Investigation on the molecular mechanism of NPC1L1-mediated cholesterol uptake

累积证据表明，Niemann-Pick Cl-like 1(NPClL1)是肠道胆固醇吸收所必需的，也是新型降胆固醇药物依折麦布的靶标。在本项目中，我们分离了人NPClL1基因，并构建了高表达人NPClL1的Caco-2细胞。NPClL1蛋白主要表达于肠顶膜，反映了NPClL1在肠刷状缘膜上的生理性表达。胆固醇转运活性与NPClL1的表达水平呈正相关，依折麦布以浓度依赖的方式抑制NPClL1的摄取。此外，由于已证实胆固醇通过多种转运体和/或受体跨质膜的转移与磷脂的转移有关，我们研究了NPClL1对胆固醇的摄取是否也发生在脂质相关的机制中。结果发现，NPClL1过表达导致胆固醇和磷脂酰胆碱从由胆固醇、磷脂酰胆碱和牛磺胆酸盐组成的胶束中摄取增加，但胆固醇的转运活性远高于磷脂酰胆碱。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。相比之下，NPClL1介导的牛磺胆酸盐摄取很少。研究还表明，胆固醇的存在不会影响胶束对磷脂酰胆碱和牛磺胆酸盐的摄取和对ezetimibe的敏感性。这些结果表明，尽管NPClL1具有较强的从胶束中摄取磷脂的活性，但胆固醇是NPClL1在胶束成分中较好的底物，而依折麦布的抑制作用对胆固醇摄取具有相对选择性。

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.