Ileal Bile Acid Transporter Metabolism and Regulation

回肠胆汁酸转运蛋白代谢和调节

• 批准号: 6865045
• 负责人: PAUL A DAWSON
• 金额: $ 23.53万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-10 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865045
• 关键词: MDCK cell; basolateral membrane; bile circulation; biological models; cholanate compound; gastrointestinal absorption /transport; genetically modified animals; high performance liquid chromatography; ileum; laboratory mouse; membrane transport proteins; messenger RNA; posttranslational modifications; protein localization; protein structure function; transfection; western blottings

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.

描述（由申请人提供）：近年来已确定了许多负责肝肠循环的主要载体。值得注意的是，该列表中没有负责胆汁酸从回肠肠上皮细胞、肾近端小管细胞和胆管细胞流出的基底外侧膜转运蛋白。这阻碍了理解胆汁酸通过肠肝循环的分子机制和调节。我们最近应用基因分析的方法，回肠胆汁酸转运蛋白基因敲除小鼠，以确定一个候选的基底外侧胆汁酸转运蛋白，Osta/B。这项工作的目的是检验Osta/B是回肠基底外侧胆汁酸转运蛋白的假设，并了解其调节。为了实现这些目标，提出了三个具体目标。目的1：验证异聚体Osta/B转运蛋白是回肠基底外侧胆汁酸转运蛋白的假设。为此，将研究以下问题。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是否促进转染的MDCK细胞（一种极化上皮细胞模型）的胆汁酸流出？4)Osta/B的表达是否与肠发育和Caco-2细胞分化过程中跨细胞胆汁酸流的出现相关？5)Osta/B的表达是否是肠极化上皮细胞模型Caco-2细胞基底膜转运所必需的？目的2：利用基因敲除小鼠模型研究Osta/B基因对小肠胆汁酸转运的相对贡献。对于本研究，将在Mrp 3和Osta缺失小鼠中检查胆汁酸代谢，包括粪便胆汁酸排泄、胆汁酸池大小和肠道胆汁酸吸收。目的3：阐明胆汁酸对回肠Osta/B的调节机制。本研究旨在阐明胆汁酸调控Osta和Ostb基因的转录机制。为此，将在体内研究通过胆汁酸喂养或消耗对小鼠Osta/B的调节。Osta基因的转录调控将在体外使用转染的启动子构建体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节，因为它与正常和疾病状态下的饮食脂质代谢有关。