PHOSPHOINOSITIDE HYDROLYSIS AND BETA CELL SECRETION

磷酸肌醇水解和 β 细胞分泌

• 批准号: 6380613
• 负责人: WALTER S. ZAWALICH
• 金额: $ 29.99万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6380613
• 关键词: Adenoviridae; biological signal transduction; body weight; cyclic AMP; dexamethasone; enzyme activity; fasting; genetic transduction; genetically modified animals; glucose; glucose metabolism; hydrolysis; inositol phosphates; insulin; isozymes; laboratory mouse; laboratory rat; pancreatic islet function; pancreatic islets; phosphatidylinositols; phospholipase C; protein kinase C; secretion; transfection /expression vector; western blottings

The regulation of glucose homeostasis depends on the secretion of insulin from the pancreatic beta-cell in amounts commensurate to satisfy peripheral tissue requirements. Playing a strategic role in the regulation of insulin release in the phospholipase C (PLC)/protein kinase C (PKC) transduction system. Islets of Langerhans contain the three major isozymes of PLC(beta1, gamma1 and delta1) and the calcium- dependent PKC isozyme, PKCalpha. Species differences in the content and activation of PLC and PKC have been identified and these differences play a major role in regulating the response to glucose. For example, rat islets contain 5 times as much PLCdelta1 as do mouse islets and in response to glucose stimulation inositol phosphates (IPs), a marker for PLC activation, increase 400%. The activation of PLC in rat islets is paralleled by a rising 30-fold increase in second phase release. In contrast, mouse islet responses to glucose are notable for the minimal activation of PLC and the small, flat second phase insulin secretory response. Rat and human islets, but not mouse islets, can be sensitized by prior short term exposure to glucose, a process termed time-dependent potentiation (TDP), or they can be desensitized by long term exposure to glucose, referred to as glucose toxicity or time-dependent suppression (TDS). The immunity of mouse islets to these multiple effects of glucose is a consequence of glucose's inability to activate PLC: significant increases in PLC activation, second phase insulin secretion and the induction of both TDP and TDS result when mouse islets are stimulated with carbachol, an agonist that significantly activates an isozyme of PLC distinct from that activated by glucose. These and other observations have led us to conclude that PLC/PKC activation regulates physiologic insulin secretion and that disordered PLC/PKC signaling culminates in disordered insulin secretion. Experiments described critically test this concept using rat and mouse islets. We will also treat cultured rat or mouse islets with adenoviral vectors containing the cDNAs for the isozymes of PLC and to determine if the increased expression and subsequent activation of these enzymes reestablishes sensitivity to glucose. Finally, the impact of the regulated over- expression of the major PLC isozymes and PKCalpha (using a reverse tetracycline-controlled transactivator system) on in vivo glucose homeostasis and in vitro sensitivity to stimulation will be determined in transgenic mice. These studies will define the role of PLC/PKC activation in regulating islet response patterns to glucose stimulation and they may facilitate the development of genetically-engineered cell lines designed to replace missing of defective beta-cells.

葡萄糖稳态的调节取决于 来自胰腺β细胞的胰岛素的量足以满足 外周组织的要求。在其中发挥战略作用 磷脂酶 C (PLC)/蛋白质中胰岛素释放的调节 激酶 C (PKC) 转导系统。朗格汉斯岛含有 PLC 的三种主要同工酶（beta1、gamma1 和 delta1）和钙- 依赖性 PKC 同工酶，PKCalpha。含量和种类的差异 PLC 和 PKC 的激活已被识别，这些差异 在调节对葡萄糖的反应中发挥重要作用。例如， 大鼠胰岛含有的 PLCdelta1 是小鼠胰岛的 5 倍，并且 对葡萄糖刺激的反应 磷酸肌醇 (IP)，一种标记物 PLC激活，提升400%。大鼠胰岛中 PLC 的激活是 与此同时，第二阶段的释放量增加了 30 倍。在 相比之下，小鼠胰岛对葡萄糖的反应显着最小 PLC 的激活和小而平坦的第二阶段胰岛素分泌 回复。大鼠和人类胰岛可致敏，但小鼠胰岛不敏感 通过之前短期接触葡萄糖，这一过程称为时间依赖性 增强作用（TDP），或者它们可以通过长期暴露而脱敏 葡萄糖，称为葡萄糖毒性或时间依赖性抑制 （总固体溶解度）。小鼠胰岛对葡萄糖的多重影响的免疫力 是葡萄糖无法激活 PLC 的结果：显着 PLC 激活、第二相胰岛素分泌和 当小鼠胰岛受到刺激时，TDP 和 TDS 都会被诱导产生 与卡巴胆碱（一种显着激活同工酶的激动剂） PLC与葡萄糖激活的PLC不同。这些和其他 观察结果使我们得出结论，PLC/PKC 激活调节 生理性胰岛素分泌和 PLC/PKC 信号传导紊乱 最终导致胰岛素分泌紊乱。实验描述 使用大鼠和小鼠胰岛严格测试这个概念。我们还将 用含有以下成分的腺病毒载体处理培养的大鼠或小鼠胰岛 PLC同工酶的cDNA并确定是否增加 这些酶的表达和随后的激活重新建立 对葡萄糖的敏感性。最后，过度监管的影响 主要 PLC 同工酶和 PKCalpha 的表达（使用反向 四环素控制的反式激活系统）对体内葡萄糖的影响 将确定稳态和体外对刺激的敏感性 在转基因小鼠中。这些研究将定义 PLC/PKC 的作用 激活调节胰岛对葡萄糖刺激的反应模式 它们可能会促进基因工程细胞的发展 旨在替代缺失的有缺陷的β细胞的细胞系。