Role of Caveolae in Signaling in Fat Cells

描述(申请人提供)：脂肪酸是哺乳动物热量最高的燃料来源。脂肪酸被吸收并储存在脂肪细胞中，当其他组织需要时，从这个储存库中动员起来。过量的循环脂肪酸是胰岛素抵抗和2型糖尿病的近端原因，因此调节细胞摄取和释放脂肪酸的因素可能与这些病理状况有直接联系。适当的燃料动态平衡要求脂肪酸快速和大量地穿过细胞表面和质膜。脂肪细胞质膜中特别丰富的结构称为小窝，这是一个小的膜内陷(“小空洞”)富含胆固醇和鞘磷脂，是由小窝-1蛋白的表达形成的。小凹是一种脂筏，其特点是抵抗脂肪酸等温和洗涤剂的洗涤。缺乏小窝蛋白-1的小鼠有胰岛素抵抗，并有异常的脂肪酸代谢。我们假设脂肪细胞中的小窝蛋白/小窝蛋白在调节脂肪酸跨质膜移动中发挥作用，并可能减轻其潜在的病理作用。已经建立了一系列不同程度表达小窝蛋白-1的模型细胞系，导致脂肪细胞中具有小窝等脂筏特征的膜胆固醇水平增加。初步数据显示，这些细胞类似脂肪细胞，因为它们增强了急性脂肪酸的跨膜摄取，以及增强了脂肪酸的储存。我们的两个具体目标是：1.确定新型小窝蛋白Cavin在小窝功能中的生理生化作用。我们发现这种蛋白质经历了化学计量泛素化，我们希望确定这种独特的修饰在小窝功能和动力学中的作用。2.研究腔泡膜脂类和蛋白质组成对脂肪酸通量和储存速率及程度的影响。为此，我们将使用多种分析方法，实时监测我们现有的细胞系、脂肪细胞和其他模型细胞系中的脂肪酸转运速率，这些细胞系是通过将与小窝、跨膜脂肪酸通量和脂肪酸储存相关的蛋白质导入而创建的。这项应用的长期目标是了解脂肪细胞的细胞分区，因为它适用于脂肪细胞作为能量储存的主要部位的角色，以及作为包括智人在内的有机体营养状态的传感器。

DESCRIPTION (provided by applicant): Fatty acids comprise the most calorie-rich source of fuel for mammals. Fatty acids are taken up and stored in adipocytes and mobilized from this depot when needed by other tissues. Excess circulating fatty acids are a proximal cause of insulin resistance and type 2 diabetes, and therefore factors that modulate their cellular uptake and release have potentially direct connections with these pathological conditions. Proper fuel homeostasis requires that fatty acids move across the cell surface, the plasma membrane, rapidly and in large amounts. The adipocyte plasma membrane is particularly abundant in structures called caveolae, which are small membrane invaginations ("little caves") enriched in cholesterol and sphingomyelin that are formed by the expression of caveolin-1 protein. Caveolae are a type of lipid raft characterized by resistance to dispruption by mild detergents such as fatty acids. Mice lacking caveolin-1 are insulin resistant and have abnormal fatty acid metabolism. We hypothesize that caveolin/caveolae in adipocytes play a role in the regulation of fatty acid movement across the plasma membrane and may mitigate their potentially pathological actions. A series of model cell lines have been created that express caveolin-1 to varying extents, resulting in the increased membrane cholesterol levels characteristic of lipid rafts such as caveolae in adipocyte. Preliminary data show that these cells are adipocyte-like in that they have enhanced acute fatty acid transmembrane uptake as well as enhanced fatty acid storage. Our two specific aims are 1. To determine the physiological and biochemical role of the novel caveolar protein, cavin, in caveolae function. We found that this protein undergoes stoichiometric ubiquitination, and we wish to determine the role of this unique modification in caveolae function and dynamics. 2. To study the effects of caveolar membrane lipid and protein composition on the rate and extent of fatty acid flux and storage. To this end, we will use a number of assays that allow real time monitoring of fatty acid transport rates in our extant cell lines, in adipocytes and in other model cell lines that we will create by transfection with proteins relevant to caveolae, transmembrane fatty acid flux and fatty acid storage. The long term goals of the application are understand the cellular compartmentalization of adipocytes as it applies to their role as the principle site of energy storage, and as sensors for the nutritional state of the organism, including homo sapiens.