Transcriptional Regulation of Metabolic and Hepatic Homeostasis by FoxO Signaling

描述(申请人提供)：这项名为“FoxO信号对新陈代谢和肝脏动态平衡的转录调控”的提案通过研究燃料激素在肝脏中的作用，阐述了糖尿病的基本机制。2型糖尿病患者血液中高血糖素水平升高和胰岛素作用缺陷是高血糖的原因，但其分子机制仍不清楚。胰升糖素和胰岛素相互控制新陈代谢和细胞内稳态，其中肝脏是执行其细胞功能的主要器官。在空腹肝脏中，胰高血糖素刺激糖异生，降解包括糖原、脂肪和蛋白质在内的大分子，并促进调节细胞器周转的自噬途径。在喂养肝脏中，胰岛素逆转了胰高血糖素的分解代谢。从禁食到进食的代谢和细胞适应需要通过这两种激素的相反作用严格控制基因转录，而这种适应的失败会导致糖尿病患者的高血糖。调节多种生物过程的叉头转录因子Foxo1被胰岛素信号抑制。胰岛素通过蛋白酪氨酸激酶B激活，使Foxo1在小鼠的Ser253或人类的S256处磷酸化，并触发Foxo1核输出和胞浆隔离以实现泛素化。相反，当胰岛素水平降低或发生胰岛素抵抗时，胰升糖素促进空腹肝脏或糖尿病患者肝脏中Foxo1蛋白的稳定性。Foxo1可以介导胰升糖素的第二信使环磷酸腺苷对糖异生酶和自噬基因表达的影响，但Foxo1在代谢中的作用及其与胰岛素抵抗的调节尤其是与胰岛素抵抗协同作用。 调控和细胞功能完全不清楚。在目标1中，我们将使用Foxo1肝脏特异性基因敲除小鼠，并研究Foxo1是否是胰高血糖素信号转导的关键介质，以调节肝脏葡萄糖的产生、糖原分解、脂肪和蛋白质稳态、线粒体的更新和功能、自噬和生存，而Foxo1的干扰阻止了由高血糖素诱导的促进糖尿病发展的生物过程。在AIM2中，我们将使用质谱学和磷酸特异性抗体来确定S153处的Foxo1被胰高血糖素和蛋白激酶PKA磷酸化是否促进核靶向性，以及S276处的磷酸化是否提高细胞的转录活性。在目标3中，我们在体内获得了模拟Foxo1水平的胰岛素抵抗的Foxo1-S253A突变小鼠。利用这一独特的小鼠模型，我们将确定胰高血糖素是否刺激去磷酸化的Foxo1的效果，扰乱代谢和细胞内稳态以及肝功能。总之，我们利用Foxo1基因功能缺失和功能获得的方法来研究Foxo1在胰升糖素作用中的生理作用，并发现Foxo1激活的新的分子机制，这将促进我们对糖尿病发病机制的理解，并有助于开发检测和抑制胰高血糖素-Foxo1途径的策略来控制疾病。

DESCRIPTION (provided by applicant): This proposal "Transcriptional Regulation of Metabolic and Hepatic Homeostasis by FoxO Signaling" addresses the fundamental mechanism of diabetes by studying fuel hormone's action in the liver. Elevated blood glucagon levels and defective insulin action in patients with type 2 diabetes are responsible for hyperglycemia, but the molecular mechanisms remain elusive. Glucagon and insulin reciprocally control metabolic and cellular homeostasis, in which the liver is a major organ that executes their cellular functions. In the fasting liver, glucagon stimulates gluconeogenesis, degrades macromolecules including glycogen, lipid, and protein, and promotes the autophagic pathway that regulates cellular organelle turnover. In the feeding liver, insulin reverses the catabolic metabolism of glucagon. The metabolic and cellular adaptation from fasting to feeding requires a tight control of gene transcription by opposing effects of the two hormones, and the failure of the adaptation causes hyperglycemia in diabetes. The forkhead transcription factor Foxo1 that regulates multiple biological processes is inhibited by insulin signaling. Insulin phosphorylates Foxo1 at Ser253 in mice or S256 in humans via PKB activation, and triggers Foxo1 nuclear export and cytoplasmic sequestration for ubiquitination. Conversely, glucagon promotes Foxo1 protein stability in the fasting liver or the liver of diabetes when insulin level is decreased or insulin resistance occurs. Foxo1 can mediate the effect of cyclic AMP, the second messenger of glucagon, on expression of gluconeogenic enzymes and autophagic genes, but the role of Foxo1 and its regulation by glucagon, particularly in concert with insulin resistance, in metabolic regulation and cellular function is completely unclear. In Aim 1, we will use Foxo1 liver-specific knockout mice and examine whether Foxo1 is a key mediator in glucagon signaling to regulate hepatic glucose production, glycogenolysis, lipid and protein homeostasis, mitochondrial turnover and function, autophagy and survival, whereas disruption of Foxo1 prevents the glucagon-induced biological processes that promote the development of diabetes. In Aim2, we will use mass-spectrometry and phospho-specific antibodies to determine whether Foxo1 phosphorylation at S153 by glucagon and protein kinase PKA promotes nuclear targeting and whether phosphorylation at S276 enhances transcriptional activity in cells. In Aim 3, we have generated Foxo1- S253A mutant mice mimicking insulin resistance at the Foxo1 level in vivo. Using this unique mouse model, we will determine whether glucagon stimulates the effect of dephosphorylated Foxo1, disrupting metabolic and cellular homeostasis and liver function. In overall, we use Foxo1 gene loss- and gain-of-function approaches to investigate the physiological role of Foxo1 in glucagon action and identify novel molecular mechanisms of Foxo1 activation, which will advance our understanding of the mechanism of diabetes and help develop strategies detecting and inhibiting the glucagon->Foxo1 pathway to control the disease.