Pterostilbene Ameliorates Streptozotocin-Induced Diabetes through

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Pterostilbene ameliorates streptozotocin-induced diabetes through enhancing antioxidant signaling pathways mediated by Nrf2 Bhakkiyalakshmi Elango, Sireesh Dornadula, Ramasamy Paulmurugan, and K.M. Ramkumar Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00378 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on December 26, 2015

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Pterostilbene ameliorates streptozotocin-induced diabetes through enhancing antioxidant signaling pathways mediated by Nrf2 Bhakkiyalakshmi Elango,† Sireesh Dornadula,‡ Paulmurugan Ramasamy,§ Kunka Mohanram Ramkumar*,‡

†

‡

§

Department of Biotechnology, SRM University, Kattankulathur-603203, Tamilnadu, India SRM Research Institute, SRM University, Kattankulathur-603203, Tamilnadu, India Department of Radiology, Stanford University School of Medicine, Palo Alto, CA, USA

Running title: Anti-diabetic potential of pterostilbene

*- Corresponding author Dr. K.M. Ramkumar, SRM Research Institute, SRM University, Kattankulathur – 603 203, Tamilnadu. E-mail: [email protected] Tel: +91-9940737854 Fax: +91-44-2745 2343

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ABSTRACT Nuclear factor erythroid 2-related factor 2 (Nrf2) remains to be a ‘master regulator’ of cytoprotective and antioxidant genes. In our present study, we investigated the anti-diabetic role of Pterostilbene (PTS) in streptozotocin (STZ)-induced diabetic model through Nrf2-mediated antioxidant mechanisms. Nrf2 activation potential of PTS in MIN6 cells was assessed by Nrf2Keap1 complex dissociation at different time points and ARE-driven downstream target genes expression. Immunoblot experiments on Nrf2 activation and phosphorylation has conferred cytoprotection against STZ-induced cellular damage. In STZ-induced diabetic mice, PTS administration significantly decreased blood glucose levels through the improvement of insulin secretion. In addition, we also observed insulin-positive cells with recovered islet architecture in pancreas of STZ-induced diabetic mice after treatment with PTS. The activation of Nrf2 and its downstream target genes expression has been observed upon PTS treatment thereby reducing oxidative damage in the pancreatic tissues. Furthermore, PTS treatment significantly reverted the key enzymes of glucose metabolism, such as hexokinase, glucose-6-phosphatase, glucose-6phosphate dehydrogenase and fructose-1,6-bisphosphatase, to near-normal levels in liver tissues of STZ-induced diabetic mice. These results clearly indicate that PTS maintains glucose homeostasis suggesting the possibility of future candidate for diabetes management. Keywords:

Antidiabetic activity, Carbohydrate metabolism, Nrf2, Pterostilbene, STZ

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INTRODUCTION A key transcription factor, Nrf2 (Nuclear factor-erythroid2-related factor 2) plays a central role in the amelioration of oxidative and electrophilic stress that regulates intracellular antioxidants, phase II detoxifying enzymes and thereby promote cell survival and maintain cellular redox homeostasis.1 NAD(P)H quinone oxidoreductase-1 (NQO1), Heme oxygenase-1 (HO-1), γ-Glutamylcysteine synthetase (γ-GCS), Glutathione Peroxidase (GPx), Catalase (CAT) and Superoxide dismutase (SOD) are the well-reported Nrf2 target genes that are upregulated through the antioxidant response element (ARE) in response to variety of stress.2, 3 Under the basal condition, Nrf2-dependent transcription is repressed by its own suppressor, the Kelch-Like ECH-Associated Protein 1 (Keap1). During oxidative/electrophilic stress, Nrf2-Keap1 complex dissociates and Nrf2 translocates into the nucleus where it activates downstream target gene expression and maintains cellular redox homeostasis.4 Inspite of its antioxidant function, Nrf2 also regulates an array of genes that defend cells against the deleterious effects of environmental insults.5, 6 Since Nrf2-dependent functions are able to protect multi-organs, its activation has been implicated in various diseases including cardiovascular and neurodegenerative diseases, acute and chronic lung injuries, inflammation and autoimmune disorders.7-9 Hence, Nrf2 regulation and its potential molecular mechanism are crucial in the fiels of drug development for therapeutic intervention. For the past few years, activation of Nrf2 remains to be a promising strategy for increasing antioxidant defenses by transcriptionally activating the antioxidant and detoxifying genes in hyperglycemia-induced oxidative stress, involved in the pathogenesis of diabetes and its complications.10, 11 Since then, the molecular mechanism behind Nrf2 activation and its impact on diabetes has been extensively studied thereby highlighting its potential role in cytoprotection.12 Few natural products, such as sulforaphane13, 14, resveratrol15, curcuminoids16, epigallocatechin-3-gallate17 and synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien28-oic acid (CDDO) and its derivatives18, Dihydro-CDDO-trifluoroethyl amide (dh404)19, glutathione peroxidase mimetic, ebselen20 have been reported as Nrf2 activators thereby involved in cell protective mechanisms. 4



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Recently, in our laboratory, a cell-based reporter system with high throughput screening (HTS) platform has been developed that identifies Nrf2 activation in cells in the form of Nrf2 and Keap1 protein-protein interaction. We also demonstrated that pterostilbene (PTS, a natural analogue of resveratrol) significantly perturb Nrf2-Keap1 interaction pattern and persists to be a potent Nrf2 activator.21 Hence the promising and accumulating line of evidences for this naturally occurring stilbene insist current researchers to explore its multifaceted role against several disorders including diabetes. Interestingly, our earlier reports demonstrated the cytoprotective effect of PTS via Nrf2 activation against STZ-induced toxicity in pancreatic betacells highlighting its antioxidant and anti-apoptotic properties.22 A recent clinical trial and reports further revealed that PTS regulates cholesterol levels, reduces blood pressure and oxidative stress conditions in dyslipidemic individuals.23 Inline, in this study we attempt to identify the molecular mechanism of anti-diabetic potential of PTS in streptozotocin (STZ)-induced diabetic mice through the activation of Nrf2 mediated antioxidant mechanism. The anti-diabetic activity of PTS was studied by estimating blood glucose and serum insulin levels of STZ-induced diabetic animals. In view of the significance of carbohydrate metabolism in diabetes, this study also aimed to examine the effect of PTS on liver glucose-metabolizing enzymes, such as hexokinase, glucose-6-phosphatase (G6P), fructose-1,6-bisphosphatase (F1,6BP) and glucose 6-phosphate dehydrogenase (G6PD) in STZ-induced diabetic mice. Furthermore, the histopathological alterations in the pancreas of diabetic animals were investigated along with the immunoblot analysis to show Nrf2 activation potential of PTS in diabetic pancreas. In addition, this study also proved the cytoprotective functions of PTS against STZ-induced toxicity through Nrf2-mediated mechanism in an in vitro model system using MIN6 pancreatic beta cells.

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METHODS Culturing of MIN6 cells In order to study the cellular and molecular mechanisms underlying the anti-diabetic effects of PTS, we used MIN6 cell line and insulin-secreting pancreatic β-cells from mice. The MIN6 cells were cultured in DMEM supplemented with 10% heat inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine in a humidified atmosphere at 37°C with 5% CO2. Cytotoxicity assay MIN6 cells (2 X 104cells/ml) plated in 96-well microplates were incubated overnight at 37°C. Pterostilbene at different concentrations (0 - 8 µM) were treated to each well and incubated for 24 h at 37°C followed by Streptozotocin (10 mM) exposure for 1 h. The cell viability was measured using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, 24 h after incubation the cells were replenished with 90 L of phenol-red free media with 10 L MTT stock (5 mM) solution and incubated for 3 h, then media was aspirated carefully without disturbing the precipitate. The precipitate of metabolically reduced tetrazolium MTT from the cells was dissolved in 50 l of DMSO by keeping at 37oC for 20 min. Absorbance of the solution was measured at 540 nm using a multi-well plate reader (Infinite 1000, Tecan, Männedorf, Switzerland). For each set of conditions, the experiments were performed in triplicate. The relative cell viability (%) compared to control cells was calculated as follows: Cell viability (%) = [Abs (sample)-Abs (blank)/Abs (control)-Abs (blank)] x 100. Measurement of Intracellular Reactive Oxygen Species (ROS) Intracellular ROS levels were measured using an oxidation sensitive fluorescent dye, 2,7dichlorodihydrofluorescein diacetate (H2DCFDA). Briefly, MIN6 cells were treated with STZ followed by PTS, and then H2DCFDA (20 μM) was added to the cells and further incubated for 30 min at 37 °C. Then the reaction was stopped with phosphate-buffered saline (PBS) containing 10% fetal calf serum (FCS). The cells were pelleted by centrifugation (800g, 10 min), washed, and resuspended in PBS. The resultant fluorescence intensity was assessed by FACS analysis 6



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(BD Biosciences, CA, USA). A greater shift in fluorescence intensity implies higher amount of DCF and greater ROS generation

Luminometric evaluation of Nrf2-Keap1Complementation System with PTS MIN6 cells transiently transfected with CLuc−Nrf2 and NLuc−Keap1 fusion proteins were exposed to PTS and experiments were performed for different time points (2, 4, 8, 12 and 24 h). Cells were harvested and assayed for luciferase activity using luminometer (Promega, Madison, WI). The developed Nrf2-Keap1 sensor system identifies the potential of PTS in dissociation of the complex. The developed sensor system showed a drop in luciferase signal, which is directly associated with Nrf2 activation (Nrf2-Keap1 complex breakage) that is, lower signal intensity corresponds to the higher propensity for dissociation of the complement system that gets activated. ARE-Luciferase Reporter Gene Assay NQO1-ARE-Luc and GST-ARE-Luc reporter gene constructs used for cell-based reporter gene assay were provided by Donna D. Zhang (College of Pharmacy, University of Arizona, Tucson, AZ). ARE-Luc construct (500 ng/well) was transiently transfected into MIN6 cells in 12-well plates using Lipofectamine 2000. After 24 h, the media was changed and different concentrations of PTS (2, 4 and 8 μM) were added, with subsequent 24 h incubation, and the luminometry assay was performed as previously described. Luciferase activities were expressed as fold induction relative to values obtained from control cells. Results represent mean values of at least three independent transfection experiments, each carried out in triplicate. Nuclear and cytosolic fractionation To study the effect of PTS on Nrf2 translocation, nuclear and cytoplasmic extracts were prepared using a commercially available nuclear extraction kit (Pierce NE-PER®) as per the manufacturer’s instructions (Pierce, Rockford, IL, USA). Briefly, cells were homogenized in CER-I buffer using a homogenizer and incubated on ice for 15 min and centrifuged at 10 000 × g for 10 min at 4°C. The supernatant solution was collected (cytoplasmic fraction), and pellets containing nuclei were suspended in NER buffer supplemented with protease inhibitors 7


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according to the manufacturer’s instructions. After thorough vortex mixing for 1 min with the break for every 10 min for 40 min, samples were centrifuged at 16000 × g for 15 min at 4°C, and supernatant solution was collected (nuclear fraction). After quantification of the protein concentration by Biorad Bradford assay, the samples were subjected to Western blot analysis. Western blotting The total proteins, and cytoplasmic and nuclear fractions from different treatment conditions of the cell lysates were collected and proteins were resolved using a 10% SDS-PAGE gradient gel (Invitrogen, Carlsbad, CA) and electroblotted onto a nitrocellulose membrane. Primary and secondary antibodies against Nrf2 (Nrf2:1/500-ab31163; pNrf2:1/500- ab76026; Goat Anti-rabbit IgG- ab97051, Abcam, Cambridge, UK), GAPDH (ab9485, Abcam, Cambridge, UK) and lamin-B (ab16048, Abcam, Cambridge, UK) were used for detecting respective proteins by a standard protocol. The enhanced chemiluminescence system (GBOX, Syngene, UK) was used for the detection of the protein bands. Animals and diet Six-weeks-old male Swiss Albino Mice weighing 20 + 5 g were used in this study. Animals were housed and maintained under laboratory conditions of temperature of 18-24 ⁰C, relative humidity of 55-75%, with a 12 h light and 12 h dark cycles. The mice were housed in a polypropylene cage that provides at least one cubic foot of space for one mouse. The mice were provided with ad libitum access to water and standard pellet diet (Hindustan Lever Ltd., Bangalore, India) with a composition of 5% fat, 21% protein, 55% nitrogen-free extract and 4% fiber (w/w) with adequate minerals and vitamins. Experimental induction of diabetes in mice The animals were acclimatized to the laboratory conditions for 2 weeks prior to the inception of experiments. The mice were given an intraperitoneal (i.p.,) injection of multiple low doses of streptozotocin (Sigma Chemical Co., St. Louis, MO) (50 mg/kg) freshly prepared in 0.1M sodium citrate buffer (pH 4.5) for five consecutive days. After a week, mice with moderate diabetes (i.e, blood glucose concentration range of 200-300 mg/dL) that exhibited glycosuria and hyperglycemia were selected for further experiments. This study was reviewed and approved by 8



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the Institutional Animal Ethical Committee (57/IAEC/2011) of SRM University, which abide by the guidelines of the Institute of Laboratory Animal Resources. Studies involving animals were reported in accordance with the ARRIVE guidelines.24 Experimental design The mice were divided into seven groups (n=6): (1) normal control; (2) normal control mice that were treated with pterostilbene (10 mg/kg BW); (3) STZ-induced diabetic mice; (4) diabetic mice that were treated with pterostilbene (5 mg/kg BW); (5) diabetic mice that were treated with pterostilbene (10 mg/kg BW); (6) diabetic mice that were treated with resveratrol (10 mg/kg BW), a known Nrf2 activator which served as positive control and (7) diabetic mice that were treated with glibenclamide (600 µg/kg BW), anti-diabetic drug control. Pterostilbene, resveratrol and glibenclamide were intraperitoneally administered to the respective groups till the end of the experimental period (5 weeks). Blood glucose and body weight of experimental animals were periodically recorded after overnight fasting. After the five-week protocol, animals were fasted overnight and were sacrificed by decapitation. Blood was collected for biochemical evaluation. The pancreas and liver were dissected, immediately rinsed in ice-cold saline, and stored for further biochemical analysis. Analytical procedure Fasting Blood glucose levels were frequently monitored by Accu-Chek Glucometer (Roche Diagnostics, Indianapolis, IN, USA). Serum insulin level was determined with an Enzyme Linked ImmunoSorbent Assay (ELISA) kit using mouse insulin as standard (Mercodia, Uppsala, Sweden). Intraperitoneal glucose tolerance test (IPGTT) was done at the end of experimental period with minor modifications of method described by Goren et al.

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. Briefly, at the end of

experimental period, after over-night fasting, 1 g/kg BW glucose was intraperitoneally injected to all the mice. The blood glucose was measured at 30, 60, 90 and 120 min after the glucose injection. Hexokinase, G6P and F1-6BP were assayed according to the protocols described by Brandstrup et al., 26 Zak et al., 27 and Gancedo et al., 28 respectively, and the inorganic phosphate (Pi) liberated was estimated by the method of Berenblum et al. 29. G6PD was determined by the 9


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method of Ells et al.

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Glycogen content was estimated by the method of Huijing31 and protein

content in tissue homogenates was measured by the method of Bradford.32 Histopathology of pancreas The pancreatic tissues obtained from all the experimental animals were subjected to Hematoxylin and Eosin staining. The number and size of islets of Langerhans in pancreas was analyzed and photographed using microscope (Carl Zeiss, Thornwood, NY, USA). To examine the expression of insulin in pancreatic tissues, an immunohistochemical analysis was performed using insulin antibody (sc-9168; Santa Cruz Biotechnology, Heidelberg, Germany). The 5 µm thick paraffin sections were deparaffinized in xylene and hydrated with ethanol. The hydrated sections were then treated with 3% H2O2 in methanol for 30 min to block any endogenous peroxidase and washed with 0.01M phosphate buffer for 10 min. The sections were further processed by an indirect immunoperoxidase technique using a One-Step Polymer-HRP Detection kit (Leica Biosystems, Newcastle, UK) with secondary antibodies. A standard concentration of Hematoxylin was added as a counter stain. Morphometric analysis The image analysis software Image-J was used to calculate islet area, insulin-positive cells and the beta cell mass. Using a total of 5 sections for each group of mice, β-cell mass was estimated using the formula: cell mass (mg) = [islet area/whole pancreas area] X pancreas weight (mg). Cells positive for insulin was quantified by the presence of a dark brown nuclear stain. Observations were made from a minimum of 50 islets and, when quantified, were expressed as a percentage of the total number of islet cells. Statistical analysis All data were expressed as mean ± SEM of three separate experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 20 (SPSS, Cary, NC, USA) followed by Tukey’s post hoc test. P

Pterostilbene Ameliorates Streptozotocin-Induced Diabetes through

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