Physiological Role of NPC1L1 in Lipid Transport and Metabolism

描述（由申请人提供）：Niemann Pick C-1 Like 1 （NPC1L1）蛋白在肠道胆固醇吸收中的重要性已得到充分认识。然而，NPC1L1是一种膜结合蛋白，作为肠细胞摄取胆固醇的转运蛋白，还是一种细胞内蛋白，负责将胆固醇从根尖质膜运送到细胞内室进行脂蛋白组装，目前仍有争议。NPC1L1在人肝脏中也有高水平表达，但在啮齿动物肝脏中的表达很少。因此，肝脏NPC1L1的重要性尚不清楚。本研究的目的是：(i)确定NPC1L1参与肠道胆固醇吸收的生理机制，以及（ii）描述肝脏NPC1L1在脂蛋白代谢和体内平衡中的作用。初步结果表明，尽管NPC1LT小鼠的胆固醇吸收存在缺陷，但NPC1L1+/+和NPC1LT小鼠的肠刷状边界膜小泡的胆固醇转运动力学没有差异。此外，在NPC1L1+/+和NPC1LT”小鼠中，荧光胆固醇都能被有效地从肠腔吸收到肠道的吸收上皮细胞中，但在使用胆固醇吸收抑制剂依折替米贝的小鼠中则没有。这些结果有力地支持了NPC1L1不是高亲和膜转运蛋白，而是细胞内胆固醇转运蛋白的假设。我们的数据还显示，抑制肝脏中NPC1L1的活性降低了VLDL的产生，抑制了高密度脂蛋白胆固醇的选择性摄取，这表明肝脏NPC1L1可能调节血浆脂蛋白稳态。Specific Aim 1将通过将NPC1L1*/+和NPC1LT“小鼠”与ABCG5G8“小鼠配对，确定NPC1L1在肠道内胆固醇积累的分布和部位，从而确定NPC1L1在细胞内脂质转运中的功能作用，进一步验证NPC1L1是一种细胞内脂质转运蛋白，在细胞顶摄取到细胞内区室进行脂蛋白组装后靶向胆固醇。Specific Aim 2将使用体外细胞培养模型来验证NPC1L1的表达是必要的，但在促进胆固醇吸收和细胞内运输时，没有细胞表面胆固醇转运蛋白如SR-BI和/或CD36的表达是不够的。Specific Aim 3将产生在肝脏中表达NPC1L1的转基因小鼠，其表达水平与其在人肝脏中的表达水平相似，以确定肝脏NPC1L1在血浆脂质稳态、VLDL生成、高密度脂蛋白胆固醇选择性摄取和胆固醇逆向转运中的功能意义。综上所述，这些研究将更好地了解NPC1L1的生理重要性和作用机制，可能为治疗高胆固醇血症和降低脂质诱导代谢紊乱的风险提供替代靶点。

DESCRIPTION (provided by applicant): The importance of the Niemann Pick C-1 Like 1 (NPC1L1) protein in cholesterol absorption by the intestine is well recognized. However, whether NPC1L1 is a membrane bound protein serving as a transporter for cholesterol uptake by enterocytes or an intracellular protein responsible for cholesterol trafficking from the apical plasma membrane to intracellular compartments for lipoprotein assembly remains controversial. The NPC1L1 is also expressed at high levels in human liver, but its expression in rodent liver is minimal. Hence, the importance of hepatic NPC1L1 is still unclear. The goals of this study are (i) to identify the physiological mechanism by which NPC1L1 participates in cholesterol absorption in the intestine, and (ii) to delineate the role of liver NPC1L1 in lipoprotein metabolism and homeostasis. Preliminary Results showed that whereas NPC1LT'' mice were defective in cholesterol absorption, no difference in cholesterol transport kinetics was observed between intestinal brush border membrane vesicles obtained from NPC1L1+/+ and NPC1LT'' mice. Moreover, fluorescent cholesterol was efficiently taken up from intestinal lumen into absorptive epithelial cells of the intestine in both NPC1L1+/+ and NPC1LT'' mice, but not in mice treated with the cholesterol absorption inhibitor ezetimibe. These results provided strong support for the hypothesis that NPC1L1 is not the high affinity membrane transporter but an intracellular cholesterol transport protein. Our data also showed that suppression of NPC1L1 activity in the liver lowered VLDL production and inhibited selective uptake of HDL-cholesterol, suggesting that hepatic NPC1L1 may modulate plasma lipoprotein homeostasis. Specific Aim 1 will identify the functional role of NPC1L1 in intestinal lipid transport by mating NPC1L1*/+ and NPC1LT'' mice to ABCG5G8''' mice and then determining the distribution and site of cholesterol accumulation in the intestine to further test the hypothesis that NPC1L1 is an intracellular lipid trafficking protein targeting cholesterol after apical uptake to intracellular compartments for lipoprotein assembly. Specific Aim 2 will use in vitro cell culture model to test the hypothesis that NPC1L1 expression is necessary but not sufficient without expression of cell surface cholesterol transporters such as SR-BI and/or CD36 in promoting cholesterol absorption and intracellular transport. Specific Aim 3 will produce transgenic mice expressing NPC1L1 in the liver, to similar extent as its expression level in human liver, to identify the functional significance of hepatic NPC1L1 in plasma lipid homeostasis, VLDL production, selective uptake of HDL-cholesterol, and reverse cholesterol transport. Taken together, these studies will provide a better understanding on the physiological importance and mechanism of action of NPC1L1, potentially offering alternative targets for therapeutic treatment of hypercholesterolemia and lowering the risk of lipid-induced metabolic disorders.