Project Summary Insulin controls hyperglycemia after feeding by stimulating glucose uptake and suppressing endogenous glucose production. The liver contributes 90% of endogenous glucose production during fasting, generated through glycogen breakdown and gluconeogenesis, thus insulin’s control of hepatic glucose metabolism is crucial for restoring whole body glucose homeostasis. Insulin acutely suppresses hepatic glucose production and promotes glucose uptake and storage within fifteen minutes of a glucose bolus. This is accomplished through direct and indirect effects on the liver. The direct mechanism by which insulin acutely controls hepatic glucose utilization is unclear, and previous studies suggest it becomes dysregulated with insulin resistance. Our lab and others demonstrated that insulin directly controls liver glucose balance through AKT, a serine/threonine kinase and an obligate insulin signaling intermediate in hepatocytes. The goal of this study is to determine 1) the metabolic pathways AKT controls to acutely regulate glucose utilization and 2) the mechanisms involved and how they becomes aberrant in metabolic disease. Preliminary stable isotope tracing experiments suggest that AKT rapidly increases glucose contribution to glycolytic intermediates and lipogenic precursors within 5 minutes, independent of changes to glycogen metabolism. I have also found that insulin stimulates phosphorylation of an allosteric regulator of glycolysis, PFK2/FBPase2, in hepatocytes at Ser469 and Ser486 in an AKT-dependent manner. Phosphorylation at these residues correlates with increased PFK2 kinase activity. Thus, I hypothesize that insulin acutely shifts glucose balance in the liver from gluconeogenesis to glycolysis postprandially through AKT-mediated PFK2 phosphorylation. I will test whether AKT suppression of gluconeogenesis allows for the increased contribution of glucose to glycolytic intermediates and lipid precursors, and the role of PFK2 phosphorylation downstream of insulin signaling in mediating AKT’s acute effects on glucose flux. Finally, I will interrogate how the AKT-PFK2 pathway contributes to in vivo hepatic glucose utilization in both healthy and insulin resistant states using diet interventions, hyperinsulinemic clamps and adeno-associated virus delivery of phosphomutant PFK2. Overall, this study will determine the acute mechanisms by which insulin directly controls hepatic glucose balance, which may reveal novel therapeutic targets for treating insulin resistance and maintaining glucose homeostasis.