The childhood obesity epidemic has been strongly associated with environmental factors. Family aggregation studies in humans and transgenerational studies in animal models show that obesity is highly heritable, and ancestral exposures can predispose offspring to the risk of various metabolic disorders. Our laboratory was the first to show the role of paternal exercise in programming “thrifty phenotype” in offspring. Our published and preliminary OXPHOS efficiency analyses using Drosophila and mouse models indicate that the “thrifty phenotype” is caused by an alteration in the offspring's mitochondrial bioenergetic efficiency. Preliminary proteomic and transcriptomic analyses revealed that developmental changes in bioenergetic efficiency were due to mitochondrial proteome remodeling associated with alterations in somatic and germ cell microRNAs (miRNAs), thereby providing a potential mechanism for the transgenerational transmission and programming of metabolic risks. The central hypothesis of the project is that miRNAs play a key role in the developmental programming of mitochondrial bioenergetic efficiency. Based on published and preliminary data, a secondary hypothesis is that changes in mitochondrial bioenergetic efficiency are inherited via spermatozoal cells as alterations in miRNAs. To test these hypotheses, we propose to take advantage of the physiological simplicity and genetic manipulability of fruit fly Drosophila. Therefore, the specific aims for this project are: Aim 1. Determine the functional consequences of altered miRNA expression in programming offspring metabolic state. Aim 2. Determine the spermatozoal epimutations responsible for the transgenerational programming of the bioenergetic phenotype. The methodological innovations of this project include the application of a multiplexed assay platform with a variable energetic clamp technique recently adopted to flies, a novel proteomics-based approach to merge mitochondrial flux data with in-house proteomics to reveal molecular markers of transgenerational phenotype. This study is expected to significantly advance our understanding of the epigenetic origins of childhood obesity. Deciphering primary molecular mechanisms of developmental metabolic programming will provide a better understanding of how childhood obesity and related metabolic diseases could be more effectively targeted.