Physiological Role of NPC1L1 in Lipid Transport and Metabolism

描述(由申请人提供)：Niemann Pick C-1 like 1(NPC1L1)蛋白在肠道吸收胆固醇方面的重要性得到了很好的认识。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，还是细胞内的蛋白，负责将胆固醇从顶端质膜运输到细胞内以组装脂蛋白，目前仍存在争议。NPC1L1在人的肝脏中也有高水平的表达，但在啮齿动物肝脏中的表达很少。因此，肝脏NPC1L1的重要性仍不清楚。本研究的目的是(1)明确NPC1L1参与肠道胆固醇吸收的生理机制；(2)阐明肝脏NPC1L1在脂蛋白代谢和动态平衡中的作用。初步结果表明，NPC1LT‘’小鼠对胆固醇的吸收存在缺陷，而NPC1L1+/+和NPC1LT‘’小鼠的肠道刷状缘膜囊泡在胆固醇转运动力学上没有差异。此外，在NPC1L1+/+和NPC1LT‘’小鼠中，荧光胆固醇都能有效地从肠腔被吸收到肠道上皮细胞，但在服用胆固醇吸收抑制剂ezetimibe的小鼠中则不能。这些结果有力地支持了NPC1L1不是高亲和力的膜转运蛋白而是细胞内胆固醇转运蛋白的假说。我们的数据还显示，肝脏中NPC1L1活性的抑制降低了极低密度脂蛋白的产生，并抑制了对高密度脂蛋白-胆固醇的选择性吸收，表明肝脏NPC1L1可能调节血浆脂蛋白的动态平衡。具体目的1将通过将NPC1L1*/+和NPC1LT“”小鼠与ABCG5G8“”小鼠交配，然后确定胆固醇在肠道中的分布和位置，以进一步验证NPC1L1是一种针对胆固醇的细胞内脂质运输蛋白的假说，该蛋白在顶端摄取到细胞内隔室以组装脂蛋白后，以胆固醇为靶标。特殊目的2将使用体外细胞培养模型来检验假设，即如果没有细胞表面胆固醇转运体如SR-BI和/或CD36的表达，NPC1L1的表达是必要的，但不足以促进胆固醇的吸收和细胞内的运输。特异性目标3将建立在肝脏中表达NPC1L1的转基因小鼠，其表达水平与其在人类肝脏中的表达水平相似，以确定肝脏NPC1L1在血浆脂质平衡、VLDL产生、选择性摄取高密度脂蛋白-胆固醇和反向胆固醇转运方面的功能意义。综上所述，这些研究将使人们更好地了解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的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.