Int J Pharm Pharm Sci, Vol 7, Issue 7, 335-339Original Article


TO STUDY THE ROLE OF EXERCISE AND DIETARY MANIPULATION IN OBESE RATS TREATED WITH ORLISTAT

SARATH CHANDRA M1, JYOTHI. Y2*, SYED IMAM RABBANI3

1,2,3Krupanidhi College Of Pharmacy, No 12/1, Carmelaram Road, Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bengaluru, Karnataka 560035
*Email: jokiran05@gmail.com

Received: 04 Apr 2015 Revised and Accepted: 30 May 2015

ABSTRACT

Objective: The present study was undertaken to evaluate the role of exercise and dietary manipulation in obese rats treated with Orlistat.

Methods: The study was conducted for 16 weeks. High-fat diet (HFD) was used to induce obesity, and once these animals were obese (eight week), manipulation in the diet, exercise, Orlistat and low dose of Orlistat were tested for 8 more weeks. During the treatment period, animals were continued with HFD except for the groups where the low-calorie diet (LCD) was used. The various manipulative options studied to include swimming test (physical exercise), Orlistat (200 mg/kg), low dose Orlistat (65 mg/kg) and low-calorie diet. The parameters were evaluated including Body weight was measured on weekly two days. Biochemical parameters such as HDL, LDL, VLDL, Triglycerides and total cholesterol were measured using diagnostic kits. Blood-glucose level was measured. On 16th week, the animals were sacrificed and their organ weights were measured. Organ weights included the Liver, Left and Right kidney and fat pad weights such as Mesenteric fat, Epididymis fat.

Results: There was a significant reduction in body weight, Triglycerides, total cholesterol, LDL, VLDL, Blood-glucose level organ weights included the Liver, Left kidney and Right kidney and fat pad weights such as Mesenteric fat, Epididymis fat and increased in HDL level of the animals treated with exercise and dietary manipulation and Orlistat.

Conclusion: The present study revealed that [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups.

Keywords: Anti-obesity, High-fat diet, Low-calorie diet, Exercise, Orlistat.

INTRODUCTION

Obesity is a medical condition in which excess body fat gets accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. Among the multiple factors contributing to its etiology, the sedentary life styles, white-collar jobs, lack of exercise, psychological factors, and the consumption of energy-rich diets are the major ones [1, 2].

Due to obscure etiology, the treatment of obesity is difficult and challenging. Further, the cause of concern is the non-availability of drugs for its treatment and the short-term efficacy and limiting side effects of the available drug [3].

It is the accumulation of body fat to the level which might have a negative effect on health. Body Mass Index (BMI) is a widely used diagnostic tool which provides a simple numeric measure of people ‘fatness’. BMI of 18.5 to 25 may indicate optimal weight a number above 25 may indicate the person is overweight; while a number above 30 suggests the person is obese and over 40, indicates morbidly obese. Indian urban population is experiencing high rates of obesity, as their work often demands less physical exertion. Even rural areas are not immune because of increased mechanization of farming activity leading to reduced physical activity [4].

The prevalence of obesity is increasing worldwide. Numerous diseases are caused or made worse by obesity. These include type two diabetes; hypertension; dyslipidemia; ischemic heart disease; stroke; obstructive sleep apnea; asthma; non alcoholic steato hepatitis; gastro esophageal reflux disease; degenerative joint disease of the back, hips, knees, and feet; infertility and poly cystic ovary syndrome; various malignancies; and depression [5].

A compound that selectively limits the intestinal absorption of dietary fat, in excess of that manageable by dietary manipulation alone, could become a useful therapeutic agent for the treatment of obesity. The inhibition of gastrointestinal lipase may be a potential method for decreasing fat absorption. Pancreatic lipase is the key enzyme required for the absorption of dietary triglyceride. Pancreatic lipase may also be indirectly responsible for an absorption of cholesterol.

Orlistat is a pharmacological agent that promotes weight loss in obese subjects via inhibiting of gastric and pancreatic lipase, an enzyme that is crucial for the digestion of the long-chain triglycerides, which at three daily doses of 120 mg reduces fat absorption by 30% and has been proven to be useful in facilitating both weight loss and weight maintenance [6].

However, because Orlistat can result in undesirable side effects, such as fecal incontinence, flatulence, and steatorrhea, its use may be limited. Therefore, it may be worthwhile to search the natural substances that show potent inhibitory activity against pancreatic lipase and have fewer side effects [7].

Increasing dietary fat increases body fat and it is unlikely that humans escape this important biological rule. Increasing dietary fat is associated with increased prevalence of obesity probably by increasing the intake of energy dense foods.

In the National Weight Loss Registry, three things were associated with weight loss: continued monitoring of food intake, lowering dietary fat intake, and increased exercise. The relation of dietary fat is most evident when physical activity is low [8].

Physical exercise can be used effectively by itself or in combination with dietary restriction to trigger weight loss [9]. Hence, the present study is designed to evaluate the effect of dietary manipulation and exercise in the management of obesity in Orlistat treated rats.

MATERIALS AND METHODS

Chemicals and reagents

Triglyceride and Cholesterol Kit (Span Diagnostics, Bangalore, India), Orlistat Capsules 120 mg (Torrent Pharmaceuticals LTD, India). All other reagents used in the experiments were of analytical grade and of the high purity.

Table 1: Composition of experimental diets

Composition of diet High-fat diet (HFD) in gm Low-calorie diet LCD) in gm
1. Fat:
a. Lard 230 0
b. Vegetable oil 50 45
2. Carbohydrate:
a. Dextrin 97 95
b. Cornstarch 98 95
c. Sucrose 130 445
3. Protein:
a. Casein 325 250
4. Vitamin mix 30 30
5. Mineral mix 40 40
6. total weight(g) 1000 1000
7. Energy density(k cals) 5.15 3.93
8. % Macronutrient(k cals)
a. Fat 50 10
b. Carbohydrate 25 65
c. protein 25 25

Ingredients expressed by weight (gm), HFD (high-fat diet); LCD (low-calorie diet)

Description of diet [10]

Animals

Female Sprague-Dawley rats were obtained from the Central Animal house, Krupanidhi College of Pharmacy, Bangalore, India. The rats were housed under 22±2ºC temperatures, 40-60% humidity and 12-12±1 h light-dark cycle. During obesity induction, rats weighing 150-200g were taken for the study, and they were broadly divided into ten groups. Group one was normal control (Normal pellet diet was fed) and nine groups were fed with HFD (High-fat diet) to induce obesity and water ad libitum. Experimental protocols were followed as per Institutional Animal Ethical committee guidelines. Animal Ethical committee clearance (CPCSEA No. KCP/IAEC-005/2013-14) was obtained for the procurement of animals.

Experimental design

The SD female rats were divided into 10 major groups. Each of the subgroups consists of six animals as follows:

  1. Normal Control-Rats will be fed on normal pellet diet
  2. HFD Control-Rats will be fed on high-fat diet [11]
  3. HFD+Exercise [12]
  4. HFD+Low-calorie diet-(Obese rats will be switched from HFD to LCD) [11]
  5. HFD+LCD+Exercise
  6. HFD+Orlistat (200 mg/kg diet) [13]
  7. HFD+Orlistat (low dose) (100 mg/kg)
  8. HFD+Orlistat (low dose)+Exercise
  9. HFD+Orlistat (low dose)+LCD
  10. HFD+Orlistat (low dose)+LCD+Exercise

Exercise protocols [12]

Swimming

The rats will be adapted to the water before the training began. The adaptation will consist of swimming for 30 minutes, once per day for five days, in water at a temperature of 31 °C. After adaptation, the rats will be trained by swimming for 60 minutes/day, five days a week for eight weeks, with a constant overload equivalent to 5% of their body weight. The water tanks will be 50 cm in height and 30 cm in diameter, and the over load (lead fish sinkers) was attached to the animal’s chest using an appropriate vest.

Measurement of body weight

Body weight was measured on every alternative day for 16 weeks.

Estimation of plasma lipid profiles

At the end of the experiment, on 16th-week blood samples were collected from overnight fasted animals under inhalation of anesthesia by retro-orbital puncture method. Plasma was separated by centrifugation at 2500 rpm for 15 min and was used for further experiments, i.e. Total cholesterol, HDL and triglyceride levels were estimated by CHOD-PAP method and GPO-PAP method. LDL levels were calculated by the method of Johnson et al.[14]. Blood glucose is estimated by using Glucometer.

Estimation of organ weight and fat pad weight

On 16th-week the animals were sacrificed by cervical dislocation and organs like Liver and kidney weights has taken. Fat pads like Mesenteric and epididymal fat are collected and dried on filter paper and weighed.

Statistical analysis

The results will be expressed as mean±SEM. Comparisons between the treatment groups and control will be performed by analysis of variance (ANOVA) followed by Turkeys’ multiple range tests. In all tests, the criterion for statistical significance will be (p<0.05).

RESULTS

Body weight

High-Fat Diet substantially increased body weights of rats in 16th-week, when compared to the normal control group. On 16th-week, when compared to HFD control, all Treatment groups showed a significant decrease in body weight. [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups (table 2).

Table 2: Effect of exercise and dietary manipulation on body weight

S. No. Experiment groups 1 Week 8 Week 16 Week
1 Normal control 150±1.91 200.16±3.24 260.83±2.7
2 HFD (high-fat diet) control 153.3±2.94 250.33±3.16a 350.5±4.52a
3 HFD+Exercise 152.17±3.1 253.67±3.40a 330±3.21a,x
4 HFD+LCD (low-calorie diet) 154.67±2.64 252.83±4.54a 345.5±3.62a
5 HFD+LCD+Exercise 151.5±3.15 255.67±3.20a 325±2.83a,x
6 HFD+Orlistat 150.17±1.85 248±2.42a 335.83±2.97a,x
7 HFD+Orlistat (low dose) 153.3±2.60 251.67±3.92a 342.17±3.15a,y
8 HFD+Orlistat (low dose)+Exercise 150.3±3.09 254.17±4.35a 322.33±2.53a,x
9 HFD+Orlistat (low dose)+LCD 152.67±3.05 250.83±2.74a 339±2.97a,y
10 HFD+Orlistat (low dose)+LCD+Exercise 154.17±2.37 253±2.11a 318.33±4.11a,x

All values are mean±SEM. aP<0.001, bP<0.01, cP<0.05 When compared to the normal control group, xP<0.001, yp<0.01, zp<0.05 When compared to HFD control.

Table 3: Effect of exercise and dietary manipulation on bio chemical parameters

S. No. Experiment groups Triglycerides Cholesterol HDL LDL VLDL Blood glucose
1 Normal control 96±2.07 67.50±1.84 50.3±2.19 35.3±1.31 20.67±0.8 100±1.63
2 HFD (high-fat diet) control 151.17±1.96a 143.17±2.14a 25.3±1.38a 60.5±0.76a 35.5±2.78a 150.1±2.02a
3 HFD+Exercise 130.8±2.41a,x 118.5±1.09a,x 37.5±1.87a,y 46.17±1.87a 25.5±1.95z 133±2.8a,x
4 HFD+LCD (low-calorie diet) 145.83±2.73b 138.17±1.01a 30.3±1.72a 55.17±1.51a 32.17±1.6b 145.5±1.95a
5 HFD+LCD+Exercise 125.17±1.6a,x 116.17±2.5a,x 38.6±0.84b,x 47.83±1.78a,x 24.3±1.5y 128.8±1.87a,x
6 HFD+Orlistat 135.8±2.75a,y 121±1.37a,x 36.3±2.23a,y 43.17±2.3x 26.6±1.97z 137.1±1.8a,y
7 HFD+Orlistat (low dose) 143.67±2.03a 135±1.24a,z 31.6±2.47a 53±2.19a 31±1.59b 144±1.57a
8 HFD+Orlistat (low dose)+Exercise 109.6±3.28a,x 111±1.50a,x 41.16±0.94c,x 39±1.65x 19.5±1.54x 126.8±1.6a,x
9 HFD+Orlistat (low dose)+LCD 140±2.16a 133.67±1.9a,y 35.3±1.6a,y 50±1.63b,x 30.8±2.57c 141.6±1.89a
10 HFD+Orlistat (low dose)+LCD+Exercise 105.3±4.41x 100.50±1.84a,x 45.83±2.03x 29.67±1.43x 18.3±1.28x 123.3±2.7a,x

All values are mean±SEM aP<0.001, bP<0.01, cP<0.05 When compared to the normal control group, xP<0.001, yp<0.01, zp<0.05 When compared to HFD control.

Table 4: Effect of exercise and dietary manipulation on organ fat pad weight

S. No. Experiment groups Epididymal Fat (gm) Mesentric Fat (gm)
1 Normal control 3.27±0.27 2.56±0.21
2 HFD (high-fat diet) control 8.25±0.23a 7.31±0.27a
3 HFD+Exercise 4.69±0.50x 4.94±0.29a,x
4 HFD+LCD (low-calorie diet) 7.57±0.22a 6.95±0.18a
5 HFD+LCD+Exercise 4.52±0.23x 4.56±0.32a,x
6 HFD+Orlistat 5.21±0.17 b,x 5.24±0.24a,x
7 HFD+Orlistat (low dose) 7.34±0.33a 6.23±0.23a
8 HFD+Orlistat (low dose)+Exercise 3.74±0.48x 4.41±0.23 a,x
9 HFD+Orlistat (low dose)+LCD 6.41±0.32a,y 5.65±0.22a,y
10 HFD+Orlistat (low dose)+LCD+Exercise 3.7±0.38 x 4.35±0.38a,x

All values are mean±SEM aP<0.001, bP<0.01, cP<0.05 When compared to the normal control group, xP<0.001, yp<0.01, zp<0.05 When compared to HFD control.

Table 5: Effect of exercise and dietary manipulation on organ weight

S. No. Experiment groups Right kidney WT (g) Left kidney WT (g) Liver WT (g)
1 Normal control 0.75±0.04 0.73±0.03 6.03±0.33
2 HFD (high-fat diet) control 1.65±0.04a 1.62±0.05a 11.3±0.3a
3 HFD+Exercise 1.22±0.03a,x 1.30±0.03a,x 9.63±0.56a
4 HFD+LCD (low-calorie diet) 1.42±0.03 a,y 1.48±0.04a 10.65±0.39a
5 HFD+LCD+Exercise 1.05±0.04a,x 1.20±0.06a,x 9.35±0.59a,z
6 HFD+Orlistat 1.29±0.03a,x 1.33±0.03a,y 10.03±0.27a
7 HFD+Orlistat (low dose) 1.41±0.02a,y 1.45±0.03 a 10.33±0.26a
8 HFD+Orlistat (low dose)+Exercise 1.02±0.06 a,x 0.26±0.07a,x 9.28±0.37a,x
9 HFD+Orlistat (low dose)+LCD 1.35±0.07a,x 1.34±0.03a,y 10.22±0.34a
10 HFD+Orlistat (low dose)+LCD+Exercise 1.15±0.04a,x 1.18±0.05a,x 923±0.4a,x

All values are mean±SEM. aP<0.001, bP<0.01, cP<0.05 When compared to the normal control group, xP<0.001, yp<0.01, zp<0.05 When compared to HFD control.

Serum lipid profiles

Rats fed with HFD showed increased levels of serum Triglycerides, LDL, VLDL and total cholesterol and decreased HDL levels, when compared normal control. All Treatments groups showed significantly suppressed the rise of Lipid profile and rise in the HDL levels was observed. [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups (table 3).

Blood glucose

Treatment groups showed a significant decrease in blood-glucose levels when compared to the HFD control group. [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups (table 3).

Organ weight and Fat pad analysis

Treatment groups showed a significant activity in decreasing the organ weight and Fat pad weights [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups (table 4 and 5).

DISCUSSION

The present study was designed to evaluate the role of exercise and dietary manipulation on the markers of obesity in rats. The study was conducted for 16 weeks High-fat diet (HFD) was used to induce obesity and once the animal, were obese (eight week), manipulation in diet, exercise, dose of Orlistat was tested for 8 more weeks. During the treatment period animals were continued with HFD except for the groups where the low-calorie diet (LCD) was used. The various manipulative options studied to include swimming test (physical exercise), low dose Orlistat (65 mg/kg) and low-calorie diet.

The parameters evaluated in this include body weight, biochemical parameters such as HDL, LDL, VLDL, triglycerides and total cholesterol measured using diagnostic kits, blood-glucose level measured by using glucometer. On 16th week, the animals were sacrificed and their organ weights were measured, such as the liver, left kidney and right kidney and fat pad weights such mesenteric fat, epididymis fat.

Obesity is a metabolic disorder characterized by an excessive accumulation of fat in the body to an extent which adversely affects the health of an individual. It is a direct consequence of perpetual imbalance between energy intake and expenditure with storage of extra calories in the form of fat in the adipose tissue. In obesity, there is an increase in intake of high-fat and high-energy food and a decrease in daily energy expenditure [4].

Diet and physical exercise remain as the main stay in obesity management; nonetheless, anti-obesity drugs may be required either to reduce appetite or to inhibit fat absorption. Various factors are found to be involved in obesity, including the genetic, environmental, behavioral, and socio-economic factors.

The present study indicated that administration of HFD to the animals for eight weeks significantly (P<0.001) increased body weight, LDL, TG, VLDL, blood-glucose level, weights of liver, left kidney, right kidney, mesenteric fat, epididymis fat and reduced the HDL level compared to normal control (table 1-4). HFD is a standard diet widely used experimentally to induce obesity in animals. The composition of HFD contains pork fat, glucose, casein, Nacl, dextrin. Earlier research indicated that expose of these ingredients to the experimental animals alters the metabolic mechanisms resulting in obesity. The data indicated that HFD induced obesity is due to hyperlipidemia, and hyperglycemia were evidence by a gain in body weight, increase in triglyceride levels and as well as increasing in the glucose levels [15]. Our observation also indicates that HFD administration for eight weeks entrances the parameters suggested for obesity. Similar mechanisms could have contributed in increased body weight, altered lipid profile, enhanced fat depositions in the present study.

When obese animals were subjected to physical exercise test for eight weeks, we found that there was significant difference (P<0.001) in body weight, lipid profile, fat deposition and blood-glucose level compared to normal control (table 1-4). Physical exercise increases the metabolic rate in the body. It enhances the digested fat to provide energy. The regular exercise was also reported to enhance the breakdown of deposited fat contributed in reduced body weight, normalize altered lipid profile, and regulate blood-glucose. The earlier research indicated that exercised animals had increased in energy expenditure through exercise training and contributed in the over body weight gain and higher free fatty acid (FFA) mobilization from other adipose tissues[16]. Our study suggests that the similar mechanism might have followed in this study too when obese animals were subjected to exercise.

The present study indicated that administration of LCD to the animals for eight weeks significantly (P<0.001) reversed the obesity parameters compared to normal control (table 1-4). The composition of LCD contains glucose, casein, Nacl, dextrin. LCD is a standard diet widely used experimentally to reduce obesity in animals. The earlier data indicated that LCD reduced obesity is due to reducing in circulating levels of glucose and lipid metabolites [17]. Our study suggests that the similar mechanism could have contributed to this study too when obese animals were substituted with LCD.

When obese animals were subjected to physical exercise and LCD test for eight weeks, we found that there was faster enhancement (P<0.001) in the reversal of obesity parameters compared to normal control (table 1-4). Physical exercise and LCD mechanism have been discussed earlier suggesting that combination of the two mechanisms could have contributed in the reduction of obesity.

The present study indicated that administration of Orlistat (200 mg/kg) to the animals for eight weeks, significantly (P<0.001) reversed the obesity parameters compared to normal control (table 1-4). Orlistat is a standard diet widely used experimentally to reduce obesity in animals. The earlier research indicated Orlistat shows pancreatic lipase inhibitor and decreased body weight pancreatic lipase inhibitor evidence by pancreatic lipase is the key enzyme for dietary fat digestion, and inhibition of the enzyme could be an effective way to alter fat absorption further data suggests that administration of Orlistat was effective in reversal of obesity parameters by the mechanism suggested in the literature [16].

Low dose of Orlistat suggests that administration of Orlistat (65 mg/kg) has also shown the significant (P<0.001) decrease in the parameters suggested for obesity (table 1-4). When obese animals were subjected to combination physical exercise and low dose Orlistat (65 mg/kg) for eight weeks, we observed that the reversal in the obesity parameters was more prominent compared to individual exercise and low dose Orlistat groups.

The results suggest that the mechanisms mentioned earlier for physical exercise, and Orlistat might have potentiated the anti-obesity action when used in combination. Similar observations were found when a combination of LCD and low dose of Orlistat was tested in obese rats, suggesting that combination in more effective than the individual treatment (table 1-4).

In addition, it was observed that triple combination of physical exercise, low dose of Orlistat (65 mg/kg) and LCD for eight weeks further suppressed the obesity parameters such as body weight, lipid profile, fat deposition and glucose metabolism in HFD rats (table 1-4). The mechanisms suggested in the earlier groups could have produced synergistic effects when used in combination. The observations indicate that exercise, low dose, Orlistat and LCD together and play a vital role in obese individual resistant to conventional anti-obesity therapies.

CONCLUSION

It can be concluded that [HFD+Orlistat (low dose)+Exercise+LCD] group was significant in reduced Body weight, Triglycerides, total cholesterol, Lipid parameters [HDL, VLDL, LDL], Blood-glucose level, organ weight [mesenteric fat and epididymal fat] and organ fat pad weight, when compared to HFD control.

Exercise shows higher free fatty acid (FFA) mobilization from adipose tissue and LCD diets exhibit little changes in circulating levels of glucose and lipid metabolites, and Orlistat shows pancreatic lipase inhibitory activity because of that reason obesity rats are reducing body weight.

The present study [HFD+Orlistat (low dose)+Exercise+LCD] group showed more potential effective than rest of all treatment groups.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Caterson ID. Obesity and its management. Australian Prescriber 1999;22:12-6.
  2. Rippe JM, Crossley S, Ringer R. Obesity as a chronic disease: Modern medical and Lifestyle management. J Am Diet Assoc 1998;98:S9-S15.
  3. Dietz WM, Goodwin NJ, Yanovski SZ. Long-term pharmacotherapy in the management of Obesity. JAMA 1996;276(23):1907-15.
  4. Srivastava S. Obesity in India. Curr Sci 2009;97:12.
  5. O’Brien PE, Dixon JB. The extent of the problem of obesity. Am J Surg 2002;184(6):S4-S8.
  6. Mahmoud RH, Elnour WA. Comparative evaluation of the efficacy of gin1er and Orlistat on obesity management, pancreatic lipase and liver peroxisomal catalase enzyme in male Albino rats. Eur Rev Med Pharmacol Sci 2013;17:75-83.
  7. Zhang J, Kang MJ, Kim MJ, Kim ME, Song JH. Pancreatic lipase inhibitory activity of Taraxacum officinale in vitro and in vivo. Nutr Res Pract 2008;2(4):200-3.
  8. Bray GA, Paeratakul S, Popkin BM. Dietary fat and obesity: a review of animal, clinical and epidemiological studies. Physiol Behav 2004;83:549-55.
  9. Gobatto CA, Mello MA, Souza CT, Ribeiro IA. The monosodium glutamate (MSG) obese Rat as a model for the study of exercise in obesity. Res Commun Mol Pathol Pharmacol 2002;111(4):89-101.
  10. Ramgopal M, Balaji M. Anti-Lipase and anti-obesity activities of Terminalia paniculata bark in high calorie diet-induced obese rats. Global J Pharmacol 2014;8(1):114-9.
  11. Dourmashkin JT, Chang GQ, Gayles EC, Hill JO, Fried SK, Julien C. Different forms of obesity as a function of diet composition. Int J Obes 2005;29(11):1368-78.
  12. Kalivianakis M, Minich DM, Havinga R, Kuipers F, Stellard F, Vonk RJ. Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption. Am J Clin Nutr 2000;72(1):174-80.
  13. Sperette GFF, Rosante MC, Duarte FO, Leite RD, Lino ADD, Andre RA. The effects of exercise modalities on adiposity in obese rats. Clinics 2012;67(12):1469-77.
  14. Levri KM, Slaymaker E, Last A, Yeh J, Ference J. Metformin as treatment for overweight and obese adults: a systematic review. Ann Family Med 2005;3(5):457-61.
  15. Borvin A, Deshaies Y. Contribution of hyperinsulinemia to modulation of lipoprotein lipase activity in the obese Zukar rats. Metabolism 2000;49:134-40.
  16. Sene-Fiorese M, Duarte FO, Scarmagnani FR, Cheik NC, Manzoni MS, Nonaka KO. Efficiency of intermittent exercise on adiposity and fatty liver in rats fed with high fat diet. Silver Spring 2008;16(10):2217-22.
  17. Gollisch KS, Brandauer J, Jessen N, Toyoda T, Nayer A, Hirshman MF. Effects of exercise training on subcutaneous and visceral adipose tissue in normal-and high-fatdiet-fed rats. Am J Physiol Endocrinal Metab 2009;297(2):E495-504.