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

共激活剂对肝细胞和 B 细胞功能的控制

• 批准号: 8010071
• 负责人: Mehboob A Hussain
• 金额: $ 2.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-04 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010071
• 关键词: Affect; Amino Acids; Animals; Binding; Binding Proteins; CREB-binding protein; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Defect; Diabetes Mellitus; Dissociation; E1A-associated p300 protein; EP300 gene; Fasting; Gene Activation; Gene Targeting; Genes; Genetic Transcription; Glucagon; Gluconeogenesis; Glucose; Glycine; Goals; Growth; Hepatic; Hepatocyte; Hyperglycemia; Hyperplasia; Hypoglycemia; Insulin; Insulin Resistance; Knock-in Mouse; Liver; Mediating; Modeling; Mus; Mutant Strains Mice; Mutate; Mutation; Nuclear; Pancreas; Patients; Phenotype; Phosphoenolpyruvate Carboxylase; Phosphorylation; Phosphorylation Site; Physiological; Play; Positioning Attribute; Process; Production; Protein Binding; Protein Dephosphorylation; Proteins; Recruitment Activity; Regulation; Relative (related person); Reporting; Resistance; Role; Serine; Signal Pathway; Signal Transduction; Site; Testing; Transducers; attenuation; base; cell growth; cofactor; forkhead protein; glucose production; hepatic gluconeogenesis; homologous recombination; human CREBBP protein; impaired glucose tolerance; in vivo; insulin secretion; mouse model; mutant; novel; programs; protein complex; protein expression; public health relevance; reconstitution; transcription factor

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.

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-细胞中环腺苷酸（cAMP）信号传导途径的机制。肝细胞中升高的细胞内cAMP增加肝葡萄糖产生，而胰腺2-细胞中的相同信号促进胰岛素分泌和增殖。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。在丝氨酸436处的胰岛素磷酸化是CREB-CBP复合物的有效解离和随后CREB依赖性基因转录的减弱所必需的。我们已经产生了一个敲入小鼠模型，其中CBP在丝氨酸436（CBP-S436 A）处突变，去除了胰岛素磷酸化的该位点。因此，CREB依赖性基因转录增加，并抵抗胰岛素的抑制性核效应。这些小鼠表现出与葡萄糖刺激的胰岛素分泌缺陷相关的肝脏葡萄糖产生增强和胰腺2细胞增生。这些作用可以由PPAR 3共激活因子11（PGC-11，cAMP靶基因）介导，其表达在这些动物的肝细胞和胰腺2-细胞中增加。PGC-11激活肝脏中的禁食基因程序并损害2-细胞中的能量产生。在该提议中，我们将尝试理解PGC-11是否介导CBP S436 A小鼠表型，因为该突变可能影响其他信号传导途径（Aim 1）。CBP和p300可能分别基于胰岛素磷酸化位点的存在或不存在而具有离散的生理作用。目的2通过研究在p300（G421 S）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剂介导的肝脏和2-细胞调节的相对重要性和机制。最后，CBP无论其磷酸化状态如何都保留在细胞核中，而叉头蛋白FoxO 1在胰岛素磷酸化后被排除在细胞核之外。在过表达小鼠模型中，FoxO 1在激活肝纤维化和抑制2-细胞生长中起主导作用。目的3将比较CBP和FoxO 1的胰岛素磷酸化突变体（在等位基因水平表达）对肝葡萄糖产生和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制理解。公共卫生关系：在这个提议中，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，在其他因素的背景下，报道介导激活的基因含有cAMP反应元件。