In lipid metabolism, bile acids play a critical role in the digestion of dietary fat and the absorption of dietary metabolites and cholesterol. To maintain their enterohepatic circulation, bile acids are conserved by active ileal uptake. Using isolated intestinal sections and enterocytes, the kinetic properties and chemical specificity of ileal bile acid transport has been examined. However, the proteins responsible for transport have not been isolated, and the process has not been studied at the molecular level. This has hindered understanding the mechanism of ileal bile acid transport, its regulation, and its role in bile acid and cholesterol metabolism. The proposed research will employ a recently identified bile acid transporter cDNA clone to examine the regulation and mechanism of the ileal bile acid transport. The following questions will be addressed: 1. What are the general properties of the ileal bile acid transporter? Bile acid transport across the ileum is believed to be mediated by a Na+- dependent transporter on the brush border membrane. To identify and characterize the transporter, my laboratory used an expression cloning strategy to isolate a candidate cDNA clone (IBAT) for the ileal bile acid transporter. To verify the identity of the candidate cDNA and extend previous studies of the transporter, IBAT's transport and expression properties will be determined. 2. Is the ileal bile acid transporter regulated by bile acid availability? Very recent in vivo studies have suggested that ileal bile acid transport activity is regulated by bile acid availability and may be a crucial determinant of bile acid and cholesterol homeostasis. To determine a) whether the transporter is regulated by changes in hamster intestinal bile acid availability (as previously hypothesized) and b) what level this reulation occurs, ileal Na+-dependent bile acid transporter activity, protein, mRNA, and gene transcription levels will be examined. 3. How does the ileal bile acid transporter function to carry bile acids across the enterocyte? To begin studying the cellular mechanism of bile acid transport, CaCo-2 cells will be used as a model system to express the ileal bile acid transporter cDNA. IBAT's transport properties will studied to test the hypothesis that the brush border membrane transporter is the rate limiting step in transcellular trafficking of bile acids across the ileal epithelium.
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