MECHANISMS AND REGULATION OF INTESTINAL CHOLESTEROL TRANSPORT

尼曼-匹克C1样1，NPC 1 L1，是胆固醇吸收的关键，是胆固醇代谢的分子靶点。 依泽替米贝，降低胆固醇的药物。以前的研究表明，NPC 1 L1表达增加， 糖尿病患者导致相关的高胆固醇血症。封闭胆固醇 已显示依折麦布与他汀类药物（胆固醇合成抑制剂）联合使用的吸收 在降低血清胆固醇方面比单独使用他汀类药物更有效。现行指南 建议积极降低高风险患者的血液胆固醇， 心血管疾病（CVD），如糖尿病患者。实现这些严格的低目标仍然是 挑战性由于依折麦布仅阻断NPC 1 L1活性，因此NPC 1 L1表达降低代表了 用于进一步降低血浆胆固醇的有吸引力的治疗方法。因此，关键是 描述参与降低NPC 1 L1表达的细胞途径， 有效降低CVD的风险。我们先前已经表明，NPC 1 L1的表达是受 DNA甲基化的表观遗传机制然而，表观遗传组蛋白修饰的作用 在调节NPC 1 L1表达中的作用尚不清楚。我们的初步数据显示HDAC抑制剂 （HDACi）丁酸盐和丙戊酸（VPA）显著降低肠Caco 2中NPC 1 L1的表达， 细胞和小鼠肠。此外，siRNA介导的HDAC 2和3的衰减，而不是其他亚型， 降低NPC 1 L1表达;然而，潜在的分子机制仍不清楚。我们 假设特异性组蛋白脱乙酰酶亚型通过修饰 染色质结构和/或改变特异性转录因子与NPC 1 L1的结合 基因我们还假设HDAC抑制剂如VPA降低胆固醇吸收， 降低高胆固醇血症小鼠模型中的血浆胆固醇。VPA是一种广泛用于 治疗癫痫和躁郁症因此，直接研究其对 人体胆固醇的吸收。然而，目前直接测量胆固醇的标准方法 吸收在人体中具有局限性，因为它依赖于使用同位素标记的胆固醇， 实验室技术。我们目前正在研究一种新的简单的测定方法， 评估胆固醇转运。我们的初步数据表明，该方法适用于评估 NPC 1 L1功能。我们假设，我们假定的简单的非放射性方法来测量 NPC 1 L1功能代表了一种新的方法，可以直接测量生活中的胆固醇吸收 动物这些研究旨在严格审查假设，并解决两个主要目标。 在具体目标1中，我们的研究旨在研究减弱HDAC同种型对 NPC 1 L1功能，以检查NPC 1 L1基因的染色质重塑和NPC 1 L1结合的改变。 在肠细胞系和类肠细胞中的转录因子，并研究HDACi对 LDL受体敲除小鼠作为高胆固醇血症模型的胆固醇吸收。研究 具体目标2中提出的方法旨在确定推定方法在测量 利用NPC 1 L1敲除小鼠和野生型同窝出生的小鼠进行胆固醇吸收。这些研究还将 在小鼠中使用新方法研究HDACi对胆固醇吸收的减少， 为将来在人类中进行转化研究奠定基础。值得注意的是，CVD的风险显著高于 与普通人相比，因此，揭示涉及组蛋白的新机制 去乙酰化以下调NPC 1 L1表达，并建立新的、最先进的方法， 测量胆固醇吸收的相关减少是及时和直接相关的， 退伍军人的健康。

Niemann-Pick C1-Like 1, NPC1L1, is crucial for cholesterol absorption and is the molecular target for ezetimibe, the cholesterol lowering drug. Previous studies showed that NPC1L1 expression is increased in patients with diabetes mellitus contributing to associated hypercholesterolemia. Blocking choelsterol absoprtion with ezetimibe in combination with statins (inhibitors of cholesterol synthesis) has been shown to be more effective than treatment with statins alone in lowering serum cholesterol. Current guidelines recommend aggressive lowering of blood cholesterol in patients with high risk for developing cardiovascular diseases (CVD), such as diabetic patients. Achieving these stringent low targets remains challenging. Since ezetimibe only blocks NPC1L1 activity, decreasing NPC1L1 expression represents an attractive therapeutic approach for further reduction in plasma cholesterol. Therefore, it is critical to delineate cellular pathways involved in decreasing NPC1L1 expression that could be exploited to efficiently reduce the risk for CVD. We have previously shown that NPC1L1 expression is regulated by an epigenetic mechanism involving DNA methylation. However, the role of epigenetic histone modifications in modulating NPC1L1 expression is not known. Our preliminary data showed that the HDAC inhibitors (HDACi) butyrate and valproic acid (VPA) significantly decreased NPC1L1 expression in intestinal Caco2 cells and mouse intestine. Also, siRNA-mediated attenuation of HDAC2 and 3, but not the other isoforms, reduced NPC1L1 expression; however, the underlying molecular mechanisms remain unclear. We hypothesize that specific histone deacetylase isoforms modulate NPC1L1 expression via modifying chromatin structure and/or altering the binding of specific transcription factors to the NPC1L1 gene. We also hypothesize that HDAC inhibitors such as VPA decrease cholesterol absorption and reduce plasma cholesterol in mouse models of hypercholesterolemia. VPA is a widely used drug for the treatment of epilepsy and bipolar disorders. Therefore, it will be important to directly study its effects on cholesterol absorption in humans. However, the current standard method to directly measure cholesterol absorption has limitations in humans as it relies on the use of isotope-labeled cholesterol and laborious laboratory techniques. We are currently inveatigating a novel simple assay that is non-radioactive to evaluate cholesterol transport. Our preliminary data indicate that this method is suitable for assessing NPC1L1 function. We hypothesize that our putative simple non-radioactive approach to measure NPC1L1 function represents a novel method to directly measure cholesterol absorption in living animals. The studies are designed to rigorously examine the hypotheses and address two main objectives. In Specific Aim 1, our studies are aimed to investigate the effects of attenuating HDAC isoforms on NPC1L1 function, to examine chromatin remodeling of NPC1L1 gene and alterations in binding of transcription factors in intestinal cell lines and enteroids, and to investigate the effects of HDACi on cholesterol absorption in the LDL receptor knockout mice as a model of hypercholesterolemia. Studies proposed in Specific Aim 2 are designed to establish the accuracy of the putative method in measuring cholesterol absorption utilizing NPC1L1 knockout mice and wild type littermates. The studies will also investigate the decrease in cholesterol absorption by HDACi using the new method in mice, providing the basis for future translational studies in humans. Notably, the risk for CVD is significantly higher in the veterans as compared to the general population. Thus, unraveling novel mechanisms involving histone deacetylation to downregulate NPC1L1 expression and establishing novel, state-of-the-art method to measure the associated decrease in cholesterol absorption are timely and directly relevant to improving the health of the veterans.