International Journal of Cardiology 179 (2015) 360369

Contents lists available at ScienceDirect

International Journal of Cardiology

journal homepage: www.elsevier.com/locate/ijcard

Effects of pioglitazone on cardiac and adipose tissue pathology in rats

with metabolic syndrome
Natsumi Matsuura a,1, Chiharu Asano b,1, Kai Nagasawa a,1, Shogo Ito a,1, Yusuke Sano a,1, Yuji Minagawa b,1,
Yuichiro Yamada a,1, Takuya Hattori a,1, Shogo Watanabe a,1, Toyoaki Murohara c,1, Kohzo Nagata a,,1
a
b
c

Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan
Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan
Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

a r t i c l e

i n f o

Article history:
Received 8 August 2014
Received in revised form 31 October 2014
Accepted 10 November 2014
Available online 13 November 2014
Keywords:
Metabolic syndrome
Thiazolidinedione
Cardiac remodeling
Inammation
Adipose tissue
Glucose metabolism

a b s t r a c t
Background: Pioglitazone is a thiazolidinedione drug that acts as an insulin sensitizer. We recently characterized
DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as a new
animal model of metabolic syndrome. We have now investigated the effects of pioglitazone on cardiac and adipose tissue pathology in this model.
Methods and results: DS/obese rats were treated with pioglitazone (2.5 mg/kg per day, per os) from 9 to 13 weeks
of age. Age-matched homozygous lean (DahlS.Z-Lepr+/Lepr+, or DS/lean) littermates served as controls. Pioglitazone increased body weight and food intake in DS/obese rats. It also ameliorated left ventricular (LV) hypertrophy, brosis, and diastolic dysfunction as well as attenuated cardiac oxidative stress and inammation, without
lowering blood pressure, in DS/obese rats, but it had no effect on these parameters in DS/lean rats. In addition,
pioglitazone increased visceral and subcutaneous fat mass but alleviated adipocyte hypertrophy and inammation in visceral adipose tissue in DS/obese rats. Furthermore, pioglitazone increased the serum concentration of
adiponectin, induced activation of AMP-activated protein kinase (AMPK) in the heart, as well as ameliorated glucose intolerance and insulin resistance in DS/obese rats.
Conclusions: Treatment of DS/obese rats with pioglitazone exacerbated obesity but attenuated LV hypertrophy,
brosis, and diastolic dysfunction, with these latter effects being associated with the activation of cardiac
AMPK signaling likely as a result of the stimulation of adiponectin secretion.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Thiazolidinediones are oral agents for the treatment of type II diabetes. These drugs increase the secretion of adiponectin in part through
activation of peroxisome proliferator-activated receptor (PPAR) in
adipocytes [1]. Adiponectin contributes to energy homeostasis by regulating glucose and lipid metabolism in adipose tissue as well as by sensitizing the liver and skeletal muscle to the effects of insulin [2]. PPAR
Acknowledgment of grant support: This study was supported by unrestricted research
grants from Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan), Ajinomoto Pharmaceuticals Co.,
Ltd. (Tokyo, Japan), Astellas Pharma Inc. (Tokyo, Japan), Mochida Pharmaceutical Co.,
Ltd. (Tokyo, Japan), Mitsubishi Tanabe Pharma Corporation (Osaka, Japan), Takeda
Pharmaceutical Company Limited (Osaka, Japan), Daiichi-Sankyo Company, Limited
(Tokyo, Japan) and Dr. Nagata (Nagoya University) as well as by Management Expenses
Grants from the Japanese government to Nagoya University.
Corresponding author at: Department of Pathophysiological Laboratory Sciences,
Nagoya University Graduate School of Medicine, 1-1-20 Daikominami, Higashi-ku,
Nagoya 461-8673, Japan.
E-mail address: nagata@met.nagoya-u.ac.jp (K. Nagata).
1
This author takes responsibility for all aspects of the reliability and freedom from bias
of the data presented and their discussed interpretation.

http://dx.doi.org/10.1016/j.ijcard.2014.11.099
0167-5273/ 2014 Elsevier Ireland Ltd. All rights reserved.

regulates the expression of several genes that affect insulin action.

Tumor necrosis factor- (TNF-), a proinammatory cytokine, has
been associated with insulin resistance [3] and attenuates insulin
signaling [4], and PPAR agonists have been shown to inhibit expression of the TNF- gene in adipose tissue of obese rodents [5]. In
addition, PPAR agonists markedly inhibit expression of the 11hydroxysteroid dehydrogenase type 1 (11-HSD1) gene in adipose tissue [6]. This enzyme, which is especially abundant in adipocytes and hepatocytes, converts inactive cortisone to active cortisol and is an
important determinant of metabolic indices including insulin resistance
[7]. White adipose tissue is specialized for the storage of excess energy,
whereas brown adipose tissue is specialized for dissipation of chemical
energy as heat. In addition, white adipose tissue can acquire characteristics of brown adipose tissue, such as expression of brown fat-specic
and thermogenic genes, in a process known as browning [8]. PPAR
agonists were recently shown to induce a brown fat gene program in
subcutaneous white adipose tissue through stabilization of the transcriptional coregulator PRDM16 [9].
We previously showed that chronic administration of the thiazolidinedione pioglitazone attenuated left ventricular (LV) hypertrophy,

N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

brosis, and diastolic dysfunction as well as inhibited phosphorylation of

mammalian target of rapamycin (mTOR) and p70 S6 kinase in the heart,
without lowering blood pressure, in Dahl salt-sensitive (DS) rats [10].
These benecial cardiac effects of pioglitazone were attributed in part to
the activation of AMP-activated protein kinase (AMPK) signaling through
stimulation of adiponectin secretion as well as to the inhibition of Akt signaling. In addition, the mild insulin resistance apparent in DS rats was
ameliorated by pioglitazone treatment. The effects of pioglitazone on cardiac injury associated with obesity or metabolic syndrome (MetS) have
remained unclear, however.
We recently established a new animal model of MetS, the DahlS.ZLeprfa/Leprfa (DS/obese) rat, by crossing DS rats with Zucker rats harboring a missense mutation in the leptin receptor gene (Lepr) [11]. When
fed a normal diet, DS/obese rats develop a phenotype, including hypertension, that resembles MetS in humans. They also develop LV hypertrophy, brosis, and diastolic dysfunction, and these changes are associated
with increased cardiac oxidative stress and inammation as well as with
activation of the cardiac reninangiotensinaldosterone system (RAAS)
[12]. We have now investigated the effects of pioglitazone on cardiac
and adipose tissue pathology as well as on glucose metabolism in DS/
obese rats.
2. Methods
2.1. Animals and experimental protocols
Animal experiments were approved by the Animal Experiment Committee of Nagoya
University Graduate School of Medicine (Daiko district, approval nos. 024-032, 025-014,
and 026-017). Eight-week-old male inbred DS/obese rats were obtained from Japan SLC
(Hamamatsu, Japan) and were handled in accordance with the guidelines of Nagoya University Graduate School of Medicine as well as with the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health publication no. 85-23, revised 1996).
The animals were fed normal laboratory chow containing 0.36% NaCl, and both the diet
and tap water were provided ad libitum throughout the experimental period. DS/obese
rats were administered pioglitazone (2.5 mg per kilogram of body weight; MetS + PIO
group) or vehicle (MetS group) orally once a day via a gastric tube from 9 to 13 weeks
of age and were compared with similarly treated (CONT + PIO and CONT groups) homozygous lean (DahlS.Z-Lepr+/Lepr+, or DS/lean) littermates. The dose of pioglitazone was
determined on the basis of our previous study [10]. Body weight as well as food and
water intake were measured weekly. For an oral glucose tolerance test (OGTT), rats
were deprived of food overnight and administered glucose (2 g/kg) orally early the next
morning. Blood glucose concentrations were measured with the use of a glucose analyzer
(Glutest Neo Super; Sanwa Kagaku Kenkyusho, Nagoya, Japan). At 13 weeks of age,
rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine
(10 mg/kg) for echocardiographic and hemodynamic analyses. They were subsequently
killed by intraperitoneal injection of an overdose of sodium pentobarbital (50 mg/kg),
and the heart, liver, kidneys, and both visceral (retroperitoneal and epididymal) and subcutaneous (inguinal) fat tissue were removed and weighed. LV tissue was also separated
for analysis.

2.2. Echocardiographic and hemodynamic analyses

Systolic blood pressure (SBP) and heart rate were measured weekly in conscious
animals by tail-cuff plethysmography (BP-98A; Softron, Tokyo, Japan). At 13 weeks of
age, rats were subjected to transthoracic echocardiography, as described previously [13].
M-mode echocardiography was performed with a 12.5-MHz transducer (Xario SSA660A; Toshiba Medical Systems, Tochigi, Japan). LV end-diastolic (LVDd) and endsystolic (LVDs) dimensions as well as the thickness of the interventricular septum
(IVST) and LV posterior wall (LVPWT) were measured, and LV fractional shortening
(LVFS), relative wall thickness (RWT), and LV mass were calculated as follows: LVFS
(%) = [(LVDd LVDs) / LVDd] 100; RWT = (IVST + LVPWT) / LVDd; LV mass (g) =
{1.04 [(IVST + LVDd + LVPWT)3 (LVDd)3] 0.8} + 0.14. LV ejection fraction
(LVEF) was calculated with the formula of Teichholz [14]. For the assessment of
Doppler-derived indices of LV function, both LV inow and outow velocity patterns
were simultaneously recorded by pulsed-wave Doppler echocardiography. For the assessment of LV diastolic function, we calculated the peak ow velocities at the mitral level during rapid lling (E) and during atrial contraction (A), the E/A ratio, the deceleration time
(DcT), and the isovolumic relaxation time (IRT). After echocardiography, a 2F
micromanometer-tipped catheter (SPR-407; Millar Instruments, Houston, TX, USA) that
had been calibrated relative to atmospheric pressure was inserted through the right carotid artery into the left ventricle [10]. Tracings of LV pressure and the electrocardiogram
were digitized to determine LV end-diastolic pressure (LVEDP). The time constant of
isovolumic relaxation (tau) was calculated by the derivative method of Raff and Glantz,
as described previously [15].

361

2.3. Histology and immunohistochemistry

LV and visceral (retroperitoneal) fat tissue was xed in ice-cold 4% paraformaldehyde
for 48 h, embedded in parafn, and processed for histology, as described [10,16]. Transverse tissue sections were stained either with hematoxylineosin for routine histological
examination or with AzanMallory solution for evaluation of brosis. For evaluation of
macrophage inltration into the LV myocardium and adipose tissue, parafn-embedded
tissue sections (thickness, 3 m) were subjected to immunostaining for the monocytemacrophage marker CD68, as described previously [16]. In brief, endogenous peroxidase
activity was blocked by exposure of the sections to methanol containing 0.3% hydrogen
peroxide, after which the sections were incubated at 4 C rst overnight with mouse
monoclonal antibodies to CD68 (clone ED1, diluted 1:100; Chemicon, Temecula, CA,
USA) and then for 30 min with Histone Simple Stain Rat MAX PO (Nichirei Biosciences,
Tokyo, Japan). Immune complexes were visualized with diaminobenzidine and hydrogen
peroxide, and the sections were counterstained with hematoxylin. All image analyses
were performed with the use of NIH Scion Image software (Scion, Frederick, MD, USA).
2.4. Biochemical analysis
Blood was collected from the right carotid artery of rats that had been deprived of food
overnight and then anesthetized by intraperitoneal injection of sodium pentobarbital (50
mg/kg). The blood was centrifuged at 1400 g for 10 min at 4 C, and the resultant serum
or plasma supernatants were maintained frozen at 80 C until analysis. The concentrations of insulin (Morinaga Bioscience Institute, Yokohama, Japan) and adiponectin (Otsuka
Pharmaceutical, Tokyo, Japan) in serum were measured with the use of mouse/rat
enzyme-linked immunosorbent assay kits. The homeostasis model assessment of insulin
resistance (HOMA-IR) and homeostasis model assessment of -cell function (HOMA-)
indices, which predict insulin sensitivity and the function of pancreatic -cells, respectively, were calculated from the serum glucose and serum insulin concentrations according to
the empirical formulae: HOMA-IR = fasting insulin (U/mL) fasting glucose (mmol/L)/
22.5; HOMA- = [20 fasting insulin (U/mL)] / [fasting glucose (mmol/L) 3.5] [17,18].
The concentration of corticosterone in plasma was measured with the use of an enzymelinked immunosorbent assay kit (Assaypro, St. Charles, MO, USA).
2.5. Superoxide production
Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production by homogenates prepared from freshly frozen LV tissue was measured with the
use of an assay based on lucigenin-enhanced chemiluminescence as described previously
[19]. The chemiluminescence signal was sampled every minute for 10 min with a microplate reader (WALLAC 1420 ARVO MX/Light; Perkin Elmer, Waltham, MA, USA), and the
respective background counts were subtracted from experimental values. Lucigenin
chemiluminescence was expressed as relative light units per minute per milligram of
protein. Superoxide production in tissue sections was examined by staining with
dihydroethidium (Sigma, St. Louis, MO, USA) as described [20]. Dihydroethidium is rapidly
oxidized by superoxide to yield uorescent ethidium, and the sections were examined
with a uorescence microscope equipped with a 585-nm long-pass lter. As a negative
control, we performed staining with dihydroethidium after incubation of sections with superoxide dismutase (300 U/mL), and we conrmed that this procedure abolished the uorescence (data not shown). The average of dihydroethidium uorescence intensity values
was calculated with the use of NIH Image (ImageJ) software [21].
2.6. Quantitative RT-PCR analysis
Total RNA was extracted from LV and visceral (retroperitoneal) fat tissue and treated
with DNase with the use of an SV Total RNA Isolation System (Promega, Madison, WI,
USA) or an RNeasy Lipid Tissue Midi Kit (Qiagen, Hilden, Germany), respectively. Portions
of the RNA (2 g) were subjected to reverse transcription (RT) with the use of a
PrimerScript RT Reagent Kit (Takara, Shiga, Japan). Quantitative PCR analysis was performed with the use of SYBR Mix Ex Taq II (Takara), a Thermal Cycler Dice Real Time System II (Takara), and specic primers for cDNAs encoding atrial natriuretic peptide (ANP)
[13], brain natriuretic peptide (BNP) [13], collagen type I or type III [22], connective tissue
growth factor (CTGF) [19], transforming growth factor-1 (TGF-1) [13], monocyte
chemoattractant protein-1 (MCP-1) [19], osteopontin [19], cyclooxygenase-2 (COX-2)
[23], TNF- [12], angiotensin-converting enzyme (ACE) [13], the mineralocorticoid receptor (MR) [13], the glucocorticoid receptor (GR) (5 -GGAAAAGCCATCGTCAAAAGG-3 and
5 -TTCTCAACCACCTCATGCATG-3 as forward and reverse primers, respectively; GenBank
accession no. NM_012576), 11-HSD1 (5 -CTCTGCTCACTATATTGC-3 and 5 -GTCTGTCT
CATGTCATTG-3 as forward and reverse primers, respectively; GenBank accession no.
NM_017080), and the p22phox [24], gp91phox [24], and Rac1 [12] subunits of NADPH oxidase. Reagents for detection of human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA (Applied Biosystems, Foster City, CA, USA) were used to quantify rat
GAPDH mRNA as an internal standard.
2.7. Immunoblot analysis
LV tissue was homogenized on ice for 1 min in cell lysis buffer (20 mM TrisHCl [pH
8.0], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, and EDTA-free protease
inhibitors [Roche Diagnostics, Mannheim, Germany]), and the homogenates were centrifuged at 20,000 g for 10 min at 4 C. Equal amounts of protein from the resulting

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

supernatants were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene diuoride membrane, as described previously [25]. The membrane was incubated rst with a 1:1000 dilution of rabbit
polyclonal antibodies to total or Thr172-phosphorylated forms of -AMPK (Cell Signaling
Technology, Beverly, MA, USA) and then with a 1:8000 dilution of horseradish peroxidase-conjugated goat antibodies to rabbit immunoglobulin G (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Detection and quantication of immune complexes were
performed as described [25].

2.8. Statistical analysis

Data are presented as means SEM. Differences among groups of rats at 13 weeks of
age were assessed by one-way factorial analysis of variance (ANOVA); if a signicant difference was detected, intergroup comparisons were performed with Fisher's multiplecomparison test. The time courses of body weight, food intake, and SBP as well as OGTT
curves were compared among groups by two-way repeated-measures ANOVA. The numbers of adipocytes in retroperitoneal adipose tissue were compared with Pearson's chisquare test. The independent or interactive inuence of rat genotype and pioglitazone
treatment on various parameters in the four experimental groups was evaluated with
two-way factorial ANOVA. A P value of b0.05 was considered statistically signicant.

3. Results
3.1. Physiological analysis, LV geometry, and cardiac function
Both body weight and food intake were increased in the MetS group
compared with the CONT group at 9 weeks of age and thereafter, and
these parameters were further increased in the MetS + PIO group
(Fig. 1A, B). SBP was signicantly increased in the MetS group compared
with the CONT group, but it was not signicantly altered by pioglitazone
treatment in either mouse genotype (Fig. 1C). At 13 weeks of age, the
ratio of LV weight to tibial length, an index of LV hypertrophy, was increased in the MetS group compared with the CONT group, and this effect was attenuated in the MetS + PIO group (Table 1). The ratios of
liver and kidney weight to tibial length were also increased in the
MetS group, and these effects were also attenuated by pioglitazone
treatment (Table 1). Whereas the ratios of visceral (retroperitoneal
and epididymal) and subcutaneous (inguinal) fat mass to tibial length

were increased in the MetS group, these effects were even more pronounced in the MetS + PIO group (Table 1).
The fasting serum glucose concentration was similar in the four experimental groups. However, the fasting serum insulin level was significantly higher in the MetS group than in the CONT group, and this
difference was attenuated by pioglitazone treatment in DS/obese rats
(Table 1). Both HOMA-IR and HOMA- were also signicantly increased
in the MetS group in a manner sensitive to pioglitazone treatment.
Moreover, an OGTT revealed that pioglitazone normalized the impaired
glucose tolerance apparent in the MetS group (Fig. 1D). The serum
adiponectin concentration was signicantly higher in the MetS group
than in the CONT group, and pioglitazone further increased this parameter in both DS/obese and DS/lean rats. The plasma corticosterone concentration was signicantly lower in the MetS group than in the CONT
group or in the MetS + PIO group (Table 1).
Echocardiography revealed that IVST, LVPWT, LVFS, LVEF, LV mass,
and RWT were signicantly increased in the MetS group compared
with the CONT group in a manner sensitive to pioglitazone treatment
(Table 2). The E/A ratio was signicantly decreased whereas DcT, IRT,
and tau were signicantly prolonged in the MetS group compared
with the CONT group, and both LVEDP and the ratio of LVEDP to LVDd
were also increased in the MetS group. All of these changes were attenuated in the MetS + PIO group compared with the MetS group
(Table 2). These data thus indicated that treatment with pioglitazone
ameliorated LV remodeling, preserved LV systolic function, and attenuated LV diastolic dysfunction in DS/obese rats.

3.2. Cardiomyocyte hypertrophy as well as cardiac brosis and gene

expression
Microscopic analysis revealed that the cross-sectional area of cardiac
myocytes was increased in the MetS group compared with the CONT
group and that this increase was attenuated in the MetS + PIO group
(Fig. 2A, B). Hemodynamic overload also resulted in signicant upregulation of the expression of ANP and BNP genes in the heart of DS/

B
600

Body weight (g)

500

400
300
200

10

CONT

CONT+PIO

MetS

MetS+PIO

11

12

Age (weeks)

Food intake (g/day)

50

13

10

160

*
*

150
100
50
0

10

CONT

CONT+PIO

MetS

MetS+PIO

11

12

Age (weeks)

13

Blood glucose (mg/dL)

200

20

D
250

30

SBP (mmHg)

40

10

CONT

CONT+PIO

MetS

MetS+PIO

11

12

Age (weeks)

13

OGTT
*

120
80

40
0

15

CONT

CONT+PIO

MetS

MetS+PIO
30

60

Time (min)

120

Fig. 1. Changes in body weight (A), food intake (B), and SBP (C) with age as well as time courses for an OGTT performed at 13 weeks of age (D) for rats in the four experimental groups. Data
are means SEM (n = 11 animals for each group). *P b 0.05 vs. CONT; P b 0.05 vs. CONT + PIO; P b 0.05 vs. MetS.

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

Table 1
Physiological and metabolic parameters for rats in the four experimental groups at 13 weeks of age.
Parameter

CONT

Body weight (g)

Tibial length (mm)
Food intake (g/day)
SBP (mm Hg)
Heart rate (beats/min)
Heart weight/tibial length (mg/mm)
LV weight/tibial length (mg/mm)
Liver weight/tibial length (mg/mm)
Kidney weight/tibial length (mg/mm)
Retroperitoneal fat weight/tibial length (mg/mm)
Epididymal fat weight/tibial length (mg/mm)
Inguinal fat weight/tibial length (mg/mm)
Fasting serum glucose (mg/dL)
Fasting serum insulin (ng/dL)
HOMA-IR
HOMA-
Serum adiponectin (g/mL)
Plasma corticosterone (ng/mL)

387.7
38.2
22.2
148.7
400.8
29.65
21.34
280.2
69.4
103.8
122.1
203.1
124.8
0.27
2.27
42.6
4.34
895.8

7.2
0.2
0.8
2.3
2.3
0.44
0.37
5.7
1.2
5.3
4.5
7.3
5.6
0.05
0.53
9.6
0.12
92.8

CONT + PIO

MetS

380.8
37.9
21.8
144.6
410.8
29.44
21.38
257.1
67.7
106.9
123.1
227.2
119.8
0.24
1.61
34.6
7.02
758.0

497.1
35.0
25.3
194.4
357.3
36.53
28.03
565.3
105.3
433.9
327.1
894.67
149.8
4.44
43.45
699.0
8.16
547.7

3.7
0.2
0.7
3.1
6.8
0.53
0.34
3.4
1.9
5.1
4.5
8.4
2.9
0.05
0.47
11.1
0.17
78.4

MetS + PIO

6.8
0.2
2.4
6.1
12.1
0.73
0.52
16.0
3.8
10.6
9.1
19.3
26.9
0.67
7.86
213.1
0.22
90.2

552.0
34.5
37.3
182.8
369.2
36.95
26.61
514.8
97.5
582.4
435.5
964.0
148.0
1.58
20.89
238.9
10.08
834.5

9.0
0.6
1.2
8.1
11.4
0.57
0.30
18.8
3.2
16.4
12.7
33.9
26.4
0.25
8.40
47.3
0.36
92.1

Data are means SEM (n = 11 animals for each group).

P b 0.05 vs. CONT.

P b 0.05 vs. CONT + PIO.

P b 0.05 vs. MetS.

obese rats, and such up-regulation was attenuated by pioglitazone

(Fig. 2C, D).
AzanMallory staining revealed that brosis in perivascular and interstitial regions of the LV myocardium was increased in the MetS
group compared with the CONT group and that this increase was attenuated in the MetS + PIO group (Fig. 2EH). The abundance of collagen
types I and III mRNAs as well as the amounts of CTGF and TGF-1
mRNAs, which correlate with cardiac brosis and growth [26], were
also up-regulated in the MetS group in a manner sensitive to pioglitazone treatment (Fig. 2IL).
3.3. Cardiac oxidative stress and AMPK activation
Superoxide production in myocardial tissue sections, as revealed by
staining with dihydroethidium, as well as the activity of NADPH oxidase
in LV homogenates were signicantly increased in the MetS group compared with the CONT group (Fig. 3AC). The expression of genes for the
p22phox and gp91phox membrane components and for the Rac1 cytosolic
component of NADPH oxidase in the heart was also up-regulated in the
MetS group compared with the CONT group (Fig. 3DF). Pioglitazone
treatment attenuated all of these effects in DS/obese rats. The amount
of phosphorylated (activated) AMPK in the left ventricle was decreased

in the MetS group compared with the CONT group, and pioglitazone
treatment increased the level of AMPK phosphorylation in both DS/
lean and DS/obese rats (Fig. 3G).

3.4. Cardiac inammation

Immunostaining for the monocyte-macrophage marker CD68 revealed that the extent of macrophage inltration in the LV myocardium
was increased in the MetS group compared with the CONT group and
that this inltration was attenuated by pioglitazone (Fig. 4A, B). The expression of MCP-1, TNF-, and osteopontin genes in the left ventricle
was also up-regulated in the MetS group compared with the CONT
group in a manner sensitive to pioglitazone treatment (Fig. 4CE).

3.5. Cardiac RAAS and glucocorticoid-related gene expression

The amounts of mRNAs for ACE, MR, 11-HSD1, and GR in the left
ventricle were increased in the MetS group compared with the CONT
group, and these effects were attenuated in the MetS + PIO group
(Fig. 4FI).

Table 2
Cardiac morphological and functional parameters for rats in the four experimental groups at 13 weeks of age.
Parameter

CONT

IVST (mm)
LVPWT (mm)
LVDd (mm)
LVDs (mm)
LVFS (%)
LVEF (%)
LV mass (mg)
RWT
E/A
DcT (ms)
IRT (ms)
Tau (ms)
LVEDP (mm Hg)
LVEDP/LVDd (mm Hg/mm)

1.80
1.77
7.28
3.99
45.48
81.18
882.3
0.49
1.90
53.58
36.84
24.36
2.84
0.40

Data are means SEM (n = 11 animals per group).

P b 0.05 vs. CONT.

P b 0.05 vs. CONT + PIO.

P b 0.05 vs. MetS.

0.02
0.03
0.12
0.14
1.04
1.39
15.1
0.01
0.09
1.47
2.24
1.76
0.72
0.10

CONT + PIO

MetS

1.81
1.80
7.57
4.48
40.83
76.63
943.2
0.48
1.99
54.56
36.51
26.06
3.59
0.47

2.14
2.11
7.63
3.66
53.06
87.62
1187.9
0.56
1.63
61.45
48.49
32.67
9.21
1.10

0.02
0.03
0.07
0.09
0.99
1.14
18.6
0.01
0.11
1.46
2.83
0.62
0.62
0.09

MetS + PIO

0.02
0.04
0.19
0.18
1.57
1.02
26.2
0.02
0.11
1.99
4.37
1.55
0.51
0.02

2.02
1.97
7.78
4.04
44.89
80.57
1082.3
0.42
1.92
52.02
29.7
25.88
7.26
0.86

0.04
0.03
0.21
0.23
1.81
1.79
50.6
0.01
0.12
1.67
2.65
1.59
1.17
0.14

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

CONT+PIO

ANP/GAPDH mRNA

CONT

CONT
+PIO

MetS

CONT

CONT+PIO

CONT

CONT+PIO

CONT
+PIO

MetS+PIO

MetS

1.0

CONT

CONT
+PIO

MetS

Collagen type /
GAPDH mRNA

Collagen type I/
GAPDH mRNA

*
3

*
2

CONT CONT MetS MetS

+PIO
+PIO

CONT CONT MetS MetS

+PIO
+PIO

CTGF/GAPDH mRNA

MetS
+PIO
8

MetS+PIO
0.0

MetS
+PIO

0.5

MetS

MetS

MetS
+PIO

CONT

BNP/GAPDH mRNA

400

MetS+PIO

800

CONT CONT MetS MetS

+PIO
+PIO

CONT CONT MetS MetS

+PIO
+PIO

TGF-1/GAPDH mRNA

MetS

Perivascular fibrosis

CONT

1200

Interstitial fibrosis (%)

Myocyte cross-sectional
area (m2)

CONT CONT MetS MetS

+PIO
+PIO

*
2

CONT CONT MetS MetS

+PIO
+PIO

Fig. 2. Cardiomyocyte size, expression of fetal-type cardiac genes, as well as cardiac brosis and brosis-related gene expression in rats of the four experimental groups at 13 weeks of age.
(A) Hematoxylineosin staining of transverse sections of the LV myocardium. Scale bars, 100 m. (B) Cross-sectional area of cardiac myocytes determined from sections similar to those in
(A). (C, D) Quantitative RT-PCR analysis of ANP and BNP mRNAs, respectively. The amount of each mRNA was normalized by that of GAPDH mRNA and then expressed relative to the mean
value for the CONT group. (E, F) Collagen deposition as revealed by AzanMallory staining in perivascular and interstitial regions of the LV myocardium, respectively. Scale bars, 100 m.
(G, H) Relative extents of perivascular and interstitial brosis, respectively, in the LV myocardium as determined from sections similar to those in (E) and (F). (I L) Quantitative RT-PCR
analysis of collagen type I, collagen type III, CTGF, and TGF-1 mRNAs, respectively. The amount of each mRNA was normalized by that of GAPDH mRNA and then expressed relative to
the mean value for the CONT group. All quantitative data are means SEM (n = 11 animals per group). *P b 0.05 vs. CONT; P b 0.05 vs. CONT + PIO; P b 0.05 vs. MetS.

3.6. Adipocyte hypertrophy as well as adipose tissue inammation and gene

expression
The cross-sectional area of retroperitoneal fat cells was increased in
the MetS group compared with the CONT group, and this effect was

attenuated in the MetS + PIO group (Fig. 5A, B). The number of small
adipocytes was also increased, whereas that of large adipocytes was reduced (P b 0.0001), in the MetS + PIO group compared with the MetS
group (Fig. 5C, Table 3). Pioglitazone did not affect the number of
small or large adipocytes in retroperitoneal adipose tissue of DS/lean

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

MetS

E3

CONT CONT
+PIO

CONT CONT MetS

+PIO

MetS
+PIO

CONT CONT MetS

+PIO

MetS

MetS+PIO

gp91phox/GAPDH mRNA

p22phox/GAPDH mRNA

D3

MetS
+PIO

100

80

60

40

20

MetS
+PIO

CONT CONT MetS

+PIO

MetS
+PIO

CONT CONT
+PIO
0.6

Relative AMPK phosphorylation

CONT+PIO

120

*
NADPH oxidase activity
(RLU/min per mg protein)

CONT

10

Rac1/GAPDH mRNA

B
Relative DHE fluorescence

MetS

MetS
+PIO

0.4

0.2

CONT

(Th172)

CONT MetS
+PIO

MetS
+PIO

p-AMPK
AMPK

Fig. 3. NADPH oxidase activity and gene expression as well as AMPK activation status in the left ventricle of rats in the four experimental groups at 13 weeks of age. (A) Superoxide production as revealed by dihydroethidium staining in interstitial regions of the LV myocardium. Scale bars, 100 m. (B) Relative dihydroethidium (DHE) uorescence intensity determined
from sections similar to those in (A). (C) NADPH-dependent superoxide production in LV homogenates. Results are expressed as relative light units (RLU) per minute per milligram of
protein. (D F) Quantitative RT-PCR analysis of p22phox, gp91phox, and Rac1 mRNAs, respectively. The amount of each mRNA was normalized by that of GAPDH mRNA and then expressed
relative to the mean value for the CONT group. (G) Immunoblot analysis of total and phosphorylated forms of AMPK in the left ventricle. Representative immunoblots and the ratio of
phosphorylated (p-) to total forms of AMPK are shown. All quantitative data are means SEM (n = 11 animals per group). *P b 0.05 vs. CONT; P b 0.05 vs. CONT + PIO; P b 0.05
vs. MetS.

rats (P = 0.32). Immunostaining of visceral fat with antibodies to CD68

revealed that the number of macrophages was increased in the MetS
group compared with the CONT group and that this effect was attenuated in the MetS + PIO group (Fig. 5A, D). The CD68-positive cells
surrounded adipocytes, forming a typical crownlike pattern, in the
MetS group (Fig. 5A). The expressions of MCP-1, TNF-, and COX-2
genes (Fig. 5EG) as well as that of 11-HSD1 and GR genes (Fig. 5H,
I) in retroperitoneal adipose tissue were also up-regulated in the MetS
group in a manner sensitive to pioglitazone treatment.

treatment increased body weight and fat mass but alleviated adipocyte
hypertrophy and inammation in visceral adipose tissue in these animals. Treatment of DS/obese rats with pioglitazone also increased the
serum concentration of adiponectin, induced activation (phosphorylation) of AMPK in the heart, as well as ameliorated glucose intolerance
and insulin resistance. To our knowledge, this is the rst to demonstrate
the benecial effect of pioglitazone on chronic inammation, a link between obesity and cardiovascular diseases.
4.1. Physiological actions of pioglitazone

3.7. Inuences of genotype and pioglitazone treatment

We also analyzed all the data of the study by two-way factorial
ANOVA in order to determine the inuences of rat genotype and pioglitazone treatment and the possible interaction of these factors (Supplementary Table 1). The results of this analysis were largely consistent
with those obtained by one-way factorial ANOVA that are presented
in the main tables and gures.
4. Discussion
We have here shown that chronic administration of the PPAR activator pioglitazone attenuated LV hypertrophy, brosis, and diastolic
dysfunction as well as LV oxidative stress and inammation, without
lowering blood pressure, in DS/obese rats. In addition, pioglitazone

The increase in body weight induced by pioglitazone in DS/obese

rats was accompanied by an increase in food intake, consistent with
previous results showing that pioglitazone increased food consumption
and body weight in Zucker fatty rats [27]. Insulin is thought to function
as a circulating satiety factor, with its ability to suppress feeding, stimulate thermogenesis, and induce weight loss being mediated by inhibition of neuropeptide Y expression in the arcuate nucleus [28,29]. The
serum concentration of insulin was markedly reduced by pioglitazone
in DS/obese rats, suggesting that this effect might contribute to overeating in these animals. The increase in the plasma corticosterone level induced by pioglitazone might also underlie this hyperphagia. A fall in
insulin and rise in corticosterone concentrations can explain most of
the changes in food intake and body weight apparent in hypoadrenal
or insulin-decient diabetic rats [30].

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

250

CD68-positive cells/mm2

200

CONT

150

CONT+PIO

100

TNF-/GAPDH mRNA

CONT CONT MetS

+PIO

CONT CONT MetS

+PIO

MetS
+PIO

8
6

CONT CONT MetS

+PIO

MetS

MetS
+PIO

2
0

MetS
+PIO

CONT CONT MetS

+PIO

MetS
+PIO

11-HSD1/
GAPDH mRNA

ACE/GAPDH mRNA

12
10

CONT CONT
+PIO

MetS
+PIO

CONT CONT MetS

+PIO

CONT CONT MetS

+PIO

MetS
+PIO

GR/GAPDH mRNA

MR/GAPDH mRNA

MCP-1/GAPDH mRNA

MetS+PIO

Osteopontin/
GAPDH mRNA

MetS

50

MetS
+PIO

CONT CONT MetS

+PIO

MetS
+PIO

Fig. 4. Macrophage inltration as well as cytokine, cardiac RAAS, and glucocorticoid-related gene expression in the left ventricle of rats in the four experimental groups at 13 weeks of age.
(A) Immunohistochemical analysis with antibodies to the monocyte-macrophage marker CD68. Scale bars, 100 m. (B) Density of CD68-positive cells determined from sections similar to
those in (A). (C I) Quantitative RT-PCR analysis of MCP-1, TNF-, osteopontin, ACE, MR, 11-HSD1, and GR mRNAs, respectively, in the left ventricle. The amount of each mRNA was normalized by that of GAPDH mRNA and then expressed relative to the mean value for the CONT group. All quantitative data are means SEM (n = 11 animals per group). *P b 0.05 vs. CONT;
P b 0.05 vs. CONT + PIO; P b 0.05 vs. MetS.

4.2. Effects of pioglitazone on dysfunctional adipose tissue

Obesity and MetS are associated with dysfunctional adipose tissue
composed of enlarged adipocytes and affected by chronic inammation
[31]. Macrophage accumulation in white adipose tissue plays a role in
the chronic inammatory response as a result of the secretion by
these cells of free fatty acids and inammatory molecules including
TNF- and MCP-1 [3,32,33]. We have now shown that pioglitazone increased visceral (retroperitoneal and epididymal) and subcutaneous
fat mass in DS/obese rats, ndings that appear to be inconsistent with
previous data showing that pioglitazone reduced visceral fat mass and
increased subcutaneous fat mass in Zucker fatty rats [27]. Pioglitazone
also attenuated adipocyte hypertrophy and macrophage accumulation
in visceral adipose tissue of DS/obese rats, effects that were accompanied by down-regulation of the expression of genes for proinammatory proteins such as MCP-1, TNF-, and COX-2 in visceral adipose tissue.
Moreover, pioglitazone increased the number of small adipocytes
and reduced the number of large adipocytes in white adipose tissue of
DS/obese rats, consistent with previous results obtained with the
thiazolidinedione troglitazone in Zucker fatty rats [34]. These observations suggest that pioglitazone might trigger apoptosis in enlarged adipocytesan effect that was previously shown to accompany the
troglitazone-induced decrease in the number of large adipocytes in
Zucker fatty rats [34]that it down-regulates inammatory cytokine

expression in adipose tissue, and that it increases the serum adiponectin

concentration possibly by inducing the differentiation of preadipocytes
into small fat cells [35].
4.3. Roles of cardiac AMPK activity and adiponectin
AMPK is a key regulator of energy metabolism and is activated by
cellular stressors that deplete ATP. The activation of AMPK by
adiponectin inhibits protein synthesis and LV hypertrophy by blocking
the mTOR-p70S6 kinase pathway [10]. Cardiac AMPK activity was decreased in DS/obese rats, consistent with previous results showing
that 2-AMPK deciency exacerbates pressure overload-induced LV
hypertrophy and dysfunction in mice [36]. Adiponectin inhibits hypertrophic signaling in the myocardium through activation of AMPK, and
adiponectin receptors were found to mediate AMPK activation by
adiponectin in cardiac myocytes in an in vitro model of hypertrophy
[37]. Adiponectin interactions with its receptors AdipoR1 and AdipoR2
are thought to mediate AMPK and p38 mitogen-activated protein kinase
activation, leading to enhanced glucose uptake and fatty acid oxidation,
in the heart [38]. Moreover, AMPK activity in the hypothalamus is
inhibited by increased glucose or insulin levels [39]. Pioglitazone was
previously shown to up-regulate AdipoR2 expression in 3T3-L1 adipocytes and to increase AMPK phosphorylation in insulin-stimulated
3T3-L1 adipocytes [40], suggesting that this drug increases insulin

367

10000
8000
6000
4000
2000
0

100

CD68-positive cells (%)

MetS+PIO

80

60
40
20

CONT CONT MetS

+PIO

MetS
+PIO

11-HSD1/
GAPDH mRNA

CONT CONT
+PIO

25

MetS

MetS
+PIO

20
15
10

5
0

CONT CONT
+PIO

MetS

MetS
+PIO

80
70
CONT

60

CONT+PIO

50

MetS

40

MetS+PIO

30
20
10

CONT CONT MetS

+PIO

MCP-1/GAPDH mRNA

MetS

90

10000

50

40
30

20
10
0

0
1000

MetS
+PIO

CONT CONT MetS

+PIO

MetS
+PIO

COX-2/GAPDH mRNA

CONT+PIO

12000

Adipocyte number

CONT

14000

TNF-/GAPDH mRNA

GR/GAPDH mRNA

Adipocyte cross-sectional
area (m2)

N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

Cell size (m2)

4

CONT CONT MetS

+PIO

MetS
+PIO

*
2

CONT CONT
+PIO

MetS

MetS
+PIO

Fig. 5. Macrophage inltration as well as inammatory and glucocorticoid-related gene expression in retroperitoneal adipose tissue of rats in the four experimental groups at 13 weeks of
age. (A) Immunohistochemical staining for the monocyte-macrophage marker CD68. Scale bars, 100 m. (B, C) Cross-sectional area of adipocytes and distribution of adipocyte size, respectively, determined from sections similar to those in (A). (D) The number of nuclei for CD68-positive cells as a percentage of total nuclei as determined from sections similar to those in (A).
(E I) Quantitative RT-PCR analysis of MCP-1, TNF-, COX-2, 11-HSD1, and GR mRNAs, respectively. The amount of each mRNA was normalized by that of GAPDH mRNA and then
expressed relative to the mean value for the CONT group. All quantitative data are means SEM (n = 11 animals per group). *P b 0.05 vs. CONT; P b 0.05 vs. CONT + PIO; P b 0.05
vs. MetS.

sensitivity in these cells, at least in part, via a PPAR-AdipoR2-AMPK

axis. In addition, pioglitazone is known to activate AMPK signaling in
adipocyte [41], and this mechanism may contribute to the observed
benecial effects. The serum adiponectin and insulin concentrations
were signicantly increased in DS/obese rats. Despite this increase in
serum adiponectin level, however, cardiac AMPK activity was reduced
in these animals. Treatment of DS/obese rats with pioglitazone further
Table 3
Morphometric analysis of retroperitoneal adipose tissue of rats in the four experimental
groups at 13 weeks of age.
Parameter

CONT

CONT + PIO

Adipocyte size (m2)

No. of small adipocytes (b3014 m2)
No. of large adipocytes (N3014 m2)

2984.7 84.2
94
71

3044.8 56.2
85
80

Parameter

MetS

MetS + PIO

increased the serum concentration of adiponectin and attenuated the

decrease in AMPK activity in the heart. Another study has shown that
insulin may inhibit AMPK signaling in the heart [42] and that activation
of AMPK signaling in human adipose tissue is one mechanism involved
in PPAR agonist-mediated restoration of insulin sensitivity [43]. Downregulation of AMPK activity in the heart may thus contribute to cardiac
remodeling and diastolic dysfunction in DS/obese rats, and the amelioration of cardiac injury by pioglitazone in these animals may be mediated through activation of AMPK in the heart.
Our nding that the circulating adiponectin concentration was signicantly increased in DS/obese rats is consistent with previous results
obtained with Zucker fatty rats [44], but it contrasts with the reduced
circulating levels of this adipose tissue-derived protein in humans
with obesity-related disorders [45]. We nevertheless found that pioglitazone further increased the serum concentration of adiponectin in DS/
obese rats, with this effect likely contributing to the amelioration of cardiac injury through activation of AMPK in the heart.

Adipocyte size (m )
11,968.5 314.9 10,189.9 315.6
No. of small adipocytes (b11,102 m2) 73
107
43
No. of large adipocytes (N11,102 m2) 89
Adipocytes were classied as small or large on the basis of the corresponding median
value for the CONT or MetS groups. Data for adipocyte size are means SEM (n = 11
animals per group).
P b 0.05 vs. CONT.

P b 0.05 vs. CONT + PIO.

P b 0.05 vs. MetS.

4.4. Cardiac inammation, oxidative stress, RAAS, and glucocorticoid

signaling
Macrophages have been implicated in brosis associated with various pathological conditions. Pioglitazone attenuated macrophage inltration into the myocardium as well as the up-regulation of MCP-1,
TNF-, and osteopontin gene expression in the heart of DS/obese rats,

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N. Matsuura et al. / International Journal of Cardiology 179 (2015) 360369

consistent with previously observed anti-inammatory effects of this

drug [46]. Our results thus indicate that pioglitazone attenuated both
cardiac inammatory responses and myocardial brosis in these animals, and they suggest that these benecial effects of pioglitazone are
attributable to an increase in the circulating concentration of
adiponectin. Increased oxidative stress has been associated with obesity
in both experimental animals and humans and has been implicated in
the development of MetS [47]. We found that pioglitazone attenuated
increases in both NADPH-dependent superoxide generation and the expression of NADPH oxidase subunit genes in the heart of DS/obese rats,
indicating that pioglitazone ameliorated cardiac oxidative stress in
these animals. The RAAS has also been implicated in the pathogenesis
of MetS [48]. We found that pioglitazone inhibited the up-regulation
of ACE and MR gene expression apparent in the heart of DS/obese rats,
indicating that PPAR signaling may contribute to inhibition of the cardiac RAAS. Pioglitazone also inhibited the up-regulation of 11-HSD1
and GR mRNA abundance apparent in the heart and adipose tissue of
DS/obese rats, consistent with previous reports that this drug attenuates
these mRNA expressions [6]. Increased expression of 11-HSD1 leads to
glucocorticoid excess [49], and adipose tissue-specic amplication of
glucocorticoid signaling induces all the characteristics of MetS [50].
Insulin-sensitizing actions of pioglitazone observed in DS/obese rats
may thus have resulted at least in part from down-regulation both of
11-HSD1 expression in adipose tissue and of consequent glucocorticoid action.
4.5. Effects of pioglitazone on cardiac hypertrophy and insulin resistance
Cardiac hypertrophy and insulin resistance are closely associated
conditions [51]. In hypertensive patients, for example, LV hypertrophy
is associated with hyperinsulinemia and a decline in insulindependent glucose uptake. TNF- is an important mediator of insulin
resistance linked to obesity, and it induces insulin resistance, at least
in part, through inhibition of the tyrosine kinase activity of the insulin
receptor [52]. Thiazolidinediones attenuate the inhibitory effect of
TNF- on the most proximal steps of insulin signaling, including
tyrosine phosphorylation of the insulin receptor and its major
substrate IRS-1 as well as activation of phosphatidylinositol 3-kinase
[53]. We have now shown that HOMA-IR was signicantly increased
in DS/obese rats compared with DS/lean rats, and that this increase
was attenuated by treatment of DS/obese rats with pioglitazone. Our
data suggest that pioglitazone ameliorated insulin resistance and glucose intolerance through down-regulation of inammatory cytokine
production as well as attenuated cardiac hypertrophy and brosis.
4.6. Study limitations
In this study, LVDd and LVDs tended to be increased with pioglitazone in both DS/obese and DS/lean rats, whereas LVFS and LVEF were
slightly but signicantly reduced in both rat strains. A recent study
showed that pioglitazone causes uid retention and edema through
PPAR stimulation of ENaC-mediated renal salt absorption [54]. Since
there is no evidence that thiazolidinediones have any direct deleterious
effect on cardiac function, reductions in LV systolic function parameters
with pioglitazone in both rat strains could be attributable chiey to increased cardiac size resulting from uid retention.
5. Conclusions
We have shown that treatment of DS/obese rats with pioglitazone
increased the serum concentration of adiponectin and induced activation of AMPK in the left ventricle, and that these effects were associated
with attenuation of LV hypertrophy, brosis, and diastolic dysfunction
as well as of cardiac oxidative stress and inammation. In addition, pioglitazone treatment increased body weight and fat mass but alleviated
adipocyte hypertrophy and inammation in visceral adipose tissue in

DS/obese rats, with these latter effects likely contributing to the observed amelioration of insulin resistance and the pathophysiology of
MetS. Further investigations are required to clarify the molecular mechanisms of these actions of pioglitazone.
Conict of interest
None.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijcard.2014.11.099.
Acknowledgments
We thank Yuuri Takeshita and Sae Ohura for the technical assistance.
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