ABCs of Intestinal Sterol Transport

肠道甾醇运输的基本知识

基本信息

批准号：
7638602
负责人：
F JEFFREY FIELD
金额：
$ 28.1万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-15 至 2010-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): ATP-binding cassette (ABC) transporters and Niemann-pick C1 like protein (NPC1L1) play important roles in cellular sterol trafficking. ABCA1 eliminates cholesterol from cells by facilitating cholesterol efflux and the formation of nascent HDL. ABCG1 facilitates macrophage cholesterol efflux. ABCG5/ABCG8 facilitate sterol secretion into bile and sterol absorption. NPC1L1 is the putative intestinal sterol transporter. ABC transporters are gene targets of the nuclear receptors, liver X receptors (LXR). LXR are expressed in intestine, as are their ABC gene targets. This proposal will address the role of ABCA1 in intestinal HDL production; the specificity of apical sterol efflux by ABCG5/ABCG8; the role of intestinal lipid rafts in apical sterol transport; and the regulation of ABC transporters and NPC1L1 in hamster intestine by dietary cholesterol, plant sterols, or fat. Human intestinal cells, CaCo-2, will be grown on semi-permeable filters separating an upper and lower well. Regulation of ABCA1 mRNA levels and mass by dietary and biliary lipids will be studied. Lipids that alter ABCA1 expression and LXR agonists that enhance ABCA1 expression will be used to address the role of ABCA1 in intestinal HDL production. Following over expression or silencing of ABCA1 and/or ABCG1 expression, HDL production will be estimated. Human intestinal explants will be used to study the regulation of NPC1L1, ABCA1, ABCG1, ABCG5/ABCG8 mRNA, mass, and HDL production by dietary lipids and/or LXR agonists. To address the specificity of sterol uptake and efflux, expressions of ABCG5, ABCG8, or both will be over expressed or silenced. Cells will then be incubated with bile salt micelles containing labeled cholesterol, cholestenol, ¿-sitosterol, or sitostanol. The rate of sterol uptake into cells, esterification, and basolateral and apical efflux will be estimated. Composition of intestinal lipid rafts will be analyzed following stimulation of apical sterol efflux or influx to ascertain whether specific proteins (caveolin-1, annexin-2, SR-B1, or others) interact to form a complex that facilitates apical sterol transport. Hamsters will be fed diets containing cholesterol or sitostanol or both, cholestyramine plus lovastatin, or different fats. The expression of ABC transporters and NPC1L1 will be estimated along the longitudinal and vertical axis of the intestine. Percent cholesterol absorption, fecal neutral sterol and bile acid excretion, and plasma and intestinal lipids will be estimated. The results of this proposal will provide new and novel information on the roles of the ABC transporters, NPC1L1, and lipid rafts in intestinal sterol metabolism

描述（由申请人提供）：ATP 结合盒 (ABC) 转运蛋白和 Niemann-pick C1 样蛋白 (NPC1L1) 在细胞甾醇运输中发挥重要作用，ABCA1 通过促进胆固醇流出和新生 HDL 的形成来消除细胞中的胆固醇。促进巨噬细胞胆固醇流出 ABCG5/ABCG8 促进甾醇分泌到胆汁中，并促进 NPC1L1 的吸收。假定的肠道甾醇转运蛋白。 ABC 转运蛋白是核受体、肝脏 X 受体 (LXR) 的基因靶标，该受体也是它们的 ABC 基因靶标。 ABCG5/ABCG8 的顶端甾醇流出的特异性；以及 ABC 转运蛋白和 NPC1L1 在顶端甾醇转运中的作用；人类肠道细胞 CaCo-2 将在分隔上孔和下孔的半透过滤器上生长，研究饮食和胆汁脂质对ABCA1 mRNA水平和质量的调节。改变 ABCA1 表达的脂质和增强 ABCA1 表达的 LXR 激动剂将用于在 ABCA1 过度表达或沉默后解决 ABCA1 在肠道 HDL 生成中的作用。和/或 ABCG1 表达、HDL 产量将用于研究膳食脂质和/或 LXR 激动剂对 NPC1L1、ABCA1、ABCG1、ABCG5/ABCG8 mRNA、质量和 HDL 产量的调节。甾醇摄取和流出的特异性，ABCG5、ABCG8 或两者的表达将被过度表达或沉默，然后细胞将与胆汁盐一起孵育。含有标记胆固醇、胆固醇、¿ -谷甾醇，或谷甾烷醇。在刺激顶端甾醇流出或流入后，将估计甾醇摄入细胞、酯化以及基底外侧和顶端流出的速率，以确定是否存在特定蛋白质（caveolin-1）。、annexin-2、SR-B1 或其他）相互作用形成促进顶端甾醇转运的复合物。仓鼠将被喂食含有胆固醇或谷甾烷醇或两者、考来烯胺加洛伐他汀或不同脂肪的饮食。ABC转运蛋白和NPC1L1的表达将沿着肠道的纵轴和纵轴、粪便中性甾醇和胆汁酸来估计。该提案的结果将估计 ABC 转运蛋白 NPC1L1 的作用。肠道甾醇代谢中的筏脂质