Metformin prevents glucotoxicity by alleviating oxidative and ER stress–induced CD36 expression in pancreatic beta cells  Jun Sung Moon, Udayakumar Karunakaran, Suma Elumalai, In-Kyu Lee, Hyoung Woo Lee, Yong-Woon Kim, Kyu Chang Won  Journal of Diabetes and Its Complications  Volume 31, Issue 1, Pages 21-30 (January 2017) DOI: 10.1016/j.jdiacomp.2016.09.001 Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 1 Metformin protected beta cells against high glucose and thapsigargin-induced apoptosis. INS-1 cells were treated with indicated concentrations of high glucose (A) for 24h with and without metformin. In case of (B), cells were treated with metformin for 1h and then exposed to thapsigargin (0.5uM) for 24h. Cell viability was measured by WST assay. Data are expressed as the mean±SEM from three separate experiments. *p<0.001 vs control; *p<0.005 vs high glucose; thapsigargin-treated group. (C, D) Caspase-3 was analyzed by Western blot analysis. Western blots were quantified from three independent culture experiments. *p<0.001 vs control; *p<0.001 vs HG; #p<0.005 vs thapsigargin-treated cells. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 2 Metformin recovered high glucose-reduced insulin (A) and Pdx1 mRNA (B). INS-1 cells were treated with high glucose (30mM) with and without metformin for 12h. Insulin and Pdx1 mRNA were measured by RT-PCR. Data represent three independent experiments. *p<0.05 vs the control; #p<0.05 vs HG-treated cells. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 3 Effects of metformin on ER stress markers. INS-1 cells were treated with high glucose (30mM) with or without metformin for 24h (A). In case of (B), cells were treated with metformin for 1h and then exposed to thapsigargin (0.5uM) for 12h. ER stress markers were detected by Western blotting. β-actin was used as a loading control. Western blots were quantified from three independent culture experiments. *p<0.001 vs the control; **p<0.05 vs HG; thapsigargin-treated cells. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 4 Metformin reduced high glucose-induced ROS generation and JNK activation. (A) INS-1 cells were treated with high glucose (30mM) with or without metformin for 12h. Intracellular peroxide levels were detected by flow cytometry using a fluorescence dye H2DCFDA. Data are expressed as the mean±SEM from three separate experiments. *p<0.001 vs control; **p<0.05 vs HG. (B) INS-1 cells were treated with high glucose (30mM) with or without metformin for 12h. In case of (C), cells were treated with metformin for 1h and then exposed to thapsigargin (0.5uM) for 12h. Representative protein expression of JNK phosphorylation. Relative amounts of protein were calculated with reference to the amount of β-actin used as a loading control. *p<0.001 vs control; **p<0.005 vs HG; **p<0.05 vs thapsigargin. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 5 Metformin inhibited high glucose-induced CD36 expression. (A) Hydrogen peroxide (H2O2; 50μM for 1h) was administered to elevate oxidative stress and N-acetyl cysteine (NAC; 10mM for 12h) was given to reduce oxidative stress. H2O2 increased CD36 mRNA expression similar to high glucose (30mM), but NAC decreased. (B) Metformin also decreased HG-induced CD36 mRNA expression. HG (C) and thapsigargin (D) treatment increased CD36 protein level, which was decreased by metformin. (E) Palmitate uptake was significantly increased following HG for 12h and normalized by treatment with metformin treatment. Data are expressed as the mean±SE from three separate experiments. *p<0.05 vs control; #p<0.05 vs H2O2; **p<0.05 vs HG; **p<0.05 vs thapsigargin. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 6 Effects of CD36-siRNA and SSO effects on cell viability, caspase-3 and JNK activation. (A–C) INS-1 cells were treated with indicated concentrations of Sulfo-N-succinimidyl oleate (SSO) for 1h and then exposed to high glucose (30mM) for another 24h. INS-1 cells were transfected with 100nmol/L of CD36-siRNA and then incubated with high glucose (30mM) for 24h. Cleaved caspase-3, JNK phosphorylation and cell viability were measured by Western blot and WST assay, respectively. Data represent three independent experiments. *p<0.001 vs control; **p<0.005 vs HG. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 7 Metformin ameliorated glucotoxicity in primary rat islets. Metformin (0.5mM) treatment recovered mRNA expression of insulin (A) and Pdx1-1 (B), which was diminished by high glucose (30mM) exposure. Data are expressed as the mean±SEM from three separate experiments. *p<0.05 vs the control; #p<0.05 vs HG. Intracellular peroxide levels were detected by flow cytometry using a fluorescence dye H2DCFDA. *p<0.001 vs control; **p<0.005 vs HG. (C) Metformin recovered glucose-stimulated insulin secretion (D) in rat islets, which were decreased with high glucose (30mM) concentrations for 3days. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 8 Possible mechanisms for the anti-apoptotic effects of metformin. Metformin promoted beta cell survival against high glucose-induced apoptosis by inhibition of oxidative and endoplasmic reticulum stress, which leads to the reduction of CD36 expression with reduction of free fatty acid uptake. Journal of Diabetes and Its Complications 2017 31, 21-30DOI: (10.1016/j.jdiacomp.2016.09.001) Copyright © 2017 Elsevier Inc. Terms and Conditions