Project Summary/Abstract Diet has a profound impact on organismal health. Fasting improves human health in part by reducing inflammation, decreasing oxidative damage and extending longevity, however, the mechanisms by which fasting improves intestinal regeneration remains poorly understood. The intestinal epithelium renews fastidiously every 5-7 days via Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5+) expressing intestinal stem cells (ISCs) found at the base of the intestinal crypt. LGR5+ ISCs balance differentiation and epithelial cell divisions to influence tissue regeneration by integrating metabolic and signaling cues from their environment like diet. Fasting has a profound effect on ISC function in young and aged mice and can improve the age-associated decline in tissue regeneration through the induction of fatty acid oxidation (FAO), a process that oxidizes fatty acids into acetyl-CoA units. In addition, LGR5+ ISCs strongly express 3-hydroxy-3-methylglutaryl-CoA synthetase 2 (HMGCS2), the rate-limiting enzyme in the ketogenic pathway whereby acetyl-CoA units are converted to ketone bodies such as beta-hydroxybutyrate (bOHB) and acetoacetate (AcAc). Mechanistically, bOHB reinforces the NOTCH signaling pathway by inhibiting class I histone-deacetylases (HDACs) to instruct ISC cell fate decisions. These findings further support a nuanced relationship between host nutritional state and stem cell function whereby dynamic control of ISC bOHB levels enable their rapid adaptation to diverse physiological states such as fasting. Other roles for ISC-derived ketone body metabolites have yet to be elucidated and, as such, we propose that bOHB and AcAc function as distinct signaling metabolites regulating ISC fasting responses (Aim 1) and have unique roles as energetic substrates (Aim 2). To test this hypothesis, we will use key genetic mouse models to understand how perturbed bOHB/AcAc ratios alter intestinal stem cell function in vivo and in vitro (Aim 1), as well as labelled substrate administration and novel techniques for rapid mitochondrial isolation to determine key ISC metabolic adaptations to fasting (Aim 2). Taken together, the experiments proposed will mechanistically delineate the signaling and energetic roles of ketone body metabolites on intestinal stemness and improve our understanding of how the fasting response via ketone bodies influences intestinal regeneration. We expect this approach will identify therapeutic options exploiting ketone bodies and the signaling and energetic pathways engaged by them to enhance intestinal regeneration in cases of injury and age-related decline of stem cell function.