Beta-cell Proliferation

描述（由申请人提供）：本提案的总体目标是定义控制胰腺细胞增殖的机制。生理和病理状态导致的胰岛素需求变化需要胰腺细胞的适应性功能改变。这些适应性变化包括胰岛素合成和分泌的增加，以及祖细胞增生和可能形成的新细胞。在这些机制中，细胞增殖和细胞周期调节在细胞质量的建立和决定中占主导地位。对代谢挑战缺乏足够的细胞反应可导致高血糖和糖尿病。胰高血糖素样肽-1 （GLP-1）刺激小鼠胰腺细胞增殖。在与细胞受体结合后，GLP-1刺激环AMP （cAMP）和磷酸肌醇3激酶（PI3K）的激活。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)，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，通过组蛋白去乙酰化酶活性和刺激RNA聚合酶刺激基因转录。另一方面，PI3激酶激活蛋白激酶B （PKB = Akt）。PKB磷酸化转录因子fox01，导致其核排斥，从而改变基因表达，包括抑制胰腺十二指肠同源盒-1 （PDX-1）的转录。PDX-1是GLP-1对细胞增殖的影响和对胰岛素抵抗的细胞群适应所必需的。我们的初步结果表明，GLP-1通过cAMP-PKA-CREB通路刺激细胞周期正调节因子cyclin A2的转录。细胞周期蛋白A2的上调足以增强小鼠体外细胞增殖。此外，在¿-细胞中，PDX-1是GLP-1刺激的cAMP产生、随后的PKA激活和cyclin 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受体信号相互依赖，PDX-1和GLP-1诱导的细胞增殖之间存在机制联系。本研究的具体目的是进一步阐明GLP-1诱导的cAMP/PKA信号和细胞周期蛋白A2在调节细胞增殖中的作用。目的1或我们的建议是通过PKA调节亚基的细胞特异性消融来研究体内cAMP/PKA信号在细胞增殖和周期调节蛋白中的作用。目的2是研究细胞特异性周期蛋白A2上调对胰岛质量和细胞功能的体内影响。目的3是评估细胞特异性周期蛋白A2消融在发育期间和成年动物中对GLP-1的反应以及对高脂肪饮食诱导的胰岛素抵抗的适应对细胞增殖的影响。这些体内细胞增殖机制的研究可能为人类糖尿病的治疗提供途径。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代谢需求，而对代谢需求的适应包括细胞增殖（即复制）。在这个建议中，我们的目标是了解控制胰腺细胞增殖的机制，特别是那些由肠促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治疗方法。

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.