Project Abstract With the increasing prevalence of obesity, the health burden of obesity-driven diseases such as type 2 diabetes (T2D) has risen markedly. T2D is now one of the most prevalent diseases worldwide and exacts a tremendous toll population-wide in terms of decreased lifespan and health-span. In T2D, hyperglycemia follows from impairment in pancreatic beta-cell insulin production and insulin resistance in the liver and other metabolically active tissues. Pioneering research from the Pajvani laboratory has implicated obesity-driven reactivation of the Notch signaling pathway, previously thought to be only active during development and organogenesis, as a crucial driver of hepatic insulin resistance. Forced activation of hepatocyte Notch pathway signaling in mouse models is sufficient to cause glucose intolerance in chow-fed mice. In contrast, overexpression of upstream components of Notch pathway signaling in hepatocytes does not cause hyperglycemia unless an obesogenic diet is also provided, implicating other, as yet unknown, nodes in the Notch pathway in the propagation of maladaptive signals in obesity. In this grant application, I propose to identify the Notch pathway receptors involved in obesity-driven metabolic disease. The work I propose builds on preliminary experimental data from mice indicating that Notch2 is responsible for Notch activity in T2D, as well an unexpected protective role for Notch1, which appears to be mediated by a paradoxical increase in Notch2 protein followed by Notch1 depletion. I will investigate the impact of hepatocyte-specific deletion of Notch2 on diet-driven development of hyperglycemia (Aim 1A) and confirm in vitro whether this occurs in a hepatocyte-autonomous fashion (Aim 1B). I will study the unexpected homeostatic role of Notch1 on Notch pathway activity in models of T2D through hepatocyte-specific Notch1 deletion and Notch1-Notch2 double deletion to determine epistasis between Notch1 and Notch2 (Aim 2A). Finally, I will use immunoprecipitation of Notch2 followed by targeted western blotting as well as mass spectrometry to dissect the mechanism by which Notch1 depletion leads to increased Notch2 protein (Aim 2B). Together, our proposed work will clarify key steps in the transmission and regulation of Notch pathway signals that drive metabolic dysfunction in obesity.