Transcriptional Regulation of Metabolic and Hepatic Homeostasis by FoxO Signaling

FoxO 信号传导对代谢和肝脏稳态的转录调节

• 批准号: 9264906
• 负责人: Shaodong Guo
• 金额: $ 19.12万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9264906
• 关键词: Transcriptional; Regulation; Metabolic; Hepatic; Homeostasis

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.

描述（由申请人提供）：该提案“FoxO 信号传导对代谢和肝脏稳态的转录调节”通过研究燃料激素在肝脏中的作用来解决糖尿病的基本机制。 2 型糖尿病患者的血糖水平升高和胰岛素作用缺陷是导致高血糖的原因，但其分子机制仍不清楚。胰高血糖素和胰岛素相互控制代谢和细胞稳态，其中肝脏是执行其细胞功能的主要器官。在禁食肝脏中，胰高血糖素刺激糖异生，降解包括糖原、脂质和蛋白质在内的大分子，并促进调节细胞器更新的自噬途径。在进食的肝脏中，胰岛素逆转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两种激素的相反作用来严格控制基因转录，而适应失败会导致糖尿病中的高血糖。 调节多种生物过程的叉头转录因子 Foxo1 受到胰岛素信号传导的抑制。胰岛素通过 PKB 激活使 Foxo1 在小鼠的 Ser253 位点或人类的 S256 位点磷酸化，并触发 Foxo1 核输出和细胞质隔离以实现泛素化。相反，当胰岛素水平降低或发生胰岛素抵抗时，胰高血糖素可促进空腹肝脏或糖尿病肝脏中Foxo1蛋白的稳定性。 Foxo1 可以介导环 AMP（胰高血糖素的第二信使）对糖异生酶和自噬基因表达的影响，但 Foxo1 的作用及其受胰高血糖素的调节，特别是与胰岛素抵抗相结合，在代谢中的作用 调节和细胞功能完全不清楚。在目标 1 中，我们将使用 Foxo1 肝脏特异性敲除小鼠，并检查 Foxo1 是否是胰高血糖素信号传导的关键介质，以调节肝葡萄糖生成、糖原分解、脂质和蛋白质稳态、线粒体周转和功能、自噬和存活，而 Foxo1 的破坏防止胰高血糖素诱导的促进糖尿病发展的生物过程。在 Aim2 中，我们将使用质谱和磷酸化特异性抗体来确定 Foxo1 在 S153 处被胰高血糖素和蛋白激酶 PKA 磷酸化是否会促进核靶向，以及 S276 处的磷酸化是否会增强细胞中的转录活性。在目标 3 中，我们培育了 Foxo1-S253A 突变小鼠，在体内模拟 Foxo1 水平的胰岛素抵抗。使用这种独特的小鼠模型，我们将确定胰高血糖素是否会刺激去磷酸化 Foxo1 的作用，从而破坏代谢和细胞稳态以及肝功能。总的来说，我们使用 Foxo1 基因丧失和功能获得的方法来研究 Foxo1 在胰高血糖素作用中的生理作用，并确定 Foxo1 激活的新分子机制，这将增进我们对糖尿病机制的理解，并有助于制定检测策略并抑制胰高血糖素->Foxo1通路来控制疾病。