The Role of MTP in Lipid Droplet Formation in Adipocytes

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），一种在肝细胞和肠细胞内组装富含甘油三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。此外，MTP在3 T3-L1细胞中表达，当这些细胞被诱导分化为脂肪细胞时，表达增加约5倍。MTP以点状图案存在于脂滴表面上，并且在相邻脂滴的接触点处特别突出。当MTP表达被沉默并且细胞被诱导分化时，仅形成非常小（<1.5 μ m）的脂滴。此外，当MTP转染到CHO细胞中并刺激脂滴形成时，与在没有MTP的细胞中形成的那些相比，形成更大的液滴。最后，使用Cre-Lox方法敲低小鼠脂肪组织中的MTP表达导致肥胖减少和比同窝对照组更瘦的小鼠。 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。具体目标1：确定在脂滴成熟过程中对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导致更瘦的小鼠，体脂减少，葡萄糖代谢改善。使用从脂肪特异性MTP敲除小鼠和3 T3-L1细胞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缺乏对分化、脂滴形成和成熟、从头脂肪生成和甘油代谢的影响。我们还将探讨MTP表达增强对原代前脂肪细胞和3 T3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，我们将定义敲除对体重、体脂组成、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试验以及高胰岛素正常血糖钳夹试验评估葡萄糖代谢。 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。将突变MTP的推定脂质结合区中的选定位点以确定MTP在脂滴成熟中是否作为“融合蛋白”起作用。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。探讨MTP与3 T3- L1细胞脂滴结合的机制。这些研究将检验这样的假设，即在内质网膜的特定区域中MTP与甘油三酯的结合启动了MTP从内质网腔向脂滴表面的移动。共聚焦和电子显微镜以及生物化学方法将被用来检查亚细胞位置的MTP在分化3 T3-L1细胞和它的关联与脂滴的比较与选定的成员的PAT家族的脂滴蛋白。 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。2000年对在退伍军人管理局医疗设施接受治疗的退伍军人进行的一项研究显示，68.4%的妇女超重（BMI 25），37.4%被归类为肥胖（BMI 30）。73%的男性超重，其中32.9%被归类为肥胖。这种流行病造成了一个主要的健康问题，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基础以及脂肪过度积累的机制知之甚少。事实上，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。拟议中的研究将为脂肪如何储存提供新的见解，并可能对理解肥胖及其许多并发症（包括心血管疾病）的相关因素非常重要。

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.