Despite decades of investigation, the biochemical pathways involved in regulation of fuel-stimulated insulin secretion are incompletely resolved. This proposal seeks to gain a better understanding of how metabolic, paracrine, and hormonal signaling mechanisms are integrated to regulate insulin secretion from islet ?-cells, and how these pathways are altered when islets fail in diabetes. Glucose-stimulated insulin secretion (GSIS) involves both a “triggering” pathway driven by a KATP channel-dependent mechanism and an “amplifying” phase that is largely KATP channel-independent. Our group has identified several novel pathways contributing to amplification of GSIS: 1) Insulin secretion is activated by mitochondrial metabolism of anaplerotic substrates to generate citrate and isocitrate, their egress from the mitochondria, and their engagement with the cytosolic, NADP-dependent isocitrate dehydrogenase (IDH1). NADPH produced in the IDH1 reaction is used to drive sequential reduction of glutathione (GSH), glutaredoxin (GRX), and sentrin/SUMO-specific protease-1 (SENP- 1), which removes SUMO peptides from secretory granule-associated proteins to potentiate insulin granule exocytosis. More recently, we have discovered that glucose stimulation induces reductive, “counter-clockwise” tricarboxylic acid (TCA) cycle flux to replenish cytosolic isocitrate for the IDH1 reaction. Molecular or pharmacologic suppression of isocitrate dehydrogenase 2 (IDH2), which catalyzes NADPH-dependent reductive conversion of 2-ketoglutarate to isocitrate in the mitochondria, results in impairment of reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. In patch clamped ?-cells from human subjects with type 2 diabetes (T2D) NAPDH, isocitrate, and GSH rescue insulin granule exocytosis in glucose- unresponsive ?-cells; 2) Collaboration between the Newgard and Campbell laboratories has revealed that in addition to its potent effect on insulin secretion, pharmacologic suppression of IDH2 alters glucagon secretion. Mice with knockout of proglucagon-derived peptide production in ?-cells have impaired insulin secretion responses to glucose and amino acids, suggesting that alteration of ?-cell function in response to IDH2 inhibition could contribute to regulation of insulin secretion; 3) Very recently, we have found that inhibition of IDH2 not only impairs GSIS, but also suppresses insulin secretion in response to glucose + the GLP-1 analog exendin- 4. Based on these findings, we propose the following specific aims: Aim 1) To define the biochemical pathway by which stimulatory glucose activates the IDH2/IDH1 metabolic cycle; Aim 2) To define the role of the IDH2/IDH1 pathway in paracrine ?- to ?-cell and incretin regulation of insulin secretion; Aim 3) To investigate signaling by the IDH2/IDH1 pathway in normal human islets, and in islets from humans and rodents with T2D. Taken together, studies described in this project have strong potential to defi...