PROTECTIVE ROLE OF GYMNEMIC ACID IN CURBING HIGH FAT DIET AND HIGH FRUCTOSE INDUCED PANCREATIC OXIDATIVE STRESS MEDIATED TYPE-2 DIABETES IN WISTAR RATS

PROTECTIVE ROLE OF GYMNEMIC ACID IN CURBING HIGH FAT DIET AND HIGH FRUCTOSE INDUCED PANCREATIC OXIDATIVE STRESS MEDIATED TYPE-2 DIABETES IN WISTAR RATS

Lakshmi Narasimhan Chakrapani and Kalaiselvi Periandavan ^*

Department of Medical Biochemistry, University of Madras, Taramani Campus, Chennai - 600113, Tamil Nadu, India.

ABSTRACT: High fat diet and High fructose intake is the predominant lifestyle change that increased the epidemic of type-2 diabetes. HFD+HF feeding leads to oxidative stress mediated T2D through production of free radicals and decrement of antioxidant enzymes. Our present study focussed on the curbing effect of gymnemic acid on high fat diet and high fructose feeding induced oxidative stress mediated Type 2 diabetes mellitus. A total of 30 rats, 18 diabetic and 12 normal wistar rats were used for this study. Rats were HFD+HF fed and confirmed diabetic then animals were orally treated with gymemic acid (150 mg/kg body weight) daily for 30 days. Gymnemic acid supplementation effectively lowered the blood glucose and lipid levels expect HDL in HFD and HF fed diabetic rats. Free radical scavenging ability of gymnemic acid lead to the decrement of free radicals and thereby preventing macromolecular damage. Islets possess significantly lesser antioxidant potential when compared to other tissues and easily susceptible to oxidative stress under T2D condition. GPx a key antioxidant which can quench hydrogen peroxides and lipid peroxides play a crucial role in islet redox balance. Under diabetic condition GPx levels are significantly lowered. So an agent which boosts the antioxidant milieu can counter the oxidative assault effectively under pathologic condition. Gymnemic acid, an active component from the leaves of Gymnema sylvestre possess such boosting capacity not only at the activity level but also at the protein level. However further experimental investigation is needed to exploit its mechanism of action in increasing GPx expression is needed to substantiate its ethnomedicinal usage.

Gymnemic acid, T2D, Oxidative stress and GPx

INTRODUCTION: The burden of diabetes is increasing globally, particularly in developing countries. The causes are rapid increases in overweight, including obesity and physical inactivity.

Diabetes is predicted to become the 7^th leading cause of death in the world by the year 2030, Total deaths from diabetes are projected to rise by more than 50% in the next 10 year ¹.

Type 2 diabetes which is characterized by glucotoxicity and lipotoxicity leads to increased production of reactive oxygen species (ROS) and/or impaired antioxidant defence systems ². Islet has the lowest intrinsic antioxidant capacity compared with other metabolic tissues. Hence disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and deoxyribonucleic acid (DNA) ³. Hyper-glycemia is the key factor behind oxidative stress of the cell. Underlying mechanism is the proton electromechanical gradient generated from the mitochondrial electron transport chain which ensuing to increased production of superoxide. Other mechanisms include glucose autoxidation, protein kinase C activation, methylglyoxal formation and glycation, hexosamine metabolism and sorbitol formation ⁴. Similar to the paradoxically deleterious effects of chronic hyperglycemia, fatty acids (FA) and chronic hyperlipidemia, which are essential β-cell fuels in the normal state, become toxic when chronically present in excessive levels. Prolonged exposure of pancreatic β-cells causes chronic oxidative damage⁵.

ROS levels need to be finely regulated to keep good radicals from going bad and thereby avoid oxidative damage to cellular processes. Antioxidant defense system plays this pivotal role in redox balance ⁶. The major antioxidant enzymes are catalase, GPx, and SODs. SOD catalyzes the reaction of superoxides with hydrogen to form hydrogen peroxide. Catalase catalyzes dismutation of hydrogen peroxide to water and molecular oxygen. GPx catalyzes the reduction of hydrogen peroxide by GS and can also reduce lipid peroxides to alcohols. . GSH is regenerated for future use from its oxidized form, GSSG, through a reaction involving GSH reductase and NaDPH ⁷. Malaisse et al., 1982 ⁸ demonstrated that β-cells in albino rats were sensitive to peroxide and that the activity of GPx was low in Beta cells. Studies showed that the levels of GPx at mRNA and protein levels were low in islets and in T2D condition the expression levels are even more decreased ⁹.

Studies by Koulaijian and his team ¹⁰ revealed that over-expression of GPx can lessen beta cell dysfunction. Even though numerous drugs available in the treatment of diabetes, they come with the cost of potential side effects. So there is always a search for plausible drug with lesser side effects. Gymnema sylvestre has been extensively reported in the treatment of diabetes ^{11, 12, 13} and gymnemic acid, a key active constituent ¹⁴ present in the leaves accounts to 80% of this potential.

Gymnemic acid has been reported to be anti-hyperglycemic ^{13, 15}, anti-hyperlipidemic agent ¹⁶. This present study aims whether gymnemic acid has any potential role in boosting antioxidant enzymes with special reference to GPx, a vital enzyme in quenching lipid peroxides and hydrogen peroxides.

MATERIAL AND METHODS:

Source of Chemicals: Gymnemic acid (90% pure, standardized to 75% gymnemic acid IV) was procured from St Clare herbals, Hungary. Bovine Serum Albumin (BSA) was procured from Sigma-Aldrich, USA. Enhanced chemiluminescence (ECL) kit was purchased from Millipore, USA. All other chemicals used were of analytical grade and were obtained from Medox Biotech, India, Sisco Research Laboratories Pvt., Ltd., (SRL), Genei, Bangalore and CDH (Central Drug House Pvt., Ltd., Mumbai, India).

Animals: Male Albino rats of Wistar strain were obtained from Central Animal House facility, University of Madras, Taramani campus and experiments were conducted in accordance with guidelines approved by the institutional animal ethical committee (IAEC No: 01/20/2014). The animals were housed two per cage in large spacious cages under conditions of controlled temperature (25 ± 2 °C) with 12/12 h light/dark cycle and were given food and water ad libitum. The animals were maintained on a commercial rat contained 5% fat, 21% protein, 55% nitrogen free extract, 4% fibre (w/w) with adequate vitamins and minerals.

Study Design: Young adult (4 months old / weighing around 130 - 150g) rats were used throughout the study. Animals were divided into five groups with six rats of each group as follows:

High fat diet (HFD) comprised of normal rat chow supplemented with 4% cholesterol, 1.5% cholic acid and 30% coconut oil. Gymnemic acid (150 mg/kg body weight / day) was dissolved in physiological saline (0.89%) and administered by oral gavage.

Isolation of Islets: Isolation of islets is carried out based by the method of Lacy and Kostianovsky ¹⁷.

Lipid and Lipoprotein Profile: Total cholesterol (TC), triglycerides (TG) and high density lipoproteins (HDL) in serum were analysed using commercial kits from spin react in semi-auto analyser (Rx Monza, Randox, U.K).

The values of total cholesterol, triglycerides and HDL are expressed as mg/dl.

• LDL (low density lipoprotein) and VLDL (very low density lipoprotein).
• LDL cholesterol was calculated according to the Friedwald formula:
• LDL = Total cholesterol- (VLDL+ HDL cholesterol).
• VLDL (very low density lipoprotein) = TG/5
• VLDL and LDL concentrations are expressed as mg/dl.

Oral Glucose Tolerance Test and Plasma Glucose: The experimental rats were subjected to oral glucose tolerance test two days prior to sacrifice. All animals were fasted overnight and the following morning, they were subjected to oral glucose tolerance test by giving an oral dose of glucose (1 ml/100 g body weight, 50% w/v glucose solution) after collecting blood by puncturing the orbital sinus with the help of heparinized micro-haematocrit capillary tubes for estimating fasting plasma glucose and insulin. Blood samples were collected subsequently at 60, 120, and 180 min and centrifuged for 10 min at 800 x g at 4 ºC within 30 min to prevent auto-glycolysis by leukocytes. Plasma glucose was estimated by glucose oxidase-peroxidase method (Randox Laboratories Ltd.,). Results are expressed as mg/dl.

HbA1c: Glycosylated haemoglobin (HbA1c) levels were analyzed by HPLC and ion exchange method. From the blood samples collected glycosylated haemoglobin was separated from total haemoglobin. HbA1c content was calculated based on the ratio of HbA1c peak area to the total haemoglobin peak areas. Results are expressed in % glycation.

Estimation of Reactive Oxygen Species (ROS): Hydrogen peroxide, superoxide and hydroxyl radicals were estimated by the method of Jiang et al., ¹⁸ Nishikimi et al., ¹⁹ and Puntarulo and Cederbaum ²⁰ respectively.

Assessment of Antioxidant Defense System: The enzyme was assayed according to the method of Marklund and Marklund ²¹. The activity of catalase was assayed by the method of Sinha ²². The activity of GPx was determined by the modified method of Rotruck et al., ²³. Reduced glutathione (GSH) was determined by the method of Moron et al., ²⁴

Immunoblotting: Estimation of Protein was carried out by the method of Lowry et al., ²⁵ Immunoblotting Samples containing 50 - 100 µg of proteins were separated by SDS-electrophoresis on 10%- 12% polyacrylamide gels and transferred to polyvinylidenedifluoride membranes. The membranes were incubated with specific primary antibodies and the antibodies used was GPx (CST 1:1000 dilution). To verify the uniformity of protein load and transfer efficiency across the test samples, membranes were reprobed with β-actin (CST, 1:1000 dilution). Immuno-reactive bands were developed with Immobilon Western-Chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA), visualized using an enhanced chemiluminescence system (Chemi-Doc, BioRad, USA) and presented in comparison to β-actin expression

Data Analysis: The results are expressed as Mean ± Standard Error of Mean (SEM). Differences between groups were analysed by one-way analysis of variance (ANOVA) using the SPSS software package for Windows (Version: SPSS 20.0). Post hoc testing was performed for inter-group comparisons using the least significant difference (LSD) test; significance at p value <0.05 has been given in tables and figures.

RESULTS:

Lipid Profile and FFA: Table 1 shows the impact of gymnemic acid and metformin on serum lipid profile of experimental groups. Assessment of serum lipid profile in the present study reveal that a significant (p < 0.05) increase in the levels of serum total cholesterol, triglycerides, LDL, VLDL and free fatty acid (FFA) with a concomitant decrease in HDL was observed in HFD+HF alone fed group when compared to the control rats. Gymnemic acid supplementation to HFD and HF fed rats demonstrated a significant decrease (p < 0.05) in serum total cholesterol, triglycerides, LDL, VLDL and FFA with a concomitant increase (p < 0.05) in HDL, when compared to HFD+HF alone fed group, more over it was observed that gymnemic acid and metformin have almost equal beneficial effect on reducing lipid levels in the serum.

TABLE 1: LIPID PROFILE AND FFA

Fasting Plasma Glucose (FBG), Oral Glucose Tolerance Test (OGTT) and Glycosylated Hemoglobin (HbA1c): Fig. 1a displays fasting plasma glucose levels estimated at various intervals during experimental period. Fasting glucose levels at the initial day of initiation were around 70 - 90 mg/dl in control and experimental animals. On HFD+HF feeding rats showed 89%, 79% and 39% increase in group 2, 4 and 5 rats when compared with control rats showing a percentile increase of 9.3% at the end of 30 day period. At the end of 60 days HFD and HF fed group 2, 3 and 5 animals showed a drastic increase in the levels of the blood glucose 225 mg, 185 mg and 192 mg/dl when compared to that of the initial day 75 mg, 83 mg and 90 mg/dl respectively.

Supplementation of gymnemic acid (group 3) and metformin (group 4) from 60^th for 30 days lead to the decrement of increased blood glucose levels to 95 mg and 102 mg/dl when compared to that of the diabetic control rats 282 mg/dl (group 2). No significant changes were seen between control (group 1) and drug control rats (group 5).

Values are expressed as mean ± SEM for six animals in each group. ‘a’: control vs other groups, ‘b’: group II vs group III and IV, values are statistically significant at the level of p<0.05

Fig. 1b depicts oral glucose tolerance test (OGTT) in control and experimental animals. group 2 rats showed delayed glucose tolerance when compared to that of control animals. Group 3 and 4 rats also improved tolerance when compared to group 2 rats. Group 5 displayed a similar tolerance like group 1 control rats.

Fig. 1c shows glycosylated hemoglobin (HbA1c) levels in control and experimental animals. HbA1c levels indicated poor glycemic control in HFD and HF fed diabetic control rats (group 2). There was a 2.4 fold increase in glycation of hemoglobin in group II diabetic rats when compared to that of control rats.

Gymnemic acid and metformin supplemented groups showed 1.7 and 1.8 fold decrease in the levels of glycation when compared to diabetic control rats (group 2).

Oxidative Stress Markers:

Free Radicals: Fig. 2a, b and c shows the level of hydrogen peroxide, superoxide in the Islets of control and experimental rats. Group II HFD+HF fed rats showed an increase of 63% in the levels of free radicals (p<0.05) when compared to control animals (Group I). Administration of gymnemic acid and metformin to HFD+HF fed rats decreased the levels of free radicals generated by 32% and 30% respectively.

Values are expressed as mean ± SEM for six animals in each group. ‘a’: control vs other groups, ‘b’: group II vs group III and IV, values are statistically significant at the level of p<0.05

Values are expressed as mean ± SEM for six animals in each group. ‘a’: control vs other groups, ‘b’: group II vs group III and IV, values are statistically significant at the level of p<0.05

Lipid Peroxide: Fig. 3a shows the effect of gymnemic acid and metformin on the levels of MDA in Islets of experimental rats. Assessment of MDA levels in experimental groups edict that MDA levels were elevated in HFD+HF fed rats due to free radical induced oxidative stress. There was a 3.5 fold increase in the MDA levels in HFD+HF fed experimental group, when compared with that of control rats. On gymnemic acid and metformin supplementation, the MDA levels were evidently brought down in HFD+HF fed rats by 3.2 and 3.0 folds respectively, when compared group 2 rats.

Protein Carbonyls: Fig. 3b shows the levels of protein carbonyls in the islets of experimental rats. Group II rats shows a significant increase (29%) in the levels of protein carbonyls when compared with control rats. Treatment with gymnemic acid and metformin significantly brought down the levels of protein carbonyls in HFD+HF fed rats by 22% and 19% correspondingly.

Enzymatic and Non Enzymatic Antioxidants: Fig. 4a - 4d shows the levels of enzymatic antioxidants in the islets of HFD+HF fed rats with or without gymnemic acid supplementation. There was a marked decrease (p<0.05) observed in the currently assayed antioxidant enzymes namely SOD, Catalase, GPx  and GSH in HFD+HF fed rats by 37%, 30%, 39%, 41% respectively, when compared to control rats. Approximately 35-60% recovery of the activities of these enzymes have been recorded in the HFD+HF fed rats when co-supplemented with gymnemic acid. In a same way, supplementation of metformin resulted in elevation of antioxidants approximately by 15-35% respectively.

FIG. 5: GPx

Values are expressed as mean ± SEM for six animals in each group. ‘a’: control vs other groups, ‘b’: group II vs group III and IV, values are statistically significant at the level of p<0.05

Protein Expression of GPx: Protein expression of GPx in HFD+HF diabetic rats supplemented with or without gymnemic acid / metformin are depicted in Fig. 5. The present study revealed that there was a significant decrease (p<0.05) in protein levels of GPx in HFD+HF fed group 2 diabetic rats when compared with group 1 control rats. Upon supplementation of gymnemic acid / metformin, the protein levels of GPx were significantly increased (p<0.05) in diabetic rats when compared to their corresponding untreated groups.

DISCUSSION: Two major causative factors of oxidative stress in islets are lipotoxicity and glucotoxicity. Our study displays increased levels of serum lipids, FFA and lipo-proteins except HDL in HFD and HF fed T2D rats (group 2) on comparison with that to control rats. Our results are in corroboration with the previously available literature ²⁶. Increase in the serum lipids on feeding High fat diet and High fructose has been well documented by the studies of Jia et al., ²⁷ and Coelho et al., ²⁸. Increase in circulatory cholesterol may be due to the presence of cholic acid in the diet. Cholic acid enhances absorption of cholesterol into the blood stream ²⁹. Significant restoration of altered lipid profile and circulatory free fatty acids are seen after administration of gymnemic acid to type-2 diabetic rats which is congruent with the studies of Singh et al., ³⁰ which states administration of gymnemic acid to Type-2 diabetic rats lowered the circulatory cholesterol, triglycerides, LDL-c and VLDL levels in serum and also up-regulated HDL levels. This lipid lowering activity may be attributed to its G3PDH inhibiting potential ³¹_, intestinal fatty acid inhibitory potential ³² and prevention of partial absorption of glucose ³³. Metformin which is well attributed for its lipid curbing potential showed a slightly better reduction of circulatory lipid levels when compared with gymnemic acid treated rats.

On the other hand, our findings show a highly significant increase in blood glucose levels and impaired glucose tolerance upon high fat diet and fructose feeding for 90 days in Group 2 rats. Our results are supported by the studies of Nampurath et al., ³⁴ and Woods et al., ³ which demonstrate that feeding rats with high fat diet and fructose leads to increased blood glucose and impaired glucose tolerance.

Administration of gymnemic acid to diabetic rats resulted in significant (p<0.05) decrease of blood glucose levels and improved glucose tolerance. Our results are coherent with the work of Sugihara et al., ¹³ who have established the anti-diabetic activity of gymnemic acid IV in STZ induced diabetic rats. Possible mechanism of action of gymnemic acid might be mediated by its ability to inhibit the enzymes involved in the digestion of carbohydrates ³⁶, block the absorption of glucose in the intestine ³⁷, modulate glycolysis ³¹ and exert incretin mimetic activity to increase insulin secretion ³⁸.

Glycosylated haemoglobin (HbA1c), a key bio-marker for diabetes, shows glycemic control over the past three month period. Our result shows a marked increase in the levels of glycosylated haemoglobin in group 2 rats. Increased glucotoxicity induced oxidative stress is the prime reason for glycosylation ³⁹. Metformin by increasing insulin sensitivity ⁴⁰ reduces blood glucose levels, which in turn might have been responsible for the observed reduction in the levels of glycosylation in group 4 rats. Oral supplementation of gymnemic acid effectively lowered the levels of glycosylation in group 3 rats when compared to group 2 rats. Gymnemic acid prevents the formation of advanced glycation end products (AGEs).

This might be due to its antioxidant potential as have demonstrated its potential to scavenge free radicals in an in vitro study by Niramala and her co-workers³⁶ and Pathan et al., ⁴¹. have observed marked increase in the antioxidant status in the hearts of the cardiomyopathy induced experimental rats on supplementation with gymnemic acid-phospholipid complexes. Glucolipotoxicity leads to generation of ROS and thereby leading oxidative stress. So we sought to analyse the oxidative radicals and stress markers.

High fat diet and high fructose feeding leads to the development of type 2 diabetes and thereby to oxidative insults ^{42, 43}. The term “oxidative stress” is frequently used to describe the imbalances in redox status of cell, such metabolic disturbances involves the overproduction of reactive free radicals ⁴⁴ or decreased levels of antioxidant enzymes ⁴⁵.

Hence we measured free radicals such as superoxide, hydrogen peroxide and hydroxyl radicals. In our study, all the free radicals demonstrated an increase in islets of the HFD+HF fed rats. Our results were in corroboration with findings of Panchal et al., ⁴⁶. The products of the action of these highly reactive free radicals are molecules that are enriched in one or more oxygen atoms. These are generally considered to be markers of oxidative stress namely lipid peroxide and protein carbonyls. Protein carbonyls are formed either by direct oxidation of amino acid side chain or through conjugation by advanced lipid end products. As expected, along with increased free radicals, the lipid peroxides and protein carbonyls are also higher in HFD+HF fed rats.

Gymnemic acid has been reported to have free radical scavenging activity in an in-vitro study ³⁶. Gymnemic acid effectively scavenged the free radicals and reduced the levels of lipid peroxidation and protein carbonylation in HFD+HF fed rats. The efficiency was more or less similar to metformin. The extent of cellular damage caused by lipid peroxidation depends on the protective efficacy afforded by its innate antioxidant system ⁴⁵. Cells have a variety of enzymic and low molecular weight antioxidants as a defense against the deleterious effects of ROS overproduction and oxidative damage. Elevated ROS production, in the absence of increased antioxidant defenses would exacerbate oxidative damage and oxidative stress ⁴⁷. The biological antioxidant defense system is a unified assortment of enzymes such as SOD, CAT, GR and GPx as first line of defense and antioxidant molecules such as glutathione as second line of defense, which has been developed by the cell to protect itself against the harmful molecular species ⁴⁸. Our current findings of antioxidant defense status of experimental groups indicate, Feeding HFD+HF has been reported to reduce antioxidant enzyme activity. Treating with gymnemic acid have improved the activity of antioxidant enzymes and GSH levels, similar observations have been made by Pathan and his colleagues ⁴¹.

Since we found the considerable increase in the activity levels of GPx on supplementation of gymnemic acid to HFD+HF group than metformin, we thought to analyse the protein level expression of GPx. High fat feeding and High carbohydrate has been reported to decrease antioxidant levels and their activity.

Our findings also should similar pattern, group 2 HFD+HF fed rats showed decreased expression of GPx when compared to normal control rats.  Generally GPx expression at the protein level has been reported to be very low in rodent islets. Gymnemic acid supplementation increased the expression levels of GPx there by corresponding to the increased activity of the enzyme. There are no earlier reports available to support our findings. However, Robertson et al., (2007) ⁹ reported that adenoviral overexpression of GPx increased GPx activity and protected islets against these adverse effects of ribose in HIT cell line.

CONCLUSION: The current study established that gymnemic acid supplementation was resistant to diet-induced anomalies in lipid metabolism, elevated first phase of GSIS in pancreatic islets and reduced oxidative stress in high fat diet and high fructose fed rat islets by modulating the protein expressions of GPx. These findings are important and serves as a gist because pancreatic beta cells play a key role in T2DM development. Further studies are warranted to understand the complete underlying mechanism.

ACKNOWLEDGEMENT: Sincere thanks to the authorities of University of Madras for the financial support in the UGC-UPE fellowship. Heartfelt thanks to the UGC authorities for the financial support in the form of BSR fellowship.

CONFLICT OF INTEREST: All the authors possess no conflict of interest in publication of this research work.

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    How to cite this article:

    Chakrapani LN and Periandavan K: Protective role of gymnemic acid in curbing high fat diet and high fructose induced pancreatic oxidative stress mediated type-2 diabetes in Wistar rats. Int J Pharm Sci Res 2018; 9(5): 2130-39. doi: 10.13040/IJPSR.0975-8232.9(5).2130-39.

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