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Investigation on the molecular mechanism of NPC1L1-mediated cholesterol uptake

NPC1L1介导胆固醇摄取的分子机制研究

基本信息

批准号：
18390047
负责人：
ITO Kousei
金额：
$ 6.98万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390047/
关键词：
lipids responses to drugs intestinal absorption transporters

项目摘要

Cumulative evidence suggests that Niemann-Pick Cl-like 1 (NPClL1) is essential for intestinal cholesterol absorption and is the target of ezetimibe, a novel cholesterol-lowering drug. In this project, we isolated human NPClL1 cDNA and constructed human NPClL1-overexpressing Caco-2 cells. NPClL1 protein was mainly detected on the apical membrane, which reflects the physiological expression of NPClL1 on the intestinal brush border membrane. It was also shown that the transport activities of cholesterol were positively correlated with the expression level of introduced NPClL1 and ezetimibe inhibited the uptake in a concentration-dependent manner. In addition, since it has also been established that the transfer of cholesterol across the plasma membrane via many kinds of transporters and/or receptors is associated with the transfer of phospholipids, we examined whether the uptake of cholesterol by NPClL1 also takes place in the lipid-associated mechanism. It was found that overexpression of NPClL1 results in an increase in the uptake of both cholesterol and phosphatidylcholine from micelles composed of cholesterol, phosphatidylcholine and taurocholate but that the level of transport activity for cholesterol was much higher than that for phosphatidylcholine. In addition, the uptake of phosphatidylcholine was much less sensitive to ezetimibe than the uptake of cholesterol. In contrast, NPClL1-mediated uptake of taurocholate was minimal. It was also demonstrated that the uptake and ezetimibe-sensitivity of phosphatidylcholine and taurocholate from the micelles were not affected by the presence of cholesterol. Taken together, the results suggested that cholesterol is a preferable substrate of NPClL1 in micellar components and the inhibitory effect of ezetimibe is relatively selective to cholesterol uptake, although NPClL1 has potent activity for uptake of phospholipids from micelles.

累积证据表明，Niemann-Pick Cl-like 1 (NPClL1) 对于肠道胆固醇吸收至关重要，并且是新型降胆固醇药物依折麦布的靶标。在这个项目中，我们分离了人NPC1L1 cDNA并构建了人NPC1L1过表达的Caco-2细胞。 NPClL1蛋白主要在顶膜上检测到，这反映了NPClL1在肠刷状缘膜上的生理表达。还表明胆固醇的转运活性与引入的NPC1L1的表达水平呈正相关，并且依折麦布以浓度依赖性方式抑制摄取。此外，由于已经确定胆固醇通过多种转运蛋白和/或受体跨质膜的转移与磷脂的转移相关，因此我们检查了NPClL1对胆固醇的摄取是否也发生在脂质相关机制中。发现NPC1L1的过度表达导致从由胆固醇、磷脂酰胆碱和牛磺胆酸盐组成的胶束中对胆固醇和磷脂酰胆碱的摄取增加，但胆固醇的转运活性水平远高于磷脂酰胆碱的转运活性水平。此外，磷脂酰胆碱的摄取对依折麦布的敏感性远低于胆固醇的摄取。相反，NPC1L1介导的牛磺胆酸盐的摄取是最小的。还证明胶束中磷脂酰胆碱和牛磺胆酸盐的摄取和依折麦布敏感性不受胆固醇存在的影响。总而言之，结果表明胆固醇是胶束成分中NPC1L1的优选底物，并且依折麦布的抑制作用对胆固醇摄取具有相对选择性，尽管NPC1L1对于从胶束中摄取磷脂具有有效的活性。