Glucose uptake through translocation and activation of GLUT4 in PI3K/Akt signaling pathway by asiatic acid in diabetic rats

Abstract

In this study, we examined the in vivo effect and the mechanism of asiatic acid (AA) on glucose uptake in an insulin target skeletal muscle. Diabetic rats showed significantly increased levels of plasma glucose, thiobarbituric acid reactive substances, and lipid hydroperoxides, decreased levels of insulin and antioxidants, and impairment in insulin-signaling proteins such as insulin receptor (IR), insulin receptor substrate (IRS)-1/2, phosphoinositide 3-kinase (PI3K), Akt, and glucose transporter 4 (GLUT4) proteins. Oral treatment with AA (20 mg/kg body weight) showed near-normalized levels of plasma glucose, lipid peroxidation products, and antioxidants and improved insulin, IR, IRS-1/2, PI3K, Akt, and GLUT4 proteins. These findings suggest that AA improves glucose response by increasing GLUT4 in skeletal muscle through Akt and antioxidant defense in plasma and it also improves glucose homeostasis.

Keywords

Asiatic acid streptozotocin glucose transporter insulin signaling protein antioxidant

Introduction

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both.¹ The International Diabetes Federation has projected that the number of people with diabetes in the world will increase from 382 million in 2013 to 592 million by 2035, with 80% of cases occurring in low-income and middle-income countries.² Insulin has several effects in skeletal muscle, but its stimulating effect on glucose metabolism is of prime importance in relation to diabetes. Upon binding to its receptor, insulin facilitates glucose uptake through a distinct signaling cascade involving multiple enzymes of which the proteins phosphoinositide 3-kinase (PI3K) and Akt (also known as “protein kinase B”, PKB) represent key nodes. Akt is activated by phosphorylation at two specific sites (Thr308 and Ser473) by a PI3K-dependent mechanism.³ Akt stimulates translocation of the glucose transporter 4 (GLUT4), and thereby glucose uptake, through the phosphorylation of Akt substrate of 160 kDa. Studies have shown that insulin stimulates glucose uptake primarily by inducing the GLUT4 translocation to the plasma membrane rather than increasing the intrinsic activity of the transporter protein.⁴ An absolute or relative lack of insulin, as in case of diabetes, leads to severe dysfunction and deregulation of insulin signaling pathway leading to a concomitant rise in blood glucose level and hence to diabetes.⁵ Many compounds that induce exocytosis and/or decreases the endocytosis will result in increased GLUT4 expression on the plasma membrane and ultimately enhance glucose absorption. In this context, several naturally occurring triterpenoids have been shown to affect glucose uptake and insulin receptor function, both of which play an essential role in diabetes. Thus, it has become clear that triterpenoids may exert their glucose-lowering effect through multitarget pathways.

Centella asiatica (L.) Urban, has been reported to possess antileprotic, antitumor, antistress, wound healing, antibacterial, and antidiabetic properties.⁶ This plant is also used as tonic in several Ayurvedic formulations.⁷ Cravotto et al. carried out clinical trials on plant extract C. asiatica and ascertained its beneficial effects on hypertension/diabetic complications.⁸ The plant C. asiatica contains huge amount of asiatic acid (AA) and its alcoholic extracts in humans predicts a detectable concentration of AA in plasma. AA is a metabolite of asiaticoside and by the hydrolytic cleavage of the sugar moiety it becomes AA, which is responsible for the therapeutic effects and it clearly delineates the pharmacokinetic nature of AA.⁹ Recent study in our laboratory reported that AA improves the level of plasma insulin, decreases glucose level, reverses the changes in the levels of the key carbohydrate-metabolizing enzymes¹⁰, prevents lipid peroxidation, and improves antioxidant status and lipid metabolic enzymes in rats with streptozotocin (STZ)-induced diabetes.^11,12 The purposes of this study were to evaluate the effects of AA on plasma glucose and insulin signaling cascade proteins and to explore the possible related mechanisms using an STZ-induced diabetic rat model.

Materials and methods

Chemicals and reagents

STZ and AA were purchased from Sigma-Aldrich Co (St Louis, Missouri, USA). Insulin receptor (IR), insulin receptor substrate (IRS-1), IRS-2, PI3K, Akt, GLUT 4, and β-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Anti-rabbit secondary antibody was purchased from GeNei (Bangalore, Karnataka, India).

All other chemicals and solvents were of analytical grade purchased from S.D. Fine Chemicals, Himedia, and Sisco Research Laboratories Pvt. Ltd (Mumbai, Maharashtra, India).

Animals

Male Wistar rats weighing 180–200 g were procured from Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, India, and maintained in an air-conditioned room (25 ± 1°C) with a 12-h light/12-h dark cycle. Food and water were provided ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital Annamalai University, India (reg no: 160/1999/CPCSEA, proposal no: 848).

Induction of experimental diabetes

Experimental diabetes was induced in 12-h fasted rats by single intraperitoneal (i.p.) injection of STZ (40 mg/kg body weight (b.w.)) dissolved in cold citrate buffer (0.1 M, pH 4.5). STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemia. STZ-injected animals exhibited hyperglycemia within a few days. Diabetic rats were confirmed by measuring the elevated plasma glucose (by glucose oxidase method), 72 h after injection with STZ. The animals with glucose levels above 235 mg/dl were selected for the experiment.

Experimental design

The experimental animals were divided into five groups; each group consisted of six rats. AA were dissolved in 5% dimethyl sulfoxide and glibenclamide was diluted in water and administered orally to experimental groups using intragastric tube daily for a period of 45 days.

Group I: normal control rats

Group II: normal + AA (20 mg/kg b.w.)

Group III: diabetic control rats

Group IV: diabetic + AA (20 mg/kg b.w.)

Group V: diabetic + glibenclamide (600 µg/kg b.w.)

After 45 days of treatment, the animals were anesthetized between 8:00 a.m. and 9:00 a.m. using ketamine (24 mg/kg b.w. i.p.) by sacrificed cervical decapitation. Blood samples were collected in tubes containing ethylenediaminetetraacetate (a salt of ethylenediaminetetraacetic acid (EDTA)). The plasma was obtained after centrifugation (313 × g for 10 min at 4°C) and used for various biochemical measurements. After the separation of plasma, the buffy coat enriched with white cells was removed and the remaining erythrocytes were washed three times with physiological saline. A known volume of erythrocyte was lysed with hypotonic phosphate buffer solution at pH 7.4. The hemolysate was separated by centrifugation at 489 × g for 10 min, and the supernatant was used for the estimation of enzymatic antioxidants. The fresh skeletal muscle tissue was excised immediately and used for performing Western blotting.

Biochemical estimation

Estimation of plasma glucose and insulin

Plasma glucose was estimated using a commercial kit (Sigma Diagnostics Pvt. Ltd, Baroda, Gujarat, India) by the method followed by Trinder.¹³ Insulin in the rat plasma was assayed by the solid phase system amplified sensitivity immunoassay using reagent kits obtained from Medgenix INS-ELISA, Biosource, Europe S.A., Nivelles, Belgium by the method of Burgi et al.¹⁴

Estimation of lipid peroxidation in plasma

Lipid peroxidation in plasma was estimated spectrophotometrically by measuring thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) by the method of Fraga et al.¹⁵ and Jiang et al.,¹⁶ respectively. In brief, plasma (0.1 ml) was treated with 2 ml of thiobarbituric acid (TBA)–trichloroacetic acid (TCA)–hydrochloric acid (HCl) reagent (0.37% TBA, 0.25 N HCl, and 15% TCA, 1:1:1 ratio) placed in a water bath for 15 min and cooled and centrifuged at room temperature; clear supernatant was measured at 535 nm against a reagent blank.

Then, 0.1 ml aliquot of plasma was treated with 0.9 ml of Fox reagent (88 mg of butylated hydroxytoluene, 7.6 mg of xylenol orange, and 0.8 mg of ammonium iron sulfate were added to 90 ml methanol and 10 ml of 250 mM sulfuric acid) and incubated at 37°C for 30 min. The color that developed was read at 560 nm colorimetrically.

Estimation of enzymatic antioxidants in plasma

Superoxide dismutase activity (SOD) was determined by the method described by Kakkar et al.¹⁷ A single unit of enzyme was expressed as 50% inhibition of nitroblue tetrazolium reduction per minute per milligram protein. Catalase (CAT) was assayed colorimetrically at 620 nm and expressed as l mol of hydrogen peroxide (H₂O₂) consumed per minute per milligram protein as described by Sinha.¹⁸ The reaction mixture (1.5 ml) contained 1.0 ml of 0.01 M pH 7.0 phosphate buffer, 0.1 ml of sample, and 0.4 ml of 2 M H₂O₂. The reaction was carried out by the addition of 2.0 ml of dichromate–acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed at a 1:3 ratio).

Glutathione peroxidase (GPx) activity was measured by the method described by Rotruck et al.¹⁹ Briefly, the reaction mixture contained 0.2 ml of 0.4 M phosphate buffer with pH 7.0, 0.1 ml of 10 mM sodium azide, 0.2 ml of sample, 0.2 ml glutathione, and 0.1 of 0.2 mM H₂O₂. The content was incubated at 37°C for 10 min. The reaction was arrested by 0.4 ml of 10% TCA and centrifuged. Supernatant was assayed for glutathione content using Ellmans reagent.

Estimation of nonenzymatic antioxidants in plasma

Reduced glutathione (GSH) was determined by the method of Ellman.²⁰ To the homogenate, 10% TCA was added and centrifuged. One milliliter of the supernatant was treated with 0.5 ml of Ellmans reagents (19.8 mg of 5,5′-dithiobis-(2-nitrobenzoic acid) in 100 ml of 0.1% sodium nitrate) and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm.

Plasma vitamin E was determined by the method described by Baker et al.²¹ A portion of the sample (0.1 ml), 1.5 ml of ethanol, and 2 ml of petroleum ether were added, mixed, and centrifuged for 160×g for 10 min. The supernatant was evaporated to dryness at 80°C, then 0.2 ml of 2,2′-dipyridyl solution and 0.2 ml of ferric chloride solution were added and mixed well. This was kept in the dark for 5 min and then 2 ml of butanol was added. Absorbance was read at 520 nm.

Ascorbic acid (vitamin C) concentration was measured using the method of Omaye et al.²² To 0.5 ml of plasma, 1.5 ml of 6% TCA was added, left aside for 5 min and then centrifuged at low speed. To 0.5 ml of supernatant, 0.5 ml of 2,4-dinitrophenylhydrazine (DNPH) reagent (2% DNPH and 4% thiourea in 9 N sulfuric acid) was added and incubated for 3 h at room temperature. After incubation, 2.5 ml of 85% sulfuric acid was added, and the color developed was read at 540 nm after 30 min.

SDS-PAGE and Western blot analysis

Western blotting was performed to analyze the expression pattern of IR, IRS-1, IRS-2 Akt, PI3K, and GLUT-4 by Laemmli.²³ The skeletal muscle tissue samples was homogenized in an ice-cold radioimmunoprecipitation assay buffer (1% Triton, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 mmol/l EDTA, 20 mmol/l Tris (pH 7.4), 150 mmol/l sodium chloride, 10 mmol/l sodium fluoride, and 0.1 mmol/l phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 1252 × g for 15 min at 4°C to remove debris. Protein concentration was measured by the method described by Lowry et al.²⁴ Samples containing 20 µl (50 µg of protein/well) of total cellular proteins were loaded and separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE). The resolved proteins were blot transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, Massachusetts, USA). The membranes were incubated with the blocking buffer containing 5% nonfat skim milk or bovine serum albumin (BSA) for 2 h to reduce nonspecific binding sites and then incubated with β-actin (rabbit polyclonal; 1:500 dilution in 5% BSA in Tris-buffered saline and 0.05% Tween-20 (TBST), anti-mouse GLUT-4 (monoclonal; 1:1000), anti-rabbit IRS-1 (monoclonal; 1:1000), anti-rabbit IRS-2 (polyclonal; 1:1000), and anti-rabbit Akt (monoclonal; 1:1000) with gentle shaking overnight at 4°C. After this, the membranes were incubated with their corresponding secondary antibodies (anti-rabbit immunoglobulin G conjugated to horseradish peroxidase) for 2 h at room temperature. Membranes were washed thrice with TBST for 30 min. Protein bands were visualized by an enhanced chemiluminescence method using an electrochemiluminescence kit. Bands were scanned using a scanner and quantitative by Image J, a public domain Java image processing software (Wayne Rasband, NIH, Bethesda, Maryland, USA), control of which was set to 1.

Statistical analysis

All the experimental data were statistically evaluated with SPSS version 16.0 (SPSS Inc, Cary, North Carolina, USA) software and expressed as mean ± standard deviation for six rats in each group. Hypothesis testing methods included one-way analysis of variance followed by least significant difference test. Values of p < 0.05 were considered to point out statistical significance.

Results

Table 1 epitomizes the levels of plasma glucose and insulin in normal and experimental rats. Diabetic rats were significantly increased in the level of plasma glucose and decreased insulin in diabetic rats compared with control rats. Oral administration of AA as well as glibenclamide to diabetic rats significantly normalized the altered levels of plasma glucose and insulin when compared with diabetic rats.

Table 1.

Effect of AA on plasma glucose and insulin in control and experimental rats.

Groups	Glucose (mg/dl)	Insulin (μU/ml)
Normal control	81.02 ± 5.24	15.02 ± 1.0
Normal + AA (20 mg/kg b.w.)	83.39 ± 6.21	13.54 ± 1.05
Diabetic control	240.36 ± 12.47^*	5.99 ± 0.47^*
Diabetic + AA (20 mg/kg b.w.)	103.11 ± 7.89^#	11.35 ± 1.22^#
Diabetic + glibenclamide (600 μg/kg b.w.)	95.74 ± 5.76^##	12.87 ± 1.43^##

AA: asiatic acid; b.w.: body weight; ANOVA: analysis of variance; LSD: least significant difference.

Data were given as mean ± standard deviation for six animals in each group. One-way ANOVA is followed by post hoc test LSD. Values are statistically significant at p < 0.05.

^*Diabetic control rats were compared with normal rats.

^#AA-treated diabetic rats were compared with diabetic control rats.

^##Glibenclamide-treated diabetic rats were compared with diabetic control rats.

Table 2 shows the levels of TBARS, LOOH, SOD, CAT, GPx, GST, vitamin C, vitamin E, and GSH in normal and experimental rats. Diabetic rats exhibited increased levels of TBARS and LOOH and significant decrease in SOD, CAT, GPx, GST, vitamin C, vitamin E, and GSH when compared with normal control. Conversely, administration of AA as well as glibenclamide to diabetic rats significantly increased the levels to near control values.

Table 2.

Effect of AA on TBARS, LOOH, nonenzymatic antioxidant in plasma and enzymatic antioxidant in erythrocytes of normal and experimental rats.

Groups	TBARS (mmoles/dl)	LOOH (mmoles/dl)	SOD (U^**/mg Hb)	CAT (U^$/mg Hb)	GPx (U^@/mg Hb)	Vitamin C (mg/dl)	Vitamin E (mg/dl)	GSH (mg/dl)
Normal control	0.20 ± 0.01	9.52 ± 0.63	6.78 ± 0.42	170.65 ± 9.16	14.28 ± 1.10	2.08 ± 0.15	1.65 ± 0.10	25.75 ± 1.81
Normal + AA (20 mg/kg b.w.)	0.18 ± 0.01	9.30 ± 0.79	6.93 ± 0.15	174.10 ± 7.24	14.49 ± 0.96	2.18 ± 0.17	1.63 ± 0.48	27.11 ± 1.56
Diabetic control	0.33 ± 0.02^*	24.91 ± 1.16^*	4.01 ± 0.18^*	101.41 ± 7.24^*	6.91 ± 0.53^*	0.95 ± 0.05^*	0.72 ± 0.15^*	14.14 ± 0.9^*
Diabetic + AA (20 mg/kg b.w.)	0.24 ± 0.02^#	11.15 ± 0.84^#	5.91 ± 0.26^#	145.84 ± 8.11^#	12.26 ± 1.20^#	1.75 ± 0.12^#	1.34 ± 0.16^#	19.90 ± 1.12^#
Diabetic + glibenclamide (600 μg/kg b.w.)	0.23 ± 0.02^##	10.35 ± 0.80^##	6.10 ± 0.28^##	151.67 ± 9.84^##	13.70 ± 1.00^##	1.83 ± 0.14^##	1.40 ± 0.13^##	21.04 ± 1.36^##

AA: asiatic acid; TBARS: thiobarbituric acid reactive substance; LOOH: lipid hydroperoxide; SOD: superoxide; CAT: catalase; GPx: glutathione peroxidase; b.w.: body weight; GSH: glutathione.; U**: enzyme concentration required to inhibit the chromogen produced by 50% in 1 min under standard condition; U^$: micromolar hydrogen peroxide decomposed per minute; U^@: micromolar GSH utilized per minute; ANOVA: analysis of variance; LSD: least significant difference.

Data were given as mean ± standard deviation for six animals in each group. One-way ANOVA is followed by post hoc test LSD. Values are statistically significant at p < 0.05.

^*Diabetic control rats were compared with normal rats.

^#AA-treated diabetic rats were compared with diabetic control rats.

^##Glibenclamide-treated diabetic rats were compared with diabetic control rats.

The expressions of IR, IRS-1, IRS-2, PI3K, Akt, and GLUT-4 proteins in skeletal muscle of control and experimental rats by Western blot analysis are illustrated in Figures 1 and 2. Densitometric analysis of Western blots are given in Figures 1 and 2. The skeletal muscles showed downregulated expression of IR, IRS-1, IRS-2, PI3K, Akt, and GLUT-4 protein in STZ-induced diabetic rats as compared to normal rat. Administration of AA to STZ-induced diabetic rats showed upregulated expression of this protein in skeletal muscles. The results are expressed as density ratio to β-actin.

Figure 1.

Effect of AA on IR, IRS-1, IRS-2 Akt and PI3K protein expression in the skeletal muscle of control and experimental rats. Histograms depicted quantitation of six independent experiments (means ± S.D), with data normalized by defining the control group with IR, IRS-1, IRS-2 Akt and PI3K protein, as 1 unit. NC: normal control; AA: asiatic acid; DC: diabetic control. *p ≤ 0.05: compared with control rats Duncan's Multiple Range Test (DMRT); ^# p ≤ 0.05: compared with diabetic rats (DMRT). IR: insulin receptor; PI3K: phosphoinositide 3-kinase.

Figure 2.

Effect of AA on GLUT4 in the skeletal muscle of control and experimental rats. Histograms depicted quantitation of six independent experiments (means ± S.D), with data normalized by defining the control group with GLUT4 protein, as 1 unit. NC: normal control; AA: asiatic acid; DC: diabetic control. *p ≤ 0.05: compared with control rats (DMRT); ^# p ≤ 0.05: compared with diabetic rats (DMRT). GLUT4: glucose transporter 4.

Discussion

Insulin-producing β cells that do not express the glucose transporters are resistant to STZ. This observation also explains the greater toxicity of STZ compared with N-methyl-N-nitrosourea in cells that express GLUT2, even though both substances alkylate DNA to a similar extent. Furthermore, STZ induces activation of polyadenosine diphosphate ribosylation and nitric oxide release. The importance of the GLUT2 glucose transporter in this process is also shown by the observation that STZ damages other organs expressing this transporter, particularly kidney and liver. Among the insulin target tissues, skeletal muscle is responsible for more than 75% of glucose disposal in response to insulin in the postprandial state.²⁵ Regulation of glucose metabolism of skeletal muscle is therefore quantitatively most important in energy balance and is the primary tissue of insulin-stimulated glucose uptake, disposal, and storage.²⁶ A wide array of plant-derived active principles has been reported with beneficial effect on glucose transport and metabolism in skeletal muscle.²⁷

It has been well established that lipid peroxidation increases under the diabetic pathological conditions, since diabetes is related to oxidative stress. Diabetes is marked by increased production of free radicals or impaired antioxidant defenses. The generation of SOD anion radicals by glucose oxidization and its dismutation to H₂O₂ leads to the formation of reactive hydroxyl radicals.²⁸ This study has revealed that the increased blood glucose and decreased serum insulin levels is closely associated with the elevated lipid peroxidation. The reactive oxygen species (ROS)-scavenging capacity by antioxidants is decreased in diabetes such that constant oxidative stress develops and oxidation of lipids, proteins, and other macromolecules such as DNA is increased. Augmentation of plasma antioxidative capacity would also attenuate lipid peroxidation through this mechanism.²⁹

Oxidative stress occurs when ROS production is excessive and/or defense mechanisms fail. ROS are kept under control by an endogenous system of protection that consists of scavenger enzymes (SOD, CAT, and GPx) and nonenzymic antioxidants (vitamin E, ascorbic acid, and GSH).³⁰ Oxidative stress is closely related with both diabetic animal models and humans.³¹ Concerning to the changes in lipid peroxidation, the diabetic tissue showed decreased activity of the key antioxidants SOD, CAT, and GPx, which plays an important role in scavenging the toxic intermediate of incomplete oxidation. The decrease in the activity of these antioxidants can lead to an excess availability of the SOD anion and H₂O₂ in biological systems, which in turn generate hydroxyl radicals resulting in initiation and propagation of lipid peroxidation.³² Nonenzymatic antioxidants such as vitamins C, vitamins E, and GSH are known to be decreased in diabetes state because of their free radical scavenging property.³³ These vitamins also directly scavenge ROS and upregulate the activities of antioxidant enzymes.³⁴ In this study, elevated lipid peroxidation levels and reduced antioxidants enzymes levels were observed in STZ-induced diabetic rats compared with the control rats. These changes may be due to the glucose oxidation, formation of free radical generation, and nitric oxide donor property of STZ.³⁵ In our study, administration of AA to diabetic rats showed restoration of the levels of enzymatic and nonenzymatic antioxidants with reduced lipid peroxidation by-products. This may be attributed to the free radical scavenging and antidiabetic activities of the AA.

Oxidative stress has been reported to impair insulin sensitivity in skeletal muscle.³⁶ STZ-induced diabetes diminution in IR protein is associated with decreased IRS-1 protein. Since degradation of IRS-1 by ROS has been reported by Newsholme et al.³⁷ STZ-induced ROS produced probably degraded the IRS-1 in skeletal muscle. Stimulation of oxidants such H₂O₂ to muscle cells blocks insulin-induced glucose uptake and GLUT4 translocation by impairment of IR activation and PI3K/Akt signaling.^38,39 Interestingly, oxidative products are elevated in the muscles of patients with type 2 diabetes.³⁶

IRS 1 and 2 play crucial and complementary roles in insulin signaling and exhibit differential regulation and roles.⁴⁰ IRS 1 is regulated at the protein level and is reported to be more closely linked to glucose homeostasis such as suppression of gluconeogenic genes, whereas IRS 2 controls lipid metabolism in liver.⁴¹ IRS 1 plays a central role in insulin action for glucose transport in skeletal muscle.⁴² Previous studies have suggested that STZ diabetes is associated with large increases in insulin-stimulated IRS-1 tyrosine phosphorylation, despite the decreased levels of total IRS-1 protein in skeletal muscle.⁴³ The reported molecular defects of diabetes in skeletal muscle include impairment in IRS phosphorylation, PI3K/Akt expression.⁴⁴

Previous reports suggest that diabetes, which could alter the plasma insulin level and its counter regulatory hormones, drastically lower GLUT4 protein expression.⁴⁵ GLUT4 expression is downregulated in STZ-induced diabetes, a state of insulin deficiency, suggesting that insulin act as a positive regulator of gene expression.⁴⁶ A decrease in the protein expression was observed in STZ-induced diabetic animals,⁴⁵ which accounts for the impaired glucose disposal. These reports support this study, which demonstrated a decrease in protein expression in the skeletal muscle of STZ-induced diabetic rats. It has been shown that the marked reduction in protein expression in the skeletal muscle of diabetic rats could be restored back with triterpenoids such as cucurbitane glycosides, momordicosides, and pongamol treatment.^27,47 Similar restoration was observed in the AA-treated diabetic rats of this study. This confirms that AA promotes the expression of GLUT-4 protein.

Chronic hyperglycemia may directly contribute to the impairment in the PI3 kinase/Akt signal pathway. Activation of PI 3-kinase and the serine–threonine PKB Akt are the important steps in insulin action.⁴⁸ Akt has been reported to be a mediator of the glucose uptake signaling pathway, which is controlled by insulin in both muscle and fat cells and hepatic gluconeogenesis. The PI3K/Akt pathway is required for insulin-dependent regulation of systemic and cellular metabolism. The PI3K/Akt pathway is activated downstream of the insulin receptor. PI3K catalyzes the addition of a phosphate molecule to the three positions of the inositol ring of phosphoinositides, converting membrane phosphatidylinositol into PIP, PIP2, and phosphatidylinositol-3,4,5-triphosphate (PIP3).⁴⁹ Akt binds to PIP3, which facilitates activation of Akt by upstream kinases. Once activated, Akt is released from the plasma membrane and translocates to cellular compartments, such as the cytoplasm, mitochondria and nucleus, where it regulates several substrates by phosphorylation events. PDK1 also activates isoforms of PKC, which are required for insulin-stimulated translocation of the GLUT4 at the plasma membrane, resulting in increased glucose uptake. AA exhibited increased cell surface levels of GLUT4 accompanied with relative expressions in PI3K and Akt in skeletal muscle tissue of diabetic rats; it implied that AA activated insulin-mediated PI3K/Akt-dependent glucose transport through the translocation of GLUT4 and subsequently by its activation. These observations suggest that muscle cells are targets for AA action. Also these results reinforce proposals that AA stimulates glucose uptake through an insulin signaling pathway involving PI3K/Akt and probably interferes with other proteins and structures related to GLUT4 translocation to the plasma membrane.

In conclusion, our results suggest that AA shows powerful antioxidant property which was evidenced by improving the oxidative stress markers. AA also increased insulin secretion in rats with sufficient insulin secreting function; furthermore, AA also exerts antihyperglycemic effect by enhancing glucose uptake into skeletal muscle in insulin-deficient STZ diabetic rats via PI3K-Akt signaling pathway. Hence, these findings provide a basis for the use of AA since they have important implications for the prevention of diabetes and related complications.

Footnotes

Funding

The authors thank the Indian Council of Medical Research (ICMR; No/3/1/2/21/2011/RHN), India, for providing financial support in the form of Senior Research Fellowship (SRF) to VR.

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