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Abstract

The consumption of a high-fat diet (HFD) is considered a risk factor for obesity development. Nonetheless, a causal role of dietary fat has never been documented, because of inadequate animal models. In our study, one group of rats was fed with standard rat diet, while other group of rats fed with high-fat diet for 4 weeks. After 4 weeks of feeding, the hemodynamic parameters in the rats fed with HFD were significantly increased as compared with control rats. Rats fed with HFD had elevated levels of serum lipids, insulin, leptin, glucose and apolipoprotein B. Lipid peroxides and caspase-3 levels were increased while serum apolipoprotein A1 and antioxidant enzymes levels in heart tissues were decreased in HFD-induced obesity in rats as compared to normal healthy control rats fed on standard rat pellet diet. This model of diet-induced obesity will be a useful tool for studying the mechanisms by which dietary fat induces the obesity in humans.

Keywords

Obesity high-fat diet insulin leptin caspase antioxidant enzymes

Introduction

Obesity has become a global health epidemic which accompanied with dyslipidemia, diabetes, insulin resistance, hypertension, stroke and arteriosclerosis. The prevalence of obesity in the United States has increased; presently, one-third of American adults are considered obese. Among other factors, obesity is an important contributor to essential hypertension in humans. Data from the Framingham Heart Study suggest that 78% of essential hypertension in men and 65% of essential hypertension in women can be directly attributed to obesity.¹ However, the mechanism that links obesity with high blood pressure and altered renal function has not been fully elucidated.

Despite considerable efforts by scientists and health care professionals to understand and successfully treat obesity, its incidence continues to rise and the obesity-related health costs are staggering.² The trend toward greater obesity in the young raises concern because infant or childhood obesity alone significantly increases susceptibility to adult chronic diseases, including cardiovascular disease, hypertension and diabetes.³ Although, it is clear that genetic factors contribute to the propensity of an individual to become obese, the striking increase in overweight that occurs as previously underdeveloped countries modernize and the continued growth in the numbers of obese individuals in developed countries indicate an important role for environmental factors as well. The consumption of a high-energy density, high-fat diet (HFD)⁴ is thought to be one of the main factors.

The epidemiological studies have showed a significant positive correlation between mean dietary fat intake and the incidence of obesity and its related complications and risk factors.^5,6 Furthermore, as the mean amount of fat in the diet increases, the incidence of obesity also increases.⁷ The association of dietary fat with the incidence of obesity is exaggerated in some genetically isolated populations, such as the Pima Indians.⁸ Recent studies have shown that consumption of dietary fats promote hypothalamic resistance to the main anorexigenic hormones, leptin and insulin, leading to the progressive loss of the balance between food intake and thermogenesis and, therefore, resulting in body mass gain.^9,10

Although diets high in carbohydrates may also predispose an individual to obesity, our focus was on the effects of diet high in fat because of the epidemiological evidence implicating fat in both obesity and public health concerns. What is not clear is whether or how dietary fat per se is the predisposing factor, in particular because the incidence of obesity has tended to increase in the face of a slightly decreased mean fat intake in the United States.¹¹

A number of studies have demonstrated that antioxidants may act as a regulator of obesity in mice or rats with HFD. Obesity has been shown to be one of the conditions that decrease antioxidant capacity.¹² Obesity seems to decrease antioxidant defense by lowering the levels of antioxidant enzymes (catalase, glutathione peroxidase [GPx] and glutathione reductase [GR]).¹³

More specifically, we described a model that controls key variables to better reach unambiguous conclusions regarding the role of dietary fat in the etiology of obesity. Identifying the regulatory processes that mediate HFD-induced obesity is of fundamental importance and will require a well-established and controlled animal model.

The experiments described in this report were to establish such a model and to determine whether the rats fed with the HFD share important characteristics with obese humans.⁴ The purpose of the present study was to document the development of animal model and to partially characterize the model with respect to some factors likely to be involved in the cardiovascular changes associated with obesity.

Materials and methods

Animals

The study was approved by the Institutional Animal Ethics Committee (IAEC) of Hamdard University, New Delhi, which is registered with Committee for the Purpose of Control & Supervision of Experiments on Animals (CPCSEA), Government of India, India (Registration No. 173/CPCSEA, 28 Jan 2000). Wistar male albino rats, weighing 150−200 g, were procured from the Central Animal House Facility, Hamdard University, New Delhi, and acclimatized under standard laboratory conditions at 25 ± 2°C, and relative humidity (50% ± 15%) and normal. The animals were kept in polypropylene cages under standard laboratory conditions (12 hour light and 12 hour dark: day: night cycle) and had a free access to tap water ad libitum.

Experimental design

After acclimatization, all the animals were randomly divided into two groups of eight animals each and were treated as follows: Group I (normal healthy control) – rats fed with normal rat pellet diet (12.5% lipids, 62.3% carbohydrate and 24.3% protein; Amrut rat feed, Mfd by: Pranav Agro Industries Ltd, Maharashtra, India) for 28 days; Group II (pathogenic control) – rats fed with HFD (42% lipids, 36% carbohydrate and 22% protein) for 28 days. HFD was purchased from National Centre for Laboratory Animal Sciences (NCLAS), National Institute of Nutrition (NIN), Hyderabad, Andhra Pradesh, India. All other chemicals used were of analytical grade. Double distilled water was used for all biochemical assays.

Body weight gain, food and water intake measurement

Body weight was measured weekly, body weight change measured from difference between final body weight and initial body weight. Food intake was estimated by subtracting the amount of food left in the cages from the total amount of food provided to each rat.¹⁴

Measurement of hemodynamic parameters

Hemodynamic parameters (systolic, diastolic, mean arterial blood pressure, heart rate) were measured by Non-Invasive Blood Pressure Recorder using rat tail-cuff method (Kent Scientific Corporation, Torrington, Connecticut, USA).

Measurement of serum parameters

Blood was collected from the retro-orbital plexus of both the groups of overnight fasted rats using microcapillary tubes on 29th day. Serum was separated by centrifugation (4000 rpm, 10 min) and transferred to Eppendorf tubes. After serum collection, all animals were killed by cervical dislocation; hearts and visceral fat pad weights (mesenteric, perirenal and epididymal fats) were dissected out and weighed. The concentrations of lactate dehydrogenase (LDH; Reckon Diagnostics Pvt Ltd, Baroda, Gujarat, India), glucose, total cholesterol (TC), triglycerides (TGs; all the three from Span Diagnostics Ltd, Surat, Gujarat, India) and high-density lipoprotein-cholesterol (HDL-C; Reckon Diagnostics Pvt Ltd, Baroda, Gujarat, India) in serum were measured with commercial kits. The concentrations of insulin, leptin and apolipoproteins-A1 and B in the serum were measured, respectively, with rat insulin ELISA kit (Alpco Diagnostics, Salem, USA), rat leptin ELISA kit (BioVendor, Czech Republic) and apo-A1 and B, immunoturbidimetric immunoaasay kit (Randox Laboratories Ltd, Antrim, UK).

Determination of antioxidant enzymes in heart tissue

All antioxidant enzyme activities were determined after six rat hearts tissues homogenized with phosphate buffer saline at a pH of 7·4. Glutathione (GSH),¹⁵ glutathione peroxidase (GPx),¹⁶ glutathione reductase (GR),¹⁷ glutathione-S-transferase (GST),¹⁸ superoxide dismutase (SOD)¹⁹ and catalase²⁰ activities were estimated, respectively.

Determination of lipid peroxidation, sodium potassium ATPase and caspase-3 activity in heart tissue

Lipid peroxidation was determined with spectrophotometric measurement of the amount of malondialdehyde equivalents with thiobarbituric acid and was expressed as thiobarbituric acid-reactive substances (TBARS; nmol malondialdehyde/mg protein)²¹ and sodium potassium ATPase activity was measured in heart tissue by method of Bonting.²²

The reaction mixture consisted of 100 µL tissue homogenate incubated in a medium containing Tris-HCl (75 mM, pH-7.5), NaCl (600 mM), KCl (50 mM), MgCl₂ (5 mM), EDTA (1 mM) and ATP (3 mM) for 30 min at 30°C. The reaction was arrested by the addition of trichloro acetic acid (TCA). Na⁺/K⁺ ATPase activity is expressed as µmoles of phosphorous liberated/min per mg of protein.

Caspase-3 activity was measured using Caspase-3/CPP32 Colorimetric Assay Kit (BioVision, Mountain View, California, USA). A total of 50 µL supernatant from homogenized tissue with cooled lysis buffer was taken from each sample and 50 μL of 2X Reaction Buffer (containing 10 mM Dithiothreitol [DTT]) was added to each sample. Then, 5 μL of the 4 mM DEVD-pNA substrate (200 μM final conc.) was added and incubated at 37°C for 1−2 hours to allow a dissociation of p-nitroanilide (pNA) from the conjugate DEVD-pNA. CPP-32 activity was measured spectrophotometrically at 405 nm using a 100-μL micro quartz cuvet (Sigma, St. Louis, MO, USA) or sample was diluted to 1 mL with Dilution Buffer. Caspase-3 activity was calculated as nmole/hour per mg protein.²³

Histopathological studies

The studied animals were killed and two rat hearts tissue samples were collected, fixed in 10% formalin buffered solution, cut into 5-µm sections and stained with haematoxylin/eosin. The sections of heart tissues were studied to determine the level of tissue damage by HFD.

Statistical analysis

Statistical analysis was carried out using Graphpad Prism 3.0 (Graphpad software, San Diego, California, USA). All of the data were expressed as mean ± SEM. Statistical analysis was performed by Dunnett’s t-test and p < 0.05 were considered to be significant.

Results

Results of our research work show that HFD-induced obesity results in an increase in systolic (from 127.83 ± 6.86 to 165.83 ± 10.33 mmHg; p < 0.01), diastolic (from 96 ± 4.65 to 116.66 ± 5.7 mmHg; p < 0.05) and mean arterial blood pressures (BP) (from 106.16 ± 5.00 to 132.66 ± 7.1 mmHg; p < 0.01) as well as increase in heart rate (from 421.83 ± 15.77 to 687 ± 67.83 bpm) in HFD-treated rats as compared to normal healthy control rats as recorded by Non-Invasive Blood Pressure recorder using rat tail cuff method (Table 1).

Table 1.

Effect of high-fat diet on hemodynamic changes in albino rats by non-invasive bp (NIBP) recorder using tail-cuff method^a

Treatment groups	Heart rate (BPM)	Systolic BP (mmHg)	Diastolic BP (mmHg)	Mean arterial BP (mmHg)
Group I (normal healthy control group)	421.83 ± 15.77	127.83 ± 6.86	96 ± 4.65	106.16 ± 5.00
Group II (HFD-treated group)	539.20 ± 37.68^b	165.16 ± 10.58^b	119 ± 4.56^c	135.33 ± 6.09^c

Abbreviations: BP: blood pressures, HFD: high-fat diet.

^a All values were expressed as mean ± SEM for eight rats in each group.

^b p < 0.05 as compared to normal healthy control group (group I).

^c p < 0.01 as compared to normal healthy control group (group I).

HFD group (group II), shows significant (p < 0.01) increase in body weight gain than those of the normal healthy control group (group I), after 4 week of treatment. Food intake in HFD group was higher than that of normal healthy control group (Table 2).

Table 2.

Effect of high-fat diet on food, water intake, body weight gain, heart weight and visceral fat pad weights in albino rats^a

Treatment groups	Food intake (g/100 g body weight/day)	Water intake (mL/100 g body weight/day)	Body weight gain (g)	Heart weight (mg/g rat)	Visceral fat pad weights (mg/g rat)
I (normal healthy control group)	12.78 ± 2.31	40.34 ± 3.81	75.13 ± 5.02	3.08 ± 0.01	5.18 ± 0.01
II (HFD-treated group)	17.65 ± 3.67^b	46.60 ± 4.71^b	126.25 ± 13.90^c	3.37 ± 0.02^b	9.34 ± 0.02^b

Abbreviation: HFD: high-fat diet.

^a All values were expressed as mean ± SEM for six rats in each group.

^b p < 0.05 as compared to normal healthy control group (group I).

^c p < 0.01 as compared to normal healthy control group (group I).

Serum TC, TGs, low-density lipoprotein-cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), atherogenic indexes (TC/HDL-C and LDL-C/HDL-C) and apolipoprotein-B levels in HFD group (group II) were significantly (p < 0.01) increased as compared to the normal healthy control group (group I), while HDL-C and apolipoprotein-A1 levels were significantly reduced in group II as compared to group I (Tables 3 and 4). Serum insulin, glucose and LDH levels were significantly (p < 0.01) increased in HFD-treated group as compared to the normal healthy control group. Serum leptin level in HFD-treated group was significantly (p < 0.05) increased as compared those in normal healthy control group.

Table 3.

Effect of high-fat diet on serum total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), very low-density lipoprotein-cholesterol (VLDL-C) and triglycerides (TGs) levels in albino rats^a

Treatment groups	Total cholesterol (mg/dL)	HDL-C (mg/dL)	Triglycerides (mg/dL)	LDL-C (mg/dL)	VLDL-C (mg/dL)	Atherogenic index
Treatment groups	Total cholesterol (mg/dL)	HDL-C (mg/dL)	Triglycerides (mg/dL)	LDL-C (mg/dL)	VLDL-C (mg/dL)	TC/HDL-C	LDL-C/HDL-C
Group I (normal healthy control group)	101.69 ± 2.00	32.26 ± 1.01	64.97 ± 2.19	56.64 ± 0.31	12.99 ± 0.43	3.25 ± 0.09	1.83 ± 0.08
Group II (HFD-treated group)	167.06 ± 4.74^b	25.28 ± 0.94^c	156 ± 11.63^b	110.61 ± 5.87^b	31.20 ± 2.32^b	6.02 ± 0.52^b	4.43 ± 0.37^b

Abbreviation: HFD: high-fat diet.

^a All values were expressed as mean ± SEM for eight rats in each group.

^b p < 0.01 as compared to normal healthy control group (group I).

^c p < 0.05 as compared to normal healthy control group (group I).

Table 4.

Effect of high-fat diet on serum leptin, insulin, glucose, apolipoprotein-a1 and b (Apo-A1 and B) and lactate dehydrogenase (LDH) levels in albino rats^a

Treatment groups	Leptin (pg/mL)	Insulin (ng/mL)	Glucose (mg/dL)	Apo-A1 (mg/dL)	Apo-B (mg/dL)	LDH (IU/L)
Group I (normal healthy control group)	159.37 ± 6.26	0.24 ± 0.02	94.14 ± 2.16	1.33 ± 0.08	4.65 ± 0.22	23.48 ± 0.64
Group II (HFD-treated group)	242.96 ± 9.32^b	1.33 ± 0.35^c	211.21 ± 4.65^c	0.20 ± 0.02^c	20.13 ± 1.93^c	208.82 ± 12.46^c

Abbreviation: HFD: high-fat diet.

^a All values were expressed as mean ± SEM for eight rats in each group.

^b p < 0.05 as compared to normal healthy control group (group I).

^c p < 0.01 as compared to normal healthy control group (group I).

HFD group shows a significant reduction in the activities of antioxidant enzymes in the cardiac tissue of rats with obesity induced by HFD. The glutathione-dependent antioxidant enzymes (GPx, GR, GST), catalase and superoxide dismutase activities were significantly (p < 0.01) reduced in the HFD group when compared with normal healthy control group (Tables 5 and 6). Lipid peroxide (TBARS) levels were significantly (p < 0.01) increased in HFD-fed rats as compared to the normal healthy control rats. Sodium potassium ATPase activity was reduced significantly (p < 0.01) in the rats fed with HFD as compared to the normal healthy control rats. The mean caspase-3 levels were significantly (p < 0.01) increased by 3.4-folds in HFD-fed rats as compared to normal healthy control rats (Table 6).

Table 5.

Effect of high-fat diet on cardiac tissue glutathione, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) levels in albino rats^a

Treatment groups	GSH (µmol of phosphorous liberated/min/mg protein)	GPx (nmol NADPH oxidized/min/mg protein)	GR (nmol NADPH oxidized/min/mg protein)	GST (nmol CDNB conjugate formed/min/mg protein)
Group I (normal healthy control group)	26.75 ± 0.97	184.81 ± 7.54	29.3 ± 3.84	434.25 ± 7.56
Group II (HFD treated group)	9.54 ± 0.47^b	142.66 ± 3.14^b	18.95 ± 0.25^b	246.43 ± 2.06^b

Abbreviation: CDNB: 1-Chloro-2, 4-dinitrobenzene; HFD: high-fat diet; NADPH: Nicotinamide Adenine Dinucleotide Phosphate.

^a All values were expressed as mean ± SEM for six rats in each group.

^b p < 0.01 as compared to normal healthy control group (group I).

Table 6.

Effect of high-fat diet on cardiac tissue catalase (CAT), superoxide dismutase (SOD), lipid peroxides (LPO), sodium potassium ATPase (Na+-K+ ATPase) and caspase-3 levels in albino rats^a

Treatment groups	Catalase (nmol H₂O₂ consumed/min/mg protein)	SOD (IU/mg protein)	TBARS (nmol MDA/ mg protein)	Na-K ATPase (µmol Pi liberated/min/ mg protein)	Caspase-3 (nmol/hr/mg protein)
Group I (normal healthy control group)	55.75 ± 1.88	1.77 ± 0.01	0.200 ± 0.003	0.725 ± 0.04	62.34 ± 9.28
Group II (HFD treated group)	24.99 ± 1.28^b	1.23 ± 0.33^b	0.729 ± 0.01^b	0.426 ± 0.01^b	209.30 ± 8.51^b

Abbreviations: HFD: high-fat diet, TBARS: thiobarbituric acid-reactive substances, MDA: malondialdehyde.

^a All values were expressed as mean ± SEM for six rats in each group.

^b p < 0.01 as compared to normal healthy control group (group I).

Heart and visceral fat pad weights (mesenteric, perirenal and epididymal fats) were significantly (p < 0.05) increased in HFD-fed rats as compared to the normal healthy control rats (Table 2).

Histopathological observation of rat’s heart tissue of HFD group showed deposition of fat globules in myocardial cells with marked fatty changes in portal region than in peripheral region as compared to normal healthy control rat’s heart tissue which showed normal architecture with regular morphology of myocardial cells and no fatty changes (Figure 1A and B).

Figure 1.

(A and B) Photomicrograph of heart tissue (n = 2) of normal healthy control rats showed normal architecture with regular morphology of myocardial cells while high-fat diet-fed rats showed deposition of fat globules in myocardial cells with marked fatty changes in portal region than in peripheral region. (Magnification ×400 and scale bar: 100 µm).

Discussion

A model of diet-induced obesity in rats is well controlled and shares many features with human obesity. Dietary fat is one of the most important environmental factors associated with the incidence of cardiovascular diseases (CVD); high cholesterol and saturated fat diets have been shown to promote atherosclerosis.²⁴ A rodent model of obesity based on the intake of HFD is advantageous in studying obesity-related cardiovascular abnormalities.²⁵ In the present study, body weight gain was higher in rats fed with HFD as compared to the normal healthy control rats. Matsuo et al²⁶ reported that body weight gain was greater in beef tallow diet group than in the other dietary groups.

Results of our research work show that HFD-induced obesity in rats resulted in a significant increase in systolic, diastolic and mean arterial blood pressures as well as increase in heart rate in HFD-treated rats as compared to normal healthy control rats. This suggests that a short period of high fat diet intake may increase Ca²⁺ channel numbers or alter channel regulation, leading to increased transmembrane Ca²⁺ influx. Elevation in Ca²⁺current density is associated with significantly elevated blood pressure.²⁷ The results of our research work showing increase in systolic BP (from 127.83 ± 6.96 mmHg to 165.83 ± 10.33 mmHg) and diastolic BP (from 96 ± 4.65 mmHg to 116.66 ± 7.1 mm Hg) corroborates with the study of Aubin et al.²⁸ They reported that in rats fed a HFD, systolic BP (171 ± 7 mmHg) and diastolic BP (109 ± 3 mmHg) were increased as compared to a standard diet-fed rats (systolic BP, 134 ± 8 mmHg; diastolic BP, 96 ± 5 mmHg).

In the present study, heart and visceral fat pad weights were significantly affected by HFD in HFD group. The visceral fat pads (i.e. mesenteric, perirenal and epididymal fat) of HFD rats weighed 50% more than those of normal healthy control rats as feeding of HFD in rats increases body weight, adiposity and visceral fat deposition.²⁹ The present study was designed to establish whether alterations due to HFD in rats would result in different levels of dyslipidemia and oxidative stress. HFD is a good source for the induction of obesity in normal healthy rats. Many studies indicated that obesity is induced by a high-energy diet containing 40% beef tallow.^30,31 We found that HFD for a period of 4 weeks produced significant increase in the serum TC, TGs and LDL-C levels in HFD-induced obesity in rats. Serum HDL-C levels were decreased in the HFD group rats as compared to the normal healthy control group rats. Our study results are supported by the work of Lavie and Milani,³² which indicated that obesity adversely affects plasma lipids, especially by increasing TC and decreasing the levels of HDL-C. The HFD might lead to an increase in the synthesis of phospholipids and cholesterol esters in rats.³³ Hyson et al³⁴ indicated that the blood levels of LDL-C and its oxidation are related to cardiovascular risk.

Another early detectable parameter in our model is lipid peroxidation. Free radicals are known to be involved in a variety of human pathologies, including atherosclerosis,³⁵ obesity³⁶ and hypertension.³⁷ A potential mechanism for the generation of free radicals may be the activation of β-adrenergic receptors reported for obesity-prone rats.³⁸ This could increase lipolysis to yield free fatty acids that are able to uncouple the mitochondrial phosphorylation and further generate free radicals.³⁹

In animal and human studies, obesity is associated with a decrease in tissue or plasma antioxidant capacity.⁴⁰ GSH constitutes the first line of defense against free radicals and is also responsible for the maintenance of protein thiols and acts as a substrate for GPx and GST.⁴¹ The present data indicate that GSH content was depleted in the rats with obesity induced by a HFD. Enzymatic antioxidants, such as superoxide dismutase, catalase or GPx, GR and GST can scavenge reactive oxygen species and free radicals or prevent their formation.⁴² The results of present study showed that antioxidant enzyme activities (SOD, catalase, GSH, GPx, GR and GST) in the HFD group (i.e. group II) were significantly reduced as compared to the normal healthy control group (i.e. group I). HFD-induced obesity is associated with insulin resistance, cardiac hypertrophy and myocardial apoptosis. Caspase-3 is a key enzyme involved in the caspase-dependant apoptotic pathway. It has been reported by Fang et al⁴³ that HFD feeding triggered elevated myocardial apoptosis, as assessed by elevated caspase-3 activity. In the present study, caspase-3 activity in heart tissue increased in HFD-fed rats, which corroborates with the findings of Fang et al.⁴³

Apolipoprotein A1 (Apo-A1) is associated with HDL, having several antiatherogenic properties. Apolipoprotein B (Apo-B) is associated with low-density lipoprotein, intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL) and chylomicrons.⁴⁴ Serum Apo-B levels were significantly increased in HFD rats as compared to the normal healthy control rats while serum Apo-A1 levels significantly decreased in HFD rats as compared to the normal healthy control rats. Apo-B secretion by the liver is regulated by factors such as rate of cholesterol biosynthesis, availability of triglycerides and cholesterol esters.⁴⁵ Measurement of the levels of the important lipoproteins such as Apo-A1 and Apo-B can throw light on lipid metabolism and the effect of various drugs on the same. Additionally, the Apolipoprotein-A1 class of lipoprotein, besides being associated with HDL particle, is known to play important antioxidant and anti-inflammatory roles in atherosclerosis.⁴⁶

The obese rats are both hyperleptinemic and hyperinsulinemic, and as occurs in humans, both plasma insulin and leptin concentrations were directly correlated with the degree of adiposity. In all likelihood, the hyperinsulinemia in the HFD-fed rats was a result of insulin resistance, a common feature of human obesity and one that is central to the development of diabetes and cardiovascular disease. Serum insulin level was significantly (p < 0.01) increased in HFD group as compared to the normal control healthy group. This may be due to insulin resistance caused by HFD. Mehta et al⁴⁷ indicated that HFD lead to insulin resistance through oxidative stress. In another study, serum insulin levels were higher in the HFD group as compared to the normal control healthy rats.²⁴ Serum leptin level in the HFD group was significantly (p < 0.05) increased as compared with those in the normal control group. Fried et al⁴⁸ indicated that basal levels of leptin are known to be strongly positively correlated with body fat on a HFD. The report indicated that leptin might contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes, so as to promote increased intracellular fatty acids.⁴⁹

HFD damages cell membrane as evident from significant decrease in levels of membrane-bound enzymes like Na⁺/K⁺ ATPase as compared to normal control (p < 0.01). Our results are in agreement with the findings of Takeuchi et al⁵⁰ who reported the activity of Na+-K+-ATPase in the liver and skeletal muscle was lower in rats fed with the lard diet. The decrease of ATPases could be due to enhanced lipid peroxidation by free radicals. Since this membrane-bound enzymes are ‘SH’ group-containing enzymes, so are they lipid-dependant. In our study, HFD decreases the sodium–potassium ATPase activity in HFD-induced obesity in rats as compared to the normal healthy control rats. Previous investigations have suggested that sodium-potassium–ATPase activity is lower in severely obese patients than in normal controls.⁵¹

In the histology study, heart tissue of HFD-fed rats showed deposition of fat globules in myocardial cells with marked fatty changes in portal region as compared to normal healthy control rat’s heart tissue, which showed normal architecture with regular morphology of myocardial cell.

The results of the present study confirmed that administration of HFD produced significant obesity in rats as indicated by increased body weight gain, hemodynamic changes and an increase in levels of serum lipids, insulin, leptin and Apo-B levels and decrease in serum Apo-A1 and HDL-C levels, cardiac Na⁺/K⁺ ATPase activity and antioxidant levels that were further supported by histopathological findings.

Conclusion

In conclusion, we partially characterized a rat model of diet-induced obesity that also develops hypertension. The advantages over other similar animal models are the close similarity to human obesity hypertension, namely activation of rennin angiotensin system (RAS), dyslipidemia and increased oxidative stress. The model may also be useful to study other pathologies associated with obesity, such as atherosclerosis.

Footnotes

The study was supported by major research project grant to Dr. Uma Bhandari by University Grants Commission, New Delhi, India.

References

Kannel

Brand

Skinner

Jr Dawber

Mc Namara

. The relation of adiposity to blood pressure and development of hypertension: the Framingham study. Ann Intern Med 1967; 67: 48–59.

Flegal

Carroll

Kuczmarski

Johnson

. Overweight and obesity in the United States: prevalence and trends, 1960–1994. Int J Obes Relat Metab Disord 1998; 22: 39–47.

Caprio

Tamborlane

. Metabolic impact of obesity in childhood. Endocrinol Metab Clin North Am 1999; 28: 731–747.

Woods

Seeley

Rushing

D’Alessio

Tso

. A controlled high-fat diet induces an obese syndrome in rats. J Nutr 2003; 133: 1081–1087.

Lichtenstein

Kennedy

Barrier

. Dietary fat consumption and health. Nutr Rev 1998; 56: S3–S19.

Roberts

Pi-Sunyer

Dreher

. Physiology of fat replacement and fat reduction: effects of dietary fat and fat substitutes on energy regulation. Nutr Rev 1998; 56: S29–S41.

Popkin

Paeratakul

Zhai

. Dietary and environmental correlates of obesity in a population study in China. Obes Res 1995; 3: 135S–143S.

Price

Charles

Pettit

Knowler

. Obesity in Pima Indians: large increases among post-World War II birth cohorts. Am J Phys Anthropol 1993; 92: 473–479.

De Souza

Araujo

Bordin

Ashimine

Zollner

. Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus. Endocrinology 2005; 146: 4192–4199.

10.

Munzberg

Flier

Bjorbaek

. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology 2004; 145: 4880–4889.

11.

Willett

. Dietary fat and obesity: an unconvincing relation. Am J Clin Nutr 1998; 68: 1149–1150.

12.

Asayama

Nakane

Dobashi

Kodera

Hayashibe

Uchida

Nakazawa

. Effect of obesity and troglitazone on expression of two glutathione peroxidases: cellular and extracellular types in serum, kidney and adipose tissue. Free Radic Res 2001; 34: 337–347.

13.

Carmiel-Haggai

Cederbaum

Nieto

. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats. FASEB J 2005; 19: 136–138.

14.

Lin

Thomas

Storlien

Huang

. Development of high fat diet-induced obesity and leptin resistance in C57Bl=6J mice. Int J Obes 2000; 24: 639–646.

15.

Ellman

. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82: 70–77.

16.

Mohandas

Marshall

Duggin

Horvath

Tiller

. Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer Res 1984; 44: 5086–5091.

17.

Calberg

Mannerviek

. Glutathione reductase levels in rat brain. J Biol Chem 1975; 250: 5475–5480.

18.

Habig

Pabst

Jakoby

. Glutthione-S-transferase. The first enzymatic step in mercaptouric acid formation. J Biol Chem 1974; 249: 7130–7139.

19.

Marklund

. Involvement of the superoxide anion radical in the auto-oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469–474.

20.

Caliborne

. Assay of catalase. In: Greenwald

, (ed.) Handbook of methods of oxygen radical research. London, UK: Bacoraton, CRC Press, 1985, p.283–285.

21.

Ohkawa

Ohishi

Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358.

22.

Bonting

. Sodium potassium activated adenosine triphosphatase and cation transport. In: Bitler

(ed.) Membrane and ion transport. Vol 1. London: Interscience Wiley, 1970, p.257–263.

23.

Gurtu

Kain

Zhang

. Fluorometric and colorimetric detection of caspase activity associated with apoptosis. Anal Biochem 1997; 251: 98–102.

24.

Hsu

Yen

. Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Br J Nutr 2007; 98: 727–735.

25.

Carroll

Zenebe

Strange

. Cardiovascular function in rat model of diet-induced obesity. Hypertension 2006; 48: 65–72.

26.

Matsuo

Takeuchi

Suzuki

. Body fat accumulation is greater in rats fed a beef tallow diet than in rats fed a safflower or soybean oil diet. Asia Pacific J Clin Nutr 2002; 11: 302–308.

27.

Wilde

Massey

Walker

Vollmer

Grekin

. High-fat diet elevates blood pressure and cerebrovascular muscle Ca²⁺ current. Hypertension 2000; 35: 832–837.

28.

Aubin

Lajoie

Clément

Gosselin

Calderone

Perrault

. Female rats fed a high-fat diet were associated with vascular dysfunction and cardiac fibrosis in the absence of overt obesity and hyperlipidemia: therapeutic potential of resveratrol. J Pharmacol Exp Ther 2008; 20: 961–968.

29.

Corbett

Stern

Keesey

. Energy expenditure in rats with diet-induced obesity. Am J Clin Nutr 1986; 44: 173–180.

30.

Han

Kimura

Zheng

Okuda

. Platycodi radix affects lipid metabolism in mice with high fat diet-induced obesity. J Nutr 2000; 130: 2760–2764.

31.

Han

Kimura

Zheng

Takahasi

Okuda

. Anti-obesity effects in rodents of dietary tea saponin, a lipase inhibitor. Int J Obes Relt Metab Disord 2001; 25: 1459–1564.

32.

Lavie

Milani

. Obesity and cardiovascular disease: hippocrates paradox? J Am Coll Cardiol 2003; 42: 677–679.

33.

Jayakumar

Nalini

Venugopal

. Effect of ginger (Zingiber officinale) on lipids in rats fed atherogenic diet. J Clin Biochem Nutr 1991; 27: 79–82.

34.

Hyson

Schneeman

Davis

. Almonds and almond oil have similar effects on plasma lipids and LDL oxidation in healthy men and women. J Nutr 2002; 132: 703–707.

35.

Steinberg

. A critical look at the evidence for the oxidation of LDL in atherogenesis. Atherosclerosis 1997; 131: 5–7.

36.

VanGaal

Zhang

Steijaert

De Leeuw

. Human obesity: from lipid abnormalities to lipid oxidation. Int J Obes Relat Metab Disord 1995; 19: S21–S26.

37.

Sagar

Kallo

Kaul

Ganguly

Sharma

. Oxygen free radicals in essential hypertension. Mol Cell Biochem 1992; 111: 103–108.

38.

Levin

Triscari

Sullivan

. Altered sympathetic activity during development of diet-induced obesity in rat. Am J Physiol 1983; 244: R347–R355.

39.

Turrens

. Superoxide production by the mitochondrial respiratory chain. Biosci Rep 1997; 17: 3–8.

40.

Ozata

Mergen

Oktenli

Aydin

Sanisoglu

Bolu

. Increased oxidative stress and hypozincemia in male obesity. Clin Biochem 2002; 35: 627–631.

41.

Prakash

Gupta

Kochupillai

Singh

Gupta

Joshi

. Chemopreventive activity of Withania somnifera in experimentally induced fibro sarcomatumors in Swiss albino mice. Phytother Res 2001; 15: 240–244.

42.

Husain

Mejia

Lalla

Kazin

. Dose response of alcohol-induced changes in BP, nitric oxide and antioxidants in rat plasma. Pharmacol Res 2003; 51: 337–343.

43.

Fang

Dong

Thomas

Heng

Leilei

Ren

. Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription. Am J Physiol Heart Circ Physiol 2008; 295: H1206–H1215.

44.

D’Souza

Mengi

Hassarajani

Chattopadhayay

. Efficacy study of the bioactive fraction (F-3) of Acorus calamus in hyperlipidemia. Indian J Pharmacol 2007; 39: 196–200.

45.

Dixon

Furukawa

Ginsberg

. Oleate stimulates apolipoprotein B containing lipoproteins from HepG2 cells by inhibiting early intracellular degradation of apolipoprotein B. J Biol Chem 1991; 266: 5080–5086.

46.

Garner

Waldeck

Wittig

Rye

Stocker

. Oxidation of high density lipoproteins II evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins A-I and A-II. J Biol Chem 1998; 273: 6088–6095.

47.

Mehta

Van Theil

Shah

Mobarhan

. Nonalcoholic fatty liver disease: pathogenesis and role of antioxidants. Nutr Rev 2002; 60: 289–293.

48.

Fried

Ricci

Russell

Laferrere

. Regulation of leptin production in humans. J Nutr 2000; 130: 3127S–3131S.

49.

Uygun

Kadayifci

Yesilova

. Serum leptin levels in patients with nonalcoholic steatohepatitis. Am J Gastroenterol 2000; 95: 3584–3589.

50.

Takeuchi

Matsuo

Tokuyama

Suzuki

. Serum Triiodothyronine concentration and Na⁺ K⁺-ATPase activity in liver and skeletal muscle are influenced by dietary fat type in rats. J Nutr 1995; 125: 2364–2369.

51.

Beutler

Kuhl

Sacks

. Sodium-potassium-ATPase activity is influenced by ethnic origin and not by obesity. N Eng J Med 1983; 309: 756–760.

The effect of high-fat diet-induced obesity on cardiovascular toxicity in wistar albino rats

Abstract

Keywords

Introduction

Materials and methods

Animals

Experimental design

Body weight gain, food and water intake measurement

Measurement of hemodynamic parameters

Measurement of serum parameters

Determination of antioxidant enzymes in heart tissue

Determination of lipid peroxidation, sodium potassium ATPase and caspase-3 activity in heart tissue

Histopathological studies

Statistical analysis

Results

Discussion

Conclusion

Footnotes

References