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

描述（由申请方提供）：肥胖是由白色脂肪组织（WAT）过度扩张引起的。我们以前确定了抑制素（Phb）作为一种蛋白质存在于内皮细胞（EC）表面上选择性地在WAT和使用的肽CKGGRAKDC，它结合到Phb和Phb介导的内吞作用，直接细胞毒性剂小鼠WAT作为实验方法肥胖逆转。为了更好地建立Phb作为一个前瞻性的肥胖治疗目标，我们已经开始表征其目前未定义的功能在WAT。我们鉴定了膜联蛋白A2，也称为膜联蛋白II（Anx 2），作为由CKGGRAKDC肽模拟的Phb结合蛋白。虽然Anx 2先前已被证明具有促血管生成功能，表明Phb /Anx 2相互作用在WAT内皮中的作用，但尚未探索Anx 2在脂肪生物学中的作用。我们的初步研究表明，Phb /Anx 2复合物不仅在WAT内皮细胞，而且在脂肪细胞膜脂筏。此外，我们发现Anx 2功能在脂肪形成过程中是必需的。基于这些观察结果，我们假设在EC和脂肪细胞中Phb和Anx 2形成一种受体复合物，在WAT发育和体内平衡中发挥重要作用。为了验证我们的假设，我们将在以下具体目标中，基于小鼠和细胞培养模型以及我们鉴定Phb /Anx 2相互作用结构域的初步数据，剖析Anx 2和Phb在WAT中的脂肪形成和血管生成功能。(1)我们将研究Anx 2在WAT开发中的作用。通过比较处于不同发育阶段和饮食诱导肥胖的Anx 2缺陷小鼠和野生型小鼠的单个WAT库，我们将通过评估WAT扩增和脂肪细胞特性以及通过分析WAT血管形成和WAT细胞群的代表性来确定Anx 2对脂肪形成和WAT血管生成的重要性。(2)我们将探讨Phb /Anx 2相互作用在脂肪形成和WAT血管生成中的作用。在子目标2A中，我们将确定Anx 2功能扰动后脂肪细胞中脂滴蓄积受损的原因。通过使用完全缺乏Anx 2或设计为Phb /Anx 2结合缺陷的分化脂肪细胞，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摄取以及脂解。在子目标2B中，我们将通过评估完全缺乏Anx 2或设计为缺乏Phb /Anx 2结合的EC的增殖、迁移和血管形成能力来测试Phb/Anx 2相互作用在WAT血管中的作用。因此，我们将建立Phb和Anx 2在WAT中的细胞表面相互作用的作用。这将有助于我们理解WAT组织扩张的机制，并提供有价值的信息，这种蛋白质复合物的生物学可能作为抗肥胖治疗的目标。

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.