Regulation of Hepatic Gluconeogenesis by the CREB:TORC2 Pathway

描述(由申请人提供)：在禁食期间，循环中的胰腺高血糖素通过CREB辅活化子TORC2诱导葡萄糖生成程序促进肝脏葡萄糖输出。在进食过程中，TORC2被隔离在细胞质中，对禁食信号做出反应，移位到细胞核，在那里它与Forkhead转录因子FOXO1一起触发糖异生基因的表达。初步研究表明，在禁食早期，TORC2通过依赖P300的乙酰化被瞬时激活，当它通过与组蛋白甲基转移酶复合体的结合来刺激糖异生程序时。当FOXO1被相互激活时，在长时间禁食期间，TORC2通过SIRT1介导的脱乙酰基被沉默。提出了三个目标：在目标I中，将确定P300在禁食早期通过乙酰化增强TORC2活性的作用。将确定TORC2中的乙酰化位点，并评估P300在催化TORC2乙酰化中的重要性。依赖NAD+的脱乙酰酶SIRT1在长时间禁食期间通过脱乙酰化沉默TORC2的作用也将被探索。在AIM II中，将评估Ser/Thr激酶SIK2通过P300的磷酸化来调节肝脏TORC2活性的重要性。将确定P300中的SIK2磷酸化位点，并分析它们在破坏P300：TORC2相互作用中的作用，从而减少TORC2乙酰化。在目标III中，将确定TORC2相关的组蛋白甲基转移酶(HMT)复合体在调节禁食期间糖异生程序的诱导中的作用。在禁食期间，TORC2对于HMT复合体的募集和糖异生启动子上的组蛋白甲基化的重要性将通过TORC2或HMT组分的耗尽以及HMT相互作用缺陷突变TORC2蛋白的表达来确定。HMT在通过甲基化TORC2调节糖异生基因表达中的潜在作用也将通过TORC2相关位点的鉴定和突变来评估。综上所述，拟议的研究将提供对调节途径的洞察，该调节途径通过一种辅助激活因子来调节空腹代谢，该辅助激活因子是葡萄糖平衡所必需的，并有助于糖尿病患者的高血糖。这一结果可能导致识别新的分子靶点，用于开发改善胰岛素抵抗患者血糖控制的治疗化合物。与公共健康相关：胰腺激素胰高血糖素在禁食期间通过开启名为TORC2的基因开关来维持循环中的血糖水平，该开关可以增加肝脏中的葡萄糖产量；胰岛素通过关闭TORC2开关来防止进食过程中血糖的异常升高。胰升糖素和胰岛素通过一组酶在TORC2蛋白中产生不同的化学变化，从而对TORC2开关起到相反的作用。通过表征TORC2中的这些化学变化并了解它们如何改变这种切换触发葡萄糖产生的能力，我们的研究可能会为糖尿病患者的治疗带来新的疗法。

DESCRIPTION (provided by applicant): During fasting, elevations in circulating pancreatic glucagon promote hepatic glucose output through induction of the gluconeogenic program by the CREB coactivator TORC2. Sequestered in the cytoplasm during feeding, TORC2 translocates to the nucleus in response to fasting signals, where it triggers gluconeogenic gene expression in concert with the Forkhead transcription factor FOXO1. Preliminary studies indicate that TORC2 is transiently activated through P300-dependent acetylation during early fasting, when it stimulates the gluconeogenic program via an association with a histone methyl-transferase complex. TORC2 is silenced through SIRT1-mediated deacetylation during prolonged fasting, when FOXO1 is reciprocally activated. Three Aims are proposed: In Aim I, the role of P300 in augmenting TORC2 activity through acetylation during early fasting will be determined. Acetylation sites in TORC2 will be identified, and the importance of P300 in catalyzing TORC2 acetylation will be evaluated. The role of the NAD+ dependent deacetylase SIRT1 in silencing TORC2 through deacetylation during prolonged fasting will also be explored. In Aim II, the importance of the Ser/Thr kinase SIK2 in modulating hepatic TORC2 activity through phosphorylation of P300 will be evaluated. SIK2 phosphorylation sites in P300 will be identified, and their role in disrupting the P300:TORC2 interaction and thereby reducing TORC2 acetylation will be analyzed. In Aim III, the role of a TORC2 associated histone methyl-transferase (HMT) complex in mediating induction of the gluconeogenic program during fasting will be determined. The importance of TORC2 for recruitment of HMT complexes and for histone methylation over gluconeogenic promoters during fasting will be determined, by depletion of TORC2 or HMT components, and by expression of HMT interaction-defective mutant TORC2 proteins. The potential role of HMTs in modulating gluconeogenic gene expression by methylating TORC2 will also be evaluated through identification and mutation of relevant sites in TORC2. Taken together, the proposed studies will provide insight into regulatory pathways that modulate fasting metabolism through a coactivator that is required for glucose balance and that contributes to hyperglycemia in diabetes. The results may lead to the identification of new molecular targets for the development of therapeutic compounds that improve glucose control in insulin resistant individuals. PUBLIC HEALTH RELEVANCE: The pancreatic hormone glucagon maintains circulating glucose levels during fasting by turning on a genetic switch, called TORC2, that increases glucose production in the liver; insulin protects against abnormal elevations in blood glucose during feeding by turning off the TORC2 switch. Glucagon and insulin exert these opposing effects on the TORC2 switch through a group of enzymes that cause different chemical changes in the TORC2 protein. By characterizing these chemical changes in TORC2 and understanding how they modify the ability for this switch to trigger glucose production, our studies may lead to new therapies for the treatment of diabetic patients.