Project Summary. Adaptive metabolic responses to hypoxia reflect essential evolutionary survival strategies in all eukaryotes. We recently identified a unique metabolite that increases in cardiovascular (CV) cells in response to hypoxia, L-2-hydroxyglutarate (L2HG). This metabolite is derived from ?- ketoglutarate, or 2-oxoglutarate (2OG), a key intermediate in the tricarboxylic acid cycle. Once formed from 2OG and NADH, L2HG has no other metabolic fate except to undergo oxidation back to 2OG by the stereospecific dehydrogenase, L2HG dehydrogenase (L2HGDH), suggesting that it accommodates (?buffers?) the increase in reducing equivalents accompanying hypoxia. L2HG has two other unique actions: it suppresses glycolysis and, as we show here, it increases pentose phosphate pathway (PPP) activity. The central hypothesis of this proposal is that L2HG suppresses glycolysis and enhances PPP activity in CV cells to eliminate reactive oxygen species (ROS), maintain cell redox potential, and preserve cell function in hypoxia. To address this hypothesis, we will focus on three specific aims. First, we will determine the molecular metabolic mechanisms underlying the effects of L2HG on glycolysis and PPP activity. In particular, we will focus on the unique role of a specific phosphofructokinase-2 isoform, PFKFB4, as a key regulatory determinant of increased flux through the PPP in hypoxia. Second, we will determine the effect of this L2HG-induced increased PPP activity in hypoxia on cellular redox potential and intra- and extracellular ROS elimination. Here, we will focus on PPP-derived NADPH and GSH as key cofactors in NADPH oxidase and glutathione peroxidase activities, respectively, in order to enhance elimination of excess ROS. Third, we will study the effects of L2HG in hypoxia or ischemia on cellular and cardiac function, respectively, using unique cellular and genetic murine models. Taken together, these studies should provide insights into the mechanisms by which L2HG promotes metabolic remodeling to preserve cell and cardiac function in oxygen-limited states.