PROJECT SUMAMRY The pursuit of an effective geroprotective strategy has been a critical goal for aging research since it has became clear that the rapidly graying population will otherwise generate an unbearable socioeconomic strain. Caloric restriction (CR) is an effective dietary intervention for healthspan and lifespan extension. Recently, other dietary interventions have been developed as means to improve health and extend lifespan that may be more sustainable than CR. Amongst these is protein restriction (PR), a dietary regimen that also boasts significant metabolic, health, and longevity benefits in diverse species. The Lamming Lab has investigated the role of individual essential amino acids in the effects of PR. Restriction of the branched chain amino acids (BCAAs; leucine, isoleucine, valine) was found to recapitulate the many metabolic benefits of PR, and extends lifespan in male mice when the dietary intervention was lifelong. Further study has found the restriction of isoleucine (Ile) to be both necessary and sufficient for the metabolic benefits of PR. In preliminary experiments, isoleucine restriction (IleR) in young adult mice improves glycemic control, reduces adiposity, and, in males, delays age- related increases in frailty and extends lifespan. In this proposal, I will first determine if IleR can effectively promote healthy aging when started in late-life. I will examine how IleR affects metabolic health, physical performance, frailty, and lifespan. We have found that IleR induces ketogenesis: the production of the ketone body β-hydroxybutyrate (BHB), which has been shown to have geroprotective properties. In fact, a ketogenic diet has been shown to extend mouse lifespan. In the second aim of this proposal, I will investigate the role of ketogenesis in the metabolic benefits of IleR using mice with the conditional deletion of Hmgcs2, the rate-limiting enzyme for ketogenesis, in the liver. In the final aim of this proposal, I will leverage pharmacological and viral approaches to manipulate the catabolic flux of BCAAs to test the hypothesis that increasing BCAA catabolism can simulate the effects of IleR by reducing the standing pool of BCAAs. These experiments will enable us to determine whether the reduction of Ile itself, or of its catabolites, are responsible for the beneficial effects of IleR, and demonstrate if the manipulation of BCAA catabolic flux can be a viable geroprotective strategy. The overarching goal of this proposal is to further define the effects and identity of the molecular mechanisms underlying the benefits of IleR. The extensive investigation described here represent the perfect project for my growth as a trainee and will push forth the development for a feasible, highly effective, and metabolically focused geroprotective strategy.