Follistatin promotes browning and influences energy metabolism

描述(申请人提供)：肥胖是一个主要的健康问题，正在以流行的速度在世界各地蔓延，没有任何减少的迹象。肥胖的发展往往与胰岛素抵抗和糖尿病有关，其结果是能量摄入超过消耗。棕色脂肪组织(BAT)具有燃烧多余卡路里、促进甘油三酯清除和葡萄糖处理的独特能力。此前，我们发现卵泡抑素(Fst)是睾酮的直接靶点，它调节骨骼肌质量并抑制转化生长因子-β(转化生长因子-β)信号转导。根据我们的初步发现，我们假设Fst通过调节整个Mst/转化生长因子-β/骨形态发生蛋白/Myf5/PRDM16信号通路，促进整体生热程序，改善肥胖和代谢紊乱的症状。我们将通过以下具体目标来验证我们的假说：目标1：我们将在体外和体内证明Fst在棕色脂肪分化过程中的重要作用以及生热程序的调节。目的2：我们将确定Fst调节整个生热程序的分子机制，目标3：我们将在脂联素或UCP1调节元件的控制下产生表达Fst的小鼠，并比较它们的代谢参数和对高脂饮食的反应与对照仔鼠。我们将利用基础和β肾上腺素受体激动剂(CL316,248)刺激条件下的体外和体内模型，确定内源性和外源性Fst对关键生热标志物的蛋白质和基因表达谱以及整体细胞生物能量学的影响。体外模型及脂联素-Fst和UCP1-Fst转基因小鼠中MYF5/PRDM16、Mst/pSmad2/3/BMP/COX-2、胰岛素受体和AMPK/PGC-1α信号通路的参与将通过Affymetrix基因表达和免疫印迹定量分析来分析。在小鼠前脂肪细胞和MEF培养中，Fst、Myf5和Smad3的表达水平将被siRNAs抑制，并将确定它们的生热能力。我们将利用脂联素和UCP1特异性启动子建立Fst转基因小鼠，并在基础和β肾上腺素能受体激动剂(CL 316,248)刺激条件下，测试高脂饮食对它们的体成分(Micro CT)、能量消耗(间接量热分析)、胰岛素敏感性和糖耐量的影响。血清FST、脂联素水平和血脂谱将采用ELISA法进行分析。评估Fst在调节整个产热过程中的关键作用，将为设计治疗肥胖症和相关代谢性疾病的新型治疗药物提供理论基础。

DESCRIPTION (provided by applicant): Obesity is a major health problem spreading at an epidemic pace throughout the world without any sign of abatement. Development of obesity, which is often associated with insulin resistance and diabetes, results from an excess of energy intake over expenditure. Brown adipose tissues (BAT) have the unique ability to burn excess calories, and facilitate triglyceride clearance and glucose disposal. Previously, we identified Follistatin (Fst) as a direct target of testosterone that regulates skeletal muscle mass and inhibits transforming growth factor-β (TGF-β) signaling. Based on our preliminary findings, we hypothesize that Fst promotes overall thermogenic program and improves symptoms of obesity and metabolic disorder by regulating overall Mst/TGF-β /BMP/Myf5/PRDM16- signaling pathways. We will test our hypothesis with the following Specific Aims- Aim 1: We will demonstrate the essential role of Fst during brown fat differentiation and the regulation of thermogenic program in vitro and in vivo. Aim 2: We will determine the molecular mechanisms by which Fst regulates the overall thermogenic program and Aim 3: We will generate mice expressing Fst under the control of adiponectin or UCP1 regulatory elements, and compare their metabolic parameters and response to high fat chow diet with control littermates. We will determine the effect of endogenous and exogenous Fst on protein and gene expression profiles of key thermogenic markers, and overall cellular bioenergetics using both in vitro and in vivo models under basal and β-adrenoceptor agonist (CL316,248) stimulated conditions. Involvement of Myf5/PRDM16, Mst/pSmad2/3/BMP/COX 2, insulin receptor and AMPK/PGC-1α signaling pathway in in vitro models as well as in adiponectin-Fst and UCP1-Fst transgenic mice will be analyzed by Affymetrix gene expression and quantitative western blot analysis. Expression levels of Fst, Myf5 and Smad3 in mouse preadipocytes and MEF cultures will be inhibited by siRNAs and their thermogenic capabilities will be determined. We will generate Fst-transgenic mice using adiponectin (Adipoq) and UCP1-specific promoters, and test the effect of high fat diet on their body composition (Micro CT), energy expenditure (indirect calorimetric analysis), insulin sensitivity and glucose tolerance under both basal and β-adrenoceptor agonist (CL 316,248) stimulated conditions. Serum levels of Fst, adiponectin and lipid profiles will be analyzed by ELISA. Evaluating the critical role of Fst during its regulation of overall thermogenic process will provide rationale for novel therapeutic drug design for the treatment of obesity and related metabolic diseases.