Abstract: Stem cell exhaustion and related metabolic dysfunction are hallmarks of aging, compromising tissue regeneration/renewal leading to eventual loss of tissue integrity and function. Understanding metabolic programs necessary for proper stem cell function and aspects affected by age could open up therapeutic targets. Our initial metabolomics analysis in the muscle stem cell (MuSC) identified glutamine metabolism as a critical pathway during activation. Glutamine has multiple metabolic fates in mammalian cells. Aside from direct incorporation into protein, glutamine can be metabolized to generate nucleotides, other amino acids, and ATP via its anaplerotic metabolism in the TCA cycle in a “clockwise” direction. We have generated preliminary data using stable isotope-enabled metabolic flux analysis to demonstrate that both glutamine- driven oxidative and reductive or “counter-clockwise” pathways are active in muscle stem cells. Moreover, our data suggest that these pathways are impaired in the aged cell, in part due to decreased glutaminase expression. These preliminary findings lead to several new questions: 1- Is glutamine metabolism necessary for stem cell function?, 2- What are the relative roles of oxidative and reductive pathways of glutamine metabolism for energy generation and cell viability? And, 3-How does age alter glutamine metabolism, and does restoration of these pathways restore cell function? We hypothesize that satellite cell function is dependent on glutamine metabolism via both the oxidative pathway for energy production and the reductive pathway, via isocitrate dehydrogenases 1 and 2 (IDH1/2), for generation of NADPH to fuel the glutathione redox pathway, ensuring redox homeostasis. Moreover, we hypothesize that impairment of these pathways contribute to age-related muscle stem cell dysfunction, which is reversible by restoration of glutamine metabolism. We will test these hypotheses through the following aims. Aim1 will test the hypothesis that glutamine-driven “bidirectional” metabolism is necessary for muscle stem cell function and myogenesis. We will use small molecule inhibitors of glutaminase and, as a specific manipulation of the reductive pathway, IDH2, to test this hypothesis. Aim2 will test the hypothesis that glutamine metabolism is impaired in old MuSCs, and that correction of this deficit, via viral or genetic glutaminase gain of function approaches, restores MuSC function and regenerative capacity to aged muscle. Aim3 will test the hypothesis that glutamine metabolism regulates MuSC resilience through maintenance of cell redox status and attenuating oxidative stress. We will test if the glutamine-driven reductive pathway generates isocitrate/citrate to serve as “stored” form of NADPH for the cytosolic glutathione redox mechanism. The proposed studies will fully test the hypothesis that glutamine metabolism is critical for muscle stem cell function, and that both oxidative and reductive TCA metabolism contribut...