MECHANISMS AND REGULATION OF INTESTINAL CHOLESTEROL TRANSPORT

肠道胆固醇转运的机制和调节

• 批准号: 7686587
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686587
• 关键词: Address; Affect; Atherosclerosis; Binding Proteins; Biochemical; Biological Assay; Caco-2 Cells; Cell Culture Techniques; Cholesterol; Cholesterol Homeostasis; Confocal Microscopy; DNA Binding; Data; Deoxyribonucleases; Development; Diabetes Mellitus; Diet; Dietary Cholesterol; Disease; Dose; Endocrine; Enterocytes; Epithelial Cells; Esterification; Functional disorder; Future; Gel; General Population; Goals; Guidelines; Health; Healthcare; Hormones; Human; In Vitro; Incidence; Individual; Insulin; Insulin Resistance; Intestinal Absorption; Intestines; Knockout Mice; Lead; Maintenance; Mediating; Mediator of activation protein; Mission; Modality; Modeling; Molecular; Molecular Biology Techniques; Molecular Target; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Plasma; Play; Prevalence; Process; Rattus; Regulation; Research Design; Response Elements; Role; Schering-Plough brand of ezetimibe; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Small Interfering RNA; Small Intestines; Sterols; Streptozocin; Therapeutic; Time; Transcriptional Regulation; Up-Regulation; Vesicle; Veterans; base; brush border membrane; cardiovascular disorder risk; cholesterol absorption; cholesterol control; cholesterol transporters; chromatin immunoprecipitation; design; diabetic patient; diabetic rat; effective therapy; ezetimibe; feeding; hormone regulation; hypercholesterolemia; improved; in vitro Model; in vivo; inhibitor/antagonist; insight; intestinal crypt; intestinal epithelium; intestinal villi; laser capture microdissection; mRNA Expression; meetings; monolayer; novel; patient population; promoter; public health relevance; response; transcription factor; uptake

DESCRIPTION (provided by applicant): Intestinal cholesterol absorption plays a critical role in the maintenance of the body cholesterol homeostasis. This is inferred from studies showing a positive correlation between efficiency of intestinal cholesterol absorption and the level of plasma cholesterol. Additionally, pharmacological inhibition of intestinal cholesterol absorption by the recently discovered drug ezetimibe (Zetia) has been shown to be an effective therapy to lower plasma cholesterol. In this regard, the Niemann-Pick Type C1-Like 1 (NPC1L1) transporter represents the major pathway for intestinal cholesterol absorption. An increase in intestinal cholesterol absorption with concomitant upregulation of NPC1L1 expression has been implicated in the pathophysiology of hypercholesterolemia associated with diseases such as diabetes mellitus. Besides recent advances in therapeutic modalities to lower plasma cholesterol, treatment of hypercholesterolemia in patients with diabetes mellitus is still a challenge. Therefore, an understanding of the molecular mechanisms regulating NPC1L1 function and expression under physiological conditions and in diseased states would facilitate development of future therapeutic modalities for hypercholesterolemia. The major pathological feature of diabetes mellitus is insulin deficiency or a defective response to insulin. Whether insulin plays a role in NPC1L1 regulation under basal conditions or in diseased conditions of altered cholesterol homeostasis observed in diabetes mellitus is not known. We hypothesized that insulin decreases intestinal cholesterol absorption via the modulation of NPC1L1 function and expression and that the increase in intestinal cholesterol uptake and NPC1L1 expression in diabetes mellitus results from disturbances in the molecular pathways involved in NPC1L1 regulation. Our preliminary data utilizing human intestinal Caco2 monolayers demonstrated that NPC1L1 function, expression and promoter activity are decreased by insulin. We also demonstrated an increase in the expression of intestinal NPC1L1 in the rat model of Streptozotocin (STZ)-induced diabetes mellitus in parallel to an increase in cholesterol uptake in isolated enterocytes. However, the molecular mechanisms underlying regulation of NPC1L1 by insulin are not known. Therefore, our proposed studies represent a systematic approach designed to extensively investigate NPC1L1 regulation by insulin utilizing an in vitro cell culture model (Specific objective 1) and to examine the mechanisms of NPC1L1 alterations in diseases associated with hypercholesterolemia utilizing an in vivo rat model of diabetes mellitus (Specific Objective 2). Studies proposed in Specific objective 1 will utilize molecular biology techniques such as promoter analysis, DNAase footprinting and chromatin immunoprecipitation assays to delineate the transcriptional regulation of NPC1L1 by insulin. Involvement of signaling molecules and transcription factors in NPC1L1 regulation by insulin will be further elucidated by siRNA and other biochemical approaches. Specific objective 2 will focus on delineating the molecular regulation of NPC1L1 function and expression in rat model of diabetes mellitus utilizing the state-of-the art approaches such as cellular uptake in isolated enterocytes, esterification of cholesterol, laser capture microdissection and confocal microscopy. The results of the proposed studies will enhance our understanding of the regulation of the processes involved in intestinal cholesterol absorption by insulin and will reveal potential targets for better and more effective therapeutic modalities to manage hypercholesterolemia associated with diseases such as diabetes mellitus. Potential Impact on Veterans Health Care: The proposed studies are highly relevant to the VA mission since the prevalence of diabetes mellitus and cholesterol homeostasis disorders such as atherosclerosis are higher in the VA patient population than in the general population. The results of our studies will aid in the development of better therapeutic modalities for the treatment of hypercholesterolemia and hence improve the management of complications associated with diabetes mellitus. PUBLIC HEALTH RELEVANCE: An increase in intestinal absorption of cholesterol occurs in diseases such as diabetes mellitus leading to high plasma cholesterol and increased risk of cardiovascular diseases. Therefore, it is important to investigate the mechanisms underlying alterations in cholesterol absorption under physiological conditions and in diseases such as diabetes mellitus. The proposed studies focus on insulin mediated regulation of the cholesterol transporter, NPC1L1 and its modulation in rat model of diabetes mellitus. These studies have direct relevance to public and veterans health because of the high incidence of diabetes mellitus in both general and VA patient population and are of great importance for designing better therapeutic modalities for the management of cholesterol-related disorders in future.

描述（由申请人提供）： 肠道胆固醇吸收在维持体内胆固醇稳态中起着关键作用。这是从显示肠胆固醇吸收效率与血浆胆固醇水平之间正相关的研究中推断出来的。此外，最近发现的药物依折麦布（Zetia）对肠道胆固醇吸收的药理学抑制已被证明是降低血浆胆固醇的有效疗法。在这方面，尼曼-匹克C1型样1（NPC 1 L1）转运蛋白代表了肠道胆固醇吸收的主要途径。肠胆固醇吸收的增加伴随着NPC 1 L1表达的上调已经涉及与疾病如糖尿病相关的高胆固醇血症的病理生理学。除了降低血浆胆固醇的治疗方法的最新进展外，糖尿病患者高胆固醇血症的治疗仍然是一个挑战。因此，了解NPC 1 L1在生理条件下和疾病状态下调节功能和表达的分子机制将有助于开发未来的高胆固醇血症治疗模式。 糖尿病的主要病理特征是胰岛素缺乏或对胰岛素的反应缺陷。胰岛素是否在基础条件下或在糖尿病中观察到的胆固醇稳态改变的疾病条件下在NPC 1 L1调节中起作用尚不清楚。我们假设胰岛素通过调节NPC 1 L1的功能和表达来降低肠道胆固醇吸收，并且糖尿病患者肠道胆固醇摄取和NPC 1 L1表达的增加是由于NPC 1 L1调节所涉及的分子途径的干扰。我们利用人肠Caco 2单层的初步数据表明，胰岛素降低了NPC 1 L1的功能，表达和启动子活性。我们还证明了在链脲佐菌素（STZ）诱导的糖尿病大鼠模型中肠NPC 1 L1的表达增加，与分离的肠上皮细胞中胆固醇摄取增加平行。然而，胰岛素调节NPC 1 L1的分子机制尚不清楚。因此，我们拟定的研究代表了一种系统性方法，旨在利用体外细胞培养模型广泛研究胰岛素对NPC 1 L1的调节（具体目标1），并利用体内大鼠糖尿病模型研究高胆固醇血症相关疾病中NPC 1 L1改变的机制（具体目标2）。 具体目标1中提出的研究将利用分子生物学技术，如启动子分析、DNA酶足迹法和染色质免疫沉淀试验来描述胰岛素对NPC 1 L1的转录调控。胰岛素对NPC 1 L1的调控中信号分子和转录因子的参与将通过siRNA和其他生物化学方法进一步阐明。具体目标2将侧重于描绘NPC 1 L1的功能和表达的分子调控，在糖尿病大鼠模型中使用最先进的方法，如在分离的肠细胞中的细胞摄取，胆固醇的酯化，激光捕获显微切割和共聚焦显微镜。 拟议研究的结果将增强我们对胰岛素对肠道胆固醇吸收过程的调控的理解，并将揭示更好和更有效的治疗方法的潜在目标，以管理与糖尿病等疾病相关的高胆固醇血症。对退伍军人医疗保健的潜在影响：拟定研究与VA使命高度相关，因为VA患者人群中糖尿病和胆固醇稳态障碍（如动脉粥样硬化）的患病率高于一般人群。我们的研究结果将有助于开发更好的治疗高胆固醇血症的治疗方法，从而改善糖尿病相关并发症的管理。 公共卫生相关性： 胆固醇的肠吸收增加发生在诸如糖尿病的疾病中，导致高血浆胆固醇和心血管疾病的风险增加。因此，重要的是要研究在生理条件下和在疾病如糖尿病中胆固醇吸收的潜在改变的机制。本研究主要集中在胰岛素介导的胆固醇转运蛋白NPC 1 L1的调节及其在糖尿病大鼠模型中的调节。这些研究与公众和退伍军人的健康直接相关，因为糖尿病在普通人群和VA患者人群中的发病率很高，对于设计更好的治疗方式来管理胆固醇相关疾病具有重要意义。