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: 8244930
• 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/8244930
• 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; public health relevance; 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，微粒体甘油三酸酯转移蛋白（MTP）是一种在肝细胞和肠细胞内富含甘油三酸酯的脂蛋白所必需的蛋白质，在小鼠，大鼠和人类的脂肪细胞中表达。另外，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，表达增加了约5倍。 MTP在脂质液滴表面以点状模式存在，并且在相邻脂质液滴的接触点尤其突出。当MTP表达沉默并诱导细胞分化时，仅形成很小的（<1 5m）脂质液滴。另外，当将MTP转染到CHO细胞并刺激脂质液滴形成中时，与没有MTP的细胞中形成的液滴相比会形成较大的液滴。最后，使用CRE-LOX方法敲低小鼠脂肪组织中的MTP表达会导致肥胖降低，而小鼠比同窝窝对照组降低了小鼠。 我们的工作假设是：MTP是脂肪细胞中脂质滴的成熟和膨胀的关键组成部分。该工作假设将被测试如下：特定目的1：定义脂质液滴成熟中MTP的需求。这些研究将检验MTP促进正常脂质液滴扩张和生长的假设，并且在体内脂肪细胞中缺乏MTP会导致体内较瘦的小鼠体内脂肪降低并改善了葡萄糖代谢。使用从脂肪特异性的MTP-KNOCKDOWN小鼠和3T3-L1细胞中分离出来的原代脂肪细胞，其中MTP表达已被siRNA保持沉默，我们将评估MTP缺乏对分化，脂质液滴形成和成熟的影响，De Novo脂肪生成，以及甘露林氏菌属分类。我们还将探讨增强MTP表达对原代脂肪细胞和3T3细胞成熟脂质滴形成和成熟的影响。使用脂肪特异性的MTP敲低小鼠，我们将定义敲低对体重，体内脂肪成分，能量消耗，食物消耗，瘦素和胰岛素的影响。还将使用葡萄糖耐受性测试以及高胰岛素葡萄糖糖夹评估葡萄糖代谢。 具体目的2。确定MTP促进脂质液滴的生长和成熟的分子机制。这些研究将检验以下假设，即MTP通过分子的结构和功能特性促进脂质液滴扩展。 MTP的假定脂质结合区域中的选定位点将被突变，以确定MTP在脂质液滴成熟中是否功能作为“ fusogenic蛋白”。 MTP抑制剂将用于确定脂质液滴膨胀是否需要脂质转移活性。 特定目的3。检查MTP与3T3-L1细胞中脂质液滴相关的机制。这些研究将检验以下假设：ER膜中特定区域中MTP与甘油三酸酯的关联启动了MTP从ER流明到脂质液滴表面的运动。与脂质液滴蛋白的PAT家族相比，将使用共聚焦和电子显微镜以及生化方法在区分3T3-L1细胞中检查MTP的亚细胞位置及其与脂质液滴的关联。 公共卫生相关性： 肥胖症在美国，即使在美国退伍军人中也提出了一个主要的健康问题。一项对2000年VA医疗机构接受护理的退伍军人的研究表明，有68.4％的妇女超重（BMI 25）分类为肥胖（BMI 30），其中37.4％。 73％的男性超重，32.9％被归类为肥胖。这种流行病构成了重大的健康问题，因为肥胖与多种并发症有关，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，关于肥胖的病理生理基础以及脂肪过度积累的机制知之甚少。确实，关于脂肪液滴形成和脂肪细胞内成熟的基本步骤以及调节脂肪沉积的机制知之甚少。拟议的研究将提供有关如何储存脂肪的新见解，并且可以证明在理解肥胖症及其许多并发症（包括心血管疾病）的因素中很重要。