Article pubs.acs.org/JAFC
Vescalagin from Pink Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Alleviates Hepatic Insulin Resistance and Ameliorates Glycemic Metabolism Abnormality in Rats Fed a HighFructose Diet Da-Wei Huang,†,∥ Wen-Chang Chang,§,∥ James Swi-Bea Wu,§ Rui-Wen Shih,# and Szu-Chuan Shen*,# †
Department of Food and Beverage Management, China University of Science and Technology, No. 245, Sec. 3, Academia Road, Taipei 11581, Taiwan § Graduate Institute of Food Science and Technology, National Taiwan University, P.O. Box 23-14, Taipei 10672, Taiwan # Department of Human Development and Family Studies, National Taiwan Normal University, No. 162, Sec. 1, Heping East Road, Taipei 10610, Taiwan ABSTRACT: This study investigates the ameliorative effect of vescalagin (VES) isolated from Pink wax apple fruit on hepatic insulin resistance and abnormal carbohydrate metabolism in high-fructose diet (HFD)-induced hyperglycemic rats. The results show that in HFD rats, VES significantly reduced the values of the area under the curve for glucose in an oral glucose tolerance test and the homeostasis model assessment of insulin resistance index. VES significantly enhanced the activity of hepatic antioxidant enzymes while reducing thiobarbituric acid-reactive substances in HFD rats. Western blot assay revealed that VES reduced hepatic protein expression involved in inflammation pathways while up-regulating expression of hepatic insulin signalingrelated proteins. Moreover, VES up-regulated the expression of hepatic glycogen synthase and hepatic glycolysis-related proteins while down-regulating hepatic gluconeogenesis-related proteins in HFD rats. This study suggests some therapeutic potential of VES in preventing the progression of diabetes mellitus. KEYWORDS: vescalagin, inflammation, insulin resistance, carbohydrate metabolism, diabetes mellitus
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INTRODUCTION The prevalence of obesity, diabetes, and some other metabolic syndromes is linked to the increased human consumption of fructose-containing foods. High-fructose diet (HFD)-induced diabetic rats are widely used as an in vivo insulin resistance model to clarify the mechanism of therapy for type 2 diabetes mellitus (DM).1 HFD may enhance oxidative stress, imbalance of the antioxidant system via the production of free radicals, promotion of inflammation, and decreased antioxidant enzyme activity in rats.2 A previous study revealed that the disorder of blood glucose control may lead to enhanced production of reactive oxygen species (ROS), inducing inflammation, carbohydrate metabolism imbalance, and gene mutation via attenuating antioxidant defense.3 Oxidation and inflammation play a role in accumulation of advanced glycation end products, resulting in diabetic complications.4,5 Liver is an insulin-sensitive organ responsible for the regulation of energy homeostasis. Liver cells have been applied as an in vitro model for evaluating and screening active compounds with anti-hyperglycemic activity from food ingredients.6 In addition, hepatocytes retain the intact characteristics of enzymes and are recognized as a proper model for metabolism or toxicity studies.7 HFD may lead to major outcomes such as elevated blood triglyceride levels, increased tumor necrosis factor-α (TNF-α), enhanced oxidative stress and ROS production, and down-regulated expression of insulin signal transduction protein, resulting in insulin resistance in rats.8 Thus, it is important to find active © XXXX American Chemical Society
components that enhance antioxidant enzymes in vivo to ameliorate carbohydrate metabolism via regulating insulin signaling in peripheral tissues. The wax apple is reported to contain an abundance of phytochemicals including ellagitannins, proanthocyanidins, flavanones, anthocyanidins, flavonol glycosides, triterpenoids, chalcones, and volatile terpenoids.9−11 The hydrolyzable ellagitannins are plant polyphenols that may exhibit functional properties involved in the effects of antioxidation, scavenging peroxide, and neuroprotection.12 Moreover, ellagitannins display anti-inflammatory, cardioprotective, and hepatoprotective effects in an antigen-induced arthritis rat model.13,14 We previously demonstrated that vescalagin (VES) isolated from young Pink wax apple fruits enhances glucose uptake in insulinresistant FL83B mouse hepatocytes and exhibits anti-hyperglycemia activity in HFD-induced diabetic rats.15 However, the mechanisms of VES in hypoglycemia and its effects on hepatic insulin resistance and glucose use in DM are unclear. The aim of this study was to investigate the mechanism of hypoglycemia by examining the effect of VES on ameliorating hepatic insulin resistance and abnormalities of carbohydrate metabolism in HFD rats. Received: November 26, 2015 Revised: January 21, 2016 Accepted: January 22, 2016
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DOI: 10.1021/acs.jafc.5b05558 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effect of vescalagin on the (A) area under the curve of oral glucose tolerance test (AUCOGTT) and (B) homeostasis model assessment for insulin resistance (HOMA-IR) index in HFD rats. Normal, normal diet; HF, high-fructose diet (HFD) (66% fructose); HF+PIO, HFD (66% fructose) + pioglitazone (30 mg/kg BW); HF+VES (H), HFD (66% fructose) + vescalagin (30 mg/kg BW); HF+VES (L), HFD (66% fructose) + vescalagin (10 mg/kg BW). Different letters (a−d) signify a statistically significant difference at p < 0.05. Values were calculated as the mean ± SD for six rats in each group.
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yielded a curve showing the increases and decreases in glucose levels over time, and the measurement of the concentration over time was expressed as an integrated AUCOGTT. Homeostasis Model Assessment-Insulin Resistance (HOMAIR) Index. The homeostasis model assessment for insulin resistance (HOMA-IR) index was calculated using the following equation reported by Matthews et al.17
MATERIALS AND METHODS
Plant Material. The fruits of the Pink wax apple [Syzygium samarangense (Blume) Merrill and L.M. Perry cv. Pink cultivars] were collected after the third week of blooming in August 2012 from Pingtung county, Taiwan. Chemicals. D-Glucose, fructose, pioglitazone hydrochloride, ethyl ether, Tris-HCl, Triton X-100, and ethyl alcohol were purchased from Sigma (St. Louis, MO, USA). All chemicals used in this study were of analytical grade. Preparation of Vescalagin. VES was extracted and purified from unripe Pink wax apple fruit according to the method of Chang and Shen.16 The recovery of VES from the fruit after extraction, filtration, concentration, centrifugation, freeze-drying, and column separation was 0.085% of the original weight of the fruit. The lyophilized powder was obtained and stored at −80 °C for further experiments. Animals and Diets. Male Wistar rats (age 5 weeks) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The rats were maintained under standard laboratory conditions at a temperature of 22 ± 1 °C and a 12 h light/12 h dark cycle, with free access to food and water for the duration of the study. The room conditions and treatment procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all of the protocols were approved by the Institutional Animal Care and Use Committee of National Taiwan University, Taipei, Taiwan. Wistar rats were fed a normal diet for 1 week to reach body weights of approximately 250 g and divided into five groups of six rats each. Group 1 was fed a normal diet for 16 weeks. Groups 2 and 3 were fed HFD (66% fructose in diet) for 16 weeks and administered VES (10 or 30 mg/kg body weight, respectively) daily for the last 4 weeks of this 16 week period. Group 4 was fed HFD for 16 weeks and administered pioglitazone (30 mg/kg) daily for the last 4 weeks as the positive control. Group 5 was fed HFD throughout the experiment as a negative control. Area under the Curve of Oral Glucose Tolerance Test (AUCOGTT). The oral glucose tolerance test (OGTT) was accomplished in overnight-fasted rats from all experimental groups at week 16. All animals were orally treated with a load of 1.5 g of glucose/kg body weight. Blood was taken from the tail vessels of conscious animals before loading (t = 0) and at 30, 60, 90, and 120 min after glucose administration. Samples were allowed to clot for 30 min and then centrifuged (3000g, 20 min, 4 °C) to obtain the plasma. Glucose content was determined using a glucose enzymatic kit (Crumlin Co., Antrim, UK). Plotting glucose concentration as a function of time
HOMA‐IR index = fasting plasma insulin (mU/L) × fasting plasma glucose (mmol/L)/22.5
Homogeneous Solution from Rat Liver Tissue for Antioxidant Enzymes. Liver tissue (0.3 g) was homogenized with 0.5 mL of 50 mM sodium phosphate buffer solution (pH 7.4) in an ice water bath using a homogenizer (1400 rpm, 30 s). A total of 200 μL of homogenate was mixed with 280 μL of 50 mM sodium phosphate buffer solution (pH 7.4) and 480 μL of 2% Triton X-100 solution and centrifuged (10000g, 5 min, 4 °C) to acquire the supernatant for further experiments. Biochemical Measurement. Antioxidant enzyme activity and thiobarbituric acid-reactive substrates (TBARS) in liver were determined using a glucose enzymatic kit, superoxide dismutase (SOD) kit, catalase kit, glutathione peroxidase (GPx) commercial kit, and TBARS kit purchased from Randox Laboratories (Crumlin Co.). Biochemical analyses were carried out according to Randox Laboratories protocols. Western Blot Analysis. Liver tissue (0.5 g) was homogenized with 20 mL of ice buffer solution (0.2% Triton X-100, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride) at 4 °C for 2 min and centrifuged (16000g, 60 min, 4 °C) to acquire the cell protein in supernatant for Western blot analysis. Aliquots of supernatant, each containing 50 μg of protein, were used to evaluate the expression of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3 kinase (PI3K), Akt/protein kinase B (Akt/ PKB), glucose transporter-2 (GLUT-2), hexokinase (HXK), phosphofructokinase (PFK), aldolase, fructose-1,6-bisphosphatase (F-1,6-BP), and glycogen synthase (GS). The samples were subjected to 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to a polyvinylidene difluoride membrane. The membrane was incubated with blocking buffer (phosphate-buffered saline containing 0.05% Tween-20 (PBST) B
DOI: 10.1021/acs.jafc.5b05558 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. TBARS and Antioxidant Enzymes Activity of Liver in High-Fructose Diet Induced Diabetic Rats Orally Administered Vescalagin during the Last 4 Weeksa treatment item/group
pioglitazone (mg/kg BW)
vescalagin (mg/kg BW)
Normal HF HF+PIO HF+VES (H) HF+VES (L)
− − 30 − −
− − − 30 10
TBARS (μM/mg protein) 115 207 149 119 66
± ± ± ± ±
2c 20 a 29 bc 15 c 7d
SOD activity (units/mg protein) 16.1 15.1 15.8 17.0 18.4
± ± ± ± ±
2.1 2.2 2.8 1.6 1.7
ab b ab ab a
catalase activity (units/mg protein) 6.1 4.1 4.9 5.9 6.4
± ± ± ± ±
0.6 0.8 0.7 1.6 1.1
a b ab ab a
GPx activity (units/mg protein) 17.9 13.3 17.9 19.6 20.3
± ± ± ± ±
1.3 1.8 2.4 2.2 2.5
a b a a a
Each value represents the mean ± standard deviation (n = 6). Normal, rats were fed a normal diet for 16 weeks; HF, rats were fed ahigh-fructose diet for 16 weeks; HF+PIO, rats were fed a high-fructose-diet for 16 weeks and orally administered pioglitazone during the last 4 weeks; HF+VES, rats were fed a high-fructose-diet for 16 weeks and orally administered vescalagin during the last 4 weeks. Different letters (a−d) in the same column signify a statistically significant difference at p < 0.05. a
Figure 2. Expression of hepatic (A) nuclear factor-κB (NF-κB), (B) cyclooxygenase-2 (COX-2), (C) monocyte chemoattractant protein-1 (MCP-1), and (D) intercellular adhesion molecule-1 (ICAM-1) in high-fructose diet induced diabetic rats orally administered vescalagin during the last 4 weeks. Normal, normal diet; HF, high-fructose diet (HFD) (66% fructose); HF+PIO, HFD (66% fructose) + pioglitazone (30 mg/kg BW); HF +VES (H), HFD (66% fructose) + vescalagin (30 mg/kg BW); HF+VES (L), HFD (66% fructose) + vescalagin (10 mg/kg BW). Relative expressions of NF-κB, COX-2, MCP-1, and ICAM-1 in each treatment group were calculated using actin as the standard. Different letters (a−d) signify a statistically significant difference at p < 0.05. Values were calculated as the mean ± SD for six rats in each group. GLUT-2, or anti-GS (Cell Signaling Technology, Beverly, MA, USA), overnight at 4 °C. The intensity of the blots probed with 1:2000 diluted solution of mouse monoclonal antibody to bind actin (GeneTex) was used as control to ensure that a constant amount of protein was loaded into each lane of the gel. The membrane was
and 5% w/v nonfat dry milk) for 1 h, washed with PBST three times, and then probed with 1:1000 diluted solutions of anti-NF-κB, antiCOX-2, anti-MCP-1, anti-ICAM-1, anti-IRS-1, anti-HXK, anti-PFK, anti-aldolase, or anti-F-1,6-BP (GeneTex, Irvine, CA, USA), with 1:2000 diluted solutions of anti-IR, anti-PI3K, anti-Akt/PKB, antiC
DOI: 10.1021/acs.jafc.5b05558 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. Hepatic (A) insulin receptor (IR), (B) insulin receptor substrate-1 (IRS-1), (C) phosphatidylinositol-3 kinase (PI3K), (D) Akt, and (E) glucose transporter-2 (GLUT-2) expression in high-fructose diet induced diabetic rats orally administered vescalagin during the last 4 weeks. Normal, normal diet; HF, high-fructose diet (HFD) (66% fructose); HF+PIO, HFD (66% fructose) + pioglitazone (30 mg/kg BW); HF+VES (H), HFD (66% fructose) + vescalagin (30 mg/kg BW); HF+VES (L), HFD (66% fructose) + vescalagin (10 mg/kg BW). Relative expressions of IR, IRS-1, PI3K (p85), Akt, and GLUT-2 in each treatment group were calculated using actin as the standard. Different letters (a−d) signify a statistically significant difference at p < 0.05. Values were calculated as the mean ± SD for six rats in each group.
apple in terms of hypoglycemia in diabetic rats;15 however, the mechanism of VES anti-hyperglycemic activity in diabetic animals is unclear. HFD-fed rats have been recognized as a model to investigate insulin resistance as well as diabetes,1,15 and the HOMA-IR index calculated by fasting plasma insulin and fasting plasma glucose is widely used in evaluating the occurrence of insulin resistance.17 Fasting plasma glucose levels in the current work significantly increased in rats fed HFD (129.7 ± 7.3 mg/dL) for 12 weeks prior to supplementation with VES compared with rats fed a normal diet (98.0 ± 2.1 mg/dL) (p < 0.05) (data not shown), indicating induction of hyperglycemia. HFD rats showed a significant increase in AUCOGTT and HOMA-IR index values compared with the normal group (p < 0.05) (Figure 1). HFD rats treated with pioglitazone or 10 or 30 mg/kg body weight VES showed significant reductions in AUCOGTT values of 28.8, 18.7, and 33.5%, respectively, and in HOMA-IR, 49.5, 74.4, and 71.7%, respectively, compared with the negative control (p < 0.05) (Figure 1). The present study illustrated that VES
washed three times for 5 min each time in PBST, shaken in a solution of horseradish peroxidase-linked anti-mouse IgG or anti-rabbit IgG secondary antibody, washed a further three times for 5 min each time in PBST, and exposed to the enhanced chemiluminesence reagent (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Autoradiography was executed on Fuji medical X-ray film (Fuji, Tokyo, Japan), which was scanned and analyzed using a UVP Biospectrum image system (Level, Cambridge, UK). Statistical Analysis. The results are expressed as the mean ± standard deviation and were analyzed by one-way ANOVA and Duncan’s new multiple-range tests with Statistical Analysis System (SAS) version 9.3 (Cary, NC, USA). All comparisons were made relative to the controls or negative control. The differences were considered statistically significant at p values of
