SUMMARY: Preservation of mobility is one of the most significant health concerns of the elderly population. Exercise is the best-established intervention that can preserve muscle mass and strength at later ages. Understanding how exercise produces metabolic benefits in muscle is very important. In addition to exercise, diet is another key determinant of muscle health. Among various dietary elements, leucine has been considered particularly important because it is actively metabolized in muscle and can function against muscle degeneration. Previously, leucine was thought to maintain muscle mass by activating mTORC1, a protein kinase upregulating general protein and lipid anabolism. However, as demonstrated by our recent work and the work of others, mTORC1 upregulation rather produced degenerative phenotypes and wasting of muscle mass, while modest mTORC1 suppression was found to be beneficial for maintaining muscle homeostasis during aging. Therefore, leucine’s role in the preservation of muscle structure and function may be independent of mTORC1 activation and needs to be clarified. Over the previous funding period, our research group has been focusing on Sestrins, a family of highly conserved proteins that are induced upon various stress inputs. Using both biochemical and genetic methodologies, we have shown that Sestrins are multifunctional proteins that control many metabolism- controlling pathways including redox signaling, protein kinase signaling (AMPK, mTORC1, mTORC2 and AKT), and autophagy. Through genetic studies in mice and flies, we have shown that Sestrins have a critical physiological role in mediating exercise benefits. Furthermore, experiments of others have shown that Sestrins can bind and sense leucine. Although it is quite evident that Sestrins are at the crossroads between exercise, leucine, and muscle homeostasis, mechanisms of how these components are connected to each other are unclear. For instance, Sestrins, which inhibit mTORC1 (muscle anabolic kinase), function to preserve muscle function and mass during aging and disuse. Leucine was suggested to antagonize Sestrin’s mTORC1- suppressing activities, activating mTORC1. Yet, both leucine and Sestrins are beneficial for muscle health despite playing opposite roles in mTORC1 signaling. We propose to solve the intricate relationships between exercise, leucine and Sestrins through innovative methodologies combining purified protein complexes, ex vivo culture of myotubes, in vivo animal models, and Seq-Scope, the state-of-the-art ultra-high-resolution spatial transcriptomic technology developed by our own team. With respect to the expected outcomes, the work proposed here could clarify the critical functions of Sestrins in integrating the metabolic benefits of leucine and exercise in muscle homeostasis. Such results would have an important positive impact, because they will provide us with a clearer picture of how leucine and exercise can improve muscle homeostasis and provide b...