Functional & molecular study of intetinal cholesterol transporters & absorption

描述（由申请人提供）：Zetia （Ezetimibe）通过抑制肠细胞中NPC1-L1介导的胆固醇转运途径来抑制胆固醇吸收。我们证明CD36也在胆固醇吸收中起作用。也有研究表明，清除受体SR-BI是肠刷状缘膜上另一种可能的胆固醇转运体。我们假设SR-BI和CD36分别是近端肠和远端肠的胆固醇转运蛋白，它们与NPC1-L1（可能在细胞内）协同工作，介导胆固醇从管腔摄取到发生乳糜微粒组装的肠细胞内腔室。目的1：我们将确定：1)缺乏SR-BI、CD36或NPC1L1是否会改变胆固醇吸收的部位和/或效率（通过改变输注部位或剂量）；2)如果在缺乏一种或另一种转运蛋白的情况下，对应转运蛋白被上调以维持胆固醇吸收。在淋巴瘘小鼠中，我们还将确定Ezetimibe是否仍然有效地抑制这些敲除动物的小肠摄取胆固醇。目的2：首先，我们将确定依zetimibe的亚有效剂量，以及熊脱氧胆酸盐或Pluronic F-68 （F-68）的亚有效剂量是否能抑制大鼠肠道胆固醇吸收。同样，我们将确定同样的现象也适用于鼠标。其次，在上述基因敲除小鼠模型中，我们将确定依折麦比、熊脱氧胆酸盐或F-68单独或联合使用是否能有效抑制小肠对胆固醇的吸收。目的3：我们将利用Ezetimibe抑制肠道胆固醇的能力，以及Pluronic L-81阻止乳糜微粒形成的能力，确定途径并量化胆固醇通过外排到肠细胞顶端以及通过基底外侧膜作为富含甘油三酯的脂蛋白分泌出肠细胞的量。使用Ezetimibe和Pluronic L-81的组合，我们将能够研究小肠对胆固醇的摄取和排出。这项研究的完成将为我们提供关于肠道中各种转运蛋白如何调节胆固醇摄取和淋巴运输的新见解。这些新数据也可能为高胆固醇患者的临床管理提供新的信息。

DESCRIPTION (provided by applicant): Zetia (Ezetimibe) inhibits cholesterol absorption via inhibition of NPC1-L1 mediated cholesterol transport pathway(s) in the enterocytes. We demonstrated that CD36 also plays a role in cholesterol absorption. It has also been demonstrated that the scavenger receptor SR-BI as another putative cholesterol transporter on intestinal brush border membrane. We hypothesize that SR-BI and CD36 are cholesterol transporters in the proximal and distal intestine, respectively, working in conjunction with NPC1-L1 (may be intracellular) to mediate cholesterol uptake from the lumen to intracellular compartments of the enterocytes where chylomicron assembly occurs. Aim 1: We will determine: 1) if the lack of SR-BI, CD36, or NPC1L1 alters the site and/or efficiency of cholesterol absorption (by varying the site or dose of infusion); and 2) if the counterpart transporters are upregulated to maintain cholesterol absorption in the absence of one or the other transporter protein. In lymph fistula mice, we will also determine if Ezetimibe remains effective in inhibiting cholesterol uptake by the small intestine in these knockout animals. Aim 2: First, we will determine if sub-effective doses of Ezetimibe, in combination with sub-effective doses of either ursodeoxycholate or Pluronic F-68 (F-68), inhibits intestinal cholesterol absorption in the rat. Also, we will determine the same phenomenon also works in the mouse. Second, in the knockout mice models described above, we will determine if Ezetimibe or ursodeoxycholate or F-68 alone or in combination is/are effective in inhibiting cholesterol absorption by the small intestine. Aim 3: We will take advantage of the ability of Ezetimibe to inhibit intestinal cholesterol, and the ability of Pluronic L-81 to prevent chylomicron formation to identify the pathway and quantify the amount of cholesterol exiting the enterocytes through efflux to the apical side as well as secreted through the basolateral membrane as triglyceride-rich lipoproteins. Using a combination of both Ezetimibe and Pluronic L-81, we will be able to study both the uptake and efflux of cholesterol by the small intestine. Completion of the proposed studies will provide us with new insights into how cholesterol uptake and lymphatic transport are regulated by the various transporters in the gut. These new data may also provide us with new information in the clinical management of hypercholestermic patients.