Regulation of Hepatic Gluconeogenesis by the CREB:TORC2 Pathway

CREB:TORC2 通路对肝糖异生的调节

• 批准号: 9017999
• 负责人: MARC R MONTMINY
• 金额: $ 73.05万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-07 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9017999
• 关键词: Acetylation; Acute; Address; Age; Agonist; BRD2 gene; Beta Cell; Binding; Blood Glucose; Brain; Bromodomain; CREB1 gene; Cell Nucleus; Cells; Complex; Consensus; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Deacetylase; Deacetylation; Defect; EP300 gene; Embryo; Equilibrium; FOXO1A gene; Family; Family member; Fasting; Fibroblasts; Future; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucagon; Gluconeogenesis; Glucose; Grant; Hepatic; Hepatocyte; Histone Deacetylase; Histones; Hormones; Hour; Hyperglycemia; Immune Sera; Insulin; Insulin Resistance; Islets of Langerhans; Kinetics; Knock-out; Knockout Mice; Leucine Zippers; Liver; Long-Term Effects; LoxP-flanked allele; Mediating; Methylation; Monitor; Mono-S; Mus; Mutant Strains Mice; Mutation; Nuclear; PCAF gene; Pathway interactions; Phosphorylation; Phosphorylation Site; Process; Protein Dephosphorylation; Protein Kinase Inhibitors; Proteins; Proteomics; RNA Interference; Receptor Signaling; Recruitment Activity; Regulation; Resistance; Role; Signal Transduction; Skeletal Muscle; Structure of beta Cell of islet; Testing; Tissues; Transactivation; Transcription Coactivator; Transferase; Ubiquitination; Up-Regulation; Work; attenuation; base; cell type; cofactor; fasting glucose; feeding; glucagon-like peptide; glucose production; hepatic gluconeogenesis; histone methylation; improved; in vivo; inhibitor/antagonist; insight; insulin secretion; knock-down; member; multicatalytic endopeptidase complex; mutant; overexpression; pancreatic islet function; paralogous gene; peptide hormone; programs; promoter; protein kinase inhibitor; response; salt-inducible kinase; transcription factor; ubiquitin-protein ligase

Under fasting conditions, increases in circulating glucagon stimulate hepatic glucose production via induction of the cAMP pathway. Conversely, increases in gut-derived glucagon-like peptide 1 (GLP1) during feeding enhance glucose clearance by promoting insulin release. The transcription factor CREB is thought to mediate long term effects of both peptide hormones, following its phosphorylation by PKA and association with CBP/P300. The transcriptional response to cAMP follows burst-attenuation kinetics; CREB activity peaks after 1 hour of stimulation, returning to baseline after 4-6 hours. In addition to their effects on CREB phosphorylation, glucagon and GLP1 also increase CREB activity by stimulating its association with the cAMP Regulated Transcriptional Coactivators (CRTCs/TORCs), latent cytoplasmic CREB cofactors that translocate to the nucleus following their dephosphorylation in response to cAMP. CRTC1 is expressed only in brain, while CRTC2 and CRTC3 are co-expressed in most tissues. The extent to which CRTC2 and CRTC3 function on overlapping or distinct subsets of CREB target genes is unclear, however. In the previous grant period, we showed that the CREB/CRTC2 pathway contributes importantly to fasting glucose production; acute depletion of CRTC2 in liver substantially lowers blood glucose concentrations and gluconeogenic gene expression, while over-expression of wild-type and to a greater extent phosphorylation-defective CRTC2 increases gluconeogenesis. By contrast with effects of acute hepatic CRTC2 knockdown, mice with a whole-body knockout of CRTC2 show only modest reductions in fasting glucose levels; and they develop an insulin secretion defect as they age. These results point to the involvement of additional CREB coactivators that compensate for loss of CRTC2 in liver, and they suggest that CRTC2 expression in pancreatic islets also modulates circulating glucose concentrations through its effects on insulin secretion. Supporting the latter, MafA, a beta cell transcription factor that is required for insulin secretion, is strongly upregulated by CREB and CRTC2. Proposed studies during the upcoming grant period focus on the hypothesis that members of the CRTC family exert overlapping effects on CREB activity. The importance of a newly identified CREB interacting protein in potentiating CREB activity and compensating for loss of CRTC2 in CRTC2 mutant mice will be tested. Finally the role of a potent CREB inhibitor, which is upregulated in pancreatic islets under hyperglycemic conditions, in promoting resistance to Gs-coupled receptor signaling, will be evaluated. Three aims are proposed; they extend the previous work by addressing the mechanisms by which the CREB pathway promotes gluconeogenesis in liver and facilitates insulin secretion from pancreatic islets. In Aim 1, we will use mice with floxed alleles of CRTC2 and CRTC3 to evaluate the relative roles of these coactivators in modulating hepatic gluconeogenesis and insulin secretion. We will generate mice with tissue specific knockouts of CRTC2 and CRTC3 in liver or pancreatic islets. Do CRTC2 and CRTC3 exert overlapping effects on gluconeogenic gene expression in liver? Do they promote insulin secretion by upregulating the leucine zipper factor MafA? In Aim 2, we will test the role of BRD2-a bromodomain protein identified in a proteomic screen for CREB associated proteins- in stimulating expression of gluconeogenic genes. We will characterize domains in BRD2 and CREB that mediate this interaction; and the role of CREB acetylation in modulating the BRD2:CREB association will also be tested. We will evaluate whether inhibition of BRD2, through administration of a selective bromodomain inhibitor, improves glucose levels in the setting of insulin resistance. In Aim 3, we will examine the mechanism by which CREB target gene expression in pancreatic islets is down-regulated in insulin resistance. In particular, we will investigate the role of Protein Kinase Inhibitor beta (PKIB) in interfering with GLP1 and other hormones, following its upregulation in response to hyperglycemia: PKIB knockout mice will be used to determine whether depletion of this inhibitor improves pancreatic islet function in the setting of insulin resistance. Taken together, the proposed studies will provide new insight into mechanisms by which glucagon and GLP1 promote glucose balance through their effects on the CREB pathway in liver and pancreatic beta cells.

在禁食条件下，循环胰高血糖素的增加通过诱导cAMP途径刺激肝葡萄糖的产生。相反，喂养过程中肠道来源的胰高血糖素样肽1（GLP1）的增加通过促进胰岛素释放来增强葡萄糖清除率。转录因子CREB被认为是在PKA磷酸化并与CBP/P300相关的磷酸化之后，介导两种肽激素的长期影响。对营地的转录反应遵循爆发累积动力学；刺激1小时后，CREB活性达到峰值，4-6小时后返回基线。 除了对CREB磷酸化的影响外，胰高血糖素和GLP1还通过刺激其与cAMP调节的转录共激活剂（CRTC/TORC），潜在的细胞质CREB辅助因子的相关性来增加CREB活性，从而将其转移到核心后，从而响应camp的磷酸化。 CRTC1仅在大脑中表达，而CRTC2和CRTC3在大多数组织中共表达。但是，CRTC2和CRTC3在重叠或CREB靶基因的不同子集上起作用的程度尚不清楚。在上一个赠款期间，我们表明CREB/CRTC2途径对禁食葡萄糖产生重要贡献。 CRTC2在肝脏中的急性消耗基本上会降低血糖浓度和糖原性基因表达，而野生型的过表达和更大程度上的磷酸化缺陷CRTC2会增加糖异生。 与急性肝CRTC2敲低的影响相反，具有CRTC2全身敲除的小鼠仅显示空腹葡萄糖水平的适度降低。随着年龄的增长，他们会形成胰岛素分泌缺陷。这些结果表明，其他CREB共激活因子参与了肝脏中CRTC2损失的介绍，它们表明胰岛中的CRTC2表达也通过其对胰岛素分泌的影响来调节循环葡萄糖浓度。支持后者，MAFA是胰岛素分泌所需的β细胞转录因子，由CREB和CRTC2强烈上调。 在即将到来的赠款期间的拟议研究集中在CRTC家族成员对CREB活动产生重叠影响的假设。新鉴定的CREB相互作用蛋白在增强CREB活性并补偿CRTC2突变小鼠中CRTC2损失的重要性将进行测试。最后，将评估有效的CREB抑制剂的作用，该抑制剂将在高血糖条件下在胰岛上上调，在促进对GS耦合受体信号的抗性中的作用。 提出了三个目标；他们通过解决CREB途径促进肝脏中糖异生的机制来扩展先前的工作，并促进胰岛中胰岛素分泌。 在AIM 1中，我们将使用与CRTC2和CRTC3的Floxed等位基因的小鼠评估这些相对作用 调节肝糖异生和胰岛素分泌方面的共激活剂。我们将在肝脏或胰岛中生成具有CRTC2和CRTC3的组织特异性敲除小鼠。 CRTC2和CRTC3是否对肝脏中的糖生成基因表达产生重叠的影响？它们是否通过上调亮氨酸拉链因子MAFA来促进胰岛素分泌？ 在AIM 2中，我们将测试在蛋白质组学筛选中鉴定出CREB相关蛋白质的BRD2-A溴结构域蛋白的作用 - 刺激糖原性基因的表达。我们将表征介导这种相互作用的BRD2和CREB中的域； CREB乙酰化在调节BRD2：CREB关联中的作用也将进行测试。我们将通过施用选择性溴化域抑制剂来评估BRD2的抑制是否可以改善胰岛素抵抗的葡萄糖水平。 在AIM 3中，我们将研究胰岛耐药性中胰岛胰岛中CREB靶基因表达的机制。特别是，我们将研究蛋白激酶抑制剂β（PKIB）在响应高血糖的上调（PKIB基因敲除小鼠）的上调后，将使用该抑制剂的耗竭是否会改善胰岛耐药的功能，以确定该抑制剂改善胰岛素耐药的功能。 综上所述，拟议的研究将提供有关胰高血糖素和GLP1通过对肝脏和胰腺β细胞CREB途径的影响来促进葡萄糖平衡机制的新见解。