The Function of Prohibitin / Annexin 2 Interaction in White Adipose Tissue

描述（由申请人提供）：肥胖是由白色脂肪组织（WAT）过度扩张引起的。我们之前确定了禁止素（Phb）作为WAT中内皮细胞（EC）表面选择性存在的蛋白质，并使用了一种肽CKGGRAKDC，它与Phb结合并经历Phb介导的内吞作用，将细胞毒性药物引导到小鼠WAT中，作为肥胖逆转的实验方法。为了更好地确定Phb作为一种前瞻性的肥胖治疗靶点，我们已经开始描述其在WAT中目前尚未确定的功能。我们确定了膜联蛋白A2，也称为膜联蛋白II (Anx2)，是一种由CKGGRAKDC肽模拟的phb结合蛋白。虽然Anx2先前已被证明具有促血管生成功能，表明Phb / Anx2在WAT内皮中的相互作用，但Anx2在脂肪生物学中的作用尚未被探索。我们的初步研究发现Phb / Anx2复合物不仅存在于WAT内皮细胞中，也存在于脂肪细胞中。此外，我们发现在脂肪形成过程中需要Anx2的功能。基于这些观察，我们假设在EC和脂肪细胞中Phb和Anx2形成一个受体复合物，在WAT的发育和体内平衡中起重要作用。为了验证我们的假设，我们将基于小鼠和细胞培养模型，以及我们确定Phb / Anx2相互作用域的初步数据，在以下具体目标中解剖WAT中Anx2和Phb的脂肪生成和血管生成功能。(1)我们将探讨Anx2在WAT发展中的作用。通过比较不同发育阶段和饮食诱导肥胖的Anx2缺陷小鼠和野生型小鼠的个体WAT库，我们将通过评估WAT扩张和脂肪细胞特性，以及分析WAT血管化和WAT细胞群的代表性，确定Anx2对脂肪形成和WAT血管生成的重要性。(2)探讨Phb / Anx2相互作用在脂肪形成和WAT血管生成中的作用。在Sub-Aim 2A中，我们将确定为什么当Anx2功能受到干扰时，脂肪细胞中的脂滴积累会受损。通过使用完全缺乏Anx2或设计为缺乏Phb / Anx2结合的分化脂肪细胞，我们将评估标记脂肪形成的蛋白质的表达和定位的变化，并将测量葡萄糖和脂肪酸的摄取，以及脂肪分解。在Sub-Aim 2B中，我们将通过评估完全缺乏Anx2或被设计为缺乏Phb / Anx2结合的EC的增殖、迁移和血管形成能力，来测试Phb / Anx2相互作用在WAT血管中的作用。因此，我们将确定Phb和Anx2之间的细胞表面相互作用在WAT中的作用。这将有助于我们理解控制WAT组织扩张的机制，并为这种蛋白质复合物的生物学提供有价值的信息，这种蛋白质复合物可能作为抗肥胖治疗的靶点。

DESCRIPTION (provided by applicant): Obesity results from excessive expansion of white adipose tissue (WAT). We previously identified prohibitin (Phb) as a protein present on the endothelial cell (EC) surface selectively in WAT and have used a peptide CKGGRAKDC, which binds to Phb and undergoes Phb-mediated endocytosis, to direct a cytotoxic agent to mouse WAT as an experimental approach to obesity reversal. In order to better establish Phb as a prospective obesity therapy target, we have begun to characterize its currently undefined function in WAT. We identified annexin A2, also known as annexin II (Anx2), as a Phb-binding protein mimicked by the CKGGRAKDC peptide. While Anx2 has been previously shown to have a pro-angiogenic function, suggesting a role for Phb / Anx2 interaction in WAT endothelium, the role of Anx2 in adipose biology has not been explored. Our preliminary studies revealed Phb / Anx2 complex in cell membrane lipid rafts not only in WAT endothelium but also in adipocytes. Moreover, we discovered that Anx2 function is required during adipogenesis. Based on these observations, we hypothesize that in EC and in adipocytes Phb and Anx2 form a receptor complex that plays an important role in WAT development and homeostasis. To test our hypothesis, we will dissect the adipogenic and angiogenic functions of Anx2 and Phb in WAT based on mouse and cell culture models, as well as on our preliminary data identifying Phb / Anx2 interaction domains, in the following Specific Aims. (1) We will investigate the role of Anx2 in WAT development. By comparing individual WAT depots from Anx2-deficient and wild-type mice at different stages of development and upon diet-induced obesity induction, we will determine the importance of Anx2 for adipogenesis and WAT angiogenesis by assessing WAT expansion and adipocyte properties, as well as by analyzing WAT vascularization and the representation of WAT cell populations. (2) We will explore the function of Phb / Anx2 interaction in adipogenesis and WAT angiogenesis. In Sub-Aim 2A, we will determine why lipid droplet accumulation is impaired in adipocytes upon perturbation of Anx2 function. By using differentiating adipocytes either completely lacking Anx2 or designed to be deficient in Phb / Anx2 binding, we will assess changes in expression and localization of proteins marking adipogenesis and will measure glucose and fatty acid uptake, as well as lipolysis. In Sub-Aim 2B, we will test the role of Phb / Anx2 interaction in WAT vasculature by assessing proliferation, migration, and vasculature formation capacity of EC either completely lacking Anx2 or designed to be deficient in Phb / Anx2 binding. As a result, we will establish the role of the cell surface interaction between Phb and Anx2 in WAT. This will contribute to our understanding of the mechanisms governing WAT tissue expansion and provide valuable information on the biology of this protein complex potentially useful as a target of anti-obesity therapeutics.