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.
Obesity is positively associated with cardiovascular disease, type 2 diabetes, and cancer, thus representing a socioeconomic and medical problem of escalating concern. Our study will contribute to the understanding of mechanisms that control adipose cell differentiation and blood vessel formation during the expansion of white adipose tissue responsible for obesity. The molecular interaction between prohibitin and annexin 2 uncovered by our work is a potential target for anti-obesity therapy, and this study is a next step toward prospective clinical trials.
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