The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids and plays a key role in maintaining bile acid, lipid, and cholesterol balance. Emerging evidence suggests that ASBT is an attractive therapeutic approach to restore metabolic functions and lower plasma cholesterol. This is particularly important in light of recent findings indicating the need to lower cholesterol to very low levels in individuals with high risk for developing cardiovascular diseases (CVDs) such as the veterans. Achieving such stringent target remains challenging in many cases despite the use of cholesterol synthesis inhibitors and blockers of cholesterol absorption. In this regard, dietary fatty acids are known to influence metabolic functions as well as lipid and cholesterol homeostasis. A recent study showed that the effectiveness of ASBT inhibition in restoring normal metabolic functions depends on the types of fatty acids present in the diet. However, specific effects of different types of fatty acids on ASBT function are not fully understood. Our recent novel data using resin-assisted capture (Acyl-RAC) and click-chemistry based metabolic labeling approaches provided evidence that ASBT protein is subject to s-acylation, a reversible post-translational modification where fatty acids attach to cysteine residues of membrane proteins governing their membrane raft localization and function. We have shown that incorporation of the unsaturated fatty acid oleate or omega-3 fatty acid eicosapentaenoic acid (EPA) into ASBT was associated with a significant decrease in its function. We have previously demonstrated that ASBT is regulated by post-transcriptional mechanisms including its association with membrane lipid raft microdomains. Our preliminary findings showed that feeding a diet rich with fish oil decreased ASBT function and association with lipid rafts in mice. Thus, identifying mechanisms that target s-acylation could be beneficial in inhibiting ASBT function as well as restoring normal lipid and cholesterol homeostasis. ASBT activity and association with lipid rafts were increased in a transgenic mouse model (ISR2tg) with intestine- specific overactivation of the Sterol Response Elements Binding Protein (SREBP2). Our recent data demonstrated that ISR2tg mice develop hypercholesterolemia and severe hepatic inflammation and fibrosis when fed a high fat high cholesterol diet and blocking bile acid absorption by cholestyramine reduced the diet-induced liver injury. ASBT function and association with lipid rafts are increased in ISR2tg mice. Based on these data, our proposed studies will test the hypothesis that acylation is critical for ASBT function and association with lipid rafts and that intestine-specific mechanisms are involved in regulating ASBT acylation (Specific aim 1). Studies in Specific Aim 2 will elucidate mechanisms mediating the effects of omega-3 fatty acids on ASBT function and s-acylation in...