PHOSPHOINOSITIDE HYDROLYSIS AND BETA CELL SECRETION

磷酸肌醇水解和 β 细胞分泌

• 批准号: 6736853
• 负责人: WALTER S. ZAWALICH
• 金额: $ 34.74万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736853
• 关键词: antisense nucleic acid; biological signal transduction; enzyme activity; enzyme inhibitors; gene expression; gene targeting; genetically modified animals; glucose; hydrolysis; hyperinsulinism; insulin; insulinlike growth factor; isozymes; laboratory mouse; laboratory rat; muscarinic receptor; noninsulin dependent diabetes mellitus; obesity; oligonucleotides; pancreatic islet function; phosphatidylinositol 3 kinase; phosphatidylinositols; phospholipase C; protein kinase C; secretion

DESCRIPTION (provided by applicant): The present proposal has four major objectives that are based on the following considerations: A. Genetically endowed with the capacity to respond to the prevailing glucose level by altering insulin secretory rates, the pancreatic beta-cell plays a preeminent role in the maintenance of glucose homeostasis. B. Phospholipase C (PLC)/protein kinase C (PKC) activation regulates insulin secretory responses to glucose. C. Pronounced species differences exist in the expression levels of several PLC isozymes and this appears responsible for the profound differences in glucose-stimulated insulin secretion that exist between rat and mouse islets. D. Muscarinic cholinergic receptor knockouts afford us the opportunity to establish how signaling via PLC activation contributes to biphasic insulin secretion, time-dependent potentiation and time-dependent suppression of secretion and the in vivo consequences of these alterations. E. Genetic manipulation of the proteins that control insulin signaling have led to a reevaluation of the autocrine effects of insulin and/or insulin-like growth factor I (IGF-I) on the beta-cell. F. Disrupted insulin secretion culminates in diabetes. These are the Specific Aims. 1. Using the rat insulin II promoter to achieve tissue-specific beta-cell expression, transgenic mice overexpressing PLCbeta1 and PLCdelta1 will be generated. Will the overexpression of these isozymes convert the minimal mouse islet glucose response to the exuberant biphasic insulin secretory response that characterizes glucose-stimulated rat or human islets? 2. Coupled to PLCbeta1 via a muscarinic receptor, cholinergic stimulation of mouse islets compensates for their minimal in vivo and in vitro responses to glucose stimulation. Muscarinic (M1 and M3) receptor knockouts have been generated. How do these alterations change the constellation of effects that cholinergic stimulation exerts on the beta-cell? Are there compensatory changes in PLC isozyme expression and how do these animals respond to diets designed to induce obesity? 3. Antisense oligonucleotides will be employed to determine the relationship between reductions in PKCalpha expression in islets and their secretory responsiveness. 4. IGF-I inhibits insulin secretion, presumably by the activation of phosphatidyinositol 3-kinase (PI3k), and reduced expression of the p85alpha regulatory subunit of PI3K is associated with augmented glucose-induced secretion. Can we identify the biochemical pathways sensitive to IGF-I signaling in the beta-cell? Considering the fact that inhibitors of PI3K are as potent as the oral secretagogues utilized to treat Type II diabetes, potentially new therapeutic targets may be identified. These aims will be accomplished by using sound principles of experimentation and employing isolated islets that retain their in vivo sensitivity to glucose.

描述（由申请人提供）：本提案有四个主要目标，基于以下考虑：A。胰腺β细胞具有通过改变胰岛素分泌速率来响应当前葡萄糖水平的遗传能力，在维持葡萄糖稳态方面发挥着卓越的作用。 B。磷脂酶C（PLC）/蛋白激酶C（PKC）激活调节胰岛素对葡萄糖的分泌反应。 C.明显的物种差异存在于几种PLC同工酶的表达水平，这似乎是导致大鼠和小鼠胰岛之间存在的葡萄糖刺激的胰岛素分泌的深刻差异的原因。D.毒蕈碱胆碱能受体敲除为我们提供了机会，以建立如何通过PLC激活信号有助于双相胰岛素分泌，时间依赖性增强和时间依赖性抑制分泌和这些改变的体内后果。 E.控制胰岛素信号传导的蛋白质的遗传操作已经导致重新评估胰岛素和/或胰岛素样生长因子I（IGF-I）对β细胞的自分泌作用。F.胰岛素分泌紊乱最终导致糖尿病。这些是具体的目标。1.使用大鼠胰岛素II启动子实现组织特异性β细胞表达，将产生过表达PLC β 1和PLC δ 1的转基因小鼠。这些同工酶的过度表达是否会将最小的小鼠胰岛葡萄糖反应转化为葡萄糖刺激的大鼠或人胰岛所特有的旺盛的双相胰岛素分泌反应？2.通过毒蕈碱受体与PLC β 1偶联，小鼠胰岛的胆碱能刺激补偿了它们对葡萄糖刺激的最小体内和体外反应。已经产生了毒蕈碱（M1和M3）受体敲除。这些改变如何改变胆碱能刺激对β细胞产生的一系列影响？PLC同工酶表达是否有代偿性变化？这些动物对诱导肥胖的饮食有何反应？3.反义寡核苷酸将用于确定胰岛中PKCalpha表达的减少与其分泌反应性之间的关系。4. IGF-I可能通过激活磷脂酰肌醇3-激酶（PI 3 k）抑制胰岛素分泌，而PI 3 k的p85 α调节亚基表达减少与葡萄糖诱导的胰岛素分泌增加相关。我们能否确定β细胞中对IGF-I信号敏感的生化途径？考虑到PI 3 K抑制剂与用于治疗II型糖尿病的口服促分泌素一样有效的事实，可以鉴定潜在的新治疗靶点。这些目标将通过使用合理的实验原理和采用保持其体内对葡萄糖的敏感性的分离的胰岛来实现。