The Role of MTP in Lipid Droplet Formation in Adipocytes

MTP 在脂肪细胞脂滴形成中的作用

• 批准号: 8141848
• 负责人: Larry L. Swift
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8141848
• 关键词: 3T3 Cells; 3T3-L1 Cells; Adipocytes; Adipose tissue; Adult; American; Biochemical; Body Weight; Body fat; Cardiovascular Diseases; Caring; Cell Differentiation process; Cells; Chinese Hamster Ovary Cell; Confocal Microscopy; Data; Deposition; Dietary Fats; Dyslipidemias; Electron Microscopy; Energy Metabolism; Enterocytes; Epidemic; Euglycemic Clamping; Family; Fatty acid glycerol esters; General Population; Glucose; Glucose Clamp; Glucose tolerance test; Glycerol; Growth; Health; Hepatocyte; Homeostasis; Human; Hypertension; Insulin; Insulin Resistance; Laboratories; Leptin; Lipid Binding; Lipids; Lipoproteins; Location; Medical; Membrane; Membrane Proteins; Metabolism; Molecular; Movement; Mus; Mutate; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Overweight; Pattern; Property; Protein Deficiency; Proteins; Publishing; Rattus; Role; Site; Small Interfering RNA; Study models; Surface; Technology; Testing; Triglycerides; United States; Veterans; Woman; Work; base; food consumption; glucose metabolism; improved; in vivo; inhibitor/antagonist; insight; knock-down; lipid biosynthesis; member; men; microsomal triglyceride transfer protein; mutant; novel; protein expression; protein function; research study

DESCRIPTION (provided by applicant): Our laboratory recently discovered that microsomal triglyceride transfer protein (MTP), a protein essential for the assembly of triglyceride-rich lipoproteins within hepatocytes and enterocytes, is expressed in adipocytes of mice, rats, and humans. In addition, MTP is expressed in 3T3-L1 cells, and expression increases ~5-fold when these cells are induced to differentiate into adipocytes. MTP is present in a punctate pattern on the surface of lipid droplets and is especially prominent at the points of contact of adjacent lipid droplets. When MTP expression is silenced and the cells induced to differentiate, only very small (<1 5m) lipid droplets are formed. In addition, when MTP is transfected into CHO cells and lipid droplet formation stimulated, larger droplets are formed compared to those formed in cells without MTP. Finally, knocking down MTP expression in mouse adipose tissue using Cre-Lox approaches leads to decreased adiposity and a leaner mouse than littermate controls. Our working hypothesis is: MTP is a critical component in the maturation and expansion of lipid droplets within the adipocyte. This working hypothesis will be tested as follows: Specific Aim 1: To define the requirement for MTP in lipid droplet maturation. These studies will test the hypotheses that MTP facilitates normal lipid droplet expansion and growth and that the absence of MTP in adipocytes in vivo results in leaner mice with reduced body fat and improved glucose metabolism. Using primary pre-adipocytes isolated from fat specific MTP-knockdown mice and 3T3-L1 cells, in which MTP expression has been silenced by siRNA, we will evaluate the effects of MTP deficiency on differentiation, lipid droplet formation and maturation, de novo lipogenesis, and glycerol metabolism. We will also explore the effects of enhanced MTP expression on lipid droplet formation and maturation in primary pre-adipocytes and 3T3 cells. Using fat-specific MTP knockdown mice, we will define the effects of the knockdown on body weight, body fat composition, energy expenditure, food consumption, leptin, and insulin. Glucose metabolism will also be assessed using glucose tolerance tests as well as hyperinsulinemic euglycemic clamps. Specific Aim 2. To determine the molecular mechanism by which MTP facilitates the growth and maturation of lipid droplets. These studies will test the hypothesis that MTP facilitates lipid droplet expansion via structural and functional properties of the molecule. Selected sites in the putative lipid binding region of MTP will be mutated to determine if MTP functions as a "fusogenic protein" in lipid droplet maturation. MTP inhibitors will be used to determine if lipid transfer activity is necessary for lipid droplet expansion. Specific Aim 3. To examine the mechanism by which MTP associates with lipid droplets in 3T3- L1 cells. These studies will test the hypothesis that association of MTP with triglyceride in specific regions in the ER membrane initiates the movement of MTP from the lumen of the ER to the lipid droplet surface. Confocal and electron microscopy as well as biochemical approaches will be used to examine subcellular locations of MTP in differentiating 3T3-L1 cells and its association with lipid droplets in comparison with selected members of the PAT family of lipid droplet proteins. PUBLIC HEALTH RELEVANCE: Obesity presents a major health problem in the United States, even among American Veterans. A study of Veterans receiving care at VA medical facilities during 2000, revealed that 68.4% of the women were overweight (BMI 25) with 37.4% classified as obese (BMI 30). Seventy-three percent of the men were overweight with 32.9% classified as obese. This epidemic poses a major health problem as obesity is associated with a number of complications include type 2 diabetes, insulin resistance, dyslipidemia, hypertension, and cardiovascular disease. Unfortunately, little is known about the pathophysiological basis of obesity, and the mechanisms that underlie the excessive accumulation of fat. Indeed, little is known about the basic steps in lipid droplet formation and maturation within the fat cell and the mechanisms that regulate fat deposition. The proposed studies will provide new insight into how fat is stored and could prove important in understanding the factors involved in obesity and its many complications, including cardiovascular disease.

描述（由申请人提供）： 我们的实验室最近发现微粒体甘油三酯转移蛋白（MTP）是肝细胞和肠细胞内富含甘油三酯的脂蛋白组装所必需的蛋白质，在小鼠、大鼠和人类的脂肪细胞中表达。此外，MTP 在 3T3-L1 细胞中表达，当这些细胞被诱导分化为脂肪细胞时，表达量增加约 5 倍。 MTP 以点状形式存在于脂滴表面，并且在相邻脂滴的接触点处尤其突出。当 MTP 表达被沉默并且细胞被诱导分化时，仅形成非常小的（<1 5m）脂滴。此外，当 MTP 转染至 CHO 细胞并刺激脂滴形成时，与没有 MTP 的细胞中形成的脂滴相比，会形成更大的脂滴。最后，使用 Cre-Lox 方法敲低小鼠脂肪组织中的 MTP 表达可以减少肥胖，并使小鼠比同窝对照小鼠更瘦。 我们的工作假设是：MTP 是脂肪细胞内脂滴成熟和扩张的关键成分。该工作假设将进行如下测试： 具体目标 1：确定脂滴成熟中 MTP 的要求。这些研究将检验以下假设：MTP 促进正常脂滴扩张和生长，体内脂肪细胞中缺乏 MTP 会导致小鼠体脂减少、葡萄糖代谢改善，从而使小鼠变得更瘦。使用从脂肪特异性 MTP 敲低小鼠和 3T3-L1 细胞中分离的原代前脂肪细胞（其中 MTP 表达已被 siRNA 沉默），我们将评估 MTP 缺陷对分化、脂滴形成和成熟、从头脂肪生成的影响，和甘油代谢。我们还将探讨增强的 MTP 表达对原代前脂肪细胞和 3T3 细胞中脂滴形成和成熟的影响。使用脂肪特异性 MTP 敲除小鼠，我们将确定敲除对体重、体脂肪成分、能量消耗、食物消耗、瘦素和胰岛素的影响。还将使用葡萄糖耐量测试以及高胰岛素正常血糖钳来评估葡萄糖代谢。 具体目标 2. 确定 MTP 促进脂滴生长和成熟的分子机制。这些研究将检验 MTP 通过分子的结构和功能特性促进脂滴扩张的假设。 MTP 假定的脂质结合区域中的选定位点将发生突变，以确定 MTP 是否在脂滴成熟中充当“融合蛋白”。 MTP 抑制剂将用于确定脂质转移活性是否是脂滴扩张所必需的。 具体目标 3. 研究 MTP 与 3T3-L1 细胞中脂滴结合的机制。这些研究将检验这样的假设：MTP 与 ER 膜特定区域中的甘油三酯的结合启动了 MTP 从 ER 内腔到脂滴表面的移动。共聚焦和电子显微镜以及生化方法将用于检查 MTP 在分化 3T3-L1 细胞中的亚细胞位置及其与脂滴的关联，并与脂滴蛋白 PAT 家族的选定成员进行比较。 公共卫生相关性： 肥胖在美国是一个主要的健康问题，甚至在美国退伍军人中也是如此。一项针对 2000 年在 VA 医疗机构接受护理的退伍军人的研究显示，68.4% 的女性超重（BMI 25），其中 37.4% 属于肥胖（BMI 30）。 73% 的男性超重，其中 32.9% 属于肥胖。这种流行病造成了重大的健康问题，因为肥胖与许多并发症有关，包括 2 型糖尿病、胰岛素抵抗、血脂异常、高血压和心血管疾病。不幸的是，人们对肥胖的病理生理学基础以及脂肪过度积累的机制知之甚少。事实上，人们对脂肪细胞内脂滴形成和成熟的基本步骤以及调节脂肪沉积的机制知之甚少。拟议的研究将为脂肪如何储存提供新的见解，并且对于理解肥胖及其许多并发症（包括心血管疾病）所涉及的因素可能很重要。