This application is in response to Notice Number NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications, and represents an expansion of our currently funded grant: 5R01 DK075607-03;Regulation and Function of Drosophila Nuclear Receptors. The specific aims of the parent grant addressed two major nuclear receptor-regulated pathways: adult maturation and metabolism. We have completed our studies of maturation and are now focusing our attention on roles for Drosophila nuclear receptors (NRs) in sensing metabolites and maintaining metabolic homeostasis, with the goal of identifying and characterizing the critical aspects of NR signaling that are conserved through evolution up to humans. In this Competitive Revision application, we propose to accelerate our studies by characterizing the metabolic functions of two Drosophila NRs: DHR3, the single fly ortholog of the mammalian ROR subclass, and DHR38, the fly ortholog of mammalian NR4A receptors. Our preliminary data indicate that each of these receptors has a critical role in metabolism that is similar to that of its vertebrate counterpart. DHR3 binds cholesterol and its activation depends on sterol levels in vivo. In addition, several key sterol metabolic genes are misregulated in DHR3 mutants, and DHR3 mutants have reduced cholesterol levels. These observations lead us to propose that DHR3 functions as a cholesterol sensor to maintain cholesterol homeostasis. For DHR38, null mutants have normal levels of circulating sugar and triacylglycerol, but display reduced levels of glycogen and has reduced expression of phosphoglucomutase, a key enzyme that acts in glycogenolysis and glycogenesis. These observations support the hypothesis that DHR38 plays a critical role in maintaining carbohydrate homeostasis. To test these hypotheses, we will conduct detailed phenotypic and metabolic characterization of DHR3 and DHR38 mutants. We will study the roles of signaling pathways and ligands in controlling NR activity. Finally, we will identify and characterize target genes that are directly regulated by each NR and that contribute to key metabolic activities of the receptor. These experiments follow along the lines of study proposed in the parent grant - using Drosophila as a model system to define the molecular mechanisms of NR regulation and function, with direct implications for how the orthologous NRs act in humans, as well as their contributions to critical human diseases associated with NR dysfunction, including cardiovascular disease, diabetes, and obesity. PUBLIC HEALTH RELEVANCE: Our studies use Drosophila as a simple model system to define the molecular mechanisms of nuclear receptor action that are conserved through evolution up to humans. This work will have an impact on our understanding of normal nuclear receptor signaling pathways and provide new directions for combating critical human diseases associated with nuclear receptor dysfunction, including cardiovascular disease, diabetes, and obesity.