Project summary/Abstract The number of overweight and obese individuals in the U.S. has increased dramatically over the last two decades. With this rise, the prevalence of a number of obesity-associated metabolic diseases, such as type 2 diabetes (T2D) and non- alcoholic fatty liver disease (NAFLD), has similarly sky rocketed. NAFLD encompasses a range of hepatocellular alterations, including simple steatosis and steatosis with inflammation (NASH), which can lead to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatic insulin resistance, changes in mitochondrial metabolism, inflammatory signaling, extracellular vesicle signaling and/or altered intestinal microbial diversity are all proposed to contribute the pathogenesis of NAFLD and NASH. However, the mechanistic basis for the pathogenesis of NAFLD remains incompletely understood and there are currently no approved treatments for NAFLD. 5'-AMP-activated protein kinase (AMPK) is a cellular energy sensor that coordinates metabolic pathways to balance energy demand with production, and growing evidence suggests that loss of AMPK activity and its downstream signaling pathways contributes to NAFLD. This proposal will test the hypothesis that increased phosphorylation-dependent, ubiquitin/proteasomal-mediated degradation of AMPK contributes to the pathogenesis of obesity-associated NAFLD. They will further establish the identity of the F-box protein that mediates the specific recognition of phosphorylated AMPK by the Skp-Cullin1-Rbx1 E3 ligase complex. Finally, studies herein will address how modulating the expression or activity of the putative F-box protein during the pathogenesis of NAFLD or after development of established NASH impacts major outcomes associated with these diseases. These goals will be achieved through a combination of approaches including cell biology, amino acid and peptide chemistry, animal physiology, metabolic isotope tracing, and homology modeling- and molecular docking-based drug design and optimization. This approach will leverage the unique and complimentary expertise of the participating investigators of this multi-principal investigator proposal, leading to new knowledge regarding the pathogenesis and a potential treatment of NAFLD.