Caveolae and adipocyte lipid metabolism

描述（由申请人提供）：小窝和脂肪细胞脂质代谢小窝是脂肪细胞质膜的突出特征，其包含钙。50%的细胞表面积，因此回避了为什么它们如此丰富以及它们在这种代谢重要的细胞类型中的功能的问题。小窝结构是50-100 nm的囊状内陷，从质膜突出到胞质溶胶中。它们由小的整合膜蛋白、小窝蛋白1和2以及小窝蛋白家族的外周膜蛋白（小窝蛋白-1-3）组成。它们浓缩了维持其结构完整性所需的高水平胆固醇和鞘脂。在体外和体内，通过基因操作/失活导致脂肪细胞代谢异常的脂肪细胞小窝组分的损失。这一提议的长期目标是了解相关蛋白质和脂质如何相互作用形成小窝，以及小窝如何影响脂肪细胞的代谢和生化功能，这对生物体具有表型影响。具体目标1将阐述在小窝缺陷的脂肪细胞中脂质代谢在体外和体内失调的机制。初步数据支持cavin-1和/或cavin-2在经尿道刺激的脂解中充当调节因子的假设，并且我们将在动物和缺乏特异性小窝组分的细胞培养模型中通过实验解决该假设。胆固醇耗竭导致小窝塌陷，同时皮质细胞骨架重塑和蛋白体降解cavin-2。在目标2中，我们将通过这个和其他协议来操纵小窝，以确定小窝如何感知胆固醇，以及小窝如何影响质膜组织和功能。我们将测试的假设，泛素化是必要的小窝的形成。在目标3中，我们建议对脂肪细胞中缺乏cavin-1的小鼠进行表型分析，以评估该组织对全局cavin-1敲除中观察到的代谢异常的贡献，并且我们将代谢表征缺乏cavin-3的小鼠，其脂肪量减少。我们还将对cavin-2基因敲除小鼠进行表型分析。小窝缺陷小鼠的多器官表型似乎与人类在小窝组分中具有失活突变的表型相同，因此这些研究与人类生物学的生理相关性相当大。脂肪细胞的研究也将告知其他相关系统和组织中对小窝感兴趣的研究团体，例如心血管生物学。

DESCRIPTION (provided by applicant): Caveolae and adipocyte lipid metabolism Caveolae are prominent features of the adipocyte plasma membrane comprising ca. 50% of the cell surface area, thus begging the question of why they are so abundant and what their functions are in this metabolically important cell type. Caveolar structures are 50-100 nm sac-like invaginations projecting from the plasma membrane into the cytosol. They are comprised of small integral membrane proteins, caveolins 1 & 2, and peripheral membrane proteins of the cavin family (cavin-1- 3). They concentrate high levels of cholesterol and sphingolipids which are needed to maintain their structural integrity. Loss of adipocyte caveolae components, in vitro and in vivo, by gene manipulation/inactivation results in abnormalities of adipocyte metabolism. It is the long-term goal of this proposal to understand how the relevant proteins and lipids interact to form caveolae, and how caveolae impact the metabolic and biochemical functions of the fat cell, which has phenotypic consequences for the organism. Specific Aim 1 will address the mechanism(s) by which lipid metabolism is dysregulated in vitro and in vivo in adipocytes deficient in caveolae. Preliminary data support the hypothesis that cavin-1 and/or cavin-2 serve as regulatory factor(s) in hormonally- stimulated lipolysis, and we will experimentally address this hypothesis in animals and in cell culture models lacking specific caveolae components. Cholesterol depletion collapses caveolae concomitantly with remodeling of the cortical cytoskeleton and degradation of cavin-2 by the proteosome. In Aim 2, we will manipulate caveolae by this and other protocols to determine how caveolae sense cholesterol, and how caveolae influence plasma membrane organization and function. We will test the hypothesis that ubiquitination is requisite for caveolae formation. In Aim 3, we propose to phenotypically analyze mice lacking cavin-1 in adipocytes to assess the contribution of this tissue to the metabolic abnormalities seen in the global cavin-1 knockout, and we will metabolically characterize mice lacking cavin-3, which have a reduced adipose mass. We will also phenotype and analyze cavin-2 knockout mice. The multi-organ phenotype of caveolae-deficient mice appears identical to that of humans harboring inactivating mutations in caveolae components, and therefore the physiological relevance of these studies to human biology is considerable. The adipocyte studies will also inform the research community interested in caveolae in other relevant systems and tissues, for example, in cardiovascular biology.