Project Summary Blood glucose homeostasis is a central metabolic challenge in vertebrates and is regulated by the coordinated action of glucagon-producing α-cells and insulin-producing β -cells within the endocrine pancreas. Disturbances in this regulatory network cause metabolic disorders such as type 2 diabetes mellitus (T2D) – whose prevalence, comorbidities, and medical costs constitute a significant public health concern. Although the defining physiological abnormalities underlying T2D are an inappropriately low insulin secretory response to elevated glucose levels and insulin resistance, patients with T2D also exhibit hyperglucagonemia which exacerbates hyperglycemia through stimulation of hepatic glucose production. While mechanisms of glucose- stimulated insulin secretion have been established, the suppression of glucagon secretion by glucose has remained poorly understood. Support for a critical role of glycolytic flux in regulating glucagon secretion comes from a recent study in which α-cell-specific ablation of glucokinase impaired glucose-induced glucagon suppression. Additionally, the Kaestner lab has found using single cell RNAseq of islets from T2D organ donors that the islet-specific glucose-6-phosphatase 2 (G6PC2), which opposes the action of glucokinase, is dramatically upregulated in -cells in T2D patients. This upregulation is functionally important, because overexpression of G6PC2 in murine pseudo-islets has been demonstrated by the Kaestner lab to increase glucagon secretion. This research proposal will test the hypothesis that α-cell glycolytic flux mediates glucagon secretion, and its dysregulation underlies hyperglucagonemia observed in Type 2 Diabetes patients. The function of G6PC2 in the regulation of glucagon secretion will be investigated in vivo using an inducible, -cell specific genetic mouse model under normal and diabetogenic conditions. Mice with -cell specific G6PC2 ablation will be evaluated using glucose tolerance tests to assess glucagon secretion under varying blood glucose levels in vivo. Pancreatic islets will then be isolated and subjected to both static and dynamic hormone secretion assays to verify the role of G6PC2 on glucagon secretion ex vivo. Additionally, - cell-enriched ‘pseudo islets’ will be prepared and analyzed for glycolysis using the Seahorse XFe96 Analyzer under glucagon stimulating and suppressing conditions to assess the impact of genetic manipulation of G6PC2 levels on -cell glycolytic flux and ATP production rate. To determine how these findings translate to humans, these studies will also be performed in human cells via complementary loss- and gain-of-function studies using the previously described ex vivo hormone secretion and glycolytic rate assays. Ultimately, the results from study will contribute to our fundamental understanding of -cell biology and T2D pathophysiology, and identify a potential therapeutic target for improving T2D hyperglucagonemia and restoring blood ...