Functional & molecular study of intetinal cholesterol transporters & absorption

描述(申请人提供)：Zetia(Ezetimibe)通过抑制NPC1-L1介导的胆固醇转运途径(S)抑制肠道细胞对胆固醇的吸收。我们证明了CD36也在胆固醇吸收中发挥作用。也有研究表明，清道夫受体SR-BI是肠道刷状缘膜上的另一种可能的胆固醇转运蛋白。我们假设SR-BI和CD36分别是近端和远端肠道的胆固醇转运体，与NPC1-L1(可能是细胞内的)一起工作，介导胆固醇从肠腔到发生乳糜粒组装的肠细胞的细胞内腔。目的1：我们将确定：1)缺乏SR-BI、CD36或NPC1L1是否改变了胆固醇吸收的位置和/或效率(通过改变输注的位置或剂量)；以及2)在缺少一种或另一种转运蛋白的情况下，对应的转运蛋白是否上调以维持胆固醇的吸收。在淋巴瘘小鼠中，我们还将确定Ezetimibe在这些基因敲除动物的小肠中是否仍然有效地抑制胆固醇的吸收。目的2：首先，我们将确定亚有效剂量的Ezetimibe是否与亚有效剂量的熊去氧胆酸盐或Pluronic F-68(F-68)联合使用，抑制大鼠的肠道胆固醇吸收。此外，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Ezetimibe、熊去氧胆酸盐或F-68单独或联合使用是否有效地抑制小肠对胆固醇的吸收。目的：我们将利用Ezetimibe抑制肠道胆固醇的能力和Pluronic L-81防止乳糜粒形成的能力来确定途径并定量胆固醇通过外流到顶端离开肠细胞以及通过基底膜以富含甘油三酯的脂蛋白的形式分泌。结合使用Ezetimibe和Pluronic L-81，我们将能够研究小肠对胆固醇的吸收和外流。拟议研究的完成将为我们提供新的见解，了解肠道中各种转运蛋白是如何调节胆固醇摄取和淋巴运输的。这些新数据也可能为我们在高胆固醇患者的临床治疗中提供新的信息。

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.