MECHANISMS AND REGULATION OF INTESTINAL CHOLESTEROL TRANSPORT

肠道胆固醇转运的机制和调节

• 批准号: 10223192
• 负责人: Waddah A. Alrefai
• 金额: --
• 依托单位: JESSE BROWN VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223192
• 关键词: Address; Aftercare; Alkynes; Animals; Anticholesteremic Agents; Attenuated; BODIPY; Binding; Biological; Biological Assay; Bipolar Disorder; Blood; Butyrates; Caco-2 Cells; Cardiovascular Diseases; Cell Line; Cells; Chemistry; Cholesterol; Cholesterol Homeostasis; Chromatin Structure; Coronary heart disease; Coumarins; DNA Methylation; Data; Development; Diabetes Mellitus; Diet; Disease; Drug usage; Enzymes; Epigenetic Process; Epilepsy; Esterification; Fiber; Funding; Future; Gene Mutation; General Population; Genes; Guidelines; HDAC2 gene; Health; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone Deacetylation; Histones; Human; Individual; Intestines; Investigation; Isotope Labeling; Knock-out; Knockout Mice; Label; Laboratories; Low Density Lipoprotein Receptor; Measurement; Measures; Mediating; Methods; Micelles; Modeling; Molecular; Molecular Target; Mus; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Plasma; Play; Property; Protein Isoforms; Regulation; Risk Factors; Role; Serum; Small Interfering RNA; Stroke; Techniques; Therapeutic; Thin Layer Chromatography; Time; Valproic Acid; Veterans; Wild Type Mouse; absorption; attenuation; base; cardiovascular disorder risk; cardiovascular risk factor; cholesterol absorption; chromatin remodeling; design; diabetic patient; effective therapy; ezetimibe; factor C; feeding; fluorophore; high risk; histone modification; hypercholesterolemia; improved; in vitro Model; inhibitor/antagonist; jejunum; mouse model; new therapeutic target; novel; patient population; therapeutic target; transcription factor; translational study; uptake

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.

Niemann-Pick C1-Like 1 (NPC1L1) 对胆固醇吸收至关重要，是胆固醇吸收的分子靶点 依折麦布，降胆固醇药物。先前的研究表明 NPC1L1 表达在 患有相关高胆固醇血症的糖尿病患者。阻断胆固醇 依折麦布与他汀类药物（胆固醇合成抑制剂）联合使用已显示出吸收 在降低血清胆固醇方面比单独使用他汀类药物治疗更有效。目前的指导方针 建议高风险患者积极降低血液胆固醇 心血管疾病（CVD），例如糖尿病患者。实现这些严格的低目标仍然存在 具有挑战性的。由于依折麦布仅阻断 NPC1L1 活性，因此减少 NPC1L1 表达代表 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 描绘了参与减少 NPC1L1 表达的细胞途径，可用于 有效降低CVD风险。我们之前已经证明 NPC1L1 的表达受 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 调节 NPC1L1 表达的作用尚不清楚。我们的初步数据表明 HDAC 抑制剂 (HDACi) 丁酸盐和丙戊酸 (VPA) 显着降低肠道 Caco2 中 NPC1L1 的表达 细胞和小鼠肠道。此外，siRNA 介导的 HDAC2 和 3 减弱，但其他异构体没有减弱， NPC1L1 表达减少；然而，潜在的分子机制仍不清楚。我们 假设特定的组蛋白脱乙酰酶亚型通过修饰来调节 NPC1L1 的表达 染色质结构和/或改变特定转录因子与 NPC1L1 的结合 基因。我们还假设 VPA 等 HDAC 抑制剂会减少胆固醇吸收， 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 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 吸收在人类中存在局限性，因为它依赖于同位素标记的胆固醇的使用并且费力 实验室技术。我们目前正在研究一种新颖的简单测定法，该测定法对 评估胆固醇转运。我们的初步数据表明该方法适合评估 NPC1L1 功能。我们假设我们假定的简单非放射性测量方法 NPC1L1功能代表了一种直接测量活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。 在具体目标 1 中，我们的研究旨在调查减弱 HDAC 亚型对 NPC1L1 功能，检查 NPC1L1 基因的染色质重塑和结合的改变 肠细胞系和类肠细胞中的转录因子，并研究 HDACi 对 作为高胆固醇血症模型的 LDL 受体敲除小鼠的胆固醇吸收。研究 具体目标 2 中提出的旨在确定测量中假定方法的准确性 利用 NPC1L1 敲除小鼠和野生型同窝小鼠的胆固醇吸收。研究还将 使用新方法在小鼠中研究 HDACi 减少胆固醇吸收的情况，提供 为未来人类转化研究奠定基础。值得注意的是，CVD 风险明显更高 退伍军人与普通民众相比。因此，揭示涉及组蛋白的新机制 去乙酰化下调 NPC1L1 表达并建立新颖、最先进的方法 测量相关的胆固醇吸收减少是及时且直接与改善 退伍军人的健康。