Ileal Bile Acid Transporter Metabolism and Regulation

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DESCRIPTION (provided by applicant): Many of the major carriers responsible for the enterohepatic circulation have been identified in recent years. Notably absent from that list is the basolateral membrane transporter responsible for the efflux of bile acids from the ileal enterocyte, renal proximal tubule cell, and cholangiocyte. This has hindered understanding the molecular mechanism and regulation of bile acid flux through the enterohepatic circulation. We have recently applied a gene profiling approach to the ileal bile acid transporter knockout mouse in order to identify a candidate basolateral bile acid transporter, Osta/b. The goal of the proposed work is to test the hypothesis that Osta/b is the major ileal basolateral bile acid transporter and to understand its regulation. To accomplish these goals, three specific aims are proposed. Aim 1: To test the hypothesis that the heteromeric Osta/b transporter is an ileal basolateral bile acid transporter. For this aim, the following questions will be investigated. 1) What tissues express Osta/b mRNA and protein? 2) What is the cellular localization of the Osta/b protein? 3) Does Osta/b promote bile acid efflux in transfected MDCK cells, a model polarized epithelial cell? 4) Does Osta/b expression correlate with the appearance of transcellular bile acid flux in intestinal development and in Caco-2 cells programmed to differentiate? 5) Is Osta/b expression necessary for basolateral membrane transport in Caco-2 cells, a model intestinal polarized epithelial cell? Aim 2: To use knockout mouse models to determine the relative contribution of Osta/b to intestinal bile acid transport. For this study, bile acid metabolism including fecal bile acid excretion, bile acid pool size, and intestinal bile acid absorption will be examined in Mrp3 and Osta null mice. Aim 3: To elucidate the mechanism responsible for bile acid regulation of the ileal Osta/b. The goal of this aim is to elucidate the transcriptional mechanisms responsible for the regulation of the Osta and Ostb genes by bile acids. For this aim, the regulation of mouse Osta/b by bile acid feeding or depletion will be investigated in vivo. The transcriptional regulation of the Osta gene will be investigated in vitro using transfected promoter constructs and transcription factor binding assays. The long-range goal of this work is to understand the mechanism and regulation of ileal bile acid transport as it relates to dietary lipid metabolism in normal and disease states.