The identification of forkhead transcription factors of the Foxo sub-family as effectors of insulin action on gene expression has filled a yawning gap in our knowledge of insulin signaling, and has identified a cellular biological mechanism linking Insulin Receptor activation to regulation of the cell's transcriptional response through sub-cellular redistribution of transcription factors. Key advances supported by this grant in the past cycle have been: i, demonstration of a key role of Foxo1 in hormonal regulation of hepatic glucose production and lipid synthesis; ii, identification of novel roles of Foxo1 in the hypothalamic arcuate nucleus, linking insulin and leptin signaling with neuropeptide synthesis and action; iii, identification of the obesity susceptibility gene Carboxypeptidase E (Cpe) as a Foxo1 target, providing insight into the pathophysiology of the recidivism of obesity following weight loss; iv, identification of acetylation-based mechanisms of regulation of Foxo1 function by nutrients, and their integration in the pathogenesis of insulin resistance; v, discovery of non-transcriptional functions of Foxo1 as a coregulator of gene expression; vi, demonstration of a functional interaction between Foxo and Notch, establishing a mechanistic link between a major developmental pathway and the nutrient- sensing pathway. We seek to continue our studies with the following aims: in Aim 1, we propose to investigate whether Foxo1 is a transcriptional sensor of nutrient and neurotransmitter levels, linking food intake with energy expenditure and hepatic glucose production in Npy/AgRP neurons. Acetylation- defective Foxo1 mutant mice developed during the past cycle will be instrumental in a critical test of this hypothesis. In Aim 2, we will use Foxo1 mutants to probe the neuroanatomy of hormone and nutrient signaling. While a budding consensus highlights the role of the CNS in diabetes and obesity, the complexity of brain endocrine signaling has thus far thwarted efforts to develop CNS-targeted medications for either condition. The central role of Foxo1 in hormone signaling, and its regulation by nutrients make it an attractive tool to probe the neuroanatomy of insulin and leptin signaling using gain- and loss-of-function mutations in mice. We propose to establish anatomic-functional relationships between sub-phenotypes engendered by Foxo1 manipulations and specific neuron subsets. In Aim 3, we will investigate metabolic functions of the Foxo/Notch axis. We have shown that Foxo1 regulates cell differentiation by interacting with the Notch pathway. The Notch pathway is generally thought of as a developmental pathway, and its metabolic functions have not been explored. We show in preliminary data that Notch1 and Foxo1 cooperate to regulate hepatic glucose metabolism. We propose to explore the mechanism of this observation, with the ultimate goal of applying Notch inhibitors to diabetes treatment. PUBLIC HEALTH RELEVANCE: Transcription factor Foxo1 is a key sensor of the organism's metabolic and hormonal status, and can induce changes in gene expression that account for the protean manifestations of insulin resistance, and its progression to overt diabetes. Based on a body of work developed during the past decade, we will endeavor to understand the integrated physiology of Foxo1 function in specific tissues, and its biochemical underpinnings. The ultimate goal of this work is to find therapeutic approaches to modify Foxo1 function. Indeed, while Foxo1 is an attractive biological target to reverse diabetes and metabolic diseases, it is largely intractable as a drug target. Therefore, it is hoped that by parsing its modes of action and interacting partners, new ways to modulate its function can be found.