Aberrant nuclear signaling in the islet beta cell under metabolic stress

John D Dingell Va Medical Center, Detroit MI

Investigators

Abstract

PROJECT SUMMARY Chronic exposure of the pancreatic islet β-cells to gluco- and glucolipotoxic conditions (metabolic stress) induces dysfunction and loss of functional β-cell mass. However, putative molecular and cellular mechanisms involved in the genesis of islet β-cell defects under the duress of metabolic stress remain partially understood. Original studies from our laboratory in rodent and human islets have demonstrated novel roles for Rac1- induced intracellular oxidative stress in β-cell demise under the duress of diabetogenic stimuli. Interestingly, however, potential impact of metabolic stress on nucleocytoplasmic shuttling of key cargo necessary for optimal β-cell function has never been examined. Based on compelling preliminary evidence, we propose to evaluate the hypothesis that metabolic stress induces abnormalities in the karyopherin-α2 (KPNA2)-Ran G protein modules within the nucleocytoplasmic shuttling apparatus culminating in aberrant import/export of signaling proteins resulting in β-cell failure. We aim to assess our hypothesis via studies described under four complementary Specific Aims in INS-1 832/13 β-cells, rodent islets, and human islets. Aim 1 will aim to understand the impact of metabolic stress on KPNA2-Ran signaling modules leading to dysregulation of the islet β-cell. Studies under Aim 2 will establish essential roles for the NADPH oxidase signaling modules (Rac1- p22phox) in the onset of nuclear oxidative stress leading to β-cell dysfunction under metabolic stress. Experiments under Aim 3 will delineate the proteomic signatures of Rac1-KPNA2 and their corresponding signaling pathways in the nuclear and non-nuclear compartments derived from β cells exposed to metabolic stress. Lastly, studies under Aim 4 will evaluate the therapeutic potential of targeting Rac1 (using novel small molecule inhibitors) in alleviating intracellular oxidative stress and β-cell dysfunction in the diet-induced obesity animal model. A variety of pharmacological, molecular biological (in/active mutants, siRNA/CRISPR/cas9 gene editing), microscopic (BiFc) and proteomics (co-IP and UPLC-ESI-MS/MS) and bioinformatic approaches will be employed to validate our hypothesis in rodent and human islets. To further enhance the translational significance of our project, we will validate our working model in islets derived from T2DM donors. Our studies are innovative and carry translational impact as they are expected to advance our current understanding of the cellular mechanisms that regulate islet β-cell dysfunction in human diabetes. Most notably, nuclear import/export of G proteins (Rac1, Ran) in islet β-cell in health and diabetes is an understudied area, and data accrued in the proposed multidisciplinary investigations are expected fill these important knowledge gaps in this field of islet biology. 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 aging Veterans.