Functions And Regulation Mechanisms Of Glycogen Synthase Kinase-3 In The Beta Cell Apoptosis Induced By Oleic Acid

Part oneProtection of pancreatic beta cells against oleic acid-induced apoptosis by inhibiting the activity of glycogen synthase kinase-3Objective:Type 2 diabetes mellitus is a disease characteristied by insulin resistance and beta cell dysfunction, and the apoptosis of beta cells plays a prominent role in the pathogenesis of type 2 diabetes. This study was designed to explore the potential roles of glycogen synthase kinase 3 (GSK-3) in INS-1 cells.Methods:Oleic acid(OA) could induce apoptosis in INS-1 cells. Apoptosis was evaluated by the PI staining analysis by flow cytometry, phosphorylated GSK-3 and total GSK-3 were analyzed by Western blot, and then lithium chloride (LiCl) was used to inhabit the acticity of GSK-3 and further studied the inhibitive effect on apoptosis of INS-1 cells.Results:Oleic acid can increase the ratio of apoptosis of INS-1 cells with a concentration-dependent manner. Simultaneously, the decreased phosphorylation of GSK-3 demonstrates the activation of GSK-3. While treated with 24mmol/L LiCl (a inhibitor of GSK-3), OA induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 increased remarkably.Conclusion:Inhibition the activity of GSK-3 plays a very important role in protecting oleic acid-induced apoptosis in pancreatic INS-1 cells. That provides a novel insight into the mechanism of type 2 diabetes and a potential drug target for clinical treatment. Objective:ER-stress induced apoptosis of beta cells is a substantial mechanism of type 2 DM. PERK, an important transmembrane protein of ER, can be activated by the overactivation of ER-stress which will induce beta cells apoptosis. Our previous data indicated that GSK-3 activated by FFAs will induce apoptosis of beta cells, while ER-stress can be caused by long-time exposure to FFAs. The purpose of this study is to reveal the role of ER-stress and GSK-3 and its potential signal pathway during beta cells apoptosis.Methods:The alterations of ER-stress related signal factors and kinases induced by OA are assessed by western blot, and the changes of PERK and AMPK are analyzed by ELISA meanwhile. The phosphorylated GSK-3βand total GSK-3 are detected by western blot after the inhibition of PERK and AMPK, while the PERK is inhibited by the transfection of P58(IPK) plasmid and AMPK is inhibited by the inhibitor Compound C. Finally, the interaction between PERK and GSK-3 are identified by co-immunoprecipi tation and direct immunofluorescence.Results:1. Activation of ER-stress related signal factors and kinases in INS-1 cells treated with OA. The expression of GRP78,ATF6,XBP1,PERK and AMPK significantly increased in the presence of 0.4mM OA(P<0.01).2. ELISA measurment of PERK and AMPK activity. Activity of PERK and AMPK significantly augmented in the presence of OA (P<0.01).3. Detections of the alterations of GSK-3 after inhibiting the activity of PERK and AMPK. (1) GSK-3βwas significant activated when treated with OA; (2) the phosphorylation of GSK-3βObviously increased after the inhibition of PERK after transfection of P58(IPK) plasmid(P<0.01); (3) the activity of GSK-3 had no change after the inhibition of AMPK by Compound C (P>0.05).4. Identification of interaction between PERK and GSK-3 by co-immunoprecipitation. Co-immunoprecipitation of PERK and GSK-3 verified the association of PERK and GSK-3.5. Co-localization of PERK and GSK-3 in INS-1 cells. Obvious co-localization was observed after merging FITC-conjugated PERK (green) and Rhodamine-conjugated GSK-3β(red).Conclusion:Activation of GSK-3 can induce beta cells apoptosis by ER-stress which is caused after long-time exposure to FFAs. PERK, a biomarker of ER-stress will activate GSK-3 directly. Collectively, PERK-GSK-3 signal pathway will play an important role during beta cell apoptosis in type 2 DM.