Ileal Bile Acid Transporter Metabolism and Regulation

描述（由申请人提供）：近年来已经确定了许多负责肠肝循环的主要载体。值得注意的是，在这一列表中缺少负责胆汁酸从回肠肠细胞、肾近端小管细胞和胆管细胞外排的基底外侧膜转运蛋白。这阻碍了对胆汁酸通过肠肝循环的分子机制和调控的理解。我们最近对回肠胆汁酸转运蛋白敲除小鼠应用了基因分析方法，以确定候选的基底外侧胆汁酸转运蛋白Osta/b。本研究的目的是验证Osta/b是主要的回肠基底外侧胆汁酸转运体的假设，并了解其调控。为实现这些目标，提出了三个具体目标。目的1：验证异质Osta/b转运体是回肠基底外侧胆汁酸转运体的假设。为此目的，将调查以下问题。1)哪些组织表达Osta/b mRNA和蛋白？2) Osta/b蛋白的细胞定位是什么？3) Osta/b在转染MDCK细胞（一种模型极化上皮细胞）中是否促进胆汁酸外排？4) Osta/b表达是否与肠道发育和Caco-2细胞程序化分化过程中胆汁酸通量的出现相关？5) Osta/b在Caco-2细胞（一种肠极化上皮细胞）的基底外侧膜运输中是否必要？目的2：利用敲除小鼠模型确定Osta/b对肠道胆汁酸运输的相对贡献。本研究将在Mrp3和Osta null小鼠中检测胆汁酸代谢，包括粪胆汁酸排泄、胆汁酸池大小和肠道胆汁酸吸收。目的3：阐明胆汁酸调节回肠Osta/b的机制。目的是阐明胆汁酸调控Osta和Ostb基因的转录机制。为此，我们将在体内研究胆汁酸饲喂或消耗对小鼠Osta/b的调节。Osta基因的转录调控将在体外使用转染启动子构建和转录因子结合试验进行研究。这项工作的长期目标是了解回肠胆汁酸转运的机制和调控，因为它与正常和疾病状态下的膳食脂质代谢有关。

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.