瘦素抵抗时抑制STAT3功能的Foxo1关键肽段的鉴定

Leptin controls food intake and energy expenditure by regulating hypothalamic neuron activities. Leptin exerts its actions through complex signaling pathways including Jak2-STAT3 mediated activation of pro-opiomelanocortin (POMC) gene. Although insufficient leptin levels leads to obesity, plasma and cerebrospinal fluid leptin levels are often higher in obese subjects, as expected from their higher fat volume compared with the lean. leptin fails to affect downstream physiological consequences in these animals because of impairment in the leptin signaling pathways, referred as leptin resistance. To understand the molecular mechanisms of leptin resistance, we previously studied the regulation of POMC gene expression by leptin. We showed that phospho-STAT3 activated POMC promoter in response to leptin signaling through a mechanism that required an SP1-binding site in the POMC promoter. Furthermore, Foxo1 bound to STAT3 and prevented STAT3 from interacting with the SP1-POMC promoter complex, and consequently, inhibited STAT3-mediated leptin action. Our study suggested that leptin action could be inhibited at a step downstream of STAT3 phosphorylation and nuclear translocation, and provided a hypothesis of leptin resistance in which an increased Foxo1 antagonized STAT3-mediated leptin signaling..On the basis of this novel hypothesis, we propose in this project to further the understanding how Foxo1 interacts with STAT3 thereby inhibiting leptin signaling in vitro and in vivo. First, we will construct a serial of mutant genes of Foxo1, co-transfect these constructs with STAT3 gene and find the key peptide of Foxo1 interacting with STAT3 by the means of immuno-precipitation; second, in cultured cell lines with established leptin signaling pathway the effect of the key peptide of Foxo1 on the STAT3 regulation of target gene POMC will be assessed with a luciferase reporter gene system; finally transgenic mice with brain-specific expression of the key peptide of Foxo1 will be generated, followed by the measurement of food intake, body weight and the levels of neuropeptides in hypothalamus of the transgenic mice exposed to designed diets to verify the role of this key peptide on the central action of leptin. .Foxo1-mediated leptin resistance may be a direct result of its expression up-regulation by pathophysiological factors but may also be due to increased nuclear accumulation of Foxo1, which may result from impaired insulin signaling. We speculated that Foxo1 may serve as a link to the so-called "diabesity." The accomplishment of this project will not only improve our new hypothesis by detailing information of Foxo1-STAT3 interaction, shedding light on the role of Foxo1 in leptin resistance, but also make it possible for the pharmaceutical scientists to synthesize chemical compounds, according to the amino acid sequences of the key peptide,to antagonist the interaction between Foxo1 and STAT3 for the purpose of treatment and prevention of diabesity.

瘦素抵抗与动物能量代谢障碍密切相关。申请者发表了Foxo1阻断瘦素信号传导，从而导致瘦素抵抗的新发现，但Foxo1和瘦素信号链中的核心分子STAT3 结合的细节尚不清楚，因此，本项目拟找出具有结合功能的Foxo1的关键肽段。首先制备一系列Foxo1突变体，用免疫共沉淀法检测它们与STAT3的结合，找出Foxo1分子上的关键肽段；然后通过检测靶基因POMC的启动子活性来了解此关键肽段对STAT3转录调控功能的影响；最后制备脑组织特异性表达此关键肽段的转基因小鼠，测定其体重、进食量和下丘脑神经肽，分析Foxo1关键肽段对下丘脑STAT3的功能，乃至对瘦素中枢作用的影响。此研究在理论上可望进一步揭示Foxo1与STAT3相互作用，从而阻碍瘦素信号传导的规律；在应用上，可望针对Foxo1关键肽段的氨基酸序列来干预Foxo1与STAT3的相互作用，阻止瘦素抵抗的发生，为防治能量代谢障碍提供新的依据。

能量代谢障碍（如糖尿病和肥胖症）越来越严重地威胁着全球人类的健康，而能量代谢障碍与瘦素抵抗(leptin resistance)有密切的关系。以往的研究表明，Foxo1蛋白可以通过结合STAT3阻断瘦素的信号传递链，其下丘脑水平的升高是产生瘦素抵抗的原因之一，因而，在下丘脑中阻止Foxo1与STAT3结合，将是逆转瘦素抵抗的策略之一。本课题组在项目实施过程中找到了Foxo1分子上的一个关键肽段，明确了其对STAT3的抑制效应。在免疫共沉淀实验中，这一肽段能与STAT3进行蛋白-蛋白方式的相互结合，在荧光素酶报告基因检测试验中，这一肽段能明显地抑制STAT3介导的POMC启动子的活性，证明了它能对瘦素信号传导的重大影响；随后，成功制备了过量表达Foxo1功能片段的转基因小鼠。通过Western杂交的方法，检测到下丘脑表达Foxo1蛋白分子的关键肽段；荧光定量PCR结果显示，下丘脑中的神经肽AgRP表达增加；表型分析结果显示，转基因小鼠的采食量增加。以上结果圆满完成了预定的实验目标。.同时，我们意外地发现，转基因小鼠采食量的增加同时伴随着棕色脂肪中UCP1基因表达的增加，意味着转基因小鼠通过产热增加能量消耗，所以，结果尽管转基因小鼠的采食量增加，但小鼠体重并不增加。以往的研究证明，瘦素对动物能量调控的作用是通过两个方面进行的，瘦素一方面抑制能量摄入，另一方面增加能量消耗。课题组正在追踪这一表型背后的分子基础，一旦搞清楚其作用机制，就可能针对此44个氨基酸的肽段开发小分子药物，增加瘦素作用的敏感性，让肥胖动物通过增加体内的热量散发来减肥。因此，继续本课题的研究有非常重要的理论和实际意义，期望得到基金委的继续支持。.自2013年项目实施以来，共培养了青年教师1名，博士生1名和硕士生5名。在《河南科学》发表了研究论文1篇（已接收），完成了硕士论文1篇，大部分的实验结果将在系统完善后发表。