Novel Regulatory Roles Of Rhog And Iqgaps In Pancreatic Islet Beta Cell Function

NOVEL REGULATORY ROLES OF RhoG AND IQGAPs IN PANCREATIC ISLET BETA CELL FUNCTION by SRI ANEESHA CHUNDRU JUNE 2020 Advisor: Dr. Anjaneyulu Kowluru Major: Pharmaceutical Sciences Degree: Master of Science Type-2 Diabetes Mellitus is a chronic metabolic disease characterized by insulin resistance and pancreatic beta-cell dysfunction, the latter possibly caused due to the defects in insulin signaling pathways in beta-cells. Published evidence implicates regulatory roles for small G proteins (Arf6, Cdc42, Rac1) in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. More recent studies suggests the novel roles for these G proteins, specifically Rac1, in the induction of metabolic dysfunction and apoptosis of islet beta-cell. However, potential upstream regulators of sustained activation of Rac1 have not been identified in the beta-cell. Herein, we examined putative roles for RhoG, a small G protein in islet beta-cell dysregulation induced by chronic exposure to glucotoxic conditions which has been identified as an upstream regulator of Rac1 under specific experimental conditions. We further extended our studies to determine the regulatory role of IQGAPs, a novel G protein scaffolding protein and an effector protein for RhoG in islet beta-cell function. Our studies indicated that RhoG is expressed in INS-1 832/13 cells, human and rat islets. siRNA-mediated knockdown of RhoG (55%) exerted no effects on GSIS in INS-1 832/13 cells. These data indicate minimal roles for RhoG in GSIS. Chronic exposure of these cells to high glucose (20mM; 24 hours) resulted in increased expression of RhoG. In a manner akin to Rac1, which has been shown to translocate to the nuclear fraction to induce beta-cell dysfunction under metabolic stress, sub-cellular fractionation studies revealed increased translocation of RhoG to the nuclear fraction in INS-1 832/13 cells. Furthermore, siRNA-mediated depletion of endogenous RhoG markedly attenuated sustained activation of Rac1 and associated mitochondrial dysfunction (e.g., caspase-3 activation in INS-1 cells exposed to glucotoxic conditions. These studies suggest the key regulatory roles of RhoG in the sustained activation of Rac1 under conditions of glucotoxicity. They also indicated minimal roles for this G protein in physiological insulin secretion. Our studies on IQGAPs reveal that IQGAP1 and IQGAP2, but not IQGAP3 are expressed in INS-1 832/13 cells, human and rat islets. siRNA mediated knockdown of IQGAPs markedly attenuated Rac1 mediated Glucose-stimulated insulin secretion which provide preliminary evidence for the regulatory role of IQGAPs in Glucose-stimulated insulin secretion and Rac1 activation. Our data also demonstrated the minimal changes in the expression and subcellular localization of IQGAPs in beta cells following exposure to glucotoxic conditions. Taken together our studies suggests the regulatory roles of RhoG and IQGAPs in pancreatic beta-cell function