Trehalose is a widely consumed, naturally-occurring biopharmaceutical and dietary supplement, which is widely purported to have health-promoting effects in retinal, neurodegenerative, vascular and metabolic disease. The mechanism of action of this promising phytochemical and disaccharide, however, remains incompletely understood. Understanding how trehalose exerts its therapeutic and preventive effects is important because it allows patients and clinicians to best predict the optimal contexts in which it should be deployed. Moreover, because we demonstrated that trehalose appears to act through broad, fasting-like, networked mechanisms to mitigate metabolic disease, dissecting fundamental actions of trehalose promises to inform next-generation compounds that leverage these same mechanisms, and to illuminate basic aspects underlying the therapeutic effects of adaptive fasting itself. Indeed the adaptive hepatic fasting response is gaining traction as a therapeutic target against non-alcoholic fatty liver disease (NAFLD), which afflicts approximately one billion individuals worldwide. Our long-term goal is to understand adaptive liver metabolism during fasting to produce new therapies against NAFLD. Our immediate research objective is to define the biological effects and mechanism of action of the natural compound and fasting mimetic, trehalose. Three key pieces of new data substantiate our approach: First, blocking hepatic glucose transport induces the hepatocyte glucose fasting response, and this is sufficient to convey the therapeutic effects of generalized fasting on NAFLD. Second, trehalose, is a hepatic glucose transport inhibitor that reduces diet-induced NAFLD in mice. Third, trehalose activates canonical fasting-like signaling pathways in association with its action to prevent and reverse diet-induced NAFLD. We will therefore directly test the central hypothesis that trehalose coordinately induces canonical fasting pathways to modulate hepatic metabolism. Our Specific Aims are to: 1) Define mechanistic contributions of hepatic fasting signaling and autophagy (both of which are central effector pathways of the fasting response) to the biological actions of trehalose, and 2) Delineate interactions between host and microbial trehalose catabolism, and the biological actions of trehalose. Completing these studies will: 1) illuminate basic principles of hepatic glucose fasting, 2) catalyze development of next-generation biopharmaceuticals which leverage these same fasting-like mechanisms, and 3) inform optimal contexts in which to deploy trehalose as a natural, health-promoting dietary supplement and preventative against metabolic disease.