist circumference; this finding was not
observed in males [51].
Adrenal Androgens and Adipose Tissue
The adrenal cortex secretes androgen hormones in both sexes, including

dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and
androstenedione. In humans, DHEA-S is the major adrenal corticosteroid, with the highest
serum concentration of any steroid hormone. Concentrations of this hormone decrease
considerably with age, parallel to the alterations in body composition that occur with aging
[52]. DHEA content in human adipose tissue is much higher –nearly 10-times higher- than
that observed in the systemic circulation [53]. Epidemiologic research suggests an association
between DHEA-S levels and obesity, insulin resistance and cardiovascular disease [54].
DHEA has been demonstrated, at least in vitro, to exert an anti-adipogenic effect on human
omental preadipocytes, suggesting that the hormone may influence adipogenesis in a fat-
depot dependent manner [55]. Exposure to DHEA in vitro as well as in rodent models has
yielded seemingly beneficial alterations in adipokine profile; treatment with the hormone was
found to significantly upregulate adiponectin expression in VAT and induce resistin gene
expression in WAT [56, 57]. Investigations of humans treated with DHEA yield conflicting
findings regarding its impact on adiposity and cardiovascular disease [58]. One randomized-
controlled trial conducted on elderly subjects demonstrated decreases in visceral and
subcutaneous fat as well as improvement in insulin action with 6 months of DHEA
supplementation [59] A possible confounding matter when analyzing these studies is the fact
that DHEA serves as a precursor hormone that is converted to testosterone and estrogens,
hormones that independently regulate the volume and distribution of adipose tissues and
exhibit separate effects on body composition [60]. There are gender-specific patterns of
distribution for receptors for sex steroids with effects to modulate synthesis of lipoprotein
lipase and leptin production [61, 62]. Specifically, sex steroids suppress adipose tissue LPL
activity in males, while leptin gene expression is regulated by 17-beta estradiol in female
rodents [61, 62].
Adipose Tissue and the Adrenal Medulla
The adrenal medullary cells operate, in effect, as modified postganglionic neurons,

releasing the catecholamine hormones epinephrine, norepinephrine, and dopamine.
Epinephrine is the primary hormone secreted by the adrenal medulla, and is released in
response to activation of the sympathetic nervous system (SNS). Catecholamines are involved
in regulation of adipocyte lipolysis [63]. A reduction in catecholamine-induced lipolysis has

been reported in obese and insulin resistant subjects; leading to speculation that this finding
could be an integral mechanism involved in the pathogenesis of obesity [64]. There is a large
amount of data demonstrating communication between adipocytes and adrenal medullary
chromaffin cells and it appears that this cross-talk between adipose tissue and the adrenal
medulla in some instances meets the definition an endocrine feedback loop. Adipocyte-
derived leptin increases catecholamine secretion from the adrenal medulla, while resistin
could possibly inhibit catecholamine secretion from chromaffin cells [65]. In turn,
catecholamines may participate in regulation of adipocyte endocrine activity. Catecholamines
have been found to synergistically suppress leptin and resistin secretion and stimulate
expression of proinflammatory cytokines by adipocytes in vitro [65].
Adrenal Medullary Function in Obesity
SNS activation is increased in obesity. It has been suggested that this over activity of the
SNS is involved in the pathogenesis of comorbidities and target-organ damage related to
obesity. A possible rationale for the amplified SNS activity of obesity is that this represents a
homeostatic adaptation or auto-regulatory response. This has been termed ―diet-induced
thermogenesis,‖ in which an increase in adrenergic lipolysis may provide defense against the
harms of further fat storage [66]. Adipose tissue has been implicated as a possible contributor
to this apparent homeostatic feedback mechanism via its secretion of adipokines which
promote SNS activation and stimulate adrenal medullary function [66, 67]. These adipokines
which may indirectly or directly regulate SNS output include leptin, non-esterified free fatty
acids, angiotensinogen, TNF-α, IL-6, and adiponectin.
The association between leptin and SNS output is especially compelling with regards to
obesity. Leptin is secreted from adipocytes and circulates at levels proportionate to the mass
of adipose tissue. Leptin increases SNS outflow to BAT, thereby increasing thermogenesis,
possibly representing a homeostatic mechanism for energy balance [67]. Conversely, acute
beta-adrenergic stimulation suppresses leptin production in humans and animals, although in
a study of patients with pheochromocytoma, leptin was not found to be suppressed or
correlated to norepinephrine levles, indicating that chronic activation of adrenergic receptors
might not inhibit leptin secretion [68].
Catecholamines and BAT
Catecholamines may have a specific role in regulation of BAT. Observational studies in

patients harboring pheochromocytoma show an increase in BAT metabolic activity, as
demonstrated by 18-fluorodeoxyglucose-positron emission tomography (FDG-PET) [69].
Pheochromocytoma patients may demonstrate an increased uptake of FDG-PET particularly
in retroperitoneal BAT corresponding with increased vascularity of the brown fat on
pathologic exam. After treatment with alpha-adrenergic and beta-blockers, the
hypervascularity in the retroperitoneal fat decreases, suggesting decreased activity of BAT
[70]. Increased expression of adiponectin mRNA in BAT occurs in patients with
pheochromocytoma, with a decline in plasma adiponectin levels after surgical resection of

pheochromocytoma together with its surrounding BAT [71].
Nonfunctioning Adrenal Adenomas and Adipose Tissue
The prevalence of adrenal masses discovered serendipitously on high-resolution

computed tomography (CT) scans is approximately 4%, and based on analyses of autopsy
series the prevalence is about 6% [72]. The majority of these incidentalomas are
nonfunctioning adenomas (NFA) of the adrenal cortex, not altering adrenal endocrine
function, however 5-9% of adrenal adenomas may result in subclinical Cushing‘s syndrome
(SCS), ensuing from the autonomous production of cortisol by the adrenal tumor at an
intensity not enough to cause the classic clinical manifestations of Cushing‘s syndrome [72].
A causal relationship between non-functioning adrenal tumors and impaired glucose
tolerance, reduced insulin sensitivity and the metabolic syndrome has been suggested. When
matched for BMI, persons with NFA are usually found to have a more central type of fat
accumulation associated with increased insulin resistance [73]. Correlating with this data is
the finding that patients with adrenal incidentalomas have increased epicardial fat thickness
and increased left ventricular mass [74]. Slightly higher levels of late night serum cortisol and
dexamethasone suppressed cortisol concentrations are observed in patients with NFA, though
often at a level not sufficient to qualify for the established criteria for SCS [73, 75].
Cortisol production occurs in a continuous range; as such, a possible reason for the
changes in body fat distribution and numerous metabolic derangements detected in patients
with NFA could be subtle hypercortisolism. Since patients with incidentally detected adrenal
tumors often exhibit decreased insulin sensitivity and are hyperinsulinemic, and given that
that insulin stimulates adrenal cancer cell lines in vitro, another prevailing theory explaining
the association of adrenal tumors with insulin resistance is that NFA may be a manifestation
of, rather than the cause of, the metabolic syndrome (75, 76).
Adipocytokine Effects on Adrenal Function
The principal regulation of adrenal hormone production (glucocorticoids and androgens

under ACTH control, mineralocorticoids by the RAAS, and catecholamines by the SNS) has
been established and detailed extensively previous chapters. Below, we review the evidence
suggesting that adipokines may also function to regulate activity of the adrenal gland.
Although a complete analysis of the impact of all adipokines on adrenal function is beyond
the scope of this chapter, we will focus on the influence of leptin and adiponectin, two
particularly significant adipocytokines with adrenotropic effects.
Leptin-Adrenal Interactions
Since leptin was first identified as a WAT-derived circulating satiety factor in 1994 [77],
the impact of this adipokine on adrenal function has been the subject of intensive research.
Studies have shown the immediate effects of leptin on adrenal steroid production in vitro and
have demonstrated the importance of this hormone as a fundamental metabolic signal acting
on the adrenal gland. In cultured bovine cells, leptin decreases adrenal synthesis of cortisol by
down regulation of the steroid producing enzyme cascade, and administration of this hormone
dulls the cortisol response to ACTH. It has been proposed that leptin modulates
transcriptional control of adrenal cortisol production [9, 78]. Similarly, leptin – in
concentrations occurring in vivo in humans - directly decreases cortisol secretion in response
to ACTH stimulation of human and rodent adrenal cell cultures, suggesting that the hormone
regulates activity of the HPA axis.
Leptin was not found to have any long-term effects on basal cortisol secretion, however
[79]. It seems that leptin also exerts regulatory effects at higher levels of the HPA axis. A
common murine model for the study of obesity is the ob/ob mouse, which is deficient in
leptin and exhibits enhanced HPA activity [80]. Chronic administration of leptin results in
correction of the excess corticosterone production characteristic of these mice, and attenuates
the CRH stress induced response [80]. Conversely, in a study involving rhesus monkeys,
short-term leptin infusion did not affect cortisol release or the cortisol response to ACTH
[81]. Studies of the association between the secretion of leptin and adiponectin within fat
depots surrounding and bordering adrenal neoplasia, provide evidence for the possible
paracrine mechanisms underlying a dynamic role of adipose tissue in primary adrenal
pathology [82]. Researchers measured mRNA expression of leptin and adiponectin in adipose
tissue surrounding adrenal neoplasm of patients with primary aldosteronism (PA), Cushing‘s
syndrome due to adenomas secreting cortisol and patients with nonfunctional adrenal
adenomas (NFA). Findings included increased expression and serum levels of leptin in
adipose tissue surrounding adrenal neoplasia.
The most elevated levels of leptin were detected in adipose tissue surrounding adrenal
neoplasia of patients with PA. Researchers in the study propose that perhaps MR blockade
might decrease leptin levels in obesity, thereby possibly mitigating hypertension and
decreasing the cardiovascular risk associated with SNS activation by leptin. Another novel
discovery in this study involves the difference in mRNA expression of leptin in distinct
locations of adipose tissue in patients with Cushing‘s syndrome. Specifically, expression was
increased in peri-adrenal tumor adipose tissue compared with subcutaneous and peri-renal fat
depots. Furthermore results of these studies suggest a discrete and direct impact of fat
surrounding adrenal neoplasia in regulation of the expression and release of leptin, unrelated
to whether or not the person is obese [82].
Above we have provided a review of the association between leptin and SNS activity,
particularly as it relates to obesity. Many studies have also demonstrated a direct impact of
leptin on the adrenal medulla. Leptin exerts a powerful secretagogue action on chromaffin
cells, directly increasing catecholamine secretion in porcine adrenal medullary cells [83].
Leptin and resistin both stimulate catecholamine secretion from rat chromaffin cells [65]. In
turn, catecholamines suppress secretion of leptin and resistin from cultured adipocytes,
further lending support for the existence of an adipo-adrenal axis.
Adiponectin-Adrenal Interactions
Adiponectin is the most abundant adipose tissue derived peptide in the circulation,
exerting multiple effects on metabolism and modulating a variety of biological functions.
Receptors for this adipocytokine are widespread throughout a diversity of tissues and organs;
adiponectin has been found to decrease inflammation and atherogenesis, and low levels of
this hormone may confer increased risk of cardiovascular diseases and is characteristic of
insulin resistant states and the metabolic syndrome.
Most adiponectin effects are mediated by the adenosine monophosphate-activated protein
kinase (AMPK) and peroxisome proliferator-activated receptor gamma (PPARγ) signaling
pathways. The existence of receptors for adiponectin in human and mouse adrenal cortex has
been demonstrated, leading to speculation that adiponectin may have a regulatory role in the
activity of the adrenal cortex [84].
Adiponectin receptors are present in all layers of the adrenal cortex and medulla in
cultured rat adrenocortical cells (8). In Y-1 murine adrenocortical cells, an ACTH-responsive
cell line, adiponectin acutely and significantly inhibited corticosterone and aldosterone
secretion via down-regulation of chief enzymes of steroidogenesis [84]. Dexamethasone was
shown to inhibit adiponectin release from subcutaneous adipocytes. ACTH also was found to
exert a pro-inflammatory adipocyte response, acutely decreasing expression of adiponectin in
WAT [85, 86]. These findings provide evidence for glucocorticoid regulation of adiponectin.
Administration of adiponectin in vitro in cultured rat adrenal cortical cells results in
stimulation of adrenocortical cell proliferation and increased corticosterone output while
production of aldosterone is unaffected [8].
In patients with various types of adrenal neoplasms there is decreased expression of
adiponectin mRNA from fat bordering the adrenal tumor compared with that from peri-renal
and subcutaneous fat, with the lowest circulating levels of adiponectin detected in patients
with Cushing‘s syndrome [82]. These findings are compatible with results of the studies
mentioned above, supporting the suppressive actions of glucocorticoids on secretion of
adiponectin.
CONCLUSION
The prevalence of obesity has risen to such an extent that it is presently recognized as a
global epidemic. The subject matter of adipocyte biology and adipose secretory functions,
particularly the relationship between adipocytokines and other endocrine organs is a growing
field of intense study.
Adrenal-adipose interactions are of special importance among these investigations, and
the impact of the adrenal glands on lipid metabolism, inflammation, and the ―stress‖ response
has long been acknowledged. In this chapter, we have analyzed the interactions between each
of the adrenal hormones and adipose tissue, highlighting the physiologic and
pathophysiologic effects of these hormones on the adipose tissues. We elaborated on, in
particular, the actions of adrenal hormones in obesity and metabolic syndrome in addition to
the possible causal role that these hormones may play in obesity-related comorbidities.
Furthermore, we have reviewed the data supporting an association between adipocyte
dysfunction and primary adrenal disorders, describing the two-way interaction or cross talk
between adrenal and adipose hormones. We conclude that further study is needed to fully
elucidate the significance of the observed derangements of adrenal function seen in obesity
and the alterations in adipokine levels and effects associated with primary adrenal
pathologies.
The essential focus of future research in this area should be to clarify whether these
observed changes represent adaptive mechanisms conferring protection from disease states or
conversely whether they are part of the pathogenesis. Finally, in our investigation into the
cross talk between adipose tissue and the adrenal gland, we have presented a theoretical
foundation for forthcoming pharmacological interventions focusing on targeting adrenal
hormones in the management of obesity.
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Chapter 5
HYPOADRENALISM: PRIMARY AND SECONDARY

ADRENAL FAILURE
Marianna Minnetti and Ashley B. Grossman

Oxford Centre for Diabetes, Endocrinology and Metabolism,
University of Oxford, Oxford, UK
ABSTRACT
Primary adrenal failure has a prevalence of around 1/20,000. In the past, the primary
cause was tuberculosis, but in the developed world most cases are now due to auto-
immune adrenal failure, diagnosed in its early stages by the presence of antibodies to the
enzyme 21-hydroxylase (although these decrease over time). This may be associated with
other auto-immune diseases such as Hashimoto‘s thyroiditis, premature ovarian failure,
hypoparathyroidism, pernicious anaemia and type 1 diabetes mellitus. There is some
evidence that the actual tissue destruction is T-cell mediated. Other causes included
metastatic adrenal infiltration, bilateral adrenal haemorrhage, lupus anti-coagulant,
histoplasmosis and various genetic disorders (familial glucocorticoid deficiency, primary
adrenal hypoplasia, adrenoleukodystrophy, etc.). The symptoms are often very non-
specific, including malaise, nausea and vomiting, but on examination the hypovolaemia is
associated with skin pigmentation due to activation of melanotrophs by the high
circulating of ACTH. Diagnosis is made by the association of low serum cortisol with
high levels of ACTH, but more rapidly by the subnormal response of serum cortisol to
Synacthen (ACTH 1-24). Plasma renin is elevated and aldosterone suppressed.
Secondary adrenal failure is due to hypothalamic or pituitary dysfunction, and the
symptoms and signs are often milder. Cortisol is low, as is plasma ACTH, but renin and
aldosterone levels are normal. Treatment is with hydrocortisone, usually best in divided
doses (typically 10, 5 and 5 mg daily), plus fludrocortisone once-daily in the case of
primary adrenal failure. There are slow-release forms of hydrocortisone becoming
available. Doses of hydrocortisone need to be increased during acute stress and an
emergency pack of hydrocortisone should be available. Care must be taken to avoid
under-replacement and consequent on-going fatigue, or over-replacement and the
metabolic syndrome. Some patients may additionally benefit from DHEA.

E-mail: ashley.grossman@ocdem.ox.ac.uk.
92 Marianna Minnetti and Ashley B. Grossman
Keywords: adrenal failure, hypoadrenalism, adrenal insufficiency, Addison‘s disease
INTRODUCTION
Hypoadrenalism, adrenal failure or adrenal insufficiency is the clinical situation
characterised by the deficient production or action of glucocorticoids, with or without
deficiency also in mineralocorticoids and adrenal androgens. It is a life-threatening condition
that can result from disorders affecting the adrenal cortex (primary adrenal failure), the
pituitary gland (secondary or central adrenal failure), or the hypothalamus (tertiary adrenal
failure) [1]. In 1855 Thomas Addison described for the first time several signs and symptoms
of hypoadrenalism: ―general languor and debility, feebleness of the heart’s action, irritability
of the stomach, and a peculiar change of the colour of the skin occurring in connection with a
diseased condition of the suprarenal capsules”[2]. If left untreated, adrenal failure is a lethal
condition, and before the availability of glucocorticoids the majority of patients with primary
hypoadrenalism died within 2 years of diagnosis [3]. This chapter will provide an overview of
the epidemiology, aetiology, pathophysiology, clinical manifestations, diagnosis and
treatment of adrenal failure and adrenal crisis.
EPIDEMIOLOGY
The estimated incidence of primary adrenal failure is 4.4-6 new cases per million
population per year [4] and the prevalence of chronic primary adrenal failure in Europe is 93-
144 cases per million population [4-8]. Clinical manifestations of primary adrenal failure
present most often between 20 and 50 years, although they can present at any age. Patients
with primary hypoadrenalism have a more than twofold increased standardised mortality
ratio, mainly due to cardiovascular and infectious disease [9, 10].
The incidence of secondary hypoadrenalism is much higher than that of primary adrenal
failure [11], and patients are mostly diagnosed in the sixth decade of life [12, 13]. Patients
with secondary adrenal failure have an increased mortality rate, principally due to
cardiovascular disease [14]. Women are most frequently affected than men by primary and
secondary adrenal insufficiency [5, 7, 12, 13, 15, 16].
Adrenal crisis is an emergency contributing to the excess mortality of patients with
adrenal failure, with an incidence of 5-10 adrenal crises per 100 patient years [17].
CAUSES OF PRIMARY ADRENAL FAILURE

The most commonly cause of primary adrenal failure in the developed world is
autoimmune adrenalitis or Addison‘s disease, although in the early 1900s the majority of
cases were associated with tuberculosis [18, 19] (Table 1).
Hypoadrenalism: Primary and Secondary Adrenal Failure 93
Table 1. Causes of primary adrenal failure

CLINICAL FEATURES IN ADDITION TO PRIMARY ADRENAL
DIAGNOSIS
INSUFFICIENCY
AUTOIMMUNE ADRENALITIS (Addison‘s disease)
Isolated autoimmune adrenalitis No other features
Hypoparathyroidism, chronic mucocutaneous candidiasis, other autoimmune
APS 1 (APECED)
disorders
APS 2 Thyroid disease, type 1 diabetes mellitus, other autoimmune diseases
APS 4 Other autoimmune diseases, excluding thyroid disease or diabetes
INFECTIOUS ADRENALITIS
Tuberculous adrenalitis Coughing, chest pain, fever, night sweats, chills
AIDS (HIV, Cytomegalovirus) Recurrent infections, weight loss, chronic diarrhoea, night sweats, skin problems
Fungal adrenalitis (Histoplasmosis, Coccidioidomycosis,
Mostly in immunocompromised, post-transplant and elderly populations
Cryptococcosis)
Syphilis Weight loss, night sweats, and left upper quadrant pain.
African trypanosomiasis Lethargy, anorexia, cold intolerance, weakness, paresthesia
GENETIC DISORDERS
Adrenoleukodystrophy Weakness, spasticity, dementia, blindness, quadriparesis.
Allgrove‘s (Triple A) syndrome Achalasia, alacrima, mental retardation, osteoporosis
Congenital Adrenal Hyperplasia
21-hydroxylase deficiency
Hyperandrogenism
11β-hydroxylase deficiency
Hyperandrogenism, hypertension
3β-hydroxysteroid dehydrogenase type 2 deficiency
Ambiguous genitalia in boys, postnatal virilization in girls
17α-hydroxylase deficiency
Pubertal delay in both sexes, hypertension
P450 oxidoreductase deficiency
Skeletal malformations, mental retardation, growth failure, hyponatraemia,
hyperkalaemia, cholesterol deficiency
XY sex reversal
P450 side-chain cleavage deficiency
Lipoid Congenital adrenal hyperplasia XY sex reversal
Adrenal hypoplasia congenita Hypogonadotropic hypogonadism with pubertal failure.
Familiar Glucocorticoid Deficiency Hypoglycaemia, hyperpigmentation and failure to thrive
Craniofacial malformations, mental retardation, growth failure, hyponatraemia,
Smith-Lemli-Opitz syndrome
hyperkalaemia, cholesterol deficiency
Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia
IMAGe syndrome
congenita and genital abnormalities
External ophthalmoplegia, retinal degeneration, cardiac conduction defects, other
Kearns-Sayre syndrome
endocrine disorders
Wolman‘s disease Bilateral adrenal calcification, hepatosplenomegaly
Xanthomata, arthritis, premature coronary artery disease, short stature, gonadal
Sitosterolemia
and adrenal failure
Primary generalised glucocorticoid resistance or Chrousos
Fatigue, hypoglycaemia, hypertension, hyperandrogenism
syndrome
ADRENAL INFARCTION
Primary antiphospholipid syndrome Deep vein thrombosis, pregnancy-related complications, stroke
Heparin-induced thrombocytopenia syndrome Patients receiving heparin, thrombocytopenia, arterial or vein thrombosis
Waterhouse-Friderichsen syndrome Petechial and purpuric rash, septic shock
BILATERAL ADRENAL METASTASES
Disease-associated clinical manifestations
Lung, breast, stomach, lymphomas, and melanoma
DRUGS
Anti-fungal agents (ketoconazole, fluconazole)
Etomidate
Tyrosine kinase–targeting drugs
Anticoagulants None, unless related to drug
Aminoglutethimide
Phenobarbital
Phenytoin, Rifampicin, Troglitazone
ADRENAL INFILTRATION
Primary adrenal lymphoma, amyloidosis,
Disease-associated clinical manifestations
haemochromatosis
BILATERAL ADRENALECTOMY
Unresolved Cushing‘s syndrome
Bilateral phaeochromocytoma Disease-associated clinical manifestations
Bilateral adrenal masses
Addison’s disease is characterised by the destruction of the adrenal cortex by immune

mechanisms. Circulating 21-hydroxylase antibodies are detected in approximately 90% of
patients [8]. The most important genetic factors that can predispose to the development of
Addison‘s disease are the major histocompatibility complex haplotypes DR3-DQ2 and DR4-
DQ8, with the recent addition of protein tyrosine-phosphatase non-receptor type 22 PTPN22,
cytotoxic T-lymphocyte antigen 4 CTA-4 and the MHC class II transactivator CIITA [1].
Environmental triggers, such as drugs, smoking, infections, food and stress, may play a role
in genetically-predisposed patients [20].
The histological picture of adrenal cortex in Addison‘s disease is dominated by a
widespread diffuse inflammatory mononuclear infiltrate consisting of lymphocytes, plasma
cells and macrophages [21].
Addison‘s disease can be isolated (40%) or may be part of an autoimmune polyendocrine
syndrome APS (60%). Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED) or autoimmune polyendocrinopathy syndrome type 1 (APS1) is an autosomal-
recessive syndrome linked to the mutation of the AIRE gene. The most frequent pathological
conditions related to the APS1 include Addison‘s disease, mucocutaneous candidiasis and
hypoparathyroidism.
Other APS1 associated diseases include autoimmune hepatitis, primary hypothyroidism,
a malabsorption syndrome such as gluten-sensitive enteropathy, vitiligo, pernicious anaemia,
alopecia, type 2 diabetes, ovarian failure, primary hypogonadism, pulmonary disease,
pericarditis, cerebellar degeneration, asplenia, encephalopathy, oesophageal cancer,
polyneuropathy, and others. The onset often occurs during childhood [22].
Autoimmune polyendocrinopathy syndrome type 2 (APS2) is the most common APS and
is characterised by presence of Addison's disease (always present), autoimmune thyroid
diseases (Graves‘ disease or autoimmune thyroiditis) and/or type 1 diabetes mellitus. Other
associated diseases are primary hypogonadism, hypopituitarism, IgA deficiency, Parkinson‘s
disease, myasthenia gravis, vitiligo, gluten-sensitive enteropathy, stiff-man syndrome,
serositis, alopecia and others. Several of the diseases of APS2 are linked with HLA antigens
HLA-DR3 or HLA-DR4. Primary adrenal failure in type 2, but not in type 1 APS, is
markedly associated with both HLA DR3 and HLADR4.
APS type 3 involves the same disorder of endocrine glands as type 2 but usually without
any defect of the adrenal cortex.
The different clinical combinations of autoimmune adrenal failure and one or more other
autoimmune diseases not included in the previous groups are characteristic of autoimmune
polyendocrinopathy syndrome type 4 APS-4 [22, 23].
Infectious adrenalitis may result in adrenal failure: the most common infections are
tuberculosis, HIV-associated infections and fungal infections such as histoplasmosis. In the
developing world, tuberculosis continues to account for about 20–30% of cases of Addison‘s
disease [24] with adrenal involvement being found in 6% of patients with active tuberculosis
in an autopsy series [25].
Adrenal insufficiency is prevalent in 17% of patients with AIDS [26]. Most common
causes of adrenal insufficiency in patients affected by HIV virus are opportunistic infections
such as Cytomegalovirus, Mycobacterium tuberculosis, Cryptococcus Neoformans,
Histoplasma Capsulatum, Pneumocystis Jirovecii and Toxoplasma Gondii [27].
Adrenal involvement is typical of disseminated chronic histoplasmosis. This fungal

infection often coexists with HIV-AIDS and is more commonly seen in the
immunocompromised, post-transplant and elderly populations [28].
Genetic mutations associated with several familial causes of adrenal failure have now
been identified. The most common one include adrenoleukodystrophy (ALD), Allgrove or the
‗triple A‘ syndrome (AAA), adrenal hypoplasia congenital (AHC), congenital adrenal
hyperplasia (CAH), Lipoid CAH, and familial glucocorticoid deficiency (FDG).
Adrenoleukodystrophy ALD is a rare X-linked recessive disorder of peroxisomal fatty
acid beta-oxidation which results in the accumulation of very-long chain fatty acids within
peroxisomes. The most strictly affected tissues in ALD are the nervous system white matter
and the adrenal cortex. Approximately 10% of affected individuals present with signs of
adrenal failure only, between age two years and adulthood [29].
Allgrove syndrome is a rare autosomal recessive disorder characterised by the triad of
adrenocortical failure due to ACTH resistance, achalasia and alacrima (absence of tear
secretion). These patients present characteristically in childhood with hypoglycaemia and
adrenal crises, but the disease has infrequently presented as late as the fourth decade [30].
Other neurological changes may occur and may dominate the clinical picture.
Congenital adrenal hyperplasia, CAH, is one of the most common cause of primary
adrenal failure in children. It is characterised clinically by adrenal failure with or without salt
wasting, prenatal virilisation and genital ambiguity in new-born girls, and post-natal
virilisation in both boys and girls. CAH is due to the defects of the enzymes involved in
adrenal steroidogenesis. The enzyme deficit results in defects in cortisol biosynthesis with
accumulation of precursors, deviation to increased androgen production, and inconstant
mineralocorticoid defects. The most common cause of congenital adrenal hyperplasia is 21-
hydroxylase deficiency, with an overall incidence of approximately 1 case in 16,000 live
births, caused by a mutation of deletion in CYP21A2 [31]. There are three different
phenotypes associated with varying degrees of enzyme activity: classic salt wasting, classic
simple virilising and non-classic or late-onset. There is an association with particular ethnic
groups, especially Ashkenazi Jews.
Lipoid congenital adrenal hyperplasia (LCAH), the most severe form of CAH, is most
common in Palestine and Japanese populations. LCAH is caused by mutations in the STAR
gene, leading to a block in the first step in steroidogenesis, complete deficiency in
glucocorticoid, sex steroid and mineralocorticoid hormones, and the pathognomonic massive
cholesterol ester accumulation in the adrenal cortex.
Adrenal hypoplasia congenita AHC is an X-linked recessive disorder characterised by
primary adrenal failure and hypogonadotropic hypogonadism with pubertal failure. AHC is
caused by a mutation in the NR0B1 gene.
Familiar glucocorticoid deficiency, FDG, or hereditary unresponsiveness to ACTH, is a
group of rare autosomal recessive disorders caused by mutations in the melanocortin type 2
receptor (MC2R) or more commonly in its accessory protein (MRAP), which together yield
the functional receptor for ACTH. In FDG, the cells of the zona fasciculata within the adrenal
cortex do not produce cortisol in response to ACTH stimulation, while mineralocorticoid
production from the zona glomerulosa remains intact. Patients usually present in infancy with
hypoglycaemia, hyperpigmentation and failure to thrive [30].
Adrenal infarction is caused by haemorrhage or adrenal vein thrombosis leading to acute
adrenal crisis, shock, and death.
Primary Antiphospholipid syndrome and heparin-induced thrombocytopenia syndrome

have been associated with adrenal failure [32, 33]. Waterhouse-Friderichsen syndrome, a
meningococcal sepsis syndrome involving bilateral adrenal haemorrhage, is not limited to
meningococcal infection, but may occur after infection with staphylococci or other pathogens
[34].
Metastases from malignant tumours elsewhere (lung, breast, stomach, lymphomas, and
melanoma) are recognised to cause chronic adrenal failure, although this is uncommon as
there has to be almost total adrenal destruction. Adrenal carcinomas does not usually cause
adrenal failure, although they may cause Cushing‘s syndrome or virilisation: surgical removal
will lead to temporary adrenal failure [35].
Drugs that may reduce cortisol synthesis include anti-fungal agents (e.g., ketoconazole),
the anesthetic etomidate (even after a single injection) and novel tyrosine kinase–targeting
drugs [18].
CAUSES OF CENTRAL ADRENAL FAILURE

Any disease process or injury that implicates the pituitary gland or hypothalamus and
interferes with ACTH secretion can cause secondary adrenal failure (Table 2, Table 3).
A tumour of the hypothalamo-pituitary region is one of the most frequent cause of
secondary adrenal failure.
It is usually associated with panhypopituitarism caused by tumour growth or treatment
with surgery (adenomas, craniopharyngiomas, meningiomas, ependymomas, etc.) or
irradiation (craniospinal irradiation in leukaemia, radiation for tumours outside the pituitary
axis, irradiation of a pituitary tumour).
Among patients with pituitary or hypothalamic disorders, especially space-occupying
lesions, few have only adrenal failure. Other hormonal axes are usually involved, and
ophthalmologic or neurologic symptoms may accompany, precede, or follow hypoadrenalism
[11, 36]. In lymphocytic hypophysitis there seems to be a particular predilection for early
involvement causing failure of ACTH secretion. This has more recently been reported after
ipilimumab treatment for cancer.
The administration of long term, high doses of glucocorticoids is the most common cause
of the tertiary adrenal failure. Chronic administration of high doses of steroids can decrease
the synthesis of corticotropin-releasing hormone (CRH) from the hypothalamus. Patients may
have hypothalamo-pituitary-adrenal axis (HPA) suppression after receiving 20 mg of
prednisone or other equivalent steroids per day for more than 3 weeks. Inhaled
glucocorticoids can also cause hypoadrenalism in a dose-dependent fashion [37].
Loss-of-function mutations of genes important for pituitary development or for synthesis
and processing of the corticotropin precursor proopiomelanocortin cause secondary adrenal
failure, albeit very rarely [11].
Isolated corticotropin deficiency generally results from an autoimmune process, as shown
by the frequent association with other autoimmune endocrine disorders [38]. Autoimmune
lymphocytic hypophyitis is less frequent, mostly affecting women during or after pregnancy
[39].
Table 2. Causes of secondary adrenal failure
CLINICAL FEATURES IN ADDITION TO

DIAGNOSIS
SECONDARY ADRENAL INSUFFICIENCY
PITUITARY TUMOURS
Adenomas, cysts,
Anterior or posterior pituitary hormone deficiencies, or
craniopharyngiomas,
both, and associated symptoms
ependymomas, meningiomas,
rarely carcinomas
PITUITARY SURGERY OR
IRRADIATION
for pituitary tumours, tumours
both, and primary disease-associated symptoms
outside the HPA axis or
leukaemia
INFECTIONS OR
INFILTRATIVE PROCESS
Lymphocytic hypophysitis,
haemochromatosis, tuberculosis, Anterior or posterior pituitary hormone deficiencies, or
meningitis, sarcoidosis, both, and primary disease-associated symptoms
actinomycosis, histiocytosis X,
Wegener‘s granlulomatosis
Abrupt onset of severe headache, visual disturbance,
nausea, vomiting; anterior or posterior pituitary
PITUITARY APOPLEXY
hormone deficiencies, or both, and primary disease-
associated symptoms
Peripartum abrupt onset of severe headache, visual
disturbance, nausea, and vomiting; anterior or posterior
SHEEHAN‘S SYNDROME pituitary hormone deficiencies or both, and primary
disease-associated symptoms

TRAUMATIC BRAIN INJURY
both, and associated symptom
GENETIC DISORDERS
Congenital proopiomelanocortin
Early-onset severe obesity, hyperphagia, red hair
deficiency
Prader-Willi syndrome Hypotonia, obesity, mental retardation, hypogonadism
Panhypopituitarism; short stature, delayed puberty,
HESX1 gene mutations
cognitive changes, septo-optic dysplasia
Mutations in gene for Panhypopituitarism, neonatal hypoglycaemia, pituitary
orthodentical homeobox 2 hypoplasia, ectopic posterior pituitary gland
Mutations in gene for LIM Panhypopituitarism; growth hormone, thyrotropin, and
homeobox 4 corticotropin deficiencies
Mutations in gene for PROP Panhypopituitarism; late-onset corticotropin deficiency,
paired-like homeobox 1 occasionally enlarged sella turcica
Mutations in gene for SRY (sex- Panhypopituitarism; infundibular hypoplasia,
determining region Y) box 3 hypopituitarism, mental retardation
Mutations in gene for T-box 19 Congenital isolated corticotropin deficiency
Table 3. Causes of tertiary adrenal failure
DIAGNOSIS
CLINICAL FEATURES IN ADDITION TO
TERTIARY ADRENAL INSUFFICIENCY
DRUGS
Glucocorticoid therapy (for more than Primary disease-associated symptoms
4 weeks)
Mifepristone Medical abortion, treatment of Cushing's
syndrome
CUSHING‘S SINDROME Weight gain, skin changes, osteoporosis

HYPOTHALAMIC TUMOURS Anterior or posterior pituitary hormone
Craniopharyngiomas or metastases deficiencies, or both, and primary disease-
from lung or breast cancer associated symptoms
HYPOTHALAMIC SURGERY OR Anterior or posterior pituitary hormone
IRRADIATION deficiencies, or both, and primary disease-
associated symptoms
INFECTIONS OR INFILTRATIVE Anterior or posterior pituitary hormone
PROCESSES deficiencies, or both, and primary disease-
associated symptoms
TRAUMA, INJURY Anterior or posterior pituitary hormone
deficiencies, or both, and primary disease-
associated symptoms
Drugs such antipsychotics, antidepressants and mifepristone, a glucocorticoid receptor

antagonist, cause resistance to glucocorticoids through impairment of glucocorticoid signal
transduction [40].
PATHOPYSIOLOGY
Under normal conditions, the secretion of glucocorticoids from the adrenal zona
fasciculata is under the exclusive control of pituitary ACTH (Figure 1).
The secretion of cortisol, the most important glucocorticoid in man, is diurnal and
pulsatile with maximum concentrations measured in the morning (06.00-08.00h) and lowest
around midnight. ACTH is synthesised and secreted by the corticotroph cells of anterior
pituitary from a large precursor molecule, proopiomelanocortin, in response to the
hypothalamic hormones CRH and vasopressin.
The adrenal androgens, androstenedione, ehydroepiandrostenedione (DHEA), and the
sulphate ester of ehydroepiandrostenedione (DHEA-S), secreted by zona reticularis, are
under ACTH regulatory control. These androgen precursors are converted in peripheral
tissues to more potent androgens such as testosterone, and oestrogens.
Figure 1. Pathophysiology of primary and central adrenal failure.
DHEA and DHEA-S secretion is age dependent, with an increase noted during the
adrenarche (6-10 years), and peak concentrations achieved around age 20-30 years.
Thereafter, DHEA concentrations steadily fall. This pattern suggests the possible existence of
ACTH-independent factors controlling the release of adrenal androgens.
Mineralocorticoids, and therefore aldosterone, are produced by the zona glomerulosa, the
outermost zone, and are primarily regulated by the renin-angiotensin system and extracellular
potassium concentrations.
The symptoms and signs of primary adrenal failure appear when the injury to
adrenocortical tissue is more than 90% [41]. The simplest model for the beginning step of
endocrine autoimmunity involves the loss of immunological tolerance to a peptide. The role
of antibodies in the pathogenesis of Addison‘s disease has not been completely established; in
fact, it appears that the destruction of adrenocortical cells mediated by T-lymphocytes is the
principal feature and the production of the antibodies against 21-hidroxylase can be secondary
to the release of peptides following the destruction of the adrenal glands. A persistent
subclinical viral infection, or excessive metabolic activity in response to stress, may
potentially lead to local activation of dendritic cells.
Proteins in the adrenal cortex, such as 21OH-derived peptides, will be presented on MHC
molecules of the dendritic cells under such conditions.
The initial phase of activation of dendritic cells may be followed by clonal expansion of
auto reactive T and B cells and the production of autoantibodies against 21-hydroxylase [21,
42]. The antibodies are of the IgG1 or IgG2a subclass, suggesting that T-helper cells are
involved in the destruction of the adrenal cortex [43, 44].
At the beginning of the chronic gradual destruction, there is a decrease in the adrenal
reserve and the secretion in response to stress is suboptimal, although basal steroid secretion
is normal. Consequently, any major or even minor stressor can precipitate an acute adrenal
crisis and lead to the clinical manifestations of the disease. Low plasma cortisol
concentrations result in the increase of production and secretion of ACTH due to decreased
negative feedback inhibition. The elevated plasma ACTH concentrations are responsible for
the well-recognised hyperpigmentation observed in these patients [1].
In secondary or tertiary adrenal failure, the loss of basal ACTH secretion cause the
atrophy of zona fasciculate and reticularis of the adrenal cortex with a decrease of basal
cortisol secretion, but a preservation of aldosterone secretion by the zona glomerulosa [45].
CLINICAL MANIFESTATIONS
Chronic Primary Adrenal Failure: the clinical features of chronic primary adrenal failure
are due to deficient concentrations of all adrenocortical hormones (glucocorticoids,
mineralocorticoids and adrenal androgens) (Table 4). The onset of chronic hypoadrenalism is
often insidious and the diagnosis may be difficult in the early stages of the disease because
most of the symptoms are non-specific.
The main symptom is fatigue, accompanied by general malaise, loss of energy, increased
irritability and reduced muscle strength. Additional symptoms are gastric pain, nausea,
vomiting, dizziness, low blood pressure and postural hypotension and other autoimmune
manifestations (eg, vitiligo). Sign and symptoms connected to glucocorticoid deficiency are
also anorexia, weight loss, myalgia and joint pain, fever, anaemia, lymphocytosis,
eosinophilia, hypoglycaemia, hypercalcaemia and increased thyrotropin release. Clinical
features especially due to mineralocorticoid deficiency are hyperkalaemia and hyponatremia,
salt craving, dehydration and raised serum urea. Clinical manifestations closely related to
adrenal androgen insufficiency are decreased axillary and pubic hair, dry and itchy skin and
loss of libido and amenorrhea in women, and an absence of adrenarche or pubarche in
children.
A more specific sign of primary hypoadrenalism is hyperpigmentation, caused by
enhanced stimulation of the skin MC1-receptor by ACTH and other pro-opiomelanocortin-
related peptides. The hyperpigmentation predominantly affects areas of skin subjected to
pressure, palmar creases, elbows, knuckles and oral mucosa [1, 11, 36, 43].
Chronic Central Adrenal Failure: The clinical manifestations of secondary or tertiary
adrenal failure are similar to those of primary hypoadrenalism (Table 4). However,
hyperpigmentation of the skin does not occur, because the secretion of ACTH is not increased
and patients may instead present an alabaster-coloured pale skin. Moreover, since the
production of mineralocorticoids by the zona glomerulosa is mostly preserved, dehydration
and hyperkalaemia are not present, and hypotension is less prominent. Hyponatraemia and
increased intravascular volume may be the result of inappropriate antidiuretic hormone
secretion, which can result from the loss of the inhibition of vasopressin release by
glucocorticoids. There might also be clinical manifestations of a pituitary or hypothalamic
tumour, such as symptoms and signs of deficiency of other anterior pituitary hormones,
headache or visual field defects [1, 11, 36].
Adrenal Crisis or acute adrenal failure may complicate chronic adrenal failure, and may
be precipitated by a physiological stress, such as trauma, surgery, serious infection, bilateral
adrenal infarction or haemorrhage. It is possibly less common in patients with secondary or
tertiary adrenal failure. In addition, an adrenal crisis can be the first expression of adrenal
failure. This disease is often referred to as the great mimic, for its capacity to look similar to
many other more frequently recognised diseases.
Table 4. Signs, symptoms and biochemical findings in adrenal failure. If prevalence is

not given, data are not available
Symptoms Comments Pathophysiological Mechanism Prevalence

FATIGUE, LACK OF
Glucocorticoid deficiency Adrenal
ENERGY, REDUCED 100
androgen deficiency
STRENGTH
ANOREXIA, WEIGHT LOSS Glucocorticoid deficiency 100
More frequent in
GASTRIC PAIN, NAUSEA, Glucocorticoid deficiency
primary adrenal 92
VOMITING Mineralocorticoid deficiency
failure
Primary adrenal
SALT CRAVING Mineralocorticoid deficiency 16
failure only
MYALGIA, JOINT PAIN Glucocorticoid deficiency 6-13
Mineralocorticoid deficiency
DIZZINESS 12
Glucocorticoid deficiency
DRY AND ITCHY SKIN In women Adrenal androgen deficiency -
LOSS OR IMPAIRMENT OF
In women Adrenal androgen deficiency -
LIBIDO
SKIN HYPER- Primary adrenal Excess of proopiomelanocortin-
94
PIGMENTATION failure only derived peptides
LOW BLOOD PRESSURE, Pronounced in
POSTURAL HYPOTENSION, primary adrenal 88-94
Glucocorticoid deficiency
DEHYDRATION failure
Deficiency of
ALABASTER-COLOURED Secondary adrenal
proopiomelanocortin-derived -
PALE SKIN failure only
peptides
FEVER Glucocorticoid deficiency -
LOSS OF AXILLARY OR
In women Adrenal androgen deficiency -
PUBIC HAIR
ABSENCE OF
ADRENARCHE OR In children Adrenal androgen deficiency -
PUBARCHE
PATHOPHYSIOLOGICAL
BIOCHEMICAL FINDINGS Comments Prevalence
MECHANISM
HYPONATRAEMIA Glucocorticoid deficiency (leading 88
to SIADH)
Primary adrenal
HYPERKALAEMIA Mineralocorticoid deficiency 64
failure only
Glucocorticoid deficiency (mostly
HYPERCALCAEMIA 6
concurrent hyperthyroidism)
RAISED SERUM Primary adrenal
Mineralocorticoid deficiency -
CREATININE failure only
ANAEMIA,
LYMPHOCYTOSIS, Glucocorticoid deficiency -
EOSINOPHILIA
HYPOGLYCAEMIA Glucocorticoid deficiency -
Primary adrenal Glucocorticoid deficiency
INCREASED TSH
failure only Autoimmune thyroid failure
The main clinical manifestation of adrenal crisis is severe hypotension and clinical
evidence of hypovolaemia, but patients may also have generic symptoms, such as nausea,
vomiting, painful abdomen, weakness, fatigue, myalgia, lethargy, confusion or coma
[17, 46, 47].
DIAGNOSIS
The first step is to assess the function of the adrenal cortex. Once adrenal failure is
confirmed, it is important to determine whether the cortisol lack is primary or secondary and
hence it is essential to establish the aetiology.
Basal morning serum cortisol concentrations and basal plasma ACTH: plasma
concentrations of ACTH and cortisol fluctuate throughout the day. Consequently, the
diagnostic usefulness of random samples is limited. Total cortisol, but not the biologically
active free fraction, can increase because of a change in hepatic cortisol- binding globulin
production. Patients with cirrhosis have low levels of cortisol-binding globulin, whereas
patients receiving oral oestrogens or in pregnancy have high levels of cortisol-binding
globulin [48].
In healthy people, serum cortisol concentrations are highest in the early morning (275-
555 nmol/L) and plasma ACTH concentration at 08.00h is 4.5-12 pmol/L. A low serum
cortisol concentration (<80 nmol/L) in a blood sample taken in the early morning strongly
suggests adrenal failure.
Measurements of cortisol and ACTH concentrations separate patients with primary
adrenal failure from those with secondary adrenal failure. In primary adrenal failure plasma,
ACTH is greatly increased (higher than 22 pmol/l), with serum cortisol generally below the
normal range) [49, 50].
Salivary cortisol concentrations at 08.00h of more than 16 nmol/ excludes adrenal
failure, whereas a value of less than 5 nmol/l indicates a high probability of adrenal failure.
This test has been used to screen for adrenal failure and might be an useful alternative [51].
Urinary free Cortisol (UFC): generally, baseline urinary measurements are not
recommended for the diagnosis of adrenal failure [1, 11, 36].
DIAGNOSIS OF PRIMARY ADRENAL FAILURE Low serum cortisol and high
plasma ACTH levels at 08.00h are extremely predictive of primary hypoadrenalism.
Primary hypoadrenalism is also associated with high plasma renin concentration or
activity, low aldosterone concentrations, hyperkalaemia and hyponatraemia [46].
Short Synacthen (ACTH) test: Traditionally, adrenal failure is diagnosed biochemically
by measuring serum cortisol before and then 30 minutes and 60 minutes after intravenous or
intramuscular administration of 250 µg synthetic ACTH [1-24] (Synacthen). This test shows
the impaired ability of the adrenal cortex to respond to ACTH. In healthy patients, synthetic
ACTH leads to an increase in serum cortisol to peak concentrations of greater than 430-500
nmol/L, depending on the cortisol assay. In patients with primary adrenal failure, exogenous
hormone administration usually does not evoke any further increase in serum cortisol
[14, 52, 53].
Measurement of serum 21-hydroxylase (anti-adrenal) autoantibodies: Adrenocortical
antibodies or antibodies against 21-hydroxylase are present in more than 90% of patients with
recent onset autoimmune adrenalitis. Autoimmune adrenalitis can be associated with other
autoimmune diseases and screening should involve measurement of fasting glucose and
thyrotrophin to exclude at least type 1 diabetes and thyroid disease, although thyrotropin may
be elevated by cortisol deficiency alone [54, 55].
If antibodies are negative, CT imaging is recommended to demonstrate haemorrhage,
tumours, the calcification typical of tuberculosis or infiltrations. The radiographic appearance
of glands destroyed by histoplasmosis or other infections is generally large (>4.5 cm), while
in autoimmune adrenalitis the glands are often reduced in size and atrophic [56, 57].
In male patients, plasma very long-chain fatty acids should be detected to check for
adrenoleukodystrophy [29].
DIAGNOSIS OF SECONDARY ADRENAL FAILURE In secondary hypoadrenalism
plasma ACTH concentrations are low or low normal. Plasma concentrations of aldosterone
and renin are generally unaffected.
The insulin-induced hypoglycaemia test (ITT) helps the investigation of the integrity of
the HPA axis and it is useful particularly in patients with suspected corticotropin deficiency
of recent origin. It is widely regarded as the gold standard. Insulin, at a dose of 0.1-0.15 U/kg,
is administered to induce hypoglycaemia, and measurements of cortisol concentrations are
determined at 30 min intervals for at least 120 min. Hypoglycaemia is a powerful stressor that
in physiologic conditions results in rapid activation of the HPA axis. The ITT also has the
advantage that GH reserve also can be estimated. Patients with cerebral or cardiac
dysrhythmias must be excluded, and a high degree of supervision is mandated [45, 58].
The short ACTH test is simpler and less invasive test than the ITT. Synthetic ACTH 250
µg is injected intravenously or intramuscularly, and serum cortisol is measured at 0, 30, and
60 min. A peak cortisol is defined as normal if greater than 430-550 nmol/L, depending on
the cortisol assay, at any time point. The rationale for the test relies on the fact that ACTH
deficiency leads to decreased cortisol synthesis, such the acute response to ACTH in patients
with chronic ACTH deficiency is attenuated. This test is particularly useful for all slow onset
pituitary deficiency states, such as after radiotherapy. The standard short corticotropin test
should not be used during the first 4-6 weeks after a pituitary or hypothalamic insult. To
increase sensitivity studies have explored the use of a low-dose test with 1 μg of ACTH [45,
52, 53], but this is cumbersome and is not in general in use.
Corticotropin-releasing hormone (CRH) test is used to differentiate between secondary
and tertiary adrenal failure, but it is rarely helpful [58].
MRI of the hypothalamic and pituitary region can reveal pituitary adenomas,
meningiomas, craniopharyngiomas, metastases and infiltrations.
THERAPY OF PRIMARY AND CENTRAL ADRENAL FAILURE

USE OF GLUCOCORTICOIDS: The challenge of management is to adapt
glucocorticoid replacement therapy to the necessities of each patient (Table 5). Monitoring of
replacement therapy to optimise the individual replacement dose or make dose adjustments is
mainly based on clinical manifestations, rather than objective biological serum markers
estimating the tissue activity of cortisol. Symptoms and signs indicating under-treatment
(i.e., fatigue, nausea) or over-treatment (i.e., weight gain, skin alterations) are not specific to
adrenal failure.
Furthermore, none of the conventional glucocorticoid treatments can perfectly imitate the
physiological cortisol rhythm, especially the fall in cortisol late evening with a nadir around
midnight [14].
Hydrocortisone is the most commonly used glucocorticoid for adrenal failure therapy. It
has high oral bioavailability, but has a short half-life between 60 and 120 minutes [59]. Dose
replacement with hydrocortisone should be customised on an individual basis and may be
guided by weight-related regimens.
Current treatment practice requires that the total daily dose of immediate release
hydrocortisone is administered two or ideally three times daily. The recommended total daily
dose is 15-25mg a day. However, the serum cortisol profile is still far from paralleling the
normal physiological cortisol circadian rhythm [14, 60].
A modified-released hydrocortisone tablet is now available in some European countries.
It is administered in the morning and contains a rapid-release coating and a timed-released
inner core of hydrocortisone, suggesting that only a once-daily dose might be necessary to
provide adequate coverage throughout the day [61-63].
Cortisone acetate is used in some European countries. Cortisone acetate can be
administers up to three times daily and the recommended total daily dose is 25-37.5 mg.
It requires conversion to hydrocortisone via the hepatic enzyme 11β-hydroxysteroid
dehydrogenase type 1 in vivo and, as such, has a lower cortisol peak and a slower onset
decline than hydrocortisone [64, 65].
Prednisolone, administered as a single morning dose, has also been used. It has more
sustained action compared with hydrocortisone. The once-daily dose should range between 3
and 5 mg [64].
A study examining the differences in health status related to the use of hydrocortisone,
cortisone acetate or prednisolone treatment in patients with adrenal failure failed to determine
any significant differences in health-related quality of life based on the type of glucocorticoid
used for replacement therapy [66].
USE OF MINERALOCORTICOIDS: Under-replacement with mineralocorticoids has
been reported in patients with primary adrenal failure (Table 5). It is important to note that
different glucocorticoids also have different mineralocorticoid activities, e.g., dexamethasone
is devoid of any mineralocorticoid activity while prednisolone has less than hydrocortisone.
In central adrenal failure, mineralocorticoid replacement is not necessary [50, 67].
Fludrocortisone is given in a dose of 0.05-0.20 mg once daily, in the morning. The dose
is titrated individually based on blood pressure, plasma renin activity concentrations and
serum sodium and potassium concentrations [14].
Table 5. Features of available steroid replacements
Recommended Recommended Dose

Steroids Comments
Total Daily Dose Frequency
Glucocorticoids
20-25 mg for Two or three doses
primary adrenal with 1/2- 2/3 of dose Physiological GC; 96%
failure in the morning and orally available; short
Hydrocortisone
15-20 mg for subsequent doses later half-life with steep
secondary adrenal in the early afternoon/ peaks and troughs
failure evening
Modified-Release
Once daily
Formulation
Lower serum cortisol
peak; requires
conversion to
Cortisone Acetate 25-37.5 mg Once in the morning
hydrocortisone, which
results in slow rise to
peak and slower decline.
Intermediate duration;
Prednisolone 3-5 mg Once daily more anti-inflammatory
than mineralo-corticoid
Mineralocorticoids
Dose adjustment needed
in hot climate, strong
Once daily in the perspiration, pregnancy
9-α-Fludrocortisone 0.1 mg
morning; or ½-0-1/2 or in patients with
concomitant
hypertension
Androgen
Once daily in the Not regarded as
DHEA 25-50 mg
morning standard replacement
USE OF ANDROGENS: Based on the evidence currently available, DHEA replacement

should not be undertaken routinely in clinical practice in patients with adrenal failure
(Table 5).
Women with hypopituitarism with concomitant adrenal failure have a more sever
androgen deficiency than those with primary adrenal failure. In this case, DHEA replacement
could be tried on an individual basis for patients with seriously and persistent impaired
quality of life and reduced libido.
The dose of DHEA used in most studies is between 25 and 50 mg once daily. The side
effects of DHEA replacement include acne, hirsutism, alopecia, an itching scalp/skin and
particularly increased sweat odour [14, 68, 69].
Anticonvulsants, such as phenytoin, phenobarbital and carbamazepine, stimulate
cytochrome P450 3A4, thereby inducing hepatic enzymes and leading to accelerated
glucocorticoid metabolism and a reduced glucocorticoid effect. Antiretroviral drugs, such as
ritonavir inhibit cytochrome P3A activity, and lead to delayed glucocorticoid metabolism and
increased glucocorticoid concentrations [1]. Growth hormone replacement in patients with
hypopituitarism can slightly lower the levels of cortisol as this inhibits the activity of 11beta-
hydroxysteroid dehydrogenase type 1, which converts cortisone to cortisol.
THERAPY OF ADRENAL CRISIS

Adrenal crisis is usually defined as acute impairment of general health with the need for
parenteral glucocorticoid administration and probable hospital admission. All patients and
their caregivers should be educated such that they are able to recognise an imminent adrenal
crisis. Treatment of patients who present in probable adrenal crisis must not be delayed by
diagnostic procedures.
Therapy should be initiated immediately with the rapid intravenous or intramuscular
administration of hydrocortisone 100 mg and intravenous saline administration.
To prevent future adrenal crisis, patient education should be reinforced to empower the
patient during several conditions:
 In cases of vigorous, sustained physical activity and deep, long-lasting psychological

stress, an additional hydrocortisone dose of 5-10 mg is recommended;
 For more minor physical and psychological stressors, such as dental procedures, it
might be also wise to double the dose for 24h;
 In case of illness and fever, the daily hydrocortisone replacement dose should be
doubled or tripled immediately to at least 30-60 mg/day;
 During a major stressor, including major surgery, severe trauma and childbirth,
diarrhoea or vomiting, hydrocortisone should be administered intravenously or
intramuscularly (100-400 mg/24 h).
It is essential that all patients with adrenal failure should receive a ―steroid emergency
card‖ which provides information as to the necessity for treatment, the current replacement
regimen and any relevant contact information for the responsible clinician.
All patients should also be provided with an emergency kit, which may contain rectal
suppositories (prednisolone-suppository), an ampoule of 100 mg hydrocortisone-21-
hydrogensuccinate i.m. or s.c. injection, injection devices, and an instruction leaflet on self-
administration in emergency situation [14, 17, 47].
STEROID REPLACEMENT IN PREGNANCY

Normal pregnancy is associated with a gradual physiologic increase in total serum
cortisol and corticosteroid binding globulin (CBG). This is a consequence of oestrogen-
induced hepatic production of CBG and increase of placenta CRH production, especially in
the third semester. Adrenal failure in pregnancy is relatively rare, but it is associated with
significant maternal and fetal morbidity and mortality if untreated. Patients should be treated
with hydrocortisone 20-30 mg/day, in three doses. At the onset of labour, the daily dose
should be doubled or tripled. If caesarean section is contemplated, stress dose of 100 mg IV
hydrocortisone should be given every 6-8 hourly [14, 70, 71].
ADRENAL FAILURE IN CRITICAL ILLNESS

Part of the physiological response to critical illness is an increase in serum cortisol [72].
Both inadequate cortisol secretion and impaired glucocorticoid receptor signalling are
involved in the pathogenetic mechanism underlying adrenal failure in critical illness.
Furthermore, proinflammatory cytokines may compete with ACTH on its receptor [73] or
induce tissue resistance to glucocorticoids [74]. Hypoadrenalism is recurrently been reported
in critically ill patients with septic shock, trauma, burns, liver failure, severe pneumonia, HIV
infection, after cardiac surgery, head injury, after the use of etomidate, and in brain-dead
organ donors [75-81]. Dysfunction of the HPA axis in critical illness could be described by
the term critical illness-related corticosteroid failure (CIRCI). CIRCI is characterised by an
exaggerated and protracted proinflammatory response. Adrenal failure should be suspected in
hypotensive patients who have responded poorly to fluid resuscitation and vasopressor agents,
mainly in the setting of sepsis. It is a dynamic process that occurs as a consequence of a
decrease in adrenal steroid production or tissue resistance to glucocorticoids [82].
CONCLUSION
Adrenal failure is an uncommon condition characterised by impaired adrenocortical
function. The main presenting symptoms such as fatigue, anorexia, and weight loss are non-
specific, thus diagnosis of adrenal failure is often delayed: failure to identify patients with
hypoadrenalism can result in adrenal crisis and death.
Most cases of primary adrenal failure are caused by autoimmunity and patients are in
particular at risk of developing other autoimmune diseases. The short ACTH test provides the
diagnosis of adrenal insufficiency in most patients and measurement of free serum or salivary
cortisol may be suitable when corticosteroid binding globulin is low, but requires
standardisation. In most cases, the aetiologic diagnosis could be restricted to measurement of
21-hydroxylase antibodies.
Although glucocorticoid therapy has permitted patients with life-threatening adrenal
insufficiency to survive, management of hypoadrenalism continues to be challenging. Serum
cortisol levels differ significantly between patients, and requirements change over the course
of the day and in response to stressful situations. This variation requires the tailoring of
therapy to individual patients and often results in the division of an overall glucocorticoid
dose into two or three doses throughout the day. Hopefully, new hydrocortisone preparations
will better mimic normal physiological cortisol levels with once-daily dosing and improve
disease control and outcomes. DHEA replacement therapy has been introduced for female
patients with persistent and seriously impaired quality of life, although they should not be
used routinely in clinical practice.
The most common cause of central hypoadrenalism is the suppression of CRH and
vasopressin synthesis and secretion that occurs as a result of exogenous steroid
administration. The diagnosis of central hypoadrenalism is typically made using the short
ACTH test or the insulin-induced hypoglycaemia test, and clinicians should be conscious of
the advantages and disadvantages of using each test. Treatment of central hypoadrenalism is
by glucocorticoid replacement therapy, and fludrocortisone is not required.
For obvious reasons patient education is considered indispensable for crisis prevention.
Critically patients, their relatives and caregivers must be taught to recognise signs of adrenal
crisis and to be prepared to administer life-saving treatment.
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Chapter 6
CLINICAL MANAGEMENT
OF HYPERALDOSTERONISM
Jun Yang and Peter J. Fuller

Hudson Institute of Medical Research, Clayton, Australia
Department of Endocrinology, Monash Health, Clayton, Australia
ABSTRACT
Primary aldosteronism, or Conn‘s syndrome, is a common cause of secondary
hypertension with a prevalence of up to 20% in patients with resistant hypertension. It is
characterized by inappropriately high aldosterone production, most commonly from a
unilateral adrenal adenoma or bilateral adrenal hyperplasia. Aldosterone synthesis in
primary aldosteronism is independent of the renin-angiotensin system and not
suppressible by volume expansion. Beyond its effect on raising blood pressure,
aldosterone excess also mediates adverse cardiovascular, renal and metabolic
consequences via its actions at the mineralocorticoid receptor. Patients with hypertension
that has an early onset, is difficult to control or is associated with hypokalemia or cardiac
dysfunction should be screened using an aldosterone/renin ratio and subsequently
confirmed with fludrocortisone or saline suppression testing. The identification of the
subtype of primary aldosteronism using radiological imaging and adrenal vein sampling
is also important as unilateral disease is curable by surgery.
The early diagnosis and targeted treatment of primary aldosteronism with either
surgery or mineralocorticoid receptor antagonists is crucial to prevent end-organ damage
mediated by aldosterone excess.
Keywords: primary aldosteronism, conn‘s syndrome, secondary hypertension, aldosterone

Corresponding author: Jun Yang. E-mail: Jun.yang@hudson.org.au.
116 Jun Yang and Peter J. Fuller
INTRODUCTION
Primary aldosteronism (PA), or Conn‘s syndrome, was first described in 1955 by Jerome
W. Conn, as a constellation of hypertension, hypokalemia, metabolic alkalosis and
neuromuscular symptoms associated with increased levels of aldosterone, a steroid hormone
which had only been purified two years earlier by Sylvia and James Tait [1, 2].
Biosynthesis of aldosterone from the adrenal zona glomerulosa is primarily regulated by
the renin-angiotensin system and extracellular potassium concentration, and to a lesser extent,
adrenocorticotropin (ACTH) [3].
The main effects of aldosterone are mediated by the mineralocorticoid receptor (MR) in
the cytosol of epithelial cells of the renal collecting duct [4]. MR activation promotes sodium
reabsorption, mainly via the epithelial sodium channel, and potassium excretion via Na+/K+-
ATPase. Hydrogen loss also occurs which can cause a metabolic alkalosis. In PA, aldosterone
production escapes control by renin or potassium, thereby leading to normal or elevated
aldosterone levels with low or suppressed renin. The development of methods for measuring
plasma renin activity permitted the differentiation of primary from secondary aldosteronism.
Secondary aldosteronism can occur in edema-forming states or renovascular hypertension
whereby hypertension is associated with elevated renin and aldosterone.
PA is now recognized as one of the most treatable causes of hypertension with a
prevalence ranging from 4.6% to 13.0% in patients with hypertension and up to 20% in those
with refractory hypertension [5-7]. A large prospective study of 1125 consecutive newly
diagnosed hypertensive patients referred to specialized centers found a prevalence of 11.2%
[7]. Of note, current studies of prevalence tend to focus on patients in referral centers or
special subgroups, the prevalence of PA in the general hypertensive population is less clear
[8]. The different subtypes of PA and their key features are summarized in Table 1. They
include aldosterone-producing adenoma (APA, 35% prevalence), bilateral adrenal hyperplasia
(BAH, 60%), unilateral adrenal hyperplasia (2%), aldosterone-producing adrenocortical
carcinoma (< 1%), ectopic aldosterone-producting adenoma (< 1%) and familial
hyperaldosteronism including Type 1 (< 1%), type II (1.2 - 6%) and type III (rare) [9, 10]. All
are characterized by inappropriate autonomous secretion of aldosterone that is not
suppressible by sodium loading or volume expansion [11]. APA and BAH constitute the
majority of cases of PA although the exact proportion attributed to each condition depends on
the availability of adrenal vein sampling (AVS), a diagnostic procedure used to lateralize
aldosterone secretion. It has been reported that the prevalence of APA is 60% in centers
which perform AVS but only 30% in centers without AVS [7]. APA can be cured surgically
and is therefore important to distinguish from BAH which is treated medically.
Undiagnosed and untreated PA is associated with increased cardiovascular, renal and
metabolic morbidity and mortality related specifically to the effect of aldosterone excess on
the MR in a range of target tissues [reviewed in 15, 16]. In particular, there is a large body of
evidence to show that activation of the MR in the cardiovascular system promotes tissue
inflammation and fibrosis, with adverse consequences for cardiac function [17]. One large
cross sectional study, involving 459 patients with PA and 1290 controls with essential
hypertension, individually matched for sex, age and office systolic blood pressure, showed an
increased prevalence of left ventricular hypertrophy, coronary artery disease, nonfatal
myocardial infarction, heart failure, and atrial fibrillation in those with PA [18].
Clinical Management of Hyperaldosteronism 117
Table 1. Summary of subtypes of primary aldosteronism
Subtype Prevalence Genetic basis Main Features

Hypertension that may be resistant to
Somatic mutations in
treatement; hypokalemia in ~ 50%
Aldosterone KCNJ5 (> 40%),
[12]; elevated aldosterone renin ratio
producing adenoma 30% - 60% ATP1A1 (5%), ATP2B3
(ARR); lateralisation of aldosterone
(APA) (2%) and CACNA1D
excess in adrenal vein sampling
(10%)
(AVS)
Bilateral adrenal As above, except AVS demonstrates
60% Unknown
hyperplasia (BAH) bilateral aldosterone secretion
Unilateral adrenal
2% Unknown As with APA
hyperplasia
Large adrenal mass > 4 cm;
Adrenocortical
< 1% Unknown prominent hypokalemia; markedly
carcinoma
elevated ARR [13]
Ectopic APA < 1%, rare Unknown Rarely described [14]
Familial
Chimeric gene
aldosteronism type I Onset of hypertension at young age;
combining promoter
(FHI or family history of PA or strokes at
< 1% region of 11β-
Glucocorticoid- young age; aldosterone level
hydroxylase and
remediable suppressed by dexamethasone
aldosterone synthase
aldosteronism, GRA)
Link with chromosomal
Familial Identical to sporadic PA apart from
region 7p22 but no
aldosteronism type II 1.2 – 6% occurrence of PA in two or more
candidate genes
(FHII) family members.
identified.
Severe hypertension in childhood
Familial
Germline mutations in (< age 7);
aldosteronism type III < 1%, rare
KCNJ5 severe hypokalemia; bilateral adrenal
(FHIII)
hyperplasia.
Another study reported a higher rate of cardiovascular events (22.6% vs 12.7%),

particularly strokes and arrythmias, in 270 patients with PA compared with 810 patients with
essential hypertension matched for BP and other risk factors [19].
However, with targeted treatment, the left ventricular changes were found to be reversible
after a mean of 36 months of followup in 180 patients with PA [20]. The fact that these
adverse events are reversible or preventable with targeted treatment of PA highlights the
importance of early case detection and management.
This chapter will cover recent advances in our understanding of the aetiology of PA as
well as its diagnosis including the initial screen, confirmation and final subtyping. The
surgical and medical management of PA will then be discussed.
The diagnosis and management of PA in pregnancy will not be discussed here as it has
recently been thoroughly reviewed by Riester and Reincke [21].
ETIOLOGY
Prior to 2011, glucocorticoid-remediable aldosteronism (GRA), or familial primary
aldosteronism type I (FH-I), was the the only form of PA with a precise known genetic defect.
It is an autosomal dominant condition first described by Sutherland et al., in a father and son
whose symptoms of PA were relieved by dexamethasone [22]. The cause lies in a chimeric
gene formed by unequal crossing over between the coding sequence of CYP11B2
(aldosterone synthase) and the promoter region of CYP11B1 (11β-hydroxylase) which causes
excessive aldosterone production in response to ACTH that is suppressible by dexamethasone
or other glucocorticoids [23]. Due to the ectopic expression of aldosterone synthase in the
zona fasciculata in GRA, there is increased production of cortisol metabolites hydroxylated at
the C-18 position including 18-oxocortisol and 18-hydroxycortisol which may aid diagnosis.
Affected individuals often develop hypertension in youth and are resistant to standard
antihypertensives although a personal or family history of hypertension is not obligate in
GRA as some members of affected GRA kindreds display normal or only mildly elevated
blood pressure [24]. Analysis of affected kindreds has demonstrated a high prevalence of
early cerebral hemorrhage, largely as a result of aneurysms [25]. The diagnosis was
previously made by dexamethasone suppression testing although that is now supplanted by
genetic testing using a long PCR technique to detect the chimeric gene [26].
Other familial forms of PA include type II (FH-II) which is also autosomal dominant but
does not harbour a known bybrid gene mutation and often presents in an identical fashion to
sporadic PA [10, 27]; and type III (FH-III) which tends to present with marked
hyperaldosteronism and massive bilateral adrenal hyperplasia [28].
The genetic basis of FH-III was recently elucidated as a result of major advances in
genomic sequencing. A gain-of-function mutation (T158A) was identified in the KCNJ5 gene
which encodes an inwardly-rectifying potassium channel (Kir 3.4) on zona glomerulosa cells
[29]. Somatic mutations in KCNJ5 have subsequently been found in approximately 40% of
sporadic APAs among Caucasian patients, especially in patients who are female, younger and
with a tendency to more severe PA [30-33]. The prevalence was even higher at 65% in a
Japanese series [34]. The reported mutations are almost always associated with a loss of
selectivity of the Kir 3.4 channels to potassium, with increased sodium conductance and cell
membrane depolarization which leads to an influx of calcium with stimulation of aldosterone
synthesis. The mechanism of the mitogenic response remains to be clearly determined.
Since the discovery of KCNJ5 mutations, somatic mutations within APAs have been
identified in ATP1A1 (encoding the α-subunit of Na+/K+ ATPase), ATP2B3 (encoding a Ca2+
ATPase calcium channel) and CACNA1D (encoding a voltage-gated calcium channel) with
prevalences of approximately 5%, 2% and 10% respectively [32, 35-37]. These mutations
tend to be more common in males with smaller APAs. Of note, germline mutations in KCNJ5
and CACNA1D are rarely detected by gene sequencing in familial cases of PA while only
somatic mutations have been described in ATP1A1 and ATP2B3. Furthermore, somatic
mutations in KCNJ5, ATP1A1, or CACNA1D genes are not limited to APAs. They are also
found in multinodular adrenals although only in one rather than all of the nodules [38].
It suggests that factors other than the recognised mutations may contribute to nodular
hyperplasia. Indeed the molecular basis for BAH remains obscure with several large series
noting an absence of germline KCNJ5, ATP1A1 and ATP2B3 mutations although genetic
analysis is limited by the absence of available adrenal tissue in cases of BAH [35, 39].
Detailed reviews of the genetics of PA have been recently published by Zennaro et al., [40],
Monticone et al., [41] and Al-Salameh et al., [42].
DIAGNOSIS – SCREENING
Despite advances in the genetic understanding of PA, current diagnostic procedures still
rely on screening for PA using the aldosterone to renin ratio (ARR) and confirmation using
suppression testing. As recommended by the US Endocrine Society Clinical Practice
Guidelines [11] as well as the Italian Society of Hypertension [43], it is prudent to screen for
PA in groups with a high prevalence of the condition, including hypertension which is:
a) Joint National Commission (JNC) stage 2 (> 160-179/100-109);

b) drug resistant (BP > 140/90 despite 3 antihypertensives);
c) associated with spontaneous or diuretic-induced hypokalemia;
d) present with an adrenal incidentaloma;
e) associated with a family history of PA, early-onset hypertension or stroke at age less
than 40-50 yr.
Of note, whilst hypokalemia can occur, it is documented in only 9 – 37% of patients with
PA [44]. Some centers advocate the screening of all hypertensive patients as it is thought that
the cost of screening is relatively small compred to the cost of lifelong antihypertensive drug
therapy and the potential cure afforded by surgical or targeted medical treatement [45].
ARR is recommended as the the initial screening test for the detection of PA on the basis
that single measurements of either plasma aldosterone concentration (PAC), direct renin
concentration (DRC) or plasma renin activity (PRA) show broad overlap between normal
patients and those with PA [46, 47]. The ratio is more robust and less affected by diurnal
variations and postural changes than individual measurements of PAC, DRC or PRA [48].
Despite its routine use, there are laboratory-based analytical issues in the measurement of
ARR. Plasma aldosterone is generally measured by radioimmunoassay which can be affected
by antibody cross-reactivity with aldosterone metabolites, especially when using automated
immunoassay platforms [49]. DRC assays may also dsiplay cross-reactivity with non-target
molecules such as prorenin while PRA assays are subject to variability in the incubation
period and non-standardized approach to angiotensinase inhibition [49].
Developments in high performance liquid chromatography and tandem mass
spectrometry (LC-MS/MS) has allowed aldosterone and renin activity to be quantified in a
more reproducible manner [50-53]. An even more high-throughput method, immuno-MALDI
(iMALDI)-based assay, has been proposed for PRA determination [54]. However, the
challenges of LC-MS assay development and calibration, as well as the substantial cost,
means radioimmunoassays remain the predominant methodology.
At present, there are no firm recommendations for ARR cutoffs due to the variability of
assays between laboratories and a range of units for measurement. PAC is reported as pg/mL,
ng/dL, ng/L and pmol/L while PRA can be reported in ng/mL/h, pmol/L/min, nmolL/h and
DRC in ng/L or mU/L. As a result, published ARR thresholds appear widely discrepant.
Furthermore, differences in patient populations and other confounding factors prevent

generalization about the most appropriate ARR with thresholds between studies varying by
more than 10-fold [55]. As a guide, suggested ranges rather than definitive values are
included in the Endocrine Society Guidelines:
a) 20 – 40 ng/dL:g/mL/h for PAC/PRA

b) 3.8 – 7.7 ng/dL:ng/L for PAC/DRC
c) 550 – 1100 pmol/L:ng/mL/h for PAC/PRA
d) 66 – 135 pmol/L:mU/L for PAC/DRC
e) 105 – 210 pmol/L:ng/L for PAC/DRC
Some centers also require an elevated PAC (usually > 410 pmol/L) as part of positive
screening for PA, arguing that the ARR is very sensitive to the renin level and may be falsely
elevated in patients with lowered renin, such as the elderly and the black African-American
populations [56-60]. However, while the addition of a threshold aldosterone level will
increase the specificity of the ARR, it will reduce the sensitivity of the screening process [61].
For example, 19% of patients with an APA and 43% of patients with BAH had PAC < 410
pmol/L amongst 63 patients with confirmed PA from the Princess Alexandra Hospital
Hypertension Unit series [62]. Therefore a threshold aldosterone level is not universally
incorporated into the screening of PA. Preanalytical effects of diet, posture, time of collection
and concomitant medications can significantly affect plasma aldosterone and renin, as
summarized in Table 2, althogh concurrent antihypertensives have not consistently been
shown to impact on the diagnosis of PA [61, 63-65].
Table 2. Causes of false positive and negative ARRs
ARR Cause Mechanism

Decrease renin
β-blocker, α-methyldopa, clonidine, non-
steroidal anti-inflammatory drugs,
Renin reduced in humans, but raised in
licorice products moxonidine [66, 67]
rodents with unclear effect on ARR
False Renin levels low due to reduced renin
Renal impairment [68]
Positive secretory mass and salt/water retention
Aging [69] Decrease renin
Induce angiotensinogen production by the
Oral estrogens [70] liver and increase angiotensin II, which
reduces renin (only affects DRC, not PRA)
Severe dietary salt restriction,
renovascular and malignant hypertension, Increase renin activity
pregnancy
Hypokalemia Decrease aldosterone secretion
False
Diuretics (including spironolactone) Increase renin activity.
Negative
Dihydropyridine calcium channel
antagonists (e.g., amlodipine), Decrease aldosterone level; Increase renin
angiotensin II receptor blockers, ACE- activity
inhibitors
Patients should be carefully prepared before having their blood taken for an ARR. The
same preparation is required before later confirmatory testing and subtype evaluation. Some
salient points include [11, 57, 71]:
a) Correct hypokalemia with an aim of achieving K+ > 4.0 mM.

b) Encourage a liberal salt intake of around 100 - 150 mmol per day (this represents the
average population intake on a normal diet; 6.4 g NaCl = 100 mmol Na+, 1 teaspoon
table salt = 2.3 g NaCl).
c) Blood should be collected in the morning before 10:00 AM and after one hour or
more of ambulation.
d) Withdraw drugs which strongly interfere with ARR for at least 2 – 4 weeks:
 spironolactone, eplerenone, amiloride (≥4 weeks)
 potassium wasting diuretics (≥2 weeks)
e) Stop the agents listed below for ≥ 2 weeks if hypertension (and other underlying
cardiovascular disease) can be safely managed with alternative medications:
 β-blockers
 central alpha-2 agonists (clonidine and methyldopa)
 non-steroidal antiinflammatory drugs
 angiotensin converting enzyme (ACE)-inhibitors
 angiotensin II receptor blockers
 dihydropyridine calcium channel antagonists (e.g., amlodipine)
 moxonidine
f) Use alternative agents which have less effect on the ARR to control hypertension:
 verapamil (90 – 240 mg daily)
 hydralazine (12.5 – 50 mg BD)
 prazosin (0.5 – 5mg BD or TDS)
Given the analytical complexities of measuring the ARR, and the numerous factors that
can influence its level including oral contraceptive agents, antidepressants, renal impairment,
pregnancy, phase of menstrual cycle and age, it is prudent to interpret the ARR in the full
clinical context with attention to the medical and family history, severity of hypertension,
resistance to antihypertensive medications, absolute aldosterone concentration and the
presence of electrolyte or acid-base disturbances [49].
DIAGNOSIS – CONFIRMATION
An elevated ARR is not diagnostic of PA. As a screening test, it is highly sensitive but
not very specific and may simply reflect a suppressed renin level [11, 72]. Confirmation of
PA requires the demonstration of at least partly autonomous aldosterone production in the
presence of manoeuvres designed to suppress aldosterone in order to reduce false positive
ARRs and thus prevent inappropriate adrenal venous sampling or surgery. A number of
confirmatory tests are available. They include the fludrocortisone suppression test (FST),
saline suppression test (SST) and oral salt loading, which all aim to expand plasma volume
and hence suppress renin; or the captopril challenge test which aims to suppress aldosterone
via inhibition of angiotensin-converting enzyme. Their characteristics, advantages and

limitations are summarized in Table 3. Of note, the intravenous SST is traditionally
performed in the recumbent position although a recent report suggested that a seated SST was
superior for detecting those with bilateral disease [73]. This study observed that all cases of
bilateral adrenal hyperplasia (n = 11 in a cohort of 24 patients with PA) which tested positive
with a FST and seated SST had post-saline aldosterone levels below the diagnostic threshold
of 140 pmol/L for a recumbent SST. Further studies are required to substantiate this result.
Currently there is no universal gold standard and the Endocrine Society‘s recommendation is
to select a test taking into account cost, patient status, compliance and local laboratory
expertise [11].
DIAGNOSIS – SUBTYPING
Once autonomous aldosterone production is confirmed, further testing is required to
identify the PA subtype. Unilateral disease in the form of aldosterone producing adenoma
(APA) is important to distinguish from bilateral disease such as bilateral adrenal hyperplasia
(BAH) as it is surgically curable. For patients who are not suitable surgical candidates,
treatment should proceed as for BAH without further testing.
All patients with PA who are potential surgical candidates should undergo a triple-phase
adrenal CT scan to assist in subtype differentiation and to exclude large masses which may
represent adrenal carcinoma. An MRI is slightly more sensitive but less specific and subject
to motion artifact [84]. However, there are limitations to the use of CT as the sole test to
differentiate between unilateral and bilateral disease as small adrenal adenomas < 1 cm may
go undetected or be interpreted incorrectly as adrenal hyperplasia, while areas of hyperplasia
may be called adenomas. Furthermore non-functioning adrenal incidentalomas are not
uncommon, especially in patients over 40 years of age and are indistinguishable from
functioning adrenal adenomas.
There is poor concordance between the findings of adrenal CT and adrenal vein sampling
(AVS) [62, 85-88], with one study showing that CT detected fewer than 25% of adrenal
adenomas smaller than 1 cm and only correctly identified the adenoma in 53% [89]. A most
recent review of 38 studies that compared localization by CT/MRI and AVS found that in
37.8% of 950 patients, the MRI/CT results disagreed with AVS results. Based on CT/MRI
alone, surgery would have been done unnecessarily for bilateral disease in 14.6% of patients,
medical treatment would have been given for unilateral disease in 19.1% and removal of the
wrong adrenal would have occurred in 3.9% [90]. Even the latest generation of multidetector
CT scanners have not been shown to offer sufficient diagnostic accuracy, correctly predicting
AVS localization in only 65% of cases [91].
Currently the only reliable way to differentiate unilateral from bilateral PA preoperatively
is by adrenal vein sampling (AVS). During AVS, adrenal and peripheral veins (usually
external iliac or inferior vena cava) are sequentially or simultaneously catheterized through a
percutaneous femoral vein approach under fluoroscopic guidance. Small amounts of contrast
are injected to guide the catheter through difficult anatomy and to confirm catheter tip
location.
Table 3. Features of four confirmatory tests for the diagnosis of primary aldosteronism
Fludrocortisone Intravenous Saline

Test Oral sodium loading Captopril challenge test
Suppression Test (FST) Suppression Test
Fludrocortisone acetate 100
2L normal saline infused
mcg QID for 4 days;
over 4 hours, starting at 0800
potassium chloride NaCl 6 – 12.8 g per day
– 0930 hr. Captopril 25 – 50 mg given
supplement to keep K+ > for 3 days;
orally after sitting/standing
4.0 mM; NaCl 1.75g TDS potassium chloride
Patient stays recumbent at for 1 hr, between 7AM –
and high sodium diet. supplement to keep K+ >
least 1 hr before and during 9AM.
4.0 mM;
Method infusion.
Inpatient (or ambulatory
Measure renin, aldosterone,
clinic) for 4 days with Measure 24 hr urinary
Measure aldosterone, cortisol at time 0 and 1 – 2
measurement of aldosterone aldosterone from
cortisol and K+ at time 0 hr after challenge with
and renin at 1000 hr of day morning of day 3 to day
(baseline) and 4 hr patient seated.
4 (patient seated) and 4.
(completion of saline
cortisol at 0700 and 1000
infusion).
hr.
K+ > 4.0;
24 hr urine Na > 3
High dietary sodium;
mmol/kg; Normal K+.
24 hr urinary Na+ > 200
0700 cortisol > 1000 Adequate Na+ (7.6g NaCl
Pre-requisites nmol/day.
cortisol; Accuracy not affected by per day or more) [74].
If outpatient, blood pressure dietary Na intake [74].
Normal K+.
and K+ should be checked
twice daily.
> 8.5 ng/dL (240 pmol/L)
> 5 ng/dL (138 pmol/L),
sensitivity 97% [80]
sensitivity 90%, specificity
Diagnostic 84% [76].
> 12 ng/dL (330 pmol/L) or
aldosterone > 6 ng/dL (166 pmol/L) at
elevated ARR post captopril;
level, unless 1000 hr on day 4 > 7 ng/dL (194 pmol/L),
but based on only 6 cases
specified sensitivity 88%, specificity24 hour urinary
[81]
otherwise, for > 8.13ng/dL (225 pmol/L) 100% [77, 78]. aldosterone > 12 ug
diagnosis (cut- after 3 days of FST may (33.3 nmol)
> 13.9 ng/dL (382 pmol/L)
offs differ at also be appropriate (n = 48) > 6.75 ng/dL (187 pmol/L)
sensitivity 69.6%, specificity
various [75]. sensitivity 82.6%, specificity
of 74% [74]
centers) 75.1% [7, 79]
Absence of suppression of
> 10ng/dL (277 pmol/L)
aldosterone by 30% from
makes PA highly likely.
baseline.
Suitable for patients with
Very specific.
severe hypertension, cardiac
Very sensitive and specific Simple; requires only 4
Advantages Inexpensive. failure, cardiac arrhythmia
[72, 76]. hours.
or severe renal insufficiency.
Inexpensive.
Inexpensive, quick, easy.
Inpatient stay is expensive; Urine collection may be
Takes 4 days; incomplete;
Diagnostic accuracy poor in
Difficult to maintain serum Poor compliance with
Not as sensitive as FST [72]. patients with low Na+ intake;
K+; salt loading;
Dis-advantages Avoid in cardiac failure or Numerous reports of false
Avoid if severe Avoid if severe
other edematous state. negative and positives [82,
hypertension, cardiac hypertension, cardiac
83].
failure, stroke, cardiac failure, renal failure,
ischemia. cardiac arrhythmia.
Rossi GP (University
Hospital, Padova, Italy);
Major centers Mulatero P (Torino, Italy); Young WF Jnr (Mayo,
Stowasser M (Brisbane,
using the Giacchetti G (Ancona, Italy); Rochester, Minnesota);
Australia); Fardella CE
procedure [44, Loh KC (Tan Tick Seng Connell JMC (Glasgow,
(Santiago, Chile)
48] Hospital, Singapore); UK)
Connell JMC (Glasgow,
UK)
Blood is gently aspirated from the veins, accurately labeled and assayed for aldosterone
and cortisol concentrations. The cortisol measurements are used to confirm successful
cannulation of the adrenal vein and to correct the aldosterone measurements for dilutional
effects. AVS is technically challening, especially catheterization of the right adrenal vein
which is smaller than the left adrenal vein and empties directly into the inferior vena cava
posteriorly at a right angle. Whilst an adrenal CT helps to define the anatomy of the adrenal
veins, there is still considerable anatomic variability [72].
Modern imaging techniques using multidetector CT venous mapping for the localization
of the right adrenal vein or image registration of cone-beam CT and contrast-enhanced CT
images may facilitate successful catheterization [92, 93]. The use of CT during angiography
to aid catheter positioning has been reported to improve cannulation success [94, 95] while
intra-procedural rapid cortisol analysis to confirm successful cannulation is also useful but
has limited availability at most centers [96-98] Another factor that can significantly improve
cannulation success rates is the focussed expertise of one or two dedicated interventional
radiologists who are given a high throughput of procedures [99-101]. Success rate improves
dramatically with the experience of the angiographer and can reach up to 96% in centers with
a significant case load and focused expertise [85, 86, 89].
The use of corticotrophin (ACTH) during AVS has also been reported to increase the
success rate of cannulation with some studies showing improved rates of lateralization [99,
102, 103]. The main rationales behind the use of ACTH are to minimize stress-induced
fluctuations in aldosterone secretion during non-simultaneous AVS, maximize cortisol
production and therefore enhance the adrenal vein to peripheral vein cortisol ratio to facilitate
confirmation of a successful cannulation, and to maximize secretion of aldosterone from an
adrenal adenoma [100]. However, there is concern that ACTH could lead to stimulation of the
contralateral gland and cause loss of laternalization [62, 88, 104]. A recent prospective study
comparing different methods of ACTH administration actually demonstrated a reduced
lateralization success rate [105]. However, in centers where AVS is not performed in the
setting of an ACTH infusion, the procedure is performed by a dedicated radiologist in the
early morning after overnight recumbency to avoid the confounding effects of posture on
aldosterone levels and to take advantage of high endogenous ACTH levels.
Successful catheterization requires evidence of elevated cortisol production from the
adrenal veins compared to the periphery. It requires the adrenal to peripheral vein cortisol
ratio, also called the selectivity index (SI), to be > 1.1 to 3 without ACTH [62, 88, 106] or > 3
to 5 in the presence of ACTH infusion [86, 99, 100]. Several recent studies have identified the
confounding effects of autonomous cosecretion of cortisol from adrenal adenomas [107, 108].
It is therefore important to exclude cortisol hypersecretion prior to AVS and to consider the
measurement of an alternative to cortisol such as metanephrines during AVS [109].
Once successful cannulation is confirmed, the lateralization index (LI) can be calculated.
Aldosterone measurements are first divided by their respective cortisol concentrations at the
same site to correct for dilutional effects of peripheral veins flowing into either adrenal vein
(Aldosterone/Cortisol Ratio, ACR). The LI is the ratio of the larger ACR (―dominant‖ side) to
the smaller ACR (―non-dominant‖ or contralateral side). Whilst there is still controversy in
the interpretation of LI for the lateralization of disease, it is generally accepted that LI > 2 to 3
without ACTH [81, 106] or > 4 in the presence of ACTH infusion [86, 89] represents
unilateral disease. LI < 2 is accepted to represent bilateral adrenal overproduction of
aldosterone.
In addition to an elevated LI, some groups also require the demonstration of suppression
of aldosterone secretion on the non-dominant side. They require the adrenal vein ACR on the
non-dominant side to be suppressed below peripheral vein ACR in order to diagnose
unilateral disease on the dominant side [88, 102, 103, 110].
The achievement of both criteria would give a positive and negative predictive value of
100% [111]. In a recent study of 29 patients with LI < 4 (in the presence of ACTH),
contralateral suppression of aldosterone (in 16) was an accurate predictor of normalisation of
ARR (100% vs 46% in 13 patients with non-suppression) after APA removal [112].
Similarly, a retrospective study of 80 patients found those with contralateral suppression (n =
66) experienced better blood pressure control (normotension in 96% vs. 64%), and
biochemical outcomes (normalisation of fludrocortisone suppression test in 88% vs 44%)
from surgery [113]. However, the lack of contralateral suppression was not associated with a
lower rate of response to adrenalectomy in 190 patients with LI > 4 in another recent study
[114]. Hence, there is still uncertainty as to whether contralateral suppression should be
incorporated into the diagnostic criteria for an APA. Pragmatically, contralateral suppression
is a useful feature in cases where AVS fails to cannulate the dominant side. An APA may be
diagnosed if a convincing radiologic lesion is present on the dominant side together with
suppressed aldosterone production from the contralateral side.
Some centers do not advocate AVS for patients aged < 40 yr with a solitary unilateral
adrenal adenoma > 1 cm on CT [89, 100, 115], citing the incidence of adrenal incidentalomas
of < 1% in those younger than 40 yr [116]. This recommendation is supported by a recent
study where all the young patients (< 40 yr) with unilateral adrenal nodules > 1 cm were
found to have APA, although only five patients displayed these characteristics [110]. This
information should be provided to young patients who may choose to proceed to surgery
without AVS. Whilst AVS is considered the current gold standard in subtyping PA, its
protocol and interpretation are not standardized around the world [117, 118]. A recent study
evaluated the consequences of using four sets of diagnostic criteria taken from experienced
institutions in Brisbane, Padua, Paris and Turin, and found discordance in subtype
differentiation in up to 40% of AVS [119]. It found almost five times more AVS procedures
were classified as unsuccessful with the strictest SI criteria than with the least strict criteria
(18% versus 4% respectively) In addition, twice as many AVS procedures were classifed as
lateralized using the least stringent LI cut-off compared to the most stringent cut-off (60% vs
26% respectively). The most serious clinical consequence of a wrong interpretation is
unjustified surgery in a patient with bilateral aldosterone excess who is wrongly diagnosed
with unilateral disease due to lenient selectivity criteria.
It may therefore be safer to adopt strict selectivity criteria for unilateral PA, when
wrongly diagnosed as bilateral disease, can still be adequately managed with medical therapy.
Alternatives to AVS which are less costly, invasive and operator dependent are being
sought. Clinical prediction scores based on the presence of a typical unilateral adrenoma on
CT plus serum potassium < 3.5 mmol/L or estimated glomerular filtration rate > 100 m?/min/
1.73m2 for the diagnosis of APA was found to be 100% specific but only 53% sensitive in
one study [120]. However, the same criteria was not sufficiently sensitive or specific in other
studies [121, 122]. Nevertheless, the prediction score may assist with clinical decision making
in those with failed AVS. Another approach to subtyping is adrenal scintigraphy using
radioactive tracers which are preferentially taken up by an adrenal adenoma after suppression
of normal glandular uptake with dexamethasone pretreatment.
Semiquantification of 131I-6β-iodomethyl-norcholesterol (NP-59) single photon emission

computed tomography (SPEC-CT) has been found to accurately predict postsurgical
outcomes of adrenalectomy in a cohort of 49 patients [123] while 11C-metomidate positron
emission tomography (PET-CT) was found to offer a sensitivity of 76% and specificity of
87% when compared to AVS [124]. The use of these techniques is limited by radiotracer
availability. Finally, a common limitation to any technique that aims to distinguish unilateral
from bilateral disease is the lack of a true gold standard for diagnosing APA. False negatives
where lateralized secretion is not diagnosed can easily go unnoticed as these patients do not
undergo surgery. False positives which undergo unnecessary surgery and experience
persistent hypertension can also be dismissed as having essential hyertension or residual
effect of prolonged vascular remodelling by aldosterone [125, 126]. Even the histopathologic
diagnosis of APA is difficult as there are no morphological criteria to identify aldosterone-
producing cells, although this may be improved by new specific antibodies that target the
adrenal steroidogenic enzymes such that APAs may be identified by immunohistochemistry
[127]. Postoperative suppression tests are necessary to formally ascertain the cure of PA but
are only routinely performed in a few centers. Therefore, each patient with PA should be
assessed within the full context of their comorbidities, family history, biochemistry, imaging
and AVS results for the best possible diagnosis, and be reassured that at least their
aldosterone excess can be specifically targeted by medical therapy even if they are wrongly
classified as having bilateral disease.
MANAGEMENT
Treatment of PA depends on the underlying etiology, patient comorbidities and other
factors such as drug intolerance. The mainstays of treatment include surgery for unilateral
disease and mineralocorticoid receptor antagonists (MRA) for bilateral disease. Two MRAs
are currently available, spironolactone and eplerenone, both of which are steroidal,
competitive antagonists [128]. In addition, a low sodium diet of less than 80 mEq per day is
recommended to help limit target organ damage as sodium status has been found to correlate
with left ventricular mass and obstructive sleep apnea as well as the severity of proteinuria in
patients with PA [129-131]. Laparoscopic adrenalectomy is the treatment of choice for
patients with unilateral disease who are suitable surgical candidates as it reduces medication-
related side effects, is more cost effective than lifelong medical therapy, especially if the life
expectancy is greater than 25 years [132], and produces better clinical outcomes for patients.
A nationwide epidemiological study conducted in Japan which analyzed data from 1706
patients with PA found that only surgical therapy was associated with amelioration of
hypokalemia and hypertension in this cohort [133]. Furthermore, a systematic review of
surgery in PA identified that surgery was associated with the use of fewer antihypertensive
medications, improved quality of life, and probable lower all-cause mortality compared with
medical treatment, although only a qualitative analysis was performed due to the
heterogeneity of protocols and reported outcomes [134]. Surgical treatment has also been
shown to reverse parameters of vascular and myocardial injury induced by aldosterone excess
[135, 136]. After unilateral adrenalectomy, the remaining contralateral gland offers sufficient
adrenal reserve although the basal cortisol level becomes sustained by a mildly elevated
ACTH and the cortisol secretory response to ACTH stimulation becomes slightly reduced
[137]. This may be an important consideration in patients who already have a degree of
adrenal suppression due to anti-inflammatory glucocorticoid treatment.
Prior to surgery, pre-treatment with a MRA is useful to correct hypokalemia and to
reverse renin suppression so that the contralateral adrenal gland may resume aldosterone
secretion to ensure a smoother perioperative course with better control of blood pressure and
plasma potassium. The MRA should be ceased post-operatively to avoid hyperkalemia and
the serum potassium monitored weekly for four weeks. All potassium replacement should be
withheld in the first 24 hours post-operatively unless plasma potassium is less than 3.0 mmol/
L as the patient‘s aldosterone levels may be very low following the removal of an APA [72].
PAC can be measured 1 – 2 days post-operatively although a normalized ARR approximately
six weeks after MRA cessation would be a more reliable measure of biochemical cure [9].
Typically the hypertension resolves in 1 – 6 months [138]. The long-term cure rates of
hypertension after surgery ranges from 33% to 85% [139-142] with older age and longer
duration of hypertension being the main negative prognostic factors. On the other hand,
favorable prognostic factors include having one or no first-degree relative with hypertension
and preoperative use of two or less antihypertensive drugs [140]. Successful treatment of PA
may also lead to a decrease in the renal glomerular filtration rate (GFR) due to removal of the
hyperfiltrating effect of aldosterone excess and unmasking of the underlying renal damage
[143, 144]. Age, male sex, low plasma potassium levels, and high plasma aldosterone
concentrations have been found to be independent risk factors associated with a lower GFR in
PA [145].
For patients with APA who have contraindications to surgery or who choose to have
medical therapy, a MRA is required. In a study examining the medical management of APA,
24 patients with APA were treated with spironolactone ± amiloride ± other antihypertensives,
and followed for 5 to 17 years [146]. Systolic blood pressure control was achieved in 75%
while diastolic blood pressure control was achieved in 83%. During follow-up, none of the
patients experienced a cardiovascular event or developed heart failure. Furthermore only five
patients experienced an increase in the size of the APA and no malignant transformation was
noted. Studies by Catena et al., have also shown that medical treatment of APAs can correct
microalbuminuria and reduce left ventricular mass to a similar extent as surgery [147, 148].
Medical management remains the mainstay of treatment for PA caused by bilateral
pathology [9, 11, 149]. MRAs such as spironolactone are the first choice as they target the
pathophysiology and protect the cardiovascular and renal systems from blood pressure-
independent injury caused by aldosterone excess. However spironolactone also acts on the
androgen and progesterone receptors and is associated with dose-dependent adverse effects
such as gynecomastia in men and menstrual disturbance in women. One study examining
spironolactone use in essential hypertension reported an incidence of gynecomastia of 6.9% at
a dose of less than 50 mg per day and 52% at a dose of more than 150 mg per day, although
no additional antihypertensive benefit was observed at this dosage [150]. A more MR-
selective antagonist, eplerenone, has far less side effects but is less potent. A recent
randomized, double blinded trial comparing the efficacy, safety and tolerability of eplerenone
(100 mg – 300 mg per day) to that of spironolactone (75 mg – 225 mg per day) found
spironolactone to offer superior antihypertensive effect albeit at the expense of increased
gynecomastia in males (21% vs 5% for eplerenone) and mastodynia in females (21% vs 0%)
[151].
If spironolactone or eplerenone are not tolerated, then amiloride, an epithelial sodium

channel antagonist, may be considered [152]. It targets the distal tubular sodium channels
which are upregulated by aldosterone, but is less effective than MR antagonists. While
spironolactone is effective as monotherapy in nearly 50% of patients with PA, about 75% of
patients on amiloride need additional antihypertensive agents to achieve blood pressure
control [153]. A suggested treatment regimen would be to commence spironolactone at 12.5 –
25 mg daily and increase to 50 – 100 mg daily; or start eplerenone at 25mg BD and increase
to 50 – 100 mg BD until normokalemia is achieved without potassium supplementation.
Amiloride can be used at 2.5 – 10 mg BD and other antihypertensives such as angiotensin
converting enzyme-inhibitors, angiotensin II receptor blockers, potassium-wasting diuretics
(e.g., hydrochlorothiazide) or calcium channel blockers may also be needed to optimize blood
pressure control.
Newer agents such as the non-steroidal dihydropyridine-based MRA finrenone (also
known as BAY 94-8862) are currently in development for the treatment of heart failure [154-
156]. They appear to display high in vitro and in vivo MR potency as well as selectivity with
respect to the other steroid hormone receptors. Trials in patients with PA are still pending.
Michael Stowasser‘s group recently examined the role of unilateral adrenalectomy in
BAH and found some benefit in highly selected patients [157]. The 40 patients either failed
medical therapy or were intolerant of MR antagonists. The adrenal gland chosen for removal
was either the one that demonstrated a higher adrenal venous aldosterone/cortisol ratio or one
that displayed the most morphological abnormality on CT.
At final follow-up 12 – 144 months after adrenalectomy, 15% of patients were cured
while 20% experienced improvement. The response rate did not differ significantly when
different lateralization criteria were applied. The study suggests that unilateral adrenalectomy
as a form of debulking surgery for BAH may still be considered in selected patients although
further research is required.
Patients with bilateral aldosterone hypersecretion who have an onset of hypertension at a
young age (e.g., < 20 years) and a family history of PA or stroke at a young age should be
tested for GRA using the long PCR test. Once diagnosed, GRA should be treated with the
lowest dose of glucocorticoid to partially suppress ACTH secretion (e.g., dexamethasone
0.125 – 0.25 mg daily or prednisolone 2.5 – 5 mg daily) and normalize blood pressure and
potassium levels [11]. The dose should be given at bedtime to suppress the early morning
surge in ACTH. Monitoring plasma renin activity, serum aldosterone and 18-hydroxylated
cortisol metabolites can provide objective measures of treatment efficacy. If the
glucocorticoid alone does not completely normalize blood pressure, a MRA may be added.
CONCLUSION
Primary aldosteronism is a common and potentially curable form of hypertension that
warrants early diagnosis and treatment. Aldosterone and renin levels should arguably be
incorporated into routine laboratory tests for newly diagnosed hypertensives given that the
prevalence of PA is around 10%. The reversibility of aldosterone-mediated end-organ damage
by targeted therapy in the form of surgery or MR antagonists provides a compelling reason
for the prompt diagnosis of this condition.
Developments in the use of LC/MS for aldosterone and renin assays will improve the
reliability and reproducibility of screening and confirmatory tests, while protocols that
promote a focussed expertise in adrenal vein sampling will improve the diagnostic accuracy
of subtyping. With advances in the understanding of its genetic basis, mutation testing may
become clinically relevant in the future if genetic mutations can be correlated with responses
to specific treatments. The identification of genetic abnormalities may also provide new
targets for novel therapeutic approaches.
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Chapter 7
CUSHING’S SYNDROME
Georgiana Alina Dobri, MD,

Divya Yogi-Morren, MD, and Betul A. Hatipoglu
Endocrinology and Metabolism Institute,
Cleveland Clinic, Cleveland, OH, US
ABSTRACT
Cushing's syndrome (CS) results from prolonged exposure to elevated endogenous
cortisol. The increased cortisol production is due to adrenal lesions (ACTH independent
CS) or driven by ACTH excess from pituitary or ectopic sources (ACTH dependent CS).
The clinical presentation has some variations depending on the etiology. The most
common findings are central obesity, hypertension, irregular periods in women and
decreased libido in men, hyperlipidemia, impaired glucose tolerance and diabetes,
hypercoagulability, muscle weakness, depression, thin skin and violaceous striae, poor
wound healing and predisposition to infections. ACTH independent CS, in the vast
majority is due to a unilateral tumor, adenoma or carcinoma (ACC). ACC may have a
mixed androgen-cortisol secretion and women may present with virilization. The bilateral
disease consists of primary pigmented nodular adrenocortical disease (PPNAD), primary
nonpigmented micronodular hyperplasia and ACTH independent macronodular
hyperplasia (AIMAH). When the source of excess ACTH is the pituitary, the disorder is
called Cushing disease (CD) and it accounts for about 70% of cases of CS. The cause is
usually a benign corticotroph microadenoma and rarely a pituitary carcinoma or
corticotroph hyperplasia due to excess CRH stimulation. Cushing disease is more
common in the young adults, especially women. The clinical signs appear insidiously
over months to years. Central obesity, glucose intolerance, menstrual irregularities and
depression are the most common features. Ectopic ACTH secretion accounts for about 8-
18% of all cases of CS. It is usually caused by neuroendocrine tumors with small cell
lung cancer being the most common. The clinical features are similar to that of CD.
However the onset and progression of clinical features are usually overt and rapid with
weight loss, hyperkalemia and hyperpigmentation as main features. The prevalence of
ectopic ACTH appears to be equal in both genders. CS due to isolated ectopic CRH
secretion is extremely rare. Commonly recommended initial testing is urinary free
cortisol, late-night salivary cortisol or 1 mg overnight dexamethasone suppression test
(DST), followed by an ACTH level.
144 Georgiana Alina Dobri, Divya Yogi-Morren and Betul A. Hatipoglu
Imaging is key to delineate etiology, complemented by Dexamethasone-CRH test

and IPSS (inferior petrosal sinus sampling) if the ACTH source is in doubt or pseudo
Cushing is suspected. When amenable, the first line in treatment is surgical removal of
the causative lesion. The pharmacological treatment is aimed at decreasing the adrenal
steroid secretion or blocking its action at the receptor level. Radiotherapy and
chemotherapy are adjunctive therapies depending on the etiology. CS has always been a
diagnostic and therapeutic challenge. Persistence and recurrence are real concerns and
lifelong follow-up is warranted.
Keywords: cushing, hypercortisolism, ACTH-dependent, ACTH-independent
INTRODUCTION
Cushing‘s syndrome (CS) is a condition resulting from prolonged exposure to
inappropriately elevated cortisol level. Exogenous Cushing's syndrome, the commonest cause
of Cushing's syndrome resulting from usage of exogenous glucocorticoids, and
hypercortisolemia without Cushing's syndrome such as seen in severe chronic illness, during
acute illness, surgery, malnutrition, anorexia, pregnancy, excess cortisol-binding globulin
states or glucocorticoid resistance syndromes are beyond the scope of this chapter. In this
section, endogenous excess cortisol production caused either by excess ACTH secretion or
adrenal overproduction is reviewed [1].
HISTORICAL OVERVIEW
The adrenal glands first have been described by Bartolomeo Eustachius in 1563 in the
Opuscula Anatomica [2, 3]. Until Thomas Addison‗s work in 1855, on patients who had
various adrenal lesions and adrenal insufficiency, not much was known about their function.
Harvey Cushing, a neurosurgeon and a researcher, born in 1869 in Cleveland, OH [4] met
Minnie G detail around 1912, who became one of his first patients with hypercortisolism
described. Cushing then accumulated twelve more cases that formed his classic 1932 paper
[5] and gave the syndrome his name as Cushing‘s syndrome [6]. Although, escaped the notice
of writers on medical history and also of Cushing himself, a paper published in Norwegian
language, around 1914 and presented at the Norwegian Medical Society in Kristiania (Oslo)
by Dr. L. Dedichen, described a case which possessed all the characteristic features of CS and
linked as being due to hyperfunction of an adrenal tumor found on autopsy [7].
EPIDEMIOLOGY
Overt CS is a rare disorder with a reported annual incidence of 2-3 per million. The true
incidence is most likely 5 to 25 per million per year [8] since approximately 10% of the
incidental adrenal adenomas are found to secrete cortisol [9].
Cushing‘s Syndrome 145
In majority of cases, CS is caused by excess ACTH production, 85-90% of the cases by

pituitary corticotroph adenoma (Cushing‘s disease CD), followed by ectopic ACTH
syndrome or very rarely ectopic CRH syndrome [10].
CLINICAL PRESENTATION
The initial clinical presentation of CS varies and can be subtle in mild hypercortisolism
(Table 1). Central obesity was and continues to be the most common finding in CS [11], seen
in approximately 95% of the adult patients and 100% of the pediatric population, with unique
fat accumulations seen over the face, neck and trunk, giving the classical ―moon‖ facies,
―buffalo hump‖ and ―truncal‖ obesity and sparing the extremities (spider appearance) [12].
Hypertension occurs in at least 80% of patients who have Cushing‘s syndrome and is a
major contributing factor to cardiovascular morbidity [13, 14]. The majority of patients with
CS present with mild-to-moderate hypertension, whereas 17% could present with a severe
form. The pathophysiology of hypertension is the glucocorticoid (GC) effect on plasma
volume, peripheral vascular resistance and cardiac output.
Hypertension usually improves with therapy directed to the cause of the disease such as
surgical removal of the tumor, but patients might require pharmacological antihypertensive
treatment, both pre- and postoperatively. In general, thiazides are common first treatment
choice for hypertension and furosemide can worsen the hypokalemia caused by excess GC
effect and therefore should be avoided. Because of the augmented renin-angiotensin system,
ARB‘s (Angiotensin II receptor blockers) and ACE (angiotensin-converting-enzyme)
inhibitors are recommended [13].
Table 1. Frequently seen signs and symptoms in CS
Symptoms Signs Disease

Depression Easy bruising Hypertension
Fatigue Facial plethora Adrenal mass
Weight gain Proximal myopathy Vertebral osteoporosis
Polycystic ovarian
Back pain Striae
syndrome
Appetite change Dorsocervical fat pad Type 2 diabetes
Decreased concentration Facial fullness Hypokalemia
Decreased libido Obesity Kidney stone
Impaired memory Supraclavicular fullness Unusual infections
Insomnia Thin skin
Irritability Peripheral edema
Menstrual abnormality Acne
Slow growth in pediatric Hirsutism/baldness
Poor healing
Short stature
Pubertal abnormalities
delayed/pseudoprecocious
exophthalmos
Virilization caused by excess cortisol and adrenal androgens [15], can be observed in
women presenting with amenorrhea or menometrorrhagia combined with hirsutism, acne and
signs of virilization (clitoral enlargement, deepening of the voice, male pattern baldness) [16].
Male patients frequently present with diminished libido or impotence associated with
subnormal testosterone [17].
Elevation of triglycerides and total cholesterol level can be seen with an increase in
circulating very-low density lipoprotein (VLDL) and LDL, but not high-density lipoprotein
(HDL) [10]. Depending on the diagnostic criteria used, total cholesterol is reported to be
elevated in 25–52% of patients, whereas high serum triglycerides were found in 7–35% of
patients and, reduced HDL-cholesterol levels in about 14.2% to 36% of patients [18]. In most
reported series these changes improve with correction of hypercortisolemia although a
complete normalization of lipid parameters is usually not achieved [19]. There are no studies
or guidelines as to how to treat hyperlipidemia in Cushing‘s syndrome. As hypercortisolism is
often associated with several other cardiovascular risk factors an aggressive management of
any cardiovascular risk factors in these patients is warranted [19].
Impaired glucose tolerance may occur in 30% to 60% of patients, and frank diabetes in
25% to 50% of patients [17]. This is not a surprise as excess GC has effect on glucose
homeostasis in many different ways: they increase glucose production by liver via genes
activation of the enzymes for gluconeogenesis, stimulation of lipolysis and proteolysis, and
potentiation of glucagon effect [20]. They reduce insulin sensitivity by inducing an
impairment of the insulin receptor signaling pathway [21], and they change insulin secretion
from pancreatic  cells [22], by affecting glucose uptake and intracellular calcium changes
[20]. Other findings are neurologic symptoms occurring around 20-39% of CS patients.
Muscular weakness and tremor are the most commonly noted abnormalities but symptoms
ranging from minor visual disturbances to hemiplegia also can be seen [11]. Changes in bone
and calcium homeostasis [23] could result in pathologic fractures, especially rib fractures
seem to be most common [17].
Less appreciated clinical problem is the hypercoagulability, especially important as it can
lead to an increased risk for thromboembolic events after surgery or even after minor
intervention [24]. In general anticoagulation should be considered in the postoperative period
in many of these patients [17]. Depression and emotional lability are estimated to reach 70%
in the literature [12]. Loss of brain volume and neurocognitive impairment have also been
reported [25]. The skin manifestations can range from the classic cutaneous features such as
facial plethora, acne, purpura, cutaneous atrophy, hirsutism, vellus hypertrichosis (lanugo
hair), to wide purplish striae over the abdomen, flanks, and upper arms [26].
Poor healing of wounds or unusual failure to localize minor infection can be seen in 40
per cent of the patients [11]. Patients with Cushing‘s syndrome are also at increased risk from
cutaneous infections due to the immunosuppressive effect of excess glucocorticoids.
Cutaneous staphylococcal, candidal, and superficial fungal infections are not uncommon.
Opportunistic infections, such as deep fungal infections with aspergillus, zygomycosis, or
phaeohyphomycosis may also occur [27].
Hyperpigmentation seen in ACTH-dependent Cushing‘s (pituitary or ectopic), is
mediated by the action of ACTH on melanocyte-stimulating receptors in areas exposed to
sunlight, friction, or trauma. Hyperpigmentation does not occur in patients with adrenal
tumors as high levels of cortisol suppress ACTH production [26]. Exophthalmus can be seen
in rare cases [28].
Cushing’s Syndrome 147
SCREENING AND ETIOLOGY DIFFERENTIATION

In suspected individuals the first step in the diagnosis of Cushing’s syndrome involves
the confirmation of hypercortisolism.
Commonly recommended and used initial tests for Cushing’s syndrome are: urinary free
cortisol (UFC) at least two occasions, late-night salivary cortisol at least two measurements, 1
mg overnight dexamethasone suppression test (DST), or 2 days low-dose DST (2 mg/d for 48
h) [29]. It is often recommended to confirm hypercortisolism with a second preferably
different method from the above choices (Figure 1).
In individuals with normal test results but with clinical features suggestive of CS
repeating the tests periodically should be considered. Each one of the recommended screening
method has its pros and cons.
UFC provides an assessment of cortisol secretion over a 24-h period. This is usually
preferred in pregnant women, in patients taking antiepileptics and cyclic Cushing’s. It is
recommended to use the upper limit of normal for the particular assay as the criterion for a
positive test, provided the creatinine shows that the collection is complete.
Figure 1. Initial screening for Cushing’s syndrome (CS).

But because UFC reflects renal filtration, values are significantly lower in patients with
moderate to severe renal impairment making this test unreliable in renal impairment [30].
One of the first recognized abnormalities in CS is loss of circadian rhythm of cortisol
secretion [31]. The measurement of salivary cortisol levels between 2300 and 0000 h has
been a popular screening test.
A value of more than 145 ng/dl is considered abnormal. The accuracy of this test is
similar to that of UFC [32]. This easily performed test has been successfully used in children
with high sensitivity (100%) and specificity (95.2%) [33]. It is not a good test in patients with
depressive illness and in night shift workers [34] and the critically ill [35, 36].
One of the gold standard screening tests is failure to suppress, ACTH and cortisol after a
dose of dexamethasone (DEX). The 1 mg DEX is usually given between 2300 and 0000 h,
and cortisol is measured between 0800 and 0900 h the following morning.
Experts have advocated a cutoff cortisol value for suppression of less than 1.8 μg/dl to
improve sensitivity (to 95%) and specificity (to 95%). Preferred cutoff for adrenal CS is
accepted to be 5 ug/dl to improve diagnostic threshold [37].
Use of 2 days, 2 mg/d DST(LDDST) as an initial screening test recommended for special
population of patients with psychiatric disorders (depression, anxiety, obsessive-compulsive
disorder), morbid obesity and alcoholism.
As estrogens increase the cortisol-binding globulin (CBG), they can cause false-positive
results with DST. Estrogen-containing drugs (like oral contraceptives) should be stopped for
6 weeks if this test will be used as screening. The enzymatic clearance of DEX is dependent
on hepatic enzyme activity that can be inhibited or induced by drug interactions. For example
some antiepileptics such as phenytoin, carbamazepine, and alcohol are known inducers of
hepatic enzymatic clearance of dexamethasone, to avoid possibility of inadequate DEX
exposure and improve reliability of the test, dexamethasone level should be also measured
[29, 38]. Once hypercortisolism is confirmed then its etiology needs to be identified. An
ACTH level is the next step to further investigate the source of CS (Figure 2).
ACTH level below 5 pg/ml at two separate occasions support the diagnosis of ACTH
independent Cushing‘s syndrome. An adrenal imaging is then recommended. If ACTH is
more than 15 pg/ml, this is most likely ACTH dependent Cushing‘s (pituitary or ectopic
source) and MRI of pituitary gland is the first step. In cases where the pituitary imaging
shows an adenoma of 6 mm or larger, further testing to confirm pituitary source is warranted.
IPSS (Bilateral Inferior Petrosal Sinus Sampling) is the preferred method if available, to
confirm the source of the ACTH and is discussed later in this chapter. If not available a CRH
(corticotrophin releasing hormone) stimulation test combined with DEX has been used to help
in confirming pituitary source. Ectopic ACTH dependent CS will not respond to CRH
stimulation. These patients will fail to suppress cortisol to less than 50% of baseline with high
dose dexamethasone suppression test as well, although this test alone is poor diagnostic tool,
it can be used if IPSS is not available. In patients with ACTH values between 5-15 pg/ml,
CRH stimulation test is indicated to identify the source, primary adrenal disease will not
response to CRH stimulation and ACTH will remain below 20 pg/ml despite CRH
stimulation. Involvement of an experienced endocrinologist is crucial during this phase of
disease evaluation.
ACTH DEPENDENT CUSHING SYNDROME OF PITUITARY ORIGIN

Although a rare disorder, excess ACTH production of pituitary origin (Cushing‘s disease
(CD)), is the most common cause for endogenous Cushing syndrome, accounting for about
70% of the cases [8].
Cushing disease is more common in the young adults, especially women. The cause is
usually a benign corticotroph adenoma (in 90% of cases). Around 80-90% of these present as
a microadenoma (defined as less than 10 mm in size). The remainder 15% can show some
aggressive behavior having a larger size, or showing extrasellar invasion and 7% of them can
even have local aggressive behavior. In most, histology does not suggest malignancy [39].
Some of the pituitary tumors initially classified as non-secreting or silent corticotrophs can
result in clinically evident hypercortisolism with time and cause CD. This transition from
silent to functioning adenoma is reported to occur from few months up to 16 years from the
diagnosis in about 9-26% of the cases [40, 41]. These tumors are usually larger and more
invasive in nature. Though in few cases radiation therapy preceded this transformation, in
larger case series this was not confirmed. Other rare reported causes for CD are pituitary
gangliocytoma [42], carcinoma [43-46] and primary multinodular corticotroph hyperplasia
that has been described on pathology specimens after total hypophisectomy. Primary
multinodular corticotroph hyperplasia is primarily a pituitary corticotroph disorder as CRH
level is reported to be low [47, 48].
Clinical Features
The clinical features of Cushing syndrome are discussed earlier in this chapter in more
detail (see section on clinical presentation and Table 1).
Clinical signs of ACTH dependent Cushing syndrome usually appear insidiously over
months to years. Central obesity, glucose intolerance, menstrual irregularities and depression
are the most common features observed. Signs of hyperandrogenism (acne and hirsutism) are
mild as ACTH excess gives rise to only minimal DHEAs elevation. Hypokalemia and
alkalosis can be present if the hypercortisolism is severe. In addition, patients with large
pituitary tumors may have evidence of mass effect, including headache, visual field deficits or
ophthalmoplegia caused by optic chiasm or cavernous sinus invasion [49, 50].
Diagnosis
After the diagnosis of ACTH dependent hypercortisolism is made, the pituitary source is
further investigated and confirmed by combination of tests and imaging such as MRI of
pituitary, IPSS or if latter is not available, a CRH stimulation with high dose Dexamethasone
test (Figure 2). As seen in about 15% the cases, hormonal secretion of either ACTH or
cortisol can be cyclical rather than constant, making the diagnosis more challenging [51].
Cyclical Cushing‘s is not limited to pituitary cases, although up to 54% of these cases are due
to a pituitary corticotroph adenoma, about 26% have an ectopic source for ACTH, about 11%
an adrenal tumor as a source and few other remains unclassified [52]. As the cycle length
varies from 12 hours to 85 days, the clinical findings follow the same rhythm of cortisol and
rarely can be constant [53]. The diagnosis is made by showing presence of three peaks and
two troughs of cortisol production [54]. Pseudo Cushing syndrome should be kept in
differential for CD. Pseudo Cushing shares common clinical findings as CS (central weight
gain, elevated blood pressure and elevated blood glucose – metabolic syndrome) along with
positive DST or elevated urinary cortisol levels.
Figure 2. Differential diagnosis.

Hypercortisolemia without Cushing's syndrome such as seen in severe chronic illness,

chronic alcoholism, psychiatric disorders, severe obesity, poorly controlled diabetes is due to
the over activity of the hypothalamic-pituitary-adrenal axis [55, 56]. The circadian rhythm in
these patients is still intact, making diagnosis possible with a normal midnight salivary
cortisol or midnight/morning ratio usually less then < 0.67 [55]. Also response to DEX-CRH
test can help distinguishing the two entities. The test is performed after eight doses of 0.5 mg
Dexamethasone are given every 6 hours. Two hours after the last dose of DEX, 100 μg CRH
are administered intravenously and cortisol level at 15 minutes after the injection determines
the interpretation value for the test. The basal cortisol level after Dexamethasone should
suppress below 1.4 - 1.8 μg/ml in pseudo Cushing.
The diagnostic cut off at 15 minutes post CRH cortisol level varies between centers from
1.4 to 3 μg/dl and continues to be debated [55, 57].
Management/Pituitary Surgery/Evaluation for Remission
The first line therapy for the majority of cases is surgical resection of the pituitary tumor.
When possible transnasal transsphenoidal approach is preferred and rarely in large and
aggressive tumors transcranial route is needed [58]. The goal for surgery is total resection of
the adenoma with preservation of the pituitary gland function, in aggressive tumors, tumor
debulking with sparing of the cavernous sinus structures could be the only possible treatment
approach. On the other hand in cases when the tumor is not visible on current imaging
techniques, careful exploration of the gland by an experienced neurosurgeon is crucial, and if
adenoma could still not be located, hemihypophysectomy is often performed by removing
usually the part which lateralized during IPSS, and rarely total hypophysectomy is needed.
Depending on the center, the cure rate after resection differs; for a microadenoma visible
on imaging it is usually between 65-90%, but rather lower for those not located on imaging
and unfortunately even lower for macroadenomas or tumors with dural or cavernous sinus
invasion [59]. Good prognostic factors for post-surgical remission are detection of the
microadenoma by an imaging study, a well-defined tumor within the sella, histological
confirmation of an ACTH-secreting tumor after surgery, low postoperative serum cortisol
levels and long-term adrenal insufficiency [59].
Post-surgical remission is not uniformly defined between centers, different cut-off values
and different biochemical tools have been used [60]. Most widely accepted and used value for
postsurgical remission is a cortisol level of less than < 5 μg/dl within 72h after surgery, but
even for patients with level between 2-5 μg/dl there is a 2.5 times more risk for recurrence
than those with a level less than 2 μg/dl [59, 60]. The suppressed HPA axis may take up to
180 days to recover after surgery in 5% of the patients and it is important to reevaluate
patients for disease persistence at different time intervals to assess need for further treatment
[61]. In patients with cortisol level between 2-5 μg/dl, a 24h urine free cortisol value above
100 μg/24h suggests persistent disease, whereas less than 20 μg/24h almost always suggests
remission, values in between are equivocal [59].
DST with a cutoff cortisol suppression level of 5 μg/dl or less does not predict initial
remission well, as recurrence rate in these patients is shown to be around 12.7% [39].
As circadian rhythm might restore quickly after a successful surgery, many expert
advocate using midnight salivary free cortisol level of less than 2 ng/ml, that seems to have
100% sensitivity and 98% specificity in defining initial remission and performs better in
picking up persistent disease than postoperative cortisol levels, 24 h urine cortisol or 1 mg
Dexamethasone suppression test [62, 63].
Radiation Therapy
Radiation therapy is used after incomplete resection of hormonally active adenomas or

for recurrent tumors and rarely as a first line therapy for patients who are poor surgical
candidates or refuse surgery.
The most frequently used techniques are conventional radiotherapy and gamma knife
stereotactic surgery. Gamma knife therapy is performed in selected cases with adenomas less
than 3cm and located at least 2-3 mm away from the optic chiasm. Both treatments offer good
local tumor control and biochemical remission. Within 2 years about 60-83% of the cases are
expected to achieve remission though it can take up to 9-12 years.
Most common side effect is hypopituitarism, observed in up to 56% of cases who receive
conventional radiation and 66% with stereotactic surgery. Other reported complications are de
novo meningiomas, optic neuropathy and brain necrosis in less than 1% of the cases [59, 64,
65].
Medical Therapy
For persistent disease after neurosurgery and/or radiotherapy medical therapy remains a
good option. It can also be used as initial treatment if other options are not possible or as a
bridge-therapy while awaiting the effects of radiation therapy.
The medical treatment used for Cushing disease can be placed in three categories
depending on their mechanism of action: ACTH inhibitors, adrenal steroidogenesis inhibitors
and cortisol receptor blockers [66, 67]. ACTH inhibitors agents targeted against the
corticotroph adenomas are emerging as promising therapies for Cushing disease.
Pasireotide is a somatostatin receptor analog which binds to subtypes 1, 2, 3 and 5, 5
being the most abundant subtype present in the corticotroph cells. The recommended dose is
600 or 900 mg twice daily. The effect of the medication on remission rate and tumor
reduction is dose dependent with reported remission rates of 15% and 26%, and observed
tumor reduction rates of 9% and 44% respectively. The response to the drug is usually
observed in the first 2 months of therapy. Better results are seen in patients with mild
hypercortisolism (baseline 24h urine cortisol of less than 5 times upper limit of normal). The
most frequent side effects are diarrhea, nausea and hyperglycemia (73%). Almost half of the
patients developed diabetes mellitus in preliminary trials [68, 69].
Pasireotide LAR is a long active form of pasireotide approved and used for treatment of
acromegaly, which has been used by some in the treatment of Nelson syndrome. More studies
are needed to see if this could be a therapeutic option for Cushing disease [70].
Cabergoline is a dopamine receptor agonist studied in couple of small studies for the
treatment of Cushing disease, as almost 80% of the corticotroph adenomas have D2 receptors.
The recommended dose is between 2-3.5 mg/week for an expected remission in about 25-
50% of the cases and a tumor reduction in 25%. Unfortunately in about a quarter of the
patients, the therapeutic effect diminishes after 1-5 years [71, 72].
The most frequent side effects are nausea, vomiting and impulsive behavior;
valvulopathy has been a concern although this is quite rare at the doses used for Cushing
disease. It is potentially a better choice for pregnant patients [73].
Retinoic acid acts on a type 2 nuclear receptor and decreases the secretion of ACTH,
possibly also causes corticotroph cell death, and decreased production of corticosterone by
adrenal cell inhibition [74]. A small study showed 24 h urine cortisol normalization in 3 out
of 7 patients with Cushing disease treated with 80 mg retinoic acid daily. ACTH initially
decreased but this was a short-lived effect of 1 month. Side effects are arthralgia and dry
mouth [75].
Temozolamide is an alkylating agent used to treat astrocytoma and melanoma. It has been
used in about 10 cases of Cushing disease and could be considered as an option in aggressive
tumor as it provides both biochemical and local tumor control [66, 76]. Octreotide,
rosiglitazone, pioglitazone, cyproheptadine and valproic acid have been reported in small
case reports and series with minimal effect and their use is not routinely recommended [67,
77]. Gefitinib (epidermal growth factor receptor inhibitor) and doxazosin (alpha 1 adrenergic
receptor blocker) have shown promising results in animal and tissue studies but their efficacy
and safety need to be proven in clinical studies [66]. Adrenal steroidogenesis inhibitors are
not FDA approved for the treatment of Cushing disease. They have no effect on the tumor
size and at times the disease escapes the initial control by increase in the ACTH level via
diminished cortisol negative feedback. They can still be used to control symptoms in patients
who are resistant to any other treatment modalities or need bridging for adrenalectomy. More
information about these agents is given in the adrenal Cushing section later in the chapter.
Bilateral Adrenalectomy
Bilateral adrenalectomy is a treatment option offered for persistent hypercortisolism after

pituitary surgery, and or radiotherapy and failed medical therapy.
The procedure has been used as last therapeutic option due to concerns about high
mortality and morbidity. Mortality rates up to 9% at 4 years have been reported, half of these
occurring within first 12 months. Patients require lifelong steroid and fludrocortisones
replacement. Recurrence of hypercortisolemia is very rare but has been reported in cases of
adrenal remnants or ectopic tissue. Reported rate of adrenal crisis is around 30% and Nelson
syndrome 20% at 5 years of follow up [78]. Nelson syndrome is a triad of hyperpigmentation,
elevated ACTH levels and aggressive corticotroph adenoma growth. It appears as a
consequence of missing glucocorticoid feedback to control adenoma cells and reported after
bilateral adrenalectomy. If not done already, radiation of the pituitary gland prior to the
adrenalectomy can be useful to prevent this complication. Treatment for Nelson syndrome is
neurosurgery combined with radiation therapy, some of the medical therapy options such as
temozolomide, octreotide, and pasireotide can be also used as adjuvants [79].
Follow-up of the patients with Cushing disease is lifelong. Recurrence of Cushing disease
has been reported after 9 years of remission [39]. During 18 years follow up of these patients
with normalization of cortisol for more than 5 years it was observed that 50% continue to
have HTN, diabetes, dyslipidemia, nephrolithiasis, and up to 30% remained with persistent
depression, cognitive deficits and osteoporosis with vertebral fractures [80]. Even after
reversal of cortisol excess, the ongoing evaluation and management of its comorbidities is
lifelong in Cushing disease.
ACTH DEPENDENT CUSHING

SYNDROME: ECTOPIC CUSHING’S SYNDROME
Ectopic Cushing‘s syndrome (CS) occurs when there is excess production of ACTH from
a non-pituitary source. It accounts for about 10% of all cases of Cushing‘s syndrome [81-85].
Cases of ectopic ACTH secretion were reported as early as 1928 before Dr. Harvey
Cushing described Cushing‘s disease caused by excess ACTH production from pituitary
tumors in 1932. However it was not until 1962 that the relationship between CS and ectopic
ACTH secretion was described by Meador et al., [86]. When the syndrome was first
described, the most common etiology was small cell lung cancer [87]. Since then many other
ACTH producing tumors have been identified, but pulmonary tumors, particularly bronchial
carcinoid and small cell lung cancer remain the most common source of ectopic ACTH
secretion. Most tumors that produce ACTH are of neuroendocrine origin and ACTH
production from thymic tumors, medullary thyroid carcinoma, pancreatic carcinoid,
pheochromocytoma and genitourinary neuroendocrine tumors have been described [85,
88, 89].
Clinical Features
The clinical features of Ectopic ACTH secretion overlap with the clinical features of
Cushing‘s disease and adrenal Cushing‘s syndrome which are covered earlier in this chapter
(see section on clinical presentation and Table 1). Among the patients with ectopic ACTH
production, those patients with small cell lung carcinomas have a more rapid onset and
progression of their symptoms compared to patients with other sources of ectopic ACTH
production [88].
Diagnostic Tests
The diagnostic testing to establish hypercortisolemia is the same as for Cushing‘s disease
and adrenal Cushing‘s syndrome as described earlier, in the section on Screening and
differentiating the etiology (Figures 1 and 2). However, there are particular features on the
biochemical evaluation that may suggest that the patient‘s source of ACTH secretion is
ectopic in nature. Patients with ectopic ACTH secretion tend to have higher ACTH levels
than those found in Cushing‘s disease and it has been suggested that an ACTH level > 200
pg/mL favors EAS over CD. Hypokalemia tends to be a salient feature in Ectopic ACTH
secretion due to the higher levels of ACTH and hence cortisol level, which has significant
mineralocorticoid activity [90].
Establishing the etiology of ACTH-dependent CS may be challenging because of the

overlap in the clinical, biochemical, and radiological features between Cushing‘s disease and
ectopic ACTH secretion. This challenge is further complicated by the fact that 5-10% of the
population harbor incidental pituitary lesions.
Magnetic resonance imaging in the preferred method of imaging to visualize the pituitary
gland if the biochemical evaluation suggests that the patient has Cushing‘s disease. It is
usually done with gandolinium contrast enhancement. On pre contrast images, the normal
pituitary gland is isointense with the brain on T1 weighted images. A focal area of
hypointensity is suggestive of an adenoma. A study by Chong et al., evaluated healthy
volunteers and patients with galactorrhea and amenorrhea and found that found that the size
and degree of hypointensity did allow some discrimination between volunteers and patients.
There was some overlap in size and degree of hypointensity between the 2 groups;
however in no case did a volunteer have a hypointense area larger than 5 mm [91, 92].
A consensus statement on CS recommends that, in a patient with a classic clinical
presentation and dynamic biochemical studies compatible with a pituitary etiology, a pituitary
tumor size of 6 mm is highly suggestive of CD and obviates the need for more invasive
testing [93]. For tumors that are < 6 mm in size, inferior petrosal sinus sampling (IPSS) is
used to differentiate between CD and ectopic ACTH secretion.
During IPSS, ACTH levels are drawn from the inferior petrosal sinuses that drain the
pituitary gland as well as the peripheral blood. These levels of ACTH in the inferior petrosal
sinuses are compared to that of peripheral blood to determine whether the source of ACTH is
pituitary or ectopic. In 1977, Corrigan et al., first described the unilateral selective
catheterization and venous sampling to localize ACTH secretion in a patient who had a
clinical and laboratory presentation that was consistent with either an ectopic ACTH secreting
tumor or pituitary-dependent Cushing syndrome [94].
Later on in 1984, Doppman et al., described simultaneous sampling from both inferior
petrosal sinuses to avoid false-negative results in the presence of a pituitary corticotroph
adenoma. This procedure was termed bilateral inferior petrosal sinus sampling [95].
Simultaneous sampling of bilateral inferior petrosal sinuses and a peripheral vein are
performed at 5 and 10 minutes before and then 2, 5, 10, and 15 minutes (25 minutes in some
cases) after CRH administration (1 μg/kg, maximum 100 μg). Placement of the catheters
during IPSS are usually confirmed by venous angiography before sampling, and intermittent
fluoroscopy is used to ensure correct localization. Pituitary drainage is usually ipsilateral,
therefore, the venous outflow on the contralateral side that does not have the pituitary
adenoma, will not have a high concentration of ACTH.
An Inferior petrosal sinus: Peripheral ACTH ratio greater than 2 before or greater than 3
after CRH administration is consistent with a pituitary origin of Cushing syndrome [94-98].
This procedure should be performed at a center with interventional radiologists who have
a high volume of patients and are experienced in performing IPSS. IPSS has a sensitivity of
96% and specificity of 100% in differentiating Cushing disease from ectopic ACTH secretion
[81, 99]. False-negative results occur and may be due to technical difficulty, abnormalities in
petrosal sinus anatomy, or properties of the tumor and it‘s ACTH secretion [100]. Several
studies have reported that prolactin measurement during IPSS can improve diagnostic
accuracy and decrease false-negative results [101-103].
Mulligan et al., found that an IPS:P prolactin ratio less than 1.8 may indicate a false-
negative result when the corresponding IPS:P ACTH ratio is less than 2 or less than 3 in pre-
or post-CRH samples, respectively. In this series, all false-negative IPS:P ACTH ratios had a
corresponding IPS:P prolactin ratio less than 1.3 [102].
Complications may occur during IPSS and the most common complication is a groin
hematoma that occurs in about 3-4% of patients undergoing the procedure [104]. Other
complications such as pulmonary embolism, obstructive hydrocephalus, subarachnoid
hemorrhage, brainstem infarction, transient sixth cranial nerve palsy and pontomedullary
junction stroke have been reported [103-107].
The risk of acute kidney injury from the iodinated contrast dye exists especially in
patients with preexisting kidney disease or hypovolemia [108].
Source Localization and Management
Following the diagnosis of Ectopic Cushing‘s syndrome, the source of ACTH must be
identified. The definitive management of Ectopic ACTH secretion is the localization and
treatment of the primary tumor. Most of the large case series of patients with ectopic
Cushing‘s syndrome describe the use of computed tomography (CT), T-1 and T2 weighted
magnetic resonance imaging (MRI) scans of the neck, chest and abdomen and pelvis,
scintigraphy with either octreotide or [131I]-metaiodobenzylguanidine, [18F] fluoro-2-deoxy-
D-glucose–positron emission tomography (FDG-PET) scans and WBC scanning to identify
the source of ectopic ACTH secretion [85, 88, 109].
Despite extensive investigation, the source of ACTH secretion remains occult in some
patients. A large NIH series of 90 patients with ectopic Cushing‘s syndrome found that CT
and MRI localized the ACTH secreting tumor in 67 of 73 patients but was negative in 9 out of
17 patients. Twenty-one of 43 octreotide scans correctly identified a source of ACTH.
Among patients with an occult tumor, three had positive octreotide scintigraphy and the
source of ectopic ACTH remained occult in 17 patients [109]. Following localization of the
tumor, definitive treatment of the tumor is essential. Curative surgery or tumor debulking
should be performed if possible. While awaiting treatment, patients can be medically treated
for rapid resolution of hypercortisolemia. Medical management is described later under
adrenal therapy section in this chapter. If the treatment of the primary tumor is unsuccessful
or if the source of ACTH secretion remains occult, bilateral adrenalectomy can be performed
to control hypercortisolism or medical treatment as aforementioned can be used.
Follow Up and Prognosis
The prognosis of ectopic Cushing‘s syndrome is poor and the published case series have
reported varying incidence of mortality. In the NIH series, 19 out of 90 patients were
deceased. Their median duration of follow up was 26 months (range 0-226 months). Patients
with an unknown or occult source of ACTH survived longer compared with those with an
identified tumor [109]. In a series of 40 patients from the UK, only 15 out of 40 patients
survived. The median duration of follow up was 60 months. Patients died of complications of
the tumor themselves or as a consequence of excessive cortisol secretion such as pancreatitis,
opportunistic infection or cardiac failure [88]. In the case series from MD Anderson, Death
occurred in 27 out of 43 patients, and the median overall survival duration was 32.2 months in
all patients. Progression of primary malignancies and systemic infections at the time of death
were the leading causes of mortality, and 2 patients died from pulmonary embolism [85].
ACTH INDEPENDENT CUSHING’S SYNDROME

Around 15-20% of CS is caused by ACTH-independent source; this includes unilateral
benign and malignant adrenal tumors, bilateral adrenal hyperplasia or dysplasia. Benign
adrenal adenomas account for 0.6 per million of cases where as the incidence for adrenal
carcinoma (ACC) is estimated to be 0.2 to 2 per million per year [8].
CS is more common in women [110]. In childhood cases, pre pubertal boys seems to be
affected more commonly then girls [111]. The most common syndrome associated with
adrenocortical tumors in adults is reported to be Cushing‘s syndrome (20%) [15].
Etiology
Around 90% of ACTH independent CS are caused by unilateral tumors [112]. Among the
unilateral tumors, adenomas are found in 60% of cases and carcinomas around 40%. ACC
present 60% of the time with a secretory syndrome, and among those almost 65% will be
mixed or pure cortisol-secreting tumors [1]. The other adrenal causes of CS include ACTH-
independent bilateral hyperplasias: primary pigmented nodular adrenocortical disease
(PPNAD), and a subtype primary non-pigmented micronodular hyperplasia and ACTH-
independent macronodular adrenal hyperplasia (AIMAH) [113]. Adrenocortical carcinoma
(ACC) is a rare disease, and is covered extensively in another chapter.
PPNAD is seen mainly in young adults and is suspected in patients with ACTH
independent hypercortisolism confirmed biochemically and with normal-sized adrenal glands
in imaging. In histopathology the gland contains several small cortical pigmented nodules.
PPNAD also can be seen as a part of multiple neoplasia syndrome, called Carney complex
(spotty skin pigmentation, heart and skin myxomas and other endocrine tumors). Majority of
cases, will show 50% paradoxical increase of UFC with dexamethasone administration
(instead of suppression of cortisol levels), this is not seen with other adrenal CS such as
AIMAH or adrenal adenoma. Germline mutations of the PRKAR1A gene are found in 50%
of cases of Carney complex as well as isolated PPNAD [114]. Somatic PRKACA
heterozygous mutations were also observed in subjects with adrenocortical adenomas causing
Cushing's syndrome [115, 116]. Another rare phenomenon is AIMAH, and can be seen with
associated with McCune–Albright syndrome. Usually, these patients have bilaterally enlarged
adrenal glands and they present during 5th or 6th decade of life. Histologically there are
nonpigmented nodules made by lipid-rich cells forming nest-like structures, and lipid-poor
cells forming small island-like structures. The role of aberrant hormone receptors has been an
exciting topic.
The production of cortisol by adrenal adenomas in the absence of ACTH was believed to
be autonomous; there is now increasing evidence, some of non-ACTH circulating hormones
via aberrant expression of receptors in the adrenocortical tissues could be responsible for the
excess hormone production.
To date few hormones have been implicated on this process such as gastric inhibitory
polypeptide, b-adrenergic agonists, vasopressin, serotonin angiotensin-II (AT-1), vasopressin
(V1-vasopressin) [117, 118], luteinizing hormone/human chorionic gonadotropin, glucagon
[119] and possibly leptin. When suspected, work up should include cortisol response to
posture change, GnRH (gonadotropin-releasing hormone agonist) and mixed meal. Surgery
can be avoided if an antagonist can be used for the responsible hormone. Successful treatment
with lupron has been described for LH mediated hypercortisolism for example, or octreotide
could be tried for GIP induced hypercortisolism [120, 121].
Briefly it should be mentioned that long term exposure to ACTH could cause ACTH
dependent bilateral nodular hyperplasia mimicking above disorders so an ACTH level before
any intervention to the adrenal gland should be done to rule out this possibility.
Treatment
Surgical Intervention
Once confirmed, preferred first line treatment of choice for ACTH dependent or
independent CS remains surgical intervention. As reviewed earlier in patients with Cushing‘s
disease, the initial treatment is selective pituitary adenomectomy by a surgeon with
experience in pituitary surgery. Post operative hypocortisolism provides a marker of surgical
success and varies between 70-90% [122]. For persistent or recurrent disease in addition to
repeat transsphenoidal surgery and radiation therapy bilateral adrenalectomy has been used as
a definitive treatment option that provides immediate control of hypercortisolism. Especially
with current minimally invasive retroperitoneal endoscopic approach adrenalectomy with a
low morbidity and mortality; bilateral adrenalectomy can be offered to patients with persistent
hypercortisolism despite medical therapy or with intolerance to medical therapy or as an
alternative to medical treatment after pituitary radiotherapy [123, 124].
Loss of popularity to this approach throughout the years is mainly due to the need of
lifelong glucocorticoid and mineralocorticoid replacement therapy and risk for Nelson
syndrome. Nelson‘s syndrome is described earlier under CD. Briefly it is growth of a pituitary
corticotroph adenoma after bilateral adrenalectomy. It can cause neurologic symptoms due to
compression from the mass effect and increased ACTH secretion. Reported rates range from
8–29%. Pituitary MRI and ACTH plasma level measurements are advised every 3–6 months
after bilateral adrenalectomy first year and then at regular intervals thereafter. Routine
preventive radiotherapy after bilateral adrenalectomy is not generally recommended [125].
Bilateral adrenalectomy, should also be considered for patients with ectopic ACTH
syndrome as reviewed above who have failed primary surgical therapy directed to specific
tumor type. It is also indicated in patients with occult ectopic ACTH syndrome or patients
with malignant disease with metastases or very severe symptoms of Cushing‘s syndrome.
Surgery remains first line treatment for all other primary adrenal tumors. In the case of
patients with unilateral benign adrenal lesions the adrenalectomy should be performed by
means of an endoscopic approach in a specialized center.
Due to their small size with a diameter of less than 5 cm, cortisol-secreting adrenal
adenomas are very suitable for endoscopic surgical techniques [123]. The most commonly
recorded complications were bleeding/hematoma and adrenal insufficiency.
Radiofrequency ablation (RFA) is a promising less-invasive intervention which has been

used for functioning adrenal adenomas measuring around 3 cm in size [126]. From the reports
it is relatively safe and effective as tumor enhancement disappeared, around 53-85% of
treated cases with improvement of hypersecretion biochemically [127]. It is also noted that
ablation is a repeatable procedure if not effective at first attempt in treating residual tumor.
Patients developed postoperative adrenal insufficiency hence in some cases transient until the
contralateral adrenal gland recovered. In terms of complications, a small retroperitoneal
hematoma, delayed abscess and pneumothorax were reported. One possible complication in
the RFA of adrenal gland tumors includes immediate hypertension.
Medical Approach
Pharmacological management of CS is usually directed at decreasing adrenal steroid
secretion.
Steroidogenesis inhibitors like metyrapone, ketoconazole aminoglutethimide, and
etomidate have been successfully used to lower cortisol level [128, 129].
Metyrapone is an old and effective drug that inhibits 11b-hydroxylase, and leads to
decreased cortisol, increased ACTH, and an accumulation of 11-deoxycortisol (cortisol
precursor). It is effective within 2 h of initiating therapy and given three times daily. Starting
dose is 250 to 500mg three times daily; the average daily dose is approximately 2 g to 4 g.
Cortisol normalization was reported in 26% to 75% of cases, depending on the study [130,
131]. Adrenal insufficiency is the major unwanted effect. The accumulation of 11
deoxycortisosterone induces hypertension and hypokalemia, and because of concomitant
elevation in DHEAs acne and hirsutism [132] which may worsen [133, 134, 135].
Ketaconazole is an oral antimycotic, in larger doses, inhibits cortisol synthesis by P450
enzymes involved with steroidogenesis, including 17,20-lysase, 11b-hydroxylase, and 17a-
hydroxylase andside-chain cleavage [136]. In contrast to metyrapone, adrenal androgen
concentrations fall, as well as cholesterol concentration. The normalization rate for the 24 h
urine cortisol is anywhere from 30 to 90% [67, 137, 135]. Ketaconazole is also given three
times daily at a dose between 200 and 1200 mg daily. Liver function must be monitored
closely. Other common adverse events associated are adrenal insufficiency, and
gastrointestinal distress. Synergistic cortisol lowering action is achieved with ketaconazole
and metyrapone given in combination.
Aminoglutethimide blocks adrenal synthesis of cortisol, aldosterone and androgens, as
well as the production of estrogens in extraglandular tissues. Recommended daily doses range
from 0.5 to 2 g in divided doses [138]. In a number of patients, cortisol deficiency may be
induced; hypoaldosteronism and hypothyroidism have also been reported [134].
Etomidate, is an imadazole used as an anesthetic, at lower doses it is a potent inhibitor of
cortisol secretion in the final pathway of cortisol synthesis and available for parenteral use
[128, 134]. It is used at 0.1-0.3 mg/kg/h, has a quick onset of action, making it particularly
useful in hospitalized patients when rapid normalization in the cortisol level is needed.
Careful monitoring is required to avoid excessive sedation [139].
Mitotane has been used widely as medical therapy after surgical treatment in adrenal
carcinoma and in inoperable adrenocortical carcinoma [134, 140]. It is an antineoplastic
medication causing destruction of adrenal tissue via apoptosis. And also alters steroid
metabolism and directly suppresses the adrenal cortex [135].
It is started at 500 mg three times daily and titration is done on clinical grounds based on
the side effects and cortisol response. Normalization of the 24 h urine cortisol is achieved in
72% of the cases after a mean time of 6 months. Gastrointestinal side effects appear in about
50%, neurological signs in 30% and lipid abnormalities in 70% of the patients. Interestingly,
a pituitary adenoma became identifiable during mitotane treatment in 25% of the patients
with initial negative pituitary imaging allowing subsequent transsphenoidal surgery [141].
The remission is usually seen after a 9 months of medication use [142]. Mitotane is usually
part of a block replacement therapy with use of hydrocortisone soon after initiation of the
mitotane and possible fludrocortisone at a later stage. The hydrocortisone dose is usually
about two to three times higher than usual replacement as the medication increases the
metabolization of the steroid.
Mifespristone is a synthetic steroid molecule with high affinity to GC and progesterone
receptor, inhibiting both the glucocorticoid receptor activation and the gene transcription. The
drug is associated with improvement in the HbA1c, oral glucose tolerance test (OGTT) and
fasting glucose parameters as well as weight loss and waist reduction. It is started at 300 mg
daily and titrated up to 1200 mg daily at doses ranging 5-25 mg/kg/day. As the aldosterone
receptors are not blocked, hypokalemia and hypertension are possible adverse effects and
spironolactone or eplerenone need to be used. Adrenal insufficiency is a potentially serious
adverse effect and it is treated with high dose dexamethasone as this is the steroid with the
highest receptor affinity. The addisonian crisis has been reported in patients treated with this
drug [143, 144]. Endometrial thickening needs to be monitored as well as TSH which was
reported to increase with the use of the medication [145].
When the biochemical control is not achieved with one medication or titration is limited
by side effects, adding a second agent is recommended. Although no combination is FDA
approved, cabergoline and ketoconazole, cabergoline and pasireotide, all above 3 agents or
ketoconazole, mitotane and metyrapone have been reported with good results [67, 146].
Mortality, Recurrence and Remission

The overall mortality from CS is about double that of the general population. In limited
reports that looked at patients in remission versus with persistent disease separately, it is clear
that in persistent disease mortality is far worse than expected and patients in remission, with
normalization of cortisol and its effect, this difference in mortality from general population
over a 10- to 20-yr follow-up period disappear [147]. Remission achieved by uni- or bilateral
adrenalectomy in patients with ACTH-independent forms.
It has been reported, although scantly, that patients with cortisol-secreting adenoma have
the same survival rate as an age-matched control population [148] the mortality being low.
After adrenalectomy, the normalization of the metabolic changes occurs very rapidly.
Although recovery of contra lateral gland can take up to one year. Also recurrences seem
to be rare but reported in the literature, on the contra lateral side [148]. This should be
included thorough postoperative follow-up for the management of patients with adrenal mass
and hypercortisolism [149]. Worldwide remission achieved with transsphenoidal surgery for
ACTH-producing pituitary adenomas are 78-97% with best results with microadenomas
[147]. Failure of initial surgery or recurrence can be dealt with promptly and effectively by
laparoscopic bilateral adrenalectomy.
A close follow-up also recommended for hypertension, obesity, diabetes seen in these
patients as 30% or more will have persisting disease despite biochemical cure. And it has
been also reported that even late in the course after cure of CS, atherosclerosis, hypertension,
and diabetes mellitus can occur more frequently than in controls [150].
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Chapter 8
PHEOCROMOCYTOMAS
AND PARAGANGLIOMAS
Anna Heeney1, Aoife J. Lowry1,

Rachel K. Crowley2 and Ruth S. Prichard1,
1
Department of Endocrine Surgery, St. Vincent‘s University Hospital,
Dublin, Ireland
2
Department of Endocrinology, St. Vincent‘sUniversity Hospital,
Dublin, Ireland
ABSTRACT
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine
tumors derived from chromaffin tissue either in the adrenal medulla (pheochromocytoma)
or the embryonic neural crest (paragangliomas). These tumors secrete catecholamines
and therefore the classic presenting symptoms are due to excess circulating levels of
norepinephrine, epinephrine or dopamine. While up to 21% of PCC may be
asymptomatic the most commonly described symptoms include sweating, palpitations
and headaches in association with intermittent hypertension. If left untreated, excess
catecholamines may result in a hypertensive crisis leading to cardiac complications, a
cerebrovascular accident or ultimately sudden death. Paragangliomas arising from
sympathetic paravertebral ganglia of thorax, abdomen, and pelvis also secrete
catecholamines however parasympathetic paragangliomas, most often located in the head
and neck region, are predominantly non-secretory and present with symptoms due to their
specific location and local effects. The majority of these lesions are benign however a
major diagnostic challenge remains the accurate recognition of tumors with the potential
for development of distant metastases. Advances in the field of molecular genetics have
led to novel diagnostic and therapeutic strategies in an attempt to address this difficult
dilemma. Surgical excision is the mainstay of treatment and offers the only potential for
cure. The discovery of germline and somatic mutations that leads to the development of
these tumors in addition to increasing knowledge of molecular mechanisms that cause

Corresponding author: Ruth S. Prichard. E-mail: ruthprichard@rcsi.ie.
172 Anna Heeney, Aoife J. Lowry, Rachel K. Crowley et al.
malignant transformation has led to exciting developments into targeted therapeutics that
are showing promising results.
This chapter focuses on recent developments in the diagnosis of PCC/PGLs,
encompassing biochemical, radiological, histological and molecular analysis. In addition
newer treatment modalities and advances in individual targeted therapies for malignant
PCC/PGLs will be discussed.
Keywords: pheochromocytoma, paraganglioma, diagnosis, management, therapeutics
INTRODUCTION
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are are (delete) rare
neuroendocrine tumors that originate from chromaffin cells in the medulla of the adrenal
gland or in the embryonic neural crest [1]. The name, meaning `dusky-colored tumor,' was
coined by Pick in 1912 although the tumor had been first recognized and described by von
Frankel in 1886 [2].
The incidence of PCCs is estimated at less than 1 per 100,000 population with the
incidence of PGLs placed at just over 1 per million [1, 3-7]. This may be an underestimation,
as a study by Beard et al., demonstrated that in 50% of cases the diagnosis was made at
autopsy [5]. The incidence may also continue to increase as incidental adrenal lesions are
found to be undiagnosed PCCs in approximately 5-7% of cases [8, 9]. In patients with
secondary hypertension, PCCs/PGLs are found in up to 1% [10, 11].
Recent advances in genetic research have confirmed the incidence of hereditary
pheochromocytomas to be over 30% and are associated with at least 10 different germ line
mutations [12, 13]. These include several tumor syndromes which are listed below in Table 1
[14]. The majority of PCCs/PGLs are benign with a normal life expectancy following
treatment [15]. The incidence of malignancy is difficult to determine but ranges from 2.4% to
50% [16, 17]. Malignant PCCs/PGLs are a subset of tumors that demonstrate local invasion,
metastases, or recurrence [18-20]. The prevalence of malignancy among PGLs is higher than
PCCs, in particular in patients with abdominal paragangliomas or mutations in the SDHB
gene [21]. The presence of malignant disease decreases survival significantly with 5 year
survival rates of 20-70% [21-24]. Tumors with malignant potential are notoriously difficult to
recognize, remaining one of the biggest diagnostic and therapeutic challenges in the
management of this disease.
PRESENTATION
The peak incidence of PCCs/PGLs is in the 3rd and 4th decades of life and there is an
equal preponderance between males and females. As mentioned, they can exist sporadically
or in conjunction with other neuroendocrine tumors in patients with underlying genetic
disorders (Table 1) [25-27].
Patients typically present with a variety of symptoms due to sympathetic over-activity
which reflect excessive secretion of norepinephrine, epinephrine or dopamine into the
Pheocromocytomas and Paragangliomas 173
circulation [28]. Symptoms can vary but include the triad of headaches, palpitations, and
diaphoresis in association with severe hypertension [29].
Hypertension is frequently paroxysmal in nature with paroxysms often occurring on a
background of sustained hypertension.
Less commonly there may be feelings of anxiety, nausea, flushing or weight loss [30].
The excess of circulating catecholamines can lead to a hypertensive crisis which can
precipitate life-threatening cardiovascular emergencies such as a myocardial infarction,
cardiomyopathy, or a cerebrovascular accident. The most common stimuli for eliciting a crisis
are exercise, especially in children or tumor manipulation and anesthesia and for this reason
optimal pre-operative and intra-operative management is essential [31].
Rarely, PCCs may present with a new diagnosis of diabetes, more commonly seen in
younger patients who have no specific known risk factors for diabetes.
Asymptomatic PCC are found in up to a fifth of cases [32, 33]. This may be due to
desensitization of the cardiovascular system to persistently high levels of circulating
catecholamines [34].
In addition, the use of advanced imaging techniques which identify adrenal
incidentalomas and routine screening of patients with suspected or known genetic mutations
has led to an increase in the detection of asymptomatic and normotensive patients with PCC/
PGL [35, 36].
Approximately 70% of PGLs are derived from parasympathetic ganglia and are found in
the head and neck region [7]. These most commonly are non-secretory and may present with
symptoms due to pressure effects on local structures [37].
Chemodectoma is the term given to a PGL originating in chemoreceptor tissue such as
the carotid body or jugulotympanic region. Jugulotympanic tumors are also known as glomus
tumors and may cause tinnitus, dizziness, or facial droop while carotid body tumors may
present as a painless submandibular mass [38]. Sympathetic PGLs are mainly found in the
abdominopelvic region and most commonly occur in the peri-aortic and peri-caval
paraganglia, as well as the organ of Zuckerkandl located at the aortic bifurcation. They are
derived from the sympathetic nervous system and present similarly to PCCs with symptoms
attributable to excess in circulating catecholamines.
PCC/PGLs were previously thought to obey the ‗rule of 10s‘; i.e., 10% malignant, 10%
bilateral, 10% hereditary, 10% extra-adrenal and 10% in children. However this rule does not
appear to hold true anymore with recent advances in genetic research showing that up to 50%
of tumors have a genetic link, up to 50% of tumors showing malignant potential and up to
25% of tumors arising in an extra-adrenal location [39].
GENETICS
Pheochromocytomas and paragangliomas have the strongest genetic component of any
endocrine tumor with up to 50% being linked to germline and somatic mutations in 17
different genes [40]. The rate of genetic association is higher in children who develop a PCC/
PGL with Cascon et al., quoting rates of up to 69% [41]. Table 1 summarizes the most
extensively studied genetic mutations associated with PCCs/PGLs.
Other genetic conditions such as the Carney triad and Carney Stratakis syndrome are
known to be associated with development of PCC/PGL but the underlying gene has not been
identified.
Multiple endocrine neoplasia type 2 (MEN2) is divided into MEN 2A and MEN 2B and
is associated with mutations in the tyrosine kinase receptor proto-oncogene RET. Patients
affected with MEN 2 typically present with medullary thyroid cancer and approximately 50%
will also have or develop a PCC. While half of these patients will have bilateral tumors,
malignancy is rare [13, 42]. Patients with known MEN2 now commonly undergo a
prophylactic thyroidectomy and have ongoing surveillance for the development of PCC.
As with MEN 2, the familial syndromes Von Hippel Lindau (VHL) and
Neurofibromatosis type 1 (NF1) are characterized by a predisposition to multiple tumor types.
The rate of PCC/PGL development in NF1 is significantly lower than in VHL or MEN2
however, the metastatic rate for NF1 tumors, at approximately 12%, is higher than with
MEN2 or VHL [42]. The VHL gene codes for a protein that regulates the activity of hypoxia-
inducible factor alpha (HIFα) which is involved in cellular processes. Patients with VHL
mutations can be subdivided into VHL type 1 and type 2. VHL1 is more common and
affected patients can develop multiple tumors including (delete) retinal angiomas, renal
carcinomas, central nervous system hemangioblastomas, islet cell tumors of the pancreas
endolymphatic sac tumors, or cysts and cystadenomas of the kidney, pancreas, epididymis, or
broad ligament, but do not develop PCC/PGL. Patients with VHL 2 are at risk of developing
PCCs/ PGLs [42]. VHL2 is further subdivided into type A,B and C. Type 2A and 2B patients
are at risk of developing any of the type 1 tumors however type 2A do not develop renal cell
carcinomas. Type 2C patients are only at risk of developing PCC/PGL without any type 1
tumors [13]. NF1 is caused by mutations in the NF1 gene which codes for a GTPase
activating protein involved in cell signaling. Although PCCs/PGLs are uncommon in patients
with NF1, it has been shown that a significant number of sporadic PCC/PGL have an
inactivating NF1 mutation [43]. It is worth noting that in patients with NF, the most common
cause of secondary hypertension is renal artery stenosis rather than PCC/PGL [44].
Another syndrome which involves the development of PCCs/PGLs is the Carney triad.
Carney first described this condition in young women in 1977 as an association of pulmonary
chondroma, gastrointestinal stromal tumors and functioning paraganglioma [45]. Carney
Stratakis syndrome is an association of familial paraganglioma and gastric stromal sarcoma. It
is considered to be a distinct condition from Carney triad as it exhibits an autosomal dominant
pattern of inheritance and is not associated with pulmonary chondroma [46].
Up to 25% of apparently sporadic cases result from a germline loss-of-function mutations
in the genes encoding the subunits A[F2], B, C and D of succinate dehydrogenase (SDH) [47-
50]. The enzyme succinate dehydrogenase is involved in the tricarboxylic acid cycle, where it
catalyzes the oxidation of succinate to fumarate, and also in the respiratory electron transfer
chain, where it transfers electrons to coenzyme Q. Germline mutations in the SDH gene
complex give rise to the familial PCC/ PGL syndrome and are divided into PGL1, PGL2,
PGL3, and PGL4, caused by mutations in SDHD, SDHAF2, SDHC, and SDHB, respectively
[51]. In addition Pasini et al., reported germline mutations in SDHB, SDHC or SDHD in eight
of 11 patients from seven unrelated families diagnosed with aforementioned Carney Stratakis
syndrome [52]. SDH-related tumors are typically extra-adrenal, although some cases of
adrenal PCCs have also been reported. SDHA mutations were initially described in autosomal
recessively inherited juvenile encephalopathy [53] and have also been implicated in adrenal
and extra-adrenal PCCs/PGLs [54, 55]. SDHB mutations are found in approximately 1.7%–
6.7% of sporadic PCCs [13] and are linked to more aggressive thoracic or intra-abdominal
PGLs with younger age of presentation, multiple tumors and higher metastatic rates [56].
Table 1. Genetic mutations associated with pheochromocytomas and paragangliomas
Frequency of PCC/PGL
Gene Syndrome Penetrance Associated tumors
malignancy Characteristics
Autosomal
Young age (mean Retinal angiomas
Dominant
VHL Von Hippel-Lindau < 10% 28) Haemangioblastoma
Variable
Bilateral/Multifocal Clear cell RCC
expression
Neurofibroma
Mean age 41
Neurofibrosarcoma
Bilateral disease
Autosomal Glioma
NF1 Neurofibromatosis < 10% common
Dominant Astrocytoma
Extra adrenal PGL
Carcinoid
rare
Leukaemia
Mean age 40
< 5% Medullary thyroid
Autosomal Hyperparathyroidism
RET MEN II Extra-adrenal cancer
Dominant No increased
PGL rare Mucosal neuromas
malignancy risk
Mean age 46
Amyloidosis
Autosomal Extra adrenal head
SDHC PGL 3 < 5% Cutaneous lichen
Dominant and neck PGLs
GISTs
Bilateral/multifocal
Autosomal Mean age 35
Papillary thyroid
Dominant with Extra adrenal head
SDHD PGL 4 < 5% cancer
parent of origin and neck PGLs
GISTs
effect Bilateral/multifocal
Extra adrenal
Autosomal Increased RCCs
SDHB PGL 1 34-70%
Dominant malignancy risk GISTs
Bilateral if adrenal
Autosomal
Uncertain
Dominant with
SDHAF2 PGL 2 Head and Extra adrenal GISTs
parent of origin
Neck PGLs
effect
TMEM Autosomal Adrenal
5%
127 Dominant Bilateral
Autosomal Adrenal
MAX 10%
Dominant Bilateral
These patients also have an increased risk of renal cell carcinoma which can have a more
aggressive phenotype in young patients [57]. SDHC and SDHD mutations were initially
associated with biochemically silent tumors especially in the head and neck [58] but have also
been shown to be present in adrenal PCCs and PGLs in other sites [59]. Due to the rapid rate
of development in molecular research techniques over the past decade, several additional
genes have been identified that contribute to hereditary PCCs/PGLs. These include myc-
associated factor X (MAX) [60], transmembrane protein 127 (TMEM127) [61] and most
recently hypoxia-inducible factor 2-alpha (HIF2A) [62]. MAX protein is involved in cell
proliferation and differentiation via the MYCMAX-MXD1 network with Burchinon et al.,
showing that MAX mutations were present in 1.12% of patients with no other known
mutations [60].
The tumor suppressor gene TMEM127 is involved in the activation of the MTOR
signaling pathway and mutations of TMEM127 are associated with adrenal PCCs which are
frequently bilateral [61, 63]. Genetic testing in patients with PCC/PGL is an important
component of management, however currently testing is both costly and time consuming. A
study using next generation sequencing which analyses multiple genes simultaneously has
proved to be highly sensitive in detecting mutations and may provide more accessible
clinically relevant data in the future [64]. Increasing numbers of patients with PCC/PGL are
being referred for next generation sequencing with the aim of optimizing surveillance
protocols depending on genetic result and identifying family members at risk of developing
PCC/PGL who can the be offered screening.
DIAGNOSIS
Biochemistry
The diagnosis of PCCs is largely made biochemically with subsequent anatomical and
functional imaging delineating disease extent. The most recent endocrine society guidelines
recommend that the measurement of plasma or urine metanephrines, the O- methylated
metabolites of catecholamines, are the most accurate tests currently available for the
diagnosis of PCC/PGL demonstrating excellent sensitivity (97%) and specificity (91%) [65-
68]. They are consistently elevated in patients with biochemically active PCC, as although
catecholamine release fluctuates, their metabolism remains fairly constant, leading to a steady
release of metanephrines. When measuring 24 hour urinary metanephrines, urinary creatinine
should also be measured to verify completeness of the urine collection. In addition, it is
recommended that when measuring plasma metanephrines, patients should be at least 30
minutes in the supine position when blood is drawn [68]. More recent studies have
demonstrated the utility of plasma methoxytyramine in diagnosing PCC, particularly for
detecting exclusively dopamine-secreting tumors, which are rare and therefore can sometimes
be overlooked by traditional measurements of metanephrines [69, 70].
Chromogranin A, a polypeptide that is secreted by chromaffin cells is the most accurate
general marker of neuroendocrine tumors. Although not used in clinical practice it is raised in
91% of PCC/PGL patients [71] and while less specific it may be a valuable tool in monitoring
response to treatment [72]. When Chromogranin A is combined with catecholamine
measurements, the sensitivity for diagnosing PCC/PGL approaches 100% and in the majority
of cases normalizes after surgery [71]. Diagnostic levels of metanephrines are defined as
levels greater than three times the upper reference limit. In patients with an elevation in
metanephrines but to a level less than the diagnostic threshold, further biochemical
investigations may be required [68]. Firstly, it is important to repeat the test after the patient
has rested supine for 30 minutes. Secondly, discontinue any substances which may cause
false positive elevations such as certain anti-hypertensives, anti-depressants and caffeine [73,
74]. In particular patients taking tricyclic antidepressants and phenoxybenzamine should stop
their medication as one study by Eisenhofer et al., showed these drugs accounted for 41% of
false positive elevation in metanephrines [73].
If investigations remain equivocal a clonidine suppression test can be carried out, in
which plasma normetanephrine levels will remain elevated in the presence of a PCC/PGL
[74]. Clonidine acts via the alpha pre-ganglionic receptors to reduce catecholamine secretion.
In normal patients, even if they are anxious, the plasma catecholamines will suppress into the
normal range 3 hours following a dose of 300g/70kg body weight clonidine hydrochloride.
This will not be seen in patients with a pheochromocytoma [75].
Imaging
Once a biochemical diagnosis has been reached, further investigation with imaging is
warranted. The aim of radiological imaging is to localize the primary tumor, to evaluate for
multi-focal or metastatic disease and to allow the surgeon to plan resection. This allows
determination of treatment strategies. Imaging is firstly focused on the adrenal gland and if
negative, imaging of additional areas of the body should be performed depending on the
individual presentation [76]. Computed tomography (CT) and magnetic resonance imaging
(MRI) are the major imaging modalities currently used for the localization of PCC‘s and
PGL‘s with functional imaging offering an additional benefit in localizing, characterizing and
staging these tumors [77]. CT (Figure 1) and MRI (Figure 2) have similar sensitivity in
detecting PCC. Recent clinical practice guidelines from the Endocrine Society recommends
CT as the first-choice imaging modality because of its excellent spatial resolution for thorax,
abdomen, and pelvis [68]. MRI is the imaging modality of choice in patients with metastatic
disease, head and neck paragangliomas, CT-contrast allergies, in pregnant females, children
and in patients in whom radiation exposure should be limited [29, 68]. Due to the high
number of adrenal incidentalomas the specificity of both CT and MRI is limited at around 70-
80% [78] and therefore additional functional imaging is often employed.
On non contrast CT imaging, PCCs/PGLs typically have a heterogeneous appearance,
with attenuation values greater than 10 Hounsfield units [79] and on dual phase contrast
enhanced CT, PCCs show higher intensity on the arterial phase at levels greater than 110
Hounsfield units [80]. Non-ionic contrast enhanced CT is safe in patients with PCC/PGL and
there is no evidence that contrast provokes catecholamine release [81] On MRI, PCCs/PGLs
typically appear as bright lesions on T2 weighted imaging and may also exhibit cystic or
necrotic components which can affect this classic appearance [79, 82].
In young patients under 40 years of age with a PCC less than 3 cm and no family history
of PCC, no further imaging workup needs to be performed if CT or MRI demonstrates an
adrenal lesion [76]. Conversely if adrenal imaging is negative imaging of additional areas of
the body should be performed. Functional imaging may be required to evaluate the extent of
disease, looking predominantly for extra-adrenal sites and to accurately stage patients.
Functional imaging encompasses metaiodobenzylguanidine scintigraphy (MIBG), positron
emission tomography and somatostatin receptor imaging (Figure 3). Functional imaging can
be performed with the catecholamine precursor 123I- or 131I-metaiodobenzylguanidine (MIBG)
scintigraphy. Human chromaffin cells express norepinephrine transporters and as MIBG is a
catecholamine precursor with a structure that resembles norepinephrine the molecule can
enter the cells through these transporters [83]. Thus MIBG imaging has been used in the
investigation of functioning PCCs and PGLs because uptake reflects adrenergic innervation
or catecholamine excretion [84]. The reported overall sensitivity and specificity of MIBG
scanning is 94% (95% CI: 91 – 97%) and 92% (95% CI: 87 – 98%) respectively (REF).
Patients who are taking certain medications including tricyclic antidepressants, labetalol and
specific calcium antagonists should temporarily discontinue these drugs prior to scanning as
they may interfere with 123I-MIBG uptake and interpretation of the imaging [85]. 123I-MIBG
has superior imaging quality than 131I-MIBG and is the modality of choice when available
[86]. However the overall sensitivity of MIBG imaging is decreased in malignant disease
[87]. This may be as a result of reduced expression of norepinephrine transporters in
malignancy or dedifferentiation. It has been shown that tumors associated with certain genetic
mutations such as VHL and SDHB may express a reduced number of noradrenaline
transporters and therefore are more likely to be negative on MIBG imaging [88, 89].
Figure 1. Axial non-contrast CT imaging of a right adrenal heterogenous mass measuring 34 hounsfield
units on the non-contrast phase. On enhanced and delayed series the mass demonstrated relatively little
washout and these CT features are consistent with a pheochromocytoma.
Figure 2. Axial MRI showing a mass (white arrow) in the medial limb of the right adrenal that is
heterogenously high signal on T2 weighted imaging consistent with a pheochromocytoma.
Figure 3. 123I-MIBG scan showing focal uptake in the right adrenal gland consistent with a
pheochromcytoma.
Alternative imaging modalities should be considered in this group of patients especially if

malignancy is suspected. MIBG may also have a role in intra-operative detection of small
tumors or metastatic disease using a - detector probe following injection of the isotope pre-
operatively [90]. Adams et al., showed that radio-isotope guided surgery using either
technetium 99m(V)-dimercaptosuccinic acid [(99m)Tc(V)-DMSA], indium 111 ((111)In)-
pentetreotide or (123)I-MIBG increased the sensitivity of detecting lymph node metastases to
97% compared with 65% for surgical palpation and conventional imaging alone.
Single photon emission computed tomography (SPECT) imaging has been used in
combination with both CT and MRI to increase accuracy in localization of the tumor as it
involves simultaneous acquisition of both morphological and functional data (Figure 4).
However SPECT imaging can miss smaller lesions due to relatively low resolution.
Positron emission tomography (PET), using biologically active tracer-labeled molecules
is increasingly employed in the diagnosis of PCC/PGL particularly in patients where the
MIBG scan is negative [76]. 18F fluro-2-deoxy-d-glucose (FDG) is the most commonly used
tracer in oncological practice that enters the cells via transported glucose which subsequently
undergoes phosphorylation to18F-FD-6P.
It is not specific for PCC/PGL and so other tumor types or inflammatory tissue may be
identified [76]. However, it has been shown to be useful in diagnosing PCC/PGLs that do not
accumulate MIBG [91] and is superior to other functional imaging techniques in patients with
disease associated with the SDHB mutation [92]. The Endocrine society guidelines have
favoured 18F-FDGPET/CT as the preferred imaging modality over 123I MIBG scintigraphy in
patients with known metastatic disease [68].
Recently, more specific tracers including 18F-DOPA, 18F-FDA (Fluorodopamine) and
11
C-HED (meta-hydroxephedrine) have been developed for use in PCCs/PGLs but are, as yet,
not as widely available. 18F-DOPA is specific to neuroendocrine tumors and has a high
sensitivity for head and neck PGLs [93]. Patient genotyping is also important to consider as a
high false negative rate has been reported for patients with SDHB mutations with 18F-DOPA-
PET imaging [94].
18
F-FDA-PET is more sensitive (88%-100%) than MIBG for detection of metastatic
disease [95] and particularly for the detection of bony metastases where it is superior to CT,
MRI, 123/121I-MIBG and 18F-FDG [96].
Figure 4. SPECT CT imaging demonstrates focal tracer uptake in the right suprarenal region (arrow)
suspicious for a right adrenal pheochromocytoma.
Imaging plays an important role in the screening of patients carrying known genetic
mutations and in the surveillance of patients with a known history of PCC/PGL.
In carriers, screening with biochemical testing and interval CT/MRI is recommended and
in those with a specific genotype such as SDHB mutations functional imaging may be used in
addition. For patients who have had a PCC/PGL resection there are no official guidelines for
surveillance but follow up with regular biochemical testing and imaging has been suggested
[40].
Histopathology
The histological diagnosis of PCCs/PGLs is straightforward. Where the difficulty arises

is in differentiating benign from malignant disease. Tumor cells in PCC/PGL demonstrate a
nested Zellballen pattern surrounded by sustentacular cells, which stain positive for S100
protein on immunohistochemistry (Figure 5).
Characteristically they exhibit immunopositivity for synaptophysin and chromogranin A
and may express neurofilament [97]. To distinguish PCC/PGL from other neuroendocrine
tumors, expression of enzymes involved in catecholamine metabolism such as tyrosine
hydroxylase are assessed using immunohistochemistry [98, 99].
There are no clear features that distinguish benign from malignant primary PCC/PGL and
therefore a major challenge in the management of this disease is the inability to predict those
patients who may develop metastatic disease. It is now possible to stain for SDH mutations
using immunohistochemistry. The loss of SDH expression in the tumor would prompt genetic
screening and may identify tumors with increased malignant potential [100].
Malignant/Metastatic Disease
The true incidence of malignancy in PCC/PGLs is difficult to accurately determine and

has traditionally been cited at approximately 10% [101]. However this can range from 2.4%
to 50%, depending on the definition of malignancy used and the specific population in
question. There are no absolute histological criteria for the diagnosis of malignancy and no
means to identify patients with PCCs/PGLs that are at risk of recurrence or metastatic spread
using standard histopathological techniques.
Figure 5. H and E staining of pheochromocytoma showing nested arrangement of cells (Zellballen).
Table 2. Pheochromocytoma of the Adrenal Gland Scoring Scale [18]
Features Score
Large nest of cells or diffuse growth > 10% of tumor volume 2
Necrosis (confluent or central in large nests) 2
High cellularity 2
Cellular monotony 2
Presence of spindle shaped tumor cells 2
Atypical mitotic figures (> 3 per 10 high power fields) 2
Extension of tumor into adjacent fat 2
Vascular invasion 1
Capsular invasion 1
Profound nuclear pleomorphism 1
Nuclear hyperchromasia 1
Several histological scoring systems have been devised to guide pathologists in the
diagnosis of malignancy. The Pheochromocytoma of the Adrenal Gland Scoring System
(PASS), devised by Thompson in 2002 is the most commonly used (Table 2) [18]. The
histological features of each tumor are given a score and the sum of the scores groups PCC‘s
into those with potential for aggressive behavior ( 4) and those likely to behave in a benign
manner (< 4). The system has been evaluated with varying results. Strong et al., found that a
threshold of 6 indicated malignant behavior but recommended that any patient with a PASS
score of 4 or more should be closely followed [19].
Disappointingly, the scoring system has also been shown to be open to significant inter-
and intra-observer variation leading to recommendations that it be used with caution [102]. In
addition a large retrospective analysis performed by Agarwal et al., found no significant
correlation between the PASS score and the risk of malignant potential [103].
As a result of these concerns regarding the PASS system, Kimura et al., devised an
alternative scoring system combining results from both adrenal PCCs and extra-adrenal
sympathetic PGLs (Table 3) [104]. This scoring system combined histological criteria, tumor
Ki67 scores along with the type of catecholamine produced by the tumor. The higher the
combined score of the individual tumor, the greater the correlation with metastatic potential
and patient survival. However, it was developed based on 146 tumors of which only 38
proved to be metastatic and therefore this model requires further validation to determine its
applicability to the clinical setting.
Although a multitude of immunohistochemical (IHC) markers of malignancy in PCCs/
PGLs have been proposed, as yet, not one has emerged that could be deemed useful in routine
clinical practice. Markers that have been investigated to date include neuroendocrine- and
catecholamine-related markers (neuropeptide Y, 3,4-dihydroxyphenylalanine) [105], granin
derived peptides (EM66, secretogranin II) [106-108], CD-44s [109], angiogenic markers and
regulators (vascular endothelial growth factor [VEGF] and VEGFR) [110], heat shock protein
90 [111] and telomerase complex proteins [112]. The Ki67 proliferative index of the tumor is
the most likely marker to be of clinical value and in several studies has been shown to
correlate with malignancy [113, 114]. A Ki67 index > 3% appears to have a high specificity
for malignant PCC as benign PCCs have not been demonstrated to have a score of > 3%.
However Ki67 has poor sensitivity with many malignant tumors exhibiting indices < 3% [19,
114-116]. In addition immunohistochemistry of PCCs/PGLs may detect genetic mutations
such as loss of SDHB expression which may predict a poorer prognosis and survival [117].
This may also allow patient stratification for mutational testing.
Table 3. Scoring system devised for both pheochromocytomas and extra-adrenal

paragangliomas
Feature Score
Histological Pattern
Uniform cell nests 0
Large irregular cell nests 1
Pseudorosettes 1
Cellularity
Low (< 150 cells/mm2) 0
Moderate (150-250 cells/mm2) 1
High (> 250 cells/mm2) 2
Necrosis (Confluent or central in large cell nests) 2
Vascular/Capsular invasion 1
Ki-67 index
< 1% or 20 cells per medium power field 0
> 1% or 20 cells per medium power field 1
> 3% or 50 cells per medium power field 2
Catecholamine phenotype
Adrenergic 0
Noradrenergic or non-functional 1
Total possible score 10
Other factors which may predict malignant potential include tumor size and location
although there is conflicting evidence in the literature regarding these physical parameters.
Several studies have reported an association between tumor size and malignancy with Feng et
al., demonstrating that tumors greater than 5 cm in diameter are more likely to be malignant
[118] and associated with a reduction in overall survival [119].
Unfortunately, malignancy has also been reported in smaller tumors and so size alone
lacks sensitivity as a predictive tool [19]. Historically, tumors in an extra-adrenal location
have been associated with a higher rate of malignancy [118, 120, 121]. However with
advances in tumor genotyping it is known that SDHB-mutated tumors are more likely to be
extra-adrenal and large in size at the time of presentation and of these approximately 50%
will be malignant [122, 123]. However, even in the absence of SDHB mutations the risk of
metastatic disease remains elevated in extra-adrenal tumors (3.4-fold) [124].
The advances in human genomic sequencing and molecular profiling has resulted in a
huge increase in our knowledge of the molecular biology of malignancy and has been applied
to PCCs/PGLs with the aim of differentiating between benign and malignant disease. Several
techniques have been applied including cDNA-based analysis, gene expression profiling and
microRNA expression profiling.
Several genetic losses associated with underlying germline mutations have been
identified using comparative genomic hybridization. Sandgren et al., reported that DNA gain
was more frequently found in malignancy with gain at 1q found in malignant PGL and gain at
19q, trisomy 12 and loss of 11q associated with malignant PCCs [125]. Further validation of
these studies is needed to exclude underlying genetic mutations confounding results.
Gene expression profiling has led to the division of PCCs/PGLs into two clusters [126]
indicating alternative tumorigenesis pathways. Studies to identify gene expression that may
differentiate benign from malignant disease have been undertaken with some of these studies
showing downregulation of the expression of genes coding for neuroendocrine factors in
association with malignancy [127, 128].
Suh et al., [129] and Waldman et al., [130] reported 5 and 132 differentially expressed
genes between benign and malignant tumors respectively using genome-wide expression
analysis, however there is little concordance between the studies and to date there is no
individual gene or molecular marker to differentiate benign from malignat PCC/PGL.
Increasing interest in microRNA (miRNA) profiling as potential diagnostic and
prognostic biomarkers of malignancy has led to investigation using microarray expression
profiling of PCCs/PGLs. Preliminary research requires further validation before adopted into
clinical use but studies by Meyer-Rochow et al., [131], Tombol et al., [132] and Patterson et
al., [133] have all shown promising results with identification of miRNAs that are
differentially expressed between benign and malignant PCCs/PGLs.
MANAGEMENT
The curative treatment of choice for PCCs/PGLs is surgical resection. However
management, especially of malignant, metastatic or recurrent disease can be multi-modal
including pharmacological control of catecholamine mediated symptoms, radiotherapy and
systemic therapy [67].
Pharmacological Management
Anti-hypertensive medications should be initiated in patients with biochemically active

PCC/PGL. This is not only to reduce symptoms but also to reduce the risk of a hypertensive
crisis which can have devastating results. Initial pharmacological treatment is with the alpha-
adrenoceptor blockers and followed by beta blockade where necessary [134]. It is important
to avoid commencing beta blockers before alpha blockade as this can lead to unopposed
stimulation of alpha-adrenoceptors resulting in a hypertensive crisis [135]. Several alpha
blockers are available including the long acting phenoxybenzamine or short acting prazosin,
terazosin, and doxazosin. If additional beta blockade is necessary to control hypertension or
to address symptoms such as tachyarrhythmia, cardioselective beta blockers such as
metoprolol or atenolol are most commonly used [134]. Other hypertensive medications
including calcium channel blockers are sometimes employed in patients with refractory
hypertension or as single agents in patients with mild hypertension or in those who can not
tolerate alpha blockade [134].
Pre-Operative Care
All patients with a known PCC/PGL who are due to undergo surgical resection should
have an extensive pre-operative work-up to identify the presence of metastatic disease which
may influence the surgical approach [136]. Knowledge of underlying genetic mutations
predisposing to bilateral disease may also influence management however genetic testing is
rarely performed in the pre-operative setting. Pre-operative blockade using appropriate anti-
hypertensive medication and careful fluid resuscitation is imperative to reduce the possibility
of intra-procedural hypertensive crises [137, 138].
Even those patients who are normotensive pre-operatively should be treated, as
unanticipated catecholamine release by the tumor during surgery or other procedures may
lead to a hypertensive crisis [135]. As the alpha blocker dose is increased, patients should
start to have symptoms like a stuffy nose, fatigue, and mild postural hypotension to ensure the
patient is adequately blocked. All patients should have a comprehensive anesthetic review
pre-operatively to assess cardiorespiratory health. Endocrine Society guidelines also
recommend a high sodium diet along with fluid intake to reverse catecholamine-induced
blood volume contraction preoperatively to prevent severe hypotension following tumor
removal [68]. Cardiovascular and blood glucose management should also be monitored peri-
operatively.
Surgery
Surgery is the curative treatment of choice for primary, recurrent or locally advanced
disease and may also have a role in debulking tumors with limited metastatic disease [29].
Surgical intervention should be undertaken in specialist centers following a comprehensive
pre-operative work-up. Minimally invasive surgery for both PCCs/PGLs via the laparoscopic
technique (Figures 6-7) is the current preferred technique when technically feasible [139-142]
and is associated with shorter length of stay, decreased analgesic requirements and increased
patient satisfaction [143]. Laparoscopic adrenalectomy via the transperitoneal approach was
first described by Gagner in 1992 [144]. One year later, the retroperitoneal approach was
described [145] and brought into clinical practice by Walz [146]. The lateral transperitoneal
approach can be used for large tumors and offers excellent exposure [147, 148]. The
retroperitoneal approach allows direct access to the gland and the option of bilateral
adrenalectomy without a requirement for repositioning if a prone approach is used [149].
Suzuki et al., favored the lateral transperitoneal method [150] for larger tumors and for
surgeons who are less experienced in laparoscopic adrenalectomy. A recent systematic review
by Chai et al., concluded that the posterior approach may be superior as shown by shorter
operation times and hospital stay however this was based on retrospective analysis with
significant selection bias and differing surgeon experience [151].
Figure 6. Overview of laparoscopic right adrenalectomy via the transperitoneal approach. The arrow A
indicates the tail of the pancreas and the arrow B indicates the right adrenal gland and. The kidney lies
inferior to the adrenal gland and is indicated by the arrow C.
Figure 7. Transperitoneal laparoscopic left adrenalectomy showing the left adrenal vein (arrow)
dissected prior to ligation.
Rubinstein et al., carried out a prospective study comparing approaches and found no
difference in operative time, blood loss, analgesic requirement, hospital stay, or complication
rates [152]. As there is no clear consensus on superior approach, the choice is determined by
the surgeon‘s experience and preference.
In patients with bilateral tumors or those at risk of bilateral disease due to known
syndromes such as MEN2 or VHL, bilateral cortical sparing adrenalectomy has been
proposed [153]. It is important to consider underlying genotype when selecting patients
suitable for cortical sparing adrenalectomy as patients with MEN-2A or VHL mutations have
a high risk of bilateral tumors yet low rates of malignancy while patients with SDHB
mutations should have a total adrenalectomy carried out due to the increased risk of
malignancy [154]. At least one third of the gland must be preserved for adequate function
[153, 155]. This may reduce the need for long term corticosteroid replacement and reduces
the risk of an Addisonian crisis following bilateral adrenalectomy [156, 157]. With subtotal
adrenalectomies, there is however a risk of recurrence varying from 10% - 60% [158, 159].
Successful repeat subtotal adrenalectomies in these patients have been described and are
possible if tumors recur [160]. More recently newer minimally invasive techniques have been
developed and include laparoscopy using single-site access [161], natural orifice transluminal
endoscopic surgery [162] via the transvaginal approach and robotic surgery [163, 164].
However these techniques are not in widespread clinical use to date.
Patients with extensive loco-regional infiltration of tumor with invasion of adjacent
organs will often require an open procedure to remove the tumor and involved organs en-
bloc. However it is appropriate to perform an initial laparoscopy primarily to assess the extent
of disease [163]. For patients in whom curative surgical excision is impossible, debulking/
cytoreductive surgery can improve symptoms caused by local invasion along with
catecholamine secretion. Long term benefits may be limited [165] with a recent study by Ellis
et al., showing that less than 10% of patients were able to stop anti-hypertensive medications
for more than 6 months [165]. Optimal management of paragangliomas mostly depends on
tumor location, local involvement of neurovascular structures, estimated malignancy risk as
well as underlying patient factors. Surgery offers the only chance of cure but may have
significant operative risks and morbidity rates depending on tumor location. In certain cases a
more conservative approach with radiotherapy is considered, In patients with head and neck
paragangliomas which are often benign, surgical excision can lead to injuries to the lower
cranial nerves [166]. In patients with abdomino-pelvic sympathetic paragangliomas, surgery
is often complicated by proximity to major vascular structures and the preponderance for
malignancy leading to local invasion. These tumors are also likely to be functioning with the
risk of hypertensive crisis as with adrenal tumors [167]. For patients with benign disease,
surgery alone is curative. However for patients with suspected or confirmed metastatic
disease alternative or additional treatment modalities may be needed.
Radiotherapy
Although malignant PCCs/PGLs have limited radio-sensitivity, patients with metastatic

disease and those who are unsuitable for surgical intervention may be candidates for
radiotherapy. Radiofrequency ablation, external beam radiotherapy and radionuclide
treatment are all modalities in current use [40].
Successful radiofrequency ablation has been reported for accessible metastatic disease
however careful per-procedural management is imperative due to the risk of a catecholamine
surge [168]. External beam radiation is often used for symptom palliation, particularly for
bony metastases [169].
It is also used in the treatment of unresectable head and neck PGLs where long-term
control can be obtained with limited toxicity [170, 171]. When used in conjunction with
radionuclide therapy (131I-MIBG), external radiation has been shown to improve response
rates in a small number of patients with widespread systemic metastases [172].
Radionuclide Therapy
Radionuclide treatment is indicated for patients with malignant disease in whom surgery
is not a viable option. It is based on the administration of radioactive compounds leading to
the emission of beta particles into tumor cells causing their destruction. These radioactive
compounds consist of a beta-emitting isotope coupled to either 131I-MIBG or somatostatin
analogues, which due to the structural similarity between MIBG and noradrenaline allows
uptake into chromaffin cells. Patients with metastatic disease with 131I-MIBG uptake on
imaging may be suitable for treatment.
Although only a minority of patients will have a complete response to MIBG treatment, a
recent systematic review has shown that over a quarter of patients had a partial response and
52% of patients demonstrated stable disease following treatment [173]. As radionuclide
therapy is well tolerated by patients with relatively few side effects it should be considered in
patients with metastatic disease that is MIBG-avid. Two strategies of delivering radionuclide
MIBG therapy have been described.
131
I-MIBG therapy given in smaller doses over a longer period of time is preferable to the
alternative of a single large dose of MIBG followed by stem cell infusion to replace bone
marrow due to lower rates of treatment related morbidity and mortality [174].
More recently, research into treatment with radiolabeled somatostatin analogues
including yttrium-90-DOTATOC (90YDOTATOC) and lutetium-177-DOTA0-Tyr3-octreotate
(177Lu-DOTATATE) has shown promising results with regards to tumor stabilization and
symptomatic relief [175]. Tumor response is predicted by the level of uptake of these agents
by tumors on pre-treatment scintigraphy [176].
Chemotherapy
Malignant PCCs/PGLs are not particularly chemo-sensitive. Chemotherapy is largely

reserved for those patients not amenable to surgical therapy and not responding to
radionuclide treatment. Most published experience has been with the regimen of
cyclophosphamide, vincristine, and dacarbazine (CVD) and in some cases this has been
shown to be of value in palliating patient symptoms, reducing the rate of tumor growth and
occasionally in shrinking tumors [169, 177-179]. A study by Huang et al., of patients who had
demonstrated response to CVD did find that at 22 years of follow up there was no survival
difference in patients who showed tumor response [180].
However this study had only 18 patients and therefore it is impossible to reach any
evidence based conclusions based on their outcomes. Experience of alternative
chemotherapeutic agents in PCC/PGLs is limited and mostly based on isolated case reports
and small series. Further clinical trials are needed before recommendations can be made on
their use in PCC/PGL.
Targeted Therapy
As a result of the fact that standard adjuvant therapy has been demonstrated to have a
limited impact on survival newer therapeutic targets are currently being explored. Increasing
knowledge of underlying genetic mutations and molecular alterations associated with
malignancy has led to improved understanding of tumorigenesis pathways. PCCs/PGLSs
have been divided into two major groups, the pseudohypoxic phenotype and RAS/RAF/ERK
signaling phenotype [181, 182] which may help in identifying appropriate targets that are
involved in the signaling pathway that modulate malignant transformation.
Such targets include the mammalian target of rapamycin (mTOR), a serine/threonine
protein kinase which may be upregulated in malignancy [183], angiogenesis-mediated growth
factors such as vascular endothelial growth factor (VEGF) [110] and heat shock protein 90
(Hsp90) that is overexpressed in malignant PCCs [112].
The PI3/Akt/mTOR pathway which is responsible for the regulation of cell growth and
survival has been implicated in the pathogenesis of malignant neuroendocrine tumors,
including pheochromocytomas [184]. Dysfunction of this pathway results from upregulation
of mTOR and subsequently disturbance of cellular proliferation and apoptosis potentiates
malignant transformation. The mTOR inhibitor, Everolimus, has been used in a number of
patients with malignant PCC/PGL but results so far have been disappointing [184]. (same
paragraph) This may be due to compensatory P13K/AKT and ERK activation in response to
mTOR inhibition [185]. Further studies aimed at targeting more than one pathway to
overcome drug resistance are in progress using a combination of mTOR inhibition with other
specific molecular drugs and results are awaited.
Like many tumors, targeting angiogenesis-mediated tumor growth by way of the VEGF
pathway has been evaluated in the treatment of malignant PCC/PGL. The anti-angiogenic
agent, Thalidomide, in combination with temozolamide has been evaluated in patients with
metastatic carcinoid tumors, PCCs, and pancreatic neuroendocrine tumors. Although only
three of the study participants had PCC 40% of patients demonstrated a biochemical response
and 25% of patients showed a radiological response. Of the 3 patients with PCC, one
demonstrated a partial response [186].
Sunitinib, a tyrosine kinase inhibitor, inhibits VEGF-R, PDGF, and c-KIT and was
originally developed as a treatment for renal cell carcinoma. In several reports it has shown
some promising results in PCC/PGLs with reduction in tumor size, catecholamine secretion
and metabolic activity on functional imaging [187, 188]. However initial results are often not
sustained and tumors appear to cease responding to the drug after a short interval [189, 190].
Heat shock proteins, thought to be involved in protein folding and degradation have also been
used as pharmacological targets in various tumors [191]. Specifically the protein Hsp90 has
been found to be over-expressed in PCC/PGL and research is undergoing to evaluate its use
as a therapeutic target.
One study investigating the role of two Hsp90 inhibitors, 17-AAG and ganetespib in vitro
in available mouse and rat PCC cell lines and in primary human PCC tissue cultures showed a
reduction in cell proliferation and migration [192]. These drugs have already been trialed in
Her2+ trastuzumab-refractory breast cancer with promising results and appear to be well
tolerated with minimal side effects [193]. Further clinical trials are required for patients with
PCC/PGL.
Additional targets for therapy of PCC/PGL will be identified with increasing
understanding of tumor pathogenesis and ongoing research at an in vitro level may
subsequently lead to effective therapies. Initial studies include an in vitro study of an insulin-
like growth factor 1 (IGF1) receptor antagonist which found significantly decreased cell
viability in mouse PCC/PGL cell lines [194]. The long chain fatty acid eicosapentaenoic acid
(EPA) has been found to induce apoptosis in rat PCC cells [195]. Finally, histone deacetylase
inhibitors have been shown to reduce cell proliferation in mouse PCC cell lines while
simultaneously increasing 131I-MIBG uptake suggesting potential use of HDAC inhibitors as
a pre-treatment enhancer for patients undergoing MIBG therapy [196].
CONCLUSION
With improvements in diagnostic imaging, greater understanding of molecular genetics
and genotype phenotype interactions an increasing number of patients with PCC/PGL are
being identified. The majority of these patients with have benign disease that can be cured by
minimally invasive surgical means. Our knowledge base of the disease is therefore (delete)
increasing significantly with promising advances in radiological imaging, targeted
therapeutics and surgical approaches. The recognition of malignant potential remains a major
diagnostic challenge and the presence of metastatic disease still carries a poor prognosis.
Several targeted treatments already identified need further evaluation and may offer
promising therapeutic options in the future.
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Chapter 9
NEW INSIGHTS OF GLUCOCORTICOIDS ACTIONS

ON THE HOMEOSTATIC CONTROL OF ENERGY
BALANCE AND STRESS-RELATED RESPONSES
Ernane Torres Uchoa1,, Silvia Graciela Ruginsk2,

Rodrigo Cesar Rorato3, Beatriz de Carvalho Borges3,
Jose Antunes-Rodrigues3 and Lucila Leico K. Elias3
1
Department of Physiological Sciences, State
University of Londrina, Londrina, PR, Brazil
2
Department of Physiological Sciences, Biomedical Sciences
Institute, Federal University of Alfenas, Alfenas, MG, Brazil
3
Department of Physiology, School of Medicine of Ribeirao
Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil
ABSTRACT
The therapeutic and prophylactic use of glucocorticoids is widespread due to their
powerful immunomodulatory and antiproliferative activities. All cellular responses to
glucocorticoids are attributed to their binding to the intracellular corticosteroid receptors,
glucocorticoid or mineralocorticoid receptors. The ligand-bound complex then
translocates to the nucleus, forming homo or heterodimers that bind to responsive
elements in the promoter region of target genes or directly interact with transcription
factors as monomers to modulate the transcription of responsive genes. More recently, it
has been suggested that nontranscriptional actions may account for the very rapid effects
observed following acute glucocorticoid treatment. Although the involvement of the
classic glucocorticoid receptors in these responses can be not ruled out, most of these
nongenomic effects seem to be mediated by the glucocorticoid binding to novel G-
coupled protein receptors and the consequent production and release of lipid-derived
mediators, the endocannabinoids (ECBs). Acting at central level, ECBs were shown to
mediate the negative feedback on the hypothalamic-pituitary-adrenal axis through the

Corresponding author: Ernane Torres Uchoa. Department of Physiological Sciences, State University of Londrina,
Londrina, PR, Brazil. E-mail: ernane_uchoa@yahoo.com.br.
208 E. Torres Uchoa, S. G. Ruginsk, R. C. Rorato et al.
interaction with the type 1 cannabinoid receptor (CB1R). As a consequence, a growing

interest has been focused during the past few years on the participation of ECBs on
glucocorticoid-related actions, such as in stress-related responses, in the central control of
food consumption and satiety, as well as in the peripheral control of adiposity, a predictor
of several chronic metabolic disorders. In the present chapter, we will discuss the main
experimental findings in this field, giving the readers the opportunity to improve their
knowledge on the increasing and novel data that have been produced over the recent
years on this clinically relevant issue, including topics as the novel aspects of the
negative feedback exerted by glucocorticoids at hypothalamic and pituitary levels, ECBs
acting as intermediates on central glucocorticoids actions, genetic mouse models for the
study of glucocorticoids in the homeostatic and stress-related responses, glucocorticoids
and stress-related responses in the interface of energy balance and immune systems, as
well as the control of energy homeostasis in the primary adrenal insufficiency.
Keywords: glucocorticoids, endocannabinoids, stress, energy homeostasis, microRNA
INTRODUCTION
The internal environment homeostasis of living organisms is constantly disrupted by
external or internal challenges [1]. Coordinated activation of multiple neuroendocrine
responses, including the activation of the hypothalamic pituitary adrenal (HPA) axis, is
required for the adaptation to these challenges [2]. The HPA axis activity is regulated by
sensors conveying signals to parvocellular neurons of the paraventricular nucleus of the
hypothalamus (PVN) [3], which release corticotrophin releasing hormone (CRH) and
vasopressin (AVP) at the median eminence into the hypothalamo-hypophysial portal blood
vessel system. Adrenocorticotropic hormone (ACTH) is then released from the anterior
pituitary into the systemic circulation [4, 5], and acts at the adrenal cortex to stimulate
glucocorticoid synthesis and release. The secretion of glucocorticoids (cortisol in humans,
corticosterone in rats and mice) is episodic, following circadian (daily) and ultradian (hourly)
rhythms, and shows marked but transient increases after exposure to stressors.
The action of glucocorticoids involves binding to intracellular receptors belonging to the
nuclear receptor family, glucocorticoid receptors type 1 or high affinity mineralocorticoids
receptors (MRs), and type 2 or low affinity glucocorticoids receptors (GRs) [6].
Following binding to the steroid, the ligand-bound complex then translocates to the
nucleus, originating homo or heterodimers that bind to responsive elements (GREs) in the
promoter region of target genes or interact with transcription factors as monomers to
modulate the transcription of responsive genes [7]. However, some biological actions of
glucocorticoids are too fast to be mediated by genomic actions, which require protein
synthesis, and they are believed to depend on membrane receptors [8].
The activation of the HPA axis is essential for stress adaptation, since glucocorticoids
control energy supply through the stimulation of some metabolic pathways as
gluconeogenesis and proteolysis.
In addition, glucocorticoids modulate the immune system, as well as the synthesis and
action of a number of hormones. Given the wide ranging effects of glucocorticoids, either
glucocorticoid excess or deficiency will produce deep effects on several functions of the
organism, including the regulation of the HPA axis.
New Insights of Glucocorticoids Actions on the Homeostatic Control … 209
In this context, an important mechanism for maintaining episodic HPA axis activation
and for limiting HPA axis activity is the negative feedback by glucocorticoids, resulting in
decreased expression and secretion of hypothalamic CRH and pituitary ACTH.
This chapter will cover new aspects about the regulation of the HPA axis and functions of
glucocorticoids, mainly the critical role of stress-induced glucocorticoid secretion,
highlighting the consequences of extreme concentrations of glucocorticoids on energy
balance, as well as showing the use of genetic mouse models for the study of glucocorticoids
effects in this field.
NOVEL ASPECTS OF THE NEGATIVE FEEDBACK

EXERTED BY GLUCOCORTICOIDS: ENDOCANNABINOIDS AS
INTERMEDIATES ON CENTRAL GLUCOCORTICOIDS ACTIONS
Stress is defined as any disruption or perceived threat to homeostasis caused by internal
or external environmental changes. The more immediate response to stress is characterized by
the activation of neural autonomic pathways, which are originated in hypothalamic
preautonomic centers and converge to motor and endocrine outputs. The second phase of
stress response is mediated by the activation of the HPA axis, which determines an increase in
the circulating levels of glucocorticoids and, consequently, an increase in the responses
triggered by these hormones in multiple target organs and systems.
Together, these coordinated actions mobilize bodily resources to cope with emergent
situations and, ultimately, eliminate the stressor. Stress-related sensory information is
conveyed to a population of neurosecretory neurons located in the PVN [3], activating the
HPA axis as described above. As further discussed in the following sections, glucocorticoids
trigger important metabolic effector responses, which primarily improve survival capacity in
the face of real or perceived challenges. Despite their major role in the control of stress-
related responses, high glucocorticoid levels can induce deleterious changes in metabolic
function. Therefore, neuroendocrine systems, including the HPA axis, share a very important
regulatory mechanism, called negative feedback, through which peripheral hormones are able
to control their own release by inhibiting the upstream components of their respective axes.
Studies performed on lesioned and transgenic animals indicate that, besides the hypothalamus
and pituitary gland, higher limbic structures are also involved in the glucocorticoid-mediated
negative feedback, particularly in response to psychogenic stressors [9, 10]. In the medial
prefrontal cortex (mPFC) and hippocampus (Hip), where GRs are densely expressed,
glucocorticoids primarily stimulate excitatory projections to inhibitory (GABAergic) relays in
the bed nucleus of the stria terminalis (BNST), resulting in the indirect inhibition of
hypothalamic CRH neurons [11]. In the hypothalamus and pituitary, however, glucocorticoids
provide the major inhibitory signal to reduce biosynthetic and secretory activities of the HPA
axis [12, 13]. In this context, it has been demonstrated that glucocorticoids have minor effects
on the direct inhibition of CRH transcription compared with the marked transcriptional
repressor activity onto AVP parvocellular neurones and pituitary proopiomelanocortin
(POMC, the precursor molecule of ACTH) cells, strongly suggesting that the major
mechanism by which glucocorticoids repress CRH transcription is indirect by modulating
neural pathways controlling CRH neuronal function [14, 15, 16].
In addition, differences between GRs and MRs activation have also been described, as
shown by the involvement of MRs in the feedback of glucocorticoids during the nadir phase
of the circadian rhythm, while GRs are occupied by increasing levels of glucocorticoids
during the reactive feedback and stressful events [16].
Interestingly, GRs have been also identified in association with neuronal membranes
[17], a mechanism which is aparently shared by other steroid receptors [18]. Therefore, as we
shall discuss in the following paragraphs, glucocorticoid signaling has several nuances that
add both redundancy and complexity to the HPA axis regulatory process. Indeed, the time
frame for glucocorticoid actions appeared as the first indicative that the negative feedback
would be the consequence of diverse mechanisms acting in parallel. In this regard, Evanson
and coworkers [19] have demonstrated that restraint-induced corticosterone secretion in rats
is inhibited by intra-PVN dexamethasone injections within 15 minutes. It has been assumed
that such very rapid actions, referred as nongenomic, would be incompatible with the
activation of the ―classical‖ pathways mediated by intracellular receptors, which demand gene
transcription and protein synthesis. The same authors also demonstrated that the conjugation
of dexamethasone to bovine serum albumin did not prevent this synthetic glucocorticoid of
inhibiting restraint-induced ACTH release, suggesting that dexamethasone-mediated feedback
response was not dependent upon binding to intracellular receptors.
Endocannabinoids (ECBs) are lipid-derived molecules that participate in a series of
physiological and pathological processes in the brain and peripheral tissues. The two most
studied endogenous compounds belonging to this family are anandamide (AEA) and 2-
araquidonoilglicerol (2-AG). They integrate a complete signaling system, also characterized
by the presence of specific receptors, named type 1 (CB1R) and type 2 (CB2R) cannabinoid
receptors, as well as by specific enzymatic apparatus for ECB production and metabolization.
ECBs have been recently demonstrated to be produced in a genomic-independent manner as
part of glucocorticoid signaling within the central nervous system. According to these
findings, ECBs would be produced by the cleavage of membrane-associated phospholipid
head groups under glucocorticoid stimulation. Then, ECBs would act as retrograde
messengers at presynaptic CB1Rs to modulate neurotransmitter release, consequently
affecting postsynaptic excitability [20]. The main known effects of CB1R activation by ECBs
are 1) inhibition of the adenylyl-cyclase-mediated pathway, 2) activation of potassium
channels and 3) inhibition of voltage-gated calcium channels [21].
CB1R and CB2R are G-protein coupled receptors that exhibit very distinct profiles of
expression: CB1Rs are found mostly in the central nervous system in neuronal and non-
neuronal populations, whereas CB2Rs are predominantly expressed by peripheral immune-
related organs [22]. The messenger RNA (mRNA) for CB1R is expressed in the
hypothalamus and in the external layer of the median eminence of rodents [23, 24], as well as
in both the anterior and intermediate lobes of the human pituitary gland [25]. Combined in
situ hybridization and immunohistochemical studies consistently demonstrated that CB1Rs
are upregulated in Cushing-associated human pituitary adenomas [25]. This study also
demonstrated that the contents of both AEA and 2-AG are increased in tumoral pituitaries,
indicating that locally produced ECBs may act at CB1Rs to directly modulate neuroendocrine
output. The pattern of CB1R expression in the hypothalamus has also been reported in the
literature [24]. According to these authors, the expression of CB1Rs occurs in neuronal
terminals and is considered only moderate when compared to other brain structures, such as
the hippocampal formation and olfactory bulb.
Within the PVN, CB1Rs are apparently expressed by both glutamatergic and GABAergic
terminals, so that CB1R activation decreases glutamate whereas increase GABA release onto
parvocellular neurons [26]. Studies employing CB1R knockout mice indicate that a disrupted
CB1R function increases the circadian HPA axis activity peak [27], suggesting that, under
steady-state conditions, the activation of the HPA is constrained by an ECB tone. In fact,
Atkinson and colleagues [28] have reported that CB1R-induced inhibitory tonus on HPA axis
activity is characterized by decreased peak amplitudes in pulsatile ACTH and corticosterone
release in rodents, particularly during the nadir of the diurnal cycle. However, there is no
evidence of an ECB tone controlling HPA axis activity within the PVN, since the local CB1R
antagonism does not potentiate the excitatory drive to CRH neurons [20] and local
administration of a CB1R receptor antagonist does not increase basal HPA axis activity [19].
These data suggest, therefore, that the tonic control upon CRH neurons originates outside the
hypothalamus.
Indeed, CB1Rs are also expressed by the Hip, mPFC and basolateral amygdaloid nucleus
(BLA) [23], where they seem to play site-specific and divergent roles on HPA axis regulation
during basal, stress and recovery conditions. In this regard, it has been recently proposed that
AEA actually represents the tonic signaling molecule of the ECB system, constraining under
basal conditions the excitability of BLA neurons through a CB1R-mediated decrease in
glutamatergic inputs to these cells.
In response to stress, however, this inhibitory AEA signal would be decreased, thus
enabling a greater glutamate release and a consequent increase the firing activity of BLA
neurons [29]. Accordingly, exposure to stress causes a reduction in amygdala contents of
AEA, possibly through a rapid induction of fatty acid amide hydrolase (FAAH)-mediated
AEA hydrolysis, an effect that inversely correlates with the magnitude of HPA axis activation
[30]. Interestingly, treatment of BLA slices obtained from previously stressed animals to
corticosterone causes the suppression of afferent glutamatergic inputs [31], suggesting that
glucocorticoid-induced effects can switch from excitatory to inhibitory depending on the
recent activity of the HPA axis, a mechanism possibly implicated in the termination of stress
response. However, the BLA does not project directly to the PVN, which also supports the
hypothesis that the HPA axis may be regulated through a multisynaptic pathway, including
other limbic structures and hypothalamic nuclei [32].
In the mPFC and Hip, glucocorticoids facilitate neuronal activity by respectively
decreasing the inhibitory [33] or increasing the excitatory drive [34] to inhibitory relays of the
BNST [35], finally culminating with the termination of stress responses within the PVN.
Differentially from the BLA, which apparently integrates a fast nongenomic pathway for
glucocorticoid action, the mPFC and Hip seem to be implicated in the delayed inhibition of
the HPA axis. Accordingly, the glucocorticoid-induced increase in 2-AG content within
mPFC is blocked by a GR antagonist and local administration of a CB1R antagonist prolongs
corticosterone secretion after acute stress [33]. Therefore, current evidence indicates that,
unlike initially proposed by the literature, the ECB system apparently contributes to both
short- and long-term mechanisms underlying glucocorticoid-mediated feedback inhibition of
the HPA axis. Based on these findings, Hill and Tasker [29] have proposed a yin-yang model
to describe how fluctuations in AEA and 2-AG levels within the hypothalamus, mPFC and
Hip affect the HPA axis activity throughout the stress response. According to these authors,
AEA inhibitory tone on HPA axis basal activity rapidly declines within the BLA after stress,
removing this inhibition over glucocorticoid secretion.
Glucocorticoids, in turn, increase 2-AG production, which apparently suppresses HPA

axis by exerting rapid (nongenomic) actions in the hypothalamus and delayed (genomic)
effects in the mPFC and possibly also in the Hip.
Basal HPA axis function would be then achieved by a parallel action of glucocorticoids
on BLA to restore ECB inhibitory tone (Figure 1).
Basal conditions Early phase of stress response Termination of stress response
Fast Delayed Delayed

PFC HIP
BLA BLA
BNST
BLA
Anandamide
2- Arachidonoylglycerol
Type 1 cannabinoid receptor PVN PVN
Glutamate CRH/AVP CRH/AVP
PVN GABA neurons neurons
CRH/AVP Glucocorticoid
neurons
Decreased Increased Inhibition Inhibition

activation activation
Figure 1. Left panel: A growing body of evidence suggests that, under basal conditions, neurons of the
basolateral amygdaloid nucleus (BLA) are under an inhibitory tone provided by anandamide (AEA),
which acts at presynaptic type 1 cannabinoid receptors (CB1Rs) to decrease glutamate release and,
therefore, BLA activation. Through multisynaptic relays, these inputs reach the paraventricular nucleus
of the hypothalamus (PVN), directly affecting the activity of parvocellular corticotrophin-releasing
hormone (CRH) and vasopressin (AVP) positive neurons. Under steady-state conditions, AEA tonus
would constrain BLA activity, thus resulting in low circulating levels of glucocorticoids. During the
early phase of stress response, however, local mechanisms within the BLA (such as increased AEA
metabolism) would be responsible for an intended disruption of this endocannabinoid tone, resulting in
an increased outflow to the parvocellular PVN and, consequently, an increased activity of the
hypothalamic-pituitary-adrenal (HPA) axis. Right panel: It has been demonstrated that circulating
glucocorticoids exert their negative feedback not only at hypothalamic (PVN) and pituitary levels, but
also at other target brain structures, such as the BLA, prefrontal cortex (PFC) and hippocampus (HIP).
Both the PVN and BLA are predominantly implicated in the fast (nongenomic) feedback mechanisms,
whereas the PFC and HIP may mediate most of the genomic actions of glucocorticoids on HPA axis
activity. Within the BLA and PVN, glucocorticoids trigger the production of 2- Arachidonoylglycerol
(2-AG), which acts at presynaptic CB1Rs to suppress excitatory (glutamatergic) and increase inhibitory
(GABAergic) inputs to CRH and AVP cells. In the PFC and HIP, glucocorticoid-induced effects would
be mostly mediated by endocannabinoid-independent mechanisms. The net effect would be an
increased outflow to GABAergic relay neurons of the bed nucleus of stria terminalis (BNST), resulting
in an increased inhibitory tonus onto PVN parvocelular neurons. The final result following the parallel
recruitment of fast and delayed glucocorticoid-mediated mechanisms would be a consistent decrease in
the activity of the HPA axis and the restoration of basal conditions, characterized by low plasma levels
of glucocorticoids and 2-AG concentrations in the PVN, and high intra-BLA AEA contents.
CLINICAL IMPLICATIONS OF
GLUCOCORTICOID NONGENOMIC ACTIONS
Several results obtained from both human and murine preparations show that
nongenomic actions underlie other important glucocorticoid-induced effects with potential
therapeutic implications. Accordingly, several clinically used glucocorticoids have already
been tested in vitro for their ability to produce genomic and nongenomic effects [36].
Apparently, nongenomic actions occur acutely after glucocorticoid treatment, which
supports the clinical use of drugs with high nongenomic potency when a rapid onset of
response is desired. However, exogenous glucocorticoid therapy rapidly downregulates in a
dose-related manner the number of [3H]dexamethasone-binding sites in humans, suggesting
that the effects of acute or prolonged therapeutic exposure to glucocorticoids should be
carefully evaluated [37].
Within this context, it has been demonstrated that glucocorticoid-induced nongenomic
actions account for the potentiation of vasoactive response to catecholamines, an effect that
has been explored for the management of cardiovascular outcomes during septic shock [38].
Zheng and colleagues [39] also demonstrated that dexamethasone produces biphasic
translation-independent effects on glucose metabolism, characterized by decreased
glycogenesis in cultured hepatocytes exposed to high doses and short periods of treatment.
Studies performed in experimental animals also showed that corticosterone administration
significantly delays exhaustion induced by forced swimming, an effect that was not blocked
by the administration of the GR antagonist RU486, indicating a nongenomic-mediated
increase in muscular tolerance during stress [40].
Furthermore, an impairment of T-cell-receptor signaling, characterized by the dissolution
of membrane-associated GR-multiprotein complexes, was shown to underlie the short-term
immunosuppressive effects produced by dexamethasone in cultured immune cells [41].
GLUCOCORTICOIDS ON THE
CONTROL OF ENERGY HOMEOSTASIS
Food intake is a fundamental behavior that is homeostatically regulated by several
factors, such as the adiposity signals leptin and insulin, and satiety signals, which includes
mechanical and chemical stimulation of stomach and small intestine, as well as hormones
released during a meal, as the cholecystokinin [42]. Leptin and insulin are classical adiposity
factors that modulate the long-term control of energy homeostasis and act primarily in
hypothalamic neurons expressing orexigenic or anorexigenic neuropeptides [42].
POMC and cocaine and amphetamine-regulated transcript (CART) in the ARC, and CRH
and oxytocin (OT) in the PVN are the main hypothalamic mediators involved in the inhibition
of food intake [42, 43]. On the other hand, neuropeptide Y (NPY) and agouti related protein
(AgRP) in the arcuate nucleus of the hypothalamus (ARC), orexins and melanin-
concentrating hormone in the lateral hypothalamic area comprise important hypothalamic
orexigenic mediators [43, 44, 45]. On the other hand, satiety signals are involved in the short-
term control of food intake and have their actions mediated by brainstem areas, the nucleus of
the solitary tract (NTS) being the most important of them, controlling the size of a meal [46].
Excitingly, it is well established that the brainstem has reciprocal connections with the
hypothalamic nuclei involved in the control of food intake [47, 48], indicating that adiposity
signals and satiety signals interact with each other [49, 50].
Glucocorticoids comprise another set of peripheral factors with crucial roles in the
regulation of energy balance [51], with their actions mediated by GRs and MRs. It is well
established that the activity of the HPA axis has a daily rhythmicity, with a peak level of
glucocorticoids preceding or at time of the onset of activity of the animal, declining its
activity over the remaining of the 24 hours of the period [52]. In addition, physiologically,
feeding has been demonstrated be one of the major synchronizers of this rhythmicity of the
HPA axis activity [52], the amount of food ingested being related with the secretion of
glucocorticoids [53]. Reciprocally, though feeding behavior to synchronize the rhythmicity
HPA activity, glucocorticoids are known to regulate food intake, being glucocorticoid
treatment in humans associated with increased food intake and body weight [54].
In fact, two human pathologies are characterized by extreme concentrations of plasma
glucocorticoids: (1) Cushing‘s syndrome results from prolonged exposure to glucocorticoids,
and it is characterized, among other features, by HPA rhythmicity abnormalities, insulin
resistance and hyperglycemia secondary to hypercortisolism. The most common cause of
Cushing‘s syndrome is the administration of pharmacologic doses of oral, parenteral or rarely,
topical glucocorticoids. Endogenous glucocorticoid excess may arise from ACTH–secreting
pituitary tumors, ectopic (nonpituitary) ACTH production, or adrenal tumors.
Hypercortisolemia is associated with increased glucose production, decreased glucose
transport and utilization, decreased protein synthesis, increased protein degradation in muscle
and body weight gain [55, 56]. (2) Addison‘s disease or primary adrenal insufficiency, first
described by Addison in 1855, is characterized by an inability of the adrenal cortex to
synthesize and secrete glucocorticoids and mineralocorticoids. The typical clinical findings of
chronic primary adrenal insufficiency include a prolonged history of malaise, fatigue,
anorexia, weight loss, joint and back pain, darkening of the skin, hyponatremia,
hypoglycemia and hyperkalemia [57]. It has been shown that glucocorticoids effects on food
intake depend on the their levels in the circulation [58], so that low doses of corticosterone in
adrenalectomized rats (resulting in corticosterone plasma levels ranging from 1 to 2 µg/dL)
were demonstrated to activate MR and to have a stimulatory effect on fat intake, body weight
gain and fat depots, which occur at the late phase of the feeding period [59, 60, 61].
Conversely, GRs are activated by higher doses of circulating corticosterone (2-10 µg/dL) just
before or during the first hours of the active feeding period, which induce carbohydrate
ingestion and metabolism [59, 60, 61, 62, 63, 64]. In addition, extremely high corticosterone
plasma concentrations (> 10 µg/dL), as in consequence of stress or food restriction, stimulate
fat and protein catabolism, mainly muscle protein catabolism, and consequently body weight
loss, increasing the availability of gluconeogenesis substrates and enhancing glucose plasma
concentration as energy source [61, 65].
Different studies have consistently demonstrated that glucocorticoids actions in the
central nervous system affect feeding behavior. Dexamethasone injection into the lateral
ventricle not only stimulated food intake but also enhanced body weight gain in rats,
accompanied by hyperleptnemia and hyperinsulinemia [66, 67].
The central effects of glucocorticoids on the regulation of food intake seem to be
mediated by the interaction of glucocorticoids with hypothalamic neuropeptides involved in
the control of energy homeostasis, as demonstrated by the presence of glucocorticoid

receptors in neurons expressing these neuropeptides [68, 69].
The study of this interaction showed that central glucocorticoid treatment increases
hypothalamic NPY levels and decreases those of CRH [67]. In addition, circulating
glucocorticoids were shown to be required for feeding-induced decrease in the expression of
orexigenic neuropeptides in the ARC as well as for the increase in the expression of the
anorexigenic neuropeptide POMC in this nucleus, being these effects consequence of 1) a
direct modulation of glucocorticoids onto ARC neurons, and 2) an indirect consequence of
changes in leptin and insulin secretion induced by food intake [70].
THE CONTROL OF FOOD INTAKE IN THE EXPERIMENTAL

MODEL OF PRIMARY ADRENAL INSUFFICIENCY
The removal of endogenous glucocorticoids in rodents, induced by bilateral
adrenalectomy (ADX), is a well-established experimental model used for the investigation of
some manifestations observed in human primary adrenal insufficiency, such as hypophagia
and body weight loss. ADX has been shown to reduce food intake and body weight gain, and
these effects are reversed by glucocorticoid replacement in rats [71, 72, 73, 74].
The hypophagic effect in response to ADX is associated with increased expression of the
anorexigenic neuropeptides CRH and OT in the PVN, as well as reduction on the expression
of the orexigenic neuropeptides NPY and AgRP in the ARC, indicating that changes in the
expression of these hypothalamic neuropeptides could account for the reduction on appetite
following ADX [70, 73, 74]. Concerning the anorexigenic neuropeptides of the ARC, ADX
was shown to reduce POMC and CART mRNA expression in the ARC, suggesting that the
anorexigenic effects in response to the removal of adrenal glands are dissociated from
elevations of these neuropeptides in the ARC [70, 75, 76].
It is well established that glucocorticoids action on food intake involve their stimulatory
drive to eat, thus ADX-induced hypophagia involves, at least in part, a reduction on this drive
to eat. However, recent evidences have highlighted the role of glucocorticoids on the short-
term control of food intake, indicating that the hypophagic effect in response to the absence of
glucocorticoids is associated with increased activation of satiety-related responses through
brainstem and hypothalamic pathways [72, 73].
Accordingly, NTS neuronal activation was further increased in response to meal in ADX
rats, indicating that this nucleus is involved the enhanced responses associated with satiety
[72]. Interestingly, not only NTS but also PVN was shown to be implicated in these effects,
since the activation of CRH and OT hypothalamic neurons was also increased subsequently to
ADX in response to meal, indicating that, besides the brainstem, the hypothalamus also
participates in the satiety-related responses. In addition, it is known that CRH and OT neurons
project from the PVN to the NTS, being OT axonal projections enhanced after ADX [77].
Interestingly, CRH and OT were shown to mediate the enhanced satiety-related responses
after ADX, as shown by the reversion of the hypophagia and feeding-induced increased in
NTS activation by the central administration of CRH-2 receptor and OT receptor antagonists
[73, 74, 77].
Thus, CRH and OT neurons of the PVN can be pointed as key mediators of the enhanced
satiety-related responses in the NTS, contributing to hypophagia in primary adrenal
insufficiency (Figure 2).
THE CONTROL OF ENERGY HOMEOSTASIS

MEDIATED BY STRESS RESPONSES
As described above, glucocorticoids have well-established roles in the regulation of
feeding behavior and energy homeostasis, stimulating the drive to eat as well as reducing the
satiety-related responses. Therefore, environmental and intrinsic challenges that activate the
HPA axis and, consequently, alter the secretion of glucocorticoids, may also disrupt energy
homeostasis. The activation of the HPA axis is the key endocrine effect responsible for
mediating the response to stress in the body. Stress also activates sympathomedullary
responses, but this issue will not be discussed in this chapter. Thus, stress consists another
experimental paradigm to study the role of glucocorticoids on the regulation of energy
homeostasis.
The experimental data concerning the impact of stress on food intake, body weight and
obesity are controversial, since some data point stress as predictor of obesity, increasing food
intake and body weight [78, 79], while others report stress as inductor of weight loss and
anorexia [80, 81]. These divergences could be justified by differences in experimental stress
protocols, which employ different kinds of stress, intensities and duration, as well as different
species and strains of animals.
It has been assumed that stress-induced suppression of food intake is mediated by CRH
and urocortins [82, 83, 84]. CRH- and analogues-induced anorexia is likely to involve a
number of brain regions including the PVN, perifornical and ventromedial regions of the
hypothalamus, lateral septum, parabrachial nucleus, the dorsal portion of the anterior BNST
and NTS [74, 84, 85].
Figure 2. Schematic illustration summarizing the hypophagia in response to adrenalectomy (ADX)

through stimulatory effect on the anorexigenic neuropeptides corticotrophin releasing hormone (CRH)
and oxytocin (OT), as well as an inhibitory effect on the orexigenic neuropeptides neuropeptide Y
(NPY) and agouti-related protein (AgRP). CRH and OT neurons, in turn, project to the nucleus of
solitary tract (NTS), where they act through specific receptors to potentiate the activation of NTS
neurons in response to satiety-related signals, thus reducing meal size in primary adrenal insufficiency.
Although stress-induced CRH release may acutely reduce appetite, glucocorticoids are
released in hours to days following a stressful challenge and they stimulate feeding, as
previously described. The reason for this seems to be that, in acute stressful conditions, these
responses are likely an adaptive phenomenon, whereas the increase on appetite as a long-term
response would be necessary to replace the lost energy and to enable stocking of energy in
advance to a possible upcoming stressing challenge [86].
On the other hand, circulating glucocorticoids may be chronically elevated if the stressing
condition persists or if there are several consecutive stressors, accounting for a chronic
stimulation of appetite, increased food intake and therefore obesity [51, 87, 88, 89]. Indeed, it
has been suggested that stress tends to stimulate appetite specifically for calorically dense
nutrients, with insulin having a key role in this regard [90, 91, 92].
Thus, it is likely that stress contribute to the propensity to obesity via glucocorticoids-
induced enhancement of appetite, especially high-energy food, in addition to the effects of
glucocorticoids in the regulation of lipid homeostasis, with actions on fat mobilization from
adipose tissue and fat deposition and storage.
It is known that short-term infusion of glucocorticoids in vivo stimulates the release of
non-esterified fatty acids from adipocytes [93], through the activation of hormone-sensitive
lipase (HSL) [94], a key enzyme responsible for enhancing fatty acid mobilization, indicating
that glucocorticoids acutely induces lipolysis. This increase in circulating free fatty acids may
limit glucose utilization and induce insulin resistance [95]. On the other hand, glucocorticoids
have also been shown to enhance adipose lipoprotein lipase (LPL) activity, which is known to
promote fat storage [96, 97]. Thus, it seems that glucocorticoids may induce lipid metabolism
through its effects on both the turnover and uptake of fatty acids in adipose tissue.
Accordingly, LPL activity is higher in visceral adipose tissue than in other adipose depots
[98], and glucocorticoids can contribute to a redistribution of fat such that it is accumulated
preferentially in visceral depots. Hence, excess of glucocorticoids arisen both from
endogenous or exogenous causes is associated with visceral fat accumulation [98-103].
Additionally, visceral fat depots are pointed to be more susceptible to glucocorticoids
actions [104], and it has been suggested that the increase in the activity of lipogenic enzymes
following glucocorticoids excess could be ascribed to the glucocorticoid-induced
hyperglycemia [105] and due to the high quantity and affinity of glucocorticoid receptors in
visceral fat depot [106]. In fact, the presence of GRs in visceral adipose tissue in greater
density than in other adipose depots [107] seems to play a key role on the relative increased
effects of glucocorticoids specifically in the visceral adipose tissue [97]. In addition, a
candidate for the specific action of glucocorticoids in this adipose depot is type 1 11-beta-
hydroxysteroid dehydrogenase (11b-HSD1), the enzyme that generates active glucocorticoid
from inactive metabolites within tissues, as shown by higher 11b-HSD1 activity in visceral
versus subcutaneous adipose tissue, with preferential visceral fat accumulation and associated
metabolic alterations [108]. In addition, the actions of 11b-HSD1 in visceral adiposity have
been evidenced by the visceral fat accumulation in mice over-expressing 11b-HSD1 gene
[109], suggesting that elevated 11b-HSD1 might be one of the causes rather than one of the
consequences of obesity, since the inhibition of this enzyme could improve metabolic
parameters and reduce weight [110].
In this context, the effects of glucocorticoids on the development of obesity have been
demonstrated in different studies, which showed that animals treated with high levels of
glucocorticoids become markedly obese, with several other physiological hallmarks of the
metabolic syndrome, such as increased plasma leptin and insulin, increased plasma
triglycerides, and impaired glucose tolerance [111, 112].
Additionally, circulating glucocorticoids are increased in different models of obesity and
ADX was shown to be effective in diminishing hyperphagia and obesity in different
experimental models [113-115]. Reciprocally, obese animals seem to have a higher sensitivity
for the metabolic effects of glucocorticoids [116, 117].
In parallel, it has been described that both stress and glucocorticoids increase the content
of hypothalamic ECBs, which have been pointed as mediators of several glucocorticoid-
induced effects, as previously described [118-120]. Accordingly, CB1R blockade efficently
reversed overweight and metabolic alterations in adult male mice exposed to early stress [78],
suggesting that the interaction glucocorticoids-ECB system in the hypothalamus is crucial for
the development of stress-induced obesity.
GLUCOCORTICOIDS IN THE INTERFACE

OF IMMUNE CHALLENGES AND FOOD INTAKE
It is well known that under an inflammatory or infectious challenge several

neuroendocrine, behavioral and immune changes are recruited to maintain homeostasis [121,
122]. This acute-phase response, characterized by HPA axis activation, fever and appetite
suppression is mediated by the increased synthesis and secretion of pro-inflammatory
cytokines, such as interleukin-1 (IL-1), IL-6 and tumor necrosis factor–alpha (TNF-α).
Examples of such challenges include infection with bacteria, viruses, fungi, parasites, tissue
damage and destruction [123].
Indeed, administration of lipopolysaccharide (LPS), a constituent of the outer part of a
Gram-negative bacterial cell membrane that are released upon bacterial lysis, which is
released upon bacterial lysis, mimics many of these responses observed during an infectious
challenge, but without causing infection in the host, and it has been widely used to investigate
the mechanisms underlying acute-phase responses [124-126].
Besides essential for preserving homeostasis, an unrestrained-inflammatory state can also
cause damage to the organism [127]. In this context, increased glucocorticoid plasma levels
observed during HPA axis activation seem to be essential to counteract an excessive immune
activation. Briefly, as already described, after exposure to an stressor, such as an immune
challenge, the HPA axis is activated, producing as the final result glucocorticoid release from
the adrenal gland [123, 128].
The classical glucocorticoid-induced immunomodulatory action is evident in ADX
animals, which exhibit increased mortality after LPS treatment or in response to other
inflammatory stimuli, but respond normally to an inflammatory challenge after glucocorticoid
replacement [129]. The anti-inflammatory glucocorticoid-induced actions are related to
peripheral and central suppression of pro-inflammatory cytokines synthesis, chemokines,
arachidonic acid metabolites, adhesion molecules and also by induced up-regulation of other
anti-inflammatory cytokines, such as IL-4 and IL-10, and cytokine receptors [130].
These glucocorticoids effects are mediated by interaction with GRs, which culminates
with the activation of NFκB and AP-1 [131, 132].
After glucocorticoid binding to the cytosolic GR, the activated complex is translocated to
the cell nucleus, where it interacts with the specific transcription factors AP-1 and NF-κB and
prevents the transcription of targeted genes, in a process called transrepression.
On the other hand, recent studies also demonstrate that glucocorticoids can increase
inflammatory response, particularly within the central nervous system. Indeed, it was reported
by de Pablos and colleagues [133] that a stronger and more distributed microglial reaction as
well as a higher increase in TNF-α mRNA occur in the cortex of LPS-injected animals
maintained under chronic variate stress. In the same study, RU486 treatment restored LPS-
induced responses to levels of non-stressed animals, confirming a glucocorticoid pro-
inflammatory actions. In addition, chronic unpredicted stress increases the effect of LPS on
NF-κB binding activity in the frontal cortex and hippocampus but not the hypothalamus
[134]. This response is blunted in the frontal cortex and hippocampus of RU486 pretreated
animals [134].
Interestingly, RU486 potentiates LPS-induced NF-κB binding activity in the
hypothalamus of stressed animals [134].
Therefore, it seems that the pro-inflammatory glucocorticoid effects occur in areas with
higher GR density, such as cortex and hippocampus, but the classic anti-inflammatory
glucocorticoid effects are observed in the hypothalamus, where GR density is comparatively
lower [134, 135]. Studies evaluating of the interface between inflammatory or infectious
responses and the regulation of energy homeostasis have demonstrated that under an acute
inflammatory or infectious challenge, the observed adaptive response is hypophagia [124,
125], which reduces nutrient availability for pathogenic microorganisms to grow and also for
safe energy from food-seeking behavior.
The inflammation-induced increased activity of CRH-expressing PVN neurons seems to
mediate this response [124, 125]. Accordingly, the use of CRH receptor antagonists partially
reverses the reduction in food intake induced by different stress paradigms [136, 137]. On the
other hand, a period of prolonged hypophagia can also have a negative effect on energy
storage, and glucocorticoids, by their anti-inflammatory effects, can indirectly restrain
hypophagia. Indeed, dexamethasone treatment attenuates the production of multiple pro-
inflammatory cytokines within the brain and liver, suggesting a potential preventive effect of
glucocorticoids on the LPS-induced hypophagia [138]. In addition, LPS-induced hypophagia
is amplified in ADX rats, as evidenced by the higher activation of hypothalamic CRHergic
cells and also by the recruitment of POMC neurons [124].
Within this context, it has also been reported that, besides the unrestrained inflammatory
response, disturbances that lead to a chronic inflammatory state, associated with chronically
elevated glucocorticoids plasma levels, can also be detrimental to the organism and can
induce a long-lasting stimulation of appetite, increasing food consumption and consequently
obesity [139, 140, 141]. Indeed, recent studies point that obesity is a chronic low-grade
inflammatory state [142, 143]. Consistent with the inflammatory backgroung, it was reported
that obese high-fat-diet fed animals have increased circulating pro-inflammatory cytokines as
well as they express high levels of TNF-α, IL-6 and IL-1β in the hypothalamus, liver and
white adipose tissue [144, 145].
Interestingly, a two to three-fold increase in plasma LPS concentrations has been reported
after a 4-week high-fat diet [146]. Accordingly, LPS is detectable in the circulation of both
healthy and obese individuals, but it may transiently rise following energy-rich meals,
possibly contributing to the development of the inflammatory process.
MICRO-RNA REGULATION AND GENETIC APPROACHES FOR

THE STUDY OF GLUCOCORTICOID ACTIONS ON HOMEOSTATIC
CONTROL AND STRESS-RELATED RESPONSES
Glucocorticoids play a fundamental role in the energy utilization and storage processes.
Abnormal glucocorticoid action contributes to the progression of metabolic syndrome, a
pathological condition characterized by chronic low grade inflammation, increased blood
pressure, leptin and insulin resistance and obesity. Glucocorticoids participate in the
development of metabolic disorders through the modulation of feeding as well as glucose and
lipid regulatory pathways; nonetheless, the precise mechanisms of these effects are still under
investigation and have been discussed in this chapter. As already mentioned, part of the
glucocorticoid effects is modulated by their cognate intracellular receptors, GRs, whose
superfamily is composed by different GR isoforms resulting from alternative RNA splicing.
Notably, all GR isoforms are further subjected to transcriptional, post-transcriptional, or post-
translational modifications, which in turn impact on GR function and glucocorticoids action
[147, 148].
Small non-coding RNA molecules (19 – 22 nucleotides), termed micro-RNAs (miRs),
function in RNA silencing and regulation of gene expression by mRNA degradation and/or
translational repression [149]. Recent studies revealed that miRs promote post-translational
modifications on GRs, regulating glucocorticoid-induced metabolic dysfunctions as well as
fat accumulation [150, 151]. As already mentioned, clinical cases of glucocorticoid excess
present enhanced fat mass and obesity through the accumulation of white adipocytes. In rats,
corticosterone decreases nonshivering thermogenesis and increases lipid storage in brown
adipose tissue [152]. According to Barclay and coworkers [153], dexamethasone treatment of
cultured human brown adipocytes stimulates the proliferation, differentiation and the
expression of uncoupling protein-1 (UCP1), a specific mitochondrial protein involved in the
regulation of thermogenesis and energy expenditure, in brown preadipocytes. Interestingly,
dexamethasone inhibited adrenergic stimulation of the functioning of brown adipocytes,
evidencing that glucocorticoids exert complex effects on development and function of human
brown adipose tissue (BAT).
Kong and coworkers [151] found a contrary effect of dexamethasone, which inhibited
brown adipose differentiation and energy expenditure in primary adipocytes. These authors
found that glucocorticoids transcriptionally upregulated miR-27b in human and mouse brown
adipocytes via a GR-mediated direct DNA binding mechanism, obstructing BAT
differentiation and reducing energy expenditure.
Antagonizing miR-27b function in vitro prevented dexamethasone-induced suppression
of the expression of BAT-specific genes. In vivo, a lentiviral system to antagonize miR-27b in
mouse demonstrated that anti-miR27b shields glucocorticoid-induced obesity and insulin
resistance and enhances oxygen consumption.
Yet, antagonizing miR-27b in dexamethasone-treated mice resulted in effective induction
of brown adipocytes within white adipocytes. The data strongly point out miR-27b as a
glucocorticoid target in negatively control browning of white adipose tissue, being a potential
molecule in preventing obesity.
Increased glucocorticoid levels are associated with hyperglycemic and fatty liver
phenotype, contributing to insulin resistance [154].
Investigating liver miRs participation on glucocorticoid-driven metabolic dysfunction,

researchers performed a large-scale miR expression profiling of livers from wild-type and
diabetic db/db mice [155], and posteriorly aligned the resultant data set with a second miR
screening from differential expression profiling between mice lacking GR specifically in
hepatocytes and controls [156]. Performing the cross-comparison, De Guia and coworkers
[150] identified 10 down-regulated miRs in response to GR deficiency. Particularly, the miR-
379/ 410 genomic cluster was found to be induced in the liver of db/db mice, as well as in
control mice under dexamethasone treatment, showing that liver miR-379/410 is a
downstream target of glucocorticoid signaling in metabolic dysfunction, reinforcing the
importance of miR-based therapeutic approaches against metabolic dysfunctions promoted by
glucocorticoids.
As already highlighted, the main biological functions of the glucocorticoids include,
among others, the stress responses as well as the suppression of inflammation. Several miRs
can modulate stress responses in different tissues. A study demonstrating the glucocorticoid
induction of a miR in the pituitary was performed by Nemoto and coworkers [157]. By
database search, the authors found 3 miRs with sequences predicted to bind to the CRH-1
receptor (CRH-R1) and demonstrated that the expression of miR-449a was significantly
higher in the anterior pituitary corticotrophs of restrained rats than in unrestrained controls.
Overexpression of miR-449a decreased CRH-R1 mRNA and CRH-R1 protein expression.
On the other hand, knockdown of miR-449a attenuated dexamethasone-induced
suppression of CRH-R1 mRNA and CRH-R1 protein expression in cultured pituitary cells.
Adrenalectomized rats presented an attenuated restraint-induced increase in miR-449a
expression in the pituitary. These results indicated that miR-449a plays a crucial role in
glucocorticoid-mediated stress response via down-regulation of CRH-R1 expression.
As previously discussed, glucocorticoids mobilization of the ECB system is essential for
many of the effects of glucocorticoids, such as negative feedback regulation of the HPA axis,
suppression of sexual behavior, and metabolic alterations.
These observations led some researchers to hypothesize that long-term exposure to
elevated glucocorticoid results in a hyperactive ECB system, which contributes to metabolic
syndrome and obesity [158]. Using a genetic mouse model to ablate the CB1R specifically in
liver (LCB1R−/−), the authors crossed mice homozygous for the CB1R-floxed allele
(CB1Rf/f) with mice expressing the bacterial Cre recombinase driven by the mouse albumin
promoter (triglyceride[Alb-cre]′21 Mgn). The study indicated that CB1R within the liver
drives the dyslipemic component of excess glucocorticoid exposure, whereas blockade of all
CB1R in the periphery attenuates all aspects of the metabolic dysregulation [158].
Opposing to the obesity process, another metabolic phenomenon regulated by
glucocorticoids is cachexia, a severe complication of chronic diseases such as cancer. A
hallmark of cachexia is a loss of skeletal muscle mass following a systemic inflammation.
Adrenalectomy or pharmacologic inhibition of GRs promotes muscle atrophy in response to
acute sepsis, starvation, metabolic acidosis and diabetes, dependently to some extent on
glucocorticoid signaling [159, 160]. To assess the role of glucocorticoid signaling in skeletal
muscle, Braun and colleagues [160], using the cre-loxP system, generated the mGRKO mice
(GR deletion exclusively in skeletal muscle cells), in order to limit the confounding effects of
global glucocorticoid blockade. Dexamethasone treatment produced equivalent increases in
food intake and decreases in body weight in both control and mGRKO mice. Interestingly,
dexamethasone-induced muscle atrophy was markedly attenuated in mGRKO mice.
Examining the ability of LPS to induce atrophy genes in the skeletal muscle in a
glucocorticoid-dependent manner, the authors found a blockade of the LPS-induced atrophic
effect in mGRKO mice, indicating a required glucocorticoid signaling for the acute induction
of catabolic genes in response to endotoxin.
CONCLUSION AND PERSPECTIVES

Important progress has been made during the last few years in understanding the
physiological actions of glucocorticoids and the mechanisms of glucocorticoid feedback
regulation of the HPA axis. Glucocorticoids have pleotropic actions regulating general
cellular metabolism and brain function. Growing evidence indicates that normal
glucocorticoids-dependent regulation depends upon the prevailing levels of these steroids,
their secretion pattern, as well as glucocorticoid interaction with neurotransmitters and
neuropeptides. Nevertheless, negative glucocorticoid feedback is essential for the fine control
of HPA axis activity and to avoid deleterious consequences of excessive CRH and
glucocorticoid production. As discussied earlier in this chapter, new evidence indicates that
glucocorticoid feedback inhibits HPA axis activity at a number of anatomical and molecular
targets, rapidly shutting-off hypothalamic and pituitary responses at the cell membrane level,
controlling the intensity and duration of stress responses at limbic sites, and inducing long-
term inhibition of the HPA axis by modulating transcription and mRNA stability at central
and pituitary levels. In spite of the recent advances in the field, a number of challenges still
remain. These include identification of peripheral metabolic signals and central sensors
impacting on the sensitivity of glucocorticoid feedback and HPA axis activity, as well as the
exact identity of the membrane-associated glucocorticoid receptor and its signaling
mechanisms. Understanding the functions of glucocorticoids and mechanisms of feedback
regulation of HPA axis activity may contribute to the development of new diagnostic and
therapeutic tools for stress-related disorders and alterations of glucocorticoid secretion.
In this context, different approaches to improve the benefit/risk ratio of glucocorticoids
also include the development of drugs that selectively target the activation of membrane-
associated GRs and its downstream nongenomic events, without evoking adverse effects,
primarily attributed to the activation of genomic pathways.
Therefore, the study of the nongenomic actions of glucocorticoids has introduced a novel
player in the complexity of the circuitries regulated by the HPA axis and the integrated
control of homeostasis. The ECB system appears as an important mediator of both central and
peripheral effects of glucocorticoids, constituting a possible target by which several aspects of
stress-mediated responses and energy homeostasis could be manipulated under diverse
physiological and pathological conditions. Finally, in addition to the clinical use of
glucocortcoids, a great number of risks associated with excessive glucocorticoid utilization
has been reported. Hence, comprehension of glucocorticoid side effects is vital to a well
succeeded therapy. In recent times, the crescent use of the genetic tools has become even
more valued. Site-specific GR deletion, in addition to discovering microRNAs regulation by
glucocorticoids in different tissues, raise new therapeutic targets in order to prevent metabolic
dysfunction, fat accumulation, suppression of inflammation and excessive stress response,
pointing out to new potential treatments for these pathological conditions.
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Chapter 10
ROLE OF ADRENAL HORMONES IN THE FETAL

PROGRAMMING OF HYPERTENSION
Sandhya Khurana1, Collin J. Byrne2, Stephanie Mercier3,

Jeremy Lamothe3, Chad R. Williamson2, Julie Grandbois1,2
and T. C. Tai1,2,3,4,*
S. K. and C. J. B. contributed equally to this work
1
Medical Sciences Division, Northern Ontario School of Medicine
2
Department of Biology
3
Department of Chemistry and Biochemistry
4
Biomolecular Sciences Program, Laurentian University,
Sudbury, ON, Canada
ABSTRACT
Hypertension afflicts approximately 40% of the adult population worldwide and is
the major contributing factor for cardiovascular disease, stroke and kidney disease.
Currently, the underlying cause of 90-95% of all cases of hypertension is not known.
Hypertension is a complex disease that is attributed to both genetic and environmental
influences. In the past decade, studies have emerged suggesting that early-life
environmental factors, which influence development of the fetus, may cause adult
diseases such as hypertension. Studies also demonstrate that glucocorticoids, known for
their role as stress hormones, are involved in fetal programming of adult hypertension.
Although the mechanisms by which glucocorticoids cause the development of adult
hypertension are not clear, literature strongly supports a dysfunction of the hypothalamus
–pituitary–adrenal axis (HPA) in prenatally programmed hypertension. The sympathetic
nervous system, in particular, the sympatho-adrenal (SA) system, is also critically
important in the regulation of the cardiovascular system and blood pressure control.
Sympathetic hyperactivity, which is involved in the pathogenesis of hypertension, may
also contribute to the mechanisms for fetal programming of hypertension. In conjunction
with fetal programming of HPA or SA dysfunction, inflammation also appears to be an
*
Corresponding author: 935 Ramsey Lake Road; Sudbury, ON P3E 2C6, Canada.Email: tc.tai@nosm.ca.
236 Sandhya Khurana, Collin J. Byrne, Stephanie Mercier et al.
important contributing factor for hypertension. Immune mediators have been shown to
influence adrenal activity directly and through the HPA and SA axes. This chapter will
explore the role of adrenal hormones in the fetal programming of hypertension with
relevance to the SA, HPA axes and the neuro-immune circuit.
Keywords: HPA, glucocorticoids, cytokine, cardiovascular, fetal programming,

catecholamines
INTRODUCTION - FETAL PROGRAMMING
The Barker Hypothesis, which established a relationship between low birth weight and
the risk of development of chronic illness in adult life, was the first foray to what is currently
recognized as ―fetal programming‖. The hypothesis proposes that adverse in utero conditions
create permanent adaptive responses including changes in physiology and metabolism,
ultimately leading to increased risk of disease in adulthood [1–4].
In 1989, Barker et al. conducted an epidemiological study on individuals born between
1911-1931 in Hertfordshire County, England where detailed birth and growth records from
birth up to 1 yr of age were systematically maintained for about 25,000 individuals. Barker
observed that the incidence of elevated systolic and diastolic blood pressure and increased
mortality from ischemic heart disease, in this cohort as adults was directly correlated to their
low birth weight. He followed with another epidemiological study in 1990 on individuals
born between 1935-1943 in Preston, Lancashire, and deduced that low birth weight was
inversely correlated with placental weight. Barker and colleagues concluded that malnutrition
and exposure to stress prior to birth has a permanent reprogramming effect in offspring [3, 5].
Barker‘s work is now widely accepted and is highly pertinent to present-day cardiovascular
health issues. In developing countries, babies and their mothers are frequently malnourished;
the same problem may be found in developed countries, where even though food is abundant,
many mothers consume nutrient-poor diets and bear malnourished children.
Since Barker‘s original work, many studies have been performed in an effort to elucidate
the biomolecular mechanisms underlying the prenatal origins of disease. Numerous
observational studies have correlated the influence of social stress and anxiety during
pregnancy with increased preterm birth rate and lower birth weights. These changes are
associated not only with cardiovascular pathologies, but also with a variety of mental health
challenges including schizophrenia, increased anxiety, irritability and developmental
disorders among others [6]. Thus, lower birth weight is an indicator not only of a
subsequently altered metabolic and cardiovascular physiology, but also of changes in the
neuroendocrine system and behavioral disorders.
In Barker‘s work, the implications for hypertension are clear: low birth weight is
correlated to a smaller nephron count in the kidneys, and hence a reduced glomerular
filtration capacity [7]. In order to maintain an adequate level of filtration, a greater volume of
blood is processed, leading to an increased rate of wear and earlier damage or death to the
nephrons. As a result of nephron damage, blood pressure increases, yet again increasing the
load on the remaining nephrons creating a cycle of nephron loss and ever increasing blood
pressure that must be carefully managed as an individual ages. With an understanding of
Barker‘s reasoning, it is not surprising that kidney failure in the adult is directly correlated to
Mechanistic Role of Adrenal Hormones in the Fetal Programming … 237
malnutrition during the developmental stage when nephrogenesis occurs in utero [8].
Hypertension is a complex disease that may involve multiple physiological systems separate
from the renal systems. While only 5-10% of all hypertension cases are classified as
secondary (or non-essential) hypertension, having a discernable underlying cause such as
pheochromocytomas or obstructive sleep apnea, 90-95% of cases are classified as primary (or
essential) hypertension, where the cause is unknown [9]. Recent studies have proposed that
altered regulation of the two stress axes, namely the hypothalamus–pituitary-adrenal (HPA)
and the sympatho-adrenal (SA) axis is the underlying mechanism responsible for changes in
cardiovascular physiology and functionality of the neuroendocrine system, and in the
programming of hypertension [10, 11].
This chapter summarizes the current state of knowledge with regard to the role of adrenal
hormones, in the context of the HPA and the SA axes, in the fetal programming of
hypertension.
ROLE OF HPA AND SA IN CARDIOVASCULAR REGULATION

In situations of stress, a number of physiological and behavioral changes are triggered in
the body that prime an individual to successfully cope with or confront an imminent threat;
this is commonly known as the ―fight-or-flight‖ response first described by Cannon (1871)
[12]. The stress response is an adaptive process which is highly regulated and includes
physiological changes such as increased respiratory rate and blood flow among others. This
stress response is mediated and regulated primarily by the HPA axis, which is composed of
the paraventricular nucleus (PVN) of the hypothalamus, the anterior pituitary gland and the
adrenal gland. In response to stress, corticotrophin-releasing hormone (CRH) and vasopressin
are produced in the hypothalamus; these activate pituitary corticotropes, leading to the release
of adrenocorticotrophic hormone (ACTH). ACTH binds to melanocortin type 2 receptors in
the adrenal cortex, inducing the production of steroidal hormones (including
mineralocorticoids and glucocorticoids) via activation of the cAMP pathway [13].
Glucocorticoids (GCs) exert numerous biological changes by signaling mediated via the
glucocorticoid receptor (GR), which is expressed on a vast number of cell types and tissues.
GCs stimulate the activity of phenylethanolamine N-methyltransferase (PNMT), the terminal
enzyme in the catecholamine biosynthesis pathway, thus increasing the production of
epinephrine (adrenaline; Epi) in adrenal chromaffin cells [14]. Epi itself increases blood
pressure through an increase in cardiac output (force and rate of ventricular contraction), as
well as by facilitating norepinephrine (noradrenaline; NE) release in a co-stimulatory role
[15–17]. Both Epi and NE bind β1 and β2-adrenoreceptors in the heart, modulating
contractile force by way of adenylate cyclase signaling [18].
Working complimentary to the HPA axis, the SA axis, a component of the sympathetic
nervous system, also plays a critical role in the regulation of blood pressure. In this case,
stress responses are initiated by transmission of signals from the rostral ventrolateral medulla,
the locus coeruleus, and the PVN to the preganglionic neurons of the intermediolateral cell
column located in the thoracolumbar spinal cord. The neural signaling finally terminates
when projections of preganglionic neurons from the splanchnic nerve interface with adrenal
medullary chromaffin cells [19].
Acetylcholine (ACh) is secreted by the sympathetic splanchnic nerve fibers, stimulating

the release of large amounts of catecholamines (mostly Epi) from chromaffin cells [20]. SA
activation also leads to increased NE release from sympathetic nerves [19]. The released
catecholamines bind to adrenergic receptors, which cause multiple cardiovascular adaptations
including α-adrenoceptor-mediated vasoconstriction and β-adrenoceptor-mediated increase in
cardiac output and vasodilation [21, 22].
Activity of the SA axis is important for blood pressure regulation and may contribute to
mechanisms causing essential hypertension. For example, complete inhibition of the SA axis
can normalize blood pressure in hypertensive animal models. One study used two methods of
SA inhibition in spontaneously hypertensive rats (SHR) to test the role of the SA axis in
contributing to hypertension and to changes in cardiovascular structure. The study used
sympathectomy in combination with α1-adrenoceptor blockade or rilmenidine (an agent that
interacts with imidazoline adrenergic receptors, inhibiting catecholamine binding) treatment
and it was found that SA inhibition both reduces blood pressure to normotensive levels and
improves cardiovascular structure in the SHR model [23]. Chronic suppression of SA activity
(via rilmenidine) in hypertensive rabbits also showed normalization of the mean arterial
pressure and decreased plasma renin activity [24]. SA axis activity may also contribute to
hypertension seen with elevated angiotensin (Ang) II. One study suggests that high levels of
Ang II (a vasoconstrictor peptide hormone that can increase blood pressure) causes chronic
elevations of the sympathetic system‘s responsiveness, including elevated activity of the SA
axis, contributing to essential hypertension as well as renovascular hypertension [25, 26].
These studies further establish the importance of the SA axis in hypertension.
In humans, the hyperactivity of the sympathetic nervous system is a primary contributor
to the pathology of essential hypertension and the ―neuroadrenergic hypothesis‖ has been
described to emphasize its role in the progression of this disease [27, 28]. Rilmenidine, the
imidazoline receptor agonist described above, has been successfully used to decrease
excessive central sympathetic activation in hypertensive patients, reducing blood pressure and
left ventricular hypertrophy.
Another target for regulating central sympathetic activation includes endogenous nitric
oxide synthase (NOS), which is inhibited by asymmetric dimethylarginine (ADMA). Nitric
oxide (NO) synthesized by neuronal NOS (nNOS) is a signaling molecule that reduces
sympathetic outflow from the brainstem, and its signaling is inhibited in the presence of
elevated ADMA. Elevated ADMA is a condition that is found with hypertension [29].
Elevated ADMA is also correlated with increased NE, the primary neurotransmitter in the
sympathetic nervous system; however, the mechanism of this action is not clearly understood
[30]. Reactive oxygen species (ROS) can also modulate endothelial NOS (eNOS), thereby
altering NO in the vasculature. ROS are elevated in hypertensive patients and ROS inhibitors
are an attractive target to reduce central sympathetic activity [27]. Statins, 3-hydroxy-3-
methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, usually prescribed for reducing
cholesterol levels, are also known to reduce sympathetic hyperactivity in humans and aid in
regulating blood pressure [31–33].
Moreover, changes in catecholamine biosynthesis, storage, reuptake and metabolism can
also alter sympathetic function and aid in the neurogenic development of hypertension [34,
35]. Supporting the role of sympathetic NE in hypertension, an analytical review comparing
78 studies on hypertensive subjects and their plasma catecholamine levels by Goldstein
(1983) found that plasma NE is elevated in younger and consistently hypertensive patients.
Tachycardiac hypertensives predominantly showed elevations in Epi, but patients who show
elevations in Epi may not show the same in NE and vice versa. Thus, the sum of NE and Epi
was a better indicator to differentiate the hypertensive population from the normotensive one
than the measure of either neurohormone alone [36]. In addition to pharmacological
interventions, lifestyle changes such as aerobic training, weight loss and reducing stress can
also aid in decreasing sympathetic hyperactivity and related hypertension [27].
There have been numerous studies on the genetic origins of spontaneous hypertension.
Hypertension is a polygenic disease and one of the main research methods to identify putative
genes linked with this disease involves linkage analysis studies between relative pairs (such
as sib-pairs) to analyze allelic association between them. The second method is the analysis of
variant gene frequency associations between normotensive and hypertensive individuals [37].
Gene loci involved in blood pressure regulation (particularly in vasoconstriction and sodium
reabsorption) have been identified on numerous chromosomes. Studies examining select
populations of closely related individuals have identified gene clusters implicated in essential
hypertension on chromosomes 1, 2, 12, 17 and 18 [38–44]. Most of these genes are
implicated in the kidney‘s regulation of sodium and the renin angiotensin system (RAS), such
as angiotensin converting enzyme (ACE) that converts Ang I to II [45]. There are other genes
implicated such as NOS, which generates the vasodilator NO, and PNMT, the enzyme
responsible for synthesis of Epi from NE [43, 46–48]. Future research directions in this area
would involve additional genome-wide scans and pharmacogenetic studies [49]. Determining
the genetic variations in hypertensive individuals may provide clues as to the effects and
extent of fetal reprogramming.
FETAL PROGRAMMING OF ADULT DISEASES

Human Studies Supporting Fetal Programming of Diseases
Numerous large-scale epidemiological studies have now shown that low-birth weight
positively correlates to chronic illnesses such as insulin resistance, obesity, diabetes mellitus,
and hypertension, further supporting the Barker Hypothesis [3, 50–54]. A longitudinal study
conducted in England found a direct negative correlation between weight at birth and systolic
blood pressure [55]. Lifestyle factors that are usual contributors to elevated blood pressure
such as obesity, smoking, lack of exercise, unemployment, and excessive alcohol
consumption were accounted for in some studies and had little bearing on the birth weight-
hypertensive relationship [2]. This provides strong evidence that changes in fetal development
are responsible for decreased birth weight which coincides with physiological adaptations,
perhaps affecting neuroendocrine development, and are altogether responsible for the
programming of hypertension later in life.
Low birth weight is often a sign of adverse gestational conditions, and can serve as a
good predictor for later hypertension and other chronic cardiac problems [3]. An increase in
plasma cortisol (Cort; a GC) levels has been shown in human adults with low birth weights,
providing convincing evidence of HPA reprogramming in humans [56]. Some studies show
that children born to malnourished mothers exhibit an increased HPA and autonomic nervous
system response to experimental psychological stress [57]. Epidemiologically, the Dutch
winter famine (1944-1945) and the Biafran famine during the Nigerian civil war (1967-1970)
are both examples of maternal undernourishment leading to intrauterine growth restriction
(IUGR), culminating in altered HPA axis, and cardiovascular and metabolic disorders [58–
60].
One study used a Tier Social Stress Test to examine changes in autonomic functions and
salivary Cort in children and reported that in low birth weight boys, arterial pressure, systemic
vascular resistance, and Cort levels were elevated, indicating modifications to the HPA axis
and sympathetic activity [61]. In contrast, girls demonstrated more activation in sympathetic
nervous cardiac function, indicating changes in the SA axis. Another study suggests that the
placenta of female fetuses may deactivate GCs more effectively than the male placenta,
offering some protection against high levels of maternal GCs and HPA reprogramming [62].
HPA deregulation was also seen in children of mothers, who were in the vicinity of the World
Trade Center during the 9/11 attacks, particularly in those whose mothers were in the third
trimester of pregnancy. The mothers that developed post-traumatic stress disorder (PTSD)
due to the trauma had lower Cort levels than controls; reduced Cort was also seen in the
infants at 1 yr of age [63, 64].
Other human studies have shown varied programming effects between different human
populations. A study comparing nephron counts in groups of white and African-American
subjects found that low nephron counts and birth weights were correlated to adult
hypertension only in the white subjects [7]. Another study examined the effect of maternal
diet on adult blood pressure, finding a complex relationship between macronutrient intakes,
decreased placental size, and increased blood pressure [51].
Mechanisms of Programming Gained from Human Studies
The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates Cort by

oxidizing it to form cortisone. The activity of 11β-HSD2 opposes that of the type 1 isoform of
11β-HSD (11β-HSD1), which converts biologically inactive cortisone into biologically active
Cort. These enzymes have important functions in the placenta and in mineralocorticoid
receptor (MR) function in sodium-transporting epithelia [65]. It has been proposed that
variants of the 11β-HSD2 gene may contribute to essential hypertension in humans by
enabling aberrant activation of MR by GCs, thus simulating mineralocorticoid excess.
MR and GR sequences are very similar and in vitro studies have shown that both Cort
and aldosterone (a mineralocorticoid) have a high affinity for MR. Under normal
physiological conditions, the activation of MR by GCs is hindered by 11β-HSD2 activity; in
vivo studies suggest that only aldosterone binds MR due to the enzymatic inactivation of Cort
and other corticoids in MR-expressing tissues [66]. When mutations of the 11β-HSD2 gene
occur or a large amount of Cort is produced, Cort binds with a higher frequency to MRs,
causing a greater retention of sodium which in turn increases water reabsorption [66].
Increased water reabsorption increases blood plasma volume, which contributes to elevated
blood pressure. Another important location of 11β-HSD2 activity is in the placenta, where
high levels of 11β-HSD2 help to metabolize maternal GCs and limit their transfer to the fetus.
The activity of placental 11β-HSD2 is a key protective mechanism for the developing fetus,
as excess GCs in the fetal blood stream have a permanent reprogramming effect on the fetal
HPA axis activity [67]. Reduced activity of the 11β-HSD2 gene in the human placenta has
been shown to cause low birth weight and a slowing of in utero growth [56]. These studies
highlight the importance of GC metabolism in blood pressure regulation through its influence
on MR function and its protective role during fetal development.
Overall, there have been relatively few human studies focusing on the fetal programming
of hypertension in comparison to the numerous animal studies. Many human studies are
predominantly epidemiological, and hence correlational. In the quantitative studies that have
been done, the results have largely mirrored findings in animal models. Factors such as sex
are essential to take into consideration, as there appear to be different programming
mechanisms at play in males and females. There is still much human research to be done in
the future, though the studies that have been completed to date show informative results and
have established a multitude of future directions to be taken.
Animal Studies Supporting Fetal programming of Adult Diseases
The use of animal models has provided a significant insight in understanding the
mechanisms underlying fetal programming of hypertension. The primary purposes of using
animal models in this field are threefold: first, the physiological effects of fetal programming
can be clearly validated under a diverse set of controlled experimental conditions; second, the
complete control of biological tissues allows researchers to analyze a greater range of
physiological phenomena; and third, the measurement of these tissues can be made at any
point in the development process from conception to adult life. As a result, the availability of
a more controlled environment using animals in fetal programming studies have helped
resolving cause and effect relationships between prenatal stress and impact on adult
physiology. Some of the most frequently used animal models in this field of study include
guinea pigs, rabbits, pigs, sheep, and most common of all, rats [68].
Collectively, these animal studies have shown that the effects of fetal programming are
highly variable between species, environments, types of maternal influences, and specific
windows of time in which the mothers are exposed to environmental insults or stressful
incidents. Thus, the process of fetal programming and its relationship to hypertension may be
a complex occurrence involving many physiological systems. The use of a variety of animal-
based models of fetal programming strengthens our understanding of the mechanisms
involved when findings are translated based on common mammalian gestational
characteristics such as stages of fetal tissue and organ development coinciding with maternal
stressors [10].
Animal Studies on Maternal Undernutrition
The association between fetal programming of adult hypertension and maternal

undernutrition, wherein the maternal diet is restricted from an adequate supply of nutrients, is
well established. Researchers have discovered that programming of adult hypertension can
result from a variety of maternal stresses such as protein restriction, overall food restriction
(using both quantities of food as well as calorie restriction), iron deficiency, and specific
vitamin deficiencies [69].
The most frequently studied mechanism of maternal undernutrition is protein restriction.

In the rat model, animals are typically fed approximately 180-200 g protein kg-1 diet. A study
using rats has demonstrated that protein-restricted maternal diets (60, 90, and 120 g protein
kg-1) can result in the development of hypertension in adult life. These findings, matching
with the Barker Hypothesis, show that maternal protein restriction induces a low birth weight
as well as a sustained elevation of blood pressure in offspring [70]. Other studies have also
confirmed these effects of a low-protein diet [71, 72]. A mechanism by which maternal
protein restriction may program for increased blood pressure in offspring is through IUGR
and impaired fetal nephrogenesis caused by the nutrient deficit. One study correlated nephron
number in offspring with birth weight of growth-restricted rats and found that reduced
nephron number is linked to impaired renal function [73].
Additionally, it has been found that a lower glomerular filtration rate (GFR) is observed
in the offspring of mother rats fed low-protein diets. The maintenance of renal haemodynamic
function with impaired nephrogenesis may require adaptations which increase systemic blood
pressure [74]. Another possible consequence of protein restriction involves the placental
enzyme 11β-HSD2. Studies in rats show decreased 11β-HSD2 activity in low-protein models
of fetal programming, thereby increasing vulnerability of the fetus to active GCs that would
be abundant with maternal stress [75]. Also, several components of the RAS, an important
system for regulating blood pressure, are differentially regulated in the offspring of maternal
low-protein diet. Some of the programming changes that influence RAS include a decrease in
renal renin and Ang II, and an increase in brain angiotensinogen and ACE [71, 76]. To
understand the processes underlying the many effects of maternal diet on fetal development, it
is important to note that the timing of the protein restriction has a strong influence on
programming effects of hypertension. For example, the only significant time period during
uterine development for the maternal protein restriction in rats is during organogenesis, which
occurs at approximately days 15-22 of gestation [77]. Finally, sex differences also have an
influence on the offspring‘s response to maternal undernutrition; male rats generally have a
higher sensitivity to maternal protein restriction than females, often exhibiting lower nephron
numbers, decreased expression of intra-renal RAS proteins, and higher prevalence of adult
hypertension [72].
Although not used as frequently as the protein-restriction method, other models of
maternal undernutrition that have been studied include caloric and whole-food restriction,
iron deficiency, and vitamin deficiencies. Calorie restriction and total food restriction in rats
throughout pregnancy have both shown lowered birth weights and increased blood pressures
in adult offspring [78, 79]. A possible mechanism for these results may involve the decreased
cell mass of β cells in the pancreas of offspring, thus contributing to a decreased insulin
production [80]. Moreover, if pregnant rats are fed iron-deficient diets, their adult offspring
exhibit lower nephron numbers, lower birth weights, and higher blood pressures with a
greater sensitivity for these predispositions in males [81]. Similar to other models of maternal
undernutrition, vitamin A-deficient maternal diet in rats resulted in lower nephron number,
lower birth weight, and higher blood pressure for the offspring [82]. Hence, it has been
clearly illustrated that many forms of maternal undernutrition can predispose offspring to
numerous physiological changes, including sustained increases in blood pressure and
potentially adult hypertension.
Maternal undernutrition also has some specific effects on the HPA and SA axes, which
lead to consequences in the cardiovascular system in adult life. Protein undernutrition and
food restriction are stressors that alter the energy balance of an organism and activate the
HPA axis to restore homeostasis [83]. In rats, maternal undernutrition leads to increases in
maternal ACTH and Cort, and also decreases in placental 11β-HSD2, consequently allowing
more maternal Cort to pass into fetal circulation. Further, a decrease in hippocampal GR, MR
and CRH, as well as reduced plasma corticosteroid-binding protein (CBG) are observed in the
offspring; CBG binds free Cort, thus limiting its bioavailability in the plasma [83].
Interestingly, maternal undernutrition can also inhibit HPA activity, as was observed when
maternal food restriction reduced HPA activity (hypo-activity) in response to ether inhalation
stress in male rats.
Conflicting reports of hypo- or hyper-activity of the HPA axis has been attributed to
differences in experimental paradigms and species specificity. Age is also an important factor
in how programming effects are manifested, and functions of the HPA axis developmaternal
undernutrition. Studies commonly demonstrate that HPA axis function is suppressed in the
fetus, relatively normal in young adults, and chronically elevated in older adults [83, 84].
Alterations in the SA axis have also been noted in prenatally malnourished rats exposed to
restraint stress, which display elevated levels of NE, ACTH, and Cort [85]. Increases in
plasma catecholamines, NE and Epi, have been attributed to alterations in neuronal and
neuroendocrine cell differentiation and structural changes in the adrenal medulla [86].
Animal Studies on Prenatal Hypoxia
Animal studies have also demonstrated a clear relationship between prenatal hypoxia
(i.e., exposure to low oxygen environment) and hypertension in adulthood. The risk of
exposure to hypoxia is seen in pregnancies complicated by preeclampsia, maternal smoking,
living in high altitudes, and preterm labour. The use of a gas chamber, as well as subjection to
high altitudes as experimental paradigms of exposure to chronic hypoxia have both
established the association between hypoxia, low birth weight, and high blood pressure [87–
89].
The protocol for exposure to chronic hypoxia in the gas chamber model, as described by
Williams et al., involves placing pregnant rats from gestational day 15 onwards in a Plexiglas
chamber saturated and continuously permeated with 12% O2 and a balance of N2. In this
model, the primary focus with regard to potential physiological changes has been in studying
vascular function in the offspring. NO plays a significant role in vasodilation, and therefore,
as a regulator of blood pressure. Studies have demonstrated a lowered NO-dependent
endothelial function in adult rats programmed from hypoxic environments but not using
protein-restriction methods [87]. Interestingly sex ual dimorphism has been reported in rats
prenatally exposed to hypoxia with the cardiovascular health of males being more greatly
impacted than females; the same study also demonstrated that the critical window of exposure
was during 15 to 22 days of gestation [89].
In another model, studying the effect of gestational intermittent hypoxia (IH), the
exposure to IH was achieved by placing pregnant rats in a hypobaric chamber with 10.8% O2
for 4h/d for the entire duration of the pregnancy. In this study, offspring were born with
significant alterations in HPA sensitivity, increased anterior pituitary CRH, elevated NE and
DA, and higher anxiety when tested in the elevated plus maze test [90]. The repercussions of
maternal hypoxia on the fetal HPA axis have been extensively studied in a sheep model. A
model of pregnant ewes maintained at high altitudes (on the White Mountain research station
in Bishop, California) from day 40 of gestation onwards mimics an exposure to moderate
continuous hypoxia.
Myers et al. have reviewed the findings of these studies detailing the impact on maternal
hypothalamus and anterior pituitary as well as changes in fetal adrenal cortex, elevated leptin
and hypoxia inducible endothelial nitric oxide synthase (eNOS), both of which can regulate
fetal Cort [91]. Modifications in the sympathetic nervous system are also seen in hypoxia-
conditioned animals; in rats this is observed as changes in sympathetic ganglionic
innervations, in tissue and circulating catecholamines, as well as in the catecholamine
biosynthetic enzymes TH and PNMT in A2, C1 and C2 regions of the brain and also in the
adrenal medulla [92–94]. In chicken embryos, hypoxia resulted in hyper-peri-arterial
innervation and increased cardiac NE [95]. Together, these studies connect hypoxia-induced
fetal programming of hypertension with mechanisms of altered HPA and SA axis function.
Animal Studies on Prenatal Glucocorticoid Exposure
GC exposure, either from endogenous sources due to stressful environments or from

exogenous sources such as injections of dexamethasone (Dex; a synthetic GC) can result in
fetal programming of hypertension.
Pregnant Sprague Dawley rats given the natural GC, corticosterone (Cort), by injection
over a short interval during pregnancy show elevated arterial blood pressure and reduced
nephron numbers in both male and female offspring; while the changes in blood pressure and
nephron count were similar in both sex es, males showed an increased glomerular
hypertrophy [96]. The effect of endogenous Cort on fetal programming has also been studied
by using restraint stress on pregnant rats. In most studies, restraint stress is administered by
placing pregnant dams in the last week of pregnancy into transparent cylinders and exposing
them to light for 45 min each thrice a day [97–100]. This external stressor increased maternal
Cort and influenced HPA activity in the offspring by reducing hippocampal Cort receptor,
thereby reducing feedback inhibition [99]. In another study with a similar stress paradigm, but
with randomised timings for the stress exposure, offspring were born with lower birth weights
while placentae showed reduced 11β-HSD2 and GLUT1 transporter [101, 102].
Numerous animal studies on GC programming have been done with the synthetic GC
Dex, injected during the sensitive period of organogenesis (gestational age 15-21 days in
rats); Dex is a poor substrate of 11β-HSD2 and can cross the placenta increasing fetal GC
exposure [103]. IUGR is observed in cases of Dex exposure with widespread effects on organ
development showing reductions in mass of the lungs, heart, kidney, and gut [104]. One of
the consequences of Dex exposure is an increased expression and activity of hepatic GR and
phosphoenolpyruvate carboxykinase (PEPCK) resulting in increased gluconeogenesis and
elevated fetal blood glucose [105]. RAS changes are also observed such as increased
expression of angiotensinogen in the hypothalamus and an increased expression of AT1R in
the medulla oblongata in studies using sheep [106]. In the kidney, RAS changes in adult
ovine subjects include increased angiotensinogen, AT1R, and AT2R [107].
Moreover, nephron number has been shown to decrease in sheep due to prenatal Dex
exposure [108]. Thus, Dex exposure appears to have a pronounced influence on RAS in
various tissues and similar to the protein-restriction model, an altered renal function may play
a part in the development of Dex-mediated adult hypertension. As for sex differences, the
GC-induced model shares similar observations in both sex es; however, differences have been
observed in the onset of blood pressure changes between sex es, where females appear to
show higher blood pressures earlier in life and males appear to show higher blood pressures
much later [109]. The HPA and SA axes are particularly sensitive to excess GC exposure, and
have been extensively studied in this model of prenatal stress. Changes in the expression of
key genes that effectuate the homeostatic functionality of these pathways, as well as
epigenetic modifications in the genome, are primarily responsible for reduced feedback
inhibition and a hyperactive HPA axis.
This list of methods of fetal programming is not exhaustive but provides basic knowledge
towards the understanding the animal models employed in this field. Other animal models
include placental insufficiencies, overnutrition, and other drug exposures as maternal
influences that can affect fetal health [110–112]. Nevertheless, it is clear that fetal
programming of hypertension depends upon factors such as altered nephron number, birth
weight, sex, RAS components, NOS activity, and HPA/SA function. Animal studies have
provided invaluable insight into the mechanisms underlying the fetal programming of
hypertension.
REGULATION OF THE CARDIOVASCULAR SYSTEM BY

CATECHOLAMINES: THE ROLE OF GLUCOCORTICOIDS AND THE
NEUROENDOCRINE-IMMUNE CIRCUIT
Role of Catecholamines in the Regulation of the Cardiovascular System
The primary catecholamines (CA) involved in the regulation of the cardiovascular system
are epinephrine (Epi), norepinephrine (NE) and dopamine. Catecholamine biosynthesis
begins with the hydroxylation of L-tyrosine by the enzyme tyrosine hydroxylase (TH),
producing L-3,4-dihydroxyphenylalanine (L-DOPA) [113]. L-DOPA is then converted into
dopamine through decarboxylation mediated by L-aromatic amino acid decarboxylase
(AAAD) [114]. NE is produced by hydroxylation of dopamine, catalyzed by dopamine β-
hydroxylase (DBH) [115]. Finally, NE is methylated by PNMT to produce Epi [116]. In the
adrenal medulla catecholamines are stored in CA storage vesicles located in the chromaffin
cells. Preganglionic nerve fibres from the sympathetic nervous system interface with
chromaffin cells and can stimulate CA release from these vesicles into circulation via Ca+2-
mediated exocytosis [117]. This sympathetic activation occurs in times of stress and is part of
the ―fight-or-flight‖ mechanism. NE is also indirectly released into the bloodstream as
spillover from its role in the sympathetic nervous system.
Following their release, catecholamines act as neurotransmitters as well as hormone
messengers, which travel through the circulation until binding to receptors at target tissues.
Multiple adrenergic receptor (adrenoceptor) types are expressed in the various target tissues
and help dictate how the cells respond to Epi and NE. These receptors include α1-, α2-, β1-, β2-
, and β3- adrenoceptors, of which the β-adrenoceptors are important for regulation of cardiac
function [118]. Each subset of β-adrenoceptor is coupled to adenylyl cyclase through G
proteins [118]. The stimulation of β1-adrenoreceptors, coupled to G stimulatory (Gs) protein,
leads to the production of cAMP from ATP, resulting in the activation of protein kinase A
(PKA) and induction of calcium channels [119].
The activation of Ca2+ channels increases Ca2+ myocardial influx during muscle
contraction. The function of α–adrenoceptors is similar to their β- counterparts; however,
their main effect on the cardiovascular system is to regulate vascular tone. Like β-
adrenoceptors, the α1-adrenoceptor is coupled to a Gs protein. Stimulation of the α1-
adrenoceptor activates phospholipase C, which catalyzes the conversion of PIP2 to DAG and
IP3, resulting in the release of Ca2+ that drives smooth muscle contraction. Contrary to the α1-
adrenoceptor, the α2-adrenoceptor is coupled to an inhibitor G protein (Gi) that is involved in
negative feedback which regulates the release of Epi and NE [120].
Catecholamines, particularly Epi, affect the function of many target tissues within the
cardiovascular system. At moderate levels, circulating Epi causes a redistribution of blood
flow via vasoconstriction (mediated by α1-adrenoreceptors) in many systemic arteries and
veins, and vasodilation (mediated by β2-adrenoceptors) in skeletal muscle, hepatic blood
vessels and coronary arteries [121–123]. Moderate levels of Epi also result in increased heart
rate and strength of contraction (inotropy) through its interaction with the β1-adrenoceptor
[124]. Similar to the induction of vasoconstriction by Epi, basal levels of NE activate
myofilament contraction in smooth muscle cells [125].
At higher concentrations of Epi, the β2 adrenoceptor acts as a negative inotrope when its
interaction with the Gs protein is replaced with an inhibitory Gi protein [126]. In spite of these
effects, high concentrations of Epi result in widespread vasoconstriction because negative
inotropy and β-adrenoceptor-mediated vasodilation are suppressed by the effects of Epi‘s
interactions with the α-adrenoceptors [127]. Dopamine also plays a role in cardiovascular
regulation via its interaction with dopamine receptors in peripheral tissues or indirectly via its
conversion to NE by DBH [35]. Some dopamine receptors located in arterial walls mediate
vasodilation and inhibition of NE release [128]. Dopamine can also regulate blood pressure
by increasing glomerular filtration and excretion of sodium [129]. Elevated levels of
circulating catecholamines can be very detrimental to the cardiovascular system. Prolonged
elevation can result in increased oxidative stress, decreased oxygen supply to tissues,
increased muscle contractions and cellular proliferation. These can lead to cellular damage,
cardiovascular dysfunction and vascular damage [118].
Regulation of Catecholamines by Glucocorticoids
GCs play an important role in regulating the production of catecholamines through

effects on key enzymes in the catecholamine biosynthesis pathway. Historically, GC
demonstrated post-translational control over catecholamine production by promoting
increased levels of S-adenosyl methionine (AdoMet). AdoMet is a key methyl donor in the
conversion of norepinephrine to epinephrine by PNMT. The binding of AdoMet to PNMT
promotes Epi production both by acting as a methyl donor and by blocking proteolytic
recognition sites on PNMT, thus preventing the enzyme‘s degradation [130].
As well as the post-translational effects, GCs have been shown to influence PNMT at the
transcript level through in vivo and in vitro studies [131, 132]. The upregulation of PNMT
mRNA levels by GCs have been well documented in vivo [132, 133]. Increases in PNMT
expression are mediated by the binding of GC to its cognate GR, which binds glucocorticoid
response elements (GRE) within the PNMT promoter, modulating its transcription [134–136].
GCs have also been shown to upregulate transcription and mRNA levels of both TH and
DBH in vitro [137–140]. Although there is some supporting evidence for this relationship in
vivo [141, 142] the effects of GCs on TH and DBH expression still remains a topic of
controversy [143]. Unlike PNMT, TH and DBH gene expression remains elevated in
hypophysectomized animals exposed to stress, indicating there may be another mechanism
behind increased expression [143]. In addition to the post-translational and transcriptional
mechanisms described above, GCs regulate catecholamine production via influences on
mRNA splicing, catecholamine secretion, and adrenoceptor expression in tissues. Lewis and
colleagues (1983) described increased translational activity of TH mRNA in the presence of
Dex in vitro [144]. Unsworth and colleagues (1999) propose that GCs may mediate alternate
splicing of PNMT in a tissue specific manner [145].
GCs can directly increase the release of catecholamines by sympathetic nerves [132,
146]. Furthermore, GCs decrease adrenoceptor mRNA and protein expression in various
tissues, limiting the downstream effects of catecholamines [146–148]. GCs may also have the
capacity to regulate catecholamine production through other mechanisms, which require
further study. For example, GCs can mediate long-term changes in expression of epigenetic
regulators such as DNMT and the demethylase methyl-CpG- binding domain protein 2
(MBD2), as well as alter their activity, potentially having significant downstream
consequences on catecholamine enzyme expression and biosynthesis although the exact
mechanism has yet to be discovered [149].
It is clear that GCs are intricately connected to the production and impact of circulating
catecholamines. Not only can GCs mediate transcriptional and translational control, but they
can also affect catecholamine release and their signalling indirectly by modulating
adrenoceptor abundance in target tissues. Additionally, by increasing expression of
biosynthetic enzymes, GCs drive increased levels of Epi, thus magnifying and prolonging
sympathetic activation.
Crosstalk between Cytokines and Catecholamines
The neuroendocrine system, its influence on hypertension and cardiovascular disease

The role of inflammation in the genesis of hypertension and accompanying organ damage
is well established [150]. Inflammation is one of the most important factors contributing to
cardiovascular risk; and it is a major part of the formation, progression and destabilization of
atherosclerotic lesions [151–153]. The link between immune and cardiovascular function is
apparent in major immune diseases including rheumatic diseases, HIV, and psoriasis.
Cardiovascular pathologies are the main cause of premature mortality in patients with
autoimmune rheumatic diseases [154]. Individuals with HIV infection have higher
cardiovascular risk, arterial stiffness, systolic and pulse pressures than matching uninfected
individuals [155]. A recent meta-analysis of observational studies concluded that psoriasis, a
chronic inflammatory skin condition, is associated with increased prevalence and incidence of
hypertension and that odds of hypertension increase with severity of psoriasis [156]. It has
been said that ‗inflammation is the sine qua non of pathology‘ and the connections between
inflammation, hypertension, and CVD lends support to inflammation‘s essential role in
disease [157].
A study by Blake et al. (2003) concluded that there is a tandem effect of hypertension and
inflammation in contributing to CVD in a prospective cohort study of 20,525 women [158].
The study found an increasing risk of cardiovascular events over time strongly and
independently associated with high BP, and with high plasma levels of the inflammatory
biomarker C-reactive protein (CRP). Further, the combined effect of BP and CRP was greater
than either determinant alone [158]. Later studies established the role of the adaptive immune
response in hypertension after finding that mice with a genetic deletion in recombinase-
-/-
activating protein (RAG-1 ), which lack T- and B-lymphocytes, experience blunted
hypertension in response to both Ang II and deoxycorticosterone acetate (DOCA)-salt;
adoptive transfer of T-cells restored the elevation in BP [159]. This study also identified the
role of the cytokine TNFα in BP elevation when mice treated with Ang II responded with
both increased BP and increased production of TNFα from T-cells; anti-TNFα therapy with
etanercept blunted the Ang II-mediated elevations in BP in these mice [159]. Also supporting
the role of cytokines in hypertension, Peeters et al. (2001) identified altered profiles of pro-
and anti-inflammatory cytokines and cytokine production capacity when comparing blood of
human patients with essential hypertension to control volunteers [160]. It is difficult to
elucidate the causal relationships between inflammation and hypertension, as the two
conditions seem to interact for mutual enhancement; however, both processes are important
contributors to CVD.
In describing a new model for how inflammation and hypertension interact, Harrison et
al. (2011) hypothesized that modest elevations in BP (to values of ~135-140 mm Hg), such as
in prehypertension, largely caused by activity of the CNS, trigger immune changes that lead
to hypertension. In this model, initial elevations in BP are responsible for neoantigen
formation from oxidation and altered mechanical forces in vasculature. Neoantigens then
induce inflammatory responses in kidneys and perivascular fat where cytokines and other
inflammatory mediators are released. These cytokines and inflammatory mediators work in
concert with catecholamines and other BP-elevating hormones leading to vascular and renal
dysfunction and initiating a more severe hypertensive state [161]. This feed-forward
explanation by Harrison et al. is very similar to recent findings by Kirabo et al. (2014), whose
work outlined a mechanism for hypertension based on an autoimmune-like reaction. In this
mechanism, initial increases in BP lead to oxidative stress and lipid peroxidation which
results in neoantigen formation, immune cell activation, and initiation of T-cell proliferation
and cytokine production, leading to further increases in BP [162]. With the support of these
and other findings, a new paradigm is being established that implicates inflammation in the
elevation of BP and progression of hypertension.
Adrenal Cytokines and their Influence on Catecholamines
The ―neuro-immune circuit‖ as described by Glaser and Kiecolt-Glaser (2005) is the

integration of the nervous and immune systems, allowing organisms to coordinate responses
to microbial, physiological, and socio-ecological threats [163]. Neural and endocrine stimuli
modulate transcription in immune cells, while cytokines and other immune mediators signal
the brain to modulate neural and endocrine activities. This bi-directional neural and immune
communication is also an important component of adrenal regulation through the HPA and
SA axes, and there is growing evidence that immune communication occurs at the level of the
adrenal medulla itself.
The ability of cytokines to signal to sympathetic neurons of the brain is well established;
however, relatively little is known of how cytokines influence the activities of the neurally-
derived catecholamine-producing cells of the adrenal medulla. Investigations into the
potential role of cytokines in regulating catecholamine production by the adrenal gland were
in part inspired from insights gained from studying depression. Depression can be induced by
alterations in norepinephrine and other neurotransmitter levels, and sympathetic hyperactivity
is a well characterized component of the condition [157]. It has also been shown that a large
proportion of patients that receive IFNα therapy for treatment of cancer or infectious disease
develop a behavioural syndrome that is very similar to major depression [157]. This finding
led to questions about the influence of cytokines on neurotransmitter production and the role
of cytokines in regulating neural activity. In sites of catecholamine production outside the
brain, the influences of cytokine signalling is only beginning to be understood.
Adrenal medullary cells may be routinely exposed to cytokines for autocrine or paracrine
signalling, supported by findings that many cytokines are produced in adrenal tissue itself
[164–171]. There is also now strong evidence that cytokines can profoundly influence the
adrenal medulla by inducing quantitative and qualitative changes in secretion, intracellular
signalling and gene transcription [172]. IFNα supresses ACh activation of Ca2+ influx and
catecholamine release in bovine chromaffin cells [173]. Exposure of isolated bovine adrenal
medullary chromaffin cells to IFNα produces a PKC-dependent suppression of NE uptake
[174]. IFNα may also influence chromaffin cell catecholamine synthesis through ERK1/2
dependent phosphorylation of TH [175]. Signalling by IFNα in chromaffin cells involves
phosphorylation and nuclear translocation of STAT1 and STAT2, and ERK1/2 activation
[175]. IL-1β has opposite activities on chromaffin cell catecholamine secretion depending on
the presence of co-stimulators. In basal conditions IL-1β increases catecholamine release
from bovine adrenal chromaffin cells, but with concurrent stimulation by ACh or K+ the
cytokine inhibited catecholamine secretion through an ERK1/2 dependent mechanism [176–
180]. IL-1β also increases protein levels of the catecholamine biosynthetic enzyme TH and,
like IFNα, induces phosphorylation of TH, in this case at the ser-40 site which is associated
with increased enzyme activity [178, 181]. IL-6 is another cytokine that has been shown to
induce changes in chromaffin cells by signalling via IL-6 receptor. In PC12 cells (a rat
pheochromocytoma cell line) recombinant IL-6 treatment decreases DA and NE release and
decreases TH protein [182]. In bovine adrenal chromaffin cells, IL-6 activates signalling
through STAT3 phosphorylation and nuclear translocation, and ERK1/2 phosphorylation
[183]. Of the cytokines examined for effects on chromaffin cell function, TNFα exerts some
of the more potent influences [184]. Like other cytokines discussed, chromaffin cells respond
to TNFα via ERK1/2 signalling, and exposure of chromaffin cells to TNFα induces PNMT
mRNA expression [184, 185]. Whether the responses of medullary cells to cytokines
primarily functions for protective action against microbial challenge or if it is specifically for
regulating catecholamine production, it is clear that signalling of cytokines to adrenal
medullary cells has potential to exacerbate dysfunctional catecholamine production if there
are local changes in cytokine signalling within the medulla.
Above, we have summarized how adrenal function can be regulated by cytokines, and
how inflammatory processes and cytokine-mediated signalling is a key component of
hypertension and resulting CVD. The bi-directional relationship of the immune and neuro-
endocrine systems conceivably provides fitness advantages to organisms in ways similar to

the ‗neuro-immune circuit‘ and may be a physiologically important part of maintaining
health, dysfunction of which may result in pathology. The neuro-immune circuit has helped to
explain perplexing phenomena such as the co-morbidity of neuropsychiatric symptoms and
inflammatory disease [186]. Similarly, integration of immune and adrenal functions provides
an explanation for the aetiology of inflammation-related hypertension and may help to
elucidate mechanisms of essential hypertension.
IMPLICATIONS OF FETAL PROGRAMMING

Changes in HPA, SA and Adrenal Function
Maternal stress during pregnancy influences blood pressure regulation and adrenal
function of offspring by inducing changes in the development and function of their HPA and
SA axes. Changes in the stress response of offspring are one of the most immediate signs of
programming of the HPA and SA axes. For example, offspring of dams stressed by light,
heat, and restraint during pregnancy demonstrated increased cardiovascular sensitivity to
subsequent stressors. These offspring showed elevated systolic and diastolic BP, and altered
cardiovascular responses upon exposure to restraint stress; they also had less effective
recovery of BP following the stressor compared to animals from control mothers [98].
Changes in an offspring‘s cardiovascular responses to stress may be the result of
programming effects observed in both HPA and SA axes. The combination of HPA and SA
programming influencing adrenal function is demonstrated by a study in rats, which found
hyperactive SA axis and enhanced stressor-induced elevations in plasma Cort and
catecholamines in prenatally programmed offspring [187]. In this study the prenatal stress
was administered by exposure of pregnant rats from day 1 of pregnancy to unpredictable
noise and light stress three times per week [187]. Two excellent reviews by Maccari and
colleagues (2007, 2008) summarized the influence of restraint stress administered to pregnant
rats on the HPA and neuroendocrine dysfunction in offspring, and the consequent learning
deficits, increased anxiety, disturbances in circadian rhythm, and immune irregularities [100,
188]. There are numerous models of inducing maternal stress and they elicit similar
physiological changes. In humans, observational studies on the influence of stress have been
undertaken in women who suffer from mood disorders and bear children with emotional,
behavioural and other psychiatric disorders [189, 190]. An almost universal characteristic of
stress models is increased plasma GC and, as discussed later, this appears to be an important
factor for programming offspring.
A useful method for studying fetal programming involves using synthetic GCs to
simulate maternal stress. During fetal development, maternal placental enzyme 11β-HSD2
limits the exposure of the fetus to maternal GCs. However, 11β-HSD2 is less effective at
metabolizing exogenous synthetic GCs such as Dex. As a result, when mothers are
administered these exogenous GCs, fetal exposure to GCs is increased. Administration of
synthetic GCs to pregnant mothers can thus mimic times of high maternal stress or nutrient
deficiency (increase in endogenous GCs), or situations when 11β-HSD2 activity is
compromised (such as from a genetic defect) or not able to keep up with increased GC
production or release. Administration of GCs to pregnant mothers has been used in studies
that demonstrate programmed changes in both SA and HPA function. A study of lambs
prenatally stressed with betamethasone (a synthetic GC) showed programming through
elevated blood pressure, reduction in baroreflex sensitivity (an important indicator of heart
rate control), and increases in mean arterial pressure (indicating enhanced sympathetic
responses) evident when these animals were pharmacologically challenged with injections of
either ovine CRH (to increase BP) or sodium nitroprusside (SNP; to decrease BP) [191]. GC
exposure in the form of Dex can also affect sympathetic innervation of cardiac tissue,
although the impact of this on function in later life is not clear [11]. In light of the
programming effects that GCs can have on sympathetic function and the SA axis, it is not
surprising that GC exposure can have immediate and long-term effects on fetal HPA axis
activity as well. Evidence of altered HPA axis function is seen in increased basal Cort levels
of programmed offspring. In sheep, betamethasone injections at gestational day 104 resulted
in elevated plasma Cort levels at 1 year of age [192]. This phenomenon is also well
documented in rats [193, 194]. Additionally, prenatal GC exposure can have significant
lasting effects on several regions of the brain including the limbic system, hypothalamus,
pituitary, amygdala, and the hippocampus, thereby contributing to the programming of
functional changes in adrenal regulatory pathways [67, 149, 195–197].
High levels of maternal GCs may drive fetal programming through multiple mechanisms.
For example, prolonged elevations in maternal GCs result in catecholamine-mediated
constriction of placental blood vessels, leading to fetal hypoxia, which activates the fetal HPA
axis [7, 176]. Among other effects, this fetal hypoxia can modify TH and PNMT expression,
promoting elevated catecholamine production in adulthood [177]. High levels of maternal
GCs can also induce increased placental CRH and increase fetal levels of 11β-HSD1 and
CBG protein in the liver. These characteristics can produce elevated levels of fetal Cort
during development, promote further GC exposure and significantly magnify HPA
programming [149, 195, 198, 199]. GCs may also drive epigenetic changes in the fetus that
can influence gene expression in its tissues, perhaps programming changes in stress response
and adrenal function. An example of epigenetic programming by GCs was shown when
premature fetal GC exposure in guinea pigs lowered global methylation status in several
tissues, and increased methylation status in the kidneys between growth days 52 and 65 [200].
Studies on mood disorders such as depression or anxiety during pregnancy have also shown
interesting findings with regard to epigenetic changes in placental GR (also known as
NR3C1) and 11β-HSD2, both of which can consequently influence fetal HPA. Increased
methylation of placental NR3C1 was seen at CpG2 sites in women that were depressed while
in women who suffered from anxiety, the increased methylation was seen in 11β-HSD2
CpG4 [190].
A decrease in placental 11β-HSD2 in the rat prenatal restraint stress model was correlated
with corresponding increases in CpG methylation of the 11β-HSD2 promoter, and an increase
in DNMT3a in the placenta; in the fetal brain cortex, DNMT1, was elevated suggesting tissue
specificity in epigenetic changes [86]. Thus, GCs are capable of mediating changes in
epigenetic regulators such as DNMTs, which can alter the methylation status of key genes in
GC metabolism and conceivably also catecholamine biosynthesis in a tissue specific manner.
During gestation, natural increases in maternal GC and resulting epigenetic changes may aid
in tissue maturation, but early or excessive GC exposure may alter epigenetic regulators
leading to programming, although the exact mechanism still remains unclear [185].
Programming of GR is particularly sensitive to environmental changes. Interestingly, not only

prenatal stress, but also early life experiences such as postnatal care, mother-pup interactions
and maternal behaviours can influence HPA programming and stress responses in the adult.
In animals exposed to high levels of postnatal care (high licking and grooming dams, high-
LG) showed a greater degree of hippocampal GR expression compared to the animals that
received low levels of licking and grooming (low-LG). The low expression of GR in the low-
LG group was associated with an increase in the methylation of exon 17 of the GR promoter,
and this could be reversed by central infusion with trichostatin A, a histone deacetylase
(HDAC) inhibitor suggesting the involvement of histone deacetylases in the epigenetic
modification of GR [67, 201].
The effects of prenatal stress are dependent on the animal model being studied and the
gestational period during which stress is experienced. Programming effects are primarily
associated with the period of organ and neuroendocrine development, the functionality of the
HPA axis, and GR and MR expression profile during fetal growth [10]. For example in sheep,
the window of programming is much earlier and shorter around day 27 of a 150 day gestation
than rats; days 14-21 of 21 day gestation [194, 202, 203]. Once again, an abundance of
research has also discussed sex differences in fetal GC programming with the majority of
findings indicating that females generally remain unaffected by the fetal insult [204]. Studies
have suggested that this sex ual dimorphism could arise very early in programming and that
the variance in male and female placental tissue could be a contributing factor [205, 206].
Mechanistically, the fetal programming of altered HPA axis is very complex and still not
fully understood.
In summary, prenatal GC exposure results in a spectrum of changes in the fetus that can
influence HPA axis function; changes can affect GR and MR receptor densities in regions of
the brain, hormones and associated biosynthetic enzymes, and altered CRH production in the
amygdala. These neuroendocrine alterations are apparent in the form of changes in stress
responses and behavioural changes in these offspring. These findings exemplify the
significance of stress and GC exposure during fetal development, which can result in
extensive life-long changes to adult physiology. More recently, the significance of epigenetic
regulators has become a key area of research in understanding GC programming.
Changes in Immune Function and Cytokine Profiles
Prenatal stress has been shown to alter immune parameters and immune function [207].
Thus, prenatal stress may lead to disrupted communication between immune and
neuroendocrine systems and to increased risk for pathologies caused by immune and
neuroendocrine dysfunction, such as CVD. A review by Merlot et al. (2008) of the fetal
programming effects of prenatal stress on immune function summarized a variety of
seemingly contradictory findings from animal studies of psychological and GC-induced
maternal stress during gestation. There appears to be little consistency in the findings from
immune parameters measured between studies and this may be due to differences in
experimental conditions such as timing, length, type, and severity of stress during gestation,
as well as the times in which immune parameters were examined following gestation. In spite
of varied observations, it is clear that maternal stress can induce lasting changes in adaptive
immune function in adult progeny.
Literature predominantly supports the conclusion that maternal stress has a general
inhibitory effect on immune function of offspring. For example, rats born from mothers that
underwent social stress during late gestation show decreased lymphocyte counts in adulthood,
predominantly in CD4+ subset, as well as attenuated mitogen-induced lymphocyte
proliferation [208]. Similarly, more recent experiments with maternal social stress in pigs
showed that prenatal stress decreased CD4+/CD8+ T-cell ratio, total white blood cells,
lymphocytes and granulocytes in piglets. The study also found that prenatal stress attenuated
mitogen-induced TNFα production [209]. Further supporting the findings that prenatal stress
can influence cytokine production, a study of rhesus monkeys found that acoustical startling
of pregnant mothers diminished endotoxin-induced IL-6 and TNFα production in whole blood
cultures from juvenile offspring [210]. The stress paradigms mentioned above have been
confirmed to significantly elevate Cort levels in the pregnant mothers [211, 212].
Direct treatment of pregnant mothers with Dex also influences offspring cytokine
profiles. A study with rats measured plasma levels of TNFα, IL-1β, and IL-6 in adult
offspring of mothers treated with Dex from day 13 of pregnancy until birth. Both male and
female offspring showed marked increases in IL-1β and TNFα levels. The authors
commented that the differential programming of the three cytokines examined is likely
reflective of their different cellular origins [213].
Interestingly, a number of recent studies have shown that maternal cytokines may also be
involved in controlling the transmission of GCs through the placenta to offspring. Elevated
levels of serum proinflammatory cytokines are found in pregnant women experiencing stress
[214, 215]. The proinflammatory cytokines IL-1β, IL-6, and TNF-α potently inhibit 11β-
HSD2 activity in explant cultures of term human placental villi. Near maximal inhibition is
reached after 2 hours of cytokine exposure and by 24 hours the cytokines almost completely
inhibit 11β-HSD2 activity. The decrease in activity was not matched by a decrease in mRNA
levels, suggesting that cytokine-meditated inhibition of 11β-HSD2 occurs through post-
translational mechanisms. The study also reported that the inhibition in enzyme activity
appears to be mediated by Ca2+ influx and by reduced adenylyl cyclase activity [216].
The attenuation of 11β-HSD2 activity by cytokines is supported by similar findings from
another in vitro study using term human placental trophoblast cells and human
choriocarcinoma JEG-3 cells [217]. In addition to 11β-HSD2, proinflammatory cytokines
have been found to influence activity of the type 1 isoform of 11β-HSD (11β-HSD1). As
mentioned earlier, the two isoforms have opposite enzymatic activities, with type 1
converting biologically inactive cortisone into biologically active Cort. The type 2 isoform
accounts for nearly all of the 11β-HSD activity in the human placenta, but the type 1 isoform
is expressed in the amnion and can influence fetal environment [218]. IL-1β increased 11β-
HSD1 mRNA and both TNFα and IL-1β enhanced Dex-mediated induction of 11β-HSD1
mRNA and enzyme activity in human term amnion fibroblasts. Potentiation of GC-induced
11β-HSD1 activity by cytokines may be important in the positive feedback loop for
increasing GC and prostaglandin production in fetal membranes or amniotic fluid. GCs and
prostaglandins are essential for fetal organ maturation and during parturition, and this
signaling may be particularly beneficial when preterm delivery is initiated because of
infection.
In summary, cytokines may be involved in the fetal programming of disease through two
distinct mechanisms: first, by modulating GC metabolism in the placenta and increasing fetal
exposure to maternal Cort; and second, by contributing to disease progression in adult
offspring through alterations in functional immune parameters. Prenatally programmed

immune changes may contribute to adulthood hypertension by interfering with
neuroendocrine-immune communication, and by exacerbating dysfunctional catecholamine
biosynthesis and secretion. As discussed above, adrenal function is powerfully influenced by
immune activity either through the direct action of cytokines on adrenal cytokine receptors or
by the impact of inflammatory signals on regulatory sites of HPA and SA axis activity in the
CNS [219]. More research needs to be completed to fully understand the consequences of
immunological changes caused by prenatal stress as they relate to adrenal function and
hypertension; however, fetally programmed alterations in immune parameters and cytokine
production are a potential component of HPA hyperactivity and disease in prenatally stressed
offspring [220].
CONCLUSION
Evidence from numerous human and animal studies suggests that prenatal stress has
profound influences on the cardiovascular and neuroendocrine system, the immune response
and metabolic pathways in adulthood. Exposure to stress and resultant excess GC during
critical windows of fetal development alter signalling via GR and components of fetal RAS
system as a mechanism for adaptation to the excess circulating hormone. Furthermore,
changes in placental 11-βHSD2 consequently leads to an altered balance of the fetal HPA
axis and increased risk for physiological diseases in their adult life. Although genetics play an
important role, prenatal stress is an independent risk factor that can impact the predisposition
to cardiovascular dysfunction. Observations from epidemiological studies in humans, and
experimental animal studies have provided information on the mechanistic pathways,
highlighting changes in gene expression and epigenetic markers that are influential in
inducing these programming effects and increasing disease risk. By means of epigenetic
modifications, fetal programmed changes may not be limited to the F1 (first) generation that
experienced the stress and can be transmissible across generations [103, 221]. Furthermore,
paternal stress and elevated GCs can alter miRNA signatures in the sperm and have HPA
consequences for the offspring [222]. Thus, both maternal and paternal stress can influence
and confound the mechanisms in reprogramming the HPA [149, 223]. A better understanding
of these complexities will aid in developing measures to limit prenatal insults, prevent
maladaptive fetal responses to stress, and reduce fetal programming of hypertension and
cardiovascular disease.
ABBREVIATIONS
11β-HSD2 11-Beta hydroxysteroid dehydrogenase isoform 2
ACE Angiotensin converting enzyme 1
ACh acetylcholine
ACTH Adrenocorticotrophic hormone
Ang II Angiotensin II
AT1R- Angiotensin II receptor 1

BP blood pressure
Cort Corticosterone (rodents and non-human primates); Cortisol (humans)
CBG corticosteroid-binding protein
CRH corticotropin-releasing hormone
CVD cardiovascular disease
DA dopamine
DBH Dopamine β-hydroxylase
Dex Dexamethasone
DNMT DNA methyl transferase
Epi epinephrine
GC(s) Glucocorticoid(s)
GR Glucocorticoid receptor
HDAC Histone deacetylase
HPA hypothalamus – pituitary - adrenal
IUGR intrauterine growth restriction
MR mineralocorticoid receptor
NE Norepinephrine
PNMT phenylethanolamine N-methyltransferase
PTSD post-traumatic stress disorder
PVN paraventricular nucleus
RAS renin–angiotensin system
SA sympathoadrenal
TH tyrosine hydroxylase
TNFα tumor necrosis factor alpha
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Chapter 11
ADRENAL CORTICAL NEOPLASMS: PERSPECTIVES IN

PEDIATRIC PATIENTS
Shahrazad T. Saab, MD
and Gregory T. MacLennan, MD
Division of Anatomic Pathology, Institute of Pathology,
Case Western Reserve University, Cleveland, OH, US
ABSTRACT
Adrenal cortical neoplasms (ACNs) in children include adrenal cortical adenomas
(ACAs) and adrenal cortical carcinomas (ACCs). These are rare pediatric tumors. At
presentation, most children show signs of virilization accompanied by additional
manifestations of hypersecretion of other adrenal hormones. Although most ACNs occur
sporadically, a few arise in the setting of syndromes, including Beckwith-Wiedemann
and Li-Fraumeni Syndromes. Various histopathologic scoring systems have been devised
to classify ACNs as adenomas or carcinomas, showing good correlation with clinical
outcome when applied to adults. ACNs in children frequently have many of the
morphologic and histologic features of adult ACCs. Thus, ACCs are disproportionately
overrepresented, comprising 90% of all pediatric cortical tumors. Subsequently, the term
adrenal cortical neoplasms/tumors has become nearly synonymous with adrenal cortical
carcinoma in the pediatric age group. However, ACCs in children do not have the same
poor prognosis as their adult counterparts, with favorable outcome in about 70% of
childhood ACCs. This observation has led to investigation of the criteria for malignancy
in pediatric ACNs. In recent years, new histopathologic criteria have been proposed to
better classify ACNs of children in accordance with their biologic behavior.
Keywords: adrenal cortical neoplasm, adrenal cortical adenoma, adrenal cortical carcinoma,
children
272 Shahrazad T. Saab and Gregory T. MacLennan
INTRODUCTION
Pediatric adrenocortical neoplasms (ACNs) include adrenal cortical adenomas (ACAs)

and adrenal cortical carcinomas (ACCs). These tumors are rare. The National Cancer Institute
data demonstrate that only 1.3% of childhood malignancies are carcinomas, with ACCs
representing less than 0.2% of all pediatric malignancies [1]. There is a bimodal age
distribution of ACCs with peaks in the first decade and again in the fourth and fifth decades
[2, 3]. The median age at presentation is approximately 3 years with a slight predominance in
girls [1-3]. The incidence in the pediatric population is similar to that of adults, ranging from
0.5 to 2.0 cases per million, except for an unusually high prevalance in children in southern
Brazil, occuring in 3.4-4.2 per 1 million children younger than 15 years. This is attributed to a
germline mutation in the TP53 gene, which is common in this subpopulation [2-4]. In
children, the vast majority of ACNs are functional, producing excess hormones that result in
clinical signs of virilization with or without cushingoid features as the most common
presentations. Other manifestations include feminization and precocious puberty [1-6]. Most
occur sporadically, but ACCs are also associated with Beckwith-Wiedemann and Li-
Fraumeni syndromes [4]. Computed tomography (CT) scans and magnetic resonance (MR)
images of ACNs typically demonstrate well defined, inhomogeneously enhancing suprarenal
masses, occasionally with areas of necrosis, hemorrhage, and calcification observed in both
ACAs and ACCs [7]. Typically, ACAs have well circumscribed margins compared to ACCs.
Short of finding evidence of metastasis or vascular invasion, it is difficult to distinguish
between ACAs and ACCs radiologically [7, 8].
Macroscopically, ACAs are well encapsulated, solitary masses with a smooth and
bosselated cut surface and are usually less than 5 cm in diameter. The color varies from tan-
grey, tan-yellow, to orange-brown [7]. ACCs tend to be larger than 10 cm [2].
Most are encapsulated, but may show extracapsular extension or extension into adjacent
vasculature. Their cut surface varies from tan-grey to orange-yellow and is often variegated
with foci of hemorrhage and soft and friable necrotic areas. It has been demonstrated that both
ACAs and ACCs can show areas of hemorrhage, necrosis, calcification, and cystic
degeneration [6]. Interestingly, and in contrast to ACNs in adults, vascular invasion and
capsular invasion can also be seen in both benign and malignant ACNs [3, 6]. This curiosity
is discussed at length below.
Histologically, ACNs are composed of cords and clusters of round, oval, or polygonal
cells with a prominent sinusoidal network surrounding groups of cells. The malignant cells
bear resemblance to the non-neoplastic adrenal cortical cells. The cytoplasm can be clear and
vacuolated due to the high lipid content, resembling the cells of the zona fasciculata.
Alternatively they can bear resemblance to the more compact, eosinophilic cytoplasm of the
cells composing the zona reticularis. Frequently, there is an admixture of these two types of
cells [9].
As will be discussed below, ACAs often exhibit numerous atypical histologic features
characteristic of adult ACCs, including elevated proliferation rates, atypical mitoses, marked
nuclear pleomorphism, confluent areas of necrosis, vascular invasion, and capsular invasion
(Figures 1 and 2).
Adrenal Cortical Neoplasms: Perspectives in Pediatric Patients 273
Figure 1. Low-risk ACN/ACA in a 16-year-old girl with cushingoid features, elevated ACTH, and
well-circumscribed 3.5cm mass weighing 8.5g with extensive gross areas of necrosis (top). Histologic
sections showing extensive necrosis (middle) and no other atypical histologic features (below). (H&E,
10x and 40x) The patient is alive and well 5 years post resection with marked resolution of cushingoid
features.
A property that is common among many different kinds of endocrine neoplasms,

including adrenocortical neoplasms, is that their biologic behavior tends not to be readily
predictable on the basis of their histomorphometric characteristics. Numerous studies have
evaluated a variety of pathologic features of prognostic significance. Some of the many
studied parameters were as follows: architectural pattern, nuclear atypia, mitotic activity,
atypical mitotic figures, necrosis and its pattern, presence of intersecting fibrous bands, and
vascular and/or capsular invasion. It has become widely accepted that no single pathologic
feature is predictive of malignant behavior, thus necessitating the development of various
grading systems that incorporate several parameters [10-12]. Though the Weiss grading
system is the most widely applied, many have been shown to have good accuracy and
reproducibility in adults [13].
Historically, the medical literature has focused on adult ACNs or has not separated
pediatric from adult tumors. This has resulted in the assumption that the various grading
criteria can be used in both populations. When these grading systems are applied specifically
to ACNs of childhood, the result is that the overwhelming majority of tumors are interpreted
to be ACCs. Subsequently, the terms ―adrenal cortical tumors/neoplasms‖ have become
synonymous with ACC in this age group [6]. Two large series of pediatric ACNs from the
International Pediatric Adrenocortical Tumor Registry (IPATR) [14] and the Armed Forces
Institute of Pathology (AFIP) [15] have reported that 90% of 254 ACNs and 89% of 83 ACNs
of children, respectively, met the pathologic criteria of ACC. A recent study of 33 ACNs in
children evaluated the tumors using the standard grading systems described in the literature
[10-12]. It showed that the Van Slooten, Weiss, and Hough systems classified the tumors as
carcinomas in 100%, 94%, and 64% of cases respectively [16].
The study from the AFIP demonstrated that of 54 ACNs in children that met the
histopathologic criteria for malignancy set by these three grading systems [10-12], thus
classifiying them as ACCs, 51 (94%) of these exhibited a benign clinical course. These
benign-behaving tumors displayed many of the defining gross and histologic features of adult
ACCs. These included confluent areas of necrosis, broad fibrous bands, capsular invasion,
vascular invasion (sinusoidal and venous), diffuse/solid pattern in >30% of the tumor,
moderate to severe nuclear pleomorphism, lipid-depleted/eosinophilic cytoplasm in >30% of
tumor cells, nuclear hyperchromasia, prominent nucleoli, increased mitoses with an average
of about 9 mitoses per 20 HPF (range of 0-59 mitoses), and atypical mitoses. Despite the
presence of many of these worrisome features, none of the children in this group had
recurrence or development of metastatic diseasease throughout their follow up [15].
Various studies have demonstrated that none of these atypical or worrisome histologic
features has prognostic significance when predicting the biologic behavior of pediatric tumors
[3, 6, 14-16]. Cagle and colleagues demonstrated that ―benign‖ ACNs in children are more
likely to be mitotically active, have moderate to severe pleomorphism, and broad fibrous
bands when compared to adults [6]. In the AFIP study, 9 of 83 cases were histologically
classified as benign by the standard grading systems [10-12] and were indeed clinically
benign. Of the 74 cases that were diagnosed as malignant 51 cases, discussed previously,
exhibited benign clinical behavior, indicating that application of the existing classification
criteria was probably inappropriate in this subset of pediatric tumors. Thus, in this series of 83
children, the vast majority (72%) were biologically indolent [15], contrary to the existing
misconception that most childhood ACNs are malignant. The accumulated literature on
pediatric ACNs indicates that the most important prognostic features include tumor weight,
extension of tumor beyond the adrenal gland, metastasis, negative surgical margins, and
clinical information such as tumor spillage or residual disease [1-3, 5, 6, 14-16, 19, 20].
Figure 2. Low-risk ACN/ACA in a 2-year-old boy who presented with new-onset acne, hirsutism,
appearance of pubic hairs, an abrupt growth spurt, elevated testosterone and hydroxyprogesterone, and
a well-circumscribed 7.5 cm mass weighing 67g (A). Histologic sections showing lipid-depleted tumor
cells with severe nuclear atypia (B), atypical mitoses (C), and necrosis (D). (H&E, 40x, 40x, and 20x)
The patient is alive and well 7 years post resection with appropriate growth for age and resolution of
acne.
Atypical histologic features characteristic of ACCs in adults are frequently observed in

both ACAs and ACCs in children and are therefore not helpful in distinguishing between
them. Cagle and colleagues regarded tumor size as ―the only reliable predictor of biologic
behavior in childhood‖ [6] (Figure 3). Numerous authors have emphasized the importance of
tumor weight and have quoted various weight cut-off points capable of distinguishing benign
from malignant tumor behavior [6, 14, 15]. Dehner and colleagues [3] have suggested three
weight ranges that categorize tumors into three risk assessment groups (Table 1). They have
proposed that ACNs weighing 200g or less be assigned to the low-risk category, those
weighing between 200 and 400g belong to the intermediate-risk category, and those greater
than 400g are in the high-risk category.
Figure 3. High-risk ACN/ACC in a 14-year-old girl who presented with virilization and a 24 cm mass
weighing 950 g (top). Histologic sections showing lipid-depleted tumor cells with severe nuclear
atypia, increased mitotic figures, necrosis (middle) and venous invasion in the tumor capsule (bottom)
(H&E, 40x each). Metastatic disease was present in mediastinal lymph nodes. The patient is deceased
despite chemotherapy and radiation therapy. (Figure 3a: reprinted with permission from
MacLennan GT, Resnick MI, Bostwick DG. Pathology for Urologists. Elsevier, 2003).
(Photograph 3a from MacLennan GT, Resnick MI, Bostwick DG. Pathology for Urologists. 2003. With
permission).
Any direct tumor invasion into surrounding tissues and/or organs or any evidence of
metastasis qualifies the tumor as an ACC regardless of size. In addition, any tumor weighing
less than 400g with microscopic invasion into the immediate surrounding tissues, but is
completely resected, should be assigned into the intermediate category.
In this suggested classification scheme, ACA is considered "low risk," atypical adrenal
cortical neoplasms are considered ―intermediate-risk,‖ and ACC is considered ―high risk.‖
According to this classification, the term ACC is reserved for those ACNs weighing in excess
of 400 g or that demonstrate direct invasion of adjacent tissues and/or organs, or metastasis,
thus constituting only 10%-30% of ACNs in children [3].
Also contributing to the difficulties with ACNs in children is the lack of a standardized
system for tumor staging, leading various authors to suggest their own. A general summary
gathered from various sources is as follows: Stage I ACNs are those weighing 200 g or less
with negative resection margins and no evidence of metastasis. Stage II ACNs are completely
resected, but have invaded adjacent tissues and/or organs with no evidence of metastasis.
Stage III and IV ACNs have metasized to regional lymph nodes or distant sites, respectively
[3].
Table 1. Proposed risk groups for adrenocortical neoplasms in children*
Risk Criteria
Low Any cortical neoplasm confined to the adrenal gland and weighing less than 200g.
Intermediate Any cortical neoplasm confined to the adrenal gland and weighing between 200 and
400g. Any cortical neoplasm weighing less than 400g with microscopic invasion into
surrounding soft tissues, completely resected, and no evidence of metastatic spread.
High Any cortical neoplasm weighing in excess of 400g or with direct gross invasion into
adjacent organs (ex. liver, spleen, or kidney) or with metastatic spread.
*Adapted from Dehner et al.3
The 5-year survival rate for ACA, as expected, is 100% [19]. Adult ACC carries a
guarded prognosis, with reported 5-year overall survival rates varying between 38 and 60%
[17, 18] versus the more favorable overall prognosis of 60-70% disease-free survival in
children [3, 19, 20]. In children, disease specific survival varies greatly with tumor stage.
Low-stage ACCs are associated with a 100% 5 year survival rate, versus 0% survival for
stage III and IV disease [19]. Children who are 5 years or older tend to have worse outcomes
because of a trend toward larger tumors and more advanced disease at presentation [1, 2, 12].
As in adults, children with high-stage ACC have a rapidly fatal course with an average
survival of several months to 3 years after diagnosis. Disease recurrence is associated with
poor outcome and typically occurrs between 4 and 16 months after initial resection [1, 6, 15,
18-20].
CONCLUSION
ACNs are rare in the pediatric age group. The majority occur in children less than 5 years
of age. Contrary to what was once believed, the great majority of pediatric ACNs are
biologically indolent and therefore can be considered benign, despite exhibiting pathologic
features that are associated with aggressive biologic behavior in similar appearing tumors of
adults. Proper classification of ACNs in children has been hampered by their traditional
inclusion in large case series that include both pediatric and adult ACNs. It is now understood
that those histologic criteria defining malignant features adult ACCs do not apply to children.
The most important features defining malignancy in children are tumor weight, invasion of
surrounding tissues/organs, and evidence of metastasis. Complete initial resection is essential
for a durable cure.
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Adrenal Cortical Tumors in Children: Factors Associated With Poor Outcome. Journal
of Pediatric Surgery. 2011;46:1201-7.
[2] McAteer JP, Huaco JA, Gow KW. Predictors of Survival in Pediatric Adenocortical
Carcinoma: A Surveillance, Epidemiology, and End Results (SEER) Program Study.
Journal of Pediatric Surgery. 2013;48:1025-31.
[3] Dehner LP, Hill DA. Adrenal Cortical Neoplasms in Children: Why So Many
Carcinomas and Yet So Many Survivors? Pediatric and Developmental Pathology.
2009; 12:284–91.
[4] Fassnacht M, Libé R, Kroiss M, Allolio B. Adrenocortical carcinoma: a clinician‘s
update. Nature Reviews Endocrinology. 2011;7:323-35.
[5] Neblett WW, Frexes-Steed M, Scott HW Jr. Experience with adrenocortical neoplasms
in childhood. American Surgeon. 1987;53:117-25.
[6] Cagle PT, Aubrey JH, Physher TJ, Page DL, Johnson EH, Kirkland RT, et al.
Comparison of Adrenal Cortical Tumors in Children and Adults. Cancer.
1986;57:2235-37.
[7] Onur MR, Turgut AT, Dogra VS, MacLennan GT. Adrenal Neoplasms. In: Dogra VS,
MacLennan GT, editors. Genitourinary Radiology: Male Genital Tract, Adrenal and
Retroperitoneum. The pathologic basis. 1st ed. London: Springer-Verlag; 2013.
[8] Martínez León MI, Romero Chaparro S, Weil Lara B, Domínguez Pinos MD, Ceres
Ruiz L, Ibáñez Cerrato F, Escobosa Sánchez O. Adrenocortical tumors in children:
imaging adenomas and carcinomas. Radiologia. 2012;54:342-9.
[9] Lacson A, deSa DJ. Endocrine system. In: Gilbert-Barness E, Kapur RP, Oligny LL,
Siebert JR, editors. Potter's pathology of the fetus and child. 2nd ed. Philadelphia:
Mosby; 2007.
[10] Hough AJ, Hollifield JW, Page DL, Hartmann WH. Prognostic factors in adrenal
cortical tumors. A mathematical analysis of clinical and morphologic data. American
Journal of Clinical Pathology. 1979;72:390–9.
[11] Weiss LM, Medeiros LJ, Vickery AL Jr. Pathologic features of prognostic significance
in adrenocortical carcinoma. American Journal of Surgical Pathology. 1989;13:202–6.
[12] Van Slooten H, Schaberg A, Smeenk D, Moolenaar AJ. Morphologic Characteristics of
Benign and Malignant Adrenocortical Tumors. Cancer. 1985;55:766–73.
[13] Blanes A, Diaz-Cano SJ. Value of mitotic figure variability. American Journal of
Clinical Pathology. 2007;127:398-408.
[14] Michalkiewicz E, Sandrini R, Figueiredo B, Miranda ECM, Caran E, Oliveria-Filho

AG, et al. Clinical outcome characteristics of children with adrenocortical tumors: a
report from the International Pediatric Adrenocortical Tumor Registry. Journal of
Clinical Oncology. 2004;22:838–845.
[15] Wieneke JA, Thompson L, Heffess CS. Adrenal cortical neoplasms in the pediatric
population. American Journal Surgical Pathology. 2003;27:867–881.
[16] Sbragia L, Oliveira-Filho AG, Vassallo J, Pinto GA, Guerra-Junior G, Bustorff-Silva J.
Adrenocortical tumors in Brazilian children. Immunohistochemical markers and
prognostic features. Archives of Pathology and Laboratory Medicine. 2005;129:1127–
1131.
[17] Ayala-Ramirez M, Jasim S, Feng L, Ejaz S, Deniz F, Busaidy N, et al. Adrenocortical
carcinoma: clinical outcomes and prognosis of 330 patients at a tertiary care center.
European Journal of Endocrinology. 2013;169:891-9.
[18] Vassilopoulou-Sellin R, Pamela N. Schultz. Adrenocortical Carcinoma Clinical
Outcome at the End of the 20th Century. Cancer. 2001;92:1113-21.
[19] Hanna AM, Pham TH, Askegard-Giesmann JR, Grams JM, Iqbal CW, Stavlo P, et al.
Outcome of adrenocortical tumors in children. Journal of Pediatric Surgery.
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[20] Mayer SK, Oligny LL, Deal C, Yazbeck S, Gagné N, Blanchard H. Childhood
adrenocortical tumors: case series and reevaluation of prognosis: a 24-year experience.
Journal of Pediatric Surgery. 1997;32:911-5.
Chapter 12
PREMATURE ADRENARCHE
Alexander K. C. Leung1,, Kam Lun Hon2

and Benjamin Barankin3
1
Department of Pediatrics, University of Calgary,
The Alberta Children‘s Hospital, Calgary, Alberta, Canada
2
Department of Paediatrics, Chinese University of Hong Kong,
Shatin, Hong Kong, China
3
Toronto Dermatology Centre, Toronto, Ontario, Canada
ABSTRACT
Adrenarche refers to puberty of the adrenal gland and is characterized by activation
of adrenal androgen production, and by impressive increases in dehydroepiandrosterone
(DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and testosterone.
Premature adrenarche is secondary to an earlier than normal isolated maturation of the
zona reticularis with an increase in adrenal androgen secretion high for prepubertal
chronological age- and sex-specific reference range but appropriate for Tanner puberty
development stage II to III. Premature or precocious adrenarche refers to an isolated
development of pubic hair (pubarche) before eight years of age in girls and nine years in
boys, without the appearance of other signs of sexual maturation. Dark, coarse, and often
curly hair is the first clinical sign. The hair is limited initially to the labia majora in girls
or to the root of the penis in boys and then extends gradually into the pubic region.
Axillary hair, adult type body odor, and oily hair or skin are not uncommonly seen. Acne
and seborrhea may also be present. Hirsutism, deepening of the voice, clitoral
enlargement, vaginal discharge, breast development, phallic or testicular enlargement,
hypertension, and other evidence of virilization are characteristically absent. A transient
acceleration of growth is common but final height is usually not affected. The onset of
puberty usually occurs at the normal age. Black children are much more frequently
affected than Caucasian children. The female to male ratio is approximately 10:1. Both
prematurity and intrauterine growth retardation might predispose to premature adrenarche
in susceptible individuals. Excess weight gain might be a trigger for adrenarche, and
obesity is reported to be associated with a higher incidence of premature adrenarche. The

Corresponding Author - Fax: (403) 230-3322, E-mail: aleung@ucalgary.ca.
282 Alexander K. C. Leung, Kam Lun Hon and Benjamin Barankin
parents and child should be reassured that, in most cases, premature adrenarche is a
benign condition and that the child will develop normally. Continued observation and
periodic re-evaluation are necessary because premature adrenarche might be the first sign
of precocious puberty. In some girls, premature adrenarche might be a forerunner of
polycystic ovary syndrome or syndrome X. Girls with higher body mass index warrant
particularly close follow-up. Early identification of these patients can allow early
treatment of the appropriate conditions with reduction in risk for early cardiovascular
disease.
Keywords: premature adrenarche, pubarche, pubic hair, adrenal androgen, polycystic ovary
syndrome, syndrome X
INTRODUCTION
Adrenarche refers to the developmental maturation of the adrenal gland and usually
occurs shortly before the onset of gonadarche [1]. The term ―adrenarche‖ was coined by
Fuller Albright and Nathan Talbort in the early 1940s when they linked the developmental
rise in adrenal androgens to the appearance of pubic and axillary hair which they referred to
as ―sexual hair‖ [2, 3]. The condition is characterized by activation of adrenal androgen
production, and by impressive increases in dehydroepiandrosterone (DHEA),
dehydroepiandrosterone sulfate (DHEAS), androstenedione, and testosterone [4]. Adrenarche
is an enigmatic phenomenon that occurs only in humans and some higher primate species
such as the chimpanzee and gorilla [5]. Premature or precocious adrenarche refers to an
earlier than normal secretion of adrenal androgens which results in an isolated development
of pubic hair (pubarche) before eight years of age in girls and nine years of age in boys,
without the appearance of other signs of sexual maturation such as breast development in girls
and testicular enlargement in boys [6]. In premature adrenarche, serum adrenal androgens (or
urinary adrenal androgen metabolite excretion) are high for prepubertal chronological age-
and sex-specific reference range but appropriate for Tanner puberty development
stage II to III [5, 7-9].
Precocious development of pubic hair can cause embarrassment to the child and anxiety
to the parents. Clinicians should be concerned since precocious development of pubic hair can
be the first overt sign of an androgen-secreting tumor of a gonad or adrenal gland, congenital
adrenal hyperplasia, or true precocious puberty [10]. Also, recent studies suggest that
premature adrenarche in some girls can be a forerunner of polycystic ovary syndrome and/or
syndrome X [5].
EPIDEMIOLOGY
In a cross-sectional study of 1,231 school girls of Eastern European origin, only 2 (0.8%)
of 255 girls aged 7 to 7.9 years were found to have premature adrenarche [11]. Black children
are much more frequently affected than Caucasian children [1, 6]. The female to male ratio is
approximately 10:1 [5, 6, 12]. Both prematurity and intrauterine growth retardation might
predispose to premature adrenarche in susceptible individuals [8, 12, 13]. Excess weight gain
Premature Adrenarche 283
might be a trigger for adrenarche, and obesity is reported to be associated with a higher
incidence of premature adrenarche [8, 12, 13]. The occurrence of premature adrenarche is
usually sporadic, although familial occurrence has also been described [7].
PATHOGENESIS
Adrenarche begins several years before the onset of gonadal maturation and correlates
with the appearance of the zona reticularis of the adrenal gland [9]. Adrenarche is
independent of gonadarche and gonadotropins and proceeds even in individuals with gonadal
dysgenesis [6, 14]. In normal puberty, adrenarche and gonadarche are closely linked.
Premature adrenarche is secondary to an early isolated maturation of the zona reticularis,
the innermost layer of the human adrenal cortex. This leads to a shift in the activity of adrenal
enzymes with accretion of 17, 20-lyase and 17-hydroxylase activities, with resultant increase
in adrenal androgen secretion for the chronological age but with normal
glucocorticoid levels [15, 16].
The principal adrenal androgens secreted are androstenedione, DHEA, and DHEAS [9,
17]. DHEA is converted to DHEAS which is biologically inactive while dihydrotestosterone
(through androstenedione and testosterone) is the most potent androgen. The effect of
androgens is mediated through the androgen receptor. Several pathophysiological
mechanisms for early isolated maturation of the zona reticularis have been proposed, namely,
dysregulation of CYP17 (P450c17) enzyme, overweight or sudden weight gain, premature
and rapid development of zona reticularis, and hyperinsulinism [14, 16]. In a Finnish study,
the androgen receptor CAG repeat, the length of which correlates inversely with androgen
sensitivity, has been shown to be shorter in girls with premature adrenarche [18]. Suffice to
say, the majority of children with premature adrenarche have idiopathic premature adrenal
androgen secretion [8].
Oversecretion of adrenocorticotropic hormone (ACTH) or corticotropin-releasing
hormone (CRH) per se cannot account for premature adrenarche because ACTH always
causes a greater increase in corticosteroids than in androgens. In patients with
hyperadrenocorticotropism and hypercortisolemia, DHEA and DHEAS are not usually
elevated. Nevertheless, ACTH and CRH might have a permissive role in the modulation of
adrenal androgen secretion [5, 6]. An increase of a central androgen-stimulating
proopiomelanocortin-derived hormone might be the primum movens of premature adrenarche
[19]. Increased sensitivity of the sexual hair follicles to androgens has also been suggested as
a mechanism because in some patients, premature pubarche is associated with normal
androgen levels [4]. This might account for the increased prevalence of premature adrenarche
among black children [1]. The association of premature adrenarche with prematurity and
intrauterine growth retardation suggests that premature adrenarche might be a component of a
fetal or neonatal programming event [16, 20].
Insulin resistance has been identified in some patients with premature adrenarche of
Spanish, Caribbean-Hispanic and African-American origin [21]. On the other hand, de Ferran
et al. could not establish such a relationship in a cohort of 52 Brazilian girls with premature
adrenarche [16].
CLINICAL MANIFESTATIONS
The frequency of premature adrenarche increases with age between 3 and 8 years of age
in girls, and between 3 and 9 years of age in boys, although cases have been reported as early
as 5 weeks of age [1]. Dark, coarse, and often curly hair is the first clinical sign. The hair is
limited initially to the labia majora in girls or to the root of the penis in boys and then extends
gradually into the pubic region. The amount and thickness of the hair might progress very
slowly or not at all. Axillary hair, adult type body odor, and oily hair or skin are not
uncommonly seen [1, 17, 22]. Acne and seborrhea may also be noted [1, 17, 22]. Acanthosis
nigricans is more common in individuals with premature adrenarche [1, 17]. Hirsutism,
deepening of the voice, clitoral enlargement, vaginal discharge, breast development, phallic
or testicular enlargement, hypertension, and other evidence of virilization or precocious
puberty are characteristically absent [7, 17].
A transient acceleration of growth is common. A substantial number of these patients are
tall and overweight [23, 24]. Final height is usually not affected, except in those with
significant bone age advancement [19, 23, 25]. Children with premature adrenarche and
significant bone age advancement tend to have a sub-optimal adult height, especially in those
with coexisting obesity [23]. The onset of puberty usually occurs at the normal age [8, 19]. In
girls with premature adrenarche, menarche tends to occur slightly (0.5 year) earlier, but
usually within normal limits [9, 26]. In girls with premature adrenarche and a history of low
birth weight, a three-fold increase in menarche before 12 years of age has been
reported [8, 27].
DIFFERENTIAL DIAGNOSIS
Premature adrenarche is a diagnosis of exclusion. The differential diagnosis of premature
development of pubic hair is shown in Table 1. Pubic hair of infancy is presumably due to
transiently elevated androgen levels in the first few months of life and increased sensitivity of
sexual hair follicles to androgens [28].
Shortly after birth, a transient surge of gonadotropins occurs and leads to a sharp increase
in testosterone levels which peaks at 1 to 3 months of age [28]. Thereafter, the gonadotropin
levels fall, and by 6 months of age, serum levels of testosterone decrease to low prepubertal
levels.
In pubic hair of infancy, pubic hair usually occurs in an atypical location such as the
scrotum in boys and the mons pubis in girls [28]. The growth of the pubic hair is slowly
progressively in the first few months of life, remains stationary for a few more months, and
then might regress.
Precocious puberty may be central (gonadotrophin-dependent precocious puberty) or
peripheral (gonadotrophin-independent; also known as pseudoprecocious puberty). Central
precocious puberty is often idiopathic and is caused by early maturation of the hypothalamic-
pituitary-gonadal axis. In isosexual precocious puberty, the changes that characterize puberty
occur in more or less the usual order, but at a much earlier age. Common findings in girls
with isosexual precocious puberty include rapid linear growth, development of breasts,
feminine body contours, pubic and axillary hair, and early onset of menses (menarche) [29].
In boys, isosexual precocious puberty is marked by rapid linear growth, testicular and penile
enlargement, development of a masculine build, acne and/or seborrhea, deepening of voice,
and growth of pubic and axillary hair [29]. Spermatogenesis has been observed as early as 5
or 6 years of age, and nocturnal emissions can occur [29]. Premature adrenarche is extremely
difficult, if not impossible, to differentiate from the early stage of constitutional precocious
puberty [1].
Table 1. Differential diagnosis of premature development of pubic hair
Condition Characteristics
Pubic hair of Pubic hair usually occurs in an atypical location such as scrotum in boys
infancy and mons pubis in girls. The growth of pubic hair is slowly progressively in
the first few months of life, remains stationary for a few months, and then
might regress.
Premature Isolated development of pubic hair without the appearance of other signs of
adrenarche sexual maturation. The onset of puberty is normal.
Precocious Concomitant appearance of pubic hair with breast development in girls or

puberty with testicular enlargement in boys.
Virilizing Acne, clitoral or phallic enlargement, hirsutism, markedly accelerated

tumors linear growth, and deepening of the voice at the same time or shortly after
the onset of pubic hair development.
Late-onset In addition to premature development of sexual hair, hirsutism and

congenital menstrual irregularities are usually present. In the male, the testes usually
adrenal remain infantile, but other secondary sexual characteristics are advanced. A
hyperplasia positive family history is suggestive.
Iatrogenic A history of androgen exposure will give clue to the diagnosis.
Sexual hair might be the first sign of virilization due to an adrenocortical or gonadal
tumor. In these conditions, other evidence of excessive androgen secretion such as acne
and/or seborrhea, clitoral or phallic enlargement, hirsutism, increased muscle mass, markedly
accelerated linear growth, and deepening of the voice usually appear at the same time or
shortly after the onset of pubic hair development. In boys with an adrenocortical tumor, the
testicular size is usually small, whereas the reverse is true for boys with a testicular tumor [7].
Exogenous androgen exposure can cause development of sexual hair as well as other signs of
virilization. A careful history about drug exposure is important.
Congenital adrenal hyperplasia, usually due to 21-hydroxylase deficiency, classically
produces prenatal virilization in females but can, in a late-onset attenuated form, present with
premature development of sexual hair, hirsutism, and menstrual irregularities. In the male, the
testes usually remain infantile but other secondary sexual characteristics are advanced [7].
The diagnosis is suspected when serum androgens are elevated in the presence of prepubertal
gonadotropins [8]. A positive family history of congenital adrenal hyperplasia is suggestive.
COMPLICATIONS
Girls with premature adrenarche have been shown to tend to perform less well on verbal,
working memory, and visuospatial tasks compared to on-time peers [30]. Precocious
development of pubic hair can cause embarrassment to the child and anxiety to the parents
[1]. Mood and behavioral problems are more common in girls with premature adrenarche than
on-time adrenarche girls [31]. These may interfere with parent-child interaction, peer
relationships, and school performance [31].
Premature adrenarche in girls is also linked to a variety of emotional and behavioral
problems such as depression, anxiety, and aggression [32]. In general, girls with premature
adrenarche with lower levels of executive functioning have higher externalization and
anxious symptoms compared to on-time peers [33]. In addition, girls with premature
adrenarche who have increases in serum cortisol are more likely to have higher externalizing
symptoms than those with stable pattern [33]. Furthermore, girls with premature adrenarche
who have decreases in cortisol are more likely to have depressive symptoms.
Girls with premature adrenarche have a higher incidence of polycystic ovary syndrome
later in life [6, 7, 17]. Hyperinsulinism, insulin resistance, and increased free insulin-like
growth factors have been suggested as the common origin of premature adrenarche and
polycystic ovary syndrome [6]. It has been shown that insulin and insulin-like growth factors
(IGF1 and IGF2) are capable to stimulate steroidogenesis in human fetal and adult adrenal
cells in vitro [34]. Polycystic ovary syndrome is characterized by menstrual irregularities,
obesity, acne, hirsutism, and polycystic ovaries and is a significant cause of female
infertility [1, 35].
In some girls, premature adrenarche may be a forerunner of syndrome X (obesity,
hypertension, insulin resistance, type 2 diabetes, and dyslipidemia), especially those with
polycystic ovary syndrome [5, 6, 17, 31]. Affected patients are at increased risk for early
atherosclerotic cardiovascular disease [5, 17, 36]. In contrast, premature adrenarche in boys
may be associated with an increased incidence of reduced insulin sensitivity but
not syndrome X [36].
LABORATORY STUDIES
Laboratory investigations should be done to exclude other pathologies and to confirm the
diagnosis. Serum concentrations of DHEA, DHEAS, androstenedione, testosterone, sex
hormone binding globulin (SHBG), and 17-hydroxyprogesterone as well as urinary 17-
ketosteroids should be measured [8].
In premature adrenarche, DHEA, DHEAS, androstenedione, testosterone levels are
usually increased for chronological age, and are in the range of those found in early puberty
or Tanner stage II to III pubic hair [8, 19, 23]. Serum androstenedione and testosterone levels
are usually mildly elevated [8]. DHEAS levels, however, might exceed those of pubertal
controls [7]. In girls, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and
estradiol should be measured if clinical examination is suggestive of gonadarche [17]. In
boys, serum β-human chorionic gonadotropin (hCG) should be obtained to rule out an hCG-
secreting tumor [8].
Children with premature adrenarche tend to have higher mean blood erythrocyte count
and hemoglobin concentrations than their prepubertal peers [37]. Presumably, the small
increases in androgen production during adrenarche are able to stimulate erythropoiesis [37].
Traditionally, a bone age X-ray is performed to identify a subset of patients with a high
risk of endocrinopathy. Previous studies have shown that in premature adrenarche, the bone
age is usually within 2 standard deviations of chronological age [26]. Recently, DeSalvo et al.
performed a retrospective study based on chart review of 266 patients (mean age 7.2 years;
82% female) with premature adrenarche [38]. Of the 122 patients with bone age available,
30.6% had bone age advanced by ≥2 years, 32.2% had bone age advanced between 1 to 2
years, and 37.2% had bone age within 1 year of chronological age. The authors conclude that
a bone age advanced by ≥2 years in children with premature adrenarche is quite common and
is not a cause for concern [38]. The finding needs to be confirmed by future well-designed,
large-scale studies.
Moderately elevated levels of serum androgen other than DHEAS, elevated early
morning 17-hydroxyprogesterone level, marked bone age advancement, or signs of atypical
premature pubarche such as cystic acne or signs of systemic virilization indicate the need for
an ACTH test to rule out congenital adrenal hyperplasia [7, 8]. An excessive increase in
serum 17-hydroxyprogesterone level to > 45 nmol/L after an ACTH stimulation test suggests
late-onset congenital adrenal hyperplasia [19].
Marked elevation of serum androgen levels and very advanced bone age suggest the
possibility of an adrenocortical or gonadal tumor [7]. Abdominal ultrasonography to rule out
an adrenal tumor should be performed if initial studies show marked elevation of DHEAS [8].
A patient with an androgen-producing adrenocortical tumor does not respond to ACTH
stimulation or dexamethasone suppression, whereas a patient with congenital adrenal
hyperplasia does [7]. Adrenal computed tomography or magnetic resonance imaging should
be performed if significant virilization occurs and ACTH stimulation does not reveal
congenital adrenal hyperplasia or if a mass is noted [7, 8].
Pelvic ultrasonography should be performed if polycystic ovary syndrome is suspected.
The presence of an enlarged ovary and multiple small follicles scattered around an echogenic
stroma establishes the diagnosis [19]. Color Doppler flow measurements might reveal
significant vascular changes within the intra-ovarian vessels in patients with polycystic ovary
syndrome [19]. Serum glucose, insulin, cholesterol, and triglyceride levels should be
measured if syndrome X is suspected. Carotid artery ultrasonography may be useful to detect
early manifestations of the cardiovascular changes seen in patients with syndrome X [36].
MANAGEMENT
Education and reassurance of the patient and family as well as psychological/emotional
support for the child and family are essential to the clinical management of such patients [7].
The parents and child should be reassured that, in most cases, premature adrenarche is a
benign condition and that the child will develop normally. Continued observation and
periodic re-evaluation are necessary because premature adrenarche might be the first sign of
precocious puberty. Periodic assessment of growth velocity, weight gain, and observation of
signs of androgen excess are indicated [8].
In some girls, premature adrenarche might be a forerunner of polycystic ovary syndrome

or syndrome X. Lifestyle interventions such as increasing physical activity and reducing
overweight are important measures in the prevention and treatment of premature adrenarche
and its possible long-term consequences [9]. Girls with higher body mass index (BMI)
warrant particularly close follow-up [7]. Early identification of these patients can allow early
treatment of the appropriate conditions with reduction in risk for early
cardiovascular disease [7].
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adrenarche in appropriate for gestational age girls with clinical outcome. J. Pediatr.,
2012; 160:308-313.
[26] Pere, A.; Perheentupa, J.; Peter, M.; et al. Follow up of growth and steroids in
premature adrenarche. Eur. J. Pediatr., 1995; 154:346-352.
[27] Ibanez, L.; Jimenez, R.; de Zegher, F. Early puberty-menarche after precocious
pubarche: relation to prenatal growth. Pediatrics, 2006; 117: 117-121.
[28] Leung, A. K.; Hegde, H. R.; Stephure, D. K. Scrotal hair in identical twin infants. Int. J.
Dermatol., 2005; 43:1042-1044.
[29] Leung, A. K. Isosexual precocious puberty. In: Leung, A. K., ed. Common Problems in
Ambulatory Pediatrics: Symptoms and Signs. New York: Nova Science Publishers,
Inc., 2011, pp253-262.
[30] Tissot, A.; Dorn, L. D.; Rotenstein, D.; et al. Neuropsychological functioning in girls
with premature adrenarche. J. Int. Neuropsychol. Soc., 2012; 18:151-156.
[31] Dorn, L. D.; Rose, S. R.; Rotenstein, D.; et al. Differences in endocrine parameters and
psychopathology in girls with premature adrenarche versus on-time adrenarche. J.
Pediatr. Endocrinol. Metab., 2008; 21: 439-448.
[32] Brooks-Gunn, J.; Petersen, A. C.; Eichorn, D. The study of maturational timing effects
in adolescence. J. Youth Adolesc., 1985; 14:149-161.
[33] Sontag-Padilla, L. M.; Dorn, L. D.; Tissot, A.; et al. Executive functioning, cortisol
reactivity, and symptoms of psychopathology in girls with premature adrenarche. Dev.
Psychopathol., 2012; 24:211-223.
[34] Mesiano, S.; Katz, S. L.; Lee, J. Y.; et al. Insulin-like growth factors augment steroid
production and expression of steroidogenic enzymes in human fetal adrenal cortical
cells: implications for adrenal androgen regulation. J. Clin. Endocrinol. Metab.,
1997;82:1390-1396.
[35] Ibanez, L.; Diaz, R.; Lopez-Bermejo, A.; et al. Clinical spectrum of premature
pubarche: links to metabolic syndrome and ovarian hyperandrogenism. Rev. Endocrin.
Metab. Disord., 2009; 10:63-76.
[36] Mathew, R. P.; Hernanz-Schulman, M.; Wang, W.; et al. Changes in carotid artery
sonogram in premature adrenarche. J. Pediatr. Endocrinol. Metab., 2012; 25: 139-145.
[37] Utriainen, P.; Jaaskelainen, J.; Voutilainen, R. Blood erythrocyte and hemoglobin
concentrations in premature adrenarche. J. Clin. Endocrinol. Metab., 2013;98:e87-e91.
[38] DeSalvo, D.; Mehra, R.; Vaidyanathan, P.; et al. In children with premature adrenarche,
bone age advancement by 2 or more years is common and generally benign. J. Pediatr.
Endocrinol. Metab., 2013; 26: 215-221.
Chapter 13
HYPERANDROGENISM OF ADRENAL ORIGIN
Dominik Rachoń*, MD, PhD

Department of Clinical and Experimental Endocrinology,
Medical University of Gdańsk, Poland
ABSTRACT
In contrast to the zona glomerulosa, the zonae fasciculata and reticularis of the
adrenal cortex, due to the 17,20-lyase activity of the cytochrome P450c17, are able to
produce the 19-carbon steroids (C-19), which include dehydroepiandrosterone (DHEA)
and androstenedione. Therefore, adrenal cortex is said to be the source of so called
―adrenal androgens‖ which actually have a very weak affinity to the androgen receptors
and function primarily as precursors for the peripheral conversion to testosterone (TST)
and dihydrotestosterone (DHT). In eugonadal males less then 5% of circulating TST
derives from the peripheral conversion of adrenal androstendione, hence adrenal
androgen synthesis in adult males is negligible. In females however, adrenal cortex
contributes to more then two-thirds of the circulating TST concentrations. In clinical
practice the most frequent cause of an excessive adrenal androgen production is due to
the congenital adrenal hyperplasia (CAH) caused by the enzymatic defects of different
severity, which impair the production of cortisol leading to an exaggerated ACTH
secretion, which in turn results in excessive production of DHEA and androstendione.
Peripheral conversion of high androstendione concentrations into TST and DHT give rise
to clinical symptoms of androgen excess, which in females usually manifests as
hirsutism, acne and alopecia but also cause fertility problems or can lead to virilisation.
Other rare causes of an excessive adrenal androgen production include Cushing‘s disease
and adrenal carcinoma.
Keywords: adrenal cortex, adrenal androgens, hyperandrogenism, dehydroepiandrosterone,

androstenedione, congenital adrenal hyperplasia
*
Email: drachon@gumed.edu.pl.
292 Dominik Rachoń
INTRODUCTION
In contrast to the zona glomerulosa, the zonae fasciculata and reticularis of the adrenal
cortex, due to the 17,20-lyase activity of the cytochrome P450c17, are able to produce the 19-
carbon steroids (C-19), which include dehydroepiandrosterone (DHEA) and androstenedione
[1]. DHEA by the activity of a reversible adrenal sulfokinase is converted to the DHEA
sulfate (DHEA-S), which turns out to be the most abundant circulating steroid hormone in
humans. Therefore, adrenal cortex is said to be the source of so called ―adrenal androgens‖
which actually have a very weak affinity to the androgen receptors and function primarily as
precursors for the peripheral conversion to testosterone (TST) and dihydrotestosterone
(DHT). Although DHEA and its sulphate (DHEA-S) are secreted in greater amounts,
androstenedione is more readily converted peripherally to TST and therefore qualitatively
more important (reviewed in Ref. 2).
As in the case of cortisol, the production of DHEA and androstenedione exhibit circadian
periodicity in concert with the ACTH secretion. In contrast, DHEA-S does not exhibit a
diurnal rhythm due to the small metabolic clearance rate. Similarly, the administration of
glucocorticosteroids inhibits the adrenal DHEA and androstenedione secretion and lowers
circulating DHEA-S levels [3]. All C-19 steroids are also secreted in an unbound state
however on entering the circulation they bind weakly to albumin. TST however is bound
more extensively to the sex hormone-binding globulin (SHBG).
In eugonadal males less then 5% of circulating TST derives from the peripheral
conversion of adrenal androstenedione, hence adrenal androgen synthesis in adult males is
negligible [1]. In females however, adrenal cortex contribute to more then two-thirds of the
circulating TST concentrations and in certain conditions can be the source of an excessive
androgen production.
CAUSES OF EXCESSIVE ADRENAL ANDROGEN PRODUCTION

Congenital Adrenal Hyperplasia (CAH)
Congenital adrenal hyperplasia (CAH) refers to an inherited group of autosomal recessive

disorders caused by the mutations in genes encoding enzymes involved in the adrenal
steroidogenesis. The most frequent is the mutation of the 21-hydroxylase genes (P450c21A
and P450c21B). Other rare causes include the mutations in the 11β-hydroxylase (P450c11B
and P450c11AS) and 3β-hydroxysteroid dehydrogenase genes. Severe cortisol deficiency in
the classic form of CAH, leads to the increased pituitary ACTH secretion via a negative
feedback loop causing hyperplasia of the adrenal cortex. In the case of 21-hydroxylase
deficiency, responsible for 90-95% of CAH cases, cortisol precursors produced proximal to
the enzymatic defect accumulate and are shunted into the androgen pathway resulting in
symptoms of androgen excess causing external genital ambiguity in newborn females and
progressive postnatal virilization in both sexes (reviewed in Ref. 4).
Lack of the 11β-hydroxylase activity also leads to androgen excess causing ambiguous
genitalia in females and signs of hyperandrogenaemia in both sexes with accompaning
hypertension. This is due to the accumulation of steroids proximal to the enzymatic blockade
Hyperandrogenism of Adrenal Origin 293
such as deoxycortisol and 11-deoxycortisol, which show strong mineralocorticoid activity

leading to the excess sodium and water retention [5]. In the case of 3β-hydroxysteroid
deficiency not only adrenal but also gonadal androgen production is impaired leading to
incomplete masculinization of the external genitalia in genetic males and various degrees of
mineralocorticoid deficiency in both sexes [6].
In so called ―non-classical CAH‖ (NCCAH) or ―adult onset CAH‖ the 21-hydroxylase
deficiency is partial (20-50% of normal activity) [7] and the affected female individuals
usually start seeking medical help in adulthood due to hirsutism, fertility problems or
menstrual abnormalities [8, 9], which often simulate the features of the polycystic ovary
syndrome (PCOS).
It turns out that NCCAH is the most common of all autosomal recessive diseases [10].
Whats more, a higher frequency of NCCAH due to 21-hydroxylase deficiency was reported in
certain ethnic groups. These include Ashkenazi Jews (1:27), Hispanics (1:40), Slavs (1:50)
and Italo-Americans (1:300) [10-14].
Whereas the diagnosis of the classical form of the CAH (―salt wasting‖ or ―simple
virilizing‖) is often made after birth according to the typical clinical picture and very high
serum concentration of 17-hydroxy-progesterone (17-OHP > 500 ng/dL) the diagnosis of
NCCAH is usually made according to elevated serm 17-OHP concentrations after the
administration of a synthetic ACTH.
In eumenorrheic women the ACTH stimulation test should be performed in the follicular
phase of the cycle (best between the days 4 and 10). In order to diagnose NCCAH due to 21-
hydroxylase deficiency serum 17-OHP concentrations must rise above 10 ng/mL 60 min after
i.v. administration of a synthetic ACTH (250 µg) [15]. Basal serum 17-OHP concentrations
greater than 8 ng/mL are also usually diagnostic of 21-hydroxylase deficiency whereas serum
17-OHP concentration of less then 2 ng/ml obtained during the follicular phase in
eumenorrheic patients usually exclude the diagnosis of NCCAH and do not warrant the
performance of an ACTH stimulation test [16].
Androgen excess of adrenal origin can also be confirmed by the measurement of serum
TST, DHEA-S or androstenedione in a 2 day suppression test with dexamethasone in which
serum concentrations of the above hormones are measured at baseline and after 2 days of
treatment with dexamethasone (0.5 mg every 6 h for 48 hours). A significant decrease
(> 50%) in serum total TST, DHEA-S or androstenedione following dexamethasone
administration, points to the adrenal origin of hyperandrogenism. Apart from being less
cumbersome, this test can be done in an outpatient or when the ACTH stimulation test cannot
be performed due to the lack of the synthetic ACTH or incapability to measure serum 17-
OHP concentrations. The concomitant measurement of serum cortisol levels will additionally
exclude the presence of Cushing‘s syndrome (described below) [17].
Hyperandrogenism Accompanying Cushing’s Disease
Cushing‘s syndrome is a clinical feature caused by cortisol excess, which per se cannot
give rise to the signs of hyperandrogenism (see also chapter 7). Whereas the most common
cause is iatrogenic, endogenous hypercortisolaemia usually results from an ACTH secreting
pituitary adenoma (Cushing‘s disease). Very rarely ectopic sources of ACTH may also be
294 Dominik Rachoń
encountered (i.e., small cell lung carcinoma). In these both cases, due to an exaggerated
production of the androgen precursors in the zonae fasciculata and reticularis of the adrenal
cortex, signs of hyperandrogenism in females are usually present. Although Cushing‘s disease
is a very rare cause of hyperandrogenaemia it must always be taken into the consideration in a
female patient with a recent onset of hyperandrogenism accompanied by signs of
hypercortisolism (facial fullness and plethora, buffalo hump, red striae, proximal myopathy,
hypertension) [17]. In this particular clinical setting, the easiest way of excluding Cushing‘s
syndrome is by performing a 1 mg dexamethasone overnight suppression test which is very
simple and convenient in an outpatient [18]. Dexamethasone is given at a dose of 1 mg orally
around 11:00 PM and serum cortisol is measured between 8:00 and 9:00 AM of the following
morning. Postdexamethasone serum cortisol concentrations less then 1.8 µg/dl (< 50 nmol/L)
usually exclude the presence of Cushing‘s syndrome.
Alternatively, a 2 mg 48 h dexamethasone suppression test can be performed with the
concomitant measurement of serum androgen precursors (androstendione or DHEA-S) and
TST concentrations. Dexamethasone is usually given in doses of 0.5 mg every 6 hours for
2 days, beginning at 9:00 AM. Serum cortisol is measured at around 9:00 AM on the third
day (6 hours after the last dexamethasone dose).
Another approach is to begin oral dexamethasone administration at 12:00 PM and obtain
serum cortisol concentrations at around 8:00 AM on the third day, exactly 2 h after the last
dexamethasone dose [19]. The cutoff value for the suppression of serum cortisol is the same
as in the 1 mg overnight suppression test. Decrease in the postdexamethasone concentrations
of androgen precursors and serum TST, which were mildly elevated at baseline, usually
points to the adrenal origin of hyperandrogenism (i.e., due to 21-hydroxylase deficiency) [17].
Hyperandrogenism due to Adrenal Adenoma or Cancer
Recent onset and rapidly progressing symptoms of hyperandrogenism in a previously

eumenorrheic woman always warrant the investigation towards an androgen-producing
tumor. Rapid onset of androgen excess in the affected female individual usually give rise to
the signs of virilization such as: temporal balding, deepening of voice, decreased breast size,
increased muscle mass, loss of female body contours, and clitoral enlargement over a short
period of time (less than a few months) [17].
The most frequent are the androgen secreting tumors of the ovaries. Androgen-secreting
neoplasms of the adrenal glands are extremely rare and usually turn out to be the adreno-
cortical cancer [20].
They usually secrete large amounts of DHEA, its sulphate (DHEA-S) and
androstenedione, which are readily converted into TST. Serum DHEA-S levels over
800 ng/mL are therefore usually suggestive of an adrenal tumor and warrant an abdominal
scan by either computed tomography or magnetic resonance imaging (MRI) [21]. In
androgen-secreting adrenal tumors a concomitant excess producton of cortisol is usually
present giving rise to clinical signs of hypercortisolaemia (described above).
Treatment of Hyperandrogenism of Adrenal Origin
Hyperandrognism of adrenal origin caused by enzymatic defects leading to the

impairment of cortisol and mineralocorticoid production with accompanying adrenal
hyperplasia and excess androgen production due to the exaggerated ACTH secretion always
warrants replacement thereapy with glucocorticosteroids. In the ―salt waisting‖ form of CAH
additional treatment with mineralocorticoids (i.e., fludrocortisone) must always be
implemented (rewieved in Ref. 22).
Whereas in children, natural cortisol (hydrocortisone or cortisol acetate) should be used,
in adults with NCCAH synthetic glucocorticoids with high ACTH suppressing potential are
preferred. These include dexamethasone at the dose of 0.25-0.75 mg per day or prednisolone
at the dose of 2.5-7.5 mg. Recently it has been however postulated not to use dexamethasone
in women of reproductive age due to its ability to pass the placenta which, in the case of
unplanned pregnancy, poses a risk of imparing proper adrenal gland development in the fetus
[23].
In these circumstances prednisolone is preferred which is being deactivated by the
placental 11β-hydroxysteroid dehydrogenase type 2 and therefore will not affect the adrenals
of the fetus in the case of unplanned pregnancy. Nevertheless, in a female patient with
menstrual irregularity and the cosmetic signs of hyperandrogenism due to NCCAH such as
hirsutism, acne and alopecia, treatment with an oral contraceptive containing a progestin with
an antiandrogenic potential (i.e., cyproterone or dienogest) can also be implemented [24].
Treatment of hyperandrogenism due to Cushing‘s disease or androgen producing adrenal
cancer is obviously always through surgical intervention. Transphenoidal removal of the
ACTH secreting adenoma in the case of Cushing‘s disease or surgical resection of the
androgen secreting adrenal tumour is always the treatment of choice. In the case of an
unoperative adrenocortical cancer palliative treatment with mitotane - an adrenal
steroidogenesis inhibitor, seems to be the optimal strategy [20].
CONCLUSION
In clinical practice the most frequent cause of an excessive adrenal androgen production
is the CAH caused by the enzymatic defects of different severity, which impair the production
of cortisol and therefore lead to an exaggerated ACTH secretion, which in turn results in
excessive production of the androgen precursors (DHEA and androstendione). In these
patients treatment with glucocorticosteroids with high ACTH suppressing potential are
preferred. Nevertheless, in a female patient with menstrual irregularity and the cosmetic signs
of hyperandrogenism due to NCCAH such as hirsutism, acne and alopecia, treatment with an
oral contraceptive containing a progestin with an antiandrogenic potential (i.e., cyproterone or
dienogest) can also be implemented. Treatment of hyperandrogenism due to Cushing‘s
disease or androgen producing adrenal tumor is obviously always through surgical resection.
296 Dominik Rachoń
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[9] Kohn B, Levine LS, Pollack MS, Pang S, Lorenzen F, Levy D, et al. Late-onset steroid
21-hydroxylase deficiency: a variant of classical congenital adrenal hyperplasia. J Clin
Endocrinol Metab. 1982 Nov;55(5):817-27. PubMed PMID: 6288753.
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Prevalence of nonclassical steroid 21-hydroxylase deficiency based on a morning
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[11] Dumic M, Brkljacic L, Mardesic D, Plavsic V, Lukenda M, Kastelan A. 'Cryptic' form
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population. Acta Endocrinol (Copenh). 1985 Jul;109(3):386-92. PubMed PMID:
2992207.
[12] Dumic M, Brkljacic L, Speiser PW, Wood E, Crawford C, Plavsic V, et al. An update
on the frequency of nonclassic deficiency of adrenal 21-hydroxylase in the Yugoslav
population. Acta Endocrinol (Copenh). 1990 Jun;122(6):703-10. PubMed PMID:
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[13] Sherman SL, Aston CE, Morton NE, Speiser PW, New MI. A segregation and linkage
study of classical and nonclassical 21-hydroxylase deficiency. Am J Hum Genet. 1988
Jun;42(6):830-8. PubMed PMID: 3259403.
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Chapter 14
FUNCTIONAL ROLES OF CORTICOSTERONE

AND STRESS IN PENILE MORPHOLOGY
Diogo B. De Souza, PhD, Dilson Silva, PhD,

Célia M. Cortez, PhD, Waldemar S. Costa, PhD,
and Francisco J. B. Sampaio, PhD
Urogenital Research Unit, State University of
Rio de Janeiro, Rio de Janeiro, RJ, Brazil
ABSTRACT
The glucocorticoids are known as the stress hormones. It is known that these
hormones plays a role on different organic systems, and take part on physiologic
regulation and adaptation to stress. However the impact of glucocorticoids and stress on
the penis is very little studied. The present manuscript aims to present published and
original information on the action of both glucocorticoids and stress stimuli on the penile
morphology. Experiments were performed in prepubertal rats which were submitted to
corticosterone injections or stress stimuli. After the experimental period, the animals
were submitted to euthanasia and the penises were collected and processed for
morphometric analysis. The impact of corticosterone administration or stress induction
was assessed on the content of collagen and smooth muscle in penile corpus cavernosum.
Data was compared using statistical methods. Both glucocorticoids administration and
stress stimuli promoted morphological alterations on cavernous tissue. Interestingly, the
numeric results was opposite for these experiment protocols. While stress stimuli
promoted penile fibrosis, with collagen deposition, the corticosterone administration
promoted a diminution of collagen tissue. Also, in the stress induced animals, it was
noted a decrease of smooth muscle fibers while in the animals of the glucocorticoid
experiment, this structure was augmented in corpus cavernosum. Both corticosterone and
stress stimuli influences the penile morphology in an important manner. These alterations
may be related to erectile dysfunction and may explain some mechanisms by how
impotence occurs in stressed men and in those using glucocorticoids. Interestingly, these
experiments showed antagonic quantitative results, thus, it is possible that glucocorticoids
secretion may be a mechanism of defense in response to stress.
300 Diogo B. De Souza, Dilson Silva, Célia M. Cortez et al.
Keywords: penis, erectile dysfunction, stress, glucocorticoids
INTRODUCTION
Glucocorticoids are hormones produced and released by adrenal cortex that plays a role
on different organic systems, and take part on physiologic regulation and adaptation to stress
[1]. Cortisol (for humans) and corticosterone (for rats) are the major glucocorticoids secreted
by the hypothalamus-hypophysis-adrenal axis and participates on the homeostasis control and
on the organic response to stress conditions [2, 3]. Because of this involvement of
glucocorticoids on the stress response, these are known as the stress hormone.
Repeated stress may act as a trigger for various diseases. Glucocorticoids released in
response to stress are directly related to the regulation of the immune system and acts in
several tissues / organs [4]. Thus, for studying the stress effects on different tissues, some
researchers commonly give glucocorticoids for experimental animals.
Kavitha et al., studied the testicles after the excessive corticosterone administration [5].
These researchers found a Leydig cells steroidogenic alteration with testosterone diminution
[5]. In another experiment, corticosterone administration altered the bladder morphology of
rats. The authors showed alterations on the vascular density, collagen, elastic fibers and
smooth muscle of bladder wall [6]. Thus, it has been previously shown that the corticosterone
administration can influence urogenital tissue, and, thus, can change the penile morphology as
well as seen in bladder and testicle.
Furthermore, several studies use experimental conditions in order to submit animal
models to stress conditions [7, 8]. For these researchers, the stress would be a condition much
more complex than only a peak secretion of glucocorticoid. For most of the experimental
models used for stress induction, no physical aggression should be induced, since stress
commonly is primarily a psychological disorder.
In the present chapter, we present two experiments that investigated the stress on the
penile tissue. On the first experiment, the administration of corticosterone was used while in
the second experiment a restraint stress model was used.
Erectile dysfunction (ED) is a very frequent condition of men. It is thought to have a
worldwide prevalence between 10 and 20% [9]. Commonly, ED is a condition of older men,
however juvenile ED is not rare. Even though the etiology of juvenile ED have not been
completely elucidated, psychogenic origin of ED has been reported in at least 52% of teenage
patients [10]. The research on ED risk factors commonly does not consider the psychosocial
variables that contribute to it. It is known that stress and anxiety are highly associated with
ED [11] even so, few researchers have studied the relation of stress and ED.
One possible link between stress and ED is the decrease of testosterone observed in
experimental models using corticosterone or submitted to stress stimuli. This has been also
related to impaired sexual function on these models [5, 8, 12]. Since testosterone is an
important hormone for penile function, it is thought that ED may be involved in stressed
individuals. It is known that the normal penile morphology is essential to normal penile
erection. Man with ED have altered proportions of the corpus cavernosus elements [13, 14].
Modifications on cavernosal structure was also demonstred in experimental models with
diminished testosterone [15, 16].
Functional Roles of Corticosterone and Stress in Penile Morphology 301
Thus, it is thought that the morphology of corpus cavernosus of rats submitted to chronic
stress may be altered. However the impact of glucocorticoids and stress on the penis is very
little studied. The present manuscript aims to present published and original information on
the action of both glucocorticoids and stress stimuli on the penile morphology.
METHODS
For the glucocorticoid experiments, 22 newborn male rats were assigned into a control
group (n = 7) or a treated group (n = 15).
The treated group received daily intraperitoneal injections of corticosterone at a dose of 2
mg/Kg [5, 6], while control groups received the same volume of saline, by the same route.
These injections were applied from the 7th to the 25th day of life. The animals were submitted
to euthanasia at the 65th day of life when the penis were dissected and fixed in 4% buffered
formaldehyde.
For the stress stimuli experiments, 15 rats of 28 days of age were were assigned into a
control group (n = 7) or a stressed group (n = 8). The stressed grouped was submitted to
immobilization in a rigid opaque plastic cylinder that restrained the movements of the rats [8].
The cylinders, with different diameters and length, were adjusted weekly depending on the
animal‘s weight. The animals did not experience any pain and were contained for 2 hours
daily in the morning from the 28th to the 63rd day of life. The animals were submitted to
euthanasia at the 64th day of life when the penis were dissected and fixed in 4% buffered
formaldehyde. All experiments were done according to the Brazilian law for scientific use of
animals, and this project was formally approved by the local Ethics Committee.
The skin-denuded middle part of the penile shaft was processed for paraffin embedding
and cross-sections of 5µm thickness were obtained. Quantitative analysis of corpus
cavernosum smooth muscle and conjunctive tissue was performed in Masson‘s trichrome
stained slices captured under 200x magnification, by the point counting method [15, 17, 18].
This was used to objectively determinate smooth muscle and conjunctive tissue surface
density, expressed as percentage. Five different fields were selected from five non adjacent
slices, therefore a total of 25 images, or 2,500 points were counted for each animal.
Picrosirius red stained slices were observed under polarized light for differentiate collagen
types III (seen in green) and I (red/orange) [19].
The Student‘s-t-test was used for mean comparisons. In all cases, significance was set at
a probability value of 0.05.
Both glucocorticoids administration and stress stimuli promoted morphological
alterations on cavernous tissue. The group treated with glucocorticoids presented an increase
of 42.4% of smooth muscle content (14.6% for controls vs. 20.8% for treated) and a decrease
of 6.9% of connective tissue content (59.3% for controls vs. 55.2% for treated), both with
statistically significant differences. These findings are illustrated in Figure 1 and 2.
The group submitted to stress stimuli showed a decrease of 36.4% of smooth muscle
content (14.0% for controls vs. 8.9% for treated) and an increase of 20.0% of connective
tissue content (53.6% for controls vs. 64.5% for treated). Again, these differences, when
tested showed to be statistically significant. These findings are illustrated in Figure 3 and 4.
Figure 1. Photomicrographs of corpus cavernosum of control (A) and treated with corticosterone rat
(B). Analyzing images A and B, we can observe a reduction of connective tissue and an increase in
smooth muscle in treated animals. Masson‘s trichrome, X200.
a b
Figure 2. Graphic representing the density of connective tissue (A) and smooth muscle (B) on corpus
cavernosum of rats treated with corticosterone. Note that the treated animals showed a reduction of
connective tissue and an increase in smooth muscle.
Figure 3. Photomicrographs of corpus cavernosum of control rat (A) and rat submitted to stress stimuli
(B). Analyzing images A and B, we can observe an increase of connective tissue and a reduction in
smooth muscle in stressed animals. Masson‘s trichrome, X200.
When observed under polarized light in picrosirius red stained slices, the corpus
cavernosum of animals submitted to corticosterone treatment showed a higher predominance
of greenish colored collagen (indicating collagen type III) while animals submitted to stress
stimuli showed a higher predominance of red / orange collagen (indicating collagen type I),
(Figure 3). Interestingly, the numeric results was opposite for these experiment protocols.
a b
Figure 4. Graphic representing the density of connective tissue (A) and smooth muscle (B) on corpus
cavernosum of rats submitted to stress stimuli. Note that the stressed animals showed an increase of
connective tissue and a reduction in smooth muscle.
Figure 5. Photomicrographs of corpus cavernosum of rat treated with corticosterone (A) and rat
submitted to stress stimuli (B). Analyzing images A and B, we can observe a higher predominance of
greenish colored collagen (indicating collagen type III) in rat treated with corticosterone while animals
submitted to stress stimuli showed a higher predominance of red / orange collagen (indicating collagen
type I). Picrosirius red under polarization, X200.
While stress stimuli promoted penile fibrosis, with collagen deposition, the corticosterone
administration promoted a diminution of collagen tissue. Also, in the stress induced animals,
it was noted a decrease of smooth muscle fibers while in the animals of the glucocorticoid
experiment, this structure was augmented in corpus cavernosum. Finally, the results of
collagen types were also opposite. Penile erection requires adequate neural impulse
transmission, blood supply, and functional erectile tissue in the corpus cavernosum. ED can
develop from a defect in one of these issues or from a combination of two or more of them.
From an anatomical aspect, the corpus cavernosum is the most important structure involved in
penile erection. It is mainly composed of connective tissue, smooth muscle fibers, and
vascular trabeculae. Connective tissue must permit elongation and rigidity during erection and
allow rapid detumescence after orgasm. By the other hand, smooth muscle fibers should relax
to allow blood inflow and maintain the erection by increasing the intracavernous pressure.
Thus, in order to achieve a normal penile function, an adequate percentage of smooth muscle
fibers and connective tissue is required [13]. In these different models used for studying the
impact of stress on the tissues, we found that the glucocorticoid peak is quite different from a
restraint stress stimuli, at least on the effects promoted on penile morphology.
It thought that the stress stimuli acts on penile tissue (and maybe in other tissues as well)
in a quite different way than that of glucocorticoid. Based on these findings, one could even
hypothesize that they act in antagonical ways. By this theory, the glucocorticoid would be
neither a cause nor a consequence of the stress stimuli, but a physiological response to stress,
which may even protect penile tissue against more damage.
CONCLUSION
Both corticosterone and stress stimuli influences the penile morphology in an important
manner. These alterations may be related to erectile dysfunction and may explain some
mechanisms by how impotence occurs in stressed men and in those using glucocorticoids.
Interestingly, these experiments showed antagonic quantitative results, thus, it is possible
that glucocorticoids secretion may be a mechanism of defense in response to stress.
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[3] Pratsinis, H., Tsagarakis, S., Zervolea, I., Stathakos, D., Thalassinos, N., Kletsas, D.
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[4] Sapolsky, R. M. Glucocorticoids and hippocampal atrophy in neuropsychiatric
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[5] Kavitha, T. S., Parthasarathy, C., Sivakumar, R., Badrinarayanan, R., Balasubramanian,
K. Effects of excess corticosterone on NADPH generating enzymes and glucose
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P. Vasculogenic erectile dysfunction in teenagers: a 5-year multi-institutional
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metabolism clinics of North America. 2007;36:521-31.
[12] Sato, Y., Suzuki, N., Horita, H., Wada, H., Shibuya, A., Adachi, H., Tsukamoto, T.,
Kumamoto, Y., Yamamoto, M. Effects of long-term psychological stress on sexual
behavior and brain catecholamine levels. J. Androl. 1996;17:83-90.
[13] Costa, W. S., Carrerete, F. B., Horta, W. G., Sampaio, F. J. Comparative analysis of the
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2006;97:567-9.
[14] Pinheiro, A. C., Costa, W. S., Cardoso, L. E., Sampaio, F. J. Organization and relative
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Cell biology international. 1996;20:15-27.
Chapter 15
GLUCOCORTICOIDS AND NEURODEGENERATION
Sheela Vyas1,*, Ana Joao Rodrigues2,3,

Joana Margarida Silva2,3, Francois Tronche1,
Nuno Sousa2,3 and Ioannis Sotiropoulos2,3
1
Laboratory of Gene Regulation and Adaptive Behaviors,
Department of Neuroscience Paris Seine, INSERM U1130, CNRS UMR 8246,
Université Pierre et Marie Curie, Paris Cedex 05, France
2
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, Campus
de Gualtar, University of Minho, Braga, Portugal
3
ICVS/3B‘s - PT Government Associate Laboratory,
Braga/Guimarães, Portugal
ABSTRACT
Glucocorticoids (GCs) exert wide-spread actions in central nervous system ranging
from gene transcription, cellular signaling, modulation of synaptic structure and
transmission, glial responses to altered neuronal circuitry and behavior through the
activation of two steroid hormone receptors, glucocorticoid receptor (NR3C1, GR) and
mineralocorticoid receptor (NR3C2, MR). These highly-related receptors exert both
genomic and non-genomic actions in the brain, which are context-dependent and essential
for adaptive responses to stress resulting in modulations of behavior, learning and
memory processes. Thus, GCs through their receptors are implicated in neural plasticity
as they modulate the dendritic and synaptic structure of neurons as well as the survival
and fate of newly-generated cells (neuro- and glio-genesis) in adult brain. GCs are also
important in fetal brain programming as inappropriate variations in their levels during
critical developmental periods are suggested to be casually related to the development of
brain pathologies and maladaptive responses of hypothalamic-pituitary adrenal (HPA)
axis to stress during adulthood. They regulate immune responses in brain, which have
important consequences for neuronal survival. In situations of chronic stress and HPA
axis dysfunction resulting in chronically high or low GCs levels, a multitude of
molecular, structural and functional changes occur in the brain, eventually leading to
*
Corresponding Author: sheela.vyas@upmc.fr. & ioannis@ecsaude.uminho.pt.
308 Sheela Vyas, Ana Joao Rodrigues, Joana Margarida Silva et al.
maladaptive behavior. In fact, clinical studies suggest a causal relation of deregulated GC

responses with development of neurodegenerative disorders such as Alzheimer´s (AD)
and Parkinson‘s (PD) diseases. AD and PD patients have high levels of circulating
cortisol while animal studies suggest that this chronic GC elevation participates in
neurodegenerative processes in both AD and PD pathologies. This chapter will focus on
the role of HPA axis and GCs on neurodegenerative processes involved in AD and PD
pathogenesis.
Keywords: glucocorticoids, neurodegeneration, Alzheimer‘s disease, Parkinson‘s disease,

epigenetics
INTRODUCTION
Glucocorticoid (GC) hormone is synthesized and released into systemic circulation from
adrenal glands following activation of hypothalamic-pituitary-adrenal (HPA) axis, which
entails synthesis of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP)
by paraventricular neurons (PVN) of hypothalamus and their release from median eminence
into portal blood. These hormones stimulate the synthesis of adrenocorticotropic hormone
(ACTH) in the anterior pituitary, which when released into general circulation binds to ACTH
receptor (melanocortin type II receptor) in adrenal glands promoting GC synthesis from
cholesterol. GC release by HPA axis is under circadian control and occurs in an oscillatory
pattern or ultradian rhythm that varies in amplitude according to the time of day (peak in the
morning and trough in the evening/night in diurnal animals including humans and vice versa
in nocturnal animals, e.g., rodents). In addition, there is a surge of GC release in response to a
stress stimulus, which can be either psychogenic (e.g., fear) or physical (e.g., cellular lesion
or pathogen invasion). In response to stress, GCs exert critical adaptive functions by
modulating most biological processes (e.g., metabolism, cardiovascular and immune systems
as well as behavior); and through feedback inhibition of HPA axis they play a role in
terminating the stress response as well as facilitating the restoration of physiological
homeostasis [1]. In addition to their role in stress response, appropriate GCs levels are
important during development, for example in cell maturation, and in the differentiation of
lungs, kidneys and brain [2-4].
It is now thoroughly established that GCs have the capacity to profoundly modulate
different brain functions, as well, increasing evidence points to their role in brain
development. The appreciation that brain is a key target of this circulating adrenal steroid
hormone emerged from the pioneering work, principally by the laboratories of McEwen and
de Kloet, on identification and biochemical characterization of two receptors in the
hippocampus to which GCs bind - the mineralocorticoid receptor (MR) and glucocorticoid
receptor (GR) [5, 6]. Since then, GR presence in brain was observed to be widespread with
every cell type expressing this receptor in contrast to MR expression, which is more
restricted. MR is expressed by the neurons of the limbic system, i.e., hippocampus, locus
coeruleus, amygdala, prefrontal cortex and nucleus of the solitary tract, as well as neurons of
hypothalamus. MR is also present in non-neuronal cells, namely in glia and in epithelial cells
of choroid plexus and ependyma [7]. In brain, 3[H] corticosterone binding assays showed that
MR has 10-fold higher affinity (Kd= 0.5 nM) for GCs compared to GR (Kd= 5 nM), which
Glucocorticoids and Neurodegeneration 309
means that at basal GC levels, MR is occupied and activated [8] whereas GR is only activated
when GC levels reach a certain level as it happens in circadian peak and during stress [9]. GC
actions are pleiotropic, the principle factors determining their functions are: a) circulating
levels with accessibility to each cell type and b) context in which the receptors are activated.
GC levels are tightly regulated at each level of HPA axis and this is important in ensuring that
stress response is correctly executed. Deregulated HPA axis resulting in sustained high or low
GC levels are implicated in different diseases, for example disorders of metabolism (e.g.,
diabetes, obesity), immune (e.g., rheumatoid arthritis) and nervous systems (e.g., depression)
[10-12].
Synthetic GCs (e.g., dexamethasone, methylprednisolone) are routinely used in clinical
situations, particularly in disorders with an inflammatory component such as rheumatoid
arthritis or brain edema as they exert powerful anti-inflammatory and immunosuppressive
actions. However, prolonged GC use suppresses HPA axis resulting in harmful side effects
such as increased risk of infection, hyperglycemia, weight gain, behavioral or cognitive
problems. Interestingly, GCs are now also used clinically in neonates, as endogenous GCs are
required for fetal lung maturation as they promote the production of lung surfactant. This
could affect the programming or subsequent responsiveness of HPA axis particularly with
regards to stress responses in adults [13]. Thus prolonged GC exposure or exposure to high
levels of GC in specific developmental windows such as the prenatal and perinatal period can
impair the HPA axis negative feedback, increasing the propensity for developing
neuropsychiatric and metabolic disorders [14].
Glucocorticoid actions through MR and GR in brain have been particularly studied in
relation to glutamatergic as well as monoaminergic (e.g., dopaminergic and serotonergic)
systems, which have wide-range consequences from mood behaviors to cognition. Several
excellent reviews already exist on our current understanding of neuronal functions of GCsin
brain via these two receptors [15-19]. Our aim in this chapter is to describe how their actions
in neurons and glia impact the neurodegenerative processes, emphasizing on Alzheimer (AD)
and Parkinson diseases (PD). One of the arguments for their implication relates to GC
functions being exquisitely dependent on environmental changes, and in this regard, both
genetic susceptibility and environmental factors are believed to play key roles in the etiology
of these neurodegenerative diseases. Most of our current understanding of GCs involvement
in brain disorders relates to the functions of GR as this receptor plays a major role in stress
responses. Thus, before describing our current knowledge of GCs in neurodegeneration, we
reiterate the regulation of GC release by HPA axis and functional activity of GR as both are
likely affected in AD and PD as discussed below.
REGULATION OF GC RELEASE AND AVAILABILITY

Paraventricular nucleus (PVN) of hypothalamus receives integrated information from
suprachiasmatic nucleus for circadian control of GCs and from the limbic system for
psychogenic stress-induced GC release [20, 21]. In stress-induced GC release, limbic
structures such as amygdala are involved in stimulating PVN neurons to synthesize CRH
whilst hippocampus plays a crucial role in negative feedback inhibition of the HPA axis [22].
The fast feedback inhibition of HPA axis following acute stress is important to prevent
depletion of GC needed for both successive stress and ultradian release, which interestingly is
impaired in aging as well as in patients suffering from depression. Both MR and GR at
hypothalamic and hippocampal levels play an important role in regulating the activity of PVN
neurons. In addition GR in anterior pituitary was found to regulate pulsatile ACTH release
[23]. HPA axis is also activated in response to cellular lesion or pathogen invasion by pro-
inflammatory cytokines such as IL-1, IL-6 or TNF- released by either peripheral immune
cells or microglia [24]. IL-6 through activation of its receptor can also stimulate ACTH
release from anterior pituitary and GC from adrenal glands [25].
The availability of GCs to neurons and non-neuronal cells in brain is controlled in two
ways. Firstly, in the blood, most GCc are bound to corticosteroid binding globulin (CBG)
whose levels are down regulated by stress thereby increasing free-circulating GC levels [26].
Secondly, once inside the cells, the availability of GC for GR activation is controlled by GC-
metabolizing enzymes: 11--hydroxysteroid dehydrogenase type I (HSD111), which
regenerates active glucocorticoids (e.g., cortisol from cortisone) thus amplifying GR
activation. In addition, 11--hydroxysteroid dehydrogenase type II (HSD112) has an
opposite function, i.e., increasing the inactive form of GC. Using mice deficient for
HSD111, previous studies have shown that these mice are protected from hippocampal
memory impairments associated with aging. However, cognitive problems arise normally
because GR activity predominates due to high GC levels catalyzed by this enzyme [27, 28].
GENOMIC AND NON-GENOMIC ACTIONS OF GLUCOCORTICOID

RECEPTOR (GR)
GR exerts both genomic and non-genomic actions in brain. The genomic actions of GR
pertain to its ligand-activated transcriptional activity. Non-liganded GR in the cytoplasm is
normally in complex with chaperone proteins such as heat shock proteins 90, 70, 40, 23 as
well as immunophilins such as FKBP51 and 52. Upon GC binding, the conformational
change of the complex results in exposure of nuclear localization signal of GR, which allows
importin-mediated translocation of GR into the nucleus. Recent studies highlight the
importance of correlation between GR transcriptional activity and ultradian pulsatile nature of
GCs for generation of appropriate response to stress stimulus [29, 30].
GR protein is comprised of N-terminal transactivation domain which is important site for
GR co-regulatory binding proteins such as cAMP-response-element binding protein binding
protein (CBP), it also contains phosphorylation sites, e.g., serine 203, serine221 and serine
226. The central zinc-finger DNA-binding domain is important for the GR binding to the so-
called Glucocorticoid Response Elements (GREs), which are present in promoters of GR
target genes. The carboxy-terminal domain is the site of GC binding to GR as well as co-
activators such as histone acetylases or co-repressors. The transcriptional regulation by GR is
both cell-type and context-dependent. GR can regulate transcription by: a) direct binding as
homo-dimers to GRE DNA sequences to stimulate transcription, e.g., mitogen-activated
protein kinase phosphatase-1 gene; b) direct binding to negative GRE elements to repress
transcription, e.g., CRH or ACTH receptor genes; c) trans-repression or ―tethering‖ i.e.,
association with other transcriptional factors to inhibit their transcriptional activity. This
mechanism is by far the most notable in immune cells where GR regulates transcription of
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), activator protein-1

(AP-1) and members of interferon regulatory transcription factors (IRFs). In brain,
identification of GR-modulated genes is difficult due to anatomical complexity and cellular
heterogeneity. Nevertheless, transcriptomic studies in the hippocampus have identified
functional classes of genes modulated by GR which include genes coding for neurotransmitter
catabolism, neurotrophic factors and their receptors, signal transduction, energy metabolism
and cell adhesion [31].
The genomic actions of GR are slow in onset and long lasting. In contrast, GR exerts
non-genomic actions at plasma membrane of neurons, which are rapid (seconds to minutes),
involve alterations in neuronal excitability and are dependent on the context of the signal. The
non-genomic actions of GR at the membranes also involve activation of down-stream
signaling pathways involving kinases such as ERK, AKT, PKC and PKA [32]. Altogether,
this provides a surprising diversity and complexity of GC modulation of gene expression and
cellular signaling.
EPIGENETIC REGULATION OF GR
Animal studies confirm earlier anecdotal observations in humans indicating that early life
adverse experience has a profound impact on adult behavior. Early life stress or exposure to
GC (endogenous or exogenous) may induce neuroendocrine programming, subsequently
altering offspring's growth, metabolism, immune system and even the stress response as
previously mentioned. These observations derive from both animal and human studies, where
an alteration in the activity of the HPA axis was found [14, 33, 34]. Such prenatal programing
may be an evolutionary mode of shaping internal characteristics of the developing organisms
in order to adapt to the environment. However, such modifications might ultimately result in
the development of long-term diseases, from metabolic syndromes to psychiatric disorders
[35-39].
This long-lasting effect of early life experiences in brain function and behavior appears to
be mediated (at least partially) by epigenetic mechanisms [14, 34, 40]. In the last years,
considerable progress has been made in untangling the epigenetic alterations induced by
stress/GC. However, most of the studies are merely correlative and the mechanism through
which stress/GC induce epigenetic programing remains completely unkown.
One way of buffering the impact of maternal GC exposure in the developing fetus is by
converting cortisol/corticosterone into inactive metabolites through the action of placental
HSD112. However, some studies indicate that maternal adversity can increase the
methylation at specific CpG sites within the HSD112 gene promoter and lead to a down-
regulation of this enzyme [41, 42], which may allow excessive levels of GC to reach the fetus
and program different organs and systems. The first evidence of brain epigenetic programing
induced by early life adversity was reported by Meaney and colleagues, which showed that
natural variations in maternal behavior were correlated with DNA methylation levels of a
neuron-specific exon 17 promoter of the GR gene.
Briefly, male rats reared by ―good dams‖ (i.e., those that presented high pup licking and
grooming) demonstrated lower levels of stress response, greater performance on cognitive
tasks and larger exploratory activity in a novel environment, compared to the offspring of
―bad dams‖; this was associated with a differential methylation of this specific region of the
GR promoter [43, 44]. Importantly, these results were later replicated in humans showing
individuals with childhood stressful experiences (abuse during childhood), presented
hypermethylation of this region, in comparison to non-abused individuals [45].
Later studies revealed an increased methylation of a CpG-rich region in the promoter and
exon1F of the GR gene in the cord blood of newborns of mothers with depressed mood
during the third trimester of gestation [46]. Importantly, this pattern on methylation of the GR
gene occurred only in the offspring (and not the mothers), correlated with levels of response
to stress in infants at 3 months of age, and persisted beyond infancy. Similarly, pregnancy-
related anxiety is associated with the methylation state of the GR gene in the child [47]. These
findings suggest a common effect of parental care in both rodents and humans on the
epigenetic regulation of hippocampal GR expression. One question that still remains is
whether these epigenetic changes are the cause of maladaptive behaviors or a mere
adaptation, in the light of evidence showing that healthy individuals with a history of
childhood adversity can also present increased GR methylation and an attenuated cortisol
response to the dexamethasone test [48]. In this perspective, such adversity-induced
epigenetic changes may predispose the individual to disease (in combination with other
genetic or extrinsic factors) but are not the cause per se.
In addition, other pivotal stress players are also affected by early life stress/GC exposure.
For example, mice, in a model of early-life stress present hypersecretion of corticosterone,
alterations in passive stress coping and memory followed by a persistent increase in arginine
vasopressin expression in neurons of the hypothalamic PVN due to sustained DNA
hypomethylation of CpG residues that serve as DNA-binding sites for the methyl CpG-
binding protein 2 (MeCP2) [49]. In addition, stress/GC exposure early in life may induce
long-lasting epigenetic changes in neurotransmission-related genes. For example, animal
studies demonstrated that prenatal GC exposure leads to differential methylation of dopamine
receptor D2 [50]. In humans, depressed mood during pregnancy leads to decreased levels of
methylation in the promoter of the SLC6A4 gene, encoding the serotonin transporter, in
maternal peripheral leukocytes and in umbilical cord leukocytes collected from their infants at
birth [51]. Such changes may affect how the individual senses/processes/responds to
environmental stimuli and may explain, in part, the increased vulnerability for
neuropsychiatric disorders later in life.
In addition to particular gene epigenetic changes, stress/GC have a strong impact in the
epigenome (elegantly reviewed in [52]. Human studies on different cohorts have shown that
early life maltreatment induces long-lasting methylation changes in the genome [53-55] while
recent animal-based evidence suggest that the epigenomic landscape is also strongly
correlated with gestational maternal adversity [56] and even with natural variations in
maternal care [57]. In addition to methylation, gene expression can be further controlled by
hydroxymethylation and diverse histone modifications, adding additional layers of
complexity to the GC-driven changes that may predispose individuals to the development of
brain pathologies.
SUSTAINED GR ACTIVATION AND

NEURODEGENERATION IN AD
Alzheimer‘s disease (AD) is an age-related neurodegenerative disorder characterized by
slow and progressive dementia while the major histopathological hallmarks are senile plaques
containing amyloid beta (Aβ) deposits and intracellular neurofibrillary tangles (NFT) made of
hyperphosphorylated forms of the cytoskeletal protein Tau [58-60]. Aβ is the proteolytic
product of the bigger transmembrane protein called amyloid precursor protein (APP), which
is sequentially cleaved by β-secretase (BACE-1) and γ-secretase (enzymatic complex of
proteins) resulting in the production of Aβ; this cellular pathway is often called APP
misprocessing. Many studies have demonstrated that APP misprocessing and Aβ trigger AD
neuropathological processes such as synaptic malfunction (impairing mechanisms of synaptic
plasticity, e.g., LTP), neuronal atrophy and synaptic loss as well as mitochondrial
dysfunction, oxidative stress and glial activation.
While still debated, it is suggested that Aβ also triggers abnormal Tau
hyperphosphorylation leading to the formation of NFTs and neuronal loss in AD brain.
Indeed, accumulating data suggest the involvement of Tau protein in the detrimental effects
of Aβ as use of Tau-KO blocked the Aβ neurotoxic effects [61-64]. Further support of the
essential role of Tau in the establishment of AD pathology is based on the clinical findings
that have consistently shown that the cognitive deficits in AD patients correlate with NFTs
rather with Aβ deposition. Indeed, hyperphosphorylated and aggregated Tau resulting in
NFTs is associated with neuronal loss. Gomez-Isla et al. [65] demonstrated that strong
correlation of neuronal loss in cerebral cortex and increased NFT burden with disease
progression; no such correlation was found with Aβ. Furthermore, reduction of hippocampal
volume in AD patients was associated with phosphorylated Tau, but not Aβ levels in cerebral
spinal fluid (CSF) [66].
Several risk factors have been suggested for AD while recent evidence supports an
etiopathogenic role of chronic stress and glucocorticoid hormones in the establishment and
development of AD pathology [67, 68]. Clinical studies report high cortisol levels, measured
in plasma, saliva or CSF, of AD patients indicative of altered HPA axis [69-73] while the
increase of cortisol levels is negatively associated with memory scores in AD patients [74,
75]. Furthermore, Hartman et al. [76] monitored the 24hr secretory pattern of plasma cortisol
in AD patients finding a higher mass of cortisol release; however, the diurnal changes in
cortisol levels were not altered. Since chronic elevation of GC levels is known to impair
memory and cognitive performance, it is speculated that GCs play a role in progressive
cognitive decline in AD. Indeed, it is unclear whether high GCs are a cause or a consequence
of the disease as one of the explanations of high GC levels in AD patients is the deregulation
of feedback inhibition of the HPA axis, particularly in relation to psychogenic stressors,
occurring at the level of the hippocampus, a region significantly damaged in AD brains.
It is noteworthy that many clinical and experimental reports suggest a reduction of adult
neurogenesis in AD hippocampus while the same is true for chronic stress conditions [77-79].
Reduction of hippocampal adult neurogenesis was shown to increase HPA activity implying
that this region is involved in hippocampal feedback regulation of HPA axis during stress
[80]. Thus, high GCs can aggravate hippocampal memory processes in AD by having a
negative effect on hippocampal neurogenesis, which may, in turn, contribute to maintenance

of deregulated HPA axis.
GC IMPACT ON AD NEURODEGENERATIVE MECHANISMS

Clinical studies show that chronic stress is a risk factor in AD pathogenesis and it also
lowers the age of onset of the familial form of AD [67, 68]. Indeed, it has been evoked that
chronic stress is among the principal factors that contributes to development of AD [77]. A
principal target of GCs is hippocampus, which is a main target area for AD pathology and
chronic stress (Figure 1). The hippocampal dysfunction in AD has significant detrimental
consequences on declarative, spatial and contextual memory processes. As hippocampal
neurons have very strong GR expression and are intimately involved in regulation of HPA
axis, there has been a great deal of interest in how high cortisol levels and stress impact the
deterioration of hippocampal functions caused by toxic Aβ and Tau hyperphosphorylation in
AD.
Previous studies show that elevated GC levels and exposure to chronic stress increase Aβ
production in AD transgenic mouse models exacerbating their memory deficits [81, 82],
Specifically, chronic immobilization stress in amyloid precursor protein (APP)V717ICT-100
transgenic mice (this APP mutation is known for aggressive early onset AD) evoked
acceleration and greater severity of memory deficits and increased extracellular Aβ deposits.
Similarly, Green at al [81] showed that prolonged treatment with the synthetic GC,
dexamethasone, triggers APP misprocessing resulting in increased Aβ levels using both in
vitro and in vivo approaches (neuronal N2A cell line and pre-pathological 3xTg-AD young
mice). In addition, the same study also demonstrated transcriptional up-regulation of APP and
β-secretase expression by GR (both contain GRE in their promoter region).
Similarly, other in vitro studies have confirmed that GCs trigger APP misprocessing
without influencing the non-amyloidogenic pathway, i.e., the other cellular cascade of APP
cleavage/processing [83]. Similar observations were made in middle-aged rats in which the
amyloidogenic potential of chronic stress (chronic unpredictable stress paradigm) and
prolonged GC treatment was demonstrated insofar that both treatments were found to drive
APP processing towards the generation of Aβ and its precursor molecule (C99), both of
which have neurotoxic and cognition-impairing properties [84]. This study also showed that
GC/stress increased β-secretase (BACE-1) levels as well members of γ-secretase complex
(Nicastrin). Given that stressful stimuli occur intermittently over the lifetime, and that their
effects may be cumulative, an important finding by Catania et al., [84] was that GC potentiate
the APP misprocessing pathway in previously stressed animals of AD model (Aβ-infused
rats).
Interestingly, clinical studies suggested that the stress-related neuropsychiatric disorder,
depression, is a risk factor for the development of AD pathology as the history of depression
is correlated with increases of amyloid plaques and NFT [85]. In addition, other studies
suggested the utility of measurements of the various APP cleavage products as biomarkers to
discriminate between subjects undergoing normal aging from those suffering from depression
or AD [86-89]. Interestingly, more recently, some studies report the influence of anti-
depressant drugs on the proteolytic cleavage of APP suggesting its anti-amyloidogenic role
[89, 90] while many antidepressants are shown to normalize the HPA axis and the resulting
GC levels which are increased in many depressed patients and models of stress-driven
depression.
Besides APP misprocessing, high levels of GC trigger the other main AD
neurodegenerative pathway, the aberrant hyperphosphorylation of Tau protein. Among the
first reports suggesting a potential connection between GC and Tau was the study by Stein-
Behrens et al. which demostrated high GC levels exacerbated neuronal loss induced by kainic
acid injection in hippocampus while in parallel increased Tau immunoreactivity. Later on, it
was shown that treatment with synthetic dexamethasone for 7 days in 3xTg AD mouse model
resulted in Tau accumulation in somatodendritic compartment of neurons in hippocampus,
amygdala and cortex [81].
Figure 1. Glucocorticoids (GCs) and Stress impact on AD neurodegenerative mechanisms. The

schematic presentation reflects the triggering role of high GC levels and chronic stress on AD cellular
mechanisms based on experimental evidence using animal and cellular AD models. Prolong exposure
to GC and/or stress activates amyloidogenic cellular pathway resulting in the sequential cleavage of
APP by β- and γ-secretase which produces Aβ. Next, the cytoskeletal protein Tau, mainly found at
neuronal axon (rdown (dark) part in the healthy neuron scheme), is aberrantly hypersphosphorylated
through the activation of different kinases (e.g., GSK3-β and cdk5) which results in Tau
somatodendritic accumulation (upper (dark) part in in diseased neuron scheme). In addition, abnormal
conformation and caspase 3-mediated truncation of Tau occurs together with a parallel dysregulation of
the molecular chaperones (e.g., Hsp90 and Hsp70) facilitating reduced Tau degradation and increased
Tau oligomerization and ultimately, aggregation (see panel on the right). The above cellular cascades
result in neuronal atrophy and loss leading to the establishment of cognitive impairment.
In addition, Sotiropoulos et al., [91] showed that chronic stress or GC treatment triggers
Tau hyperphosphorylation in different epitopes implicated in cytoskeletal pathology and
synaptic loss in AD patients (e.g., pSer262) [92, 93]; note that these epitopes are correlated
with hippocampal atrophy in AD patients (e.g., pThr231) [94]. Indeed, clinical studies report
a strong correlation between the extent of Tau hyperphosphorylation (e.g., Thr231 and Ser262
residues) and severity of impairments of memory, speed of mental processing, and executive
functions [95-97].. Futhermore Tau hyperphosphorylation is associated with synaptic loss and
memory impairment in experimental animals [98] that could be also related with the stress-
induced synaptic and memory loss.
Albeit specific Tau phosphoepitopes maybe differentially regulated by chronic stress and
prolonged GC treatment, the overall in vitro and in vivo evidence [83] clearly implicates GCs
as a key mediator of the cellular response to stress. Nevertheless, other studies have also
suggested the contribution of other stress-related molecules, e.g., corticotrophin-releasing
hormone [99, 100]. Furthermore, in vitro studies suggest the mediation of glycogen synthase
kinase 3 (GSK3) or CDK5 in the above GC- and stress-triggered Tau hyperphosphorylation,
both known to lead to microtubule disruption as well as formation of NFTs [83]. In parallel,
GC were also shown to increased Tau accumulation by affecting turnover of the protein [83],
which may involve reduced degradation through dysregulation of molecular chaperones
responsible for Tau proteostasis (e.g., Hsp90, Hsp70 [101]). Interestingly, Hsp90 and Hsp70
serve to maintain the glucocorticoid receptor (GR) in a high affinity state (as previously
discussed) and thus, offering a clear cross-point between GC/GR cellular signaling and Tau
degradation machinery. This reduced degradation could facilitate the increased aggregation of
Tau into insoluble forms triggered by stress in P301L-Tau Tg mice [mice expressing human
Tau carrying the most common Tau mutation (P301L-Tau)]. In addition, chronic stress also
promotes C-terminal truncation of Tau by caspase-3 and, abnormal conformation of Tau in
the hippocampus of the same animals. Indeed, both truncation and abnormal conformation of
Tau precede its aggregation and formation of neurofibrillary tangles [99, 102, 103] thus
serving as early markers of disease. The Tau-C3 species have been suggested to contribute to
misfolding of Tau into a conformation that can nucleate and recruit other Tau molecules into
aggregates [99, 103, 104], which are shown to be neurotoxic and related to neuronal loss
[105].
GLUCOCORTICOID ROLE IN THE ONSET AND PROGRESSION OF

PARKINSON’S DISEASE
Parkinson‘s disease (PD), the most common neurodegenerative movement disorder, is
characterized by preferential loss of dopaminergic neurons in the substantia nigra pars
compacta (SNpc) and dopamine depletion in striatum that underlies the appearance of motor
symptoms such as akinesia, resting tremor, rigidity and postural instability. The main
histopathological characteristic in PD brain is Lewy bodies (LBs), which are proteinaceous
inclusions containing the presynaptic protein, alpha-synuclein, and are found in many
different brain regions far beyond SN and striatum; e.g., cerebral cortex, limbic system,
hypothalamus as well as the autonomic nervous system that are also affected in PD brain
[106-108]. Thus, in addition to motor symptoms due to SN and striatum neurodegeneration
and lessions, PD patients with cortical LBs also suffer from dementia and visual
hallucinations [109].
While several gene mutations have been identified in the familial forms of PD, the
majority of PD cases are sporadic with unknown etiology. Different cellular mechanisms
have been suggested to be involved in PD neurodegeneration and dopaminergic neuronal loss
such as oxidative and nitrative stress, mitochondrial dysfunction and deregulated intracellular
calcium levels, damaged proteostasis related to alpha-synuclein aggregation [110]. Like in
AD, deregulated HPA activity is also reported in PD patients. Specifically, previous studies
[76, 111-113] including our work [114] show that plasma cortisol levels are significantly
higher in idiopathic PD patients compared to control subjects; however the cortisol levels are
not related to disease duration or to L-3,4-dihydroxyphenylalanine (L-DOPA) treatment.
Interestingly, the diurnal mode of cortisol secretion in PD patients, in particular the normally
quiescent nocturnal cortisol secretory pattern, is affected [76].
Furthermore, monoaminergic neurotransmission in hypothalamus, the first compartment
of HPA axis, is also affected in PD patients who exhibit reduced levels of dopamine,
serotonin and noradrenaline in this brain area [115, 116] followed by reduced density of
dopamine receptors [117]. Notably, this reduction was not altered by dopamine medication,
which is often used in PD patients. Future studies are necessary to clarify whether the
deregulation of HPA axis in PD patients is situated at the hypothalamic and/or the adrenal
level as Lewy body pathology is observed in both regions.
THE NEURODEGENERATIVE POTENTIAL OF

GC IN PD PATHOLOGY
The deregulated HPA axis and the subsequent elevated GC levels in PD patients reflects
the role of stress which was suggested as one of the earliest proposed causes of PD. Although
it may not be a major etiological factor, there are clinical reports showing that chronic stress
triggers the appearance of PD symptoms or exacerbates the motor symptoms [118, 119].
Furthermore, experimental studies demonstrate that stressors such as food deprivation or
tailshock aggravate motor deficits in the 6-hydroxydopamine (6-OHDA) PD model (6-
hydroxydopamine local injections lesions the nigrostriatal pathway) [120]. Using the same
model, Smith et al. [118] showed that chronic stress exposure (restraint) before the 6-OHDA
injection worsened the 6-OHDA-driven motor deficits, aggravated the neurodegeneration of
nigrostriatal system and completely blocked compensatory recovery of motor tasks.
How does high stress level of GC-GR exacerbate motor impairments following
nigrostriatal lesions? GCs are known to profoundly shape the dopaminergic neurotransmitter
system, exerting differential or heterogeneous effects depending on whether the dopaminergic
projections arise from the ventral tegmentum area (VTA) or the SNpc. While plethora of
studies have monitored the impact of GC on the limbic arm of dopamine neuronal circuitry
related to behavioral changes as well as neuropsychiatric diseases, our knowledge about the
exact GC influence on motor-related dopamine neuronal networks is very limited. There is
lack of evidence about the impact of chronic GC elevation on nigral and striatal neurons or
glia and how this contributes to nigrostriatal degeneration and motor impairments. Analysis
of GR in PD brain revealed that global GR levels were lower in SNpc and higher in putamen
compared to control subjects and these results were recapitulated in MPTP (1-methyl 4-
phenyl 1,2,3,6-tetrahydropyridine)-treated mice [114].
However, the cell types in which GR changes occur have not been identified.
Interestingly, high GR levels in putamen of PD patients raises the possibility that
dopaminergic nerve terminal degeneration induces upregulation of GR in striatal neurons
and/or glia. In a study by Barrot et al. [121], GCs in SNpc or in dorsolateral striatum were
found not to modify either tyrosine hydroxylase levels or dopamine transporter activity. On
the contrary, adrenalectomy and the subsequent loss of corticosterone resulted in reduced D1
dopamine receptor in dorsolateral striatum suggesting that neurons expressing dopamine
receptors may represent a target of GC-GR actions for basal ganglion regulation of
movement. While the molecular mechanisms by which high GC through GR activity
exacerbate motor deficits are not well understood, it is possible that they alter glutamatergic
synapses in striatum that are under dopamine regulation.
ROLE OF GLUCOCORTICOID RECEPTOR IN REGULATION OF

INFLAMMATION IN PARKINSON’S DISEASE
Chronic inflammation mediated principally by activated microglia, astrocytes and
infiltrating T cells is a major neuropathological characteristic of PD. Evidence from recent
genome-wide studies point to involvement of the immune system in the etiology of idiopathic
PD. A number of susceptibility loci identified relate to genes expressed in immune cells such
as HLA-DQB1, LRRK2 or BST-1 [122, 123]. In addition, identified PD risk factors [such as
age, environmental toxins (e.g., heavy metals or pesticides,) traumatic brain injury, bacterial
or viral infections] activate immune responses in periphery and brain.
Using radiolabelled ligand 11C-PK-11195 for translocator protein, Positron Emission
Tomography (PET) studies in PD patients revealed an early activation of microglia in many
brain regions including basal ganglia and substantia nigra [124, 125]. Furthermore, post-
mortem studies as well as analyses of serum and cerebrospinal fluid from PD showed high
levels of pro-inflammatory mediators such as TNF-, IL-1, iNOS, IFN- and COX-2 [126].
In line with observations in PD patients, presence of inflammatory mediators and glial
reactivity in striatum and substantia nigra is a key feature in many of the experimental animal
models of PD. For example, treatment of mice or monkeys with neurotoxin 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) which selectively induces degeneration of
nigrostriatal pathway, 6-hydroxydopamine lesion of nigro-striatal pathway in rodents or
toxicity induced by alpha-synuclein injection [126, 127].
Among all the brain regions, substantia nigra has one of highest density of microglia.
Activated microglia functioning as innate-immune competent cells are likely involved in
releasing the above inflammatory molecules, thereby inducing dopamine neurodegeneration.
Indeed the important role of these pro-inflammatory mediators in promoting degeneration of
dopaminergic neurons of substantia nigra was demonstrated using mice with specific
knockout of these genes [128-131]. Many of the pro-inflammatory mediators found in PD
patients are transcriptional targets of GR. The synthetic analogue of GCs, dexamethasone,
was shown to attenuate dopamine neuronal loss by precluding activated microglia from
releasing toxic inflammatory molecules [132, 133]. In adrenalectomized mice (lacking
endogenous production of GCs), dopamine neuronal loss was augmented following MPTP
intoxication indicating that endogenous GCs do play a role in protecting dopamine neurons
[134] Examination of GR in microglia revealed an increase in nuclear localization of GR
following MPTP treatment in mice, which coincided with rise in systemic corticosterone
levels indicating that GR is activated in microglia during degeneration of dopamine neurons
[114]. The unequivocal evidence that GR in microglia normally protects dopamine neurons
appeared in a study using mice in which GR gene is deleted in microglia/macrophages. MPTP
treatment in these mice resulted in increased dopamine neuronal loss as well as increased
microglial activation and expression of pro-inflammatory mediators [114]. Indeed, the
absence of GR in microglia resulted in sustained activation of NF-B as was shown in these
microglial GR mutants. The above finding has a significant relevance for PD pathogenesis as
nuclear expression of p65 subunit of NF-B, indicative of transcriptional activity, was found
in substantia nigra microglia of PD post-mortem [135].
Chronic inflammation and sustained activation of glia in PD suggests that processes
involved in regulation of glial activation and expression/secretion of inflammatory mediators
are likely compromised. Chronic inflammation, an important component of pathology in
neurodegenerative diseases, is suggested to be a maladaptive response of homeostasis as
successful inflammatory response has a resolution phase which is an active process that
enables restoration of homeostatic set points [136, 137]. Inflammation mediated by immune-
competent cells including microglia is normally a very tightly regulated process. The
immune-regulatory processes are affected in aging leading to increased susceptibility to
infections and immune activation. Thus in aging, microglia show enhanced sensitivity to
inflammatory stimuli - a process called ―priming‖ which could be also induced by chronic
stress and deregulated HPA axis. In this regard, there are several studies showing that
chronically elevated GC levels in response to different stressors cause pro-inflammatory
cytokine production and sensitization or ―priming‖ of microglia. Importantly, subsequent
inflammatory or toxic stimulus results in aggravatation of neuronal injury [138-140]. Aging is
associated with chronically high GC levels and immuno-senescence exemplified by a
sustained low production of pro-inflammatory molecules [141]. Thus, in contrast to their
well-known anti-inflammatory actions, in fact high and sustained GCs can exacerbate
inflammation. However, it is currently not known whether GR transcriptional activity
regulating inflammatory response of microglia is compromised in AD and PD pathological
conditions where deregulated HPA axis and sustained high GC levels of are found.
GC-DRIVEN BRAIN PROGRAMMING AND

NEURODEGENERATIVE PATHOLOGIES
Although Alzheimer‘s disease (AD) is often seen as an age-related neurodegenerative
disorder, recent evidence suggests that early life events may play a role in the onset of the
disorder (Borenstein, A.R.; Early-life risk factors for Alzheimer disease. Alzheimer Dis.
Assoc. Disord., 2006). In this perspective, AD is probably not determined by a single
etiologic factor, but results from the interplay between genetic and environmental factors
throughout life, being a possible explanation why monozygous twins can be discordant for
AD.
Albeit there is still controversy and the literature is sparse, it has been suggested that
early life adverse events such as maternal stress, intrauterine infections, poor maternal and
perinatal nutrition can potentially predispose to AD eventually by epigenetic programing of
specific genes/pathways related to AD neurodegeneration. For example, early-life lead
exposure of older rats and primates induces overexpression of the amyloid precursor protein
and its amyloid beta (Aβ) product, both characteristically found in AD brain as will be
discussed later in this chapter. One interesting finding was that cognitive impairment was
only observed in mice exposed to lead [142], highlighting the relevance of the ―window of
opportunity‖ for some environmental factors to trigger the disease.
Similarly, Tau hyperphosphorylation and accumulation, the other main histopathological
characteristic of AD pathology) was elevated in both aging rodents and primates previously
exposed to lead at younger age, [143] suggesting the potential impact of early-life stress
exposure to the precipitation of AD neurodegeneration later in life. Interestingly, a recent
study has also highlighted the GC-related epigenetic drive in the establishment of AD
pathology in the brain of CK-p25 AD mouse model (exhibiting Tau pathology). These Tg
mice exhibit increased levels of HDAC2 associated with cognitive impairment, which seems
to be mediated through glucocorticoid receptor induced HDAC2 transcription [144].
Furthermore, the role of early life stressful events in the etiopathogenesis of another
neurodegenerative disorder, Parkinson‘s disease (PD) has emerged in the last years. In an
interesting study, pups of female animals exposed to lipopolysaccharide (LPS), a bacterial
endotoxin, during pregnancy, showed loss of dopaminergic neurons. This suggests that high
LPS levels in mothers might interfere with the dopaminergic neurons in the fetus enhancing
the susceptibility to PD [145].
Accordingly, different stressful stimuli could act cumulatively with the developmental
stress exposure representing the first imprint in the developing brain, determining the PD
phenotype characterized at the pathological level by a deficient substantia nigra with a low
burden of DA neurons at birth corresponding to a limited nigro-striatal neurochemical reserve
[146]. The low number of DA neurons in the substantia nigra reflecting the developmental
damage may remain subclinical during life. Thus, later exposure to the same or other DA
neuron-targeted toxicants might attack the few residual neurons leading to insurgence of PD.
CONCLUSION
Accumulating evidence suggests the neurodegenerative potential of chronic stress and
elevated GC levels in triggering clinical symptoms and participating in neuropathological
mechanisms and processes in AD and PD, two devastating age-related neurodegenerative
disorders. High circulating GC (cortisol) levels and deregulated HPA axis observed in
patients of both disorders imply that GR activity in the affected regions is most likely
compromised but the cause-consequence interrelationship between elevated GC levels and
development of neurodegenerative pathology remains unclear. While the ramifications of
prolonged exposure to GC stress are many, being causally implicated in immunosuppression,
metabolic syndrome, diabetes and others, our current understanding of the exact actions of
GC on these neurodegenerative diseases, although limited, opens a window of opportunities
to identify the various parameters that contributes to stress/GC-driven brain pathology. As
both context and cell type determine GR functions, future works using, e.g., cell-specific
mouse models of GR activation/inactivation should shed light on their roles in pathological
brain aging and onset of neurodegenerative disorders such as AD and PD.
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Jul;22(1):16-32. PubMed PMID: 22215534.
Chapter 16
DISORDERS OF THE ADRENAL GLANDS:

THE NEUROLOGISTS’ POINT OF VIEW
Tulio Bertorini* and Lihong Shen

Department of Neurology and Pathology, University of Tennessee,
Health Science Center, Memphis, TN, US
ABSTRACT
Disorders of the adrenal glands could be primary or secondary to abnormal
hypothalamic pituitary function, caused either by a decreased hormonal production such
as in Addison‘s disease or increased production such as in Cushing‘s disease which
symptoms could also be caused by therapeutic use of corticosteroids. In order to acquire
proper knowledge and understanding of these disorders, the anatomy and physiology of
the adrenal glands must be understood. The adrenal cortex has 3 defined functional areas,
the zona reticularis adjacent to the medulla secretes sex hormones, and the middle zona
fasciculata secretes corticosteroids. Both of these are under the control of the
hypothalamic-pituitary axis. The outer area on zona glomerulosa secretes
minerocorticoids and is controlled by the adrenal-angiotension system. The adrenal
medulla produces catecholamines from secreting chromaffin cells. Diseases of the
different areas adrenal glands are very important for neurologists as they can manifest
with neurological symptoms particularly those caused by excessive or deficient
corticosteroid production. There are other disorders that affect both the adrenal glands
and the nervous system independently and their neurological manifestations are not
caused by adrenal dysfunction such as, for example, adrenoleukodystrophy, which is a
metabolic hereditary disorder of the central nervous system and adrenal myeloneuropathy
that also affects the peripheral nervous system. Some mitochondrial diseases like Kearns
Sayre syndrome have multisystem manifestations that includes a myopathy and endocrine
dysfunction. This chapter discusses the disorders of the adrenal glands, their clinical
manifestations, diagnosis and management.
Keywords: adrenal glands, Addison‘s disease, Cushing‘s disease, corticosteroids,

adrenoleukodystrophy, adrenal myeloneuropathy
*
Corresponding author: tbertorini@aol.com.
334 Tulio Bertorini and Lihong Shen
INTRODUCTION
Many of the spectrum of diseases associated with adrenal dysfunction have neurological
manifestations. The clinical and physio-pathological aspects of this as well as the diagnosis
and management are covered in this chapter.
The anatomy of the adrenal gland was initially described by Bartholomeo Eustachius [1].
Their function was defined by Thomas Addison in 1855 and from this the disease acquired
his name [2]. Charles Brown-Sequard demonstrated the role of the glands for survival by
performing adrenal adrenalectomies in animals and William Osler was the first physician to
treat a patient with Addison‘s disease with adrenal extracts [3].
In 1901 Takamine and Aldrich isolated adrenaline [4]. Harvey Cushing reported a
polyglandular syndrome in 1912, and later in 1932 he associated this with adrenal
hyperactivity [5]. Several other studies demonstrated dependence of the adrenal gland from
the pituitary, leading to the isolation of the adrenocorticotrophic hormone (ACTH) by Li,
Evans and Simpson in 1943 [6]. Harris et al. described the neural control of ACTH by the
corticotrophin-releasing hormone (CRH) in 1940 [7].
The first rate-limiting step in adrenal steroidogenesis is the transport of cholesterol
intracellularly from the outer to the inner mitochondrial membrane for conversion to
pregnenolone by the cytochrome P450 system. Pregnenolone is then converted to
progesterone by type II isozyme 3β-hydroxysteroid dehydrogenase. The 21-hydroxylation of
progesterone and 17-OH-progresterone by 21-hydroxilase produces deoxycortisol and
deoxycorticosterone (DOC), respectively. In the final step of cortisol biosynthesis,
deoxycortisol is converted to cortisol, which is subsequently converted to cortisone in the
peripheral tissues.
Most of the secreted cortisol is bound to corticosteroid-binding globulin (CBG), and only
5% to 10% accounts for the biological active hormone. Increased levels of cortisol occur in
physiological states such as stress, excessive exercise, hypoglycemia, fever, or surgery [8].
The corticotrophin-releasing hormone is synthesized in neurons of the paraventricular
nucleus of the hypothalamus, and its secretion triggers the pituitary production of ACTH
which promotes glucocorticoid secretion by the adrenal cortex.
CUSHING’S SYNDOME
Cushing‘s syndrome (CS) is a metabolic disorder caused by chronic high levels of
endogenous cortisol or by therapeutic exposure to corticosteroids. This impairs carbohydrate,
protein, and lipid metabolism. Cushing‘s syndrome defines all causes of hypercortisolism,
while Cushing‘s disease (CD) defines pituitary-dependent CS. Harvey Cushing first described
a woman with obesity, hirsutism, and amenorrhea, and later he reported 12 additional patients
with the syndrome, and in 1932 he postulated this ―polyglandular syndrome‖ caused by a
primary pituitary abnormality resulting in adrenal hyperplasia [5].
Endogenous CS can be ACTH dependent or independent [9]. ACTH-dependent CS
occurs in approximately 80% to 85% of patients [10], and is most often caused by hyperplasia
of the adrenal glands secondary to increased secretion of ACTH by pituitary adenomas. This
could also be produced by ectopic production of ACTH by small cell carcinomas such as lung
Disorders of the Adrenal Glands: The Neurologists‘ Point of View 335
or other organs. Very infrequently, ectopic production of CRH can also cause CS. The
ACTH-independent syndrome is caused by adenomas, carcinomas or macronodular
hyperplasia of the adrenal glands.
The most common cause of CS, however, is the exogenous administration of ACTH or
corticosteroids, and because medroxyprogesterone and progesterone might also have intrinsic
glucocorticoid activity, their administration might also cause the syndrome.
Clinical Manifestations of Cushing’s Syndrome
CS usually affects all body systems, characterized by abnormal fat distribution with a
―buffalo hump‖, temporal wasting, central obesity, moon face, weight gain, acne, purple
striae, hirsutism (Figure 1), cataracts, menstrual irregularities, arterial hypertension, and
hyperglycemia, and hyperkalemia [11].
Osteoporosis may result in pathological fractures, and patients may complain of back
pain caused by osteoporotic vertebral compression (Figure 2A). Thinning of the skin with
easy bruising and poor wound healing are also common, and there is a tendency for
infections, hypercholesterolemia, and hypertriglyceridemia, all of which could eventually
trigger cerebrovascular disorders.
Osteonecrosis of the femoral head is a dreaded complication of the chronic use of
corticosteroids (Figure 2B). Visual abnormalities may result from compression of the optic
chiasm by a tumor. Psychiatric manifestations develop frequently [10, 12], and this might
include insomnia, emotional labiality, irritability, anxiety, panic attacks, and paranoia, and
some patients are depressed, while others are manic, and they have weight gain, increased
appetite; some have anorexia and weight loss. Learning, cognition, and memory, especially
short-term memory, can be impaired in CS [13]. Cerebral atrophy has been reported with
reduction in hippocampal volumes [14, 15]. Cognitive disorders and intracranial hypertension
occur in children with CS [16].
A ―steroid myopathy‖ is characterized by proximal weakness [11, 12], more prominent in
the legs with sparing of sphincter and lack of sensory loss, unless the patient also has
secondary diabetes. The serum CK and other muscle enzymes are not elevated, and nerve
conduction tests are also normal. The EMG shows small polyphasic motor unit action
potentials, but there are no denervation potentials. Characteristics of muscle biopsy shows
selective atrophy of type II, particularly type IIB fibers (Figure 3) and electron microscopy
may show aggregations of mitochondria and vacuolization that are nonspecific findings [17-
19].
Steroid myopathy can be caused by several mechanisms related to abnormal carbohydrate
and protein balance. Protein synthesis is affected as well as protein degradation. Patients
might also have hypokalemia producing weakness, and lack of activity also contributes to this
[12].
There is a paralytic condition that occurs predominantly in patients in the intensive care
units who have received high doses of corticosteroids and neuromuscular blocking agents;
this is called critical illness myopathy (CIM) [20-23]. These patients have diffuse weakness
more prominently in the proximal muscles, somewhat decreased reflexes and may also have a
neuropathy. This so-called neuropathy of the gravely ill might have other causes such as
microcirculatory abnormalities, metabolic derangements, toxic effects of medications and/or
bioenergetics dysfunction secondary to systemic illness. Serum CK is normal or mildly

elevated in CIM.
Figure 1. Clinical features of Cushing‘s syndrome. (A) Centripetal and some generalized obesity and
dorsal kyphosis in a 30-year old woman with Cushing‘s disease. (B) Same woman as in (A), showing
moon face, plethora, hirsutism and enlarged supraclavicular fat pads. (C and D) Typical centripetal
obesity with livid abdominal striae. (Reproduced from Stewart, 2003, with permission).
Figure 2. (A) Vertebral compression at T8 and T10 levels (arrows), and diffuse osteoporosis of an 80-
year-old woman with iatrogenic Cushing‘s syndrome. (B) MRI demonstrating bilateral osteonecrosis of
the femoral head in a patient with Cushing‘s syndrome.
Muscle biopsy in CIM shows atrophy of type II fibers with basophilic splitting, necrosis,
and reduction of ATPase activity and electron microscopy showing loss of thick filaments
(Figure 4). The exact etiology of CIM is unclear, but in some patients this could be related to
systemic inflammatory response with cytokine-induced muscle injury and activation of
proteinases [20-25]. There seems to be an upregulation of interleukin 6 and serum amyloid
A1, and this might play a role in the pathogenesis [26].
Diagnosing Cushing’s Syndrome
The first step in the diagnosis is the demonstration of elevated cortisol levels in a 24-hour
urine collection. If there is elevation of urine cortisol, a dexamethasone suppression test
should then be done to confirm the diagnosis, and then finally determination of the cause of
hypercortisolism has to be established to determine if it is ACTH dependent or independent.
The dexamethasone test is based on the fact that dexamethasone suppresses ACTH
released from the pituitary gland leading to reduction of serum cortisol levels and therefore,
decreased urinary secretion of cortisol and its metabolites [11]. Dexamethasone is usually
given at 11:00 p.m. and serum cortisol is checked at 8 a.m. Cortisol levels of over 3 mcg
suggests hypercortisolism, but the values could be affected by some drugs. Serum
measurement of salivary cortisol level is also of great diagnostic value.
Once CS is diagnosed, the clinician needs to determine if this is ACTH dependent or
independent by measuring ACTH levels. Concentrations below 5 pg/mL are diagnostic of
ACTH independent CS whereas if it is above 15, this is diagnostic of ACTH dependent CS
[11].
A standard high dose of dexamethasone suppression can also be done in patients with
suspected ACTH dependent CS from pituitary causes, and if suppression of cortisol secretion
is obtained, CD should be considered. Once this is determined, CT scans and MRI should
help to find the presence of the adenoma. In patients with inadequate suppression, an
octreotide scan and chest and abdominal CT could be done to rule out tumors causing the
syndrome.
Figure 3. (A) Selective atrophy of type II muscle fibers (ATPase stain, PH 9.4 X 100). (B) Atrophic
fibers are mainly type IIB that stain intermediate with ATPase at PH 4.6 (X 100). (Reproduced from
Bertorini et al., 2008, with permission).
Figure 4. Electron microscopy of a muscle biopsy from a patient with critical illness myopathy showing
loss of thick filaments (X 7500). (Reproduced from Bertorini, 2008, with permission).
Treatment of Cushing’s Syndrome
The treatment choice of CD consists in transphenoidal microadenolectomy, and patients

are required to have daily glucocorticoid replacement from the time of surgery until their
adrenal function recovers [8, 12].
Most cases of primary adrenal hyperplasia respond to bilateral adrenalectomy, while
unilateral adrenalectomy should be performed in those with adrenal adenoma or carcinoma
[27]. Adrenal enzyme inhibitors such as aminoglutethimide, metyrapone, and etomidate
should also be considered, particularly in patients with carcinoma. Ketoconazole, cabergoline
and mifepristone are also effective for long-term control [8] and a combination of drugs can
be used if monotherapy fails [28]. The treatment of steroid myopathy and CIM consists in
reduction of steroids and increased physical activity.
CIM and neuropathy have no nonspecific therapy other than tight glycemic control,
physical therapy [29], and reduction of corticosteroid dosage. IVIG infusions have not shown
significant benefit [30].
ADRENAL INSUFFICIENCY
Adrenal insufficiency (AI) refers to inadequate adrenocortical function that causes
reduction of corticoid secretion. This could be primarily caused by disorders of the adrenal
gland or Addison disease (AD), while a secondary form is caused by pituitary or
hypothalamic disorders causing ACTH deficiency.
Thomas Addison was the first to describe the disorder at the South London Medical
Society describing patients with anemia, weakness, languor, restlessness, paleness, and
decreased muscle strength with decreased mental function [4]. Addison also described
progressive and severe paleness, tachycardia, dyspnea, and edema, and generalized weakness.
Three of his patients were autopsied, and all had suprarenal capsular disease. In 1856, Charles
Brown-Sequard performed classic experiments by removing the adrenal gland from animals
and concluded that all had died of Addison disease. Later in 1899, Kippel introduced the
concept of encephalopathy addisonienne emphasizing that neuropsychiatric symptoms are
common in these patients [4].
AD can be caused by several disorders such as destructive lesions of the adrenal glands,
developmental defects of the glands characterized by psychomotor retardation, and impaired
steroidogenesis secondary to defects in cholesterol metabolism or mitochondrial DNA
abnormalities [31-32].
Most cases of AD are autoimmune. These occur either in isolation or as part of an
autoimmune polyglandular syndrome. Around 50% of the cases have other autoimmune
diseases [33, 34]. These include thyroid disease such as Hashimoto thyroiditis or Graves‘
disease, type I diabetes, premature ovarian failure, celiac disease and autoimmune gastritis
[33, 34].
The rare autoimmune polyendocrine syndrome type I or APS-I is also associated with
hypoparathyroidism and chronic mucocutaenous candidiasis. APS-II describes patients that
have a combination of autoimmune thyroid disease and type I diabetes. APSIII describes
cases without involvement of the adrenal glands [8] while APS-IV is characterized by AD
without thyroid involvement or type I diabetes, but patients with a combination with
autoimmune disorders.
Among other destructive causes of AI include infections such as tuberculosis, with or
without HIV infection [35]. Other mycobacterium can also cause the disorder, and this could
also be caused by toxoplasma, pneumyocystis and cytomegalovirus. Histoplasmosis,
cryptococcosis and paracoccidioidomycosis have a predilection for infecting the adrenal
glands and should be included in the differential diagnosis.
Destruction of the adrenal glands from hemorrhages is a rare cause of AI. A classical
etiology of acute of adrenal insufficiency is meningococcal septicemia (Waterhouse-
Friderichsen syndrome). This also occurs in heparin treatment and coagulopathies [36]. Other
causes include metastasis, amyloidosis, sarcoidosis, lupus, hemochromatosis, hemorrhagic
infarction, surgical removal of the glands, adrenoleukodystrophy, congenital hypoplasia and
other hereditary disorders [31, 38, 39]. A relative AD could occur in ischemic stroke
increasing morbidity in these patients [40].
In autoimmune AD, the main target of pathogenic auto-antibodies is the steroidogenic
enzyme 21-hydroxylase [41]. About 95% of cases with the disease have elevation of these
antibodies diagnosed [33, 42]. AD has been associated with certain MHC haplotypes, in
particular, DR-DQ2 and DR4-DQ8 [43].
Cytotoxic T lymphocyte antigen 4 inhibits T cell activation, and it seems to be involved
in disease susceptibility [44]. There is also an association with the gene protein tyrosine
phosphatase nonreceptor type 22 [45] while an association with polymorphisms in vitamin D
receptors has also been reported [46].
Disorders Caused by Developmental Defects
Developmental defects from genetic diseases causing AI are very rare. In adrenal
dysgenesis there are mutations of the dosage-sensitive sex reversal-adrenal hypoplasia gent 1
on Xp21 (DAX-1) [47] and mutations of steroidogenic factor-1 gene (SF-1) has also been
reported [48]. In congenital adrenal hypoplasia due to DAX-1 mutations, there is arrested
development of the adrenal cortex. This X-linked disorder manifests at birth, and the patient
has hypogonadotrophic hypogonadism. Another X-linked disorder has been associated with
glycerol kinase deficiency [49] and these patient‘s disease also present at birth and have
psychomotor retardation associated with muscular dystrophy due to the vicinity of the gene
with the dystrophin gene. They also have hypertelorism, strabismus, droopy mouth, anorchia,
cryptorchidism, short stature, and osteoporosis [50]. The so-called Zellwager spectrum
disorders can also be associated with AI. This includes a group of autosomal recessive
diseases with impaired peroxisome function causing congenital malformations [51].
Familial glucocorticoid deficiency (FGD) is a rare disorder characterized by deficiency of
cortisol and androgen secretions. The Allgrove or triple A syndrome is also an autosomal
recessive disorder that maps to chromosome 12q13 and consists of triad of ACTH resistance,
alacrima, and achalasia with gradual neurological dysfunction with neuropathy, deafness,
mental retardation [52].
Disorders Causing Impaired Steroidogenesis
Smith-Lemli-Opitz syndrome, congenital adrenal hyperplasia, lipoid congenital adrenal

hyperplasia, abetalipoproteinemia and homozygous familial hypercholesterolemia are
included in these disorders, because they have adrenal insufficiency [8, 32]. These patients
might have neurological manifestations.
Congenital adrenal hyperplasia, is a particularly important childhood disease because
patients with this disorder frequently have neurological, and particularly behavioral
manifestations. Rarely the disease has a late onset [53]. This is an autosomal recessive
condition caused by mutations of the CYP21 gene that encodes 21-hydroxylase, which
deficiency affects cortisol biosynthesis, and is associated with corticotrophin hypersecretion
and androgen over production. These patients have defects of the working memory [54]
associated with temporal lobe atrophy, and might also show white matter abnormalities on
MRI and on functional MRI. Females, but not males, may activate the amygdalae more than
healthy controls [55]. Females may also show masculine-like behavior and boy-like activities
and may have genital abnormalities such as enlarged clitoris [56-58].
Mitochondrial diseases can also manifest with endocrine disturbances, for example, the
Kearns-Sayre syndrome, which presents with myopathy, ptosis, ophthalmoplegia, deafness,
and endocrine dysfunction, hypothyroidism, mitochondrial myopathy, cardiac arrhythmia,
adrenal insufficiency [59].
Finally, several drugs can affect cortisol biosynthesis; for example, aminoglutethimide,
metyrapone, and etomidate, which are used to treat CS, while other drugs can accelerate
cortisol metabolism.
Secondary AI
Secondary AI is caused by sudden cessation of chronic exogenous glucocorticoid

therapy, and may also occur after surgical removal of adenomas. Preserved mineral-corticoid
secretion and decreased CRH or ACTH synthesis and secretion are typical.
Isolated ACTH deficiency is a rare disorder of possible autoimmune etiology in which
there is no ACTH-secretory response to CRH [60].
Pituitary apoplexy is characterized by an acute onset of headache, visual disturbances,
altered mental status, and hormonal dysfunction caused by hemorrhages or infarctions of the
hypophysis. Usually a pre-existing pituitary adenoma is found [8]. Sheehan syndrome is
caused by severe vascular insufficiency of the pituitary gland from a massive postpartum
hemorrhage. The clinical picture varies from nonspecific symptoms to coma, and there is
decreased or absent postpartum lactation and failure to resume menses after delivery. Patients
have hypogonadism and growth hormone and prolactin deficiencies. Approximately 90%
have secondary hypothyroidism and 55% have adrenal failure [61]. Because most patients
have mild disease, this disorder is often misdiagnosed. The treatment consists of hormone
replacement [61, 62].
Clinical Manifestations of Adrenal Insufficiency
The clinical presentation of AI depends on the rate and degree of the deficiency, and AD
is associated with glucocorticoiod and mineralocorticoid deficiencies while secondary AI has
an intact renin-angiotensin-aldosterone system.
Figure 5. Pigmentation in Addison‘s disease. (A) Hands of an 18-year-old woman with autoimmune
polyendocrine syndrome and Addison‘s disease. Pigmentation in a patient with Addison‘s disease
before (B) and after (C) treatment with hydrocortisone and fludrocortisone. (D) Similar changes also
seen in a 60-year-old man with tuberculous Addison‘s disease before and after corticosteroid therapy.
(E) Buccal pigmentation in the same patient. (B and C, courtesy of Professor C.R.W. Edwards.)
(Reproduced from Stewart, 2003, with permission.)
The onset of symptoms is usually insidious and is characterized by nonspecific symptoms

of corticosteroid deficiency, such as weakness, fatigue, general malaise, dizziness, weight
loss, fever, anorexia, and orthostatic hypotension, which is more prominent in AD [11].
Other symptoms include headaches, visual disturbances, craving for salt, abdominal
cramps, nausea, vomiting, and diarrhea alternating with constipation. Patients complain of
fatigue and sleepiness [62].
The most significant finding to differentiate the types of AI is the presence of
hyperpigmentation of the skin and mucous surfaces in the primary form (Figure 5). This
hyperpigmentation is caused by high plasma corticotrophin concentrations from decreased
cortisol feedback, consisting in increased melanocyte-stimulating hormone (MSH) levels [8,
35].
Since secondary AI is also associated with hypopituitarism, the clinical presentation may
be caused by other hormone deficiencies, and the symptoms are usually not as severe as in
AD.
Acute adrenal crisis or ―Addisonian crisis‖ it is a medical emergency in which patients
become hypotensive and may develop acute circulatory failure, hypoglycemia, severe
abdominal pain, confusion, convulsions, lethargy, coma, and death. This could be caused by
adrenal hemorrhage, intercurrent illness, stress, infection, trauma, and surgery in patients with
Addison‘s disease. The most common cause, however, is the abrupt withdrawal of steroids in
patients receiving long-term oral glucocorticoid treatment [8, 32].
The neurological manifestations of AI include neuropsychiatric symptoms, myopathy,
flexion contractures of the legs and rarely seizures [11]. Muscle pain and generalized
weakness can occur particularly in the upper extremities. Muscle biopsy and EMG studies are
normal or nonspecific. The flexion contractures syndrome is a rare disorder associated with
AD and hypopituitarism and rarely with isolated ACTH deficiency [64-66]. This has been
reported as a paraneoplastic manifestation [67]. This is characterized by progressive painful
flexion contractures of the pelvic girdle, hips, and knees without involvement of the extensor
muscles. There have also been cases associated with frozen shoulders [68, 69]. The exact
causative mechanism is unknown.
Neuropsychiatric manifestations of AI consist of attacks of giddiness, anxiety, and
delirium, mood and behavioral symptoms are common; psychosis and marked cognitive
changes including delirium appear to occur more rarely, and are associated with severe
disease [70-72]. There have been cases of catatonia. In patients with neuropsychiatric
symptoms, EEG studies are frequently abnormal showing diffuse slowing [73, 74].
Metabolic abnormalities such as hyponatremia may produce cognitive changes and
encephalopathy [75]. Severe hypoglycemia can also precipitate these, and hypoxia secondary
to severe hypotension may be responsible for acute changes in mental status.
Glucocorticoid receptors are distributed throughout the brain and are particularly
abundant in the hippocampus. It has been demonstrated that adrenalectomy produces massive
granular cell death in the dentate gyrus and hippocampus [76-79] and it is considered that this
could interrupt the hippocampal trisynaptic circuit, producing memory impairment and
cognitive changes [79, 80].
Glucocorticoids are essential for maintaining prefrontal cortical cognitive function [80],
and it has been proposed that a reduction of glucocorticoids may result in enhanced ability to
detect sensory inputs [81-83], and it is possible that patients receive abnormally high sensory
signals and may have a tendency to develop hallucinations.
Finally, an increase in endorphin levels has been proposed in AD [72, 84], and this might
cause psychosis.
Diagnosis of Adrenal Insufficiency
Several methods are used to diagnose adrenal insufficiency. A useful screening test is the
measurement of serum cortisol. An intact adrenocortical reserve is confirmed by basal
morning cortisol levels of over 500 nmol/L [31, 85, 86].
After screening, the most widely used test is the Synacthen or Cortrosyn test (synthetic
ACTH) for the diagnosis and differentiation between primary and secondary adrenal
insufficiency [87]. Baseline cortisol concentrations are obtained and then 30 and 60 minutes
after 250 µV of Synacthen is administered. Values over 500 indicate normal adrenal reserve
[88]. The corticotropin test is useful in the diagnosis of adrenal insufficiency in critical illness
[89].
The insulin tolerance test is considered the gold-standard for assessment of HPA reserve,
and is particularly useful in the detection of early secondary AI and growth hormone
deficiency.
The metyrapone test is based on the fact that metyrapone inhibits the adrenal cortex
enzyme 11β-hydroxylase, thus inhibiting the conversion of 11-deoxycortisol to cortisol. The
drug is administered at midnight, and cortisol levels are measured in the morning. In normal
individuals, postmetyrapone cortisol levels are low and 11-deoxycortisol rise.
ACTH concentrations above 100 ng/L are usually observed in primary AI [85], while
elevated ACTH concentrations accompanied by normal cortisol may represent a state of
subclinical AI that could progress to overt clinical AI.
In secondary AI, the CRH test can be used to differentiate pituitary from hypothalamic
etiologies [90]. In this test CRH is administered and serum cortisol is measured at baseline
and at 15, 30 and 60 minutes. In primary AI, aldosterone concentrations typically are low, and
are associated with elevated renin concentrations.
In clinically apparent autoimmune AI, adrenocortical autoantibodies are detected in over
90% of patients with AD [91]. Radiological tests are usually not necessary in patients with
autoimmune abnormalities, but for other forms of AI, imaging with MRI or CT could be
useful. CT-guided adrenal biopsy can be used in patients with suspected metastases and in
whom a known primary tumor remains unidentified [31].
Treatment of Adrenal Insufficiency
Treatment of AI consists of replacement of glucocorticoids and mineralocorticoids in

conjunction with fluid and electrolyte replacement. Patients should receive hydrocortisone or
cortisone. The oral dose of hydrocortisone is 25 mg/day in divided doses or using time-
release preparations [92] while cortisone could be given in dosages of 25 mg in the morning
and 12.5 mg in the evening. Morbidity and mortality, however, is high using the standard
treatment [93].
In primary AI, oral fludrocortisone is added in doses of 0.05-2 mg daily for
mineralocorticoid replacement [8, 90]. Steroid substitution therapy in adults with congenital
adrenal hyperplasia should be closely monitored [94]. Physical therapy is important to regain
function, and patients should be advised regarding the risk of an Addisonian crisis,
particularly in times of stress or surgery when the dosage of hydrocortisone should be
doubled or tripled [31]. During a crisis, patients should receive hydrocortisone IV in a dosage
of 100 mg q 8 hours.
ADRENOLEUKODYSTROPHY AND ADRENOMYELONEUROPATHY

Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) are allelic X-linked
genetic diseases with different clinical manifestations. In both there are accumulations of
saturated very long chain fatty acids (VLCFA) in the brain and adrenal cortex. They are
caused by a mutated gene known as ABCD1 which encodes a protein that transfers VLCFA
into the peroxisome to undergo β-oxidation. Their exact pathogenesis is unclear.
Adrenoleukodystrophy was first described by Siemerling and Creutzfield in 1923 [95]
and the disease was initially called Schilder-Addison disease because of the presence of
features of Addison disease such as ―bronze‖ dark skin changes along with findings of
Schilder disease which is a rare condition that affects children and is characterized by a
severe and acute diffuse demyelinating cerebral sclerosis with deafness and cortical blindness
[96]. Blaw coined the name of adrenoleukodystrophy in 1970 because of the association of
leukodystrophy with adrenal insufficiency [95].
In 1975, Schaumburg et al. performed extensive neuropathological analyses of ALD
patients, and later Budka and Griffin recognized that a form of this disease occurred in young
adults with progressive spastic paraparesis, neuropathy, and adrenal insufficiency. They
named this form of disorder adrenomyeloneuropathy [98, 99]. In 1973, Powers and
Schaumburg demonstrated the presence of trilamellar inclusions in the adrenocortical cells
[100] and 1976 Igarashi et al. showed that the cholesterol ester lipid fraction from the ALD
brain and adrenal cortex contained high levels of saturated very long chain fatty acids [101].
Clinical Manifestations of Adrenoleukodystrophy and

Adrenomyelomyopathy
Adrenoleukodystrophy has a variable age of onset with different phenotypes.

Neurological symptoms usually precede adrenal insufficiency in about 90% of the cases. The
patients have melanodermia and increased ACTH levels [95].
The clinical characteristics correspond to demyelinating lesions in the white matter of the
parieto-occipital lobes (Figure 6) [95]. Patients may have moderate dystonia and pyramidal
tract findings with marked behavioral changes occurring between the ages of 4 and 8 years.
Some patients have attention deficit disorders and may be diagnosed as having psychiatric
disease, and neuropsychiatric disorders can precede the onset of clinical and MRI alterations
[102].
Patients may lose the ability to read and to understand the oral language, and some also
develop seizures. Visual impairment and word deafness as well as apraxia, asterognosia, and
agraphasthesia are described. Gait disturbances with features of cerebellar and pyramidal tract
involvement are often seen. On rare occasions, the disease may have a remitting course [95,
96].
Adrenomyeloneuropathy usually manifests at the ages of 20-30 with an abnormal gait,
paraparesis, spasticity as well as proprioceptive deficits and neuropathy [95]. Patients also
have urinary incontinence, and impotence; cognitive function, however, is normal, and there
are no cerebellar or extrapyramidal tract findings. AI can be manifested clinically before the
diagnosis of the disease or may be evident biochemically at the time of diagnosis [95].
Patients may also have balding and appear older than their chronological age. They may have
hypogonadism and testicular atrophy. Heterozygote females may also manifest the disease
[103, 104], and the manifestations increase with age. Adrenoleukodystrophy should be
considered in the differential diagnosis in patients with myelopathy and neuropathy [105].
Both ALD and AMN can be seen in the same family. There could be a variety of
malformations whose cause is not clear [106].
Figure 6. T2-weighted MRI scans showing the progression of demyelinating lesions in an occipital
cerebral form of adrenoleukodystrophy (ALD) over the course of 4 years in a 6-year-old boy with ALD.
Demyelination initially struck the splenium of the corpus callosum (E, arrows). The patient developed
neuropsychological deficits associated with the progress of the demyelinating lesions. (Reproduced
from Aubourg, 1996, with permission).
Figure 7. T2-weighted MRI scans showing normal intensity of the posterior internal capsules (A,
arrows) and abnormal intensity of pyramidal tracts in posterior internal capsules of a case of
adrenomyeloneuropathy (B, arrows). Spinal cord atrophy is shown in (C). (Reproduced from Aubourg,
1996, with permission).
Diagnosis of Adrenoleukodystrophy and Adrenomyeloneuropathy
The initial laboratory diagnosis of both disorders is based on measurement of plasma

VLCFA and all affected and asymptomatic males have markedly elevated C26:0 levels with
an increased ratio of C24:0/C22:0 and C26:0/C22:0. Very rarely, patients may have normal
levels of C26, but all have abnormal ratios [107]. All of these should be performed in males
with idiopathic AI and in patients with undiagnosed paraparesis. Heterozygous female
carriers may also have elevated VLCFA, but false-negative tests can be seen. DNA
sequencing analysis is recommended for diagnosis [105, 108].
Once a mutation is identified, other family members can be rapidly tested. Screening
methods measuring C26.0 in newborn blood spot is a very significant test in neonatal
diagnosis [109]. Prenatal diagnosis can be made by measuring VLCFA levels in cultured
amniocytes and chorionic villus cells, but this should be confirmed by DNA analysis [110,
111].
The spinal fluid of these patients show marked elevation of protein content particularly in
patients with ALD, and may demonstrate increased immunoglobulin secretion with the
presence of oligoclonal bands similar to MS. In AMN, however, the spinal fluid is usually
normal.
MRI in ALD characteristically shows involvement of the occipital lobes and the splenium
and genu of the corpus callosum. The lesions may also involve the corticospinal tracts (Figure
7) and frontal lobes. Patients might also have evidence of hydrocephalus later, and the spinal
cord in AMN is atrophic. Some central demyelination may also be observed [107, 108, 110].
PET scanning using flurodroxyglucose might reveal abnormalities independent of
morphological characteristics, and could be a useful technique to evaluate the disease [112].
In ALD visual evoked responses may be normal initially, but they become abnormal
when the occipital and parietal white matter are affected. The brainstem auditory evoked
responses are abnormal and might present after 10 years of the disease, and even in those
patients without demyelinating lesions on the MRI. There is a severely delayed wave V as
well as delayed wave III-V and I-V interpeak latencies. Somatosensory evoked responses
may show large parietal waves with delayed N11-P14 and P14-N20 interpeak latencies
bilaterally. The EEG shows slowing of posterior waves [113].
In AMN, the VERs are usually normal, but the BAER abnormalities are found more
frequently than in ALD with delayed I-V interpeak latency, and somatosensory evoked
potential abnormalities are similar to those of ALD [95]. In AMN conduction velocities may
be slow in the legs.
Pathological findings in ALD consist of demyelination with loss of oligodendrocytes and
reactive gliosis, and PAS-positive macrophages and mononuclear cells. Inflammation is
usually found around demyelinating foci. In AMN, the pathological findings occur mainly in
the spinal cord with loss of myelin in axons with a distal axonopathy pattern involving mainly
the corticospinal tracts. In some patients cerebral demyelination could be seen.
Sural nerve biopsy in AMN shows both small and large myelinated fibers with
endoneural fibrosis and sometimes trilamellar bodies in Schwann cells [97].
Treatment of Adrenoleukodystrophy and Adrenomyeloneuropathy
Because most patients with ALD and AMN develop adrenal insufficiency, corticosteroid
replacement is critical. Hydrocortisone 10-40 mg a day in divided doses is usually given.
Testosterone replacement may also be helpful. Treatment of spasticity with physical therapy
and mobilization as well as muscle relaxants are useful.
Lorenzo‘s oil has proven to be effective in preventing or delaying the onset of symptoms
in boys with ALD and may be beneficial in patients with AMN. The oil is a 4:1 mixture of
two long chain fatty acids, glyceryl tioleate and glyceryl trieruricate. These fatty acids inhibit
endogenous VLCFA synthesis. When patients take this medication the levels of VLCFA
normalizes [95]. It was thought that this treatment may slow the progression and decrease
MRI abnormalities. Many trials in symptomatic patients have not shown benefit. However,
Moser et al. [114] found that 74% of asymptomatic ALD boys had normal neurological and
MRI examination after 7 years of this treatment, and 24% had abnormal MRI and 11%
developed clinical and radiological abnormalities. For this reason, this treatment should be
used early in asymptomatic ALD patients.
The only treatment shown to produce clear benefit in early symptomatic X-ALD is
hematopoetic stem cell therapy, and with this approach, 50% to 75% of boys show
improvement or stabilization in clinical and/or radiological findings [115-117]. HCT,
however, did not prevent the development of myelopathy in adrenal leukodystrophy [117].
Bone marrow transplantation may also be a good optional therapy [118]. Adeno-associated
gene therapy reduces VLCFA, and it could be a post therapeutic alternative [119].
Other drugs that have been tried for patients with ALD and AMN include lovastatin, 4-
phenylbutyrate, which are compounds that upregulate the expression of ABCD2 protein,
which potentially compensates for the loss of ABCD1 protein [118].
HYPERALDOSTERONISM
This condition is caused by selective increase of mineralocorticoid secretion by a primary
disorder of the zona glomerulosa of the adrenal glands. This is relatively uncommon and may
be caused by adrenal hyperplasia or less commonly by adenoma or carcinoma.
In 1954, Conn described a patient with a 7 year history of muscle spasms, tetany,
weakness, and arterial hypertension, severe hypokalemia, mild hypernatremia, and alkalosis
without evidence of excess glucocorticoids or androgen production, and this patient had
secretion of adrenal corticoids confirming his suspicion that this patient had excessive
secretion of the adrenal salt retaining corticoid, and he called the disorder primary
aldosteronism [120].
Primary hyperaldosteronism is caused by aldosterone-secreting adenomas or carcinoma
or bilateral hyperplasia of the gland, or primary unilateral hyperplasia [121]. Familial
hyperaldosteronism (FH) is a rare autosomal dominant disorder with two typical
presentations. Type I is associated with different degrees of hyperaldosteronism and high
level of 18-hydroxycortisol and 18-oxocortisol and responds to exogenous glucocorticoid
administration. These patients usually are hypertensive with weakness and failure to thrive
and have an increased incidence of intracranial aneurysm. Type II may be caused by
aldosterone-producing adenoma with bilateral idiopathic hyperaldosteronism or both [122].
Secondary hyperaldosteronism results from overactivation of the renin-angiotensin
system and occurs in patients with severe accelerated hypertension, renovascular
hypertension, estrogen administration, renin-secreting tumors, and Bartter syndrome which is
a rare disorder causing hypokalemia, metabolic alkalosis, hyperreninemia, hyper-
aldosteronism and hyperplasia of the juxtaglomerular apparatus [12].
Kearns-Sayre syndrome may also cause hyperaldosteronism with symptoms similar to
Bartter syndrome in addition to its multiple organ manifestations caused by mitochondrial
dysfunction[123]. There are several other causes of hyperaldosteronism including potassium
sodium-wasting nephropathy, renal tubular acidosis, diuretic or laxative abuse, chronic
edematous state, congenital absence of adrenal enzymes as well as administration of licorice,
carbenoxolone, flucrocortisone or other steroids [8].
Clinical manifestations of hyperaldosteronism are hypertension, hypokalemia, metabolic
alkalosis, causing fatigue, nocturia, and headaches. Severe hypokalemia results in polyuria,
polydipsia, paresthesias, and sometime temporary weakness or a persistent myopathy [124,
125]. Thus, hyperaldosteronism should be considered in patients presenting with muscle
weakness associated with low potassium, and patients may also have leg cramps, tetany
manifesting with Trousseau or Chvostek signs [126]. Patients may also have tremor,
encephalopathy, and syncope. Rarely, they could have idiopathic intracranial hypertension
[127]. Ischemic optic neuropathy is also an unusual presentation, and depression can occur
[10].
Diagnosis of Hyperaldosteronism
The diagnosis of hyperaldosteronism is suspected in patients with arterial hypertension

with hypokalemia and weakness. To diagnose the disease, one should measure aldosterone
and plasma renin activity (PRA) in the early morning hours. Serum aldosterone to PRA ratio
over 20 ng/dL and serum aldosterone levels of over 15 ng/dL suggest the diagnosis of
primary hyperaldosteronism [8, 12].
The second step consists in determining the cause such as adrenal adenoma or bilateral
adrenal hyperplasia. In this the postural test, aldosterone levels that increases upon standing
suggests bilateral hyperplasia. With adenomas, the baseline levels are over 20 ng/dL and
decrease upon standing as a result of decreased stimulation of ACTH. After this, imaging
would be necessary [8].
Treatment of Hyperaldosternonism
The treatment of hyperaldosteronism consists of removal of the adenoma by laparoscopic

adrenalectomy [128]. Ablative procedures using percutaneous acetic acid injection may also
be effective [129]. Hypertension may persist after treatment in as many as 40% to 65%
caused by nephrosclerosis [8, 130].
Mineralocorticoid receptor antagonists such as spironolactone and eplerenone, may be
used to normalize blood pressure and potassium levels. If patients fail to tolerate any of these
treatments, amiloride has been shown to reduce the systolic and diastolic blood pressure and
increase plasma levels [131]. Hydrochlorothiazide may be added to this to treat hypertension.
DISORDERS OF THE ADRENAL

MEDULLA (PHEOCHROMOCYTOMAS)
The adrenal medulla is the area of the adrenal gland that produces catecholamines from
secreting cells called chromaffin cells or pheochromocytes because they stain dark with
chromium salts. Tumors affecting the adrenal medulla cause increased secretion of
norepinephrine and epinephrine, and are called pheochromocytomas which are rare tumors
that can be single or multiple.
The name pheochromocytoma was proposed by Pick in 1912 [8], and the name comes
from the Greek words phaios or ―dusky‖ and chroma or ―color‖. In 1926 Roux in Switzerland
and Charles Mayo in Rochester performed successful surgical removal of adrenal
pheochromocytomas [8]. In 1929, it was discovered that pheochromocytomas contained an
excess of a pressor substance and subsequently catecholamines were isolated from these
tumors [132].
Catecholamine-secreting tumors arising from the adrenal medulla are called
pheocromocytomas. Those tumors rise in the sympathetic ganglia and are called extra-adrenal
paragangliomas [133]. Both have similar presentations and are treated similarly.
There are a number of genetic conditions associated with pheochromoctyomas which
might represent more than a third of the cases. These could include:
a. Multiple endocrine neoplasia type 2A (MEN 2A), or Sipple syndrome which is an

autosomal dominant disease characterized by primary hyperparathyroidism,
medullary carcinoma of the thyroid and bilateral pheochromocytomas [134, 135].
b. Multiple endocrine neoplasia type 2B (MEN 2B) is also an autosomal dominant

disorder and is phenotypically characterized by the association of
pheochromcytomas, mucosal neuromas and thickening of the optic nerves. These
tumors produce predominantly epinephrine and metanephrine. Many patients with
MEN 2A or 2B have mutations of the RET proto-oncogene, and it can be tested
commercially [8]. Men-1 or Wermer‘s syndrome affects the endocrine system
manifesting with disorders of pancreas, pituitary and parathyroid glands, and is
caused by different genetic mutations.
c. Von Hippel-Lindau disease is another autosomal dominant disorder characterized by
the presence of paragangliomas, pheochromocytomas, retinal angiomas, cerebellar
hemangioblastomas, renal and pancreatic cysts, and renal cell carcinomas. Most of
these tumors also produce predominantly norepinephrine and normetanephine [8].
They are all associated with mutation of the VHL, tumor suppressor gene [136].
d. Neurofibromatosis type 1 (NFI) is an autosomal dominant disease characterized by
neurofibromas, optic nerve gliomas, sphenoid dysplasia, café au lait spots, iris
hamartomas, and axillary/inguinal freckling. This disorder is caused by mutation of
the NF1 gene located in chromosome 17, which causes a decreased production of
neurofibromin [137].
e. Familial paraganglioma is an autosomal dominant disorder characterized by
paragangliomas localized in the head and neck. These tumors are usually benign.
f. Finally, other neurocutaenous disorders associated with catecholamine-secreting
tumors include ataxia-telangiectasia, tuberous sclerosis complex, and Sturge-Weber
syndrome.
Clinical Manifestations of Pheochromocytoma
Stressful stimuli such as anesthesia, hypoglycemia, or heart attacks increase the secretion
of catecholamines, which have a very active role in the cardiovascular system and metabolic
processes, increasing heart rate and blood pressure, myocardial contractibility and cardiac
abnormalities.
The clinical characteristics of pheochromocytomas are related to the type of
catecholamines that they secrete manifesting most frequently by hypertension, headaches,
palpitations and diaphoresis [138].
Tumors secreting norepinephrine usually produce severe sustained hypertension, whereas
those that secrete epinephrine produce episodic hypertensive crisis. The triad of episodic
headaches, diaphoresis, and palpitations has a specificity of over 60% for
pheochromocytomas [139].
Neurological manifestations are usually caused by changes in blood pressure, and include
episodic headaches, and sometimes ischemic or hemorrhagic cerebrovascular events [140-
142]. The patient may also have musculoskeletal pain with radiculopathy caused by bone
metastasis. Patients may also have seizures, severe hypertension, and stroke [143]. Recurrent
syncopal episodes may result from hypotension due to downregulation of catecholamine
receptors caused by chronic exposure to the neurotransmitter and volume depletion secondary
to inhibition of the renin-angiotensin system [144].
The differential diagnosis of pheocromocytoma is very extensive and includes

hyperthyroidism, anxiety, panic attacks, migraines, and drug abuse. Pheocromocytomas may
also produce somatostatin and ACTH causing symptoms resembling those in Cushing‘s
syndrome [8, 12].
Diagnosis of Pheochromocytooma
The diagnosis is based on measurement of catecholamines, particularly plasma and urine

metanephrines [143].
Other disorders can raise levels of catecholamines and metanephrines and they should be
considered in the interpretation of the tests. These include alcohol, withdrawal from
medications like clonidine, subarachnoid hemorrhage, migraines, preeclampsia, and the use of
medications such as tricyclic antidepressants, levodopa, buspirone, prochlorperazine, and
acetaminophen [144].
The clonidine suppression test is highly sensitive to distinguish between
pheochromocytoma and other causes of increased plasma catecholamines. Clonidine is a
centrally acting α-adrenergic receptor agonist that normally suppresses the release of
catecholamines from neurons, but does not suppress those secreted by tumors. Catecholamine
and metanephrine are measured before and after clonidine is administered orally in dose of
0.3 mg [145]. In patients with essential hypertension, their concentrations decrease
significantly while in patients with pheochromocytomas, this does not change [146].
Imaging techniques are very important to localize the presence of a tumor. Particularly
radioactive iodine with metaiodobenzylguanidine scan [147].
Treatment of Pheochromocytomas
The treatment of pheocromocytomas consists of complete surgical resection of the

tumors and management of hypertension, pre and post operatively is very important with
presurgical preparation which includes α-adrenergic blockers like phenoxybenzamine for
control of blood pressure and prevention of arrhythmia. A β-adrenergic antagonist also is
used after α-adrenergic blockage is obtained.
Metyrosine is also used in this setting because it is a catecholamine synthesis inhibitor
with antihypertensive properties. Hypertensive crisis should be treated acutely and
aggressively [8].
ACKNOWLEDGMENTS
Special thanks to Ms. Kay Daugherty for editorial assistance; Ms. Ginger Lindsey and
Ms. Cindy Burchfield for secretary assistance.
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Chapter 17
ADRENOCORTICAL CANCER
Amudhan Pugalenthi, MD and Eren Berber, MD,

Department of Endocrine Surgery, Cleveland Clinic, Cleveland, OH, US
ABSTRACT
Adrenocortical Carcinoma (ACC) is a rare tumor with a dismal prognosis. Currently,
radical surgical excision is the only potentially curative treatment available. In this
chapter, we will review the current data on the management of ACC and highlight recent
advances in the treatment.
Keywords: adrenocortical cancer, surgery
INTRODUCTION
Adrenocortical Carcinoma (ACC) is a rare endocrine tumor with a dismal prognosis.
Currently radical surgical excision is the only potentially curative treatment available.
However in the last two decades, significant advances in understanding the molecular
pathways that are dysregulated at the cellular level have increased hope of developing new
drugs for ACC in the near future [1]. In this chapter we will provide a current overview of
ACC and also highlight recent advances in the treatment of ACC.
DEMOGRAPHICS
ACC affects 0.72 persons per one million population and is considered an orphan disease
[2]. Women are affected more commonly than men in the ratio of 1.5:1 [3]. Although it
affects mainly adults, children can also be affected. There is a bimodal distribution of age

Eren Berber M.D., Associate Professor of Surgery, F20, Cleveland Clinic Main Campus, Cleveland, OH- 44195,
Phone: 216-445-0555; Fax: 216-636-0662, Email:berbere@ccf.org.
362 Amudhan Pugalenthi and Eren Berber
with the first peak at < 5 years of age and a second peak in the fourth and fifth decades [4, 5].
The median age of diagnosis is 46 years [6]. Females are more likely to have functional
tumors while males tend to have functional tumors before 20 years and non-functional tumors
after the age of 40 [4].
Majority of the ACCs are diagnosed when they had already grown > 10 cm. Although
there is an increased use of cross-sectional imaging in recent times, there has not been a
decrease in median size of the ACC at the time of initial presentation. Only minorities of
patients are diagnosed early when the tumor is < 5 cm [7]. ACCs most commonly arise
sporadically but some tumors are associated with hereditary syndromes such as Li-Fraumeni,
Beckwith-Wideman, Multiple Endocrine Neoplasia type1, Familial adenomatous polyposis,
Lynch syndrome and Carney complex [8].
Two-thirds of the patients have symptoms or signs related to excessive hormonal
secretion. Most commonly (50%), they present with corticosteroid excess with signs of
Cushing‘s syndrome, 20% have virilizing symptoms due to androgen excess in women, 10%
have feminizing symptoms due to estrogen-only secretion in men and rarely (2%) produce
aldosterone. Those patients with non-functional tumors present either with a mass effect due
to their large size or incidentally discovered by imaging when they are small. As the
incidentally discovered tumors are increasing, in the future we may detect tumors at an earlier
stage and smaller size. Some factors that increase the likelihood of ACC in an adrenal mass
are age < 20, Cushing‘s syndrome associated with an adrenal mass, adrenal mass associated
with increased urinary 17-ketosteroid or 17-OH corticosteroid, lack of high dose
dexamethasone suppression, fever, anemia, virilization or feminization signs. Paraneoplastic
syndromes are however very rare, the most notable being the tumor-induced hypoglycemia
secondary to excessive production of insulin like growth (IGF) factor 2. Metastatic spread
occurs to lungs (45%), liver (42%), lymph nodes (24%) and less commonly to other areas like
bone, pancreas, spleen and diaphragm [9].
PATHOGENESIS
ACC most commonly occurs sporadically. The pathogenesis of sporadic cases is not well
understood. Whether it develops de novo or arises from a pre-existing adenomatous or
hyperplastic lesion is not clearly known. Studies of genetic changes in ACC show similarity
with mutations associated with named familial syndromes. Studies of chromosomal
alterations done with comparative genomic hybridizations have revealed loss of
heterozygosity or allelic imbalances at 2p16, 11q13 and 17p13. More than 85% of malignant
tumors have these underlying genetic changes [8, 10, 11].
One of the most commonly associated mutations in ACC involves overexpression of IGF
receptor gene. IGF is involved both in the normal development of adrenal gland as well as
tumor pathogenesis. Alterations in growth factor receptors such as epidermal growth factor,
Adrenocortical Cancer 363
fibroblast growth factor, vascular endothelial growth factor have been implicated in the
development of ACC [12].
PATHOLOGY
If the clinical presentation and imaging show signs of local invasion or metastasis, the
diagnosis of ACC is obvious. In the absence of these, differentiating a benign tumor from a
malignant tumor is very difficult. There is limited data on the role of preoperative fine needle
aspiration in diagnosing incidental adrenal lesions without a prior history of cancer [13]. Due
to high false negative rates, a benign cytological diagnosis does not rule out cancer. But
computed tomography (CT) guided biopsy may be useful in a patient with previous history of
cancer that has a high propensity to metastasize to adrenal gland (i.e., lung, kidney, breast).
Post-resection, the Weiss criteria (Table 1) are utilized to differentiate between a benign and
malignant adrenal pathology [14].
Table 1. Weiss criteria*
Nuclear grade - (grade 3 or 4) as per Fuhrman criteria [58]

Mitotic rate-> 5 per 50 HPF (x40 objective, counting the greatest numbers of mitotic figures
in areas with greatest number of mitoses
Atypical mitotic figures
Clear cells 25% or less
Diffuse architecture 33% surface or more
Confluent necrosis
Presence of venous invasion
Presence of Sinusoidal invasion
Capsular invasion
Nine parameters are assessed on Hematoxylin& Eosin stained sections from representative areas of
tumor. Each parameter is scored zero when absent and 1 when present, HPF- high power field.
* Presence of 3 or more histological features has been traditionally associated with malignancy
A score of 1 is given for each of features if present. A total score of ≤2 is classified as an

adenoma. A score of >3 is suggestive of ACC. But a score of 2-3 is ambiguous and some feel
that this classification system may be inadequate [2, 15, 16]. Therefore additional markers
such as increased Ki-67 proliferative index, IGF overexpression, differential expression of
genes and microRNAs may in the future serve as important predictors of malignancy and
prognosis [17-19].
WORKUP AND DIAGNOSIS

Biochemical Tests
ACCs can be functional or non-functional. Two-thirds of patients diagnosed with ACC

present with clinical symptoms. The symptoms are usually related to excess secretion of
corticosteroids, androgen, estrogen or mineralocorticoids. These tumors are inefficient in

mature steroidogenesis. Therefore it is important to measure hormone precursor steroid
excess such as dehydroepiandrosterone sulfate (DHEAS) in the blood and urine before
classifying them as non-functional. Sub-clinical Cushing‘s syndrome is the most common
among the functional tumors.
The most reliable screening test to rule out a Cushing‘s syndrome is an abnormal urinary
free cortisol value >100 mg over 24 hours. An abnormal overnight dexamethasone
suppression test will confirm the diagnosis. A complete work up is necessary to rule out
hyperfunctionality in the form of subclinical Cushing‘s syndrome, hyperaldosteronism, or
hyperandrogenism and catecholamine excess, which may suggest the presence of a
pheochromocytoma or extra-adrenal paraganglioma.
Imaging Studies
Size and the appearance of adrenal lesions on imaging are very helpful to determine risk
of malignancy. Size of the adrenal gland is an important predictor of malignancy. At the time
of presentation, most ACCs are very large measuring about 10-15 cm. Only 2% of the tumors
≤ 4 cm are found to be ACC. If this size is in between 4.1 and 6 cm, the risk is 6%. The risk
increases to 25% when the adrenal size is > 6 cm [16, 20]. In addition to tumor size greater
than 4-6 cm, any increase in size over a period of 6 months should raise suspicion of ACC
[21].
Computed Tomography
CT scan is the most useful study to determine resectability and relationship of the tumor
to adjacent structures. Although size is important, certain morphological features of ACC on
CT scan include tumor heterogeneity, tumor necrosis/hemorrhage, ill-defined borders, a low-
attenuation central scar and calcification [22] (Figure 1 and 2).
In addition, tumor density based on Hounsfield units (HU) and washout characteristics
can distinguish between benign and malignant lesions. Adrenal lesion with an unenhanced
tumor density of HU <10 has a sensitivity and specificity of 71% and 98% to be considered as
a benign lesion [23]. ACC is suspected in any lesion which is > 10 HU on non-contrast CT, if
the delayed wash out is <50% and a delayed attenuation of >35 HU. CT scan of a locally
advanced ACC may have signs of nodal involvement or invasion into surrounding structures.
Evidence of extra-adrenal disease is evident in > 56% of CT scans performed at the initial
work-up (Figure 3). Intracaval thrombus is detected in 14-17% of CT scans [22, 24], which is
very helpful in preoperative planning and staging (Table 2). Distant metastasis to lung, liver
and bone is seen in 15-39% of patients [25].
Figure 1. CT scan showing a 4 cm left-sided ACC.
Figure 2. CT scan showing a 7 cm right-sided ACC.
Figure 3. (Continued)
Figure 3. Top: CT scan showing a large right-sided ACC invading the right lobe of the liver (axial
view); Bottom: ACC nvolving the inferior vena cava (coronal view). CT- computed tomography; ACC-
Adrenocortical Cancer.
Table 2. AJCC (TNM) staging for Adrenocortical Cancer
TX The primary tumor cannot be evaluated

T0: There is no primary tumor
T1: tumor is ≤ 5 cm and confined to the adrenal gland
T2 tumor is > 5 cm but confined to the adrenal gland
T3 tumor of any size with local invasion but absence of invasion into adjacent organs
T4 tumor of any size with local invasion into organs such as kidney, diaphragm, great
vessels, pancreas, spleen and liver
NX- regional nodes cannot be evaluated
N0- no regional lymph node metastasis
N1- regional lymph nodal metastasis
M0- No distant metastasis
M1- distant metastasis
Stage group
I T1 N0 M0
II T2 N0 M0
III T1-2 N1 M0
T3 N0 M0
IV T3 N1 M0
T4 N0-1 M0
Any T, any N, M1
Adapted from AJCC cancer staging manual (7th edition) [59]
AJCC- American Joint Commission for Cancer; T- Tumor size; N- nodal status; M- metastasis.
MAGNETIC RESONANCE IMAGING (MRI)

Both CT and MRI are equally efficacious in diagnosing ACC. MRI has a sensitivity
ranging from 81%-89% and a specificity ranging from 92%-99% in distinguishing benign and
malignant adrenal masses [26-28]. Generally adenomas have a large amount of intracellular
lipid content and MRIs differentiate adenomatous from nonadenomatous lesions based on the
lipid content. ACCs are isointense to the liver on T1-weighted images and have intermediate-
to-increased intensity on T2 weighted images.
Features suggestive of ACC in MRI include central necrosis, hemorrhage and
peripherally enhancing nodules. Contrast-enhanced MRI with Gadolinium is associated with
a sensitivity of 81%-89% and a specificity of 92%-99%. MRI is also better in diagnosing
inferior vena caval involvement in case of right-sided adrenal tumors and renal vein
involvement in case of left-sided tumors [25].
OTHER DIAGNOSTIC STUDIES

Fluorodeoxyglucose-positron emission tomography (FDG-PET) has been found to be
useful as an adjunct diagnostic tool in cases of indeterminate adrenal lesions in situations
where the CT findings are suspicious. Groussin et al., reported a sensitivity of 100% and
specificity of 88% in the ability of FDG-PET to differentiate between adenomas and ACCs
when they used an adrenal/liver standardized uptake ratio of 1.45 as a cutoff [29]. Metser et
al., analyzed 175 adrenal lesions in their retrospective study and found that PET had
misdiagnosed 9 lesions. However when non-contrast CT was further utilized to analyze these
lesions the number decreased to 3. Further studies are required to substantiate the role of
FDG-PET to classify adrenal lesions. Until then PET can be used to provide additional
information to complement other imaging studies like CT or MRI.
Metomidate and Iodometomidate (IMTO) bind to both 11 β-hydroxylase and aldosterone
synthase and are taken up by adrenocortical cells. Radiolabelled Metomidate with either 11C
for PET or 123I for single-photon emission CT (SPECT) is accepted as a more specific agent
for detecting adrenocortical tumors [30]. In a prospective study, Kreissl et al., reported their
study of 58 patients comparing Metomidate based imaging to diagnose ACC to histological
diagnosis as a gold standard. Only 30% of the proven lesions showed strong uptake, 8% had
moderate uptake and 62% did not show any tracer accumulation. They concluded that IMTO
based SPECT/CT detected ACC primary and metastases with a specificity of 100%, but had a
low sensitivity of 30%. Further studies are needed to validate the utility of Metomidate based
functional imaging as a tool for accurate diagnosis and staging of ACC.
MANAGEMENT
Surgery
Surgery remains the only curative treatment for resectable ACC. Any adrenal lesion with
the following features should be considered for resection: functional tumor, non-functional
but size >4 cm, HU >20, washout of <40-50% on delayed contrast enhanced imaging, PET
positivity, MRI showing internal necrosis/hemorrhage, peripherally enhancing nodules and
obviously invasive features on imaging. An open resection of ACC via a subcostal incision is
recommended. Although some studies have reported laparoscopic approach to ACC, the use
of minimally invasive approach is controversial [31]. Routine removal of kidney is not
advised. But if loco-regional involvement is present, en bloc removal of kidney, adrenal, and
lymph nodes should be considered.
A thoracoabdominal incision may be utilized if lung is involved. Very rarely larger
resection may be attempted in case of functional tumors to alleviate endocrine symptoms.
Rarely resection of ACC with inferior vena cava (IVC) involvement on the right side may
require resection of infra renal IVC or cardiopulmonary bypass if suprahepaticcaval or right
atrium is involved. Postoperative adrenal insufficiency is a major cause of morbidity and it
must be prevented [32]. To avoid this careful monitoring and replacing of both
glucocorticoids and mineralocorticoids is essential.
In 1992, Gagner demonstrated the laparoscopic approach for adrenalectomy [33]. Since
then it has gained widespread popularity. Some studies have reported laparoscopic approach
for malignant adrenal tumors as well. Brix et al., [31] compared 117 who underwent OA and
35 patients who underwent LA for tumors ≤ 10 cm. They found no difference in recurrence-
or disease free survival for stage I and stage II ACC.
Table 3. Outcomes comparing Open Adrenalectomy (OA) and Laparoscopic

Adrenalectomy (LA)
Author, Year Type (%) n (%) Tumor Size Recurrence (%) Survival Median F/U
Country
Brix, 2010 [31] OA 117 8 69 -- 32 months
Germany LA 35 6.2 77 -- 64 months
Porpiglia, 2010 OA 25 10.5 64 84% 38 months
[34]
Italy LA 18 9 50 100% 30 months
Leboulleux, 2010 OA 58 13.7 27 66% 4 year rate
[60]
France LA 6 6.9 67 28%
Miller, 2012 [36] OA 110 12 40 (103/44) ★ 30 months
USA LA 46 7.4 85 (51/28) ★ 19 months
Lombardi, 2012 OA 126 9.0 6 38% 40 months
[35]
Italy LA 30 7.7 4 58% 50 months
Cooper, 2013 [37] OA 46 12.3 59 53★ 38 months
USA LA 46 8 76 110★ 29 months
Donatini, 2014 OA 21 6.8 24 81% 57 Months
[61]
USA LA 13 5.5 31 85% 80 Months
- Median size in cm; F/U- follow-up; ★- median survival in months; - Stage II/III.
Similar results were reported by Porpiglia et al., and Lombardi et al., [34, 35]. However,
Miller et al., reported lesser recurrence rate and better survival after OA [36]. Cooper et al.,
reported superior recurrence free survival and overall survival after OA [37] (Table 3). The
Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) guidelines for the
minimally invasive treatment of adrenal pathology, recommends that ACC requires an en
bloc resection of any contiguous structures and regional lymphadenectomy. In addition to
recommending the open approach, it states that ―if a minimally invasive approach is chosen,
conversion to open surgery is strongly recommended‖ [38].
To summarize, although the benefits of LA are well known for benign adrenal
pathologies, including lesser morbidity, decreased pain, shorter hospital stay, OA for ACC
has better oncological outcome. In case of suspected or malignant adrenal masses OA can
accomplish superior R0 resection, lesser recurrence and better overall survival.
Role of Lymphadenectomy
Although nodal status has ben incorporated into the ACC staging system, regional
lymphadenectomy is not routinely performed as a part of resection of ACC. The National
Cancer database and German ACC registry reports node positivity rates of approximately
26% in resected specimens although rates can be as high as 68% as reported from autopsy
specimens. Some studies report that only 17-18% of patients undergo some form of
lymphadenectomy at the time of initial resection [2, 39], but the exact details of the lymph
node station and number of nodes removed in the specimen are unclear. The majority (60%)
of recurrences after surgery for ACC occur in the lymph nodes. Poor loco-regional control of
disease might contribute to the high rate of recurrence in the lymph nodal basin [40].
Most common lymph nodal station involved during recurrence are the
ipsilateralaortocaval lymph nodes [40, 41]. Based on this Gaujoux et al., suggested that
perirenal, celiac, ipsilateralaortocaval lymph nodes from the aortic hiatus to renal vein must
be removed as part of regional lymphadenectomy at the time of initial resection [42]. This is
justified, as node positivity is associated with poorer outcomes. In an analysis of 3982
patients from the National Cancer Data Base, factors associated with increase risk of death
include age > 55 years, poorly differentiated tumors, margin positivity, adjacent organ
involvement and presence of nodal and distant metastasis [2].
Based on the available data, we recommend routine regional lymphadenectomy for all
patients with ACC at the time of initial resection.
Adjuvant Therapy
Even when complete resection is achieved, up to 85% of patients with ACC will
eventually recur. Therefore there is a strong need for adjuvant therapy. Although the number
of adjuvant therapies for ACC is growing, due to the rarity of the disease and limited
experience in various centers, the true efficacy of these treatment modalities is difficult to
ascertain.
Mitotane
Mitotane (o, p‘-DDD or 1,1-dichloro-2 (o-chlorophenyl)-2-(p-chlorophenyl) ethane) is an

isomer of the pesticide, DDD, and is directly toxic to adrenocortical cells. Bergenstal et al.,
first reported the use of mitotane for ACC [43]. Mitotane is utilized regularly in the adjuvant
setting and also as a first line treatment for metastatic ACC. Although the exact mechanism of
action is not clearly understood, mitotane is an adrenolytic substance that acts through an
apoptotic process by disrupting the mitochondria [44].
A majority of patients (80%) experience some form of side effects mainly gastrointestinal
symptoms. Although in some (40%) neurological symptoms including depression and
suicidal ideations have been reported [25]. The therapeutic window is narrow; therefore the
serum concentration is targeted to be 14 mg/l.
In a multi-center retrospective study from Europe the target concentration was achieved
and maintained in 63 patients and the median follow-up in the study was 36 months. In this
study, the group that received mitotane achieved a lower recurrence rate (35% vs 61%) and
prolonged recurrence-free survival (Hazard Ratio 0.4, 95% Confidence Interval-0.22-0.79)
when compared to patients who did not reach target concentration [45]. The average dose for
response is 8.5 g and a response is usually seen within 4 weeks. The mean duration of
response is 10.2 months [9]. The ADIUVO trial is currently comparing patients with low to
intermediate risk of recurrence receiving mitotane to observation alone (http://www.adiuvo-
trial.org). Combination of mitotane along with streptozocin, or combination of etoposide,
doxorubicin, and cisplatin (EDP) have are been tried as adjuvant therapies. Khan et al. [46]
reported improved disease-free survival using a combination of mitotane and streptozocin.
Radiotherapy
The role of adjuvant radiotherapy is controversial. ACC is considered as a tumor that is

relatively insensitive to radiation. Additionally, toxicity to adjacent organs like small bowel,
kidney and spinal cord limit the dose that can be given. Habra et al., in a recent retrospective
study did not find any difference in the 5-year local recurrence rate between patients who
received adjuvant radiotherapy within 90 days and who did not receive radiation following
resection [47]. But, Polat et al., recommend adjuvant radiotherapy for all patients who are
R1/Rx status after resection, for tumors > 8 cm, or a Ki-67 index > 10% and stage III disease.
Radiotherapy plays an important role for palliation of symptoms and to prevent complications
from metastasis in brain, bone and those causing caval obstruction [41].
Other Options
Radiofrequency Ablation and recently Microwave ablation have been utilized to treat
tumor recurrences or as an adjunct to surgery in select patients. Ablation is very effective for
tumors less than 5 cm and helps in local control. Ripley et al., showed that selected patients
who had liver metastasis from ACC that underwent RFA had similar overall survival in
patients who underwent liver resection [48].
Arterial embolization or chemoembolization has been reported to help in controlling

metastatic liver disease to achieve symptom control either alone or combined with ablation
modalities. Soga et al., reported response rates of approximately 21% [49].
Treatment of Recurrent or Metastatic Disease
Mitotane alone or in combination with standard chemotherapy remains the standard of

care for unresectable or metastatic disease. Gonzalez et al., reported a recurrence rate of 74%
during follow-up after resection and when further treated with mitotane, a 19% response rate
was observed [50]. In case of complete resection of recurrence or metastatic disease, a median
survival of 74 months was reported compared to 16 months for incomplete resection [5].
The FIRM-ACT trial (the first international randomized trial for adrenocortical cancer
treatment) [51] by the Collaborative group for Adrenocortical Cancer for advanced ACC,
demonstrated treatment with mitotane-EDP had a better overall survival of 17.1 months vs
4.7 months with streptozocin-EDP combination. Based on this mitotane-EDP is the first-line
cytotoxic therapy for advanced ACC [52].
RECENT ADVANCES
Understanding the molecular basis of ACC is important. Recent studies have highlighted
that multiple pathways are noted to be dysregulated in ACC at a cellular level. The epidermal
growth factor receptor (EGFR) and insulin like growth factor 1 receptor (IGF 1R) tyrosine
kinases‘ signaling cascades have been found to be dysregulated. Targeting these signaling
molecules and proliferative pathways that they activate are of ongoing interest.
However the use of tyrosine kinase inhibitors includingimatinib or erlotinib for treatment
of ACC has produced poor responses [53]. But a phase II trial using Sunitinib has shown
some response [54, 55]. IGF 1R monoclonal antibodies like figitumumab or cixutumumab
have produced responses and achieved stable disease in patients with metastatic refractory
ACC [56].
Fassnacht et al., reported that Linsitinib (OSI-906), potent inhibitors of IGF-1R and
insulin receptor not improve overall survival in patients with advanced ACC [57]. The major
players in the pathogenesis are still unknown. The ongoing international efforts and ―-omic‖
approaches and next generation sequencing will throw more light and improve our
understanding of pathogenesis and novel treatment strategies.
CONCLUSION
ACC is an extremely rare and aggressive disease. Management of ACC continues to be a
clinical challenge. At present an open approach for suspected ACC is recommended to
achieve R0 resection along with lymphadenectomy at the time of original surgery. Adjuvant
mitotane based therapy is essential to reduce local recurrence. In future, there is a need for
exploring alternative treatment options including targeted therapies.
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Chapter 18
ADRENAL LEIOMYOMAS
Prashant B. Joshi, MS, MCh, FMAPS

Consultant Pediatric Surgeon, Sir HN Reliance Foundation Hospital
& Research Centre, Mumbai, India
ABSTRACT
Adrenal leiomyomas are rare benign tumors. Amongst all the adrenal incidentalomas
they form one of the uncommon differential diagnoses in miscellaneous tumors. Adrenal
incidentalomas are clinically unapparent adrenal masses discovered inadvertently in the
course of diagnostic testing or treatment for other clinical conditions that are not related
to adrenal disease. Leiomyomas are soft tissue tumors, which arise from smooth muscle
fibers and the residual embryonic blood vessel tissue. Less than twenty-five cases of
primary adrenal leiomyoma are reported in literature so far. Most of them are
nonfunctioning, incidentally discovered, unilateral masses but few bilateral cases have
also been reported. Majority of patients with adrenal leiomyomas have HIV and/or latent
Epstein-Barr virus infections. There are individual case reports of association with
autoimmune disease as well. Hence, immunological work up and surveillance is
important. Complete surgical excision either by open or laparoscopy is curative.
Although recurrence is uncommon, follow-up is recommended in multilobed& multifocal
tumors, especially in immunocompromised patients.
Keywords: adrenal, leiomyoma, incidentaloma, tumor, HIV, Epstein-Barr,

immunodeficiency
INTRODUCTION
Leiomyomas are soft tissue tumors, which arise from smooth muscle fibers and the
residual embryonal blood vessel tissue [1]. Leiomyomas arising in the adrenal gland are very
rare [1, 2]. Less than twenty-five cases of primary adrenal leiomyoma (AL) have been

Email: prashant.b.joshi@hnhospital.com.
378 Prashant B. Joshi
reported in the literature so far. Incidental adrenal masses are often discovered during
investigative studies for unrelated abdominal or thoracic pathology [1, 2, 3].
An adrenal mass, generally 1 cm or more in diameter, which is discovered inadvertently
during a radiological examination performed for indications not related to adrenal gland, is
called adrenal incidentaloma [4, 5, 6]. Adrenal incidentalomas are increasingly found
recentlybecause of the widespread use of imaging modalities [4, 7]. The majority of adrenal
incidentalomas are either (pheochromocytomas), adrenal cortical tumours (adenomas or
carcinomas) or adrenal medullary tumours.A benign, clinically nonfunctioning adrenocortical
adenoma is the most common cause of an incidentally discovered adrenal mass [8].
Table 1. Rare Adrenal Masses
Pathology Differentials Hormone Diagnosis

Secretion
Adrenal Benign adrenocortical Non-secreting Asymptomatic (picked up incidentally
incidentaloma adenomas on CT scan or Ultrasound)
Adrenal cysts
Adrenal lymphomas
Miscellanous Schwannomas Non-secreting

Myelolipomas
Metastases
Leiomyoma
Amongst all the adrenal incidentalomas, AL form one of the least common differential
diagnoses in miscellaneous tumors as mentioned in Table 1. Leiomyomas are benign in nature
and are mainly non-hormone secreting tumors. However, the adrenal gland can give rise to a
broad range of other tumor types. Although rare, adrenal leiomyomas need to be considered
as an important differential diagnosis amongst evaluating adrenal gland masses. After an
extensive literature search, all the reported cases of adrenal leiomyoma and its characteristics
are discussed in detail in this chapter.
Leiomyomas are benign, smooth muscle tumors that can originate anywhere in the body
where smooth muscle layers exist such as the capsule and blood vessel walls [2, 3, 9]. They
mostly arise fromuterine and gastrointestinal tissue [2, 10].
Primary leiomyoma of the adrenal gland is a rare tumor arising from the smooth muscle
of the adrenal vein and its tributaries [3]. The adrenal leiomyomas reported in the literature
were large on diagnosis (a range of 3-11 cm in diameter) and were found over a wide age
range, from early childhood to late adulthood (an age range from 2-72 years). Most (16 of 21)
were solitary (unilateral), adrenal masses with equal distribution on both right and left side.
However, five cases of bilateral involvement have also been reported.
Adrenal leiomyoma has been reported to occur at all ages. Although more common in
adult population, it has also been reported in pediatric age group [3]. A total of six cases of
Adrenal Leiomyomas 379
pediatric ALs have been reported in English literature so far [3, 11, 12, 13, 14, 15]. With
respect to the current literature, adrenal leiomyomas are reported more in females than males.
Females were more commonly affected (four out of six cases) in pediatric age group as well
[3, 11, 12, 13, 14, 15]. Most ofthe tumors reported in literature (20 of 21) were non-
functional, except one which was reported to be functional. However this is very unusual as
leiomyomas are metabolically inactive [3].
Hence by review of literature most common modality of presentation is asymptomatic
adrenal mass found on radiological investigations [1-15]. Some patients might present with
history of dull aching or mild abdominal pain [3]. There may be a history of
immunodeficiency [3].
On examination the patients are normotensive without any stigmata of Cushing‘s
Syndrome. Patient may have scars of healed cutaneous viral infection on the body [3]. The
abdomen is generally soft, non-tender with or without a palpable lump depending on the size
of the tumor.
ASSOCIATIONS
Adrenal leiomyomas have been documented in immunosuppressed states such as AIDS,
organ transplant or chemotherapy. An altered immune response has been suggested to explain
the increased prevalence of soft tissue tumors in immunodeficiency states. Multiple organ
involvement &multifocalityhave also been noted in immunodeficient patients [16–18].
Leiomyomas have been found in different sites, including the adrenals, in both children
and adults with HIV [3, 16, 17, 18]. The link between the immune system and smooth muscle
tumors of the adrenal gland is unclear. It has been postulated that infection with the human
immunodeficiency virus (HIV) may promote smooth muscle tumors. HIV may have a direct
or indirect oncogenic stimulatory effect [11, 19].
It has been suggested that both Kaposi‘s sarcoma and smooth-muscle tumors might arise
from a common stem cell under the influence of some unknown factor that is produced during
HIV infection [17]. The association between low CD4 counts and adrenal leiomyomas has not
been studied.
Strong association of adrenal leiomyomas with Epstein-Barr virus hasbeen noted [12, 20,
21, 22]. The pathological reason for this association is not clear. However interestingly,
association between Epstein-Barr virus infection and an increased incidence of smooth
muscle tumors has been observed mostly in immunocompromised patients but not in
immunocompetent patients [17].
There is only one case reported of adrenal leiomyoma & associated Autoimmune disease
(Hashimoto‘s Thyroiditis), out of 21 reported cases. This is an unusual association [23].
INVESTIGATIONS & EVALUATION

As mentioned, the term ―adrenal incidentaloma‖ is used to describe an adrenal mass that
is discovered during imaging, for indications unrelated to the adrenals. The widespread use of
MRI & CT scan is expected to increase the number of incidentally discovered adrenal masses
[24]. Adrenal masses have been identified in up to 8.7% of individuals in an autopsy [25].
An adrenal leiomyoma is a rare cause of an adrenal mass. There are less than 25 reported
cases in the literature worldwide. They are usually well defined on imaging both Ultrasound
and Contrasted CT Scan [1, 3, 24]. Heterogenous contrast enhancement in post contrast films
on CT may be found in adrenal leiomyoma however, it might be difficult to rule out
malignancy, radiologically [1, 3].
On computed tomography (CT) imaging, malignancy is suggested by a diameter that is
greater than 4 cm, an irregular border, inhomogeneity, high pre-contrast Hounsfield units
(HUs) (> 10) and limited washout of contrast after 10-15 minutes (< 50%) [4, 5, 7, 24].
FDG- PET positive AL have been reported in literature [3]. It is quite unusual as
leiomyomas are benign tumors with low proliferation rate. The possible explanation as given
by Chura et al., suggested that increased vascularity may be the cause of PET scan positivity
in benign leiomyomas [26].
The laboratory evaluation is aimed to exclude subclinical Cushing‘s syndrome primarily,
as well as an asymptomatic pheochromocytoma. A late-night serum cortisol, a serum
dehydroepiandrosteronesulphate level, 24-hour urine total metanephrines and 24-hour urine
cortisol determinations need to be performed, all of which may be within normal limits. A
normal serum cortisol undertaken at 0800hrs (after 1 mg betamethasone the night before)
effectively rules out the presence of a cortisol-producing adenoma [24].
Table 2. Reported Cases of Adrenal Leiomyoma
N Ref Age Sex Tumor Bilateral Size (cm) AIDS+ Epstein-Barr virus-
(y) Function positive
1 [29] 53 F No No 5.5 x 4.5 x 3.5
2 [28] 49 F No No 3 x 3.5 x 3 Yes
3 [31] 65 F No No 5 x 3. x 4.2
4 [19] 35 F Unknown No 3.5 Yes
5 [11] 2 M Unknown No 7x5x5 Yes Yes
6 [10] 48 F No No 5.5 x 5 x 4.5
7 [33] 72 M No No 9x7x6
8 [32] 32 M No No 3 Yes
9 [15] 10 F Unknown No 5x4x3
10 [13] 11 F Unknown Yes Left: 5; R: 3 Yes
11 [16] 15 M No Yes Left: 4 x 5 x 3.5
Right: 8x 5 x 3
12 [34] 40 F No No Unknown
13 [35] 56 M No No 7.2
14 [2] 31 F No No 11 x 9 x 7 Yes
15 [23] 38 F No No Unknown
16 [17] 28 M Unknown No 3 Yes
17 [24] 40 M No No 4.9 x 5.5 x 6 Yes Yes
18 [3] 11 F Yes Yes Left:3.5 x 3Right 9 Yes
x7x4
19 [1] 26 F No No 9x7x5
20 [14] 7 F No Yes Unknown Yes
21 [27] 55 F No Yes Left:10 x 6 x 4
Right 10 x 6
Adrenal calcification is an uncommon clinical entity [27]. Only one case out of the 21
reported cases of adrenal leiomyoma (Table 2) presented as calcification during
investigations. Adrenal hemorrhage and tuberculosis are the most common causes of adrenal
calcifications [27].
Adrenal leiomyomas co-exist in patients diagnosed with human immunodeficiency
virus/acquired immunodeficiency syndrome (HIV/AIDS) and/or latent Epstein-Barr virus
(EBV) infection [3, 11, 24, 28]. Hence ELISA testing for HIV is to be done [3, 11, 24, 28].
In case of Epstein-Barr Virus, EBV-encoded RNA protein (EBER-1) indicative of co-
infection with EBV, is required to be done [3, 28]. The EBV genome has to be detected by
polymerase chain reaction [28].
Other Immunodeficiency work up includes nitro blue tetrazolium (NBT) test, absolute
lymphocyte and differential lymphocyte counts [3].
MANAGEMENT
Main modality of management is surgery [3]. Complete surgical excision of AL is
usually curative. This can be approached by laparoscopic [28], hand-assisted laparoscopic
[29] and open techniques [3].
Open surgical approach with laparotomy and excision of adrenal mass is generally
practiced for adrenal tumors. However in adrenal leiomyomas especially, it is imperative to
look for multifocal and multilobed tumors, intra-operatively to facilitate complete excision
[3].
Laparoscopic adrenalectomy has become the treatment of choice recently inpatients with
adrenal tumors [11, 27]. Laparoscopic procedures are associated with shorter hospital stay,
less postoperative discomfort and a lower rate of complications. Well-encapsulated adrenal
masses like without evidence of local invasion radiologically, can be removed
laparoscopically [27]. Several authors limit the laparoscopic adrenalectomy to lesions less
than 6 cm in size, whereas some have performed laparoscopic adrenalectomy on tumors up to
13 cm [27].
Transperitoneal approach is recommended. Nguyen et al., reported their 10-year
experience with laparoscopic adrenalectomy in 150 patients to be safe and effective [30].
However, they also recommended referral to a specialized center for optimal outcomes.
Laparoscopic adrenalectomy is safe and effective in carefully selected cases. Size is a
criterion, but in cases without local invasion, the laparoscopic approach can be a viable option
[27, 30].
Hand assisted Laparoscopic Adrenalectomy done in a case of adrenal mass which later
turned out to be Leiomyoma on histopathological examination has also been reported [29].
HISTOPATHOLOGY
Macroscopically, the tissue has a white in color on appearance with no bleeding,
ulceration or necrosis. Microscopically it is a well-circumscribed and encapsulated smooth
muscle tumor comprising bland, spindle-shaped cells. There will be no features of mitosis,
areas of necrosis or pleomorphism [3, 24].
In addition to hematoxylin and eosin (H&E) staining, Immunohistochemistry (IHC)
assays which shows positive for smooth muscle actin and desmin, confirmsthe smooth muscle
origin of this tumor [3]. Hence, it is advisable to carry out IHC [1].
However, other benigntumors of smooth muscle origin, particularly inflammatory-
myofibroblastic tumors should be ruled out. The latter also has an inflammatory component
rich in lymphoplasma cells [1]. In addition, other IHC markers such as CD10, CD34, CD117,
ALK-1, ER, and PR also need to be assessed [1].
Result will be negative for Adrenal Leiomyoma. But, Inflammatory myofibroblastic
tumorsare positive in 50% of cases [17, 18] and are sometimes positive forCD117 [19, 20].
Additional IHC stains have also been used by someauthors [i.e., S100, desmin, epithelial
membrane antigen (EMA), CD34, Bcl-2, and CD117 (c-kit)] [1, 2].
PROGNOSIS & FOLLOW-UP

On follow-up, radiological investigations mostly ultrasound help to confirm complete
excision [3]. Close follow-up is recommended in cases of multifocal and multilobed tumors,
especially in immunocompromised patients [3].
CONCLUSION
Adrenal location of leiomyomas is very rare. It should be considered in the differential
diagnosis of adrenal incidentalomas. These tumors can vary greatly in size and can display
radiological features that are indistinguishable from other nonadenomatous adrenal lesions,
such as carcinoma and pheochromocytoma.
ALs should be suspected in immunocompromised patients presenting with adrenal
masses. Immunological workup and surveillance is important. Intraoperatively and
radiologically, one should be vigilant for multifocal tumors. Complete surgical excision is
curative. Close follow up is recommended post operatively.
ACKNOWLEDGEMENTS
The Author thanks Dr. Jalpan P. Joshi for critical insights.
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[11] Jimenez-Heffernan JA, Hardisson D et alAdrenal gland leiomyoma in a child with
acquired immunodeficiency syndrome. Pediatr. Pathol. Lab. Med. 1995:15:923–929.
[12] Monforte MH, Kapoor N et al. Epstein-Barr virus-associated leiomyomatosis and post
transplant lymphoproliferaltive disorder in a child with severe combined
immunodeficiency: case report and review of the literature. Pediatr. Dev. Pathol. 2003:
6: 449–457.
[13] Rosenfeld DL, Girgis WS et al. Bilateral smooth muscle tumors of the adrenals in a
child with AIDS. Pediatr. Radiol. 1999: 29: 376–378.
[14] Petrilli G, Lorenzi L et al. Epstein-Barr virus associated adrenal smooth muscle tumors
and disseminated diffuse large B-cell lymphoma in a child with common variable
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[15] Mouchet F, Ninane J et al. Leiomyoma of the suprarenal gland in a child with ataxia
telangiectasia. Pediatr. Hematol. Oncol. 1991: 8: 235–241.
[16] Demirel S, Erk O et al. Multiple vascular leiomyomas involving bilateral adrenal
glands, spleen, and epicardium, associated with bilateral testicular microlithiasis and
emptysellaturcica. J. Pediatr. Surg. 1997: 32: 1365–1367.
[17] Radin DR, Kiyabu M Multiple smooth muscle tumours of the colon and adrenal gland
in an adult with AIDS. AJR Am. J. Roentgenol. 1992: 159:545–546.
[18] Wachsberg RH, Cho KC et al. Two leiomyomas of the liver in an adult with AIDS: CT
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[19] Parola P, Petit N, Azzedine A, et al., Symptomatic leiomyoma of the adrenal gland in a
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immunodeficiency syndrome: report of two cases of this unusual location with evidence
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[27] Santosh Kumar, Bhuvanesh N et al. Large Bilateral Adrenal Leiomyomas Presenting as
Calcified Adrenal Masses: A Rare Case Report. Korean J. Urol. 2014; 55: 363-366.
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J. Chir. (Paris). 1994; 131: 391.
Chapter 19
RARE TUMORS OF THE ADRENAL GLAND
Shahrazad T. Saab1,*, Liang Cheng2

and Gregory T. MacLennan1
1
Division of Anatomic Pathology, Institute of Pathology, Case Western Reserve
University, Cleveland, OH, US
2
Department of Pathology and Laboratory Medicine,
Indiana University School of Medicine, Indianapolis, IN, US
ABSTRACT
The adrenal gland is essentially composed of two endocrine organs, the cortex and
the medulla. Classically, the nosology of primary adrenal tumors is based on their site of
origin; whether they arise in the cortex or medulla and whether they are benign or
malignant. In general, primary adrenal gland neoplasms have a low prevalence. The most
commonly encountered primary neoplasms of the cortex are adrenal cortical adenoma
and adrenal cortical carcinoma. Those of the medulla are pheochromocytoma and
neuroblastoma, in children. In addition, the adrenal gland has a rich vasculature and
peripheral nerve network. Potentially, any of the constituant tissues comprising the
adrenal gland can give rise to a neoplasm, either benign or malignant. This chapter will
focus on those, sometimes exceedingly, rare primary tumors of the adrenal gland. A
common theme for these tumors is that they are commonly asymptomatic and non-
secreting, as they are commonly discovered serendipitously during a work-up for
unrelalated causes. Often, the radiologic appearance is non-specific, making the
discernment of their nature more challenging without the help of a histologic diagnosis.
Historically these lesions were discovered at the time of autopsy. Currently the more
common mode of discovery is by radiology as increased medical screening and advances
in imaging modalities give rise to more frequent detection of these incidentalomas.
Keywords: adenomatoid tumor, schwannoma, cavernous hemangioma, lymphangioma,

Solitary fibrous tumor, sarcoma
*
Corresponding author: Email: Shahrazad.Saab@UHhosptials.org.
386 Shahrazad T. Saab, Liang Cheng and Gregory T. MacLennan
ADENOMATOID TUMOR
Adenomatoid Tumor (AT) is a benign neoplasm of mesothelial origin that usually arises
in the genital tracts, occurring equally in both men and women. In women, its characteristic
locations are the uterus, fallopian tube, and rarely the ovary. In men, it is most frequently seen
in the epididymis, testicular tunica, and spermatic cord. Extra-genital AT has been reported to
occur in the urinary bladder, omentum, umbilical skin, retroperitonium, mesentary of the
small intestine, pancreas, mediastinal lymph nodes, pleura, and the heart [1, 2]. AT rarely
occurs in the adrenal glands. To date there are 32 reported cases of adrenal gland AT in the
literature [3-6].
Adrenal gland AT shows a marked male predominance, with 97% of all cases reported in
men. The mean age is 40 years (range of 22 to 64). Most ATs are incidental surgical or
autopsy discoveries, while some are discovered on radiologic studies as part of a diagnostic
work up for a variety of likely unrelated symptoms including painless hematuria,
hypertension, abdominal pain, and Cushing‘s syndrome [4]. There have been two reports of
adrenal gland AT in patients with HIV/AIDS [4, 7]. No associations with other diseases have
been described.
The adrenal glands are retroperitoneal organs devoid of a mesothelial lining, making the
appearance of AT in that location particularly puzzling. AT is presumed to arise either from
mesothelial rests, pluripotent Müllerian mesenchyme, or coelomic epithelial inclusions [11].
These remnants or rests are likely derived from the embryologic proximity of the adrenal
glands and the gonads [1]. In addition, AT-like sturctures can be experimentally induced by
the introduction of sex steroids in the sub-peritoneal area, suggesting that sex steroids play a
role in histogenesis, possibly explaining the male predominance of these tumors [12].
Regardless of their origin, it is well established by immunohistochemical and ultrastructural
studies that ATs are of mesothelial derivation [1, 5,8-9].
The radiological features of ATs are widely variable and non-specific. The usual finding
is of a solitary, well-circumscribed, non-functioning adrenal mass ranging in size from 0.5 cm
to 17 cm [3, 5]. They can be solid, cystic, or a combination of these, usually with
calcifications. The predominantly cystic ATs bear close resemblance to vascular tumors such
as lymphangiomas and are frequently mistaken for them [6, 8]. An added difficulty is that
occasionally ATs can extend through the adrenal capsule into surrounding adipose tissue
mimicking invasive malignancies [3, 8,10-11]. Thus, the differential diagnosis can include
benign and malignant primary adrenal tumors or metastatic carcinoma [2, 10].
Grossly ATs are well-circumscribed, firm masses that can be predominantly solid, cystic,
or a combination [6, 8] (Figure 1a). Histologically, ATs are well-circumscribed and contained
by a well-defined capsule or infiltrative, penetrating into the periadrenal tissue [8]. The tumor
cells vary from plump and epithelioid with abundant eosinophilic cytoplasm to flat
mesothelial-like cells. They frequently have intracytoplasmic vacuoles giving them a signet
ring appearance (Figures 1b and c). Various histologic patterns have been described and
include: adenoid (adenomatoid), solid (packing of tubules and vacuolated cells),
lymphangiomatoid, microcystic-cavernous, and papillary [8-9]. Usually, there is a mixture of
these patterns within one tumor [6]. Nearly all have dystrophic calcifications, and lymphoid
aggregates within the tumor or around its periphery. Occasionally, there is adipose tissue
within the tumor [9]. Nuclear pleomorphism, mitotic activity, and necrosis are universally
Rare Tumors of the Adrenal Gland 387
absent [1-13]. The background stroma has a mucoid appearance, but there is no
intracytoplasmic mucin [7-8].
Figure 1a. Adenomatoid tumor presenting as a well circumscribed, solid, solitary mass with a pale and
firm cut surface. 1b, irregular tubules can be poorly formed or slit-like and anastomosing; 1c, the
stroma is fibrotic and the cells may have intracytoplasmic vacuoles resembling signet-ring cells; 1d,
tumor cells show positive immmunohistochemical staining for mesothelial markers, such as calretinin,
shown here.
The immunohistochemical profile is that of mesothelial cells with expression of

calretinin, D2-40, WT1, mesothelial cell antigen, thrombomodulin, vimentin, and
cytokeratins such as CKAE1/3, CAM5.2, and CK7 (Figure 1d). Their low proliferation index
is demonstrated by the MIB-1 immunohistochemical stain, which highlights less than 3% of
nuclei [10-11]. Ultrastructurally, the tumor cells have the classic microvilli of the coelomic
type, characteristic of normal mesothelial cells [8].
The relative rarity of ATs in addition to their architectural and cytologic variability,
infiltrative growth pattern, and lack of characteristic radiologic features result in a differential
diagnosis that includes adrenocortical adenoma or carcinoma, hemangioma, lymphangioma,
angiosarcoma, pheochromocytoma, or metastatic carcinoma, especially signet-ring
carcinoma. This diagnosis is particularly difficult to reach on frozen section or fine needle
aspiration where the cystic nature of the tumor can lead to its interpretation as an adrenal cyst
or pseudocyst when cyst fluid is aspirated [5, 9,13]. In addition, the presence of signet ring-
like cells on fine needle aspiration smears can be misleading, resulting in a diagnosis of signet
ring carcinoma [11]. Importantly, that they can extend into surrounding tissues does not imply
malignant behavior. No recurrences or metastases have ever been reported after primary
resection of AT [9-10]. Local resection alone is curative [8, 10].
SCHWANNOMA
Adrenal schwannoma is a rare primary tumor of the adrenal medulla. Only 35 cases have
been reported in the literature [14-16]. Schwannomas commonly arise in other sites including
the cranial nerves, head and neck, extremities, spinal nerves, retroperitoneum, and viscera
such as the gastrointestinal tract, heart, liver, pancreas, and kidney [17]. Retroperitoneal
schwannomas account for only 0.5%-5% of all schwannomas and 1% of all retroperitoneal
tumors [17-18].
Reported cases of adrenal schwannoma in the literature demonstrate a wide age range of
14 to 89 years, with a median age of 49. There is a slight female predominance, with a 1.2:1
female-to-male ratio [14].
Schwannoma is a benign, slow-growing, encapsulated neoplasm in which the chief
component arises from the Schwann cells of cranial and peripheral nerves. The adrenal
medulla is innervated by the phrenic nerve, vagus nerve, and sympathetic trunk. The tumor is
thought to arise from the constituent Schwann cells of these nerves [14]. Adrenal
Schwannomas represent <0.2% of all incidental adrenal tumors [19].
The majority of adrenal schwannomas are asymptomatic and incidentally detected.
Occasionally, they can be associated with dull flank or abdominal pain, likely attributable to
mass-effect as those symptomatic tumors tend to be greater than 5.0 cm in diameter.
Endocrinological and biochemical studies are typically negative [14].
There are no characteristic features of adrenal schwannomas to distinguish them form
other tumors on either computed tomography (CT) or magnetic resonance (MR) images.
Radiologic imaging findings are of a solitary, well circumscribed, homogenous solid or cystic
mass. Calcifications can often be present, especially in long-standing lesions. The
preoperative differential diagnosis for a non-functional adrenal mass is wide and includes
adrenocortical adenoma or carcinoma, pheochromocytoma, leiomyoma, neurofibroma,
myelolipoma, ganglioneuroma, ganglioneuroblastoma, malignant fibrous histiocytoma, and
metastasis [14, 17].
The gross and histologic features of adrenal schwannomas are no different than those of
schwannoma in any other location. Grossly, they are firm, well-circumscribed, rounded
masses with a homogenous, lobulated, gray-white cut surface (Figure 2a). Larger tumors can
have degenerative features such as cystic areas, calcifications, and hemorrhage [17, 20].
Histologically, they are well circumscribed and may be surrounded by a fibrous capsule.
Both conventional and cellular histologic variants may be observed. Conventional
schwannomas consist of cellular areas composed of compactly arranged spindle cells
alternating with a more paucicellular loose arrangement of the same spindle cells, termed
Antoni A and Antoni B areas, respectively. The spindle cells are cytologically bland with
elongated nuclei and faintly eosinophilic cytoplasm with indistinct cell borders in a
collagenous stroma (Figure 2b). Verocay bodies, nucleus-free zones in areas of nuclear
palisading, can be seen in addition to thick-walled, hyalinized blood vessels and reactive
inflammatory cells arranged in aggregates or dispersed [14, 17]. (Figure 2c) Mitotic figures
and necrosis are usually absent in conventional schwannomas [20].
Figure 2a. Adrenal schwannoma presenting as a 10 cm adrenal mass weighing 990g and incidentally
discovered on radiologic imaging; 2b, the adrenal gland cortex is seen surrounding the schwannoma
which is composed of spindle cells arranged in intersecting fascicles; 2c, alternating hypercellular
(Antoni A) and hypocellular (Antoni B) areas are present with medium-caliber vessels showing
hyalinized walls and sparse chronic inflammatory cell infiltrates.
Cellular schwannomas consist entirely of Antoni A areas with intersecting fascicles of

spindle cells. They are devoid of Antoni B areas and Verocay bodies and may have a mildly
increased mitotic activity [20]. Long-standing tumors can have mild to severe nuclear atypia,
a phenomenon referred to as ancient change. This atypia should not be misinterpreted as
indicating malignancy [14, 20].
Immunohistochemical studies of schwannoma characteristically show strong and diffuse
staining with S100 protein as well as staining with collagen IV and laminin [15]. There is no
staining with HMB-45, Melan-A, CD34, CD117, desmin, actin, chromogranin, or
synaptophysin, thus distinguishing it from melanocytic, smooth muscle, or fibrohistocytic
tumors as well as gastrointestinal stromal tumor and pheochromocytoma [14]. MIB1
demonstrates the low proliferation rate of these tumors with staining of <5% of nuclei [15].
The diagnosis of adrenal gland schwannoma is often made upon resection. The utility of
preoperative fine needle aspiration is variable and may offer a wide differential diagnosis
including benign or malignant entities [21]. Furthermore, biopsy of incidental adrenal lesions
is often not desired in many institutions due to the risk of hemorrhage, infection, and seeding
of tumor [17]. Given the rarity of this tumor, and lack of definitive non-histologic diagnostic
modalities, adrenal schwannoma remains a diagnosis of exclusion. The differential diagnosis
for a solitary, non-functional, incidentally discovered adrenal mass is a list of benign and
malignant primary and secondary adrenal lesions. Surgical excision is required to reach the
diagnosis. Clinical follow up for adrenal schwannomas show no evidence of recurrence or
metastasis after excision, as is expected for these benign neoplasms [14, 20-21].
CAVERNOUS HEMANGIOMA
Cavernous hemangioma is a benign, congenital venous malformation that can occur
within any body site, with the adrenal gland being no exception. Most often it is incidentally
discovered and can appear to slowly enlarge over time due to progressive vascular ectasia,
causing symptoms occasionally secondary to mass effect [22-23]. Thus it is usually
discovered between the ages of 50-70 years, with rare cases reported in the 2nd and 3rd
decades [23-24]. There is a female predilection with a male to female ratio of 1:2 [25].
Although most are unilateral, three cases of bilateral hemangiomas of the adrenal glands
have been reported [26]. As common sense would predict, the majority of cavernous
hemangiomas are non-functional adrenal gland lesions. Curiously, three cases of functional
adrenal cavernous hemangiomas have been reported, two with mineralocorticoid excess and
one with glucocorticoid excess, with normalization of hormone status post-operatively in all
three cases [27-28]. There have been rare reports of presentations with abdominal pain or
discomfort [23], consumptive coagulopathy, and hypovolemic shock due to hemorrhage [22].
Though fewer than 70 cases have been reported in the literature [22-24], there is some
speculation that adrenal cavernous hemangiomas are more prevalent than is reported.
Pathologically they can be misdiagnosed as necrotic, nonfunctioning adrenocortical
carcinoma or as adrenal cysts due to loss of architecture secondary to limited sampling or the
presence of extensive necrosis and hemorrhage [22].
Their radiologic appearance can at best suggest the possibility of a cavernous
hemangioma, but a broad differential diagnosis usually results and includes adrenocortical
carcinoma, hemorrhage, tuberculosis, neuroblastoma, and metastatic carcinoma.
Ultrasonography is generally not helpful in differentiating cavernous hemangiomas from
other adrenal lesions. On CT, they tend to be heterogeneous, hypodense lesions with rim
enhancement due to the presence of vascular spaces at the lesion‘s periphery [22, 24]. The
characteristic speckled pattern of calcifications has been reported in 28-87% of cases. The
distribution of calcifications is either diffusely throughout the lesion, peripherally crescentic,
or centrally stellate and branching. The latter pattern is due to the presence of multiple
phleboliths and is the most characteristic and helpful radiologic feature in suggesting the
correct diagnosis; however, calcifications are not specific to this entity [22].
Cavernous hemangiomas of the adrenal gland average 11 cm in size and range from
approximately 1 cm to 30 cm [23] and up to 5000 g [22]. Grossly, they appear circumscribed,
multicystic or spongy with blood and blood clots. The gross appearance corresponds
histologically to a collection of abnormally clustered veins of various sizes, often massively
dilated, with intraluminal blood and fibrin (Figure 3a). Papillary endothelial hyperplasia and
luminal recanalization are encountered and are the result of organizing thrombosis [27, 29]
(Figure 3b). The intervening stroma is often fibrous with variable degrees of edema,
hemorrhage, and calcification [25, 29]. As is expected, the endothelial cells demonstrate
immunohistochemical reactivity with factor VIII [28], CD31, and CD34 [23].
Figure 3a. Cavernous hemangioma on histologic section that was incidentally discovered in the adrenal
gland in radical nephrectomy for renal cell carcinoma. The hemangioma was a well-defined 0.5 cm
hemorrhagic mass histologically composed of dilated venous spaces lined by unremarkable
endothelium with central blood and fibrin. Normal adrenal gland tissue is seen on the right; 3b, foci of
papillary endothelial hyperplasia. The hemangioma intercalates between islands of adrenal cortex on
the left.
Cavernous hemangiomas are benign vascular malformations with no tendency toward

malignant transformation. Surgical resection is necessary for lesions exceeding 3.5 cm, even
when suspected to be angiomatous in nature, due to the propensity of these lesions to
hemorrhage and to exclude the possibility of malignancy [24]. None have recurred after
excision, confirming the benign nature of this lesion [22, 29].
LYMPHANGIOMA
Adrenal lymphangioma (AL), also known as cystic adrenal lymphangioma, is a benign
malformation of lymphatic vessels. Lymphangiomas are commonly encountered in infancy
and childhood with 95% of lesions occurring in the neck, axilla, and mediastinum, with the
remaining 5% in the abdominal cavity [30]. It is debatable whether these lesions result from
continued growth of ectopic or malformed lymphatic vessels, result from a hyperplastic
reaction to inflammation, are hamartomas, or proximal dilations of traumatized vessels.
Fewer than 60 cases of AL have been reported in the literature. They can occur at any age,
with a peak between the third and sixth decades of life [31].
ALs are usually incidentally discovered, solitary, non-secreting adrenal cysts with a
female predisposition (1:2 male:female ratio) [31-33]. Their size at presentation is variable
and can reach a diameter of up to 20 cm [33]. Bilaterality and functional features such as
hypertension have been described in a few cases. The latter finding is peculiar and has no
known cause [31-32]. AL can be associated with Gorlin-Goltz syndrome [31].
Though ALs can achieve a large size and remain asymptomatic in the majority of cases, a
few patients have presented with abdominal discomfort, gastrointestinal disturbance, or a
palpable mass. Occasionally, a more acute symptomatic presentation such as fever or pain is
due to hemorrhage, rupture, or infection of the cysts [31, 34].
As is the case with other cystic adrenal neoplasms, the radiologic appearance of AL is
non-specific and often generates a broad differential diagnosis as both benign and malignant
primary adrenocortical neoplasms can undergo prominent degenerative, cystic changes. Thus,
AL shares a similar radiologic appearance with other vascular lesions, epithelial cysts,
pseudocysts, infectious cysts, cystic pheochromocytoma, and adrenal cortical carcinoma [31,
35]. The diagnosis of AL is usually made histologically upon resection [31].
Grossly ALs appear as multiloculated cystic masses composed of thin-walled cysts filled
with clear to straw-colored serous, hemorrhagic, or gelatinous fluid [35, 37] (Figure 4a).
Occasionally, calcified areas can be present [32, 36]. The typical histologic appearance is of
irregular, dilated, thin-walled lymphatic vessels lined by flattened, bland, simple endothelium
(Figure 4b). The dilated lumina contain homogeneously eosinophilic, proteinaceous material.
There is often a lymphocytic infiltrate within the cyst walls that varies from sparse to dense
and is arranged either diffusely or in aggregates that protrude into the cyst lumina. Atypia is
generally absent, though the lymphocytes can occasionally exhibit atypical cytology, which is
a degenerative change and are not to be confused with lymphoma [31-32]. That ALs can have
an infiltrative periphery is noteworthy; however, this does not indicate malignant behavior
[38].
The endothelial cells lining the cystic spaces show immunohistochemical expression of
CD34, CD31, factor VIII, and D2-40. D2-40 is specific to the endothelium of lymphatics and
distinguishes them from arterial and venous vessels (Figures 4c-d). The lack of expression of
cytokeratins aids in the distinction from cysts of epithelial or mesothelial origin [31-32].
The prognosis for ALs is excellent, with no reported recurrences after resection [31].
Most ALs have been treated with adrenalectomy since the diagnosis is usually unknown at the
time of surgery. Due to the usually broad pre-operative differential diagnosis, a biopsy is
occasionally sought when the clinical suspicion for malignancy is high. An excisional biopsy
is preferred over fine needle aspiration as non-diagnostic cyst content is most commonly
obtained from these lesions [31, 35]. When the preoperative suspicion for malignancy is low,
asymptomatic lesions can be observed [31].
Figure 4a. Adrenal lymphangioma presenting as a 7 cm adrenal cyst with a homogeneously thin wall;
4b, histology reveals an endothelium lined cyst surface that shows positive staining with CD31, an
endothelial marker and D2-40, a marker of lymphatic endothelium, 4c and d, respectively.
SOLITARY FIBROUS TUMOR

Solitary fibrous tumor (SFT), previously called hemangiopericytoma, is a rare neoplasm
of fibroblastic/myofibroblastic origin that was formerly thought to arise exclusively in
intrathoracic sites [39]. It has since been recognized in the meninges, orbit, upper respiratory
tract, salivary glands, thyroid, peritoneum, liver, gastrointestinal tract, kidney, spermatic cord,
urinary bladder, prostate, skin, bone and soft tissue [39-40]. SFT is usually asymptomatic in
deep locations, but associated generalized fatigue and mild anemia have been observed at
presentation [41]. Only 5 cases have been reported in the adrenal gland, making SFTs among
the most infrequent of adrenal gland neoplasms [41-43].
Adrenal SFT is usually a solitary, painless mass that is incidentally discovered by
radiologic imaging; however one case of bilateral adrenal gland SFT has been reported. The
size is variable and can be up to 15 cm in greatest dimension [43]. The biological potential of
this tumor is variable and difficult to predict [40, 44], though most are slow growing with a
benign clinical course [43]. Malignant tumors are locally infiltrative [39]. The endocrine and
biochemical workup is normal in all cases. Several variants of SFT are recognized and
include atypical, cellular and malignant variants [44].
On CT, SFT of the adrenal gland is a solitary, solid mass with heterogeneous
enhancement. MRI shows a low T1-weighted signal intensity and intermediate to high
intensity on T2 [41, 45].
Figure 5a. Adrenal solitary fibrous tumor showing a spindle cell proliferation arranged in haphazard
and intersecting fascicles with a so-called ―patternless pattern‖ and several thin-walled and dilated
―hemangiopericytoma-like‖ blood vessels; 5b, malignant solitary fibrous tumor with increased mitotic
activity, and; 5c, moderate to marked cytological atypia.
Macroscopically, they are solid white or tan, well-circumscribed, and encapsulated [43].
The typical microscopic appearance is of a bland spindle cell proliferation arranged in a so-
called ―patternless pattern‖, referring to the mixture of histologic patterns in each tumor
including fascicular, storiform, and haphazard arrangements. The cellularity and stromal
collagen are variable. In more densely cellular areas, collagen forms a lace-like network
whereas in other areas, increased connective tissue forms dense collagen bundles, which can
occasionally have a myxoid appearance. The blood vessels within the tumor frequently adopt
a hemangiopericytoma-like growth pattern where they appear elongated, branching, and
dilated with thin walls (Figure 5a). The cells are usually bland in appearance with ovoid
nuclei having occasional grooves and pale chromatin. The cytoplasm is pale and elongated
with indistinct borders. The malignant variant is defined by having greater than 4 mitotic
figures per 10 high-power fields, with or without increased cellularity, cytological atypia,
tumor necrosis, and an infiltrative growth pattern [39] (Figures 5b-c). Most metastasizing
SFTs have some or all of these features; however, even histologically benign-appearing SFT
can also rarely give rise to metastasis [44]. The cellular and atypical variants are defined by
diffuse cellularity or nuclear atypia, respectively, without increased mitotic activity [39].
The spindle cells of SFT are immunohistochemically positive for CD34, CD99, and Bcl-2
in most cases [40]. This immunohistochemical profile is not specific to SFT and often does
not aid in the distinction between SFT and its histologic differential diagnosis, which includes
desmoid-type fibromatosis, monophasic synovial sarcoma, and dedifferentiated liposarcoma.
A new immunohistochemical marker, STAT6, has been shown to have nuclear staining in
SFT with a high sensitivity and specificity for this tumor [44].
Extrathoracic and intrathoracic SFTs appear to behave similarly [40]. The biologic
behavior is difficult to predict on histology. In both types, malignant behavior can be seen in
histologically benign or malignant appearing tumors. Complete local resection is curative in
most cases, with local recurrence or distant metastasis in approximately 6-10% of
extrathoracic SFT based on the literature [40, 44]. Long-term follow-up is necessary
following resection, as SFT cannot be regarded as absolutely benign [40].
LEIOMYOSARCOMA
Leiomyosarcoma is a malignant tumor of smooth muscle origin that is commonly
encountered when it arises from the myometrium, gastrointestinal tract, soft tissue, or
retroperitoneum [46-47]. It is the most common malignant tumor of blood vessels,
predominantly the veins [46, 48]. Primary adrenal leiomyosarcoma (PAL) is a rare lesion that
presumably arises from the smooth muscle layer of the central adrenal vein and its tributaries
[48]. Smooth muscle tumors are usually prevalent in immunocompromised individuals. A
possible etiologic association with HIV and EBV infection has been suggested in that setting
[48-50]. EBV can be detected in all tumors by in situ hybridization [50].
To date, twenty-eight PALs have been reported [48-49]. Most patients are adults with
ages ranging from 29 to 76 years [51]. An exception to this trend is a 14-year-old girl with
acquired immunodeficiency syndrome (AIDS) who reportedly had bilateral PALs [52]. There
is no gender predilection as the twenty-eight reported cases of PALs have shown a 1:1
male:female ratio. The lesions are generally larger than 10 cm at presentation and unlike other
non-functional adrenal tumors, PALs usually have a symptomatic presentation. Symptoms
include abdominal, flank, back or groin pain secondary to local organ involvement. Leg
edema, swelling, or cold feet are manifestations of inferior vena cava obstruction [48, 51].
Figure 6a. Adrenal leiomyosarcoma exhibiting a solid, white nodular and whorled cut surface; 6b,
histologic section showing leiomyosarcoma on the left with spindle cells exhibiting nuclear atypia and a
focus of necrosis. Normal adrenal gland tissue is present on the right; 6c, the tumor cells show strong
positive immunohistochemical staining with desmin.
By ultrasound, PALs appear as solid, hypoechoic, hypovascular masses [53]. By CT and

MRI they are heterogeneous with foci of necrosis and calcification. Extension into the inferior
vena cava is occasionally present [51]. Concurrent liver, bone, and lung metastases are
common [48]. Grossly, they are soft to rubbery white masses with a whirling cut surface that
can have well-circumscribed or invasive borders and measure at least 10 cm [49, 51] (Figure
6a).
Histologically, PALs resemble leiomyosarcomas of other body sites. Two histologic
variants are recognized: conventional and pleomorphic, with the former being more common.
They are composed of intersecting fascicles of spindle cells with deeply eosinophilic
cytoplasm and the ―cigar-shaped‖ nuclei that are characteristic of smooth muscle cells (Figure
6b). These cells can span the spectrum of differentiation, ranging from mature-appearing
smooth muscle cells to poorly differentiated, histiocytoid or epitheloid cells with anaplasia
and osteoclast-like giant cells. The latter are features of the pleomorphic variant [47, 54-55].
PALs can exhibit subtle features of malignancy by having as few as 1-4 mitotic figures
per 10 high power fields and at least some degree of atypia and coagulative necrosis. This
subtlety has made it difficult to identify independent prognostic factors in this tumor [50, 56].
Like other sarcomas, PALs are histologically graded using the Federation Nationale des
Centres de Lutte Contre le Cancer (FNCLCC) sarcoma grading system, which has proven to
be a good predictor of metastasis-free and overall survival [51].
The smooth muscle origin of PALs is demonstrated by positive immunohistochemical
staining for smooth muscle actin, desmin, and calponin [54, 57] (Figure 6c). Electron
microscopy and molecular genetics currently have little diagnostic utility [48], however
numerous chromosomal aberrations have been observed to correlate with varying degrees of
associated tumor aggressiveness [58].
Histologic evaluation is imperative for diagnosis and prognostication. Complete resection
with histologically negative margins is the best chance for cure [51]. This histologic grade
correlates with clinical behavior of PAL and prognosis. Metastasis is frequently to the lungs
and liver and rarely to regional lymph nodes. Post-operative adjuvant radiotherapy is
recommended for locally advanced disease. Chemotherapy has limited effectiveness and is
used in inoperable tumors, incomplete resections, and in the cases of metastasis. The
prognosis is generally poor, but can vary according to tumor size, ability to achieve complete
resection, and the morphologic grade of the tumor [49]. Venous thrombosis, adjacent organ
invasion and distant metastasis are correlated with the poorest prognosis [47, 49]. The longest
reported survival time is 21 months in a patient with low-grade PALs [54].
ONCOCYTIC ADRENOCORTICAL TUMORS

Oncocytic adrenocortical tumors (OATs) are rare group of adrenal neoplasms with 147
cases reported in the literature [59]. In recent years, there has been much interest in these
tumors as their very unique clinical and morphological features are now being recognized
[59-61]. Their characteristic and defining feature is that they are predominantly or exclusively
composed of oncocytes, epithelial cells with abundant granular, eosinophilic cytoplasm that
ultrastructurally corresponds to a copious number of mitochondria [59].
Until recently, the idea of OATs being distinctive neoplasms of the adrenal gland was
debated and reference to them in the literature was limited to scattered case reports. Since
2004 several relatively large series have been published [60, 62] with emphasis on the
correlation between histologic features and clinical behavior. It has been observed that the
accepted systems evaluating the histomorphologic features of malignancy in adrenal cortical
neoplasms, such as the Weiss criteria, do not accurately apply to OATs. It is now understood
that this distinctive category of neoplasia merits recognition not only as an independent group
of tumors, but that modified histologic criteria stratifying them according to their biologic
behavior are necessary [61].
Figure 7a. Oncocytic adrenocortical carcinoma measuring 30 cm and weighing 3.5 Kg is entirely
encapsulated with a brown cut surface and a central area of white scar and extensive necrosis. The
kidney, seen at the bottom of the photograph, was grossly entirely separate from the mass; 7b,
histologically the tumor is composed entirely of oncocytes and shows venous invasion within the
capsule; microscopic foci of necrosis and atypical mitoses are readily evident, 7c and d, respectively.
The majority of OATs are incidentally detected since only 17% are hormonally
functional [mearini]. They occur in all ages (mean of 47 years, ranging from 19 to 72) with no
specific distribution pattern [59, 63] and a female predominance (1:2.2 male:female ratio).
Reported tumor sizes (3-20 cm) and weights (12-2415 grams) are variable [61].
No risk factors have as yet been identified. The proposed mechanisms leading to
oncocyte morphology are many. Some suggest it to be a cellular response to toxins that leads
to excessive mitochondrial proliferation. Another hypothesis is that oncocytic differentiation
is a result of a defective physiological response secondary to faulty energy production
machinery, while other investigators have proposed that mutations in the mitochondrial DNA
give rise to these tumors [59]. Radiologic imaging is non-specific for OATs. The central scar
consistently found in renal oncocytoma has never been described in adrenal cases. Grossly,
however, they resemble oncocytic tumors in other locations with the majority having well-
circumscribed, encapsulated boundaries and a tan to brown cut surface (Figure 7a). Areas of
focal microcystic change and hemorrhage can also be seen. Necrosis is usually absent in
OATs [61].
On histologic evaluation, excluding those frankly malignant tumors with grossly apparent
invasion into periadrenal tissues, OATs are generally well-circumscribed and encapsulated.
The architectural arrangement of cells can be diffuse and sheet-like, trabecular, papillary,
microcystic or alveolar [59-61]. The constituent cells are predominantly classic oncocytes,
which are large (1-2 times the size of a normal adrenal cortical cell) round or ovoid to
polyhedral with abundant eosinophilic and granular cytoplasm, central nuclei, and prominent
nucleoli. A striking degree of nuclear atypia is often present. Bizarre multinucleated tumor
giant cells can be seen in both benign and malignant tumors [62]. Eosinophilic nuclear
pseudoinclusions, lymphocytic infiltrates, and areas of spindled tumor cells are common [61-
62].
In addition to the classic oncocytes, smaller cells, termed ―small oncocytes‖ with the
same diffusely granular eosinophilic cytoplasm, but reduced in cytoplasmic volume, resulting
in a high nucleus-to-cytoplasm ratio [62]. The presence of >10% clear cells in an
adrenocortical tumor is well associated with adverse clinical outcome. The suggested
categories of OATS are based on the degree of oncocytic differentiation, in an effort to
distinguish them from other adrenocortical tumors. Those tumors with 90% or greater
oncocytes are designated ―pure oncocytic tumors.‖ Those with 50-90% oncocytes are ―mixed
oncocytic tumors.‖ Tumors with oncocytic differentiation in less than half of the mass are
―ordinary adrenocortical tumors with focal oncocytic changes‖ [61].
OATs often show positive immunohistochemical staining for vimentin, wide-spectrum
cytokeratin, synaptophysin, neuron-specific enolase, Melan-A, calretinin, and inhibin-alpha
[61-62]. This immunohitochemical staining profile is similar to other adrenocortical tumors
and relates to a shared embryological origin of adrenocortical cells and coelomic epithelium.
In addition, mES-13, an antibody to a mitochondrial antigen by the same name, shows
cytoplasmic staining in all OATs [61].
OATs have overlapping features with adrenocortical carcinoma as dictated by the
standard grading criteria, with the Weiss system being the most widely used. Under this
system, all OATs would be classified as malignant as it defines a malignant tumor as having 3
or more of the following features: Marked nuclear atypia, mitotic rate >5 per 50 HPF, atypical
mitoses, clear cell composition ≤25% of the tumor mass, diffuse architecture, necrosis,
venous invasion, sinusoidal invasion, or capsular invasion [61]. The standard Weiss system
does not correctly predict the behavior of OATs, since many have exhibited a favorable
biologic behavior despite their qualification as malignant neoplasms as it would any other
non-oncocytic adrenal cortical tumor [61]. This is attributed to the finding that most oncocytic
tumors possessed three of the 9 criteria described by the standard classification system. Thus
a modified system was proposed specifically for classifying OATs, known as the Lin-Weiss-
Bisceglia (LWP) criteria (see Table 1) [60, 62].
Table 1. LWB criteria
Major Criteria
Mitotic rate >5 per 50 HPF
Atypical mitotic figures
Venous invasion
Minor Criteria
Size >10 cm and/or weight >200 g
Necrosis (microscopic)
Capsular invasion
Sinusoidal invasion
This modified classification system includes some of the parameters described in the
original Weiss system and categorizes them as major and minor criteria. Any OAT possessing
any one of the major criteria designates it as malignant using the terminology of oncocytic
adrenocortical carcinoma (Figure 7 a-d). The presence of any of the minor criteria in the
absence of any major criteria defines a tumor of borderline malignant potential, or oncocytic
adrenal tumor with borderline malingnant potential. The absence of major and minor criteria
is indicative of a benign oncoctyoma. Thus by omitting nuclear atypia, diffuse architecture,
and having clear cells comprising ≤25% of the tumor mass, features inherently found in most
oncocytic tumors, the LWP system is able to better classify OATs according to their
biological behavior [62]. By these criteria, most OATs are benign.
COLLISION AND COMPOSITE TUMORS

Collision and composite tumors represent the coexistence of two distinct neoplasms in a
single tumor mass. A collision tumor consists of two coexisting, independent neoplasms
without significant tissue admixture. A composite tumor is one in which the co-existing
neoplasms show considerable admixture of the two cell types [64]. These tumors are unusual
and can occur in various organs.
In the adrenal gland numerous variations of collision and composite tumors have been
reported, in various combinations. One interesting combination reported with unusual
frequency has adrenal cortical and medullary constituents, namely adrenocortical adenoma
and pheochromocytoma (Figures 8a-b). Their frequent co-existence has led some to believe
that their association is more than coincidental. Catecholamines are known to result in
increased plasma renin via the β1 adrenergic receptor, activating angiotensin II and elevating
aldosterone concentrations. Presumably, the excessive catecholamine production from a
pheochromocytoma can lead to over-stimulation of the adrenal cortex, such that an
adrenocortical adenoma might eventually develop. In addition, pheochromocytomas have
been known to secrete renin and aldosterone directly [65]. Conversely, an adrenocortical
adenoma may over-produce glucocorticoids that flow centripetally into the adrenal vein to up-
regulate the activity of phenyl-ethanolamine-N-methyl-transferase, an enzyme essential for
epinephrine production. This excessive production can potentially lead to the development of
pheochromocytoma [65-66]. This dual interaction has been proposed as the initiating factor
for collision tumors of these two types [65].
Pheochromocytoma can arise in conjunction with other tumors of adrenal medullary, and
thus neural crest, origin resulting in the development of an entity known as ―composite
pheochromocytoma‖, a term encompassing their common embryologic derivation. The
components of such tumors may include ganglioneuroma (Figures 8c-e), ganglioneuro-
blastoma (Figure 8f), malignant peripheral nerve sheath tumor, and neuroendocrine
carcinoma. To date, fewer than fifty cases have been reported in the literature with
pheochromocytoma-ganglioneuroma comprising 71% of these [67-68]. Patients range in age
from 5 to 82 years and usually have unilateral tumors. Bilateral tumors can present in
neurofibromatosis patients. Approximately 76% of tumors were endocrinologically
functional. Only tumors with neuroblastoma, malignant peripheral nerve sheath, or
neuroendocrine carcinoma were found to be malignant [67]. In cases with metastasis, the
pheochromocytoma component alone or in conjunction with the associated tumor could be

found at the metastatic focus [68].
Figure 8a. Pheochromocytoma (right) and adrenal cortical adenoma (left) collision tumors. The
pheochromoctyoma is typically red to pink-white and the adrenal cortical adenoma is golden yellow;
8b, pheochromocytoma (right) and adrenal cortical adenoma (left) separated by a central area of fibrous
tissue; 8c, Pheochromocytoma-ganglioneuroma composite tumor presenting as a single mass within the
adrenal medulla; 8d, Pheochromocytoma (left)-ganglioneuroma (right) composite tumor; 8e,
Pheochromocytoma (right) is composed of nests of cells resembling adrenal medulla whereas the
ganglioneuroma is composed of large, eosinophilic ganglion cells with a background of Schwann cells;
8f, Adrenocortical adenoma (x)-ganglioneuroblastoma collision tumor (*); 8g, Myelolipoma and
hemangioma with normal adrenal cortex in the lower left corner, myelolipoma on the right and portions
of the hemangioma in the middle; 8h, high-power view of the hemangioma component.
Other collision and composite tumors of the adrenal gland frequently have adrenal
cortical adenoma or myelolipoma as one of the components, but the possibilities are limitless.
Numerous examples in various combinations exist and include adrenal adenoma-metastases
[69], adrenal hemangioma-adenoma [70], adrenal hemangioma-myelolipoma (Figures 8g-h),
adrenal myelolipoma and Hodgkin lymphoma [72], primary adrenal carcinosarcoma-
metastatic colorectal carcinoma [73].
ACQUIRED ADRENAL HYPERPLASIA

Adrenal cortical hyperplasia may be diffuse or nodular. In both adults and children
simple, or diffuse, hyperplasia is most common. Nodular hyperplasia and hyperplasia with
sboth adults and children and is characterized by the presence of one or more prominent
nodules greater than 0.5 cm in diameter with background adrenal cortex showing hyperplasia.
By definition micronodules are less than 0.5 cm in diameter while macronodules are greater
than 0.5 cm in diameter [74].
The spectrum of nodular hyperplasia of the adrenal cortex can range from the
autonomous black micronodules of primary pigmented nodular adrenocortical disease
(PPNAD) through the ACTH-dependent macronodular hyperplasia of Cushing's disease to
the, sometimes, massively enlarged autonomous glands of macronodular hyperplasia.
PPNAD and macronodular hyperplasia are rare causes of autonomous adrenocortical
hyperfunction [75].
PPNAD is a rare cause of hypercortisolism presenting in children, adolescents, and young
adults [75]. Synonyms include adrenocortical dysplasia and bilateral micronodular
hyperplasia. The etiology is unknown. It can occur in a familial form with autosomal
dominant inheritance and in association with Carney's complex [74]. It is characterized by
ACTH independent Cushing's syndrome and numerous round, unencapsulated micronodules
ranging from 1-4 mm in diameter that are commonly black in color, but some can be tan or
yellow (Figure 9a). The average combined adrenal weight is 9.6 g. The nodules can extend
into the periadrenal fat or involve the entire thickness of the cortex. The internodular cells are
usually compact and eosinophilic (Figures 9b-c). Frequent lipomatous or myolipomatous
change can be present and is associated with increased activity of the glands [74].
In most cases of ACTH-dependent nodular hyperplasia, the adjacent adrenal cortex
shows diffuse or simple hyperplasia and clinical evidence of Cushing's syndrome. The
adrenal glands are rarely greater than 15 g each. Nodular hyperplasia and diffuse hyperplasia
are commonly found together and are considered to be along the same morphologic spectrum
of this disease [74].
Macronodular hyperplasia is also known as massive macronodular hyperplasia and
macronodular adrenal dysplasia. The hallmark of this disease is ACTH-independent
hypercortisolism, distinguishing it from the more common ACTH-dependent nodular
hyperplasia in patients with pituitary-dependent Cushing's disease [75]. The clinical features
are those of Cushing's syndrome with elevated plasma cortisol levels and low or undetectable
ACTH levels [74]. The mean age of diagnosis is approximately 50 years [75].
Figure 9a. Primary pigmented nodular adrenocortical disease presenting as autonomous hypercortisolism
with a 4.8 g adrenal gland containing multiple dark brown nodules and a normal appearing contralateral
adrenal gland (not pictured); 9b, microscopic appearance at low power showing numerous nodules ( *); 9c, At
high power the cells composing the nodules have abundant eosinophilic cytoplasm and intracytoplasmic
lipofuscin pigment; 9d, Macronodular hyperplasia with marked adrenal enlargement presenting as
autonomous hypercortisolism and bilateral adrenal gland enlargement, 39g (adrenal sectioned at the top) and
79g (adrenal sectioned on the bottom), with innumerable nodules of various sizes; 9e, Histologically, various
hyperplastic nodules, some showing lipomatous metaplasia; 9f, Adrenal medullary hyperplasia presenting in
a patient with severe, drug-resistant hypertension as diffuse enlargement of the adrenal medulla with
extension into the adrenal alae and a normal-appearing cortex. The adrenal weighed 5.9g; 5g, Histologically,
the medulla is diffusely expanded without discrete nodules.
The gender distribution is approximately equal. CT images often show bilateral adrenal
nodules or massively enlarged glands [74]. The internodular cortex, when identified, is either
normal appearing or atrophic, but not hyperplastic. The adrenal gland enlargement can be
very pronounced with combined adrenal glands weights exceeding 60 g (Figures 9d-e). The
nodules can completely replace the gland, can measure up to 4 cm in diameter, and can
extend below the level of the renal hilum with marked distortion of the adrenal gland [75].
Adrenal medullary hyperplasia (AMH) is currently considered a non-neoplastic
proliferation characterized by a clinical history of paroxysmal hypertension, elevated serum
and/or urinary catecholamine levels, and adrenal medullary expansion that can be nodular or
diffuse [76-77]. AMH is closely associated with pheochromocytoma. Both lesions are
frequently found in patients with multiple neroendocrine neoplasia-type 2 (MEN-2) [76]. It
has been widely accepted that AMH is a precursor lesion for pheochromocytoma. The
distinction between the two lesions has been arbitrarily set at a cut off of 1 cm where AMH is
below the cut off and pheochromocytoma is above [78].
Histologically, in AMH the medulla shows preservation of the medullary architecture,
contrasting with pheochromocytoma, but with a decreased corticomedullary ratio, which
normally is approximately 10:1. Another histologic feature is extension of medullary tissue
into the adrenal alae, where medullary tissue would not be normally found. Finally, there is
increased mitotic activity in AMH, unlike normal adrenal medullary tissue [77]
(Figures 9f-g).
The current treatment for AMH is observation, while pheochromocytomas are usually
resected. For patients with MEN–2 and bilateral pheochromocytomas, the larger tumor is
treated with adrenalectomy and the contralateral side is treated with cortex-sparing
adrenalectomy [76].
Recently, the diagnostic distinction between AMH and pheochromocytoma being based
on an arbitrarily set size criterion has been challenged [79, 80]. Several researchers have
investigated the genetic aberrations of AMH and pheochromocytoma. Loss of heterozygosity
at 1p13, 1p36, 3p12, and 3q24 were frequently found in both AMH and pheochromocytoma
alike. In addition, amplification of a mutated RET allele was found in AMH and a
contralateral pheochromocytoma in one patient. This aberration has been exclusively
described in pheochromocytoma. These findings have provided ample evidence for some to
suggest a strong link between the two lesions and to recommend that the distinction by size
be abandoned. The term micro-pheochromocytoma was recommended for nodular or diffuse
medullary lesions less than 1 cm [76]. In addition, the treatment by cortex-sparing
adrenalectomy should be considered for cases of synchronous or metachronous contralateral
micro-pheochromocytoma [76].
CONCLUSION
Adrenal gland neoplasms are aremarkably diverse in their origin, morpholog, functional
status and biologic behavior. The rare neoplasms discussed here often have non-specific
clinical presentations. In fact, most are asymptomatic and are discovered incidentally.
Surgical resection is often necessary to arrive at the diagnosis. As more of these have been
encountered and their biologic behavior has been better understood, some tumors have
undergone one or several rounds of reclassification since their original discovery. This trend
is to be expected in the future as these lesions become better understood. It is important that
the treating physician and the pathologist be aware of these uncommon neoplasms and their
pathophysiology.
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EDITOR CONTACT INFORMATION
Dr. Gaetano Santulli, MD, PhD

Russ Berrie Medical Science Pavilion
R513 Columbia University Medical Center
College of Physicians and Surgeons
1150 St. Nicholas Avenue, 10032 New York, NY, US
gs2620@columbia.edu
INDEX
Adrenal gland, 193, 388, 407

# adrenal hyperplasia, 5, 98, 118, 119, 124, 159, 169,
170, 284, 287, 289, 293, 297, 298, 299, 336, 340,
11β-HSD2, 242, 244, 245, 246, 252, 253, 255, 256
342, 346, 350, 356, 357, 358
11β-hydroxysteroid dehydrogenase type 2, 242, 297
Adrenal insufficiency, 97, 115, 116, 161, 162, 340
17-hydroxy-progesterone, 295
adrenal medulla, 4, 5, 55, 56, 57, 58, 59, 60, 61, 63,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
64, 65, 67, 69, 70, 71, 73, 74, 80, 81, 83, 90, 173,
(MPTP, 320
200, 239, 245, 246, 247, 251, 258, 262, 263, 265,
21-hydroxylase, 93, 95, 97, 98, 102, 106, 111, 113,
268, 269, 335, 351, 380, 390, 402, 403, 406, 411
114, 287, 294, 295, 296, 298, 299, 341, 342
adrenal steroidogenesis, 98, 154, 271, 294, 297, 298,
336
A adrenal vein sampling, 117, 118, 119, 124, 132, 136,
139, 140
ABCD1 protein, 349 adrenalectomy (ADX), 217, 218
Abnormalities, 78, 139 adrenals, 62, 75, 120, 297, 381, 385
Addison disease, 340, 346 adrenarche, 102, 103, 283, 284, 285, 286, 287, 288,
Addisonian crisis, 188, 344, 346 289, 290, 291
Adenomatoid Tumor, 388 adrenergic, 55, 56, 57, 61, 63, 67, 69, 70, 81, 155,
adipocyte, 49, 75, 76, 77, 78, 79, 81, 84, 86 160, 180, 222, 240, 247, 258, 265, 353, 402
Adipocyte, 73, 78, 81, 89 adrenergic antagonist, 353
adipocytes, 73, 74, 75, 76, 77, 78, 79, 81, 83, 84, 85, Adrenocortical adenoma, 403
86, 91, 219, 222, 231, 234 adrenocortical carcinoma, 15, 37, 52, 118, 161, 375,
Adipocytes, 75, 86 376, 377, 392, 400, 401, 402
adipocytokines, 74, 82, 84 Adrenocorticotropic hormone (ACTH), 210
adipogenesis, 77, 78, 80, 85, 87, 89 Adrenoleukodystrophy (ALD), 346
adipokines, 73, 74, 81, 82, 86, 87, 89, 234 adrenomyeloneuropathy (AMN), 346, 348
adiponectin, 73, 74, 77, 79, 80, 81, 82, 83, 84, 85, ADX, 217, 220, 221
86, 87, 88, 89, 90, 91 age, 5, 14, 35, 98, 102, 118, 119, 121, 124, 130, 131,
adipose, v, 31, 52, 73, 74, 75, 76, 77, 78, 79, 80, 81, 177, 179, 238, 242, 246, 253, 273, 274, 276, 277,
82, 83, 84, 85, 87, 88, 89, 90, 91, 219, 221, 222, 279, 283, 284, 285, 286, 289, 291, 292, 297, 303,
231, 234, 263, 388 316, 327, 330, 333, 346, 364, 371, 380, 388, 390,
adrenal adenoma, 4, 5, 8, 17, 82, 83, 117, 124, 127, 402, 404
128, 142, 146, 159, 160, 340, 351, 404 aldosterone, 4, 37, 52, 63, 71, 75, 76, 77, 84, 86, 93,
adrenal androgens, 94, 101, 102, 103, 148, 284, 285, 102, 103, 106, 117, 118, 119, 120, 121, 122, 123,
293, 294 124, 125, 127, 128, 129, 130, 131, 132, 133, 134,
adrenal cortex, 4, 20, 36, 37, 56, 59, 63, 74, 80, 82, 135, 136, 137, 138, 140, 142, 143, 161, 162, 242,
84, 94, 97, 98, 102, 103, 105, 106, 161, 210, 216, 343, 345, 350, 351, 358, 364, 369, 402, 410
239, 246, 285, 293, 294, 296, 302, 335, 336, 342, Aldosterone, 5, 75, 76, 86, 117, 119, 127, 131, 132,
345, 346, 359, 393, 402, 403, 404 133, 134, 135, 136, 137, 140, 141, 143
414 Index
aldosterone synthase, 77, 120, 133, 369 228, 231, 234, 239, 283, 286, 290, 296, 319, 327,
aldosterone/renin ratio, 117 337, 354, 380, 381, 392, 398
alpha-synuclein,, 318
Alzheimer, 310, 311, 321, 327, 328, 329, 330
AMH, 406 C
amyloid beta, 36, 51, 315, 322, 327
CA, 49, 52, 64, 65, 247, 263, 266, 298, 327, 374
androgen(s), 4, 74, 80, 82, 98, 101, 103, 104, 109,
carcinoma, 7, 15, 24, 37, 44, 52, 90, 118, 145, 151,
130, 145, 161, 163, 283, 284, 285, 286, 287, 289,
159, 161, 165, 177, 193, 273, 340, 350, 351, 374,
290, 292, 293, 294, 296, 297, 299, 342, 350, 364,
375, 376, 377, 384, 388, 389, 392, 394, 400, 401,
366
402, 404
androgen precursors, 101, 296, 297
cardiovascular, 47, 48, 59, 63, 69, 74, 76, 79, 80, 83,
androstenedione, 80, 101, 283, 284, 285, 288, 293,
84, 85, 89, 94, 118, 119, 123, 130, 137, 144, 147,
294, 295, 296
148, 175, 192, 215, 234, 237, 238, 239, 240, 242,
angiotensin, 37, 56, 57, 62, 71, 74, 102, 117, 118,
244, 245, 247, 248, 249, 250, 252, 256, 257, 258,
122, 123, 131, 147, 240, 241, 257, 259, 262, 264,
261, 263, 267, 271, 284, 288, 289, 290, 310, 352
267, 350, 352, 402
catatonia, 344
AP-1, 42, 220, 221, 233
CD4+, 255
APS-I, 341
chaperone, 21, 22, 40, 41, 312
APS-II, 341
Charles Brown-Sequard, 336, 341
APSIII, 341
CHOP, 22, 23, 24, 25, 27, 32, 33, 34, 35, 36, 37, 41,
APS-IV, 341
42, 45, 51
arcuate nucleus of the hypothalamus (ARC), 215
chromaffin cell(s), 55, 56, 57, 58, 60, 61, 62, 63, 64,
associations, 241, 355, 388
65, 66, 67, 68, 69, 70, 81, 83, 90, 174, 178, 179,
astrocytes, 320
189, 239, 240, 247, 251, 266, 267, 268, 269, 335,
asymptomatic, 12, 173, 175, 194, 348, 349, 359, 381,
351
382, 387, 390, 394, 395, 407
chromogranins, 56, 200
ataxia-telangiectasia, tuberous sclerosis complex,
circulatory, 56, 344
and Sturge-Weber syndrome., 352
clonidine, 123, 179, 197, 353, 361
ATF6, 22, 23, 27, 33, 34, 41, 45
complications, 154, 158, 160, 163, 354, 372
Atypical mitotic figures, 183
computed tomography, 1, 82, 129, 139, 141, 158,
autocrine, 56, 59, 60, 67, 77, 79, 251, 268
181, 289, 296, 365, 382, 390
autoimmune, 94, 95, 97, 99, 103, 106, 111, 112, 113,
congenital adrenal hyperplasia, 98, 287, 289, 293,
114, 249, 267, 341, 343, 345, 358, 379, 386
298, 346, 356
autophosphorylation, 22
Congenital adrenal hyperplasia, 95, 98, 287, 294,
298, 299, 342, 356, 357
B Conn, 5, 42, 77, 86, 117, 118, 132, 139, 141, 191,
200, 350, 360
bacterial, 26, 220, 223, 232, 320, 322 corticosteroids, 285, 323, 335, 336, 337, 354, 358,
Barker Hypothesis, 238, 241, 244 366
Bartholomeo Eustachius, 336 corticotrophin-releasing hormone (CRH), 214, 239,
Bartter syndrome, 350 336
basal, 21, 56, 70, 83, 102, 103, 105, 114, 129, 153, Cortisol, 4, 76, 78, 82, 88, 93, 106, 127, 139, 161,
213, 214, 226, 229, 248, 251, 253, 311, 320, 345 257, 302, 327, 331, 339
bed nucleus of the stria terminalis (BNST), 211 cortisone, 75, 76, 78, 108, 109, 242, 255, 312, 336,
beta blockade, 186 345
bilateral adrenal hyperplasia, 117, 118, 119, 120, critical illness myopathy, 337, 340, 355
124, 159, 351 CT, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
biochemical, 4, 12, 16, 104, 128, 130, 139, 153, 154, 17, 18, 106, 124, 127, 128, 139, 141, 158, 179,
155, 156, 157, 162, 178, 179, 182, 190, 197, 205, 180, 181, 182, 197, 198, 339, 345, 366, 367, 368,
231, 232, 310, 356, 359, 361, 390, 395 369, 375, 380, 382, 385, 398, 406, 411
body, 3, 37, 55, 61, 73, 74, 75, 77, 79, 80, 82, 86, 88, CYP11B2, 37, 120
114, 118, 163, 175, 179, 214, 216, 217, 218, 223, cysts, 14, 176, 392, 394
Index 415
cytokine(s), 74, 75, 77, 81, 110, 220, 221, 232, 250, expansion, 23, 78, 102, 118, 406
251, 255, 256, 267, 268, 269, 271, 312, 321, 323
F
D
failure, 61, 63, 69, 71, 77, 93, 94, 95, 97, 98, 99, 102,
defects, 98, 103, 293, 297, 327, 341, 342 103, 104, 105, 106, 107, 108, 109, 110, 111, 126,
deficiency, 24, 29, 37, 50, 51, 94, 98, 99, 103, 104, 131, 148, 150, 158, 238, 343, 350, 355
105, 106, 107, 109, 113, 114, 161, 210, 229, 252, Familial, 119, 132, 134, 195, 342, 350, 352, 360, 364
259, 294, 295, 298, 324, 342, 343, 344, 357, 360 Familial paraganglioma, 195, 352
dehydroepiandrosterone, 80, 88, 283, 284, 293, 294, fasciculata, 37, 98, 101, 120, 293, 294, 296, 335
366 features, 8, 12, 15, 16, 17, 103, 112, 113, 118, 145,
dexamethasone, 78, 80, 82, 88, 109, 119, 120, 128, 146, 148, 149, 151, 156, 157, 165, 171, 180, 182,
131, 134, 145, 149, 150, 159, 162, 164, 166, 212, 183, 201, 232, 273, 274, 275, 276, 277, 280, 295,
215, 221, 222, 223, 227, 233, 246, 264, 266, 270, 338, 346, 347, 355, 357, 365, 366, 369, 375, 384,
289, 295, 296, 297, 299, 311, 314, 316, 317, 320, 388, 389, 390, 394, 399, 401, 402, 404, 410
331, 332, 339, 364, 366 feedback, 23, 25, 30, 45, 48, 59, 63, 65, 79, 81, 103,
differential, 152, 167, 168, 169, 202, 223, 255, 286, 155, 209, 211, 212, 213, 214, 223, 224, 225, 226,
314, 319, 323, 341, 347, 353, 365, 379, 380, 383, 246, 247, 248, 255, 294, 310, 311, 315, 328
384, 388, 389, 390, 391, 392, 394, 397 fetal, 56, 60, 64, 67, 68, 110, 237, 238, 239, 241,
dopamine, 57, 58, 66, 74, 80, 154, 167, 173, 174, 242, 243, 244, 245, 246, 247, 252, 253, 254, 255,
178, 197, 247, 248, 257, 264, 266, 314, 318, 319, 256, 257, 258, 261, 262, 264, 267, 270, 271, 285,
320, 324, 331, 333 288, 292, 309, 311, 323, 325, 359
dopamine neurons, 321, 324, 333 Fetal programming, 243, 257, 258, 261, 323
Drugs, 99, 101, 137 fibrous, 33, 276, 387, 390, 393, 395, 396, 403, 409
dynamic(s), 10, 39, 57, 58, 79, 83, 110, 157, 225, flexion contractures syndrome, 344, 357, 360
266 fludrocortisone, 93, 111, 114, 117, 123, 128, 137,
dyslipidemia, 164, 235 162, 297, 343, 345
focal, 157, 179, 181, 182, 400, 401
E
G
encephalopathy addisonienne, 341
endocannabinoids (ECBs), 209 GADD34, 25
endocrinology, 16, 64, 65, 66, 112, 113, 114, 115, ganglioneuroma, 402, 403
116, 169, 192, 193, 195, 196, 197, 199, 200, 201, gastrointestinal, 7, 161, 176, 372, 380, 390, 391, 394,
202, 203, 207, 299, 374, 375, 376, 377 395, 397
enzymatic, 75, 150, 212, 242, 255, 258, 293, 294, GC, 44, 108, 147, 148, 162, 241, 243, 246, 247, 248,
297, 315 252, 253, 254, 255, 256, 257, 260, 310, 311, 312,
enzyme, 20, 21, 29, 31, 37, 50, 57, 60, 68, 75, 83, 93, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322
98, 108, 114, 123, 150, 176, 199, 208, 219, 239, glucagon, 148, 160
241, 242, 244, 247, 249, 251, 252, 255, 256, 260, glucocorticoids receptors (GRs), 210
312, 313, 340, 341, 345, 402 GRP78, 21, 22, 23, 26, 33, 34, 35, 37, 41, 50
Epi, 239, 240, 241, 245, 247, 248, 249, 257, 269, 326
epigenetic, 247, 249, 253, 254, 256, 261, 262, 270,
313, 314, 322, 326, 327, 333 H
epinephrine, 4, 55, 62, 71, 74, 80, 173, 174, 239,
HAC15, 37, 52
247, 248, 257, 260, 265, 351, 352, 402
Harvey Cushing, 146, 156, 163, 336
ER stress, 19, 20, 21, 22, 23, 24, 25, 26, 32, 33, 34,
heparin, 341
35, 36, 37, 38, 39, 41, 42, 43, 50, 51
hippocampus(Hip), 36, 51, 52, 211, 214, 221, 233,
ERAD, 23
253, 270, 310, 311, 313, 315, 316, 317, 318, 323,
etiology, 76, 150, 156, 157, 302, 311, 320, 339, 341,
330, 333, 344
343, 404
hirsutism, 161, 287, 299, 337, 338
exchange, 44, 45, 58
Histology, 64
416 Index
hormonal, 1, 2, 4, 74, 78, 88, 99, 151, 170, 225, 231,

290, 307, 335, 343, 364, 408
J
hormone-sensitive lipase (HSL), 28, 219
JNK, 34, 35
hydroxysteroid dehydrogenase, 74, 75, 76, 87, 88,
juvenile, 176, 255, 270, 302
95, 108, 109, 231, 256, 261, 264, 270, 271, 294,
298, 312, 324, 336
hyperaldosteronism, vii, 77, 86, 87, 118, 120, 132, K
134, 138, 139, 144, 350, 351, 360, 366
Hyperaldosteronism, v, 77, 117, 132, 135, 137, 138, Kearns-Sayre syndrome, 96, 342, 350, 360
139, 142, 350 knockout, 33, 35, 50, 60, 79, 213, 233, 320
hyperfunction, 12, 146, 404
hyperkalemia, 130, 145, 216, 337
hyperplasia, 5, 118, 119, 120, 124, 145, 151, 159, L
160, 169, 170, 289, 294, 297, 298, 299, 336, 340,
346, 350, 356, 392, 404, 405, 406, 411 laboratory, 1, 124, 131, 157, 192, 348, 358, 382
Hypertension, vi, 64, 71, 86, 87, 119, 121, 122, 132, Leiomyosarcoma, 397, 409
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, leptin, 73, 74, 77, 79, 80, 81, 82, 83, 85, 86, 88, 89,
143, 147, 163, 175, 237, 239, 241, 257, 258, 259, 90, 160, 215, 217, 220, 222, 228, 230, 232, 246,
260, 261, 264, 265, 267, 270, 351, 354 290
hypoaldosteronism, 161 Lewy bodies, 318, 331
hypoglycemia, 216, 344, 364 lipopolysaccharide (LPS), 220, 322
hypoplasia, 95, 96, 98, 341, 342, 356 lipoprotein lipase (LPL), 219
hypothalamic pituitary adrenal (HPA) axis, 210 localization, 5, 41, 65, 124, 127, 158, 167, 179, 181,
hypothalamus, 94, 99, 211, 212, 213, 214, 217, 218, 198, 199, 226, 312, 321, 361
220, 221, 226, 228, 229, 234, 237, 239, 246, 253, long chain fatty acids (VLCFA), 346
257, 263, 302, 310, 311, 318, 319, 331, 336 lovastatin, 4-phenylbutyrate, 349
LPS, 220, 221, 224, 322, 332
I
M
IL-1,, 312, 320
IL-6, 74, 75, 81, 220, 221, 251, 255, 268, 312 Male, 148, 270, 280
incidence, 94, 98, 111, 112, 128, 130, 146, 158, 159, manifestations, 4, 82, 94, 102, 103, 113, 148, 197,
174, 182, 191, 192, 238, 249, 274, 283, 285, 288, 217, 273, 274, 289, 335, 337, 344, 347, 350, 352,
350, 354, 381, 408 354, 355, 359, 397
incidentaloma, 380, 385 masses, 1, 2, 4, 5, 7, 8, 11, 12, 14, 15, 16, 17, 18, 82,
infection, 98, 99, 148, 158, 220, 249, 341, 381, 383, 124, 274, 369, 371, 374, 375, 379, 380, 382, 383,
394, 397 385, 388, 390, 394, 398, 410
inflammation, 50, 75, 76, 77, 84, 85, 118, 221, 222, maternal undernutrition, 243, 244, 245, 261, 262
223, 224, 233, 234, 235, 237, 249, 250, 252, 267, medial prefrontal cortex (mPFC), 211
269, 271, 320, 321, 331, 332, 333, 349, 355, 394 medical treatment, 124, 130, 154, 158, 160, 166, 167
insulin, 31, 48, 50, 73, 74, 77, 78, 79, 80, 81, 82, 84, medulla, 4, 55, 56, 58, 59, 60, 61, 62, 63, 65, 67, 71,
86, 87, 89, 90, 91, 106, 111, 115, 148, 164, 165, 81, 84, 173, 174, 200, 245, 246, 247, 251, 258,
191, 215, 216, 217, 219, 220, 222, 229, 230, 231, 262, 263, 335, 351, 387, 390, 403, 406
234, 235, 241, 244, 258, 288, 285, 289, 291, 292, Medullary, 81, 177
345, 358, 364, 373 MEN, 176, 177, 351, 352
internodular, 404, 406 MEN 2A or 2B, 352
intrauterine growth restriction, 242, 257, 325 metabolic syndrome, 77, 79, 82, 84, 87, 90, 223, 232,
invasion, 6, 151, 153, 188, 274, 276, 278, 279, 280, 292
312, 365, 366, 368, 383, 399, 400, 401 metabolites, 30, 34, 37, 40, 120, 131, 178, 219, 313,
invasive, 107, 128, 151, 157, 160, 186, 188, 191, 339
204, 370, 371, 376, 388, 398, 409 meta-iodobenzylguanidine, 198
ion, 225 Metaiodobenzylguanidine, 2
IUGR, 242, 244, 246, 257 metanephrine, 202, 353
Index 417
Metastases, 99, 192 ovarian, 93, 97, 114, 147, 289, 290, 292, 341
microglia, 312, 320, 321, 331 oxytocin (OT), 215, 218
micro-RNAs (miRs), 222
mineralocorticoid, 37, 73, 74, 75, 76, 79, 85, 86, 87,
98, 103, 109, 117, 118, 129, 132, 144, 156, 160, P
209, 242, 257, 295, 297, 298, 309, 310, 324, 343,
paracrine, 56, 61, 67, 77, 79, 83, 251, 268
345, 350, 392
paraganglioma, 176, 194, 195, 196, 198, 199, 206,
Mineralocorticoid(s), 73, 74, 75, 85, 86, 94, 103,
352
104, 105, 109, 133, 142, 210, 216, 225, 226, 227,
paraneoplastic, 344, 357
239, 261, 297, 325, 345, 351, 366, 370
Paraventricular nucleus, 311
mineralocorticoids receptors (MRs), 210
paraventricular nucleus of the hypothalamus (PVN),
miRNA, 32, 256
210, 214
misfolded, 21, 22, 23
Parkinson, 311, 332
Mitochondrial diseases, 342
penis, 283, 286, 301, 303, 307
Mitotic rate, 401
PET, 3, 11, 12, 15, 16, 17, 81, 181, 199, 331, 348,
molecule, 162, 179, 211, 213, 222, 240, 316
359, 369, 370, 382, 386
morphology, 113, 231, 262, 301, 302, 303, 306, 400
phenylethanolamine N-methyltransferase, 206, 239,
mothers, 238, 241, 242, 243, 252, 255, 261, 314, 322
257, 260, 263, 265, 266
MRI, 2, 5, 6, 13, 16, 107, 124, 150, 151, 158, 160,
pheochromocytes, 351
179, 180, 181, 182, 338, 339, 342, 345, 346, 347,
physiology, 39, 56, 57, 68, 73, 74, 238, 239, 259,
348, 349, 369, 370, 382, 396, 398, 409
261, 335
Multiple endocrine neoplasia, 176, 351, 352, 361
Pick, 19, 28, 29, 30, 42, 46, 47, 174, 351
myelolipoma, 403, 404
plasma renin activity (PRA), 121, 350
myopathy, 335, 337, 340, 350, 355, 360
PNMT, 57, 60, 239, 241, 246, 247, 248, 251, 253,
257, 260, 265, 266
N polyglandular syndrome, 336, 341
posture, 127, 160
nAChRs, 58 potassium, 102, 109, 118, 120, 123, 125, 128, 130,
Necrosis, 183, 184, 400, 401 131, 212, 350, 351
Negative, 71, 211 PRA, 121, 350
neurodegeneration, 36, 319, 322, 331, 332 pregnant, 149, 155, 179, 244, 245, 246, 252, 255,
neurofibrillary tangles, 315, 318, 327 271
Neurofibromatosis type 1 (NFI), 352 premature, 93, 96, 112, 249, 253, 283, 284, 285, 286,
neuro-immune circuit, 238, 250, 252 287, 288, 289, 290, 291, 292, 341
neurologic, vii, 99, 148, 160 prenatal hypoxia, 245, 263
neuropeptide Y (NPY), 57, 67, 215, 218 prenatal stress, 243, 247, 252, 254, 255, 256, 258,
neurotransmitter, 57, 60, 64, 65, 68, 212, 240, 251, 261, 263, 264, 269, , 270, 271, 323
313, 319, 327, 352 Primary aldosteronism, 77, 117, 118, 131, 132, 133,
NF-B, 313, 321 136, 138, 140, 143
normetanephrine, 179 prognostic factors, 130, 153, 399
normotensive, 175, 186, 194, 240, 241, 307, 381 prostaglandin, 255
O Q
OATs, 399, 400, 401, 402 quadriplegic, 355

offspring, 238, 244, 245, 246, 252, 255, 256, 262,
263, 270, 271, 313, 314
oncocytic, 400, 401, 402, 410
R
oncocytoma, 400
RAS, 190, 241, 244, 246, 247, 256, 257
oral, 88, 103, 106, 107, 114, 115, 123, 150, 161, 162,
Reactive oxygen species, 240
232, 296, 297, 344, 345, 347
renal, 2, 4, 14, 75, 83, 84, 117, 118, 123, 126, 130,
Osteonecrosis, 337
133, 137, 150, 164, 176, 177, 190, 195, 239, 244,
418 Index
246, 250, 259, 262, 350, 352, 369, 370, 371, 377,
385, 393, 400, 406, 409
T
reserve, 102, 107, 129, 142, 322, 345
T cell(s), 267, 320, 341
resistant hypertension, 117, 132, 406
Takamine and Aldrich, 336
retention, 37, 48, 242, 295
Tau, 315, 316, 317, 318, 322, 327, 330
RNase, 22
test, 2, 12, 15, 88, 106, 107, 111, 114, 121, 123, 124,
ROS, 75, 240
128, 135, 136, 138, 145, 146, 149, 150, 151, 153,
Roux in Switzerland, 351
154, 162, 166, 178, 179, 196, 197, 240, 245, 289,
295, 296, 299, 303, 314, 339, 345, 348, 353, 358,
S 361, 366
Thomas Addison, 85, 94, 111, 146, 336, 341
saline infusion, 138 TNFα, 250, 251, 255, 257
saline suppression test, 117, 123, 137 transmembrane, 20, 21, 22, 29, 177, 315
salt, 98, 103, 122, 123, 126, 295, 297, 350 tunicamycin, 26, 33, 35, 44
scan, 124, 181, 296, 339, 353, 366, 367, 368, 380, type 1 11-beta-hydroxysteroid dehydrogenase (11b-
382 HSD1), 219
Schaumburg, 346, 359, 360 type 1 cannabinoid receptor (CB1R), 210
Schilder-Addison, 346
Schwann cells, 349, 390, 403
Schwannoma, 390, 408
U
screening, 106, 121, 122, 123, 132, 135, 136, 138,
unfolded, 19, 20, 21, 22, 23, 39, 40, 41, 42, 44, 51,
149, 150, 164, 175, 182, 223, 298, 345, 356, 359,
52
366, 387
unilateral, 5, 63, 77, 117, 118, 124, 127, 128, 129,
sex, 80, 88, 89, 95, 98, 118, 130, 243, 244, 245, 246,
131, 140, 141, 142, 145, 157, 159, 160, 340, 350,
247, 254, 288, 294, 335, 342, 388, 408
379, 402
SFT, 395, 396, 397
UPR, 19, 20, 21, 22, 23, 24, 25, 27, 33, 35, 36, 38,
Sheehan syndrome, 343
50
SHR, 240
urine, 61, 79, 125, 153, 154, 155, 161, 178, 339, 353,
Siemerling and Creutzfield, 346
366, 382
Sipple syndrome, 351
Smith-Lemli-Opitz syndrome, 96, 342
solitary, 5, 12, 128, 215, 218, 229, 274, 310, 380, V
389, 396, 409
spontaneously hypertensive rat, 137, 240 variant, 70, 241, 298, 326, 359, 360, 397, 399
staging, 12, 179, 366, 368, 369, 371, 377 virilization, 95, 145, 148, 273, 274, 278, 283, 286,
steroid, 20, 28, 34, 36, 37, 38, 39, 40, 73, 74, 80, 82, 287, 289, 294, 296, 364
88, 89, 90, 98, 102, 108, 110, 111, 115, 118, 131, Von Hippel-Lindau, 177, 352, 361
146, 155, 161, 162, 171, 212, 225, 228, 233, 292,
294, 298, 299, 309, 310, 323, 337, 340, 355, 356,
366 W
steroidogenesis, 20, 21, 29, 34, 36, 39, 52, 73, 76,
84, 85, 90, 98, 155, 161, 171, 288, 341, 366 weight, 20, 73, 74, 76, 77, 86, 87, 95, 103, 107, 111,
stimulated, 33, 37, 57, 86, 90, 216, 268, 358 145, 152, 162, 175, 179, 216, 217, 218, 219, 223,
stimulating, 19, 34, 43, 50, 62, 148, 218, 233, 240, 232, 235, 238, 241, 242, 243, 244, 258, 260, 271,
285, 288, 311, 344 277, 283, 284, 285, 289, 290, 311, 337, 344, 401,
substantia nigra, 318, 320, 322 404
surgery, 99, 117, 124, 128, 129, 130, 131, 142, 143,
148, 153, 158, 160, 162, 166, 170, 178, 181, 186, Z
188, 189, 198, 205, 336, 340, 344, 346, 371, 372,
375, 383 zipper, 22, 23, 30
sustained, 57, 108, 109, 129, 175, 190, 244, 266, zona fasciculata, 37, 98, 101, 120, 335
311, 314, 321, 352
Synacthen or Cortrosyn, 345

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ENDOCRINOLOGY RESEARCH AND CLINICAL DEVELOPMENTS

Additional books in this series can be found on Nova‘s website

Additional e-books in this series can be found on Nova‘s website

GAETANO SANTULLI, MD, PHD

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

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Published by Nova Science Publishers, Inc. † New York

Chapter 10 Role of Adrenal Hormones in the Fetal Programming of Hypertension 235

The present book provides a state-of-the-art overview of the adrenal gland

Gaetano Santulli, MD, PhD

IMAGING OF ADRENAL GLAND

Michelle Tsang Mui Chung, MD,

In this chapter we provide an overview of the contemporary techniques utilized in adrenal

IMAGING MODALITY SELECTION

NORMAL ADRENAL GLAND

HYPERFUNCTIONING ADRENAL MASSES

Cortisol-producing adrenal adenomas are responsible for 20% of Cushing‘s syndrome,

Aldosterone-producing adrenal adenomas account for approximately one third of Conn‘s

Figure 2. Aldosteronoma in 60-year-old man with hypertension. Axial contrast-enhanced CT image

Hyperfunctioning adrenal adenoma is favored over adrenal hyperplasia in the setting of

Pheochromocytoma is an uncommon catecholamine-producing tumor of adrenal medulla.

Figure 3. Pheochromocytoma in 35-year-old woman. Coronal T2 weighted MR image shows a 4.1 cm

Pheochromocytomas are highly vascular and avidly enhance following contrast

Figure 4. Pheochromocytoma in 19-year-old woman. Axial contrast-enhanced CT image shows a

Figure 5. Adrenocortical carcinoma in 86-year-old woman. Contrast-enhanced axial T1 weighted MR

ADRENAL MASSES IN PATIENTS WITH AN ONCOLOGIC HISTORY:

Imaging of Intracellular Lipid: CT and CS-MR

Perfusion Imaging: CT Washout

A value of 40% or greater is considered diagnostic of an adenoma. As contrast washout is

Functional Imaging: PET and PET/CT

Masses with Specific Imaging Features

Figure 10. Myelolipoma in 74-year-old woman. Coronal reformatted contrast-enhanced CT image

Cyst and Pseudocyst

Masses without Diagnostic Imaging Features

MECHANISMS INTEGRATING ENDOPLASMIC

Zhi-qiang Pan2,3, Yi-yi Zhang4, Vivek Choudhary1,2

Keywords: cholesterol, endoplasmic reticulum (ER), ER stress, steroidogenesis, unfolded

The UPR and Some Key Signaling Molecules

GRP78 Is a Pivotal Regulator of the Unfolded Protein Response

Three Key Regulators Involved in the Activation of UPR Signaling Pathways

The Consequences of Severe or Persistent ER Stress

CHOP Is a Key Regulator of ER Stress-Induced Apoptosis

Caspases As Crucial Regulators of ER Stress-Induced Apoptosis

(1) The ER/Mitochondria-Independent Cell Death Pathway

(2) The ER/Mitochondria-Dependent Cell Death Pathway

Crosstalk of Various Apoptotic Pathways

Common Pharmacological Agents Capable of Inducing ER Stress

Experimentally, to study the mechanism of ER stress responses in different cells, a

Calcium Ionophores (A23187 and Ionomycin)

The Regulation of Cholesterol Homeostasis

Cholesterol is an important component of mammalian cell membranes and is required for

Cholesterol Transport in Cells

Key Mediators of Intracellular Cholesterol Homeostasis

Crosstalk Exists between Cholesterol Levels and ER Stress

In addition, in transgenic mice with an eIF2α phosphorylation defect, the expression of

Atherosclerosis and Macrophages

Macrophages from atherosclerosis-susceptible DBA/2 ApoE knockout (-/-) mice show

The Adrenal Gland

expressing human ApoE under a cholesterol-independent promoter and incubated with

[45] Oyadomari S, Mori M 2004 Roles of CHOP/GADD153 in endoplasmic reticulum

[60] Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y 2002 An

Bhanot S, Monia BP 2006 miR-122 regulation of lipid metabolism revealed by in vivo

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