Project Summary/Abstract The link between dietary intake/metabolism and long-term health and disease was first established nearly a century ago. Dietary restriction (DR), defined as a decrease in caloric intake without malnutrition, remains the most potent and reproducible intervention to improve health and longevity across multiple species. Unfortunately, long-term DR is both relatively untested and very difficult to implement in humans, leading scientists to better define the mechanisms through which DR improves health in an effort to mimic the benefits in the absence of true DR. This project focuses on a family of xenobiotic metabolizing enzymes, flavin- containing monooxygenases, or FMOs, that are induced downstream of DR and were recently reported to be both necessary and sufficient to increase health, stress resistance, and longevity in the nematode C. elegans. Interestingly, previous reports also show induction of FMO homologs in mammalian systems under DR and other conditions known to increase longevity. Unfortunately, the mechanism(s) for the effects of these well- conserved FMO proteins on health and longevity are largely unknown, as their primary role in phase I xenobiotic detoxification is not clearly linked to the observed effects on health and longevity. This project will build upon recently published and preliminary data that support a role for FMO enzymes in regulating endogenous metabolism. Utilizing recently developed tools, including a novel food source for nematodes to better measure their metabolism and a metabolomics based technique to use oxygen isotopes and identify substrates of oxygenases, this project will identify the mechanisms and implications for FMO activity within the simple nematode, Caenorhabditis elegans. The preliminary data clearly establish one-carbon metabolism (OCM) as the key intermediate metabolic network affected by FMO-2 to improve health and increase longevity. The data produced by this project will provide evidence as to 1) what the key endogenous target(s) of FMO-2 are and how they connect to OCM, 2) how OCM flux is modified by FMO-2 expression and activity and how this may be replicated through exogenous metabolites, and 3) what mechanisms are downstream of OCM and how they play into understanding the intertwined nature of stress response and longevity. To ensure the success of this project, all assays will be performed by experts in nematode biology and aging in collaboration with experts in metabolomics profiling and data analysis. The resulting data will provide a model for the metabolic impact of FMO enzymes that can then be further interrogated in mammalian systems. In addition, since the pathways focused on are important for multiple age-associated diseases, they may lead to approaches that improve health with or without exploiting the mechanism(s) of FMO activity.