Control of hepatic and b-cell function by co-activators

描述（由申请人提供）：本提案的总体目标是定义肝脏和胰腺2细胞中环AMP （cAMP）信号通路的机制。肝细胞内升高的cAMP增加了肝糖的产生，而胰腺2细胞中同样的信号促进胰岛素的分泌和增殖。cAMP信号激活蛋白激酶A (PKA)，其磷酸化核cAMP反应元件结合蛋白（CREB）。磷酸化的CREB募集核共激活物CREB结合蛋白（CBP）和相关蛋白p300。CBP，而不是p300，在丝氨酸436处含有胰岛素磷酸化位点。胰岛素磷酸化丝氨酸436是有效解离CREB-CBP复合物和随后的creb依赖性基因转录衰减所必需的。我们已经建立了一个敲入小鼠模型，其中CBP在丝氨酸436 （CBP- s436a）上发生突变，去除了胰岛素磷酸化的这个位点。结果，creb依赖性基因转录增加，并抵抗胰岛素的抑制核效应。这些小鼠表现出肝脏葡萄糖生成增强和胰腺2细胞增生，并伴有葡萄糖刺激的胰岛素分泌缺陷。这些作用可能是由PPAR3共激活因子11（pgc - 11， cAMP靶基因）介导的，其在这些动物的肝细胞和胰腺2细胞中的表达均增加。PGC-11激活肝脏中的禁食基因程序，损害2细胞的能量产生。在本提案中，我们将尝试了解pgc - 11是否介导CBP S436A小鼠表型，因为这种突变可能影响其他信号通路（目的1）。CBP和p300可能分别根据胰岛素磷酸化位点的存在或不存在而具有不同的生理作用。Aim 2将通过研究在p300 （G421S）中引入人工胰岛素磷酸化位点的小鼠模型，建立由这些密切相关的共激活因子介导的肝脏和2细胞调节的相对重要性和机制。最后，CBP无论磷酸化状态如何都留在细胞核中，而叉头蛋白fox01在胰岛素磷酸化后被排除在细胞核之外。据报道，基于过表达小鼠模型，fox01在激活肝脏糖异生和抑制2细胞生长中起主导作用。Aim 3将比较胰岛素磷酸化突变体CBP和fox01在等位基因水平上表达对肝脏葡萄糖生成和2细胞调节和/或生长的影响。这些研究具有重要意义，因为它们将在体内建立正常调节肝细胞和胰腺2细胞的信号通路，并为糖尿病患者的病理生理变化提供机制理解。公共卫生相关性：在本提案中，我们将尝试了解CBP和p300在肝细胞和胰腺2细胞中的生理重要性，在其他因子介导含有cAMP反应元件的基因激活的背景下。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define the mechanism of cyclic AMP (cAMP) signaling pathways in the liver and pancreatic 2-cell. Elevated intracellular cAMP in the hepatocyte increases hepatic glucose production, while the same signal in the pancreatic 2-cell promotes insulin secretion and proliferation. The cAMP signal activates protein kinase A (PKA), which phosphorylates the nuclear cAMP response element binding protein (CREB). Phosphorylated CREB recruits the nuclear co-activators CREB binding protein (CBP), and the related protein p300. CBP, but not p300, contains an insulin phosphorylation site at serine 436. Insulin phosphorylation at serine 436 is required for efficient dissociation of the CREB-CBP complex and consequent attenuation of CREB-dependent gene transcription. We have generated a knock-in mouse model where CBP is mutated at serine 436 (CBP-S436A), removing this site of insulin phosphorylation. As a result, CREB-dependent gene transcription is increased and resistant to the inhibitory nuclear effects of insulin. These mice show enhanced hepatic glucose production and pancreatic 2-cell hyperplasia associated with a defect in glucose-stimulated insulin secretion. These effects may be mediated by the PPAR3 co-activator 11(PGC-1 1, a cAMP target gene) whose expression is increased in both the hepatocyte and pancreatic 2-cell of these animals. PGC-11 activates a fasting gene program in liver and impairs energy production in the 2-cell. In this proposal, we will attempt to understand if PGC-1 1 mediates the CBP S436A mouse phenotype, since this mutation may affect other signaling pathways (Aim 1). CBP and p300 may have discrete physiological roles based on the presence or absence, respectively, of an insulin phosphorylation site. Aim 2 will establish the relative importance and mechanism of hepatic and 2-cell regulation mediated by these closely related co- activators by studying a mouse model where an artificial insulin phosphorylation site is introduced in p300 (G421S). Finally, CBP remains in the nucleus regardless of its phosphorylation state, while the forkhead protein, FoxO1, is excluded from the nucleus after insulin phosphorylation. FoxO1 has been reported to play a dominant role in activating hepatic gluconeogenesis and inhibiting 2-cell growth based on over-expression mouse models. Aim 3 will compare the effects of insulin phosphorylation mutants of CBP and FoxO1, expressed at allelic levels, on hepatic glucose production and 2-cell regulation and/or growth. These studies are significant because they will establish signaling pathways that normally regulate the hepatocyte and pancreatic 2-cell in vivo and provide a mechanistic understanding of pathophysiological changes found in patients with diabetes mellitus. PUBLIC HEALTH RELEVANCE: In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic 2-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements.