ABSTRACT In obesity and type 2 diabetes mellitus (T2D), polyhormonal dysregulation can culminate in a relative insulin-deficient state. Indirect evidence has recently accumulated for an enteric hormone (called a decretin) that suppresses insulin and enhances glucagon output to promote T2D. A fundamental advance for the field would be the identification of native hormones that normally regulate insulin and glucagon production and secretion by pancreatic islet cells. We recently identified an enteroendocrine decretin signaling pathway in the fruit fly Drosophila melanogaster regulated by nutrient availability, and essential for lipid, glucose and insulin balance. We demonstrated from in vitro studies that the mammalian peptide hormone Neuromedin U (Nmu) and its cognate G protein-coupled receptor, Nmu Receptor 1 (NmuR1) have similar functions. Nmu is produced in enteroendocrine cells concentrated in the stomach, duodenum and ileum. In this proposal we outline several original and novel approaches to investigate how the ligand-receptor pair Nmu/NmuR1 regulate gastrointestinal hormone output. To address these fundamental questions in both in mice and humans, we propose Specific Aims to: 1. Assess the requirement for enteric Nmu in mouse metabolic homeostasis. 2. Decode Nmu signal transduction mechanisms by G proteins and NmuR1 in pancreatic islets. 3. Identify mechanisms regulating NMU signaling in human islets This proposal is also notable for technical innovations in each Aim, including construction of multiple novel mouse strains, and use of new ELISAs to investigate Nmu regulation in mice and humans. Collectively, these state-of-the-art approaches will generate major new insights into the cellular and molecular mechanisms controlling enteroendocrine regulation of metabolism in physiological settings. At a fundamental level, our work should establish regulatory paradigms that interconnect metabolic signaling and regulation of crucial gastrointestinal hormones. Thus, our work should have broad impact by suggesting strategies to diagnose, stratify risk, and treat subsets of humans with diabetes mellitus or important pre-diabetic conditions like obesity and impaired glucose tolerance.