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

描述（由申请人提供）：肥胖症的上升趋势代表了一场公共卫生危机，因为它与一系列疾病状况密切相关，特别是包括导致2型糖尿病的胰岛素抵抗。虽然肥胖和胰岛素抵抗的机制仍然存在争议，但一个有吸引力的假设是，肥胖的过量脂质使脂肪细胞的处理能力紧张，然后通过各种提出的机制，引起胰岛素抵抗。利用脂肪细胞燃烧自身的能力 近年来，由于对成人棕色脂肪组织的研究重新产生兴趣以及发现白色脂肪组织可经历“布朗宁”变化，多余脂质已变得普遍。该项目旨在建立一个更好地了解关键的胰岛素反应转录因子FoxO 1在调节脂肪细胞布朗宁的作用。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，FoxO 1的过表达选择性地抑制“棕色”基因的表达--这些基因是参与产热脂质氧化所必需的--而不影响“泛脂肪细胞”基因。为了确定这些观察结果是否在体内及其潜在的医学应用，我已经产生了脂肪细胞中缺乏FoxO 1的小鼠，称为“A-FoxO 1”模型。因此，我在本提案中描述了两个以A-FoxO 1模型表征为中心的具体目标。首先，我提出了一个彻底的代谢表型的A-FoxO 1小鼠，包括生物特征（例如，体重、身体组成、脂肪组织贮库重量）和在全身水平（例如，葡萄糖耐量试验，胰岛素耐量试验）和细胞（例如，胰岛素信号传导途径组分的活化）。接下来，我将测试A-FoxO 1小鼠的脂肪细胞布朗宁反应，方法是将动物置于寒冷（4°C）、室温（22°C）或热中性（30°C）下不同时间段，然后对白色和棕色脂肪组织库进行基因表达和组织学分析。一个潜在的差异布朗宁反应的情况下，FoxO 1表明一个潜在的应用饮食诱导的肥胖症。因此，我将在高脂肪喂养A-FoxO 1和WT小鼠后再次进行温度暴露实验，并确定A-FoxO 1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，即使在室温下也表现出胰岛素敏感性的潜在差异，这表明对人类肥胖症的实际应用。最后，我将离体分离和培养A-FoxO 1和WT原代前脂肪细胞，以便对FoxO 1对脂肪细胞布朗宁的假定调节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，不仅对我作为科学家的发展有重大贡献，而且可能在与肥胖的持续斗争中证明是有价值的。

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 (4°C), room temperature (22°C), or thermo neutrality (30°C) 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