Role of Follistatin during Androgen Regulation of Body Composition

描述（由申请人提供）：在使用健康年轻男性、性腺功能减退男性和血清T水平低的老年男性进行的各种临床试验中，补充睾酮（T）可增加肌肉质量并减少脂肪质量。然而，T调节身体组成的分子机制还不清楚。我们发现T通过雄激素受体（AR）介导的途径增加间充质多能细胞中的肌肉质量并减少脂肪质量，并激活前脂肪细胞中的AR/β-连环蛋白/TCF-4途径以抑制与卵泡抑素（Fst）上调相关的脂肪生成。我们假设T在体外和体内均激活AR/β-catenin/TCF-4通路以激活Fst，Fst通过抑制Mst/TGF-β信号传导调节身体组成。我们将采用两种经验证的成体间充质干细胞体外模型来测试T和DHT是否通过激活AR/β-catenin/TCF-4通路来激活Fst以调节细胞分化，并且通过SiRNA抑制β-catenin消除这些作用。我们将通过免疫沉淀法确定AR、β-连环蛋白和TCF-4之间的相互作用，并通过免疫荧光和细胞分离法确定它们的核转位。将通过使用TCF-4荧光素酶报告基因测定来测量响应于雄激素的TCF-4转录激活。我们将确定这些雄激素是否通过上调Fst在体外抑制TGF-β/Mst信号传导。我们将测试T和DHT是否在体外抑制Mst生物活性、Smad 2/3磷酸化和激活Smad 7，以及通过Fst SiRNA或使用抗Fst抗体抑制Fst水平是否阻断这些雄激素对肌源性分化的作用。我们将研究去势诱导的C57/BL 6 J小鼠体成分的影响是否伴随着Fst水平的平行降低，以及在这些去势小鼠中补充T或重组卵泡抑素是否显著阻断去势诱导的对去脂质量和整体体成分的影响。这些研究将为雄激素作用机制提供新的见解，并与单独使用Fst或联合低剂量T治疗HIV和衰老相关肌肉损失直接相关，因为高超生理剂量的T与其多效性效应相关，包括通过升高前列腺特异性抗原增加前列腺癌风险。

DESCRIPTION (provided by applicant): Testosterone (T) supplementation increases muscle mass and decreases fat mass in a variety of clinical trials using healthy young men, hypogonadal men, and in older men with low serum T levels. However, the molecular mechanisms by which T regulates body composition are not well known. We identified that T increases muscle mass and decreases fat mass in mesenchymal multipotent cells via androgen receptor (AR)-mediated pathway and activates AR/beta-catenin/TCF-4 pathway in preadipocyte cells to inhibit adipogenesis that was associated with up-regulation of follistatin (Fst). We hypothesize that T activates AR/beta-catenin/TCF-4 pathway both in vitro and in vivo to activate Fst, which regulates body composition by inhibiting Mst/TGF-beta signaling. We will employ two validated in vitro models of adult, mesenchymal stem cells to test whether T and DHT activate Fst via activation of AR/beta-catenin/TCF-4 pathway to regulate cell differention, and inhibition of beta-catenin by SiRNA abolishes these effects. We will determine the interaction between AR, beta-catenin, and TCF-4 by immunoprecipitation and their nuclear translocation by immunofluorescence and cell fractionation. TCF-4 transcriptional activation in response to androgen will be measured by using TCF-4 luciferase reporter assay. We will determine whether these androgens inhibit TGF-beta/Mst signaling in vitro through up-regulation of Fst. We will test whether T and DHT inhibit Mst bioactivity, Smad2/3 phosphorylation and activate Smad7 in vitro and inhibition of Fst levels either by Fst SiRNA or use of anti-Fst antibody blocks the effects of these androgens on myogenic differentiation. We will investigate whether castration-induced effects on body composition in C57/BL6J mice are accompanied by parallel decrease in Fst levels and supplementation of T or recombinant follistatin in these castrated mice significantly block castration-induced effects on fat-free mass and overall body composition. These studies will provide novel insights into the mechanisms of androgen action and have direct relevance to the application of either Fst alone or in combination of low doses of T for the treatment of HIV and aging-associated muscle loss as high supraphysiological doses of T are associated with its pleotropic effects including increased risk of prostate cancer by elevating prostate-specific antigen.