Project Summary/Abstract Dysregulated endogenous glucose production is the central cause of type 2 diabetes. Recently published work from the lab of the sponsor determined that lactate carries the highest circulatory turnover flux and is an important TCA substrate. During these studies, a general observation made from intravenous infusion of 13C- glucose in freely moving mice was surprisingly low labeling of glycolytic intermediates in all tissues in both fed and fasted mice. What is the source of glycolytic intermediates if not circulating glucose? We have two hypotheses: 1) glycolytic intermediates come from glycogen and 2) gluconeogenesis produces tissue glycolytic intermediates. If either of these are true, these pathways are active more often and, in more tissues, than previously thought. Here, we will utilize state of the art LC-MS metabolomics in combination with intravenous infusion of 13C-metabolite tracers to measure these pathways in fed and fasted mice in a wide array of tissues. LC-MS offers advantages when measuring flux through these pathways including the ability to 1) measure labeling in glycolytic intermediates and 2) to determine quantitative labeling patterns. Utilizing this methodology, we aim to gain a comprehensive understand of glycogen metabolism and gluconeogenesis in all tissues. This work will not only deepen our understanding of these important metabolic pathways, but it has the potential to uncover novel roles for these pathways in maintaining tissue and organismal health.