Free Radical Biology & Medicine 50 (2011) 363–370

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Original Contribution

Impact of obesity on hypertension-induced cardiac remodeling: role of oxidativestress and its modulation by gemfibrozil treatment in rats

Randhir Singh a, Amrit Pal Singh a,b,⁎, Manjeet Singh a,†, Pawan Krishan a

a Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, Indiab Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143005, India

Abbreviations: HFD, high-fat diet; PPARα, peroreceptor-α; PAAC, partial abdominal aortic constrictioTBARS, thiobarbituric acid-reactive substances; SAG, supnitroblue tetrazolium; GSH, reduced glutathione; MABPLVW/BW, ratio of left ventricular weight to body weigthickness; NF-κB, nuclear factor κ-light-chain-enhancer onecrosis factor-α; IL, interleukin; TGFβ, transforming groactivated protein kinase; RAS, renin angiotensin system; R⁎ Corresponding author. Department of Pharmaceuti

University, Amritsar 143005, India. Fax: +91 183 2258E-mail address: apsingh82@gmail.com (A.P. Singh).

† Deceased.

0891-5849/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.freeradbiomed.2010.11.020

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 July 2010Revised 5 November 2010Accepted 15 November 2010Available online 29 November 2010

Keywords:Cardiac hypertrophyCardiac fibrosisObesityOxidative stressGemfibrozilFree radicals

This study investigated the possible synergistic role of obesity in hypertension-induced cardiac remodelingand its modulation by gemfibrozil treatment in rats. Male Wistar rats were fed a high-fat diet (HFD) for90 days. Normal rats were subjected to hypertension by partial abdominal aortic constriction (PAAC) for28 days. In the HFD+PAAC control group, rats on HFD were subjected to PAAC on the 62nd day and weresacrificed on the 90th day. HFD and PAAC individually resulted in significant cardiac hypertrophy and fibrosisalong with increased oxidative stress andmean arterial blood pressure (MABP) in rats as evidenced by variousmorphological, biochemical, and histological parameters. Moreover, the HFD + PAAC control group showedmarked cardiac remodeling compared to rats subjected to HFD or PAAC alone. The HFD+gemfibrozil andHFD+PAAC+gemfibrozil groups showed significant reduction in cardiac remodeling along with reductionin oxidative stress and MABP. Hence, it may be concluded that oxidative stress plays a key role in obesity-mediated synergistic effects on induction and progression of PAAC-induced cardiac remodeling, and itsdeleterious effects could be reversed by gemfibrozil treatment in rats through its antioxidant activity.

xisome proliferator-activatedn; WAT, white adipose tissue;eroxide anion generation; NBT,, mean arterial blood pressure;ht; LVWT, left ventricular wallf activated B cells; TNFα, tumorwth factor-β; MAPK, mitogen-OS, reactive oxygen species.cal Sciences, Guru Nanak Dev819.

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

Introduction

Heart failure is a clinical syndrome characterized by progressive left-ventricular systolic and/or diastolic dysfunction and is one of the majorreasons for mortality in both developed and developing countries [1].Myocardial remodeling involves histopathological and structural changessuch as cardiac hypertrophy characterized by an increase in cardiomyo-cyte size and fibrosis as adaptivemeasures against increased biomechan-ical stress. If left untreated, it subsequently leads to cardiac dysfunctionand failure [1,2]. Obesity is a fast-growing problem reaching epidemicproportions worldwide and has been considered an independent riskfactor for various cardiovascular disorders that finally result in heartfailure [3]. Obesity is well documented to be associated with significantneuroendocrine activation and increased proinflammatory cytokines and

oxidative stress [3,4]. A wide range of animal models are available tounderstand the pathophysiology of obesity and associated complications.However, the induction of obesity using high-fat diet (HFD) in rats has anadvantage over genetic models as it truly reflects obesity and inducedcomplications such as hyperglycemia, hyperinsulinemia, and cardiachypertrophy as observed in human obesity [5,6].

The peroxisome proliferator-activated receptor α (PPARα) belongsto the class of nuclear receptors and is expressed in tissues with highfatty acid metabolism. The ligands for PPARα include long-chain fattyacids and fibrates that are used clinically to control dyslipidemia. In theheart, the PPARα receptor plays an important role in regulating fuelhomeostasis and induces the expression of genes encoding mitochon-drial fatty acid oxidation enzymes involved in fatty acid uptake. ThePPARα receptors are reported to be down-regulated during cardiachypertrophy thus leading to reduced fatty acid oxidation and impairedcellular lipid homeostasis that finally results in cardiac dysfunction andfailure because of insufficient production of adenosine triphosphate[7,8]. The fibrates have been considered as possible therapy fortreatment of cardiac hypertrophy and fibrosis [9,10]. Gemfibrozil is afibric acid derivative and is widely used as a triglyceride- andcholesterol-lowering agent. Clinically, obesity rarely exists in isolationand is often associated with hypertension, dyslipidemia, and hyper-insulinemia [11]. Hence, this study aimed to investigate the role of HFDindividually and in combination with partial abdominal aorticconstriction (PAAC) in the induction and progression of cardiacremodeling and its possible reversal by gemfibrozil treatment in rats.

364 R. Singh et al. / Free Radical Biology & Medicine 50 (2011) 363–370

Materials and methods

The experimental protocol used in this study was duly approved bythe institutional animal ethics committee. Young male Wistar rats 12–14 weeks of age weighing 250–275 g were selected for this study. Therats were fed either standard laboratory feed (5% fat, 75% carbohydrates,and 20% proteins as macronutrients) or a high-fat diet containingpowdered chowand lard (30% fat, 50% carbohydrates, and20%proteins).The powdered chow was mixed with melted lard to get a dough-likeconsistency and was air dried and used as feed. The rats were exposedto a 12-h light/dark cycle in the institutional animal house and weremaintainedongroup-specific rat feedandwater ad libitum. Theratswererandomly divided into seven groups each comprising 12–14 animals. Ingroup 1 (control group), the rats were kept on normal diet and no drugtreatmentwas given. In group2 (PAAC control), the abdominal aortawaspartially constricted and rats were allowed to survive for the next28 days. In group 3 (HFD control), the ratswere kept onHFD for 90 days.In group 4 (HFD+PAAC control), the rats kept on HFD for 90 days weresubjected to PAAC on 62nd day and sacrificed on the 90th day after28 days of PAAC. In group 5 (gemfibrozil per se), the rats on normal dietwere treated with gemfibrozil (30 mg kg−1, by mouth) for 90 days. Ingroup6 (HFD+gemfibrozil), the rats subjected toHFDwere treatedwithgemfibrozil (30 mg kg−1, by mouth) for 90 days. In group 7 (HFD+PAAC+gemfibrozil), the rats subjected to HFD+PAAC were treatedwith gemfibrozil (30 mg kg−1, by mouth) for 90 days.

The PAACwas performed as per established procedure [9,12]. Briefly,the ratswere anesthetizedwith thiopentone sodium(35 mg/kg ip) and amidline incision of 3–4 cmwasmade in the abdomen to expose the aortabetween the diaphragm and the celiac artery. The 4-0 silk suture wasplaced around the middle of the aorta and was tightened along with a0.7-mm diameter needle. The needle was withdrawn to leave the vesselpartially constricted and the midline incision was sutured in layers.Neosporin antibiotic powder (GlaxoSmithKline, Mumbai, India) was ap-plied locally on the suturedwound and the rats were allowed to recover.

Morphological assessment of obesity and cardiac remodeling

The adiposity index and obesity index were measured to assessobesity. The adiposity index was calculated as the sum of the weightsof perirenal white adipose tissue (WAT), retroperitoneal WAT, andepididymalWAT divided by body weight×100. The obesity index wascalculated by taking the cubed root of the rat's weight (g) and dividingby the nasoanal length (mm)×104. The left ventricle including theinterventricular septum was weighed and values were expressed asmilligrams per gram of body weight. The left ventricle was dividedinto three equal slices and thewall thickness of each slice was noted atnine different points using an ocular micrometer. The mean values ofall three slices were calculated and noted.

Estimation of left-ventricular collagen content

The left-ventricular collagen content was determined by measur-ing the hydroxyproline concentration as described earlier [13]. Thedry left-ventricular mass was hydrolyzed in 6 M hydrochloric acid at100 °C for 16–20 h followed by addition of phosphate buffer,Chloramine T, and freshly prepared p-dimethyl aminobenzaldehyde(Ehrlich's reagent). The concentration of hydroxyproline was deter-mined using a spectrophotometer (DU 640B; Beckman Coulter,Fullerton, CA, USA) at 558 nm. The hydroxyproline concentration(taken as an index of left-ventricular collagen) was expressed asmilligrams per gram of left-ventricular dry weight.

Estimation of left-ventricular protein content

The left ventricle from freshly excised heart was minced andhomogenized in 0.1 M Tris–HCl buffer (pH 7.4, 10% w/v) using a

Teflon homogenizer. The clear supernatant of the homogenate wasused to estimate total protein content, lipid peroxidation, and reducedglutathione after centrifugation at 800 g for 10 min. Left-ventricularprotein content was determined using bovine serum albumin as astandard according to Lowry et al. [14]. Briefly, 100 μl of dilutedsupernatant was made up to 1 ml using distilled water followed byaddition of 5 ml of Lowry's reagent and 0.5 ml of Folin–Ciocalteu andincubated at room temperature for 30 min. The protein content wasdetermined spectrophotometrically at 750 nm and expressed asmilligrams per gram of blotted left-ventricular weight.

Serum analysis

Estimation of serum cholesterol and serum high-density lipopro-tein was done using a commercially available kit (Bayer DiagnosticsLtd., India). The serum triglycerides and serum glucose weremeasured using commercially available diagnostic kits (TransasiaBiomedicals Ltd., India).

Estimation of thiobarbituric acid-reactive substances (TBARS)

The quantitative measurement of TBARS, an index of lipidperoxidation in heart, was performed according to a methoddescribed earlier [15]. In brief, 0.2 ml of homogenate was pipettedinto a test tube, followed by the addition of 0.2 ml of 8.1% sodiumdodecyl sulfate, 1.5 ml of 30% acetic acid (pH 3.5), and 1.5 ml of 0.8% ofthiobarbituric acid and the volume was made up to 4 ml with distilledwater. The test tubes were incubated for 1 h at 95 °C, then cooled, and1 ml of distilled water was added, followed by the addition of 5 ml ofn-butanol:pyridine mixture (15:1, v/v). The tubes were centrifuged at4000 g for 10 min. The absorbance of the developed color wasmeasured at 532 nm. A standard calibration curve was preparedusing 1–10 nM 1,1,3,3-tetramethoxy propane. The TBARS value wasexpressed as nanomoles per milligram of protein.

Estimation of superoxide anion generation (SAG)

The left-ventricular SAG was estimated in terms of reducednitroblue tetrazolium (NBT) [16]. Weighed amounts of left-ventric-ular tissue were taken in phosphate-buffered saline containing NBTand incubated at 37 °C for 1.5 h, and NBT reduction was stopped byadding hydrochloric acid. The samples were homogenized in amixture of 0.1 M sodium hydroxide and 0.1% sodium dodecyl sulfatein water containing 40 mg/L diethylenetriaminepentaacetic acid andcentrifuged at 20,000 g for 20 min. The resultant pellets weresuspended in 1.5 ml of pyridine and kept at 80 °C for 1.5 h to extractformazan. The mixtures were again centrifuged at 10,000 g for 10 minand absorbance of formazan was measured at 540 nm. The amount ofreduced NBT was calculated using the formula

amount of reduced NBT = A × V = T × W × � × l;

where A is absorbance, V is volume of pyridine (1.5 ml), T is time thetissue was incubated with NBT (1.5 h), W is blotted wet weight of thetissue (25 mg), ε is the extinction coefficient (0.72 L/mmol/mm), andl is length of light path (1 cm). Results were expressed as reduced NBTpicomoles per minute per milligram of wet tissue.

Estimation of reduced glutathione (GSH)

The GSH in tissue was estimated using an establishedmethod [17].The supernatant of the homogenate was mixed with trichloroaceticacid and centrifuged at 1000 g for 10 min at 4 °C and the resultingsupernatant was mixed with 0.3 M disodium hydrogen phosphatefollowed by addition of 0.001 M freshly prepared 5,5′-dithiobis-2-nitrobenzoic acid dissolved in 1% w/v citric acid and absorbance was

Table 1Effects of gemfibrozil treatment on morphological parameters in rats.

Parameter Group

Control PAAC control HFD control HFD+PAAC Gemfibrozil per se HFD+gemfibrozil HFD+PAAC+gemfibrozil

Final weight (g)(% increase in body wt)

315.9±6.5 (19.9) 318.1±4.7 (17.0) 407.6±7.9a (57.9) 390.8±7.1b (50.3) 311.3±5.4 (21.3) 403.7±6.1 (50.1) 364.4±5.2 (42.8)

Obesity index 325.67±2.82 318.31±1.08 373.20±2.84a 358.54±3.23b 320.81±2.27 354.56±1.43 348.23±3.19Adiposity index 1.84±0.17 1.80±0.06 3.02±0.06a 2.60±0.06b 1.91±0.07 2.82±0.12 2.40±0.05LVW/BW (g) 2.06±0.11 3.18±0.16a 2.02±0.04 2.99±0.15b 2.14±0.13 2.13±0.15 2.42±0.24d

LVWT (mm) 2.45±0.09 3.77±0.04a 3.92±0.04a 3.96±0.04 2.56±0.06 3.65±0.04c 3.69±0.04

n=12 per group. Values are expressed as means±SEM.a pb0.05 vs control.b pb0.05 vs PAAC control.c pb0.05 vs HFD.d pb0.05 vs HFD+PAAC.

Fig. 1. Effect of gemfibrozil treatment on left-ventricular collagen content (n=6). Thevalues are expressed as means±SEM. apb0.05 vs control, bpb0.05 vs PAAC control,cpb0.05 vs HFD, and dpb0.05 vs HFD+PAAC.

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noted at 412 nm. A standard curve was plotted using 5–50 μMreduced-form glutathione and results were expressed as micromolesof GSH per milligram of protein.

Estimation of mean arterial blood pressure (MABP)

The MABP in carotid artery (mm Hg) using a pressure transducer(BIOPAC System, CA, USA) was recorded in anesthetized rats beforesacrifice for morphological and histological studies.

Histopathological studies

The left-ventricular cardiomyocyte diameter was measured aspreviously described [13]. The heart was excised and immediatelyimmersed in 10% buffered formalin, dehydrated in graded concentra-tions of ethanol, immersed in xylene, and then embedded in paraffin.Sections of 4 μm were cut and stained with hematoxylin–eosin andpicrosirius red to determine cardiomyocyte diameter and collagendeposition, respectively. The slides were observed at 100-foldmagnification to analyze red collagen deposits and by using animage analysis program (National Institutes of Health Scion imageanalyzer, Bethesda, MD, USA). The diameter of at least 100cardiomyocytes was determined in randomly selected visual fieldsat 400-fold magnification and the mean cardiomyocyte diameter wasexpressed in micrometers [13].

Drugs and chemicals

Gemfibrozil was obtained from Nicolas-Piramal Research Labs(Mumbai, India). Folin–Ciocalteu reagent was procured from SRL(Mumbai, India) and bovine serum albumin was obtained fromSigma–Aldrich (St. Louis, MO, USA). All other reagents used in thestudy were of analytical grade.

Statistical analysis

The results were expressed as means±standard error of the mean(SEM). The data obtained from various groups were statisticallyanalyzed using one-way analysis of variance followed by Tukey'smultiple range test. The p value b0.05 was considered statisticallysignificant.

Results

Effects of gemfibrozil treatment on morphological parameters

Nosignificant change inbodyweight, adiposity index, orobesity indexwas observed in the PAAC control group and gemfibrozil per se groupcompared to the control group. The HFD significantly increased the body

weight, adiposity index, and obesity index in the HFD control and HFD+PAAC control groups. However, no significant changes in body weight orobesity parameters were observed in the HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups compared to the HFD control and PAACcontrol groups, respectively (Table 1). The PAAC control andHFD+PAACcontrol group showed a significant increase in left-ventricular weight-to-body weight ratio (LVW/BW). However, it was insignificant in theHFD control group and gemfibrozil per se group compared to the con-trol group. The HFD + PAAC+gemfibrozil group showed a significantdecrease in LVW/BW compared to the HFD+PAAC control group.However, HFD+gemfibrozil did not show any significant changecompared with the HFD control group. A significant increase in left-ventricular wall thickness (LVWT)was observed in the PAAC control andHFD control group compared to the normal control group. However, thechange was insignificant in the HFD+PAAC control group compared tothe PAAC control group. The gemfibrozil per se group showed aninsignificant change in LVWT compared to the control group. The HFD+gemfibrozil group showed a significant decrease in LVWT compared tothe HFD control. The HFD+PAAC+gemfibrozil group did not show anysignificant change compared to the HFD+PAAC control group.

Effects of gemfibrozil treatment on left-ventricular collagen content

The PAAC control and HFD control groups showed a significantincrease in left-ventricular collagen content but the gemfibrozil per se

Table 2Effects of gemfibrozil treatment on various biochemical parameters in rats.

Parameter Group

Control PAAC control HFD control HFD+PAAC Gemfibrozil per se HFD+gemfibrozil HFD+PAAC+gemfibrozil

Total protein content (mg/g) (6) 103.94±2.25 159.02±4.41a 167.76±4.76a 178.44±2.84b 110.34±4.04 150.84±5.14c 148.72±3.14d

Cholesterol (mg/dl) (12) 46.48±2.74 43.29±0.82 68.65±2.02a 66.33±1.59b 44.31±1.91 59.25±1.77c 58.26±1.62d

Triglycerides (mg/dl) (12) 71.17±3.36 70.12±1.98 96.23±2.78a 99.17±1.66b 67.22±2.36 73.02±2.66c 75.36±2.04d

HDL (mg/dl) (12) 33.56±1.45 35.02±1.17 32.86±1.03 31.7±1.39 32.61±1.59 38.99±0.95c 38.60±0.67d

Glucose (mg/dl) (12) 110.18±8.82 104.49±3.01 157.59±6.88a 151.62±5.24b 112.73±4.21 133.37±4.84c 127.73±2.97d

TBARS (nmol/mg protein) (6) 1.91±0.12 4.14±0.17a 4.13±0.12a 4.91±0.19b 1.73±0.11 3.34±0.14c 3.38±0.18d

SAG (pmol/min/mg tissue) (6) 40.87±1.76 54.94±1.85a 56.23±2.03a 66.35±2.42b 37.56±2.02 46.63±1.93c 53.86±2.72d

GSH (μmol/mg protein) (6) 0.35±0.01 0.28±0.01a 0.27±0.01a 0.23±0.03b 0.37±0.02 0.33±0.01c 0.31±0.01d

Numbers in parentheses represent sample size. Values are expressed as means±SEM.a pb0.05 vs control.b pb0.05 vs PAAC control.c pb0.05 vs HFD.d pb0.05 vs HFD+PAAC.

366 R. Singh et al. / Free Radical Biology & Medicine 50 (2011) 363–370

group showed an insignificant change compared to the normal con-trol group (Fig. 1). A significant increase was observed in the HFD+PAAC control group compared to the PAAC control group. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed a signifi-cant decrease in collagen content compared to the HFD control andHFD+PAAC control groups, respectively.

Effects of gemfibrozil treatment on left-ventricular protein content

The PAAC control and HFD control groups showed a significantincrease in left-ventricular protein content but the gemfibrozil per segroup showed an insignificant change compared to the normalcontrol group (Table 2). A significant increase was observed in theHFD+PAAC group compared to the PAAC control group. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed a signifi-cant decrease in protein content compared to the HFD control andHFD+PAAC control groups, respectively.

Effects of gemfibrozil treatment on serum parameters

The HFD resulted in a significant increase in serum total cho-lesterol, triglycerides, and glucose levels along with a decrease in HDL

Fig. 2. Effect of gemfibrozil treatment on MABP measured using the BIOPAC system(n=12). The values are expressed as means±SEM. apb0.05 vs control, bpb0.05 vsPAAC control, cpb0.05 vs HFD, dpb0.05 vs HFD+PAAC.

in the HFD control and HFD+PAAC control group. However, thegemfibrozil per se and PAAC control groups did not show anysignificant change compared to the normal control group. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed signifi-cantly reversed changes in serum parameters compared to the HFDcontrol and HFD+PAAC control groups, respectively.

Effects of gemfibrozil treatment on TBARS

The PAAC control and HFD control groups showed a significantincrease in lipid peroxides measured in terms of TBARS in left-ventricular tissue compared to the normal control group. However,the gemfibrozil per se group did not show any significant change inTBARS compared to the control group (Table 2). Moreover, asignificant increase in TBARS was observed in HFD+PAAC controlscompared to the PAAC control group. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed a significant decrease in TBARScompared to the HFD control and HFD+PAAC control groups,respectively.

Effects of gemfibrozil treatment on SAG

The PAAC control and HFD control groups showed a significantincrease in SAG measured in left-ventricular tissue compared to the

Fig. 3. Effect of gemfibrozil treatment on cardiomyocyte diameter of left-ventriculartissue (n=6). The values are expressed asmeans±SEM. apb0.05 vs control, bpb0.05 vsHFD, and cpb0.05 vs HFD+PAAC.

Fig. 4. Hematoxylin–eosin staining of transverse sections of left ventricle showing cardiomyocyte diameter (bar, 100 μm). (A) Normal control, (B) PAAC control, (C) HFD control,(D) HFD+PAAC control, (E) HFD+gemfibrozil, (F) HFD+PAAC+gemfibrozil.

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normal control group, whereas the gemfibrozil per se group did notshow any significant change in SAG levels compared to the controlgroup (Table 2). Moreover, a significant increase in SAG was observedin HFD+PAAC controls compared to the PAAC control group. TheHFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed asignificant decrease in SAG compared to the HFD control and HFD+PAAC control groups, respectively.

Effects of gemfibrozil treatment on GSH

The PAAC control and HFD control groups showed a significantdecrease in GSH level in left-ventricular tissue but the gemfibrozil perse group did not show any significant change compared to normalcontrols (Table 2). Moreover, a significant reduction in GSH wasobserved in HFD+PAAC controls compared to the PAAC controlgroup. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groupsshowed a significant increase in GSH compared to the HFD control andHFD+PAAC control groups, respectively.

Effects of gemfibrozil treatment on MABP

A significant increase in MABP was observed in the HFD controland PAAC control groups compared to normal controls but thegemfibrozil per se group did not show any significant change in MABPcompared to the control group. The HFD+PAAC group showed asignificant increase in MABP compared to the PAAC control group. TheHFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed asignificant decrease in MABP compared to the HFD control and HFD+PAAC control groups, respectively (Fig. 2).

Effects of gemfibrozil treatment on histological parameters

The cardiomyocyte diameter in left-ventricular sections showed asignificant increase in the HFD control and PAAC control groupscompared to normal controls. However, the HFD+PAAC groupshowed an insignificant change in cardiomyocyte diameter comparedto the PAAC group. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed a significant decrease in cardiomyocytediameter compared to the HFD control and HFD+PAAC controlgroups, respectively (Figs. 3 and 4). A significant increase in collagendeposition was observed in the HFD control, PAAC control, and HFD+PAAC control groups. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed a significant decrease in left-ventricularcollagen content compared to the HFD control and HFD+PAACcontrol groups, respectively (Figs. 1 and 5).

Discussion

Obesity is an independent cardiovascular risk factor and accountsfor considerable cardiovascular threat in association with hyperten-sion and diabetes [3,18]. The HFD model is widely employed tounderstand the pathophysiology of obesity and associated complica-tions, as it has an edge over genetic models in closely mimickinghuman obesity [5,6]. Clinically, it is observed that obesity rarely existsindividually and is often associated with hypertension. Hence, thisstudy employed the HFD model alone and in combination with thePAAC-induced systemic hypertensionmodel to checkwhether obesityexerts any synergistic detrimental effect on heart when it is combinedwith hypertension. An earlier study conducted to understand the

Fig. 5. Picrosirius red staining of transverse sections of left ventricle showing collagen deposition (bar, 25 μm). (A) Normal control, (B) PAAC control, (C) HFD control, (D) HFD+PAAC control, (E) HFD+gemfibrozil, (F) HFD+PAAC+gemfibrozil.

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modulatory role of obesity in hypertension using spontaneouslyhypertensive rats and Wistar Kyoto rats had the limitation ofmarkedly different responses of the strains to HFD because of theirdifferent genetic makeups [19]. The present study waived thislimitation and, to best of our knowledge, it is the first study toevaluate the impacts of obesity and hypertension individually andtheir combination in cardiac remodeling using a nongenetic rat strain.

The HFD resulted in obesity as indicated by the increase inadiposity and obesity indexes. The HFD-fed rats showed a significantincrease in LVW/BW, LWVT, and cardiomyocyte diameter, indicatingconcentric cardiac hypertrophy. Moreover, significant cardiac fibrosismeasured in terms of collagen deposition was observed in the HFD-fed rats. A significant increase in MABP is consistent with earlierfindings, thus supporting the fact that obesity-induced hypertensioncould be the main culprit behind obesity-induced cardiac remodeling[20,21]. Similarly, the PAAC control group showed significant cardiachypertrophy and fibrosis without any effect on obesity parameters.The HFD-fed rats subjected to PAAC showed a significant increase incardiac hypertrophy and fibrosis compared to PAAC controls,suggesting a synergistic role of obesity with hypertension in theinduction and progression of cardiac remodeling. The gemfibrozil perse treatment in normal rats did not show any significant change in anyparameter employed in this study.

The PPARα receptors belong to class of nuclear receptors and areexpressed in tissues with high rates of fatty acid metabolism such asliver, kidney, heart, and muscles. The fibric acid derivatives such asfenofibrate and gemfibrozil are potential PPARα agonists and havebeen reported to ameliorate cardiovascular complications such as

atherosclerosis, endothelial dysfunction, and cardiac hypertrophy[22]. Moreover, the PPARα agonists are reported to exert anti-inflammatory activity through multiple mechanisms including inhi-bition of nuclear factor κ-light-chain-enhancer of activated B cells(NF-κB), activator protein-1, interleukins, and tumor necrosis factor-α (TNFα) [22,23]. An earlier study from our laboratory by Rose et al.reported a beneficial role for fenofibrate in PAAC-induced cardiachypertrophy and fibrosis [9]. In the present study, the HFD-fed ratsshowed a significant increase in serum cholesterol, triglycerides, andglucose levels. These results are consistent with earlier studiesindicating altered substrate metabolism associated with HFD[20,21]. The increase in lipid levels is associated with a decrease inPPARα expression [24]. Moreover, the pressure overload alsoproduces a reduction in PPARα expression [25]. Hence, we hypoth-esize that reduction of PPARα activity due to HFD and PAAC may beresponsible for their synergistic impact on cardiac remodeling. This issupported by our results showing amelioration of cardiac remodelingwith gemfibrozil treatment in the HFD and HFD+PAAC groups.

Obesity is associated with activation of the renin angiotensinsystem (RAS) and proinflammatory cytokines such as TNFα,interleukins (IL), and transforming growth factor-β (TGFβ) thatare involved in the induction of cardiac hypertrophy and fibrosis[20,26]. TNFα induces the NF-κB/IL-6 pathway to induce cardiachypertrophy [27]. The overexpression of TNFα is also associatedwith cardiac fibrosis [28]. Moreover, overexpression of TGFβ isobserved in obesity and it is the key culprit in proliferation offibroblasts and collagen deposition along with cardiac hypertrophythrough the mitogen-activated protein kinase (MAPK) signaling

369R. Singh et al. / Free Radical Biology & Medicine 50 (2011) 363–370

pathway [26,29]. A recent study suggested that adipose tissue itselfacts as a source of renin and can activate the RAS to induce hypertension[30].

The free radicals generated within the cell react with lipids of thecell membrane, thereby forming lipid radicals and lipid peroxidesthat are measured in terms of TBARS. Therefore, an increase in TBARSindicates oxidative stress. GSH is intracellular antioxidant defenseparticipating in the neutralization of free radicals and is regeneratedusing glutathione reductase. However, in severe oxidative stress thiscompensatory mechanism to restore the redox status of the cell failsas evidenced by a decrease in GSH levels. A significant increase inleft-ventricular TBARS and SAG and reduction in GSH indicateoxidative stress in the HFD control and PAAC control groups and asynergistic increase in the HFD+PAAC group. This study showedhyperlipidemia and hyperglycemia in the HFD and HFD + PAACgroups. The high rate of fatty acid oxidation increases reactive oxygenspecies (ROS) through the overexpression of NADPH oxidase [31].Moreover, the SAG is increased by elevated levels of glucose-6-phosphate dehydrogenase-derived NADPH in obese and hyperglyce-mic rats [32]. Furthermore, hyperglycemia reduces the levels ofintracellular antioxidant protein thioredoxin, thereby increasing SAG[33]. ROS are involved in the induction of cardiac hypertrophy andfibrosis by activating tyrosine kinase, protein kinase C, MAPK, andTGFβ [34–37]. PPARα null mice show severe cardiac dysfunctionassociated with a decrease in superoxide dismutase (SOD), anenzyme responsible for scavenging superoxide anion [38]. Thegemfibrozil treatment increases the level of SOD and reduces theexpression of genes encoding superoxide anion-generating NADPHoxidase subunits p47 phox, gp91 phox, and Rac-1 [39,40]. In ourstudy, the treatmentwith gemfibrozil inHFD andHFD+PAAC animalsresulted in a significant decrease in oxidative stress, hypertension, andcardiac remodeling.

A few studies suggest that hypertension plays a limited role inobesity-induced cardiac remodeling [26]. However, a significantreduction in MABP along with a reduction in cardiac remodeling bygemfibrozil treatment in HFD and HFD+PAAC rats suggests thathypertension has significant role to play in HFD-induced cardiacremodeling.

In conclusion, obesity plays a synergistic role to produce cardiacremodeling when combined with other models of hypertension.Oxidative stress plays an important role in the obesity-mediatedcardiac remodeling. Treatment with gemfibrozil reduced oxidativestress, MABP, and cardiac remodeling in the HFD and HFD+PAACgroups, thereby indicating that it can be a potential therapeutic optionfor patients with both obesity and hypertension.

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