Contributions of Hepatic and Intestinal Pathways to Cholesterol Excretion

7. Project Summary Reverse cholesterol transport (RCT) is process by which excess cholesterol is eliminated from the body. 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 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. 4) Multiple disease-causing mutations outside of the nucleotide binding domain of G5G8 form mature heterodimers. We hypothesize that hepatic and intestinal G5G8 are post- translationally regulated by enterohepatic bile acid signaling and are essential to promote cholesterol excretion in response to dietary cholesterol or pharmacological stimuli of RCT. Aim I 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. These strains will also be administered ligands for FXR and LXR, two well-established stimuli of Abcg5 Abcg8 transcription, reverse cholesterol transport (RCT), and cholesterol excretion. Aim II 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-B9 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 and select variants associated with clinical phenotypes. Aim III will establish structure-function relationships in G5G8 regulation and activity using variants and mutants of ABCG5 and ABCG8 known to impact gall bladder bile composition and other relevant clinical phenotypes. Adeno-associated viral vectors will deliver normal and clinical variants of G5 and G8 to the liver of mice lacking hepatic G5G8. Gallbladder bile composition, rates of biliary cholesterol secretion, and coupling of cholesterol to phospholipid and bile acid secretion determined.