7. Project Summary Reverse cholesterol transport (RCT) is the process by which excess cholesterol is eliminated from the body. Disruptions in RCT contribute to a variety of metabolic diseases including atherosclerosis, insulin resistance and non-alcoholic fatty liver disease (NAFLD). The ABCG5 ABCG8 (G5G8) sterol transporter facilitates sterol secretion in the hepatobiliary and transintestinal pathways. Our new data indicate that G5G8 is indispensable in opposing dietary cholesterol accumulation, however, the relative contribution of hepatic vs. intestinal G5G8 in opposition cholesterol accumulation is unknown. Despite its discovery over 20 years ago, little known about the post-translational regulation of the transporter. We've recently developed key biological reagents and approaches that will allow for the investigation of endogenous G5G8 post-transcriptional regulation. Preliminary experiments revealed several surprises. 1) G5G8 is localized to an intracellular compartment in vivo and in polarized hepatocytes. 2) G5G8 translocates to the apical domain in response to stimulation with the intestinal hormone FGF15/19 in vivo. 3) G5G8 is degraded in lysosomes following heterodimer formation. We hypothesize that hepatic G5G8 is post-translationally regulated to promote biliary cholesterol secretion but dependent on intestinal G5G8 to prevent dietary cholesterol accumulation, mechanisms that are disrupted by clinical variants that contribute to MetS phenotypes. Aim I will determine molecular mechanisms of G5G8 post-translational regulation in vitro and in vivo. We will interrogate the biosynthetic itinerary and steady state distribution of the G5G8 heterodimer in WIF-B polarized hepatocytes. The sub-apically located structures in which it resides in unstimulated cells will be identified. Apical translocation in response to stimuli of biliary cholesterol secretion (bile acids, FGF15/19, etc.) will be determined and results confirmed in vivo. These studies will be replicated with human G5G8 expressed in liver-specific G5G8-deficient mice. Aim II will determine the impact of hepatic and intestinal G5G8 on cholesterol excretion. Control and mice and lacking G5G8 in liver, intestine or both organs will be challenged with cholesterol-containing diets. Plasma and fecal sterols and measures of RCT will be analyzed. Hepatic gene expression and metabolic phenotypes including insulin sensitivity and measures of NAFLD will be assessed. Aim III will investigate the impact of human variants of ABCG5 and ABCG8 on basal and bile acid-stimulated biliary cholesterol secretion using novel adenoassociated viral vectors and tissue-specific G5G8-deficieny mice. We will also investigate the effect of these variants on the development of obesity-related phenotypes in response to cholesterol-containing diets.