It is well established that chronic exposure of the pancreatic islet β-cells to metabolic stress (e.g., high glucose, palmitate and ceramide) induces metabolic dysfunction and loss of functional β-cell mass. Original and ongoing investigations from our laboratory have defined novel roles for Rac1, a small G-protein, in the pathogenesis of islet β-cell dysfunction under glucolipotoxic conditions. Based on published and compelling preliminary evidence, we now propose to test the overall hypothesis that metabolic stress promotes functional and transcriptional activation of Rac1 to promote intracellular oxidative stress, mitochondrial damage and eventual demise of the effete β-cell. We propose methodical investigations to identify key signaling proteins/factors in the Rac1 activation-deactivation cycle that might contribute to the metabolic and functional defects in the pancreatic β-cell. Studies designed herein will demonstrate regulatory roles and cross-talk between novel of guanine nucleotide dissociation inhibitors, namely GDI1 (RhoGDI) and GDI2 (LyGDI) (Aim 1) and scaffolding proteins/guanine nucleotide exchange factors (α4/β-PIX; Aim 2) in the functional activation of Rac1 in islet β-cells exposed to metabolic stress. Studies outlined in Aim 3 will investigate putative (NF-kB- mediated) mechanisms underlying transcriptional activation of Rac1 in islet β-cells under the duress of metabolic stress. Our goal in studies under Aim 4 is to demonstrate that aberrant transcriptional and functional activation of Rac1 leads to mitochondrial damage and islet dysfunction in an animal model of diet-induced obesity (DIO). Complementary studies will affirm contributory roles for intracellularly generated ceramide in the induction of islet dysfunction in our in vitro (Aims 1-3) and in vivo (Aim 4) model systems. Stated goals are accomplished via pharmacological, molecular biological, microscopic and lipidomics approaches in clonal β- (INS-1 832/13) cells, rodent, and human islets. Translational relevance of our project is enhanced by validation of our hypothesis in islets derived from T2DM human donors. The proposed studies are innovative and carry translational impact since they will identify putative mechanisms that dictate islet β-cell dysfunction in human diabetes. The long-standing expertise of the PI and his collaborators in this field will provide a unique opportunity to address these important aspects of islet function in health and diabetes. The data accrued from these investigations are also expected to provide actionable insights that will impact the prevention and treatment of T2DM in humans including our Veterans.