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确定肝脏和胰腺2-细胞中环磷酸腺苷(CAMP)信号通路的机制。肝细胞内cAMP的升高增加了肝脏葡萄糖的产生，而胰腺2细胞中的相同信号促进了胰岛素的分泌和增殖。CAMP信号激活蛋白激酶A(PKA)，使核内cAMP反应元件结合蛋白(CREB)磷酸化。磷酸化的CREB招募核共激活因子CREB结合蛋白(CBP)及其相关蛋白p300。CBP，而不是p300，在丝氨酸436位含有胰岛素磷酸化位点。丝氨酸436位的胰岛素磷酸化是CREB-CBP复合体有效解离和CREB依赖的基因转录减弱所必需的。我们已经建立了一个敲入小鼠模型，其中CBP在丝氨酸436位发生突变(CBP-S436A)，移除了这个胰岛素磷酸化位点。结果，依赖CREB的基因转录增加，并抵抗胰岛素抑制的核效应。这些小鼠表现出肝脏葡萄糖生成增加和胰腺2-细胞增生与葡萄糖刺激的胰岛素分泌缺陷相关。这些作用可能是由PPAR3共激活因子11(PGC-11，cAMP靶基因)介导的，它在这些动物的肝细胞和胰腺2细胞中的表达都增加。PGC-11激活肝脏中的空腹基因程序，并损害2-细胞的能量产生。在这项建议中，我们将试图了解PGC-11是否介导CBP S436A小鼠的表型，因为这种突变可能会影响其他信号通路(目标1)。CBP和p300可能分别基于胰岛素磷酸化位点的存在或缺失而具有不同的生理作用。目的2通过研究p300(G421S)中引入人工胰岛素磷酸化位点的小鼠模型，确定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，无论CBP处于何种磷酸化状态，CBP都留在细胞核内，而叉头蛋白FoxO1在胰岛素磷酸化后被排除在细胞核之外。在过度表达的小鼠模型上，Foxo1被报道在激活肝脏糖异生和抑制2-细胞生长方面发挥主导作用。目的3比较在等位基因水平表达的CBP和FoxO1的胰岛素磷酸化突变体对肝脏葡萄糖产生和2-细胞调节和/或生长的影响。这些研究意义重大，因为它们将建立正常调节体内肝细胞和胰腺2-细胞的信号通路，并提供对糖尿病患者病理生理变化的机械性理解。公共卫生相关性：在这项提案中，我们将试图了解CBP和p300在肝细胞和胰腺2-细胞中的生理重要性，以及其他已报道的介导包含cAMP反应元件的基因激活的因素。

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.