Role of Follistatin during Androgen Regulation of Body Composition

描述(申请人提供)：在各种临床试验中，补充睾酮(T)可以增加肌肉质量和减少脂肪质量，这些临床试验使用的是健康的年轻男性、性腺功能减退的男性，以及血清T水平低的老年男性。然而，T调节体成分的分子机制还不是很清楚。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的肌肉质量和降低脂肪质量，并激活前脂肪细胞的AR/β-连环蛋白/TCF-4途径，从而抑制与卵泡抑素(Fst)上调相关的脂肪生成。我们假设T在体外和体内都激活AR/β-catenin/TCF-4途径来激活Fst，Fst通过抑制MST/TGF-β信号来调节体成分。我们将使用两个成熟的成人间充质干细胞的体外模型来测试T和DHT是否通过激活AR/β-catenin/TCF-4途径来激活FST来调节细胞分化，而siRNA抑制β-catenin可以消除这些作用。我们将通过免疫沉淀来确定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级来确定它们的核转位。我们将使用TCF-4荧光素酶报告基因检测雄激素对TCF-4转录激活的影响。我们将确定这些雄激素是否在体外通过上调Fst来抑制转化生长因子-β/MST信号。我们将测试T和DHT是否在体外抑制MST的生物活性、Smad2/3的磷酸化和Smad7的激活，以及通过Fst siRNA或使用抗Fst抗体抑制Fst的水平是否阻断了这些雄激素对成肌分化的影响。我们将研究去势对C57/BL6J小鼠身体成分的影响是否伴随着Fst水平的平行下降，以及在这些被去势的小鼠中补充T或重组卵泡抑素是否显著阻止了去势诱导的去脂体重和整体身体成分的影响。这些研究将为雄激素的作用机制提供新的见解，并与单独应用FST或与低剂量T联合应用于治疗艾滋病毒和衰老相关的肌肉损失直接相关，因为高生理剂量的T与其促性腺激素作用相关，包括通过提高前列腺癌特定抗原而增加前列腺癌的风险。

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.