Beta-cell Proliferation

描述(由申请人提供)：本提案的总体目标是定义胰腺细胞增殖的调控机制。生理和病理状态引起的胰岛素需求的变化需要胰腺细胞的适应性功能变化。这些适应性变化包括胰岛素合成和分泌增加，以及增殖，并可能从祖细胞形成新的细胞。在这些机制中，细胞增殖和细胞周期的调节在建立和确定细胞质量方面占主导地位。细胞对代谢挑战的反应不足会导致高血糖和明显的糖尿病。胰升糖素样肽-1(GLP-1)可刺激小鼠胰腺细胞增殖。GLP-1与细胞上的GLP-1受体结合后，可同时刺激环磷酸腺苷(CAMP)和磷酸肌醇3激酶(PI3K)激活。CAMP和PI3K信号通路都与细胞增殖有关。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)和相关蛋白P300，通过组蛋白脱乙酰酶活性和刺激RNA聚合酶来刺激基因转录。另一方面，PI3激酶激活蛋白激酶B(PKB=Akt)。PKB使转录因子FoxO1磷酸化，导致其核排斥，从而改变基因表达，包括下调胰腺十二指肠同源盒-1(PDX-1)的转录。PDX-1是GLP-1对细胞增殖和细胞群适应胰岛素抵抗所必需的。我们的初步结果表明，GLP-1通过cAMP-PKA-CREB途径刺激细胞周期正调控因子Cyclin A2的转录。在体外，细胞周期蛋白A2的上调足以促进小鼠细胞的增殖。此外，在细胞中，PDX-1是GLP-1刺激的cAMP产生、随后的PKA激活和细胞周期蛋白A2表达所必需的。因此，我们的研究结果表明，cAMP和PI3K介导的GLP-1受体信号是相互依赖的，PDX-1和GLP-1诱导的细胞增殖之间存在机制联系。该建议的具体目的是进一步阐明GLP-1诱导的cAMP/PKA信号转导和细胞周期蛋白A2在调节细胞增殖中的作用。目的1或我们的建议是通过细胞特异性的PKA调节亚基的消融，在体内检测cAMP/PKA信号在细胞增殖和周期调节蛋白中的作用。目的2研究细胞特异性细胞周期蛋白A2上调对胰岛质量和细胞功能的影响。目的3是评估细胞特异性细胞周期蛋白A2消融对发育过程中的细胞增殖的影响，以及成年动物对GLP-1的反应和对高脂饮食诱导的胰岛素抵抗的适应。这些体内细胞增殖机制的研究可能为人类糖尿病提供治疗方法。公共卫生相关性：糖尿病是由于产生胰岛素的细胞不能满足新陈代谢需求所致，而对需求的适应包括细胞增殖(即复制)。在这项建议中，我们旨在了解(S)胰腺细胞增殖的机制，特别是由胰岛素激素胰高血糖素样肽-1介导的机制。本文提出的研究具有重要意义，因为它们可能导致治疗人类糖尿病的方法。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define mechanisms governing pancreatic ¿-cell proliferation. Variations in insulin demand resulting from physiological and pathological states require adaptive functional changes in pancreatic ¿-cells. These adaptive changes include increased insulin synthesis and secretion as well as hyperplasia and possibly formation of new ¿-cells from progenitor cells. Of these mechanisms, ¿-cell proliferation and regulation of cell cycle appear predominant in establishing and determining ¿-cell mass. Inability of adequate ¿-cell response to metabolic challenges results in hyperglycemia and frank diabetes mellitus. The incretin hormone glucagon-like peptide-1 (GLP-1) stimulates murine pancreatic ¿-cell proliferation. Upon binding to its receptor on ¿-cells, GLP-1 stimulates both cyclic AMP (cAMP) and phosphoinositol 3 kinase (PI3K) activation. Both cAMP and PI3K signaling pathways are implicated in ¿-cell 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, which stimulate gene transcription through histone deacetylase activity and stimulating RNA polymerase. On the other hand, PI3 kinase activates protein kinase B (PKB = Akt). PKB phosphorylates transcription factor FoxO1, which leads to its nuclear exclusion, thereby altering gene expression including derepressing transcription of pancreas duodenum homeobox-1 (PDX-1). PDX-1 is required for GLP-1 effects on ¿-cell proliferation and for ¿-cell mass adaptation to insulin resistance. Our preliminary results indicate that GLP-1 stimulates, via the cAMP-PKA-CREB pathway, transcription of the cell cycle positive regulator cyclin A2. Cyclin A2 upregulation is sufficient to augment mouse ¿-cell proliferation in vitro. Furthermore, in ¿-cells, PDX-1 is required for GLP-1 stimulated cAMP production, subsequent PKA activation and cyclin A2 expression. Thus, our findings suggest an interdependence of cAMP and PI3K mediated GLP-1 receptor signaling and a mechanistic link between PDX-1 and GLP-1 induced ¿-cell proliferation. The specific aims of this proposal are to further elucidate the role of GLP-1 induced cAMP/PKA signaling and of cyclin A2 in regulating ¿-cell proliferation. Aim 1 or our proposal is to examine in vivo cAMP/PKA-signaling effects in ¿-cell proliferation and cycle regulatory proteins by ¿- cell-specific ablation of the PKA regulatory subunit. Aim 2 is to examine the in vivo effects of ¿-cell specific cyclin A2 upregulation on islet mass and ¿-cell function. Aim 3 is to assess the consequences of ¿-cell specific cyclin A2 ablation on ¿-cell proliferation during development and in adult animals in response to GLP-1 and as adaptation to high-fat diet-induced insulin resistance. These studies of in vivo mechanisms of ¿-cell proliferation may provide therapeutic approaches for diabetes mellitus in humans. PUBLIC HEALTH RELEVANCE: Diabetes mellitus results from failure of insulin-producing ¿-cells to meet metabolic demands, and adaptation to the demands includes ¿-cell proliferation (i.e., duplication). In this proposal we aim to understand mechanism(s) governing pancreatic ¿-cell proliferation, in particular those mediated by the incretin hormone glucagon-like peptide-1. The studies proposed herein are significant because they may lead to therapeutic approaches for treating diabetes mellitus in humans.