Regulation of Hepatic Gluconeogenesis by the CREB:TORC2 Pathway

在禁食条件下，循环中的高血糖素通过诱导cAMP途径刺激肝脏葡萄糖的产生。反之，喂食过程中肠道来源的胰升糖素样肽1(GLP1)的增加会促进胰岛素的释放，从而提高葡萄糖的清除能力。转录因子CREB在被PKA磷酸化并与CBP/P300结合后，被认为介导了这两种多肽激素的长期效应。对cAMP的转录反应遵循猝发-衰减动力学；CREB活性在刺激1小时后达到峰值，4-6小时后恢复到基线水平。 除了对CREB磷酸化的影响外，GLP1和GLP1还通过刺激CREB与cAMP调节的转录共激活因子(CRTCs/TORCS)的联系来增加CREB的活性。CRTCs/TORCS是潜在的细胞质CREB辅助因子，在cAMP反应中去磷酸化后移位到细胞核。CRTc1只在脑内表达，而CRTC2和CRTC3在大多数组织中共同表达。然而，CRTC2和CRTC3在CREB靶基因的重叠或不同亚集上的作用程度尚不清楚。在之前的资助期间，我们发现CREB/CRTC2途径对空腹血糖的产生有重要作用；肝脏中CRTC2的急性耗竭显著降低了血糖浓度和糖异生基因的表达，而野生型和更大程度上磷酸化缺陷的CRTC2的过度表达增加了糖异生。 与急性肝脏CRTC2基因敲除的影响相比，全身CRTC2基因敲除的小鼠在空腹血糖水平方面仅显示出轻微的下降；随着年龄的增长，它们会出现胰岛素分泌缺陷。这些结果表明，额外的CREB辅活化子参与补偿肝脏中CRTC2的丢失，它们表明，CRTC2在胰岛的表达也通过影响胰岛素分泌来调节循环中的葡萄糖浓度。支持后者的是Mafa，一种胰岛素分泌所需的β细胞转录因子，被CREB和CRTC2强烈上调。 在即将到来的赠款期间，拟议的研究集中在CRTC家族成员对CREB活动产生重叠影响的假设上。将测试一种新发现的CREB相互作用蛋白在增强CREB活性和补偿CRTC2突变小鼠CRTC2丢失方面的重要性。最后，将评估一种有效的CREB抑制物在促进对Gs偶联受体信号的抵抗中的作用，该抑制物在高血糖条件下在胰岛上调。 他们提出了三个目标：他们通过解决CREB途径促进肝脏糖异生和促进胰岛胰岛素分泌的机制来扩展先前的工作。 在目标1中，我们将使用具有CRTC2和CRTC3等位基因的小鼠来评估它们的相对作用 调节肝脏糖异生和胰岛素分泌的辅活化子。我们将在肝脏或胰岛中产生组织特异性CRTC2和CRTC3基因敲除的小鼠。CRTC2和CRTC3是否对肝脏中糖异生基因的表达产生重叠作用？它们是否通过上调亮氨酸拉链因子Mafa来促进胰岛素分泌？ 在目标2中，我们将测试BRD2在刺激糖异生基因表达中的作用。BRD2是在CREB相关蛋白的蛋白质组筛查中发现的一种溴域蛋白。我们将表征BRD2和CREB中介导这种相互作用的结构域；也将测试CREB乙酰化在调节BRD2：CREB结合中的作用。我们将评估通过给予选择性溴域抑制剂抑制BRD2是否能改善胰岛素抵抗时的血糖水平。 在目标3中，我们将研究在胰岛素抵抗中胰岛CREB靶基因表达下调的机制。特别是，我们将研究蛋白激酶抑制因子β(PKIB)在高血糖反应中上调GLP1和其他激素方面的作用：PKIB基因敲除小鼠将被用来确定在胰岛素抵抗的背景下，该抑制物的耗尽是否改善胰岛功能。 综上所述，这些研究将为研究GLP1和GLP1通过影响肝脏和胰腺β细胞的CREB途径促进葡萄糖平衡的机制提供新的见解。

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.