Insulin signaling regulates aging in many organisms, including the fruit fly Drosophila melanogaster. The adult fly produces four insulin like peptides (DILP) in specialized neurons of the brain. Knocking out these neurons extends lifespan, as do systemic mutations of the insulin signal transduction system. Activated insulin receptor represses the transcription factor FOXO. FOXO is thus thought to be a key modulator of the insulin control of lifespan and as expected its overexpression extends fly lifespan. This renewal aims to understand how particular insulin peptides work through a single insulin receptor to direct FOXO to genes that regulate aging. It will identify which of the adult insulin is most responsible for control of aging by knocking out each insulin encoding gene individually and in combination. Through the method of chromatin immunoprecipitation and full genome microarray analysis it will identify the genes that are directly regulated by FOXO when low insulin signaling slows aging. It will test the relevance of pathways involving these genes in the control of aging by conducting epistasis tests between their loss-of-function genotypes and insulin signaling mutants. Finally, the project will use a very unique resource of synthetic, recombinant Drosophila insulin peptides DILP1, DILP2 and DILP5 to study how these proteins individually and together affect signal transduction and FOXO targeting in a fly cell culture system. Together these studies will discover how specific insulin like peptides modulate aging through their control of FOXO. PUBLIC HEALTH RELEVANCE: The work proposed in this renewal aims to understand how the several different insulin like proteins of Drosophila work to regulate aging and alternatively, metabolism. It will use new, advanced genetic methods to individually knockout each of the fly's insulin genes and study how this affects life span. It will also study how each of these insulin proteins controls the binding of the essential transcription factor FOXO to downstream genes and test whether these specific genes are required for reduced insulin signaling to slow aging.