Caveolae and adipocyte lipid metabolism

描述（由申请人提供）：小泡和脂肪细胞脂质代谢小泡是脂肪细胞质膜的突出特征，约占细胞表面积的50%，因此提出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具有什么功能的问题。腔泡结构是50-100纳米的囊状内陷，从质膜向细胞质溶胶中突出。它们由小的完整膜蛋白，cavin 1和2以及cavin家族的外周膜蛋白（cavin-1- 3）组成。它们浓缩了高水平的胆固醇和鞘脂，这是维持其结构完整性所必需的。在体外和体内，通过基因操作/失活导致脂肪细胞小囊成分的丢失，导致脂肪细胞代谢异常。本研究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泡，以及小泡如何影响脂肪细胞的代谢和生化功能，从而对生物体产生表型影响。具体目标1将解决脂质代谢失调的机制(s)体外和体内脂肪细胞缺乏的小泡。初步数据支持cavin-1和/或cavin-2在激素刺激的脂肪分解中作为调节因子的假设，我们将在缺乏特定小窝成分的动物和细胞培养模型中进行实验验证这一假设。胆固醇耗竭使小泡塌陷，同时伴有皮质细胞骨架的重塑和蛋白体对cavin-2的降解。在Aim 2中，我们将通过这种和其他方案来操纵小泡，以确定小泡如何感知胆固醇，以及小泡如何影响质膜组织和功能。我们将检验泛素化是小泡形成所必需的假设。在Aim 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，以评估该组织对全球cavin-1敲除中所见的代谢异常的贡献，我们将对缺乏cavin-3的小鼠进行代谢表征，这些小鼠的脂肪量减少。我们还将对cavin-2敲除小鼠进行表型分析。小囊泡缺陷小鼠的多器官表型似乎与小囊泡成分失活突变的人类相同，因此这些研究与人类生物学的生理学相关性是相当大的。脂肪细胞的研究也将为其他相关系统和组织（例如心血管生物学）中的小泡感兴趣的研究团体提供信息。

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.