The Role of FoxO1 in the Regulation of the Adipocyte Browning Response

描述(申请人提供)：由于肥胖与多种疾病密切相关，尤其是导致2型糖尿病的胰岛素抵抗，因此肥胖症的上升趋势代表着一种公共健康危机。尽管肥胖和胰岛素抵抗之间的机制仍然存在争议，但一个有吸引力的假说认为，肥胖的过量脂肪抑制了脂肪细胞的处理能力，然后通过各种已提出的机制，引发胰岛素抵抗。利用脂肪细胞燃烧自己的能力的想法 近年来，由于人们对成年人棕色脂肪组织的研究重新产生了兴趣，以及发现白色脂肪组织可以发生褐变，多余的脂肪组织变得越来越受欢迎。这项拟议的项目旨在更好地了解关键的胰岛素反应转录因子FoxO1在脂肪细胞褐变调节中的作用。我已经在棕色脂肪细胞系和培养的白色原代脂肪细胞中发现，FoxO1的过度表达选择性地抑制了“棕色”基因的表达--那些参与致热脂质氧化所必需的基因--而不影响“泛脂肪细胞”基因。为了确定这些观察结果在体内是否成立及其潜在的医学应用，我培育了脂肪细胞中特异性缺失FoxO1的小鼠，称为“A-FoxO1”模型。因此，我在这项提案中描述了围绕A-FoxO1模式的特征的两个具体目标。首先，我建议对A-FoxO1小鼠进行彻底的代谢表型分析，包括生物计量学(例如体重、身体组成、脂肪组织库重)以及在全身水平(例如葡萄糖耐量测试、胰岛素耐量测试)和细胞水平(例如胰岛素信号通路组件的激活)的胰岛素敏感性分析。接下来，我将测试A-FoxO1小鼠的脂肪细胞褐变反应，方法是将动物置于冷(4摄氏度)、室温(22摄氏度)或热中性(30摄氏度)不同的时间段，然后对白色和棕色脂肪组织进行基因表达和组织学分析。在缺乏FoxO1的情况下，一种潜在的不同的褐变反应表明，它可能适用于饮食诱导的肥胖。因此，我将在高脂肪喂养A-FoxO1和WT小鼠后再次进行温度暴露实验，并确定A-FoxO1小鼠对胰岛素的敏感性是否存在显著差异。特别令人感兴趣的是胰岛素敏感性的潜在差异，即使在室温下也能表现出来，这表明它在人类肥胖症方面的实际应用。最后，我将在体外分离和培养A-FoxO1和WT原代前脂肪细胞，以便对FoxO1可能调节脂肪细胞褐变的机制进行详细的生化和细胞生物学研究。这些研究代表了一系列具有挑战性但合理的目标，这些目标不仅将对我作为一名科学家的发展做出重大贡献，而且可能对继续与肥胖作斗争有价值。

DESCRIPTION (provided by applicant): The rising tide of obesity represents a public health crisis due to its strong association with panoply of disease conditions, notably including insulin resistance leading to type 2 diabetes. Although the mechanism linking obesity and insulin resistance remain controversial, an attractive hypothesis posits that the excess lipid of obesity strains the handling capacity of adipocytes, which then, through various proposed mechanisms, foments insulin resistance. The idea of harnessing the ability of the adipocyte to burn off its own surplus lipid has gained popularity in recent years due to a renewed interest in the study of brown adipose tissue in adults as well as the finding that white adipose tissue can undergo "browning" changes. The proposed project seeks to establish a better understanding of the role of key insulin-responsive transcription factor FoxO1 in the regulation of adipocyte browning. I have already found in a brown-adipocyte cell line as well as in cultured white primary adipocytes that overexpression of FoxO1 selectively represses the expression of "brown" genes - those necessary for engaging in thermo genic lipid oxidation - while leaving unaffected "pan-adipocyte" genes. In order to determine whether these observations hold up in vivo and their potential medical applications, I have generated mice lacking FoxO1 specifically in adipocytes, termed the "A-FoxO1" model. I therefore describe in this proposal two Specific Aims centering on the characterization of the A-FoxO1 model. First, I propose a thorough metabolic phenotyping of the A-FoxO1 mouse, including biometrics (e.g., body weight, body composition, adipose-tissue depot weights) and assays of insulin sensitivity at both the levels of whole-body (e.g., glucose tolerance testing, insulin tolerance testing) and the cell (e.g., activation of insuin signaling pathway components). Next, I will test the adipocyte browning response in A-FoxO1 mice by subjecting animals to cold (4oC), room temperature (22oC), or thermo neutrality (30oC) for various time periods and then performing gene-expression and histologic analyses of white and brown adipose tissue depots. A potentially differential browning response in the absence of FoxO1 suggests a potential application to diet-induced obesity. I will therefore perform the temperature exposure experiments again following high- fat feeding of A-FoxO1 and WT mice and determine whether A-FoxO1 mice differ significantly in their sensitivity to insulin. Particulary of interest is a potential difference in insulin sensitivity that manifests even at room temperatur, suggesting a practical application to human obesity. Finally, I will isolate and culture both A-FoxO1 and WT primary preadipocytes ex vivo in order to perform detailed biochemical and cell- biologic studies of the mechanism underlying the putative regulation of adipocyte browning by FoxO1. These studies represent a set of challenging but reasonable goals that will not only contribute significantly to my development as a scientist but may also prove valuable in the continuing struggle against obesity