Role of Caveolae in Signaling in Fat Cells

描述（由申请人提供）：脂肪酸是哺乳动物最富含热量的燃料来源。脂肪酸被吸收并储存在脂肪细胞中，当其他组织需要时，脂肪酸从脂肪细胞中被动员出来。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，因此调节其细胞摄取和释放的因素与这些病理状况有潜在的直接联系。适当的燃料稳态需要脂肪酸快速大量地穿过细胞表面，即质膜。脂肪细胞质膜在称为小窝的结构中特别丰富，小窝是富含胆固醇和鞘磷脂的小膜内陷（“小洞穴”），由小窝蛋白-1蛋白的表达形成。小窝是一种脂筏，其特征在于对温和去污剂如脂肪酸的破坏具有抗性。缺乏小窝蛋白-1的小鼠具有胰岛素抵抗性，并且具有异常的脂肪酸代谢。我们推测，小窝蛋白/小窝在脂肪细胞中发挥作用，在调节脂肪酸运动通过质膜，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，导致脂筏特征性的膜胆固醇水平增加，如脂肪细胞中的小窝。初步数据显示，这些细胞是脂肪细胞样的，因为它们具有增强的急性脂肪酸跨膜摄取以及增强的脂肪酸储存。我们的两个具体目标是1。研究新的细胞膜小窝蛋白cavin在细胞膜小窝功能中的生理生化作用。我们发现，这种蛋白质进行化学计量的泛素化，我们希望确定这种独特的修饰在小窝功能和动力学的作用。2.研究膜脂和蛋白质组成对脂肪酸流出和储存速率和程度的影响。为此，我们将使用许多测定法，这些测定法允许真实的实时监测我们现存的细胞系、脂肪细胞和其他模型细胞系中的脂肪酸转运速率，我们将通过用与小窝、跨膜脂肪酸通量和脂肪酸储存相关的蛋白质转染来创建这些模型细胞系。该应用程序的长期目标是了解脂肪细胞的细胞区室化，因为它适用于它们作为能量储存的主要部位的作用，以及作为生物体（包括智人）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.