PROJECT SUMMARY/ABSTRACT Impaired skeletal muscle regeneration and associated pathological tissue remodeling (loss of muscle, gain of fibrotic and adipose tissues) following injury underlie functional and metabolic decline—hallmarks of aging. Regeneration is dependent on a well-orchestrated myogenic program that includes the activation and expansion of skeletal muscle stem cells/progenitor cells (MPCs) and terminal differentiation of MPCs into mature multinucleated muscle cells. We previously demonstrated that MPCs rely on extracellular availability of the nutritionally, non-essential amino acids L-serine (Ser) and glycine (Gly). Decreased availability of Ser/Gly impairs MPC expansion, induces intramuscular adipocytes following injury, and induces toxic deoxysphingolipid accumulation in the muscle. Further, we demonstrated that endogenous Ser/Gly levels decline with age. The metabolic product (i.e. requirement) of Ser/Gly for MPC expansion and the efficacy of dietary Ser/Gly for muscle regeneration and the cell (MPC)-extrinsic environment need to be resolved. We propose to use isotope tracing of Ser and Gly to define the metabolic requirement of Ser/Gly for MPC population expansion. Further, using models that we have demonstrated reduce (depleted diet) or enhance (supplemented diet) endogenous Ser/Gly levels, we will quantify the effects of Ser/Gly availability on age- and injury-related muscle regeneration and the cell (MPC)-extrinsic muscle environment. Based on preliminary data, we hypothesize that MPCs require glutathione synthesis, from extracellular Ser and Gly, to mitigate oxidative stress. Additionally, we hypothesize that diet-induced reduction of endogenous Ser/Gly exacerbates age-related (i) impairments in muscle regeneration and (ii) toxic non-canonical sphingolipids in the cell-extrinsic muscle environment. Further, we expect Ser/Gly supplementation will counter these effects. To capture the efficacy of dietary Ser/Gly to modulate age-related impairments in muscle regeneration and remodeling we will use novel sphingolipidome profiling and transcriptomics. Successful completion of this project will transform the fundamental understanding of the metabolic essentiality of Ser and Gly for skeletal muscle regeneration and the relationship of this loss to age- related muscle deterioration. The results will enable testable scientifically grounded therapies to improve the regenerative capacity in populations that have impaired muscle regeneration, such as older adults.