PROJECT SUMMARY/ABSTRACT O-GlcNAc transferase (OGT) is a nutrient sensor protein which is highly expressed in pancreatic β-cells. This enzyme exclusively catalyzes the post-translational glycosylation of target cytosolic and nucleic proteins (O- GlcNAcylation). β-cell OGT knockout mice develop severe diabetic phenotype, suggesting that OGT is crucial in shaping glucose homeostasis. The long-term goal of this research is to understand the mechanisms of how protein O-GlcNAcylation shape β-cell health and function. Our data suggest a strong relationship between OGT and mechanistic target of Rapamycin complex 1, mTORC1, a key nutrient-sensor kinase in β-cells. While aberrant autophagy and chronic mTORC1 activation is observed in islets from human diabetic patients, the mechanisms of how these signaling pathways become dysregulated is unknown. We propose that in response to nutrient stress, protein O-GlcNAcylation directly regulates mTORC1 activity and subsequent downstream mitochondrial function through the process of autophagy. Our central hypothesis is that in nutrient stress conditions, decreased O-GlcNAcylation leads to insulin secretion deficits through hypo-activation of mTORC1 and hyper-activation of autophagy. The main objective of this proposal is to delineate the signaling between OGT and mTORC1-autophagy and their contribution to β-cell health and function. Specific Aim 1 will investigate how O-GlcNAcylation regulates mTORC1 activity and β-cell mass and function. Specific Aim 2 will test whether O- GlcNAcylation modulates autophagy in β-cells and to determine the consequences of dysregulated autophagy in organelle homeostasis. This F31 proposal is innovative because there are currently no studies linking OGT and mTORC1 crosstalk in pancreatic islets and their regulation of autophagy-dependent β-cell mass and insulin secretion. Successful completion of the proposal will reveal the mechanisms linking nutrient stress conditions such as hyper-glycemia and -lipidemia to aberrant changes in organelle dysfunction and identify specific β-cell nodes of regulation between OGT and mTORC1 signaling that can be targeted for therapeutics to prevent the development of type 2 diabetes. The proposal incorporates new and timely-needed training in conceptual knowledge in islet biology, autophagy, and mitochondria, and technical skills including perifusion based insulin secretion assay, specialize imaging techniques (TEM, confocal, live cell), and Seahorse XF mitochondrial respiration assay. Scientific communication in oral and written presentation, as well as mentoring leadership will be an integral part of training to become a successful academician. All the technical resources as well as dedicated mentors at University of Minnesota and the department of Integrative Biology and Physiology are available to assist the trainee to successfully complete his F31 proposal and to assist him achieved his career and academic goals.