Gymnemic acid, a potent antidiabetic agent protects skeletal muscle from hyperglycemia mediated oxidative stress and apoptotic events in High fat and High fructose diet fed adult rats

Type 2 diabetes is delineated by impaired metabolic lexibility, and intramyocellular lipid accumulation, causing insulin resistance, particularly in skeletal muscle by reducing insulin-stimulated glucose uptake. High-fat diet and high fructose (HFD and HF) administration in rodents bestows a model for hyperlipidemia, insulin resistance, and Type 2 diabetes. The current study is focused on elucidating the role of Gymnemic acid in combating hyperglycemia mediated oxidative stress and apoptotic events in the skeletal muscle of HFD and HF induced Type 2 diabetes in Wistar albino rats by boosting antioxidant defense system. Gymnemic acid, a saponin of triterpene glycoside contained in leaves ofGymnemaSylvestre, has potent anti-diabetic properties. Treatment with Gymnemic acid restored the antioxidant status (Gpx, SOD, CAT, GR, Vit C & Vit E) with signi icant (p<0.05) decrease in free radical levels and reinvigorated the expression of apoptotic and antiapoptotic proteins in Type 2diabetic rats. Histopathological data demonstrate that oral administration of Gymnemic acid protects skeletal muscle ibers from an oxidative niche in HFD and HF in Type 2 diabetic rats. In accordance with this, Gymnemic acid might be regarded as a promising therapeutic agent against Type 2 diabetes, thereby restoring skeletal muscle integrity and function.


INTRODUCTION
Diabetes mellitus (DM), a metabolic disorder pronounced by a rise in the concentration of glucose in the blood because of absolute (type 1) or proportionate (type 2) lack of insulin leading to hyper-glycemia. This is associated with the aberrant metabolism of protein, fat and carbohydrates (Sk, 2017). DM is one amongst the most important world health emergencies of the twenty-irst century. According to WHO estimates universally, 422 million adults over eighteen years of age group had diabetes in 2014 (World Health Organizations, 2016) of which, 90% are type 2 diabetic. This alarming increase of diabetes occurring in developing countries, which is mostly due to rising living standards, sedentary habits, lifestyle changes like obesity and unhealthy diets (Jayaprasad et al., 2018). Many factors are responsible for the cause of DM, including genetics, age, physical inactivity, obesity, autoimmunity, resistance to insulin, pancreatic β-cell defects, and low-grade in lammation (Leach, 2007).
Insulin resistance is broadly de ined as a decline in the ability of the body to utilize glucose from the blood in response to insulin (Turcotte and Fisher, 2008). Resistance by target tissues (Liver, Skeletal muscle, Adipose tissue) to insulin (Sampath et al., 2012) mediated by oxidative stress (Tiwari et al., 2014) contributed by hyperglycemia and hyperlipidemia (Lim et al., 2011) is the main physiological event causing type 2 DM. However, different types of orally ingested hypoglycemic agents (Biguanides, thiazolidinediones, sulphonylureas) are used along with insulin for DM treatment. Among them, Metformin, a biguanide antidiabetic drug potentiates insulin action, has been widely used for the treatment. The adverse effects of the above-mentioned agents instigate the researchers to identify naturally occurring products for their antidiabetic activity with little or no side effects (Dholi et al., 2014). One such plant herb is G. Sylvestre (Asclepiadaceae), is a perennial, woody climber with distinct medicinal properties found predominantly in the middle and peninsular regions of India. Sweet taste suppressing factor, a property unique to its leaves and therefore called as 'Gurmar' (sugar destroying), has been utilized for more than 2000 years in DM treatment in the form of the crude extract (Kapoor, 1990;Dixit and Pandey, 1984;Gupta, 1961;Jain and Sharma, 1967) in India. (Reddy et al., 1989). G. Sylvestre leaves are exploited as a diuretic, digestive, hypoglycemic, hypolipidemic, antiallergic, antiviral and antiobesity agent and also to treat dental caries (Leach, 2007).
The leaves contain the active compound Gymnemic acids, a triterpenoid Oleanane saponins which may possess the predicted antidiabetic property through inhibition of glucose absorption in the small intestine. The Gymnemic acids, a deacylgymnemic acid (DAGA) derivative, contain many acylated groups (methylbutyroyl, tiglolyl, etc.) (Gulab and Thakur, 2012), which has been described for its multifaceted antihyperglycemic agent with antiobesity effect (Zarrelli et al., 2013).
Diseases that adversely impact on skeletal muscle health such as DM may negatively affect progenitor cell quantity and/or functionality. Functional impairments, as shown by a reduction in muscle strength (Sugihara et al., 2000;Anderson et al., 2009), are strongly related to intramuscular fat storage. The impact of Gymnemic acid on skeletal muscle has not been investigated so far during the diabetic condition. Hence the current study is aimed to elucidate the protective activity of Gymnemic acid in skeletal muscle of HFD+HF induced Type 2 diabetic rats.

Laboratory Animals
Male Wistar Albino rats were acquired from Central Animal House facility, University of Madras, Taramani campus and experiments were conducted in consonance with guidelines authorized by the Institutional Animal Ethical Committee (IAEC No: 01/10/2016). Two animals per large cavernous cage were housed under controlled temperature (25 ± 2 • C) conditions with 12/12 h light/dark cycle and were provided with water and food ad libitum. The animals were kept on commercial rat feed (GOLD MOHUR RAT FEED) manufactured by Hindustan Lever Ltd., Mumbai. The feed contained 21% protein, 5% fat, 55% nitrogen-free extract, 4% ibre (w/w) with suf icient vitamins and minerals.

Experimental Design
Young rats of 4 months old weighing around 130-150g were used for this study. Animals were sorted out into 5 groups with six rats in each group as follows,

Group 1
Rats fed with normal water and standard rat chow ad libitum served as normal healthy control.

Group 2
HFD and HF (25%) in drinking water-fed rats for 75 days and the disease was con irmed by the elevated glucose levels on day 45, served as diabetic control.

Group 3
HFD and HF(25%) in drinking water-fed rats for 75 days and Gymnemic acid (150mg/kg body weight/day) was supplemented for the last 30 days orally.

Group 4
HFD and HF(25%) in drinking water-fed rats for 75 days and Metformin (50mg/kg body weight/day) was supplemented for the last 30 days orally served as drug positive control.

Group 5
Normal water and standard rat chow adlibitum fed rats and supplemented with Gymnemic acid (150mg/kg body weight/day) for 30 days orally served as drug control.
HFD comprised of normal rat chow and 4% cholesterol, 1.5% cholic acid and 30% coconut oil supplementation. Gymnemic acid (150 mg/kg body weight /day) was dissolved in physiological saline (0.89%) and administered by oral gavage. As the experimental period ended, rats were made anesthetized with ketamine (22mg/kg bw/ip) and soleus muscle was immediately excised, drenched in physiological saline (ice-cold) and 10% homogenate of the muscle tissue was prepared using 0.01M Tris-HCl buffer with a pH of 7.4 and centrifugation is done for about 10 min at 12,000 rpm and analysis of various parameters were carried on the supernatant. The rest of the tissue was stored at -80 • C for protein expression studies.

Fasting Blood Glucose Measurements
Measurement of blood glucose levels at overnight fasting state on lateral tail vein blood samples were carried out weekly with the help of a One-touch select simple meter and system (Life scan, Scotland) all over the treatment period.

Determination of Glycosylated Hemoglobin (HbA1c)
Glycosylated hemoglobin (HbA1c) levels were analyzed by hemoglobin analyzer (D-10-Biorad) based on the ion exchange HPLC method and the absorbance changes measured at 415nm. From the collected blood samples, the glycosylated haemoglobin were separated and its content was calculated by the glycosylated haemoglobin peak area to the total haemoglobin peak area ratio. Results are expressed in gm%.

Lipoprotein Pro ile
Triglycerides (TG), Total cholesterol (TC) and highdensity lipoproteins (HDL) in serum were analyzed using commercial kits purchased from spin react in semi-auto analyzer (Rx Monza, Randox, U.K). The amount of total cholesterol, triglycerides and HDL are indicated as mg/dl. Friedwald formula was used to calculate LDL cholesterol: LDL = Total cholesterol-(VLDL+ HDL cholesterol). VLDL (very low density lipoprotein) = TG/5. VLDL and LDL concentrations are shown as mg/dl.

Biochemical Assessment of Protein Carbonyl and Lipid Peroxidation
Protein carbonyl in skeletal muscle tissue was determined using 2,4-dinitrophenyl hydrazine (DNPH) with slight modi ications of Levine et al. (1990) method and expressed as nmols of protein carbonyl/mg protein/mL. The lipid peroxide levels was assayed by Devasagayam and Tarachand (Levine et al., 1990) method and expressed as nmols of MDA released/g of tissue.

Assessment of Enzymic and nonenzymic Antioxidants
The activity of the enzyme SOD was assessed by Marklund and Marklund (1974); Devasagayam and Tarachand (1987) method. The activity of SOD is the amount of enzyme inhibiting 50% pyrogallol auto-oxidation rate and was conveyed as Units/mg protein/min. Sinha (1972) method was used to assess the catalase activity and expressed as moles of H 2 O 2 consumed /min/ mg of protein. The modiied methodology of Rotruck et al. (1973) was used to assess the GPx activity and expressed as µmoles of GSH utilized/ minute. Determination of Reduced glutathione (GSH) was carried out by Moron et al. (1979) method and was expressed as µg/mg protein. Vitamin C and Vitamin E were estimated by Roe and Kuether (1943); Jargar et al. (2012) method, respectively and expressed in µg/mg of protein.

Immuno Blot Analysis
Skeletal muscle tissue samples were homogenized in Tris-HCl buffer (0.01 M, pH 7.4) with 1mM Phenylmethylsulfonyl luoride as a protease inhibitor and protein concentrations were estimated using Lowry et al. (1951). Immunoblotting samples containing 50µg of proteins were separated by SDS−PAGE on 10% Polyacrylamide gels, followed by its transfer to polyvinyl di luoride membrane (PVDF). The membranes were incubated with primary antibodies (Bax, Bcl-2, and Cytochrome C) overnight at 4 • C and incubated 1 hour with horseradish peroxidase (HRP) conjugated secondary antibodies next day. Detection of Protein bands were done by standard, enhanced chemiluminescence (ECL) method in a Biorad Chemidoc XRS imaging system.

Statistical Analysis
The results are presented as the mean ± standard error of the mean (SEM) obtained from the average of three or six independent experiments. Analysis of Variance with one way ANOVA was done to analyze the differences between segregated groups using the Graph pad prism version 5.0 software package. Intergroup comparison testings were performed by Tukey's Post hoc testing. Values are signi icant sta- tistically at the level of p < 0.05.

RESULTS AND DISCUSSION
In luence of Gymnemic acid on body weight changes, fasting blood glucose (FBG), and glycosylated hemoglobin (HbA1c) in Type 2 diabetic rats Figure 1 A shows a signi icant increase (26%) of body weight in DM induced (group II) animals when compared to that of normal control rats (group I) (p<0.05). Treatment with Gymnemic acid or metformin (group III or group IV) profoundly show a signi icant decrease (18% and 14%) in body weight compared with that of group II diabetic rats (P<0.05). Figure 1 B shows fasting blood glucose levels estimated at various intervals during the experimental period. Levels of fasting blood glucose at the initial day were around 70 -90 mg/dl in control and experimental animals. At the end of 45 days HFD and HF fed group II, III and IV animals showed a striking increase in the levels of the blood glucose 161 mg, 145 mg and 150 mg/dl when compared to that of the initial day 75 mg, 84 mg and 79 mg/dl respectively. Upon treatment with Gymnemic acid (group III) or metformin (group IV) from 45th day for 30 days lead to the decrement of blood glucose levels to 92 mg and 98 mg/dl signi icantly (P<0.05) when compared to that of diabetic control rats with 254 mg/dl (group II). No marked changes were noticed between normal control (group I) and drug control rats (group V). Figure 1 C depicts the glycosylated Hb levels in control and experimental animal models. About 50% increase in glycosylated Hb was recorded in HFD+HF fed animals at the end of the experimental period, while Gymnemic acid or metformin-treated groups explicitly showed a signi icant decrease in HbA1C levels, even though they did not revert to the normal range due to 30 days of short term treatment.  Table 1 depicts the in luence of Gymnemic acid or metformin on the serum lipid pro ile of control and experimental animal groups. Assessment of serum lipid pro ile reveals that a signi icant (p < 0.05) increase in the levels of serum triglycerides, LDL, VLDL, total cholesterol and a concomitant decrease in HDL was observed in DM induced group II compared to that of normal control rats. Gymnemic acid treatment to HFD and HF fed rats demonstrated a striking decrease (p < 0.05) in serum triglycerides, LDL, VLDL, total cholesterol with a concomitant increase (p < 0.05) in HDL, when compared to group II, moreover it was observed that Gymnemic acid was almost equally bene icial on reducing lipid levels in the serum compared to metformin.

Effect of Gymnemic acid on Lipid Pro ile in Type 2 diabetic rats
In Table 1 shows, Group I-control rats; Group IIdiabetic rats; Group III-Gymnemic acid-treated diabetic rats; Group IV-Metformin treated diabetic rats; Group V-Gymnemic acid alone treated rats. Values are expressed as mean ± SEM for six animals in each group. Values are statistically signi icant at the level of p < 0.05. Where a, compared with Group I; b, compared with Group II rats. HFD and HF fed rats (group II) demonstrated a 2 fold elevation in the free radicals level (p<0.05) when compared to control animals (group I). Administration of Gymnemic acid or metformin to HFD and HF fed rats decreased the levels of free radicals generated by 1.5 fold and 1.4 fold, respectively. Figure 3 A shows the impact of Gymnemic acid or metformin

Figure 1: In luence of Gymnemic acid on body weight, fasting blood glucose levels and serum glycosylated Hb in Type 2 diabetic rats.
on the levels of MDA in the muscle of control and experimental rats. Assessment of MDA levels were elevated in HFD and HF fed rats by about 3.5 fold when compared with that of control rats. On comparison with group II rats, the MDA levels were evidently brought down by 3.2 and 3.0 folds, respectively, upon Gymnemic acid or metformin treatment (group III or group IV). Figure 3 B shows the protein carbonyl levels in the skeletal muscle of control and experimental rats. HFD and HF (Group II) fed rats showed a signi icant increase (1.9 fold) in the protein carbonyls level when compared to that of control rats. Treatment with Gymnemic acid or metformin signi icantly brought down the protein carbonyl levels in HFD and HF fed rats by 1.4 and 1.2 fold correspondingly.   experimental groups showed a decreasing trend in group II rats by 60%, 44%, 46% respectively comparing with control rats (group I). Gymnemic acid or metformin-treated rats had shown a signi icant increase (p<0.05) in these antioxidant status.  (Group III) show narrower intercellular spaces between the skeletal muscle ibers, which appear relatively normal on comparison with the control group. Metformin treated rats (Group IV) display narrower intercellular spaces between the skeletal muscle ibers with a central nucleus. Gymnemic acid alone treated rats (Group V) show longitudinal muscle ibers exhibiting peripheral nuclei, which is similar to that of control rats. ing narrower intercellular spaces (arrow) between the skeletal muscle ibers with the central nucleus (arrow). Group V (Gymnemic acid control) showing longitudinal muscle ibers exhibiting peripheral nuclei (arrows) look like control.

Antiapoptotic potency of Gymnemic acid in HFD and HF fed Type 2 Diabetic Rats
ROS act as potential mediators of glucose-induced apoptosis, which involves the response of apoptotic proteins (Bax and Bcl-2) and initiated by caspase 3 cleavage activation. Immunoblot analysis depicts that Bax expression (2.2 fold) and released cytosolic Cyt C levels (2.8 fold) were signi icantly high in the HFD and HF fed (group II) diabetic skeletal muscle ( Figure 6). However, treatment with Gymnemic acid or metformin signi icantly decreased the expression of Bax by 1.7 and 1.6 fold, respectively; Cyt C by 1.5 fold and 1.6 fold, respectively (Figure 6 A and Figure 6 C). The current study exhibited that HFD and HF induced diabetic conditions significantly decreased Bcl-2 expression to 1.6 fold (Figure 6 B), which upon Gymnemic acid or metformin treatment showed a 1.5 fold signi icant increase. Figure 6 shows, (A) Bax (B) Bcl-2 and (C) Cytochrome C protein expressions are shown in the blot. β-actin was used as an internal control. * Represent the non-speci ic bands of Bcl-2. Densitometry analysis of each blots are shown. Group I-control rats; Group II-diabetic rats; Group III-Gymnemic acid-treated diabetic rats; Group IV-Metformin treated diabetic rats; Group V-Gymnemic acid alone treated rats. Values are expressed as mean ± SEM for three animals in each. Values are statistically signi icant at the level of p < 0.05.

Figure 6: Impact of Gymnemic acid on the protein expression of Bax and Bcl-2 in skeletal muscleof Type 2 diabetic rats
Where a, compared with Group I; b, compared with Group II rats.
Skeletal muscle employs both glucose and free fatty acid (FFA) as sources of fuel for energy production (Lowry et al., 1951). Thus, glucolipotoxicity causes obesity-related Type 2 diabetes in skeletal muscle. Our study showed raise in body weight, fasting blood glucose, glycosylated hemoglobin and elevation of lipids and lipoproteins except for HDL in HFD and HF fed rats when compared to that of control rats establishing glucolipotoxicity. These results also demonstrate that the rats are hyperglycemic and hyperlipidemic, a typical biochemical picture associated with Type 2 diabetes. Our results are supported by Singh et al. (2017); Abdul-Ghani and Defronzo (2010), who have demonstrated that HFD fed rats for four weeks provoked obesity and dyslipidaemia. Gymnemic acid or metformin treatment in group III or group IV rats in our study showed a signi icant decrease in body weight, fasting blood glucose and lipid pro ile. (Kim et al., 2017;Singh et al., 2017) have proved that mice fed with G. Sylvestre extract (500 mg/kg) demonstrate less food and energy ef iciency ratios and less body weight gain when scrutinized with the HFD alone fed group.
Our results showed a remarkable increase in blood glucose levels upon HFD and HF feeding for 75 days in Group II rats, which is favored by the studies of (Nampurath et al., 2008;Woods et al., 2003;Kim et al., 2017). Gymnemic acid lowered blood glucose levels, which might be mediated by its ability to inhibit the enzymes involved in the digestion of carbohydrates and delaying of absorption of glucose in the intestine (Nirmala et al., 2016). In a study by (Baskaran et al., 1990;Nakamura et al., 1999) 22 DM patients (type 2) were administered orally with GS 4 extract (ethanolic extract of G. Sylvestre leaves) at 400 mg/day dose showed a substantial fall of glucose in the blood, glycosylated plasma proteins and glycosylated hemoglobin. The current study shows the signi icantly elevated levels of Glycosylated hemoglobin in HFD and HF (group II) rats and after Gymnemic acid treatment, these levels were reverted back to the normal.
We observed the signi icant restoration of amended lipid pro ile and circulating free fatty acids after administration of Gymnemic acid (group III) to type 2 diabetic rats is consistent with our previous studies by (Narasimhan and Kalaiselvi, 2018), which supported that Gymnemic acid administration to HFD and HF diet-induced Type 2 diabetic rats lowered the circulatory cholesterol, triglycerides, LDL and VLDL levels and also up-regulated HDL levels in serum. These results exhibit the antihyperlipidemic effect of Gymnemic acid. Wang et al. (1998) have also shown that Gymnemic acid inhibited potently oleic acid absorption in the small intestine.
Oxidative stress at the molecular level occurs when elevated ROS overcome the antioxidant defense capabilities systems. Chronic hyperglycemia increases the polyol pathway, formation of advanced glycation end products (AGE) and free radical generation rates. Hence we measured free radical levels such as hydroxyl radicals, hydro-gen peroxide and superoxide radicals. Our study established a notable increase in free radicals level in skeletal muscle of the HFD and HF fed rats. Our results were in corroboration with indings of (Selvaraj et al., 2016;Wang et al., 1998) in which diabetic rats exhibited elevated levels of H 2 O 2 .OH, and LPO in the liver due to HFD and dihydroxy gymnemic triacetate treatment reduced the free radical levels. Hence our study represents that Gymnemic acid prevents hyperglycemia-induced advanced glycation end products (AGE) formation. Similarly, (Kang et al., 2012;Shafey et al., 2013) have reported a decline in the level of plasma lipid peroxidation and increase in superoxide dismutase level in rats treated with G. Sylvestre leaves extract after STZ-diabetic induction when compared to untreated diabetic rats by either directly decomposing the reactive oxygen species or by increasing the generation of antioxidant molecules. In order to authenticate the ef icacy of Gymnemic acid on treating oxidative stress, we assessed the histopathology of skeletal muscle tissue. The histopathological observation showed a wide separation of skeletal myocytes and fragmentation of the skeletal muscle ibers in HFD and HF fed (group II) rats. This might be due to increased lipid peroxidation and protein carbonyls leading to myo ibrillar fragmentation. Treatment with Gymnemic acid narrowed intercellular myo ibrils and reduced signi icant pathological changes induced by hyperglycemia.
Our current indings shows that the antioxidant defense of control and experimental groups indicates that feeding HFD and HF reduce antioxidant enzyme activity and there was a marked increase in the activity of enzymes treated with Gymnemic acid suggesting the high antioxidant potential of this bioactive compound. Ohmori et al. (2005); Vasi and Austin (2009) have demonstrated that G. Sylvestre exhibited antioxidant potential over free radicals and oxidation of LDL in healthy volunteers. Supplementing diets incorporated with G. Sylvestre to experimental Wistar rats led to a signi icant increase in GSH levels, indicating that the leaves and bark have a free radical scavenging capacity, thereby reducing oxidative stress (Ohmori et al., 2005).
The surplus production of ROS triggers hyperglycemia mediated apoptosis. Apoptosis is inhibited by Bcl-2, a major anti-apoptotic protein by forbidding the mitochondrial discharge of cyt C, inally resulting in caspase activity inhibition (Chauhan et al., 2014). Lipid deposition in skeletal muscle can promote lipotoxicity and, as a result, lipoapoptosis. Our results demonstrate signi icantly increased expression of Bax and released cytosolic cyt C and decreased expression of Bcl-2 in the skeletal mus-cles of HFD and HF fed (group II) rats. (Li et al., 2004;Kirkin et al., 2004) have shown that increased Bax expression in the kidney cortex of diabetic experimental animal models. Sekiguchi et al. (2004) have elucidated apoptosis in endothelial cells in vitro by high glucose induction. Mizutani et al. (1998) have indicated low expressions of Bc1-2 in adult retinas of diabetic humans. Upon Gymnemic acid or metformin treatment in group III or group IV in the present study, the expressions of Bax, Bcl-2 and Cyt-C were reversed. These indings were coherent with the following observation. The antiapoptotic activity of Gymnemic acid by (Mizutani et al., 1998;Pathan et al., 2012) have explicated that Gymnemic acid phospholipid Complex's supplementation ef iciently suppressed cardiomyopathy induced by Doxorubicin. Our study showed prominently the antiapoptotic potency of Gymnemic acid in skeletal muscle against HFD and HF induced type 2 diabetes.

CONCLUSIONS
The above indings indicate that Gymnemic acid attenuated Type 2 diabetes induced by HFD and HF through the amelioration of oxidative stressmediated hyperglycemia-induced apoptosis. The Gymnemic acid maintains normoglycemia during diabetes, triggers not only liver and kidney to revert to their normal metabolic homeostasis but also skeletal muscle. Hence, Gymnemic acid may be considered as an adjuvant therapeutic agent for DM. However, studies are greatly needed further to establish its molecular mechanism of antidiabetic potency.