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Clinical Science (2009) 116, 539–564 (Printed in Great Britain) doi:10.1042/CS20080253 539

R E V I E W

Metabolic disturbances in non-alcoholic

fatty liver disease

Christopher D. BYRNE∗ , Rasaq OLUFADI†, Kimberley D. BRUCE∗ ,

Felino R. CAGAMPANG‡ and Mohamed H. AHMED†
∗
Endocrinology & Metabolism Unit, Institute for Developmental Sciences, University of Southampton and Southampton
University Hospitals Trust, Southampton General Hospital, Southampton SO16 6YD, U.K., †Chemical Pathology Department,
Southampton University Hospitals Trust, Southampton General Hospital, Southampton SO16 6YD, U.K., and ‡Centre for
Developmental Origins of Health and Disease, Department of Maternal, Fetal & Neonatal Physiology, University of

Clinical Science
Southampton School of Medicine, Princess Anne Hospital, Southampton SO16 5AY, U.K.

A B S T R A C T

NAFLD (non-alcoholic fatty liver disease) refers to a wide spectrum of liver damage, ranging from
simple steatosis to NASH (non-alcoholic steatohepatitis), advanced fibrosis and cirrhosis. NAFLD
is strongly associated with insulin resistance and is defined by accumulation of liver fat > 5 %
per liver weight in the presence of < 10 g of daily alcohol consumption. The exact prevalence of
NAFLD is uncertain because of the absence of simple non-invasive diagnostic tests to facilitate
an estimate of prevalence. In certain subgroups of patients, such as those with Type 2 diabetes,
the prevalence of NAFLD, defined by ultrasound, may be as high as 70 %. NASH is an important
subgroup within the spectrum of NAFLD that progresses over time with worsening fibrosis and
cirrhosis, and is associated with increased risk for cardiovascular disease. It is, therefore, important
to understand the pathogenesis of NASH and, in particular, to develop strategies for inter-
ventions to treat this condition. Currently, the ‘gold standard’ for the diagnosis of NASH is liver
biopsy, and the need to undertake a biopsy has impeded research in subjects in this field. Limited
results suggest that the prevalence of NASH could be as high as 11 % in the general population,
suggesting there is a worsening future public health problem in this field of medicine. With a
burgeoning epidemic of diabetes in an aging population, it is likely that the prevalence of NASH
will continue to increase over time as both factors are important risk factors for liver fibrosis.
The purpose of this review is to: (i) briefly discuss the epidemiology of NAFLD to describe the
magnitude of the future potential public health problem; and (ii) to discuss extra- and intra-hepatic
mechanisms contributing to the pathogenesis of NAFLD, a better understanding of which may
help in the development of novel treatments for this condition.

Key words: diabetes, inflammation, mitochondrion, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis
(NASH), obesity.
Abbreviations: ACC, acetyl-CoA carboxylase; ALT, alanine aminotransferase; AMPK, AMP-activated kinase; AP-1, activated
protein-1; aRR, adjusted relative risk; ASO, antisense oligonucleotide; AST, aspartate aminotransferase; BMI, body mass index;
BP, blood pressure; CCT, CTP:phosphocholine cytidylyltransferase; CHD, coronary heart disease; ChREBP, carbohydrate-
responsive-element-binding protein; CI, confidence interval; CKD, chronic kidney disease; CPT, CDP-choline:1,2 diacylglycerol
choline phosphotransferase; CPT-1, carnitine palmitoyltransferase-1; CVD, cardiovascular disease; DAG, diacylglycerol; DGAT-2,
diacylglycerol acyltransferase-2; ELF score, European Liver Fibrosis score; ER, endoplasmic reticulum; FAS, fatty acid synthase;
Fox, forkhead box; FRS, Framingham risk score; GFR, glomerular filtration rate; GGT, γ -glutamyltransferase; GLUT4, glucose
transporter 4; HbA1c , glycated haemoglobin; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; IL, interleukin; IMT,
intima-media thickness; INSIG-1, insulin induced gene-1; IRE-1, inositol-requiring enzyme-1; IRS, insulin receptor substrate; IU,
international units; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; L-PK, liver-specific pyruvate kinase; LPS, lipo-
polysaccharide; LXR-α, liver X receptor-α; MAPK, mitogen-activated protein kinase; MCD, methionine- and choline-deficient;
MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; mtDNA, mitochondrial DNA; mTOR, mammalian
target of rapamycin; MUFA, mono-unsaturated fatty acid; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic
steatohepatitis; NEFA, non-esterified fatty acid (‘free fatty acid’); NF-κB, nuclear factor κB; (continued overleaf)
Correspondence: Professor Christopher D. Byrne (email cdtb@southampton.ac.uk).


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540 C. D. Byrne and others

INTRODUCTION
NAFLD (non-alcoholic fatty liver disease) refers to a
wide spectrum of liver damage, ranging from simple ste-
atosis to steatohepatitis, advanced fibrosis and cirrhosis.
NAFLD is strongly associated with insulin resistance
and is defined by accumulation of liver fat > 5 % per
liver weight in the presence of < 10 g of daily alcohol
consumption. The characteristic histology of NAFLD
resembles that of alcohol-induced liver injury, but occurs
in people who consume minimal alcohol. NAFLD is
regarded as the most common cause of increased liver
enzymes in the U.S.A., associated with Type 2 diabetes,
obesity and hyperlipidaemia [1]. The reported prevalence
of obesity with NAFLD varies between 30 and 100 %,
whereas the prevalence of NAFLD within Type 2 diabetes
varies between 10 and 75 %. In routine clinical practice,
most cases of fatty liver disease are attributable to
alcohol excess; however, fatty liver disease can also
occur in association with a wide range of toxins, drugs
and diseases, such as morbid obesity, cachexia, Type 2
diabetes, hyperlipidaemia and after jejunoileal bypass
surgery. As important risk factors for NAFLD, such as
obesity and Type 2 diabetes, are increasing in prevalence
it is likely that there will be a marked increase in NAFLD
with important consequences for healthcare providers.
NAFLD is rapidly becoming an important public
health problem. Undiagnosed, this condition may pro-
gress silently and results in cirrhosis, portal hypertension
and liver-related death in early adulthood. Interestingly,
NAFLD is a hepatic component of the metabolic
syndrome and is an independent risk factor for CVD
(cardiovascular disease). Importantly, NAFLD is associ-
ated with an increased risk of all-cause death and predicts
future CVD events, independently of age, gender, LDL
(low-density lipoprotein)-cholesterol, smoking and the
cluster of features of the metabolic syndrome. Currently,
there are no biochemical markers for NAFLD, and an
increase (or decrease) in ALT (alanine aminotransferase)
is often used as a biochemical marker to monitor
progression (or amelioration) of NAFLD (despite the
fact that ALT concentrations can be misleading and do
not reflect the severity or the outcome). Mass screening
for significant liver injury in patients with NAFLD will
be an important medical challenge in the years to come
because of the epidemics of obesity and diabetes.
EPIDEMIOLOGY
NAFLD is very common and occurs in individuals of all
ages and ethnic groups. Adjusted prevalence of NAFLD
using MRS (magnetic resonance spectroscopy) in 2287
individuals (U.S.A.) was found to be approx. 34 %, and
90 % of these cases of NAFLD were attributed to non-
alcoholic causes [2]. Furthermore, the Dallas Heart Study,
which was a population-based cohort study performed
in an ethnically diverse community in U.S.A. using
MRS, reported that the prevalence of NAFLD is approx.
33.6 % [3]. On the other hand, the use of ultrasound in
the Dionysos nutrition and liver disease study in Italy
found that the prevalence of NAFLD was approx. 25 %
and was associated with most features of the metabolic
syndrome [4]. Findings from the same study showed that
the prevalence of steatosis was increased in heavy drinkers
{46.4 % [95 % CI (confidence interval), 34–59%]} and
obese persons [75.8 % (95 % CI, 63–85 %)] compared
with controls [16.4 % (95 % CI, 8–25 %)]. Steatosis was
found in 94.5 % (95 % CI, 85–99 %) of obese heavy drink-
ers. Compared with controls, the risk for steatosis was
2.8-fold higher (95 % CI, 1.4–7.1-fold) in heavy drinkers,
4.6-fold (95 % CI, 2.5–11.0-fold) in obese people, and 5.8-
fold (95 % CI, 3.2–12.3-fold) in people who were obese
and drank heavily. In heavy drinkers, obesity increased
the risk of steatosis 2-fold (95 % CI, 1.5–3.0-fold;
P < 0.001), but heavy drinking was associated with only a
1.3-fold (95 % CI, 1.02–1.6-fold) increase in risk in obese
persons (P = 0.0053). It was concluded that steatosis is
frequently encountered in healthy persons and is usually
present in obese persons who drink more than 60 g of
alcohol per day. Steatosis is more strongly associated
with obesity than with heavy drinking, suggesting a
greater role of overweight than alcohol consumption in
accumulation of fat in the liver [4]. It is likely that there is
an additive effect of obesity and alcohol consumption to
worsen the NAFLD phenotype. Moreover, these findings
raise important questions about the safety of any alcohol
consumption in obese people who are insulin-resistant
because obesity and alcohol are both likely to contribute
to the NAFLD phenotype. Importantly, the prevalence
of NAFLD as determined by ultrasound increased from
16.4 % among individuals with normal BMI (body mass
index) to 75.8 % among obese people. The prevalence
of NAFLD is even higher with morbid obesity and

Abbreviations cont.
IκB, inhibitor of NF-κB; IKK, IκB kinase; NEMO, NF-κB essential modulator; OR, odds ratio; PC, phosphatidylcholine;
PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; PERK, PKR (double-stranded-RNA-
dependent protein kinase)-like ER kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PP2A, protein phosphatase
2A; PPAR, peroxisome-proliferator-activated receptor; PTEN, phosphatase and tensin homologue deleted on chromosome 10;
PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; SAM, S-adenosylmethionine; SCD-1, stearoyl-CoA desaturase-1;
SHIP-2, SH2-containing inositol phosphatase-2; SREBP-1c, sterol-regulatory-element binding-protein-1c; TAG, triacylglycerol;
TGF, transforming growth factor; TNF-α, tumour necrosis factor-α; UDCA, ursodeoxycholic acid; VLDL, very-LDL.


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among morbidly obese undergoing bariatric surgery the
prevalence may be as high as 96 % [5]. Interestingly,
the prevalence of NAFLD in the Japanese population
rises with increasing degrees of hyperglycaemia, being
approx. 27 % in people with normal fasting glucose
levels, increasing to 43 % among those with impaired
fasting glycaemia, and 62 % among newly diagnosed
diabetes [6]. On the other hand, in non-diabetic and
non-obese individuals, the OR (odds ratio) for metabolic
disorders (insulin resistance, hypertriacylglycerolaemia
and hyperuricaemia) in subjects with NAFLD, compared
with those without NAFLD in the normal-weight group,
was higher than that in the overweight group. Multiple
logistic regression analysis showed that gender, waist
circumference, TAG [triacylglycerol (triglyceride)] level
and insulin resistance were independently associated with
NAFLD in the normal-weight group [7].
Among the Asian population (known to have high
visceral fat), the prevalence of NAFLD using ultrasound
has been estimated to vary between 5 and 40 % [8].
In Japan, it has been estimated that the prevalence of
increased ALT attributable to NAFLD has increased
from less than 10 % in 1984 to 25 % in 2001 [9]. In China,
the prevalence of NAFLD has been estimated to vary
from 5 to 24 %, and this variation may be attributed to
lifestyle differences between rural and urban populations
[10,11]. An increase in the prevalence of obesity and
diabetes may also have resulted in an increase in the
prevalence of NAFLD in the Middle East. However, in
a study by el-Hassan et al. [12] in 1992, the prevalence of
NAFLD was approx. 10 %, whereas in contrast a small
study in diabetic Saudi individuals has suggested the
prevalence of NAFLD is approx. 55 % [13].
Children may also develop NAFLD. In a retrospective
review of 742 children between the ages of 2 and 19 years
who had an autopsy performed from 1993 to 2003, fatty
liver was present in 13 % of subjects [14]. In a small
study of 44 obese children, aged 6–16 years, with a BMI
above the 97th centile, hepatic fat content was measured
by phase-contrast MRI (magnetic resonance imaging)
and an increased hepatic fat fraction was identified in
14 subjects (31.8 %) [15].
There are few studies of the natural history of NAFLD;
however, recent findings strongly suggest NAFLD is not
a harmless condition and depends critically on disease
stage. A Danish study (215 patients with biopsy-proven
NAFLD) concluded that NAFLD has a benign course
without excess mortality [16]. This view was endorsed
further by 10-year follow-up data from the Dionyosis
study [17]. However, a recent large study by Adams et al.
[18] in 420 patients with NAFLD, between 1980 and
2000, concluded that the overall death rate is higher than
expected [standardized mortality ratio, 1.34 (95 % CI,
1.003–1.76); P = 0.03], and this increase in mortality was
associated with age, impaired fasting glucose/diabetes
and cirrhosis. Importantly, Adams et al. [18] confirmed
Metabolic disturbances in non-alcoholic fatty liver disease 541
the early observation of both the Danish and Dionyosis
studies that simple steatosis had benign natural history.
Generally speaking, the natural history of NASH (non-
alcoholic steatohepatitis) is associated with the possibility
of progression to cirrhosis. Progression of liver fibrosis
was found in one-third of 106 NASH patients 4.3 years
after the first liver biopsy, and obesity and BMI were
the only associated factors with such progression [19].
Furthermore, in a large study of the natural history of
the disease, follow-up biopsies were performed after
12–20 years in 772 patients with NAFLD [20]. Of
132 patients for whom data were complete, 4 % of sub-
jects with fatty liver progressed to cirrhosis compared
with 22 % of patients with NASH and fibrosis on the first
biopsy [20]. All liver-related morbidity and mortality
were also noted in the later group with NASH, rather than
simple hepatic steatosis. Thus the evidence, albeit rather
limited, suggest that NASH is not a harmless condition.
Importantly, Adams et al. [18] showed that the 8 % liver-
related mortality in biopsy-proven NASH is similar to
the 10 % mortality reported by Matteoni and co-workers
[20], and there is general agreement that once cirrhosis
develops in patients with NAFLD the prognosis is poor
[18–23].
DIAGNOSIS AND BIOCHEMICAL MARKERS
At present, a liver biopsy remains the only reliable
way to diagnose NAFLD and establish the presence of
fibrosis. The sampling variability has the potential to
alter significantly the diagnosis and staging of NAFLD.
It is not practical to offer liver biopsy as a test for the
diagnosis of NAFLD, as the prevalence of the condition
would overwhelm service provision. Consequently
non-invasive markers of NAFLD are urgently needed
that can be used for diagnosis and monitoring responses
to therapy. Table 1 shows a summary of biochemical
markers and the potential advantages and disadvantages
of different tests for identification of NAFLD. As can
been seen from Table 1, no single biochemical test has,
to date, shown sufficient sensitivity and specificity to be
used in clinical practice for the diagnosis or monitoring of
NAFLD.
It is beyond the scope of this review to discuss
biomarkers for NAFLD in detail; however, it is
important to realize that many studies investigating the
aetiology and pathogenesis and investigating potential
treatments for NAFLD have inevitably not been able
to use liver biopsy, but have used proxy markers for
the disease. This unfortunate problem has made it very
difficult to establish which risk factors are aetiologically
linked to the different components of the liver disease and
which treatments improve liver fat, or liver inflammation
or liver fibrosis within the spectrum of NAFLD.
To date, these difficulties have impeded progress in

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Table 1 Summary of the use of simple biochemical markers in NAFLD

CDT, carbohydrate-deficient transferrin; CK-18, cytokeratin 18; hsCRP, high-sensitivity C-reactive protein; oxLDL, oxidized LDL; RT–PCR, reverse transcriptase–PCR; TBARS,
thiobarbituric acid-reacting substances.

Biochemical marker Potential advantages and disadvantages References

ALT Treatment with UDCA and vitamin E decreased serum ALT, but failed to show a parallel improvement in [215–217]
histology. Importantly, the entire histological pattern of NAFLD can be seen in the presence of normal
ALT. The sensitivity of ALT for the diagnosis of NASH in large cohort was found to be only 40 %.
AST/ALT ratio Relatively poor sensitivity and specificity in NAFLD. [218]
GGT Increased with NAFLD; the increase is also seen with high alcohol intake. [1]
hsCRP Increased in patients with histologically verified NAFLD. Multiple regression analysis revealed that in [219,220]
comparison with cases of steatosis, hsCRP was significantly increased in cases of NASH and fibrosis. The
results of the RT–PCR showed that intrahepatic mRNA expression of CRP was increased in NAFLD and
NASH.
TNF-α and adiponectin TNF-α is increased in NAFLD, whereas adiponectin is decreased. A low adiponectin level in NAFLD patients [1]
was reported and may represent a pathogenic mechanism in NAFLD. High adiponectin levels have been
reported to protect against both alcoholic fatty liver disease and NAFLD. Further studies are needed
before adiponectin can be utilized as a biochemical marker for NAFLD.
NEFAs Increased NEFA concentrations are associated with NAFLD independently of classic measures of insulin [221]
resistance. Elevated NEFA concentrations have been associated with impaired glucose utilization,
impaired suppression of glucose production and impaired insulin secretion; however, there are problems
in utilizing NEFA concentrations as a biochemical marker for NAFLD because of the marked variation in
levels throughout the day.
Impaired glucose tolerance In two large separate studies of patients undergoing anti-obesity surgery (551 patients by Marceau et al. [18,63,222,223]
[63] and 505 patients by Luyckx et al. [223]), the severity of NAFLD was closely related to impaired
glycaemia. Haukeland et al. [222] concluded that abnormal glucose tolerance independently predicts
both fatty liver and fibrosis in 88 patients. Adams et al. [18], in a study of 420 patients with NAFLD,
showed that the overall death rate in patients with NAFLD is higher than expected, especially those with
impaired fasting glucose or diabetes.
oxLDL and TBARS Unreliable biochemical markers for NAFLD. [224]
CK-18 CK-18 is a major intermediate filament protein in the liver which is associated with morphological changes [225]
in apoptosis and is increased in NAFLD. Sensitivity and specificity of CK-18 in NAFLD has been reported
to be more than 90 %, and CK-18 was independently associated with NASH in multivariate analysis.
CDT Used to differentiate between NASH and alcoholic hepatitis. [226]
Apolipoprotein A1, haptoglobin, No markers are specific for NAFLD. [227]
a2 -macroglobulin, hyaluronic
acid and procollagen peptides,
such as I, III and VI

understanding the aetiology of NAFLD in humans and
have hampered progress in assessing new treatments for
the condition. Despite these problems, progress is being
made in developing non-invasive diagnostic markers for
investigating aetiology and for testing new treatments
for NAFLD. Recently, the ELF score (European Liver
Fibrosis score) has been tested in a small subgroup of
patients with NAFLD recruited with liver fibrosis [24].
The ELF score comprises three simple biochemical
measurements fitted into a proprietary algorithm and
shows promise with good sensitivity and specificity for
NASH with fibrosis, although further research is needed.
Another simpler algorithm has recently been developed
and published in full utilizing simple anthropometric
measurements and biochemical tests [25]. This test is also
showing considerable promise with good sensitivity and
specificity for NASH. A concentrated research initiative
is now needed to address the pressing need of sensitive
specific tests for NAFLD. The challenge remains to
establish biomarkers that are simple, reproducible,
inexpensive and with high sensitivity and specificity
for NAFLD and can differentiate simple steatosis from
NASH. Identification of simple inexpensive biomarkers
would facilitate achieving reliable estimates of prevalence
worldwide and would provide diagnostic tools for the
monitoring of responses to therapeutic interventions.
Ultrasound, CT (computer tomography) scanning
and MRI have all been used in the diagnosis of NAFLD.
Ultrasound has a sensitivity of 89 % and specificity
of 77 % and is commonly used in a clinical practice


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setting. Quantitative assessment of fatty infiltration is
best achieved with MRI [1].
RELATIONSHIP WITH CVD
Recent findings now suggests that NAFLD may also
be linked to increased CVD risk in Type 2 diabetes.
CVD risk varies markedly between individuals with
Type 2 diabetes and we have shown that, using traditional
algorithms for risk prediction, CVD events may be under-
estimated by approximately one-third in Type 2 diabetes
[26]. We have also shown that there is a > 4-fold and
statistically significant increase in risk of CHD (coronary
heart disease) death if patients have all five features of
the metabolic syndrome [central obesity, high TAG,
low HDL (high-density lipoprotein)-cholesterol, high
BP (blood pressure) and insulin resistance] compared
with having only one feature (e.g. hyperglycaemia in
isolation) [27]. Importantly, given the heterogeneity of
cardiovascular risk in people with Type 2 diabetes, we
have shown that patients with NAFLD and obesity are
markedly more insulin-resistant in muscle and fat than
those subjects with obesity alone [28].
Prospective studies have reported associations between
increased liver enzymes [particularly the serum GGT
(γ -glutamyltransferase) level as surrogate markers of
NAFLD) [29,30] and the occurrence of CVD events in
both non-diabetic subjects and Type 2 diabetic patients.
In a study of 14 874 middle-aged Finnish men and women,
a small increase in GGT levels were independently
associated with an increased risk of ischaemic stroke in
both sexes [31]. Among 7613 middle-aged British men
followed for 11.5 years, increased GGT levels were inde-
pendently associated with a significant increase in mortal-
ity from all causes and from CHD [32]. Recent epidemio-
logical studies have suggested the increase in ALT may be
linked to increase in risk of CVD. The Hoorn Study is a
population-based cohort of Caucasian men and women
aged 50–75 years at baseline. The 10-year risk of all-
cause mortality, fatal and non-fatal CVD and CHD
events in relation to ALT baseline was assessed in 1439
subjects [33]. Subjects with prevalent CVD/CHD and
missing data were excluded from these analyses. When
compared with the first tertile, the age- and gender-
adjusted hazard ratios (95 % CIs) for all-cause mortality,
CVD events and CHD events were 1.30 (0.92–1.83),
1.40 (1.09–1.81) and 2.04 (1.35–3.10) respectively, for
subjects in the upper tertile of ALT. After adjustment for
components of the metabolic syndrome and traditional
risk factors, the association between ALT and CHD
events remained significant for subjects in the third
tertile relative to those in the first tertile, with a hazard
ratio (95 % CI) of 1.88 (1.21–2.92). Thus the Hoorn
Study shows that ALT predicts cardiovascular events
independently of traditional risk factors and the features
Metabolic disturbances in non-alcoholic fatty liver disease 543
of the metabolic syndrome [33], suggesting that NAFLD
is associated with CHD independently of other features
of the metabolic syndrome.
Interestingly, another study from the U.S.A. has
examined the association between elevated serum ALT
activity and the 10-year risk of CHD as estimated
using the FRS (Framingham risk score) [34]. Among
participants without viral hepatitis or excessive alcohol
consumption, those with increased ALT activity [> 43 IU
(international units)/l] (n = 267) had a higher FRS than
those with normal ALT activity (n = 7259), both among
men [mean difference in FRS, 0.25 (95 % CI, 0.07–0.4);
hazard ratio for CHD, 1.28 (95 % CI, 1.07–1.5)] and
women [mean difference in FRS, 0.76 (95 % CI, 0.4–1.1);
hazard ratio for CHD, 2.14 (95 % CI, 1.5–3.0)]. The
ALT threshold for an increased risk of CHD was
higher in men (> 43 IU/l) than in women (> 30 IU/l)
[34]. Recently, the FIBAR (Firenze Bagno a Ripoli)
study concluded that increased GGT or AST (aspartate
aminotransferase) is an independent predictor of CVD.
An increase in GGT levels above the reference range, or
also in the upper reference range, was also an independent
predictor of incident diabetes [35].
In a recent study by Targher et al. [36] (n = 2839)
NAFLD and CVD were the main outcome measures in
Type 2 diabetic patients. The authors showed that the
unadjusted prevalence of NAFLD was 69.5 % among
participants, and NAFLD was the most common cause
(81.5 %) of hepatic steatosis detected by ultrasound.
The prevalence of NAFLD increased with age (65.4 %
among participants aged 40–59 years, and 74.6 % among
those aged  60 years; P < 0.001) and the age-adjusted
prevalence of NAFLD was 71.1 % in men and 68 % in
women. NAFLD patients had a higher age (P < 0.001)
and gender-adjusted prevalence of coronary (26.6 com-
pared with 18.3 %), cerebrovascular (20.0 compared
with 13.3 %) and peripheral (15.4 compared with
10.0 %) vascular disease than their counterparts without
NAFLD. In logistic regression analysis, NAFLD was
associated with prevalent CVD, independent of classical
risk factors, glycaemic control, medication and the
features of the metabolic syndrome [36].
The Valpolicella Heart Diabetes Study is a prospective
nested case-control study in 2103 Type 2 diabetic
patients who were free of diagnosed CVD at baseline.
During 5 years of follow-up, 248 participants (case
subjects) subsequently developed non-fatal CHD
(myocardial infarction and coronary revascularization
procedures), ischaemic stroke or cardiovascular death.
After adjustment for age, gender, smoking history,
diabetes duration, HbA1c (glycated haemoglobin),
LDL-cholesterol, liver enzymes and use of medication,
the presence of NAFLD was significantly associated
with an increase in CVD risk [OR, 1.84 (95 % CI, 1.4–
2.1); P < 0.001]. Additional adjustment for the metabolic
syndrome (as defined by National Cholesterol Education

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Program Adult Treatment Panel III criteria) appreciably
attenuated, but did not abolish, this association [OR,
1.53 (95 % CI, 1.1–1.7); P = 0.02] [37]. Thus the above
two large studies have demonstrated that NAFLD is
associated with an increased risk of future CVD events
among Type 2 diabetic individuals and, importantly, this
association was independent of classical risk factors, liver
enzymes and the metabolic syndrome.
Studies have shown a link between NAFLD
and increased carotid IMT (intima-media thickness).
Fracanzani et al. [38] concluded (in a series of normal
and NAFLD subjects) that independent risk predictors
of increased IMT were the presence of hepatic steatosis
(OR, 6.9), age (OR, 6.0) and increased systolic BP (OR,
2.3). More interestingly, Targher et al. [39] suggested that
the severity of liver histopathology among 85 NAFLD
patients was strongly associated with early carotid
atherosclerosis, independent of age, gender, BMI,
smoking, LDL-cholesterol, insulin resistance and the
presence of the metabolic syndrome. In addition, a large
population study has shown that NAFLD is associated
with an increase in IMT [40]. On the other hand, in 100
dietcontrolled Type 2 diabetic individuals, the significant
increase in carotid IMT in the presence of NAFLD has
largely been explained by insulin resistance. Currently,
it is not clear how NAFLD leads to an increase in risk
of CVD. One explanation may be the fact that NAFLD
appears to be associated with all metabolic risk factors
that are features of the metabolic syndrome. In addition,
NAFLD may accelerate the progression of dyslipidaemia,
insulin resistance, atherosclerosis, endothelial dysfunc-
tion, inflammation and oxidative stress. Interestingly,
NAFLD may also be associated with a detrimental effect
on other organs that may have a direct or indirect influ-
ence on CVD, or organs that may accelerate presentation
of CVD, at least in people with Type 2 diabetes. For
example, NAFLD patients with Type 2 diabetes had
higher (P < 0.001) age- and gender-adjusted prevalence
rates of both non-proliferative (39 compared with 34 %)
and proliferative/laser-treated (11 compared with 5 %)
retinopathy, and CKD (chronic kidney disease) (15
compared with 9 %) than counterparts with Type 2 dia-
betes but without NAFLD. In these subjects, using
logistic regression analysis, NAFLD was shown to be
associated with increased rates of CKD [OR, 1.87 (95 %
CI, 1.3–4.1); P = 0.020] and proliferative/laser-treated
retinopathy [OR, 1.75 (95 % CI, 1.1–3.7); P = 0.031]
independently of age, gender, BMI, waist circumference,
hypertension, diabetes duration, HbA1c , lipids, smoking
status and medication use [41]. Furthermore, in that
study, NAFLD was associated with an increased incid-
ence of CKD [42], independent of gender, age, BMI, waist
circumference, BP, smoking, diabetes duration, HbA1c ,
lipids, baseline estimated GFR (glomerular filtration
rate), microalbuminuria and medication (hypoglycaemic,
lipid-lowering, antihypertensive or antiplatelet drugs).

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Interestingly, NAFLD has been shown to be associated
with the development of CKD in Korean individuals
[crude relative risk, 2.18 (95 % CI, 1.75–2.71], and this
relationship remained significant even after adjustment
for age, GFR, TAG and HDL-cholesterol [aRR (adjusted
relative risk), 1.55 (95 % CI, 1.23–1.95)] [43]. The
association between NAFLD and incident CKD was
evident in the NAFLD group with elevated serum GGT
[aRR, 2.31 (95 % CI, 1.53–3.50)], even after adjustment
for age, GFR, TAG and HDL-cholesterol, but not
in the NAFLD group without elevated GGT [aRR,
1.09 (95 % CI, 0.79–1.50); P = 0.008 for interaction)]
[43].
PATHOGENESIS
From the evidence presented above, it is important to
understand the pathogenesis of NASH because of the
risk of progression to more severe end-stage liver disease.
It is also important to understand the relationship
between NASH and CVD. With respect to the latter, it is
plausible that factors outwith the liver are contributing to
NASH and also to CVD. Changes in the liver associated
with NASH may then compound the problem further
and amplify risk for CVD. For these reasons, we will
discuss the contribution of factors outwith the liver
to the pathogenesis of NAFLD and we will discuss
mechanisms within the liver contributing to NAFLD.
There is a strong link between insulin resistance and
excessive deposition of TAG in hepatocytes, which
is the hallmark for diagnosis of NAFLD [44–46].
The excessive/ectopic fat depositions in the liver
could be due to increased fatty acid delivery from
adipose tissue, increased synthesis of fatty acid via
the de novo pathway, increased dietary fat, decreased
mitochondrial β-oxidation, decreased clearance of
VLDL (very-LDL) particles or these factors in combin-
ation. It is still a matter of debate whether insulin
resistance causes NAFLD or whether excessive accumul-
ation of TAG, or precursors on the synthetic pathway,
precede and promote insulin resistance [47].
Extrahepatic mechanisms contributing
to the pathogenesis of NAFLD
Lipolysis and NEFAs [non-esterified fatty acids
(‘free fatty acids’)]
There is evidence that increased delivery of NEFAs to the
liver from the peripheral (adipose) tissue is fundamental
to the development of NAFLD. In support of this notion,
studies in humans and rodents have shown that increased
NEFA delivery from adipose tissue is a significant source
of fat accumulation in hepatocytes [48]. Some investigat-
ors [49] have reported that approx. 60 % of fat deposited
in hepatocytes is generated from adipose tissue sources.

Figure 1 Interactions between liver, muscle and adipose
tissue in the control of VLDL secretion
(A) Normal state; (B) insulin-resistant state, showing an increased flux of NEFAs
from adipose tissue to the liver. FFA, NEFA; LPL, lipoprotein lipase.
In insulin-resistant subjects, there is a failure of insulin-
mediated suppression of HSL (hormone-sensitive lipase),
resulting in uncontrolled lipolysis in the adipose tissue
[48,50] (Figures 1A and 1B). HSL-knockout mice have
decreased plasma NEFA and TAG concentrations, and
have increased hepatic insulin sensitivity [51,52]. Peri-
pheral (subcutaneous) fat constitutes a major proportion
of the fat mass; however, it is not an absolute requirement
for developing fatty liver. Patients with lipodystrophy,
who are also insulin-resistant with no peripheral and
intra-abdominal fat, develop fatty liver and severe hepatic
insulin resistance [53]. In this situation, the stimulus for
fatty liver may be increased NEFA flux to the liver
stimulating lipogenesis, combined with inadequate com-
pensatory fat oxidation, leading to fat accumulation over
time. Furthermore, there is some contribution to NEFA
flux to the liver from intra-abdominal fat in insulin-
resistant subjects [54]. Intra-abdominal fat is strongly
associated with reduced insulin sensitivity even in lean
subjects [55], and has been shown to correlate positively
with hepatic fat and hepatic insulin resistance [56–58].
Metabolic disturbances in non-alcoholic fatty liver disease 545
Dietary macro- and micro-nutrients
Saturated fatty acids
The role of diet in the pathogenesis of NAFLD has
been investigated in humans and animal models [59,60].
Subjects on a high-fat diet develop fatty liver. Subjects on
a low-fat/high-carbohydrate diet also develop fatty liver
via increased de novo fatty acid synthesis [61]. In addition
to the above effect, high dietary fat intake is associated
with obesity and insulin resistance. As a consequence
of insulin resistance, there is impaired suppression of
lipolysis by insulin, leading to increased NEFA delivery
to the liver [62,63]. There is also reduced glucose uptake
in the fed state by adipose tissue and skeletal muscle,
resulting in hyperglycaemia and diversion of glucose to
the hepatic de novo pathway [64].
An increase in hepatic fatty acid is capable of exacerbat-
ing hepatic insulin resistance at the insulin receptor level
[65]. The mechanism has not been fully elucidated but is
thought to be due to translocation of the PKCδ (protein
kinase Cδ) isoform from the cytosol to the membrane
compartment, leading to impaired IRS (insulin receptor
substrate)/PI3K (phosphoinositide 3-kinase) activation
[55]. High dietary saturated fatty acids have been shown
to be associated with insulin resistance, NAFLD and
CVD [66]. In rats, high dietary saturated fatty acids
have been shown to promote ER (endoplasmic reticulum)
dysfunction and hepatocyte injury [67]. In another study
in humans [68], dietary consumption of saturated fat was
reported to be higher in overweight patients with NASH
compared with age- and BMI-matched control subjects
(14 compared with 10 % respectively).
n − 3 PUFAs (polyunsaturated fatty acids)
There is increasing interest in the role of n − 3 PUFAs in
NAFLD. The benefits of DHA (docosahexaenoic acid)
and EPA (eicosapentaenoic acid) following the high con-
sumption of fish oil have been shown in insulin-resistant
animal models [66], including (i) decreased plasma NEFA,
TAG, glucose and insulin concentrations; (ii) decreased
de novo lipogenesis, VLDL export and TAG con-
centrations; (iii) increased utilization and storage of gluc-
ose by skeletal muscle; (iv) increased insulin-mediated
glucose uptake by adipose tissue; and (v) decreased
adipocyte size and visceral fat content. Other potential
benefits of PUFAs have also been reported in other animal
experiments and these include: (i) decreased Kupffer cell
activation [69]; (ii) decreased NF-κB (nuclear factor κB)
macrophage activation [70–72]; and (iii) decreased gene
expression and production of inflammatory cytokines
[70–73]. Similar findings have also been reported in hu-
mans. In a small study of patients with NAFLD [74], there
was decreased TAG and glucose concentrations, liver
enzyme activities and hepatic steatosis following con-
sumption of 1g of fish oil for 12 months. Another
study [75] showed similar results and decreased TNF-α

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546 C. D. Byrne and others
(tumour necrosis factor–α) concentrations after 2 g of
daily supplementation with fish oil for 6 months. The
authors [75] suggested that consumption of n − 3 PUFAs
could ameliorate NAFLD by improving steatosis,
inflammation and hepatocyte injury.
There is now some evidence that dietary n − 3 fatty
acids have a role in the pathogenesis of NAFLD and could
be a potential therapeutic target [76]. For example, there is
an association between PUFA deficiency and the develop-
ment of hepatic steatosis in animal models [66]. SREBP-
1c (sterol-regulatory-element binding-protein-1c) is a
key transcription factor for de novo lipogenesis (see
below) and is negatively regulated by n − 3 PUFAs [77].
Consequently, the activities of key enzymes for fatty acid
and TAG synthesis are down-regulated with decreased
hepatic fat deposition. PUFAs also negatively regulate
the activity of a glucose-responsive transcription factor
[ChREBP (carbohydrate-responsive-element-binding
protein); see below] by increasing ChREBP mRNA
decay and disrupting translocation of ChREBP from
the cytosol to the nucleus [78]. As a result of these effects,
it is plausible that high concentrations of dietary n − 3
PUFA supplementation could be a promising treatment
for the management of NAFLD, and randomized
controlled trials in this area are urgently needed.
Carbohydrate and protein
Excessive consumption of simple sugars has been
reported to promote the development of NAFLD. In
one report, there was a 2–3-fold increase in de novo lipo-
genesis in both lean and obese subjects following a 4 day
overfeeding with either a glucose or sucrose drink [79].
Similarly, high dietary intake of fructose also increases
de novo lipogenesis in both animal models and humans
[80,81].
The precise role of dietary protein in the pathogenesis
of NAFLD is uncertain. There is a need for further
investigation as whether there is a causal relationship
between excess dietary protein and NAFLD.
Micronutrients
The relationship between micronutrients and NAFLD is
now attracting considerable interest. PC (phosphatidyl-
choline), the most abundant phospholipid in the mam-
malian cell, is formed from dietary-dependent and
-independent pathways. In the presence of CCT
(CTP:phosphocholine cytidylyltransferase) and CPT
(CDP-choline:1,2 diacylglycerol choline phospho-
transferase) enzymes, PC is formed from dietary choline
and DAG (diacylglycerol). In the dietary-independent
pathway, PC is formed from SAM (S-adenosylmethion-
ine) and PE (phosphatidylethanolamine) in the presence
of PEMT (phosphatidylethanolamine N-methyltrans-
ferase), suggesting that a supply of methyl donors
is important for the synthesis of PC (Figure 2). There is
some evidence that PC, from these different pathways,

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Figure 2 Potential control of dietary methyl donor
regulation of gene expression relevant to NAFLD
Dietary-independent pathway synthesis of PC is formed from SAM and PE in the
presence of PEMT, suggesting that a supply of methyl donors is important for the
synthesis of PC.
has a distinct molecular composition with different
physiological functions dependent on the biochemical
pathway leading to PC generation [82,83]. PC from the
dietary-independent pathway consists mainly of long-
chain PUFAs, whereas PC of dietary origin consists of
medium-chain saturated fatty acids [82]. PEMT activity
has been shown to be important in the mobilization of
essential fatty acids from liver [83].
In animal models, PC generated from the PE pathway
has been shown to be essential for the incorporation of
neutral lipids into VLDL particles [84], and for the normal
concentrations and structural composition of plasma
VLDL [85,86]. Additionally, it has been shown that
PEMT-knockout mice have gender-specific differences
in the plasma concentrations of PC, cholesterol esters
and TAGs [85]. In one study [87], PEMT-knockout mice
were fed an MCD (methionine- and choline-deficient)
diet resulting in significantly decreased hepatic PC
concentrations, severe liver damage and death. Animal
models (mice) that are fed an MCD diet develop NASH
and this has provided a good model for studying the
liver in NASH, although the mice tend to be thin and are
insulin-sensitive.
Dietary deficiency of methionine in animal models
is associated with reduced glutathione deficiency, with
secondary free-radical-induced hepatocyte damage and
inflammation [83]. As PC is essential for the synthesis of
VLDL particles, PC deficiency is associated with hepatic
fat deposition secondary to the impaired export of VLDL
particles. PEMT-knockout mice had a significant increase
in hepatic TAG and cholesterol ester concentrations [83],
and there was almost a total reversal of these effects
following dietary choline supplementation, perhaps
suggesting a role for methyl donors in the generation of
PC and NASH.

Intrahepatic mechanisms
De novo lipogenesis
Role of key enzymes
Under basal conditions, the contribution to hepatic
fat from the de novo pathway is less than 5 % [88];
however, under pathological conditions, hepatic de novo
lipogenesis also has been reported to be a significant
source of fat deposited in the liver (approx. 30 %) [47,48].
In the de novo pathway, dietary glucose is converted into
acetyl-CoA by L-PK (liver-specific pyruvate kinase).
Acetyl-CoA is then converted into malonyl-CoA
and, subsequently, to fatty acids by actions of ACC
(acetyl-CoA carboxylase) and FAS (fatty acid synthase)
respectively.
ACC: The activity of ACC is regulated by the cell
energy status, insulin and the availability of NADPH.
ACC is inactivated by phosphorylation via AMPK
(AMP-activated kinase), resulting in decreased conver-
sion of acetyl-CoA into malonyl-CoA, and PP2A
(protein phosphatase 2A; an insulin-regulated enzyme)
reverses this action by dephosphorylation of ACC.
When cell energy is low with a high AMP/ATP ratio,
ACC is kept in its inactive phosphorylated form and,
consequently, acetyl-CoA is channelled to β-oxidation
and ketogenesis for energy production. ACC is also
under hormonal control by insulin and glucagon. Insulin
stimulates PP2A, which dephosphorylates ACC thereby
activating this enzyme and promoting lipogenesis.
There are two isoforms of ACC namely ACC1 and
ACC2. ACC1, which is in the cytosol, is highly expressed
in liver and adipose tissue. ACC2, the mitochondrial
isoform, is expressed mainly in muscle and, to a lesser ex-
tent, liver. ACC2 is involved in the negative regulation of
mitochondrial β-oxidation, hence increased β-oxidation
seen in ACC2-knockout animals. This is due to decreased
malonyl-CoA, which derepresses CPT-1 (carnitine
palmitoyltransferase-1) activity [89]. Fatty acid synthesis
takes place in the cytosol and, therefore, only the ACC1
isoform is important in the de novo pathway for fatty
acid synthesis. This occurs because there is no access to
malonyl-CoA produced in the mitochondria by ACC2
[90,91]. In a LACC1KO (liver-specific ACC1-knockout)
model, Mao et al. [92] showed a 70 % reduction in hepatic
malonyl-CoA relative to control, a 50 % reduction in
de novo lipogenesis and a 40 % decrease in TAG
concentrations. Paradoxically, this mouse model was
not protected from high-fat high-calorie diet-induced
obesity, fatty liver and insulin resistance, despite a
significant decrease in fat deposition. This effect may
be due to increased compensatory activity of liver-
specific ACC2 [93]. In contrast, in vitro ASO (antisense
oligonucleotide) inhibition of both ACC1 and ACC2 led
to a significant decrease in hepatic malonyl-CoA concen-
trations, decreased hepatic lipids and increased hepatic
Metabolic disturbances in non-alcoholic fatty liver disease 547
insulin sensitivity [94], suggesting that targeting ACC
activity could improve insulin resistance and ameliorate
fatty liver.
FAS: FAS is the last key enzyme in de novo fatty
acid synthesis and a total lack of FAS activity due to
gene deletion is not compatible with intra-uterine life.
Although this is a key lipogenic enzyme, FASKOL (liver-
specific FAS knockout) mice were also not protected
from developing hepatic steatosis. There was a 3-fold
increase in malonyl-CoA, which would presumably
inhibit CPT-1 and decrease mitochondrial β-oxidation,
leading to increased hepatic NEFAs [95]. Increasing
liver fatty acids would presumably then lead to TAG
synthesis and fat accumulation, worsening the NAFLD
phenotype, rather than ameliorating the phenotype.
SCD-1 (stearoyl-CoA desaturase-1): MUFAs (mono-
unsaturated fatty acids) are the major components of
TAGs, cholesterol esters and membrane phospholipids.
The synthesis of MUFAs is catalysed by SCD-1.
Therefore modulating the activity of this enzyme may
significantly decrease TAG synthesis. In a previous study
[96], SCD-1-knockout mice had decreased de novo lipo-
genesis with increased mitochondrial fat oxidation. These
mice were protected from diet-induced obesity, fatty liver
and insulin resistance when fed a high-fat and high-calorie
diet. ASO inhibition of SCD-1 in liver and adipose tissue
also showed similar results with the SCD-1-knockout
mice [97]. In another study [98], liver-specific SCD-1-
knockout mice were shown to have decreased gene
expression for key lipogenic enzymes (ACC and FAS),
with decreased de novo lipogenesis, and were also pro-
tected from diet-induced hepatic steatosis. Additionally,
the reduction in SCD-1 activity had a marked decrease
in the nuclear content of two key transcription factors
for lipogenesis SREBP-1c and ChREBP (see text below).
DGAT-2 (diacylglycerol acyltransferase-2): There is
now increasing interest in the role of DGAT-2, the
enzyme that catalyses the final step in TAG synthesis.
Theoretically, inhibition of DGAT-2 should prevent the
synthesis of TAG and NAFLD, although this appears
not completely true as, in a recent experiment in db/db
mice [99], inhibition of DGAT-2 with ASOs produced
surprising results. Feeding mice an MCD diet caused the
development of a fatty liver after 4 weeks. Although there
was marked improvement in hepatic steatosis following
inhibition of DGAT-2 with ASO, paradoxically, there
was worsening/progressive inflammation, hepatocyte
injury and fibrosis in these mice. Histological sections
of the liver in the DGAT-2 arm revealed marked inflam-
matory changes and hepatocyte necrosis. Additionally,
there was worsening liver fibrosis with raised mRNA
for most of the markers of fibrosis and hepatic stellate
cell activation. Although an MCD diet has been
shown to increase the expression of fibrosis markers

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548 C. D. Byrne and others
Figure 3 Effect of dietary fatty acid intake and de novo fatty acid synthesis in regulating lipid synthesis
Potential interactions are shown between hepatic lipid metabolism, DNA methylation and gene expression, inflammation, VLDL secretion and hepatic triacylglycerol
accumulation in the pathogenesis of NAFLD. FACoA, fatty acyl-CoA; UCP-2, uncoupling protein-2.
[TGF-β 1 (transforming growth factor-β 1 ), α-SMA
(α-smooth muscle actin), TIMP-1 (tissue inhibitor
of metalloproteinases-1) and collagen], there was a
failure of inhibition of most of these markers after
ASO inhibition of DGAT-2. The plausible explanation
for the progressive inflammation and fibrosis may
be due to increased hepatic NEFAs and the gene-
ration of hepatotoxic free radicals [99]. Mice in the
treatment arm had significantly higher hepatic fatty acids.
Although an MCD diet inhibits mitochondrial fat oxid-
ation, there was amplification of this effect in the
ASO-inhibition mice with a diversion of fatty acids
to microsomal and peroxisomal fat oxidation. Conse-
quently, in response to increased microsomal fat oxid-
ation, there was increased microsomal enzyme [Cyp2E1
cytochrome P450 2E1)] activity with increased (micro-
somal) production of ROS (reactive oxygen species).
Interestingly, there was improved systemic insulin
resistance after ASO inhibition of DGAT-2. There was a
reduction in weight, and reduced serum NEFAs, glucose
and insulin concentrations. There was also reduced
TNF-α and increased serum adiponectin concentrations.
In view of this evidence, the authors [99] postulated that
TAG itself may not be harmful, but could instead protect
the liver from lipid toxicity and hepatotoxic free-radical
damage by buffering hepatic fatty acids into the synthesis

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of TAG. Further research in this area is clearly needed
to elucidate which molecule(s) on the TAG synthetic
pathway are able to trigger inflammatory pathways.
Role of key transcription factors
SREBP-1c: Evidence is now emerging that SREBP-
1c is a positive transcription factor for ACC and FAS
genes [100], and we have suggested that abnormalities in
SREBP-1c function may play an important pathogenetic
role in contributing to the NAFLD phenotype (see
Figure 3) [101]. Overexpression of SREBP-1c in adult
rats causes a 26-fold increase in fatty acid synthesis and
fat deposition. Apart from modulating the key lipogenic
enzymes (ACC and FAS), SREBP-1c also stimulates gene
expression for fatty acid elongation and TAG synthesis.
However, SREBP-1c is by itself insufficient for full tran-
scription activity of fatty acid synthesis. In a knockout
experiment, only a 50 % reduction in fatty acid synthesis
was reported following SREBP-1c knockout [102].
Current evidence indicates that the effect of insulin on
lipogenesis is mediated via SREBP-1c activity [103,104].
In an insulin-resistance state with hyperinsulinaemia,
the transcriptional activity of SREBP-1c is up-regulated
and both insulin and SREBP-1c synergistically stimulate
key lipogenic genes, thereby promoting de novo
fatty acid synthesis. Insulin-resistant ob/ob mice have

increased concentrations of SREBP-1c and also develop
spontaneous fatty liver. Increased SREPB-1c in these
mice increases lipogenic gene expression, increases fatty
acid deposition and accelerates TAG deposition in
hepatocytes [105,106]. Administration of leptin, which
opposes the action of SREBP-1c, reverses these metabolic
derangements [107]. In contrast, in the fasting state, when
insulin production is low but glucagon concentrations are
increased, the quantity of SREBP-1c in liver is decreased,
although this effect is reversed by re-feeding [108,109].
Forkhead transcription factors: Decreased mitochon-
drial fatty acid oxidation is also contributory to excessive
fat deposition in the liver in insulin-resistant subjects.
A plausible mechanism for this effect could be mediated
via phosphorylation and dephosphorylation of the
transcription factor Foxa2 (forkhead box a2) [110].
Under physiological conditions, active (dephos-
phorylated) Foxa2 promotes mitochondrial fatty acid
oxidation, whereas insulin reverses this effect via phos-
phorylation by IRS-1 and IRS-2. Paradoxically, in
insulin-resistance, Foxa2 is still predominantly in its
inactive form because of residual sensitivity to insulin in
the liver resulting in decreased β-oxidation and increased
hepatic fat deposition.
INSIG-1 (insulin induced gene-1), insulin signalling,
PTEN (phosphatase and tensin homologue deleted on
chromosome 10) and mTOR (mammalian target of rapa-
mycin): There is now some interest on the role of
INSIG-1 and fatty liver. INSIG -1 is highly expressed in
hepatocytes and adipocytes, and has been suggested to
act as the ‘brake’ for lipogenesis in fat and limiting TAG
deposition in hepatocytes [110a]. The gene may also
play a modulatory role in preadipocyte diffentiation. It
is possible that the benefits of PPAR-γ (peroxisome-
proliferator-activated receptor-γ ) agonists in NASH are
due to the regulation of INSIG-1 by these drugs.
Under physiological circumstances, the phosphoryl-
ation of IRS, PI3K, Akt and the downstream proteins
are essential for the action of insulin in insulin-sensitive
organs. However uncontrolled phosphorylation would
lead to amplification of insulin action with insulin hyper-
sensitivity, whereas a lack of phosphorylation of the
insulin substrate proteins could cause insulin resistance.
There are key negative regulatory proteins (phosphatases)
that physiologically terminate the action of insulin by
dephosphorylation, and constitutive action of these
proteins could lead to decreased insulin sensitivity.
Other insulin signalling counter-regulatory mechanisms
include: (i) serine or threonine phosphorylation of the
insulin receptor; (ii) activation of tyrosine phosphatases;
(iii) inhibition of insulin receptor and IRSs by the SOCS
(suppressor of cytokine signalling) family; and (iv) activ-
ation of phosphoinositide phosphatases [PTEN, SHIP-2
(SH2-containing inositol phosphatase-2) and myotubu-
larin] [111].
Metabolic disturbances in non-alcoholic fatty liver disease 549
SHIP-2 is an insulin-signal-regulatory phosphatase,
but the absence or reduced expression and activity of
this enzyme has not been reported to be associated with
fatty liver [112–114].
PTEN is a tumour-suppressor protein, but with phos-
phatase activity, and has a regulatory effect on the insulin
signalling pathways [111,114,115]. The substrate proteins
for this enzyme are PtdIns(3,4)P2 and PtdIns(3,4,5)P3 .
As a phosphatase, the physiological function of PTEN
is to dephosphorylate the second messengers generated
by the activation of PI3K, thereby down-regulating or
terminating insulin signalling downstream of PI3K [111].
Overexpression of PTEN has been shown to have in-
hibitory effects on insulin signalling, including decreased
Akt activation and GLUT4 (glucose transporter 4) trans-
location to the cell membrane [116,117]. Overexpression
of PTEN in muscle from obese fa/fa Zucker rats had been
shown to contribute to muscle insulin resistance in these
animals [118]. In contrast, down-regulation of PTEN has
the opposite effect, with increased glucose uptake in fat
and muscle in response to insulin [119]. In mice, liver-
specific deletion of PTEN has been shown to increase
insulin sensitivity, but paradoxically causes fatty liver
disease and hepatocellular cancer [120,121]. The mech-
anism for this paradox is yet to be clarified, but there has
been a number of hypotheses suggested, some of which
highlight the lack of negative regulation on the insulin
signalling pathways by PTEN. In PTEN-knockout mice,
it has been shown that there was increased synthesis and
storage of TAG in hepatocytes due to the up-regulation
of PI3K/Akt activity [119,121]. As a consequence of the
lack of PTEN activity, there is increased hepatocyte fatty
acid uptake, increased fatty acid synthesis and increased
esterification of fatty acids to TAG. Taken together, these
findings suggest that decreased PTEN activity would lead
to excessive fat deposition in the liver.
There is evidence that reduced or absent expression
of liver-specific PTEN is associated with hepatic
steatosis, inflammation, fibrosis and neoplasm [115]. In
one study, PTEN-deficient mice were shown to have
biochemical and histological evidence, including fibrosis
of NASH, present at 40 weeks age [115]. The mechanism
for NASH in this animal model has been reported to be
due to increased expression of the PPAR-γ , SREBP-1c
and downstream genes, including Akt and Foxo1,
resulting in increased lipogenesis, inflammation and
fibrosis [115]. The reason for the increase in fat deposi-
tion in the liver could be partly due to the increased
expression of SREBP-1c. As SREBP-1c is a key
transcription factor for lipogenesis with increased ACC,
FAS and SCD-1 enzyme activities, all of these act syner-
gistically to promote fatty acid synthesis.
As a consequence of increased PPAR-γ expression,
there is also a secondary induction of key enzymes
involved in mitochondrial β-oxidation. As a result,
the increase in fat oxidation with a marked increase in the

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550 C. D. Byrne and others
Figure 4 Interaction between NEFAs, mTOR and NF-κB
Schematic diagram showing the effects of increased availability of fatty acids in
hepatocytes resulting in activation of mTOR. The activation of mTOR leads to
activation, dissociation and then translocation of NF-κB from the cytosol to the
nucleus, where it regulates PTEN at the level of transcription. As a consequence,
there is amplification of insulin signalling with increased hepatic lipogenesis.
∗ Activation of mTOR leads to activation of the downstream proteins including S6
Indicates activation of the protein. FFA, NEFA.
generation of oxidative free radicals leads to inflammation
and fibrosis via activation of the NF-κB pathway
[122,123]. For example, there is a 7-fold increase in the
hepatic concentration of H2 O2 in PTEN-deficient mice
compared with the wild type [115]. There is also evidence
of transcriptional down-regulation of PTEN in the liver
with decreased enzyme protein levels in fatty liver disease,
and this has been shown in the liver of obese insulin-
resistant ZDF (Zucker diabetic fatty) rats with diabetes
and hepatic steatosis [114]. This raises the question of
cause or effect. Furthermore, only unsaturated NEFAs
have been shown to decrease the expression of PTEN
in hepatocytes, suggesting that this is a specific effect
of unsaturated fatty acids. For example, the down-
regulation of PTEN expression by the unsaturated fatty
acid oleic acid is via the activation of mTOR and/or
NF-κB pathways. Similar findings have been reported
in humans in a small study of morbidly obese subjects
(n = 20), where there was a down-regulation of PTEN ex-
pression with a biopsy-proven decrease in immunohisto-
chemistry staining for PTEN in subjects with hepatocytes
steatosis of > 80 % [114].
In insulin-resistant subjects, there is an increase
in plasma NEFA concentrations with an increase in
delivery and uptake of NEFAs by hepatocytes. This
results in the activation of mTOR, which in turn leads to
activation of NF-κB [114]. As both mTOR and NF-κB
exist as a complex, there is a subsequent dissociation and
then translocation of NF-κB into the nucleus, where it
down-regulates the expression of PTEN at the level of
transcription (Figure 4). The precise mechanism of how
NF-κB transcriptionally down-regulates PTEN is not
fully understood, but could be through the sequestration
of the CBP [CREB (cAMP-response-element-binding

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Figure 5 Regulation of the mTOR signalling pathway by
nutrients and insulin
Schematic diagram showing the up-regulation of mTOR by insulin and
nutrient-sensing mechanisms and the down-regulation of mTOR by AMPK activation.
ribosomal protein resulting in serine phosphorylation of IRS-1 and consequent
decreased insulin action.
protein)-binding protein)]/p300, the transcriptional
activator for PTEN [124].
mTOR is a downstream target of the PI3K signalling
pathway, but the activation of this protein down-regulates
insulin signalling in insulin-responsive tissue [125,126].
The mechanism is through serine phosphorylation of
IRS-1 [127]. The regulation of the mTOR signalling path-
way is complex. This pathway is activated by hormonal-
and nutrient-sensing factors, whereas inhibitory regul-
ation is through AMPK activation. Insulin via IRS
activates PI3K, which, in turn, activates Akt. Akt then ac-
tivates mTOR via phosphorylation. The activation of
mTOR leads to phosphorylation and activation of down-
stream proteins, including p70S6 kinase and S6 ribosomal
protein. As a consequence of S6 ribosomal protein phos-
phorylation, serine phosphorylation of IRS-1 occurs at
Ser636 /Ser639 [128]. Because of the serine phosphorylation
of IRS-1, this key insulin substrate protein becomes un-
responsive to insulin signals and, instead, undergoes rapid
degradation and there is a decreased interaction between
the serine-phosphorylated IRS-1, the insulin receptor
(upstream) and PI3K [129], resulting in decreased insulin
action (Figure 5).
The mTOR signalling pathway is also regulated via
a nutrient-sensing mechanism. An increase in plasma
leucine concentrations is a potent activator of the mTOR
pathway through class III PI3K activation (class I PI3K
being the substrate protein of the insulin/IRS pathway)
[130]. A high-fat diet is associated with obesity, hepatic
fat deposition, hepatic and whole-body insulin resistance
and, in an animal experiment, the mTOR signalling
pathway has been shown to be up-regulated in mice fed

a high-fat diet compared with controls [131]. As insulin
activates the mTOR pathway, we can assume that up-
regulation of this pathway may be due to increased fasting
insulin concentrations associated with high dietary fat
and insulin resistance. Under normal conditions, this
action of insulin would be mediated via tyrosine
phosphorylation of the insulin receptor; however, in this
animal experiment, there was no difference in tyrosine
phosphorylation of the insulin receptor or the down-
stream target proteins in high-fat-fed mice and the control
group. The authors [131] hypothesized that the activation
of the mTOR pathway was through a poorly understood
insulin/IRS-independent pathway, probably due to acute
fat deposition in the liver. However, in the same report
[131], the investigators were not able to show that the
up-regulation of the mTOR pathway was due to acute
hepatic steatosis. Pharmacological induction of acute hep-
atic steatosis in mice showed a 5-fold increase in hepatic
TAG content, but failed to show a significant increase in
the activation of mTOR and the downstream proteins,
leading to the authors concluding that acute hepatic
steatosis is, by itself, insufficient for sustained activation
of the mTOR signalling pathway [131].
Increased fatty acid delivery has been shown to up-
regulate the mTOR pathway, thereby exacerbating hep-
atic insulin resistance. In a recent study [132], in the ab-
sence of insulin, palmitic-acid-cultured hepatocytes had
activation of the mTOR pathway with a 4-fold increase
in p70S6 kinase activity. In the presence of insulin, there
was an amplification of this effect. The antidiabetic agent
metformin, via activation of AMPK, was shown to reverse
this effect. In insulin-resistant subjects with increased
fatty acid delivery to the liver and hyperinsulinaemia,
we postulate that worsening insulin resistance may be
due to sustained or enhanced activation of the mTOR
pathway.
ChREBP: The gene for l-pyruvate kinase, a key com-
ponent of the de novo pathway for fatty acid synthesis,
is not regulated by SREBP-1c [133], but exclusively by
glucose [134].
A glucose-responsive transcription factor, ChREBP,
has been described and thus illustrates how glucose
affects gene transcription [135]. ChREBP is controlled
by glucose and regulates the translocation of this protein
from cytosol to the nucleus [136]. In the nucleus, glucose
promotes the binding of this protein to the carbohyd-
rate-response element in the promoter region of target
genes [137].
As part of deranged insulin signalling in obese insulin-
resistant subjects, there is decreased GLUT4 transloca-
tion to the cell membrane in muscle and adipose tissue.
The investigators suggested that fatty acyl-CoA and other
NEFA derivatives and TNF-α impair the activation of
IRS and PI3K [138,139]. Consequently, there is activation
of JNK (c-Jun N-terminal kinase) pathways, leading to
Metabolic disturbances in non-alcoholic fatty liver disease 551
serine instead of tyrosine phosphorylation and insulin
resistance [138,140]. ROS and TNF-α are also capable of
activating JNK. The overall effect is decreased glucose
uptake with persistent hyperglycaemia without or with
hyperinsulinaemia. This will exacerbate de novo hepatic
lipid synthesis via activation of SREBP and ChREBP
being driven by insulin and glucose respectively.
LXR-α (liver X receptor-α): ChREBP is also regulated
by LXR-α, and important ligands for LXR-α are oxy-
sterol, insulin and glucose. LXR-α is a member of the
nuclear receptor family that plays an important role in
lipogenesis. LXR-α exerts transcriptional control on
SREBP-1c and, therefore, indirectly on ACC and
FAS [141–143]. ob/ob mice that are insulin-resistant
develop fatty liver, although this is unlikely to be due to
insulin-mediated effects on ACC and FAS. The explan-
ation for this paradox may be that not all insulin-sensitive
metabolic pathways are uniformly affected by insulin
resistance. A plausible mechanism is via glucose control.
The genes for the gluconeogenic enzymes PEPCK (phos-
phoenolpyruvate carboxykinase) and G6Ptase (glucose-
6-phosphate translocase) are up-regulated in the liver of
insulin-resistant ob/ob mice [144]. High hepatic glucose
output is a consequence of increased gluconeogenesis.
We may then postulate that the enhanced transcriptional
activities of the glycolytic and lipogenic genes are regu-
lated by the co-ordinated activity of ChREBP, LXR-α
and SREBP-1c.
Inflammation, oxidative stress, fibrosis
and ER stress
Fat is now considered a metabolically active endocrine
organ producing pro-inflammatory cytokines, including
TNF-α, IL (interleukin)-6 and IL-8, and there is evi-
dence to support the activation of other inflammatory
pathways, oxidative stress and the de novo pathway by
TNF-α [145–147]. It has recently been hypothesized
that NAFLD is a consequence of a ‘two hit’ insult. As
a consequence of insulin resistance, there is excessive
accumulation of TAG in hepatocytes, which is the
‘first hit’. Oxidative stress from β-oxidation, increased
expression of inflammatory cytokines by NF-κB-
dependent pathways and adipocytokines are potential
synergistic factors acting in concert as the ‘second hit’,
resulting in hepatocyte injury, inflammation and fibrosis.
In contrast, all subjects with NAFLD have insulin
resistance, even in lean subjects and those with normal
glucose tolerance [59,148,149]. The core metabolic
derangement is due to insulin resistance resulting
in increased lipolysis in adipose tissue, increased
NEFA uptake by hepatocytes and increased TAG
synthesis in the liver. Mitochondrial fat oxidation and
export of VLDL particles are not able to match TAG
synthesis, leading to net fat deposition in the hepato-
cytes. As a consequence of abnormal fat accumulation

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552 C. D. Byrne and others
in the hepatocytes, there is marked derangement in the
insulin signalling pathways in the liver via activation of
NF-κB, IKK-β [IκB (inhibitor of NF-κB) kinase-β],
atypical PKC and JNK pathways by NEFAs [150,151].
There is evidence that increased mitochondrial fat
oxidation, a consequence of hepatic insulin resistance, is
linked to hepatic injury, inflammation and fibrosis. This
is because NEFAs and their metabolites are ligands for
PPAR-α. This transcription factor regulates a number
of genes, including those involved in mitochondrial,
peroxisomal and microsomal fat oxidation, and there is
now evidence to support the up-regulation of these genes
in NAFLD.
As a result of increased fat acid oxidation in the
mitochondria and peroxisome, there is increased
generation of hepatotoxic oxygen free radicals. Because
of oxidative stress, there is lipid peroxidation and severe
mitochondrial dysfunction with structural and functional
abnormalities. There is ATP depletion due to the failure
to generate ATP via oxidative phosphorylation [152,153],
and this could be due to increased expression of UCP-2
(uncoupling protein-2) by PPAR-α activation [154,155].
Patients with NASH also have structural mitochondrial
abnormalities [156–160], and, under-expression of some
genes necessary for normal mitochondrial function have
also been reported in these patients [161].
Inflammation is the link between obesity and insulin
resistance, and may have an important role in the
pathogenesis of hepatic and systemic insulin resistance.
For example, serine phosphorylation of IRS-1 by inflam-
matory cytokines is a mechanism mediating insulin
resistance in obese subjects with chronic low-grade
inflammation. In support of an association between
insulin resistance and inflammatory cytokines, there is
increased production of TNF-α in obese animal models
and in obese insulin-resistant subjects [162]. In contrast,
knockout mice for either TNF-α or the TNF-α receptor
were reported to be insulin-sensitive [162]. In obese
subjects with previously increased TNF-α expression,
decreased TNF-α expression following weight loss and
exercise was observed [163]. The molecular mechanism
responsible for TNF-α-induced insulin resistance is
most probably serine phosphorylation of IRS-1, which
has an inhibitory effect on the downstream propagation
of insulin signals [163,164].
Another mechanism for TNF-α-induced insulin
resistance is through the activation of the IKK-β
pathway [165]. Overexpression of IKK-β is associated
with reduced insulin signalling in cultured cells [165].
In contrast, liver-specific deletion of IKK-β resulted in
improved hepatic insulin sensitivity even on a high-fat
diet [165]. As a result of the activation of IKK-β, there
is downstream activation of the NF-κB pathway with
worsening inflammation and hepatic insulin resistance.
At the level of transcription, the activation of NF-κB
also has an inhibitory effect on PPAR-γ agonism.

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However, it is uncertain whether inflammatory
cytokines cause NAFLD or occur as a consequence of
the developing liver condition and NF-κB activation. In
a previous review [166], increased inflammatory cytokine
production was reasoned to be a consequence of IKK-β
and NF-κB activation and, in support of this reasoning
in NAFLD, increased hepatic NF-κB activity in high-
fat-fed mice was associated with increased inflammatory
cytokine expression, with attenuation of this effect with
specific inhibition of NF-κB activity [150]. Additionally,
activation of the upstream protein IKK-β was also
associated with inflammation, hepatic and systemic insu-
lin resistance, and there was also a reversal of this effect
using a hepatic-specific inhibitor of NF-κB. In NAFLD,
it is plausible that hepatic NEFAs have a toxic effect on
hepatocyte lysosomal membranes with the consequent
release of proteolytic enzymes that directly activate the
IKK-β and NF-κB pathways with increased TNF-α
expression [167].
JNK is a member of the MAPK (mitogen-activated
protein kinase) family that regulates cell function via the
control of AP-1 (activated protein-1), and there is a strong
association between increased JNK activity and insulin
resistance. In obesity with insulin resistance, increased
NEFAs and inflammatory cytokines are accompanied
by increased JNK activity in insulin-sensitive tissue,
thereby causing insulin resistance [162]. Although a
knockout model for JNK1 did not develop insulin
resistance [168], a possible role for the JNK family of
MAPKs in NAFLD is presently uncertain.
The ER is a cellular organelle for synthesis, storage and
transport of proteins. Because of the presence of some
chaperone proteins, the ER ensures proper folding of pro-
teins. IRE-1 (inositol-requiring enzyme-1), PERK [PKR
(double-stranded-RNA-dependent protein kinase)-like
ER kinase), ATF-6 (activating factor-6) and XBP-1
(X-box-binding protein-1) are subcellular proteins
necessary for ER function. Although these protein are
important for normal ER function, abnormal activation
may occur as a consequence of a ‘stressed’ environment,
for example hypoxia, leading to inflammation and insulin
resistance [169]. Evidence is beginning to emerge for the
pathogenic role of hepatic ER ‘stress’ in inflammation
and insulin resistance. For example, ER ‘stress’ has been
shown to directly activate the IKK-β/NF-κB and the
JNK/AP-1 pathways via interactions with IRE-1 and
PERK, thereby inducing or exacerbating inflammation
and insulin resistance [162,166] (Figure 6). Hepatic ER
‘stress’ has also been shown to occur with high-fat
feeding and in a genetically induced obese animal model
[170].
Because of the modulatory effect of endogenous bile
acids and their derivatives on ER function, the therapeutic
potential of these pharmacological agents have been
explored in both humans and animal models. Treatment
of NASH in insulin-resistant subjects with UDCA

Figure 6 Interaction between inflammatory cytokines, ER
stress, JNK, NF-κB pathways and hepatic inflammation
Schematic diagram showing activation of JNK/AP-1 and IKK-β/NF-κB pathways
by ER stress and inflammatory cytokines resulting in exacerbated inflammation.
(ursodeoxycholic acid) has so far been disappointing
[168], although, in obese mice, these authors have shown
improved hepatic and whole-body insulin sensitivity
with UDCA treatment.
NEMO (NF-κB essential modulator/IKK-γ ) is the
regulatory component of the IκB complex. The two cata-
lytic components of this complex are IKK-1/IKK-α and
IKK-2/IKK-β [171]. NF-κB has an important cellular
function in the regulation of immune and inflammatory
responses, and is maintained in its inactive form in the
cytosol by binding inhibitory proteins [171]. Although
the physiological function of NF-κB in adult liver is
not known, deletion of either the catalytic component
(IKK-2) or the regulatory component (NEMO) is not
compatible with intra-uterine life [172].
Hepatocellular carcinoma is a well-known complic-
ation of NAFLD and the molecular mechanism in this
group of patients in beginning to be better understood.
There is evidence to support the tumour-suppressive
effect of NEMO in hepatocytes. In one animal
experiment [171], NEMO-knockout mice developed
spontaneous hepatocellular carcinoma at 12 months of
life. In addition, there was histological evidence of pre-
malignant lesions in the liver of these mice by 9 months.
In the same study, there was biochemical and histological
evidence of NASH prior to the development of cancer
in this mouse model. In contrast, specific deletion of
IKK-2 (a catalytic component) was not associated with
chronic liver disease or spontaneous liver cancer [171].
These findings led the authors to suggest that NEMO-
dependent NF-κB activation has a physiological role in
the prevention of spontaneous hepatic cancer.
LPS (lipopolysaccharide) is a potent inducing agent of
TNF-α and other inflammatory cytokines [173,174], and
bacterial antigens entering between tight junctions in the
intestine could have an impact on liver damage in
the pathogenesis of NAFLD. There is a failure in NF-
Metabolic disturbances in non-alcoholic fatty liver disease 553
κB activation following injection of LPS in the liver
of NEMO-knockout mice, and the administration of
TNF-α in these mice also showed a similar effect [171].
As a consequence of the absence of NF-κB activation,
these mice developed severe liver failure due to exposure
to LPS and increased cytokine drive. Thus LPS-induced
activation of TNF-α could contribute to liver damage
in the pathogenesis of NAFLD, particularly if NEMO-
dependent NF-κB activation is inadequate to protect
the liver from damage exacerbated through activation
of the JNK pathway. It is possible that oxidative stress
may be a contributory factor to hepatocyte necrosis
observed in NEMO-knockout mice. In an animal
study [171], NEMO-knockout mice were treated with
a dietary antioxidant supplement, BHA (butylated
hydroxyanisole), and were shown to have biochemical
and histological improvements in chronic liver disease.
There was also reduced hepatic JNK activity in the
antioxidant group, leading the authors to postulate
that antioxidant therapy could ameliorate or normalize
chronic liver disease in this NEMO-knockout model.
Adiponectin
There is emerging interest in the role of adipocytokines
in the pathogenesis of NAFLD. Adiponectin has been
shown to decrease de novo fatty acid synthesis but en-
hance fat oxidation. Stimulation of adiponectin receptors,
which are expressed in hepatocytes and myocytes, leads
to PPAR-α and AMPK activation [175]. As a result,
adiponectin increases β-oxidation but decreases TAG
synthesis. Adiponectin also has direct anti-inflammatory
effects by decreasing hepatic TNF-α production. In
support of a pathogenetic role for adiponectin in affecting
inflammation in NAFLD, patients with NASH have
decreased expression of adiponectin receptors in the
liver and decreased serum adiponectin concentrations
compared with patients with simple steatosis [176,177].
Endocannabinoids
The psychotropic actions of the plant Cannabis sativa
were first documented approx. 4000 years ago. The
endocannabinoid system contributes to the physiological
regulation of energy balance, food intake, and lipid and
glucose metabolism through both central and peripheral
effects. Cannabinoid receptors, named CB1 receptor and
CB2 receptor, participate in the physiological modulation
of many central and peripheral functions. The CB2
receptor is mainly expressed in immune cells, whereas the
CB1 receptor is the most abundant G-protein-coupled
receptor expressed in the brain. The CB1 receptor is
expressed in the hypothalamus and the pituitary gland,
and its activation is known to modulate all the endocrine–
hypothalamic–peripheral endocrine axes. Recent exciting
new results suggest that inhibition of the endocannabin-
oid system might be beneficial in the treatment of the
NAFLD. Interestingly, a new class of anti-obesity med-
ication, such as rimonbant (a CB1 receptor antagonist),

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554 C. D. Byrne and others
was shown to be beneficial in an animal model of hepatic
steatosis [178]. Treatment of obese (fa/fa) rats with
rimonabant (30 mg/kg of body weight) daily for 8 weeks
abolished hepatic steatosis, decreased hepatomegaly,
decreased ALT and GGT levels, decreased dyslipidaemia
and, importantly, increased the level of the anti-
inflammatory hormone adiponectin [178]. At the hepatic
cell level, activation of hepatic CB1 receptors by stellate
cell endocannabinoid 2-AG (2-arachidonoylglycerol) is
responsible for ethanol-induced steatosis by increasing
lipogenesis and decreasing fatty acid oxidation [179].
Recently, rimonabant has been shown to be effective in
reducing weight [180], and randomized clinical trials are
now needed to establish whether rimonabant produces
therapeutic benefit in the treatment of NAFLD.
Mitochondrial dysfunction and ROS
The mitochondrial genome synthesizes 13 respiratory
chain polypeptides and is very sensitive to oxidative
damage, probably because of (i) the proximity of mtDNA
(mitochondrial DNA) to the inner mitochondrial
membrane (a source of ROS), (ii) the absence of
protective histones; and (iii) the lack of effective DNA
repair mechanisms.
There is evidence that increased production of ROS,
pro-inflammatory cytokines and hepatocyte necrosis
secondary to mitochondrial dysfunction is important
in the pathogenesis of NAFLD. Although the sequence
of pathophysiological events are yet to be clarified, it is
plausible that oxidative stress is an early event in NAFLD
and could provide the ‘trigger’ for inflammation and
fibrosis. Oxidative stress is undoubtedly a major feature
of NASH, and potential sources of ROS are hepatocyte
parenchymal cells, chronic inflammatory cells within the
hepatocytes and, in obese subjects, adipose tissue with
infiltrating activated macrophages [181,182].
There is some evidence that increased generation of
ROS could enhance progression from simple steatosis
to steatohepatitis and fibrosis [183]. The potential
mechanisms are through lipid peroxidation, induction
of pro-inflammatory cytokines and induction of FAS
ligand. The plasma and mitochondrial membranes are
vulnerable to free radical damage, and ROS-induced
lipid peroxidation of membranes can cause cell necrosis
or apoptosis. Lipid peroxidation can also cause immuno-
logical dysfunction, which could lead to hepatic fibro-
genesis. It is possible that, as a result of lipid peroxid-
ation, there is release of MDA (malondialdehyde) and
4-HNE (4-hydroxynonenal), which bind hepatocyte
proteins forming new antigen(s) and, thereby, provoking
a harmful immunological response. An immunological
response could induce neutrophil chemotaxis and activate
hepatic stellate cells to promote collagen synthesis and
deposition [184,185].
Oxidative stress (ROS) has also been shown to
increase production of pro-inflammatory cytokines

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[TNF-α, TGF-α, IL-6 and IL-8] via the activation of
NF-κB [186]. NEFAs accumulate in the hepatocytes in
NAFLD and are also capable of activating the NF-κB
pro-inflammatory pathway [166,187].
In addition to the NF-κB-dependent pathways,
Kupffer cells (liver-specific activated macrophages) are a
source of inflammatory cytokines and, in an obese indi-
vidual, adipose tissue with infiltrating macrophages is also
a source of pro-inflammatory cytokines. Because of the
increased production of pro-inflammatory cytokines in
NAFLD, these cytokines (e.g. TNF-α) could exacerbate
systemic and hepatic insulin resistance with worsening
inflammation and fibrosis. Inflammatory cytokines
cause impaired insulin signalling, cell damage, neutrophil
chemotaxis, hepatic stellate cell activation and apoptosis
[188–190], and there is increased expression of TNF-α
and its receptor in liver and adipose tissue in obese
individuals with NASH, which correlates with the
degree of fibrosis [191].
Under physiological conditions, ketogenesis is ex-
quisitely sensitive to insulin but, paradoxically, mito-
chondrial fat oxidation and ketogenesis are increased
in NAFLD with hyperinsulinaemia [192,193]. Even
obese leptin (ob/ob)-deficient mice with fatty liver also
have increased fat oxidation [194], perhaps explained
by increased concentrations of long-chain fatty acids
activating PPAR-α, a nuclear receptor and a positive
transcription factor for the CPT-1, MCAD (medium-
chain acyl-CoA dehydrogenase) and HMG (3-hydroxy-
3-methylglutaryl)-CoA synthetase genes. Some investig-
ators have postulated that long-chain fatty-acid-mediated
PPAR-α activation is specific to fatty acids from the
de novo pathway [95], and there is increased expression
of PPAR-α in ob/ob mice with steatosis [195]. Long-
chain fatty acids increase the activity and expression of
CPT-1 and, thereby, increase fat oxidation [196]. Addi-
tionally, there is a decreased affinity of CPT-1 activity for
malonyl-CoA, a potent inhibitor of CPT-1 [197].
Activation of AMPK also enhances mitochondrial fat
oxidation via phosphorylation (inactivation) of ACC.
As a result, there is a reduced conversion of acetyl-CoA
into malonyl-CoA and, therefore, decreased inhibition
of CPT-1 by malonyl-CoA, resulting in increased
β-oxidation [198].
In mitochondria oxidative phosphorylation, NADH
and FADH2 are re-oxidized to NAD+ and FAD via
the electron transport chain. As part of oxidative phos-
phorylation, this is a co-ordinated flow of electrons down
an electrochemical gradient from complex I to IV with
simultaneous extrusion of protons, creating a large elec-
trochemical gradient. Consequently, a large gradient
across the mitochondrial membrane drives ATPase syn-
thase resulting in ATP production. Under physiological
conditions, most of the electrons provided to the
respiratory chain ‘transit’ through to the last complex,
where they combine with oxygen and protons to

form water. However, upstream of the production of
water, electrons react with oxygen to form free radicals
(superoxide anions). These radicals are dismutated by
SOD (superoxide dismutase) to H2 O2 and then to
water by glutathione peroxidase. Glutathione peroxidase
requires GSH to function, and decreased GSH may
be accompanied by mitochondrial dysfunction and/or
cell death [199]. In pathological conditions, it is
plausible that increased free radical production results in
further mitochondrial damage and dysfunction, with a
consequent decrease in oxidative capacity. A decrease in
mitochondrial oxidative capacity could potentially result
in an imbalance between fat oxidation and lipogenesis,
leading to hepatic fat accumulation and NAFLD.
‘DEVELOPMENTAL ORIGINS’: ALTERED
EARLY DEVELOPMENT IN THE
PATHOGENESIS OF NAFLD
Our group has published extensively in recent years
showing that modification of intra-uterine nutrition
during gestation affects epigenetic regulation of key meta-
bolic genes and has the potential to modify the disease
phenotype [200–208]. Specifically, our group has shown
for the first time that prenatal nutrition induces differen-
tial changes to the methylation of individual CpG dinuc-
leotides in the hepatic PPAR-γ promoter, altering mRNA
levels of the PPAR-α gene, downstream target genes and
phenotype [202]. We have recently developed a mouse
model of human NAFLD which shows that exposure to
a high-fat diet in utero and during lactation exacerbates
the NAFLD phenotype exhibited in offspring that were
also fed a high-fat diet post-weaning (K. D. Bruce,
F. Cagampang and C. D. Byrne, unpublished work).
Thus with our findings showing an influence of intra-
uterine nutritional exposure to affect the risk of the
NAFLD phenotype we suggest that intra-uterine nutri-
tion may have the potential to modify hepatic lipogenesis
through an influence of PPAR-γ and modifying SREBP-
1c function. We speculate that increased dietary expo-
sure to a high-fat diet in the pregnant mother results in
increased placental transfer of fatty acids to the fetus.
It is possible that increased placental transfer of fatty
acids to the fetus increases hepatic lipogenesis and
oxidative stress in the vulnerable fetal liver, perhaps also
increasing the demand for methyl donors to promote cell
growth and the subsequent development of NAFLD in
adulthood. It is conceivable that, if the increased demand
for methyl donors is not met, epigenetic changes in gene
expression would result that predispose to conditions of
fat accumulation and inflammation (such as NAFLD) in
the developing offspring.
The mitochondrial genome is particularly susceptible
to oxidative damage due to the absence of protective
histones and incomplete repair mechanisms in mitochon-
Metabolic disturbances in non-alcoholic fatty liver disease 555
dria [209]. Mitochondria and mtDNA are attractive
vectors for the transmission of early environmental stress,
as they can harbour environmental damage for many cell
divisions before cell dysfunction becomes evident [210].
Mitochondria depend on both nuclear and mtDNA to
function. mtDNA is sensitive to environmental changes
during oocyte maturation and during perimplantation
development [211]. Therefore mitochondrial dysfunction
caused by changes in mtDNA or altered expression of
nuclear genes could cause both immediate and delayed
increases in oxidant stress which may alter nuclear gene
expression and cell function.
As mentioned above, we have developed a mouse
model showing that manipulating the maternal dietary
exposure during pregnancy worsens development of the
adult offspring NAFLD phenotype. We have obtained
results from this model of the developmental origins of
NAFLD of decreased mitochondrial complex activity.
To investigate the effect on the offspring of manipulating
the maternal diet during pregnancy, we have determined
whether there were changes in electron transport chain
enzyme complex activity in the liver from mouse
offspring in adulthood which had been exposed to a
high-fat or to a control diet both pre- and post-natally
(K. D. Bruce, F. Cagampang and C. D. Byrne, unpub-
lished work). Female C57 BL/6J black mice were
randomly assigned to either a high-fat diet (HF, 45 %
kJ from fat, 20 % kJ from protein and 35 % kJ from
carbohydrate; n = 10) or to a standard laboratory chow
diet (C, 21 % kJ from fat, 18 % kJ from protein and 63 %
kJ from carbohydrate; n = 10). Dams were fed the diets
for 4 weeks prior to conception, during gestation and
lactation. At weaning, the offspring were assigned either
the high-fat or control diets, generating four experimental
groups: HF/HF (n = 12), HF/C (n = 12), C/HF (n = 12)
and C/C (n = 12), which represents the pre-natal/post-
natal diet respectively. In both male and female offspring,
body weights, total fat mass and systolic BP measure-
ments were significantly higher (P < 0.01) in offspring
from HF/HF compared with the C/C or the C/HF
groups. Hepatic electron transport chain complex activity
was decreased in offspring from HF/C and HF/HF
groups compared with the C/C group, and the greatest
decrease was observed in Complex I activity, where a
3.2- and 3.7-fold reduction in activity levels was seen
in HF/HF offspring (P < 0.05) and the HF/C offspring
(P < 0.05) respectively, compared with C/C offspring.
These findings suggests that the pre-natal exposure to a
high-fat diet impairs complex activity and mitochondrial
function. Steatosis was visible in liver sections (most
marked in the HF/HF animals (Figure 7), HF/HF Oil red
staining), and there was a marked inflammatory infiltrate
in sections from the HF/HF group (Figure 7, HF/HF
H&E staining) that is characteristic of human NASH.
Thus these preliminary results provide evidence to
suggest that exposure to a high-fat diet in utero combined

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556 C. D. Byrne and others
Figure 7 Liver histology of a mouse model of the ‘developmental origins’ of NAFLD
Sections were stained with either (upper panels) haematoxylin and eosin (H&E) or (lower panels) Oil Red. Magnification, ×40. C, control diet; HF, high-fat diet.
with post-natal high-fat exposure contributes to a more
florid disease progression of fatty liver to NASH.
Our group has also shown in human studies that
fatty liver is associated with increased NEFA supply to
the liver [28,212,213], and we have shown in our mouse
model that hepatic TAG is strongly correlated with CPT-
1 expression (r = − 0.55, P = 0.033) [214], which mediates
the transfer of long-chain fatty acyl-CoAs across the
mitochondrial membrane. CPT-1 is regulated by PPAR-α
activity and, thus, our finding that altered early nutrition
influences PPAR-α methylation [200,202] suggests that
altered early nutrition may influence the flux of fatty
acids into the mitochondria for β-oxidation. We suggest
that increased flux of fatty acids to the developing fetal
liver increases susceptibility of the liver to damage
(NAFLD) induced by a second post-natal exposure to a
high-fat diet.
CONCLUSIONS
Precise estimates for the prevalence of NAFLD (and
the different subgroups such as NASH) are difficult to
obtain, because of an unmet need for simple diagnostic
tests. Recent progress has been made in this field, and in
the near future it may be possible to use simple algorithms
to replace liver biopsy for the diagnosis of NAFLD. It
has been shown recently that NASH is associated with
CHD, and in Type 2 diabetes NAFLD may be associated
with microvascular disease. To date there are no licensed
treatments for NAFLD and the development of novel
therapies has been hampered by a poor understanding
of the molecular mechanisms contributing to NAFLD.

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This problem is now being addressed and there has
been a marked improvement in an understanding of
the pathogenesis of NAFLD over the last decade. It
is now clear that factors operating outwith the liver
(e.g. uncontrolled adipocyte lipolysis) have a powerful
impact to increase liver fat accumulation. There has been
a marked improvement in our understanding of the role
of key transcription factors and the importance of three
transcription factors SREBP-1c, LXR-α and ChREBP
operating in the liver to co-ordinate the regulation of
glycolysis and fatty acid synthesis. In the future, it
may prove possible to modify the function of these
transcription factors to ameliorate the development and
progression of NAFLD. At the present time, there is a
need to understand better the link between components
of TAG synthesis and inflammation, as decreasing factors
contributing to inflammation are likely to attenuate the
development of fibrosis and progression of disease. Work
from our laboratory suggests that factors affecting early
fetal liver development, such as increased maternal dietary
fat consumption, may act to increase the vulnerability of
developing fetal liver to fat accumulation in adulthood. If
the rapid progress in understanding of the pathogenesis of
NAFLD is maintained, it is likely that we will have novel
treatments for NAFLD and the tools for monitoring ther-
apy without resorting to liver biopsy in the near future.
ACKNOWLEDGEMENTS
We thank Lucinda England for her help with the
manuscript.

FUNDING
The unpublished work relating to Figure 7 was supported
by the Biotechnology and Biological Sciences Research
Council [grant number BB/D001633/1].
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Received 27 June 2008/27 August 2008; accepted 7 October 2008
Published on the Internet 2 March 2009, doi:10.1042/CS20080253