Despite decades of investigation, the biochemical pathways involved in regulation of insulin secretion by glucose are incompletely understood. The overall goal of this project (DK046492-22), now entering its 23rd year of funding, is to gain a better understanding of metabolic coupling mechanisms in the ?-cell, and how they are altered when islets fail in type 2 diabetes (T2D). Since the last competitive renewal of the program in 2011, we have identified two metabolic pathways that stimulate insulin granule exocytosis, the first involving anaplerotic metabolism of pyruvate, export of mitochondrial substrates and their engagement with the cytosolic, NADP- dependent isoform of isocitrate dehydrogenase (ICDc), and activation of the glutathione/glutaredoxin (GRX) system. This pyruvate/isocitrate pathway connects to glutathione metabolism in two important ways. First, the NADPH produced in the ICDc reaction is used to maintain glutathione (GSH) and GRX in their reduced states, thereby activating GRX-mediated granule exocytosis via SENP-1. Second, the ?-ketoglutarate produced by the ICDc reaction contributes to maintenance of the GSH pool via transamination to glutamate. Using patch- clamped ?-cells from human T2D subjects, we find that NAPDH, isocitrate, and GSH all rescue insulin granule exocytosis in otherwise glucose-unresponsive beta-cells. We also identified the purine/nucleotide pathway intermediate adenosuccinate (S-AMP) as a glucose-regulated metabolite that stimulates exocytosis in normal human ?-cells and rescues secretory function in human T2D ?-cells. Thus, our work has identified two novel pathways of GSIS, and demonstrated that intermediates from both pathways rescue secretion in dysfunctional human ?-cells. Based on these findings, we propose the following new specific aims: 1. To study the pyruvate/isocitrate/GSH and S-AMP pathways via metabolic flux analysis; 2. To investigate manipulation of the NAD/NADP salvage (NAMPT) pathway as a strategy for enhancing flux through the pyruvate/isocitrate/GSH and S-AMP pathways of insulin secretion, and for reversing ?-cell dysfunction in T2D; 3. To test potential additive effects of co-activation of the S-AMP, pyruvate/isocitrate/GSH and NAMPT pathways, and to define targets within these pathways for rescue of ?-cell dysfunction in T2D.