MECHANISMS AND REGULATION OF INTESTINAL CHOLESTEROL TRANSPORT

Niemann-Pick C1-like 1，NPC1L1，是胆固醇吸收的关键，也是 依折麦布，降胆固醇药物。先前的研究表明，NPC1L1的表达在 糖尿病患者会导致相关的高胆固醇血症。阻断胆固醇酯 依折麦布与他汀类药物(胆固醇合成的抑制剂)联合应用已被证实。 在降低血清胆固醇方面比单用他汀类药物更有效。当前指导方针 建议积极降低高发风险患者的血胆固醇 心血管疾病(CVD)，如糖尿病患者。实现这些严格的低目标仍然存在 很有挑战性。由于ezetimibe只阻断NPC1L1的活性，因此减少NPC1L1的表达代表着 进一步降低血浆胆固醇的有吸引力的治疗方法。因此，至关重要的是 描绘与降低NPC1L1表达有关的细胞途径，可利用这些途径 有效降低心血管疾病风险。我们之前已经证明，NPC1L1的表达受 一种涉及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 在调控中，NPC1L1的表达尚不清楚。我们的初步数据显示，HDAC抑制剂 (HDACi)丁酸和丙戊酸(VPA)显著降低肠上皮细胞NPC1L1的表达 细胞和小鼠肠道。同样，siRNA介导的HDAC2和3的衰减，但不是其他异构体， 降低NPC1L1的表达；然而，其潜在的分子机制仍不清楚。我们 假设组蛋白脱乙酰基酶亚型通过修饰调控NPC1L1的表达 染色质结构和/或改变特定转录因子与NPC1L1的结合 吉恩。我们还假设，HDAC抑制剂，如VPA，可以减少胆固醇吸收和 降低高胆固醇血症小鼠模型的血浆胆固醇。VPA是一种广泛使用的药物 癫痫和双相情感障碍的治疗。因此，直接研究其对环境的影响具有重要意义。 人体对胆固醇的吸收。然而，目前直接测量胆固醇的标准方法 人体的吸收是有限的，因为它依赖于使用同位素标记的胆固醇和费时费力 实验室技术。我们目前正在研究一种新的、简单的、非放射性的检测方法 评估胆固醇的转运。我们的初步数据表明，该方法适用于评估 NPC1L1函数。我们假设我们假定的简单的非放射性测量方法 NPC1L1函数代表了一种直接测量人体胆固醇吸收的新方法 动物。这些研究旨在严格检验假设，并解决两个主要目标。 在特定的目标1中，我们的研究旨在探讨减弱hdac异构体对 NPC1L1功能，检测NPC1L1基因染色质重塑和结合的变化 转录因子在肠道细胞系和肠样体中的表达，并探讨HDACi对其表达的影响。 低密度脂蛋白受体基因敲除小鼠作为高胆固醇血症模型的胆固醇吸收。研究 在具体目标2中提出的目的是为了确定假定方法在测量中的准确性 利用NPC1L1基因敲除小鼠和野生型小鼠吸收胆固醇。这些研究还将 使用新方法研究HDACi对小鼠胆固醇吸收的减少，提供 为未来的人类翻译研究奠定了基础。值得注意的是，患心血管疾病的风险在 退伍军人与普通民众相比。因此，解开涉及组蛋白的新机制 去乙酰化下调NPC1L1的表达并建立新的、最先进的方法来 衡量相关的胆固醇吸收减少是及时和直接相关的改善 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.