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Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats
Pharmacological Research 66 (2012) 260–268
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Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats Pankaj K. Bagul, Harish Middela, Saidulu Matapally, Raju Padiya, Tanmay Bastia, K. Madhusudana, B. Raghunath Reddy, Sumana Chakravarty, Sanjay K. Banerjee ∗ Division of Pharmacology and Chemical Biology, Indian Institute of Chemical Technology (IICT), Hyderabad 500607, India
a r t i c l e
i n f o
Article history: Received 7 February 2012 Received in revised form 28 April 2012 Accepted 13 May 2012 Keywords: Type-2 diabetes Resveratrol Metformin Metabolic syndrome Oxidative stress NRF2
a b s t r a c t Metabolic syndrome and oxidative stress are common complications of type 2 diabetes mellitus. The present study was designed to determine whether resveratrol, a widely used nutritional supplement, can improve insulin sensitivity, metabolic complication as well as hepatic oxidative stress in fructosefed rats. Male Sprague Dawley rats (180–200 g) were divided into four groups with 8 animals each. Fructose-fed insulin resistant group (Dia) animals were fed 65% fructose (Research diet, USA) for a period of 8 weeks, whereas control group (Con) animals were fed 65% cornstarch (Research Diet, USA). Resveratrol, 10 mg/kg/day (Dia + Resv) or metformin 300 mg/kg/day (Dia + Met) were administered orally to the 65% fructose-fed rats for 8 weeks. At the end of the feeding schedule, Dia group had insulin resistance along with increased blood glucose, triglyceride, uric acid and nitric oxide (NO) levels. Significant (p < 0.05) increase in hepatic TBARS and conjugated dienes, and significant (p < 0.05) decrease in hepatic SOD and vitamin C was observed in Dia group compared to Con group. Administration of metformin or resveratrol significantly (p < 0.05) normalized all the altered metabolic parameters. However, a marked insulin sensitizing action was only observed in the Dia + Resv group. Similarly, while metformin administration failed to normalize the increased TBARS levels and decreased SOD activity, resveratrol showed a more promising effect of all oxidative stress parameters measured in the present study. Attenuation of hepatic oxidative stress in fructose-fed rat liver after resveratrol administration was associated with significant (p < 0.05) increase in nuclear level of NRF2 compared with other groups. The present study demonstrates that resveratrol is more effective than metformin in improving insulin sensitivity, and attenuating metabolic syndrome and hepatic oxidative stress in fructose-fed rats. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes causes a wide range of health complications, affecting almost every organ of the body. Long-standing diabetes leads to vascular, retinal, renal, hepatic and neurological complications that include structural and functional alterations [1]. These complications are the consequence of various pathophysiological mechanisms, including metabolic syndromes and oxidative stress. The primary features of metabolic syndrome include weight gain, hypertension, cholesterol abnormalities, hyperuricemia and insulin resistance [2]. Oxidative stress is currently the unifying factor in the development of diabetes complications. Changes in oxidative stress, either stemming from glucose-mediated increased free-radical generation and/or reduction of endogenous antioxidants, are strong contenders for the title of “root cause” of diabetic
∗ Corresponding author. Tel.: +91 4027191618; fax: +91 4027193189. E-mail addresses: [email protected], [email protected] (S.K. Banerjee). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.05.003
complications [3], leading to oxidative damage of DNA, protein, and lipid membranes [3,4]. Metabolic syndrome also plays an active role in the development of diabetic complications with increased risk of oxidative stress in diabetes. The long term consequences of type 2 diabetes make it imperative to focus on the development of novel treatment strategies for the management of insulin resistance and metabolic syndrome along with reduction of oxidative stress. Resveratrol, a polyphenolic compound with a strong antioxidant activity, is found in grapes and red wine and has shown numerous beneficial effects in a range of diseases including diabetes [5]. Resveratrol is proven to be effective in different animal models of diabetes such as streptozotocin, Zucker fatty rat, NOD mice and other genetic animal models [6–10]. In addition, resveratrol is found to be effective in a variety of disorders through its antioxidant properties [9]. However, more evidence on the antidiabetic activity of resveratrol along with its diverse biological action that directly or indirectly reduce diabetic complications in fructosefed animals is required. Consumption of fructose in the form of high fructose corn syrup (HFCS) is increasing every day. Longterm fructose intake induces diabetes along with insulin resistance
P.K. Bagul et al. / Pharmacological Research 66 (2012) 260–268 Table 1 Composition of diet obtained from Research diet, USA. Ingredients (g)
High fructose diet (D11707)
Control diet (D11708B)
Casein, 30 mesh DL-methionine Fructose Corn starch Maltodextrin Cellulose Corn oil Salt mix, S1001 Vitamin mix V10001 Choline bitartarate Blue dye, FD&C# 1 Total
200 3 650 0 0 50 50 35 10 2 0 1000
200 3 0 525 125 50 50 35 10 2 0.1 1000.1
and metabolic syndrome in human as well as experimental animals [11,12]. As yet there is no published study on the role of resveratrol on insulin resistance, its metabolic complications and hepatic oxidative stress in high fructose-fed rats. The present study was designed to determine whether resveratrol provides any additional antioxidant benefit in comparison to metformin, a standard antidiabetic drug, in attenuating insulin resistance and metabolic syndrome in fructose-fed rats. 2. Materials and methods 2.1. Experimental animals All animal experiments were undertaken with the approval of Institutional Animal Ethical Committee of Indian Institute of Chemical Technology (IICT), Hyderabad, India. Male Sprague-Dawley (SD) rats weighing (180–200 g) were purchased from the National Institute of Nutrition (NIN), Hyderabad, India. The animals were housed (2–3 rats/cage) in BIOSAFE, an animal quarantine facility of IICT. The animal house was maintained at temperature 22 ± 2 ◦ C with relative humidity 40 ± 15% and 12 h dark/light cycle throughout the study. Animals were randomly divided into four groups (N = 8). Control group (Con) was fed 65% corn starch diet (Research Diet, USA), fructose-fed insulin resistant group (Dia) was fed 65% fructose diet (Research Diet, USA), third group (Dia + Resv) was fed with 65% fructose along with a single dose of 10 mg/kg/day of resveratrol orally, whereas fourth group (Dia + Met) was fed with 65% fructose along with a single dose of 300 mg/kg/day of metformin (Sigma, USA) orally for a period of 8 weeks. The composition of both diets is enlisted in Table 1. Resveratrol was supplied by Kumar Organic Products Ltd., Bangalore, India. The dose of resveratrol and metformin was selected based on the previous literature [13–16]. Resveratrol and metformin were prepared in 25% of DMSO. Accordingly Con and Dia groups were treated with 25% DMSO to nullify the effects of DMSO. 2.2. Body weight gain and food intake Changes in body weight and food intake patterns of rats in all groups were noted throughout the experimental period. The weight of each rat was recorded on day 0 and at weekly intervals throughout the course of the study. The quantity of food consumed by each group was recorded weekly, and the food consumption per rat was calculated for all groups. 2.3. Sample collection and biochemical assay After 8 weeks of feeding and drug administration, the animals in all groups were sacrificed with fed condition at 10:00 AM. Liver tissues were collected and stored at −80 ◦ C for further biochemical
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evaluation. At the time of sacrificing, blood was collected by cardiac puncture, serum was separated by centrifugation at 4000 rpm (4 ◦ C) for 15 min and serum parameters were analyzed by auto blood analyzer (Bayer diagnostic). Each liver tissue was homogenized with 20 times volume of liver weight (100 mg tissue in 2.0 mL buffer) in ice cold 0.05 M potassium phosphate buffer (pH 7.4) and treated separately for different measurement as described [17]. 2.3.1. Estimation of blood glucose, uric acid and triglyceride levels Blood glucose was measured using glucometer (One Touch Horizon, Singapore). Serum samples were analyzed for estimation of triglyceride and uric acid using an auto blood analyzer (Bayer Corp., USA). Triglyceride and uric acid kits were obtained from Siemens, India. 2.3.2. Estimation of serum insulin levels Quantitative estimation of serum insulin was done by rat insulin ELISA kits (Mercodia, USA) [11]. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation, insulin in the sample reacts with peroxidaseconjugated anti-insulin antibodies and anti-insulin antibodies bound to microtitration well. The bound conjugate was detected by reaction with 3,3 ,5,5 -tetramethylbenzidine. The reaction was stopped by adding acid and read using a spectrophotometer at 450 nm. 2.3.3. Estimation of intraperitoneal glucose tolerance test Intraperitoneal glucose tolerance test (IPGTT), was performed according to the method described by Padiya et al. [11]. Rats from all four groups were injected intraperitoneally with a freshly prepared glucose load of 2 g/kg of body weight. A drop of blood was withdrawn from tail vein by a small puncture using needle to analyze blood glucose using glucometer at (0 min) and at 5, 10, 30, 60 and 120 min after insertion of glucose load. 2.3.4. Estimation of serum nitric oxide levels Nitric oxide was determined by a commercially available kit (Assay design, USA). Assay is based on reduction of NO3− into NO2− using nitrate reductase enzyme. The azo dye is produced by diazotization of sulfanilic acid with NO2− and then subsequent coupling with N-(1-napthyl)-ethylene diamine. The azo dye was measured calorimetrically at 540 nm. A standard curve was prepared with sodium nitrite concentration range from 0–100 M (100, 50, 25, 12.5, 6.25, 3.13 and 1.56, 0 M). The absorbance of a purple/magenta color formed after adding sample was measured after interpolated to a standard curve. The serum NO level was expressed as mol/L. 2.4. Estimation of hepatic oxidative stress Liver samples from each rat were homogenized in freshly prepared ice cold potassium phosphate buffer saline with 20 times dilution. Tissue homogenate was used for the estimation of TBARS. Remaining volume of homogenate was put for centrifugation at 15,000 × g for 30 min at 4 ◦ C. The supernatant was collected and used for the estimation GSH, SOD, catalase, Vitamin C [17]. Thiobarbituric acid reactive substances (TBARS) [18] and conjugated diene [19] were measured as a markers of lipid peroxidation, and reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [20] were estimated as levels of endogenous antioxidants. Vitamin C was measured according to the method described by Roe and Kuether [21]. All of the compounds used for the estimation of different biochemical parameters were obtained from Sigma Inc., USA.
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Fig. 1. Effect of resveratrol and metformin on food intake and body weight gain. (A) Curve showing average daily food intake in each week throughout the experimental time period. (B) Bar graph showing average daily food intake considering food intake from the first day to the end of 8 weeks. (C) Bar graph showing initial and final body weight of rats from all groups. (D) Bar graph showing body weight gain of rats from all groups during the experimental period. Data shown as mean ± SEM, (N = 8) * p < 0.05 and ** p < 0.01 vs. Dia; † p < 0.05 and †† p < 0.01 vs. Con.
2.5. Analysis of protein expression by immunoblotting Extraction of nuclear and whole tissue protein fractions was done with liver tissue samples as described by Das et al. [22]. Quantity of protein was measured by the Bradford method. An equal amount (30 g) of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein was transferred to PVDF membranes (Amersham Biosciences). The membranes were then blocked by 5% non-fat dry milk in Tris-buffered saline Tween-20 (TBS-T; 10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, and subsequently washed and incubated with primary antibody NRF2 (1:500 dilution, Abbiotec) in TBS-T and 5% non-fat dry milk at 4 ◦ C overnight. After washing with TBS-T, membranes were incubated with anti-rabbit horseradish peroxidase conjugated secondary antibody (1:8000 dilution, Amersham) for 1 h. Signal was detected by chemiluminescence using the ECL detection system (Amersham). Gel staining with Coomassie Blue was used as an internal control for equal loading of protein. Quantification of bands was performed using Image J Software (NIH).
2.6. Histopathology Liver tissues (N = 2/group) were fixed in 10% neutral buffered formalin for 48 h. The fixed tissue was mounted on the section stage with the appropriate adhesive, 10 m thin sections were cut on Oscillating Tissue Slicer (Model no. OTS-4500, Harvard Apparatus, USA). Only selected good sections were mounted on positively charged superfrost plus slides (Fisher Scientific, USA). Then sections were stained with hematoxylin and eosin (H&E) or Masson’strichome reagent, dehydrated with graded series of alcohol and mounted with DPX. The stained slides were observed using
Axioplan Imaging System (MC200, Carl Zeiss Inclusive, Germany) and results were analyzed.
2.7. Statistical analysis All values were expressed as mean ± SEM. Data were statistically analyzed using one-way ANOVA for multiple group comparison followed by Newman–Keuls test using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA. Difference between two groups was compared by student’s t-test. Significance was set at p ≤ 0.05.
3. Results 3.1. Food Intake and body weight gain There was very slight variation of food intake among all four groups when we measured average per day food intake in each week throughout the experimental time period (Fig. 1A). However, when we measured average food intake per day considering food intake from the first day to the end of 8 weeks, we noticed some changes. There was no significant change of daily food intake between Con and Dia group of animals (Fig. 1B). This indicates no effect of high fructose diet on normal food intake. However, resveratrol and metformin administration (Dia + Resv and Dia + Met group) significantly (p < 0.05) reduced daily food intake when compared to Con and Dia group (Fig. 1B). Rats from all four groups showed significant increase in body weight gain during the experimental period (Fig. 1C). There was no significant change in body weight gain after 8 weeks of fructose feeding in Dia group compared to Con group (Fig. 1D). However, administration of resveratrol (Dia + Resv group) and metformin
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(Dia + Met group) showed significant (p < 0.01) decrease in body weight gain compared to Con and Dia group (Fig. 1D).
3.2. Blood glucose levels and intraperitoneal glucose tolerance test (IPGTT) After 8 weeks of feeding, rats from the Dia group showed a significant (p < 0.01) increase in blood glucose levels compared to Con group. However, this increase in serum glucose levels in fructose-fed rats was significantly (p < 0.01) decreased after chronic administration of resveratrol (Dia + Resv group) and metformin (Dia + Met group), respectively (Fig. 2A). Intraperitoneal glucose tolerance test was carried out to check insulin resistance in fructose-fed rats after 8 weeks. An intraperitoneal glucose load led to a marked increase in blood glucose levels in diabetic group at 5, 10 and 30 min, compared to the Con group. However, chronic administration of resveratrol (Dia + Resv group) and metformin (Dia + Met group) prevented this rise in serum glucose levels. Among the two treated groups, resveratrol was more efficient in reducing the elevated blood glucose levels than metformin (Fig. 2B).
3.3. Serum insulin, triglyceride, uric acid and nitric oxide levels
Fig. 2. Effect of resveratrol and metformin on blood glucose and intraperitoneal glucose tolerance test. (A) Effect of resveratrol and metformin on blood glucose after 8 weeks of fructose feeding (N = 8). (B) Effect of resveratrol and metformin in intraperitoneal glucose tolerance test (N = 3). Data shown as mean ± SEM. †† p < 0.01 vs. Con, ** p < 0.01 vs. Dia.
After 8 weeks of fructose feeding, rats of Dia group showed a significant (p < 0.01) increase in serum insulin, triglyceride, uric acid and nitric oxide levels (Fig. 3A–D). However, administration of resveratrol and metformin significantly (p < 0.01) reduced elevated levels of serum triglyceride, uric acid and nitric oxide (Fig. 3B–D). Significant (p < 0.05) decrease in serum insulin levels was observed after metformin administration but not with resveratrol administration (Fig. 3A). Resveratrol was found to be more effective in reducing the elevated triglyceride levels compared to metformin.
Fig. 3. Effect of resveratrol and metformin on serum insulin (A), triglyceride (B), uric acid (C) and nitric oxide (D) levels after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8) † p < 0.05 and †† p < 0.01 vs. Con, * p < 0.05 and ** p < 0.01 vs. Dia.
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metformin (Dia + Met group) significantly (p < 0.01) increased hepatic vitamin C levels in fructose-fed rats when compared to the Dia group (Fig. 5C). There was no change in hepatic GSH levels in Dia group compared to Con group. Administration of resveratrol (Dia + Res group) significantly (p < 0.05) increased hepatic GSH levels in fructose-fed rats compared to Dia group. However, metformin (Dia + Met group) administration fails to show any increase in GSH levels compared to Dia group (Fig. 5D). 3.6. NRF2 protein expression in rat liver Protein expression study with NRF2 using SDS–PAGE electrophoresis revealed that there was no significant change in NRF2 level in liver total protein extract among all groups except Dia + Met group (Fig. 6A and B). Significant (p < 0.05) reduction of NRF2 levels in total protein extract was observed in Dia + Met group compared to Con and Dia group. To confirm the nuclear translocation of NRF2, we extracted nuclear protein from all groups of rat livers and performed immunobloting. Whereas nuclear level of NRF2 was not altered in fructose-fed liver, significant (p < 0.01) increase in nuclear NRF2 level was observed in fructose-fed liver after resveratrol administration (Dia + Resv group). However, no change in nuclear NRF2 level was observed after metformin administration (Dia + Met group) (Fig. 6A and B). 3.7. Histopathology
Fig. 4. Effect of resveratrol and metformin on hepatic conjugated dienes (A) and TBARS levels (B) after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8). † p < 0.05 and †† p < 0.01 vs. Con; * p < 0.05 and ** p < 0.01 vs. Dia.
3.4. Hepatic conjugated dienes and TBARS Hepatic conjugated dienes were significantly (p < 0.05) increased in Dia group when compared to Con group. However, this increased conjugated dienes after fructose feeding was significantly (p < 0.01) normalized with chronic resveratrol (Dia + Resv group) as well as metformin (Dia + Met group) administration (Fig. 4A). Similarly, hepatic TBARS levels were significantly (p < 0.01) increased in Dia group compared to Con group. Chronic administration of resveratrol (Dia + Resv group) showed significant (p < 0.05) reduction in the increased TBARS levels in fructose fed animals. However, chronic administration of metformin (Dia + Met group) failed to show any decrease in TBARS levels (Fig. 4B). 3.5. Hepatic catalase, SOD, vitamin C and GSH levels There was no change in hepatic catalase activity in Dia group compared to the Con group. However, administration of resveratrol and metformin (Dia + Resv and Dia + Met group) significantly (p < 0.05) increased hepatic catalase activity compared to Dia group (Fig. 5A). After 8 weeks of fructose feeding, hepatic SOD activity was significantly (p < 0.05) reduced in Dia group when compared to Con group. However, this decreased SOD activity in fructose-fed rats was significantly (p < 0.05) increased after chronic administration of resveratrol (Dia + Resv group). However, metformin (Dia + Met group) administration fails to show any increase in SOD activity compared to Dia group (Fig. 5B). Vitamin C is known to be an important antioxidant to maintain redox balance in different tissues. Vitamin C levels were reduced significantly (p < 0.01) in Dia group compared to Con group. However, chronic administration of resveratrol (Dia + Resv group) and
The H&E stained liver sections of control rats (Con group) examined by light microscope (Axioplan Imaging System) revealed the normal hepatic structure, formed of hepatic lobules. Briefly, each lobule is made up of radiating plates, strands of cells forming a network around a central vein (Fig. 7A). The liver strands are altering with narrow sinusoids which have irregular boundaries composed of only a single layer of fenestrated endothelial cells and large irregularly phagocytic cells, which are known as Kupffer cells. By contrast, the liver histology of fructose-fed rats (Dia group) showed some discernible changes. However, the response was not uniform; both shrunken as well as distended veins were observed in different lobules of the same section of the liver. In some lobules, there are remarkable loss of normal architecture including disarrangement of hepatocytes, shrunken central vein (Fig. 7B1 ) including cord-like pattern of normal hepatocytes were not well distinct and a considerable amount of cells with pycnotic appearance, whereas in other lobule of the same section cells are in better condition except distended vein with a little fibrosis and some leucocytic infiltrations (Fig. 7B2 ). Interestingly, the liver sections from fructose-fed rats treated with either resveratrol (Dia + Resv group) and metformin (Dia + Met group) were seems to be in recovery stage though central vein were found to be still a little dilated but cells as such were looking healthy (Fig. 7C and D). Mason’s Trichrome stained sections did not show (Fig. 8A–D) much changes in collagen fiber pattern except a little thinning of connective tissue around the portal tracts of diabetic liver (Fig. 8B) as compare to control (Fig. 8A). Interestingly, Dia group treated with resveratrol or metformin did not show any such thinning effect (Fig. 8C and D). 4. Discussion Type 2 diabetes mellitus (T2DM) is a metabolic disorder, influenced by a variety of lifestyle factors including diet, stress, lack of physical activity and alcohol consumption. Current antidiabetic treatment strictly focuses on the management of glycaemia along with reduction of associated diabetic complications including organ damages. Our study focuses on therapeutic interventions that can reduce hyperglycemia as well as metabolic complications along
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Fig. 5. Effect of resveratrol and metformin administration on hepatic catalase activity (A), SOD activity (B), vitamin C level (C) and GSH level (D) after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8) † p < 0.05 vs. Con; * p < 0.05 and ** p < 0.01 vs. Dia.
with reduction of hepatic oxidative stress in the fructose-fed rats. In T2DM, liver is the primary organ which comes under stress because of the metabolic abnormalities and hepatic insulin resistance [1]. We evaluated and compared the effect of resveratrol, a widely used nutritional supplement with metformin, a commonly used first line drug, on diabetes and hepatic oxidative stress in fructose-fed rats. Consumption of fructose in the form of high fructose corn syrup (HFCS) is increasing every year in most countries including USA, India and China. High fructose intake over long periods is wellknown risk factor for diabetes and obesity [23]. In the present study, we used a high fructose rich diet which is much higher than physiological human dietary fructose, for the induction of type 2 diabetes mellitus, which is characterized by insulin resistance, metabolic
syndrome and oxidative stress. Previous studies including ours have shown that long-term fructose feeding induces diabetes associated with insulin resistance and metabolic syndrome in experimental animals such as rats and mice [11,24–26]. Previous study showed that fructose consumption causes metabolic alterations in liver that leads to abnormalities including oxidative stress [27]. Based on the increased consumption of fructose and other sugar based product in humans, we chose high fructose-fed rat model for the induction of insulin resistance along with metabolic syndromes and increased oxidative stress in liver. In the present study, we found that feeding of high fructose diet for a period of 8 weeks induces prediabetes as measured by induction of insulin resistance, increase in postprandial blood
Fig. 6. Immunoblots of NRF2 expression in liver. (A) Immunoblot of NRF2 from hepatic total protein extracts. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblot. (N = 3). (B) Densitometry analysis of NRF2 level from total protein extract (N = 3). Data shown as mean ± SEM, * p < 0.05 vs. Dia. and † p < 0.05 vs. Con. (C) Immunoblot of NRF2 from hepatic nuclear protein extracts. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblot. (D) Densitometry analysis of NRF2 level from nuclear protein extract (N = 3). Data shown as mean ± SEM, * p < 0.05 vs. Dia. and † p < 0.05 vs. Con.
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Fig. 7. Photomicrograph of control liver, H&E, 20×: illustrating central vein (CV) with endothelial lining (dotted arrow), hepatocytes and sinusoidal spaces with Kupffer cells (7A). Liver section of diabetic rat showing loss of the normal architecture with shrunken (7B1 ) or distended central vein (7B2 ). Note some nuclei were pycnotic (7B1 , arrows) and leucocytic infiltrations into the vein (7B2 , arrows). The liver sections from diabetic animals treated with either resveratrol or metformin showed almost normal histology except a little dilated CV and slightly more sinusoidal spaces (7C and D) than control.
Fig. 8. Photomicrograph of liver, Masson’s Trichrome stain, 20×: diabetic liver sections did not show much changes in collagen fiber pattern except a little thinning of connective tissue around the portal tracts (PV) (Fig. 8B) as compare to control (Fig. 8A). Note neither resveratrol nor metformin group showed any such thinning effect (Fig. 8C and D).
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glucose levels and other metabolic disturbances. Fructose (65%) rich diet feeding for 8 weeks showed significant but mild increase in postprandial blood glucose levels along with increased serum triglyceride, uric acid and insulin levels. Intraperitoneal glucose tolerance test also showed a marked insulin resistance in diabetic group animals. Administration of resveratrol and metformin reduced the increased blood glucose levels along with serum triglyceride and uric acid levels. Although both resveratrol and metformin showed improvement in insulin sensitivity, resveratrol showed greater improvement of insulin sensitivity compared to both control and metformin-treated animals. Resveratrol administration is known to increase insulin secretion from beta cells [28,29] and it is possible that higher levels of insulin due to resveratrol might be responsible for improvement of insulin sensitivity. In the present study also higher insulin level along with improved insulin sensitivity was observed in resveratrol treated rats compared to control and metformin treated groups. Although there was no change in food intake and body weight gain in fructose-fed animals, both resveratrol and metformin administration resulted in a significant decrease in food intake and body weight gain, which might also responsible for improved insulin sensitivity. Serum triglyceride and uric acid levels, which may be also responsible for the increase in insulin resistance in fructose-fed rats, was significantly reduced after resveratrol and metformin administration. Nitric oxide is an important signaling gaseous molecule which regulates wide range of physiological and pathological processes [30–32]. Increased serum NO levels have been reported in diabetic patients as well as fructose-fed diabetic rats [11,33]. Increased NO levels can also induce oxidative damage through the formation of peroxynitrate [34]. In the present study, serum NO levels were significantly higher in fructose-fed rats compared to the control rats. Chronic administration of resveratrol and metformin normalized the NO level in fructose-fed rats. The reduction of NO level in serum might be due to reduction of oxidative stress in fructose-fed rats after administration of both drugs tested in the present study. Both experimental and clinical studies indicate that oxidative stress plays a major role in the development and complications of type 2 diabetes [35]. Free radicals are generated in diabetes by glucose oxidation. The oxidative stress may be amplified by diabetes-induced metabolic stress, tissue damage, and cell death, leading to increased free radical production and compromised free radical scavenger systems, which further exacerbate the oxidative stress. Oxidative stress can also lead to damage of cellular organelles, and development of insulin resistance [36]. In the present study, high fructose feeding increased oxidative stress as evidenced by elevation of TBARS and conjugated dienes levels, and reduction of SOD activity and vitamin C levels in diabetic liver in comparison to control group. Unlike metformin, resveratrol reduced hepatic TBARS and conjugated dienes levels and increased SOD activity and vitamin C levels in the fructose-fed liver. While resveratrol administration increased both hepatic GSH and catalase activity, metformin only increased the hepatic catalase activity compared to fructose-fed liver. Our study showed that resveratrol was more effective than metformin in reducing oxidative stress in liver. This beneficial antioxidant effect might be responsible for improved insulin sensitivity in fructose-fed rats after resveratrol administration. Similarly to ensure the hepatic manifestations of metabolic syndrome and oxidative stress, we did histopathological examination in our prediabetic model. Presence of inflammation, non-alcoholic steatohepatitis and fatty liver was reported earlier after high fructose feeding in rat liver [37]. Unlike previous study, we found only a mild inflammation in fructose-fed liver. However, this change was not observed when fructose-fed rats were treated with either resveratrol or metformin.To provide insights into the molecular mechanism behind the beneficial effect of resveratrol in fructose-fed rats, we measured hepatic NRF2 protein level from
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all groups. Nuclear factor E2-related factor 2 (NRF2) is a transcription factor important in the protection against any oxidative stress. During oxidative stress, NRF2 is released from sequestration in the cytoplasm and translocates to the nucleus. NRF2 binds antioxidant response elements (AREs) in the regulatory regions of target genes and activates transcription of several antioxidant enzymes. Several NRF2 activators have already been developed for treating diseases involving oxidative stress [38]. Interestingly, NRF2 activators have also been shown to modulate insulin action [39]. Hyperglycemia-induced endothelial dysfunction, vascular complications and cardiomyocyte damage was prevented by NRF2 activation by reducing oxidative stress [38]. Similar to previous studies, the present study demonstrated that beneficial antioxidant effect of resveratrol was associated with increased nuclear translocation of NRF2 in fructose-fed liver. Increased nuclear NRF2 level might have a significant protective role against high fructose induced oxidative stress in liver as observed in our study, possibly through augmentation of hepatic antioxidant defense enzymes. In conclusion, there was an improvement of hyperglycemia and metabolic parameters after metformin and resveratrol administration in fructose-fed prediabetic rats. However, resveratrol showed a more marked insulin sensitizing action than metformin. In addition, resveratrol showed additional beneficial effect by reducing the hepatic oxidative stress in prediabetic rat through activation of NRF2. Conflict of interest The authors declare that they have no competing interest. Acknowledgments Financial support was provided by Ramalingaswami fellowship funds to SKB and SC from Department of Biotechnology (DBT), Govt. of India and in part, IICT institute fund. SKB has grant support from DBT (SR/SO/HS-110/2008). PKB is a Senior Research Fellow from Council of Scientific and Industrial Research (CSIR). We wish to thank Kumar organic products Ltd., Bangalore for providing gift sample of Resveratrol. We wish to thank Dr. K. Praveen Kumar for his excellent technical assistance and Dr. J. S. Yadav, Director, IICT, Hyderabad for providing all kind of support for this work. We would like to gratefully acknowledge Dr. Mohua Maulik for her suggestions and critical review of the manuscript. References [1] Cade WT. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting. Physical Therapy 2008;88(11):1322–35. [2] Johnson RJ, Perez-Pozo SE, Sautin YY. Hypothesis: could excessive fructose intake and uric acid cause type 2 diabetes? Endocrine Reviews 2009;30(1):96–116. [3] Tooke JE. Possible pathophysiological mechanisms for diabetic angiopathy in type 2 diabetes. Journal of Diabetes and its Complications 2000;14(4):197–200. [4] Dominguez C, Ruiz E, Gussinye M, Carrascosa A. Oxidative stress at onset and in early stages of type 1 diabetes in children and adolescent. Diabetes care 1998;21:1736–42. [5] Pirola L, Fro S. Critical review resveratrol: one molecule, many targets. Cell 2008;60:323–32. [6] Lee SM, Yang H, Tartar DM, Gao B, Luo X, Ye SQ, et al. Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1 diabetes. Diabetologia 2011;54:1136–46. [7] Lekli I, Szabo G, Juhasz B. Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin. American Journal of Physiology and Heart Circulation Physiology 2008;294:H859–66. [8] Sharma S, Misra CS, Arumugam S, Roy S, Shah V, Davis JA, et al. Antidiabetic activity of resveratrol, a known SIRT1 activator in a genetic model for type-2 diabetes. Phytotherapy Research 2010;25:67–73. [9] Su HC, Hung LM, Cheng JK. Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats. American Journal of Physiology and Endocrinology Metabolism 2006;290:1339–46.
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Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats Pankaj K. Bagul, Harish Middela, Saidulu Matapally, Raju Padiya, Tanmay Bastia, K. Madhusudana, B. Raghunath Reddy, Sumana Chakravarty, Sanjay K. Banerjee ∗ Division of Pharmacology and Chemical Biology, Indian Institute of Chemical Technology (IICT), Hyderabad 500607, India
a r t i c l e
i n f o
Article history: Received 7 February 2012 Received in revised form 28 April 2012 Accepted 13 May 2012 Keywords: Type-2 diabetes Resveratrol Metformin Metabolic syndrome Oxidative stress NRF2
a b s t r a c t Metabolic syndrome and oxidative stress are common complications of type 2 diabetes mellitus. The present study was designed to determine whether resveratrol, a widely used nutritional supplement, can improve insulin sensitivity, metabolic complication as well as hepatic oxidative stress in fructosefed rats. Male Sprague Dawley rats (180–200 g) were divided into four groups with 8 animals each. Fructose-fed insulin resistant group (Dia) animals were fed 65% fructose (Research diet, USA) for a period of 8 weeks, whereas control group (Con) animals were fed 65% cornstarch (Research Diet, USA). Resveratrol, 10 mg/kg/day (Dia + Resv) or metformin 300 mg/kg/day (Dia + Met) were administered orally to the 65% fructose-fed rats for 8 weeks. At the end of the feeding schedule, Dia group had insulin resistance along with increased blood glucose, triglyceride, uric acid and nitric oxide (NO) levels. Significant (p < 0.05) increase in hepatic TBARS and conjugated dienes, and significant (p < 0.05) decrease in hepatic SOD and vitamin C was observed in Dia group compared to Con group. Administration of metformin or resveratrol significantly (p < 0.05) normalized all the altered metabolic parameters. However, a marked insulin sensitizing action was only observed in the Dia + Resv group. Similarly, while metformin administration failed to normalize the increased TBARS levels and decreased SOD activity, resveratrol showed a more promising effect of all oxidative stress parameters measured in the present study. Attenuation of hepatic oxidative stress in fructose-fed rat liver after resveratrol administration was associated with significant (p < 0.05) increase in nuclear level of NRF2 compared with other groups. The present study demonstrates that resveratrol is more effective than metformin in improving insulin sensitivity, and attenuating metabolic syndrome and hepatic oxidative stress in fructose-fed rats. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes causes a wide range of health complications, affecting almost every organ of the body. Long-standing diabetes leads to vascular, retinal, renal, hepatic and neurological complications that include structural and functional alterations [1]. These complications are the consequence of various pathophysiological mechanisms, including metabolic syndromes and oxidative stress. The primary features of metabolic syndrome include weight gain, hypertension, cholesterol abnormalities, hyperuricemia and insulin resistance [2]. Oxidative stress is currently the unifying factor in the development of diabetes complications. Changes in oxidative stress, either stemming from glucose-mediated increased free-radical generation and/or reduction of endogenous antioxidants, are strong contenders for the title of “root cause” of diabetic
∗ Corresponding author. Tel.: +91 4027191618; fax: +91 4027193189. E-mail addresses: [email protected], [email protected] (S.K. Banerjee). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.05.003
complications [3], leading to oxidative damage of DNA, protein, and lipid membranes [3,4]. Metabolic syndrome also plays an active role in the development of diabetic complications with increased risk of oxidative stress in diabetes. The long term consequences of type 2 diabetes make it imperative to focus on the development of novel treatment strategies for the management of insulin resistance and metabolic syndrome along with reduction of oxidative stress. Resveratrol, a polyphenolic compound with a strong antioxidant activity, is found in grapes and red wine and has shown numerous beneficial effects in a range of diseases including diabetes [5]. Resveratrol is proven to be effective in different animal models of diabetes such as streptozotocin, Zucker fatty rat, NOD mice and other genetic animal models [6–10]. In addition, resveratrol is found to be effective in a variety of disorders through its antioxidant properties [9]. However, more evidence on the antidiabetic activity of resveratrol along with its diverse biological action that directly or indirectly reduce diabetic complications in fructosefed animals is required. Consumption of fructose in the form of high fructose corn syrup (HFCS) is increasing every day. Longterm fructose intake induces diabetes along with insulin resistance
P.K. Bagul et al. / Pharmacological Research 66 (2012) 260–268 Table 1 Composition of diet obtained from Research diet, USA. Ingredients (g)
High fructose diet (D11707)
Control diet (D11708B)
Casein, 30 mesh DL-methionine Fructose Corn starch Maltodextrin Cellulose Corn oil Salt mix, S1001 Vitamin mix V10001 Choline bitartarate Blue dye, FD&C# 1 Total
200 3 650 0 0 50 50 35 10 2 0 1000
200 3 0 525 125 50 50 35 10 2 0.1 1000.1
and metabolic syndrome in human as well as experimental animals [11,12]. As yet there is no published study on the role of resveratrol on insulin resistance, its metabolic complications and hepatic oxidative stress in high fructose-fed rats. The present study was designed to determine whether resveratrol provides any additional antioxidant benefit in comparison to metformin, a standard antidiabetic drug, in attenuating insulin resistance and metabolic syndrome in fructose-fed rats. 2. Materials and methods 2.1. Experimental animals All animal experiments were undertaken with the approval of Institutional Animal Ethical Committee of Indian Institute of Chemical Technology (IICT), Hyderabad, India. Male Sprague-Dawley (SD) rats weighing (180–200 g) were purchased from the National Institute of Nutrition (NIN), Hyderabad, India. The animals were housed (2–3 rats/cage) in BIOSAFE, an animal quarantine facility of IICT. The animal house was maintained at temperature 22 ± 2 ◦ C with relative humidity 40 ± 15% and 12 h dark/light cycle throughout the study. Animals were randomly divided into four groups (N = 8). Control group (Con) was fed 65% corn starch diet (Research Diet, USA), fructose-fed insulin resistant group (Dia) was fed 65% fructose diet (Research Diet, USA), third group (Dia + Resv) was fed with 65% fructose along with a single dose of 10 mg/kg/day of resveratrol orally, whereas fourth group (Dia + Met) was fed with 65% fructose along with a single dose of 300 mg/kg/day of metformin (Sigma, USA) orally for a period of 8 weeks. The composition of both diets is enlisted in Table 1. Resveratrol was supplied by Kumar Organic Products Ltd., Bangalore, India. The dose of resveratrol and metformin was selected based on the previous literature [13–16]. Resveratrol and metformin were prepared in 25% of DMSO. Accordingly Con and Dia groups were treated with 25% DMSO to nullify the effects of DMSO. 2.2. Body weight gain and food intake Changes in body weight and food intake patterns of rats in all groups were noted throughout the experimental period. The weight of each rat was recorded on day 0 and at weekly intervals throughout the course of the study. The quantity of food consumed by each group was recorded weekly, and the food consumption per rat was calculated for all groups. 2.3. Sample collection and biochemical assay After 8 weeks of feeding and drug administration, the animals in all groups were sacrificed with fed condition at 10:00 AM. Liver tissues were collected and stored at −80 ◦ C for further biochemical
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evaluation. At the time of sacrificing, blood was collected by cardiac puncture, serum was separated by centrifugation at 4000 rpm (4 ◦ C) for 15 min and serum parameters were analyzed by auto blood analyzer (Bayer diagnostic). Each liver tissue was homogenized with 20 times volume of liver weight (100 mg tissue in 2.0 mL buffer) in ice cold 0.05 M potassium phosphate buffer (pH 7.4) and treated separately for different measurement as described [17]. 2.3.1. Estimation of blood glucose, uric acid and triglyceride levels Blood glucose was measured using glucometer (One Touch Horizon, Singapore). Serum samples were analyzed for estimation of triglyceride and uric acid using an auto blood analyzer (Bayer Corp., USA). Triglyceride and uric acid kits were obtained from Siemens, India. 2.3.2. Estimation of serum insulin levels Quantitative estimation of serum insulin was done by rat insulin ELISA kits (Mercodia, USA) [11]. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation, insulin in the sample reacts with peroxidaseconjugated anti-insulin antibodies and anti-insulin antibodies bound to microtitration well. The bound conjugate was detected by reaction with 3,3 ,5,5 -tetramethylbenzidine. The reaction was stopped by adding acid and read using a spectrophotometer at 450 nm. 2.3.3. Estimation of intraperitoneal glucose tolerance test Intraperitoneal glucose tolerance test (IPGTT), was performed according to the method described by Padiya et al. [11]. Rats from all four groups were injected intraperitoneally with a freshly prepared glucose load of 2 g/kg of body weight. A drop of blood was withdrawn from tail vein by a small puncture using needle to analyze blood glucose using glucometer at (0 min) and at 5, 10, 30, 60 and 120 min after insertion of glucose load. 2.3.4. Estimation of serum nitric oxide levels Nitric oxide was determined by a commercially available kit (Assay design, USA). Assay is based on reduction of NO3− into NO2− using nitrate reductase enzyme. The azo dye is produced by diazotization of sulfanilic acid with NO2− and then subsequent coupling with N-(1-napthyl)-ethylene diamine. The azo dye was measured calorimetrically at 540 nm. A standard curve was prepared with sodium nitrite concentration range from 0–100 M (100, 50, 25, 12.5, 6.25, 3.13 and 1.56, 0 M). The absorbance of a purple/magenta color formed after adding sample was measured after interpolated to a standard curve. The serum NO level was expressed as mol/L. 2.4. Estimation of hepatic oxidative stress Liver samples from each rat were homogenized in freshly prepared ice cold potassium phosphate buffer saline with 20 times dilution. Tissue homogenate was used for the estimation of TBARS. Remaining volume of homogenate was put for centrifugation at 15,000 × g for 30 min at 4 ◦ C. The supernatant was collected and used for the estimation GSH, SOD, catalase, Vitamin C [17]. Thiobarbituric acid reactive substances (TBARS) [18] and conjugated diene [19] were measured as a markers of lipid peroxidation, and reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [20] were estimated as levels of endogenous antioxidants. Vitamin C was measured according to the method described by Roe and Kuether [21]. All of the compounds used for the estimation of different biochemical parameters were obtained from Sigma Inc., USA.
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Fig. 1. Effect of resveratrol and metformin on food intake and body weight gain. (A) Curve showing average daily food intake in each week throughout the experimental time period. (B) Bar graph showing average daily food intake considering food intake from the first day to the end of 8 weeks. (C) Bar graph showing initial and final body weight of rats from all groups. (D) Bar graph showing body weight gain of rats from all groups during the experimental period. Data shown as mean ± SEM, (N = 8) * p < 0.05 and ** p < 0.01 vs. Dia; † p < 0.05 and †† p < 0.01 vs. Con.
2.5. Analysis of protein expression by immunoblotting Extraction of nuclear and whole tissue protein fractions was done with liver tissue samples as described by Das et al. [22]. Quantity of protein was measured by the Bradford method. An equal amount (30 g) of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein was transferred to PVDF membranes (Amersham Biosciences). The membranes were then blocked by 5% non-fat dry milk in Tris-buffered saline Tween-20 (TBS-T; 10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, and subsequently washed and incubated with primary antibody NRF2 (1:500 dilution, Abbiotec) in TBS-T and 5% non-fat dry milk at 4 ◦ C overnight. After washing with TBS-T, membranes were incubated with anti-rabbit horseradish peroxidase conjugated secondary antibody (1:8000 dilution, Amersham) for 1 h. Signal was detected by chemiluminescence using the ECL detection system (Amersham). Gel staining with Coomassie Blue was used as an internal control for equal loading of protein. Quantification of bands was performed using Image J Software (NIH).
2.6. Histopathology Liver tissues (N = 2/group) were fixed in 10% neutral buffered formalin for 48 h. The fixed tissue was mounted on the section stage with the appropriate adhesive, 10 m thin sections were cut on Oscillating Tissue Slicer (Model no. OTS-4500, Harvard Apparatus, USA). Only selected good sections were mounted on positively charged superfrost plus slides (Fisher Scientific, USA). Then sections were stained with hematoxylin and eosin (H&E) or Masson’strichome reagent, dehydrated with graded series of alcohol and mounted with DPX. The stained slides were observed using
Axioplan Imaging System (MC200, Carl Zeiss Inclusive, Germany) and results were analyzed.
2.7. Statistical analysis All values were expressed as mean ± SEM. Data were statistically analyzed using one-way ANOVA for multiple group comparison followed by Newman–Keuls test using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA. Difference between two groups was compared by student’s t-test. Significance was set at p ≤ 0.05.
3. Results 3.1. Food Intake and body weight gain There was very slight variation of food intake among all four groups when we measured average per day food intake in each week throughout the experimental time period (Fig. 1A). However, when we measured average food intake per day considering food intake from the first day to the end of 8 weeks, we noticed some changes. There was no significant change of daily food intake between Con and Dia group of animals (Fig. 1B). This indicates no effect of high fructose diet on normal food intake. However, resveratrol and metformin administration (Dia + Resv and Dia + Met group) significantly (p < 0.05) reduced daily food intake when compared to Con and Dia group (Fig. 1B). Rats from all four groups showed significant increase in body weight gain during the experimental period (Fig. 1C). There was no significant change in body weight gain after 8 weeks of fructose feeding in Dia group compared to Con group (Fig. 1D). However, administration of resveratrol (Dia + Resv group) and metformin
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(Dia + Met group) showed significant (p < 0.01) decrease in body weight gain compared to Con and Dia group (Fig. 1D).
3.2. Blood glucose levels and intraperitoneal glucose tolerance test (IPGTT) After 8 weeks of feeding, rats from the Dia group showed a significant (p < 0.01) increase in blood glucose levels compared to Con group. However, this increase in serum glucose levels in fructose-fed rats was significantly (p < 0.01) decreased after chronic administration of resveratrol (Dia + Resv group) and metformin (Dia + Met group), respectively (Fig. 2A). Intraperitoneal glucose tolerance test was carried out to check insulin resistance in fructose-fed rats after 8 weeks. An intraperitoneal glucose load led to a marked increase in blood glucose levels in diabetic group at 5, 10 and 30 min, compared to the Con group. However, chronic administration of resveratrol (Dia + Resv group) and metformin (Dia + Met group) prevented this rise in serum glucose levels. Among the two treated groups, resveratrol was more efficient in reducing the elevated blood glucose levels than metformin (Fig. 2B).
3.3. Serum insulin, triglyceride, uric acid and nitric oxide levels
Fig. 2. Effect of resveratrol and metformin on blood glucose and intraperitoneal glucose tolerance test. (A) Effect of resveratrol and metformin on blood glucose after 8 weeks of fructose feeding (N = 8). (B) Effect of resveratrol and metformin in intraperitoneal glucose tolerance test (N = 3). Data shown as mean ± SEM. †† p < 0.01 vs. Con, ** p < 0.01 vs. Dia.
After 8 weeks of fructose feeding, rats of Dia group showed a significant (p < 0.01) increase in serum insulin, triglyceride, uric acid and nitric oxide levels (Fig. 3A–D). However, administration of resveratrol and metformin significantly (p < 0.01) reduced elevated levels of serum triglyceride, uric acid and nitric oxide (Fig. 3B–D). Significant (p < 0.05) decrease in serum insulin levels was observed after metformin administration but not with resveratrol administration (Fig. 3A). Resveratrol was found to be more effective in reducing the elevated triglyceride levels compared to metformin.
Fig. 3. Effect of resveratrol and metformin on serum insulin (A), triglyceride (B), uric acid (C) and nitric oxide (D) levels after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8) † p < 0.05 and †† p < 0.01 vs. Con, * p < 0.05 and ** p < 0.01 vs. Dia.
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metformin (Dia + Met group) significantly (p < 0.01) increased hepatic vitamin C levels in fructose-fed rats when compared to the Dia group (Fig. 5C). There was no change in hepatic GSH levels in Dia group compared to Con group. Administration of resveratrol (Dia + Res group) significantly (p < 0.05) increased hepatic GSH levels in fructose-fed rats compared to Dia group. However, metformin (Dia + Met group) administration fails to show any increase in GSH levels compared to Dia group (Fig. 5D). 3.6. NRF2 protein expression in rat liver Protein expression study with NRF2 using SDS–PAGE electrophoresis revealed that there was no significant change in NRF2 level in liver total protein extract among all groups except Dia + Met group (Fig. 6A and B). Significant (p < 0.05) reduction of NRF2 levels in total protein extract was observed in Dia + Met group compared to Con and Dia group. To confirm the nuclear translocation of NRF2, we extracted nuclear protein from all groups of rat livers and performed immunobloting. Whereas nuclear level of NRF2 was not altered in fructose-fed liver, significant (p < 0.01) increase in nuclear NRF2 level was observed in fructose-fed liver after resveratrol administration (Dia + Resv group). However, no change in nuclear NRF2 level was observed after metformin administration (Dia + Met group) (Fig. 6A and B). 3.7. Histopathology
Fig. 4. Effect of resveratrol and metformin on hepatic conjugated dienes (A) and TBARS levels (B) after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8). † p < 0.05 and †† p < 0.01 vs. Con; * p < 0.05 and ** p < 0.01 vs. Dia.
3.4. Hepatic conjugated dienes and TBARS Hepatic conjugated dienes were significantly (p < 0.05) increased in Dia group when compared to Con group. However, this increased conjugated dienes after fructose feeding was significantly (p < 0.01) normalized with chronic resveratrol (Dia + Resv group) as well as metformin (Dia + Met group) administration (Fig. 4A). Similarly, hepatic TBARS levels were significantly (p < 0.01) increased in Dia group compared to Con group. Chronic administration of resveratrol (Dia + Resv group) showed significant (p < 0.05) reduction in the increased TBARS levels in fructose fed animals. However, chronic administration of metformin (Dia + Met group) failed to show any decrease in TBARS levels (Fig. 4B). 3.5. Hepatic catalase, SOD, vitamin C and GSH levels There was no change in hepatic catalase activity in Dia group compared to the Con group. However, administration of resveratrol and metformin (Dia + Resv and Dia + Met group) significantly (p < 0.05) increased hepatic catalase activity compared to Dia group (Fig. 5A). After 8 weeks of fructose feeding, hepatic SOD activity was significantly (p < 0.05) reduced in Dia group when compared to Con group. However, this decreased SOD activity in fructose-fed rats was significantly (p < 0.05) increased after chronic administration of resveratrol (Dia + Resv group). However, metformin (Dia + Met group) administration fails to show any increase in SOD activity compared to Dia group (Fig. 5B). Vitamin C is known to be an important antioxidant to maintain redox balance in different tissues. Vitamin C levels were reduced significantly (p < 0.01) in Dia group compared to Con group. However, chronic administration of resveratrol (Dia + Resv group) and
The H&E stained liver sections of control rats (Con group) examined by light microscope (Axioplan Imaging System) revealed the normal hepatic structure, formed of hepatic lobules. Briefly, each lobule is made up of radiating plates, strands of cells forming a network around a central vein (Fig. 7A). The liver strands are altering with narrow sinusoids which have irregular boundaries composed of only a single layer of fenestrated endothelial cells and large irregularly phagocytic cells, which are known as Kupffer cells. By contrast, the liver histology of fructose-fed rats (Dia group) showed some discernible changes. However, the response was not uniform; both shrunken as well as distended veins were observed in different lobules of the same section of the liver. In some lobules, there are remarkable loss of normal architecture including disarrangement of hepatocytes, shrunken central vein (Fig. 7B1 ) including cord-like pattern of normal hepatocytes were not well distinct and a considerable amount of cells with pycnotic appearance, whereas in other lobule of the same section cells are in better condition except distended vein with a little fibrosis and some leucocytic infiltrations (Fig. 7B2 ). Interestingly, the liver sections from fructose-fed rats treated with either resveratrol (Dia + Resv group) and metformin (Dia + Met group) were seems to be in recovery stage though central vein were found to be still a little dilated but cells as such were looking healthy (Fig. 7C and D). Mason’s Trichrome stained sections did not show (Fig. 8A–D) much changes in collagen fiber pattern except a little thinning of connective tissue around the portal tracts of diabetic liver (Fig. 8B) as compare to control (Fig. 8A). Interestingly, Dia group treated with resveratrol or metformin did not show any such thinning effect (Fig. 8C and D). 4. Discussion Type 2 diabetes mellitus (T2DM) is a metabolic disorder, influenced by a variety of lifestyle factors including diet, stress, lack of physical activity and alcohol consumption. Current antidiabetic treatment strictly focuses on the management of glycaemia along with reduction of associated diabetic complications including organ damages. Our study focuses on therapeutic interventions that can reduce hyperglycemia as well as metabolic complications along
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Fig. 5. Effect of resveratrol and metformin administration on hepatic catalase activity (A), SOD activity (B), vitamin C level (C) and GSH level (D) after 8 weeks of fructose feeding. Data shown as mean ± SEM (N = 8) † p < 0.05 vs. Con; * p < 0.05 and ** p < 0.01 vs. Dia.
with reduction of hepatic oxidative stress in the fructose-fed rats. In T2DM, liver is the primary organ which comes under stress because of the metabolic abnormalities and hepatic insulin resistance [1]. We evaluated and compared the effect of resveratrol, a widely used nutritional supplement with metformin, a commonly used first line drug, on diabetes and hepatic oxidative stress in fructose-fed rats. Consumption of fructose in the form of high fructose corn syrup (HFCS) is increasing every year in most countries including USA, India and China. High fructose intake over long periods is wellknown risk factor for diabetes and obesity [23]. In the present study, we used a high fructose rich diet which is much higher than physiological human dietary fructose, for the induction of type 2 diabetes mellitus, which is characterized by insulin resistance, metabolic
syndrome and oxidative stress. Previous studies including ours have shown that long-term fructose feeding induces diabetes associated with insulin resistance and metabolic syndrome in experimental animals such as rats and mice [11,24–26]. Previous study showed that fructose consumption causes metabolic alterations in liver that leads to abnormalities including oxidative stress [27]. Based on the increased consumption of fructose and other sugar based product in humans, we chose high fructose-fed rat model for the induction of insulin resistance along with metabolic syndromes and increased oxidative stress in liver. In the present study, we found that feeding of high fructose diet for a period of 8 weeks induces prediabetes as measured by induction of insulin resistance, increase in postprandial blood
Fig. 6. Immunoblots of NRF2 expression in liver. (A) Immunoblot of NRF2 from hepatic total protein extracts. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblot. (N = 3). (B) Densitometry analysis of NRF2 level from total protein extract (N = 3). Data shown as mean ± SEM, * p < 0.05 vs. Dia. and † p < 0.05 vs. Con. (C) Immunoblot of NRF2 from hepatic nuclear protein extracts. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblot. (D) Densitometry analysis of NRF2 level from nuclear protein extract (N = 3). Data shown as mean ± SEM, * p < 0.05 vs. Dia. and † p < 0.05 vs. Con.
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Fig. 7. Photomicrograph of control liver, H&E, 20×: illustrating central vein (CV) with endothelial lining (dotted arrow), hepatocytes and sinusoidal spaces with Kupffer cells (7A). Liver section of diabetic rat showing loss of the normal architecture with shrunken (7B1 ) or distended central vein (7B2 ). Note some nuclei were pycnotic (7B1 , arrows) and leucocytic infiltrations into the vein (7B2 , arrows). The liver sections from diabetic animals treated with either resveratrol or metformin showed almost normal histology except a little dilated CV and slightly more sinusoidal spaces (7C and D) than control.
Fig. 8. Photomicrograph of liver, Masson’s Trichrome stain, 20×: diabetic liver sections did not show much changes in collagen fiber pattern except a little thinning of connective tissue around the portal tracts (PV) (Fig. 8B) as compare to control (Fig. 8A). Note neither resveratrol nor metformin group showed any such thinning effect (Fig. 8C and D).
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glucose levels and other metabolic disturbances. Fructose (65%) rich diet feeding for 8 weeks showed significant but mild increase in postprandial blood glucose levels along with increased serum triglyceride, uric acid and insulin levels. Intraperitoneal glucose tolerance test also showed a marked insulin resistance in diabetic group animals. Administration of resveratrol and metformin reduced the increased blood glucose levels along with serum triglyceride and uric acid levels. Although both resveratrol and metformin showed improvement in insulin sensitivity, resveratrol showed greater improvement of insulin sensitivity compared to both control and metformin-treated animals. Resveratrol administration is known to increase insulin secretion from beta cells [28,29] and it is possible that higher levels of insulin due to resveratrol might be responsible for improvement of insulin sensitivity. In the present study also higher insulin level along with improved insulin sensitivity was observed in resveratrol treated rats compared to control and metformin treated groups. Although there was no change in food intake and body weight gain in fructose-fed animals, both resveratrol and metformin administration resulted in a significant decrease in food intake and body weight gain, which might also responsible for improved insulin sensitivity. Serum triglyceride and uric acid levels, which may be also responsible for the increase in insulin resistance in fructose-fed rats, was significantly reduced after resveratrol and metformin administration. Nitric oxide is an important signaling gaseous molecule which regulates wide range of physiological and pathological processes [30–32]. Increased serum NO levels have been reported in diabetic patients as well as fructose-fed diabetic rats [11,33]. Increased NO levels can also induce oxidative damage through the formation of peroxynitrate [34]. In the present study, serum NO levels were significantly higher in fructose-fed rats compared to the control rats. Chronic administration of resveratrol and metformin normalized the NO level in fructose-fed rats. The reduction of NO level in serum might be due to reduction of oxidative stress in fructose-fed rats after administration of both drugs tested in the present study. Both experimental and clinical studies indicate that oxidative stress plays a major role in the development and complications of type 2 diabetes [35]. Free radicals are generated in diabetes by glucose oxidation. The oxidative stress may be amplified by diabetes-induced metabolic stress, tissue damage, and cell death, leading to increased free radical production and compromised free radical scavenger systems, which further exacerbate the oxidative stress. Oxidative stress can also lead to damage of cellular organelles, and development of insulin resistance [36]. In the present study, high fructose feeding increased oxidative stress as evidenced by elevation of TBARS and conjugated dienes levels, and reduction of SOD activity and vitamin C levels in diabetic liver in comparison to control group. Unlike metformin, resveratrol reduced hepatic TBARS and conjugated dienes levels and increased SOD activity and vitamin C levels in the fructose-fed liver. While resveratrol administration increased both hepatic GSH and catalase activity, metformin only increased the hepatic catalase activity compared to fructose-fed liver. Our study showed that resveratrol was more effective than metformin in reducing oxidative stress in liver. This beneficial antioxidant effect might be responsible for improved insulin sensitivity in fructose-fed rats after resveratrol administration. Similarly to ensure the hepatic manifestations of metabolic syndrome and oxidative stress, we did histopathological examination in our prediabetic model. Presence of inflammation, non-alcoholic steatohepatitis and fatty liver was reported earlier after high fructose feeding in rat liver [37]. Unlike previous study, we found only a mild inflammation in fructose-fed liver. However, this change was not observed when fructose-fed rats were treated with either resveratrol or metformin.To provide insights into the molecular mechanism behind the beneficial effect of resveratrol in fructose-fed rats, we measured hepatic NRF2 protein level from
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all groups. Nuclear factor E2-related factor 2 (NRF2) is a transcription factor important in the protection against any oxidative stress. During oxidative stress, NRF2 is released from sequestration in the cytoplasm and translocates to the nucleus. NRF2 binds antioxidant response elements (AREs) in the regulatory regions of target genes and activates transcription of several antioxidant enzymes. Several NRF2 activators have already been developed for treating diseases involving oxidative stress [38]. Interestingly, NRF2 activators have also been shown to modulate insulin action [39]. Hyperglycemia-induced endothelial dysfunction, vascular complications and cardiomyocyte damage was prevented by NRF2 activation by reducing oxidative stress [38]. Similar to previous studies, the present study demonstrated that beneficial antioxidant effect of resveratrol was associated with increased nuclear translocation of NRF2 in fructose-fed liver. Increased nuclear NRF2 level might have a significant protective role against high fructose induced oxidative stress in liver as observed in our study, possibly through augmentation of hepatic antioxidant defense enzymes. In conclusion, there was an improvement of hyperglycemia and metabolic parameters after metformin and resveratrol administration in fructose-fed prediabetic rats. However, resveratrol showed a more marked insulin sensitizing action than metformin. In addition, resveratrol showed additional beneficial effect by reducing the hepatic oxidative stress in prediabetic rat through activation of NRF2. Conflict of interest The authors declare that they have no competing interest. Acknowledgments Financial support was provided by Ramalingaswami fellowship funds to SKB and SC from Department of Biotechnology (DBT), Govt. of India and in part, IICT institute fund. SKB has grant support from DBT (SR/SO/HS-110/2008). PKB is a Senior Research Fellow from Council of Scientific and Industrial Research (CSIR). We wish to thank Kumar organic products Ltd., Bangalore for providing gift sample of Resveratrol. We wish to thank Dr. K. Praveen Kumar for his excellent technical assistance and Dr. J. S. Yadav, Director, IICT, Hyderabad for providing all kind of support for this work. We would like to gratefully acknowledge Dr. Mohua Maulik for her suggestions and critical review of the manuscript. References [1] Cade WT. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting. Physical Therapy 2008;88(11):1322–35. [2] Johnson RJ, Perez-Pozo SE, Sautin YY. Hypothesis: could excessive fructose intake and uric acid cause type 2 diabetes? Endocrine Reviews 2009;30(1):96–116. [3] Tooke JE. Possible pathophysiological mechanisms for diabetic angiopathy in type 2 diabetes. Journal of Diabetes and its Complications 2000;14(4):197–200. [4] Dominguez C, Ruiz E, Gussinye M, Carrascosa A. Oxidative stress at onset and in early stages of type 1 diabetes in children and adolescent. Diabetes care 1998;21:1736–42. [5] Pirola L, Fro S. Critical review resveratrol: one molecule, many targets. Cell 2008;60:323–32. [6] Lee SM, Yang H, Tartar DM, Gao B, Luo X, Ye SQ, et al. Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1 diabetes. Diabetologia 2011;54:1136–46. [7] Lekli I, Szabo G, Juhasz B. Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin. American Journal of Physiology and Heart Circulation Physiology 2008;294:H859–66. [8] Sharma S, Misra CS, Arumugam S, Roy S, Shah V, Davis JA, et al. Antidiabetic activity of resveratrol, a known SIRT1 activator in a genetic model for type-2 diabetes. Phytotherapy Research 2010;25:67–73. [9] Su HC, Hung LM, Cheng JK. Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats. American Journal of Physiology and Endocrinology Metabolism 2006;290:1339–46.
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