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REGULATION OF THE TWO PHASES OF INSULIN SECRETION

胰岛素分泌的两个阶段的调节

基本信息

批准号：
3153153
负责人：
WALTER S. ZAWALICH
金额：
$ 14.74万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-07-01 至 1987-06-30
项目状态：
已结题

项目摘要

There is considerable evidence to show that alterations in the pattern of insulin secretion from the beta cell of the endocrine pancreas may play a pathogenic role in type II diabetes. Normally, the pattern of insulin release in response to an increase in glucose concentration is a biphasic one. Recent work done by the principla investigator and his collaborators has led to the hypothesis that the two phases of insulin secretion are regulated by different biochemical branches within the calcium messenger system: the first branch operates via a rise in the calcium concentration of the cell cytosol activating calmodulin-dependent reactions; the second branch via a rise in diacylglycerol content activating C-kinase-dependent reactions. The purposes of the proposed studies are to establish this hypothesis more fully by studying the biochemical events which underline each phase of insulin secretion. First phase secretion will be induced by either glucose, the ionophore, A23187, or phospholipase A2. Examination of the effects of these agents on the following processes will be undertaken: a) the turnover of phosphoinositides and the production of diacylglycerol and inositol triphosphate; b) the concentration of Ca2+ in the cell cytosol; c) the phosphorylation of cytosolic proteins; and d) the production of arachidonic acid and its metabolism via the lypoxygenase and epoxygenase pathways. In addition, the products of these pathways will be employed to determine their effects on insulin release, islet cell calcium metabolism, and protein phosphorylation. Second phase secretion will be induced by either the phorbol ester, TPA, or the diglycerides 1-acetyl, 2-arachidonyl glycerol and 1-oley1 2-acetyl glycerol. Their effects on insulin secretion, cellular calcium metabolism, protein phosphorylation, and various aspects of lipid metabolism will all be studied. The basic method to be employed is the perifused rat pancreatic islet. Insulin will be measured by radioimmunoassay. The turnover of lipids is assessed by a variety of methods, but in particular, high pressure liquid chromatography. Protein phosphorylation will be examined using labeling with [32p]-HPO4= and separating protein by SDS-polyacrylamide slab gel electrophoresis either in one or two dimensions. Free calcium will be measured by the HOST-aequorin method of Borle and Snowdowne.

有大量证据表明，模式的改变内分泌胰腺β细胞分泌的胰岛素可能起到在 II 型糖尿病中的致病作用。正常情况下，胰岛素的模式响应葡萄糖浓度增加的释放是双相的一。主要研究者及其合作者最近完成的工作导致了这样的假设：胰岛素分泌的两个阶段是受钙信使内不同生化分支的调节系统：第一个分支通过钙浓度的升高来运作细胞胞质激活钙调蛋白依赖性反应；第二个通过二酰甘油含量的增加激活 C 激酶依赖性分支反应。拟议研究的目的是确定这一点通过研究强调的生化事件更全面地假设胰岛素分泌的各个阶段。第一相分泌将被诱导葡萄糖、离子载体、A23187 或磷脂酶 A2。考试这些试剂对以下过程的影响将是进行：a) 磷酸肌醇的营业额和生产二酰基甘油和三磷酸肌醇； b) Ca2+浓度细胞质； c) 胞质蛋白的磷酸化；和 d) 花生四烯酸的产生及其通过脂肪加氧酶的代谢环氧化酶途径。此外，这些途径的产物将用于确定它们对胰岛素释放、胰岛细胞钙的影响代谢和蛋白质磷酸化。第二期分泌将由佛波酯、TPA 或甘油二酯 1-乙酰基诱导， 2-花生四烯基甘油和1-油基1 2-乙酰基甘油。它们的影响胰岛素分泌、细胞钙代谢、蛋白质磷酸化、脂质代谢的各个方面都将被研究。基本的所采用的方法是灌注大鼠胰岛。胰岛素会通过放射免疫测定法测定。脂质的周转率通过评估多种方法，但特别是高压液体色谱法。将使用标记检查蛋白质磷酸化与[32p]-HPO4=并通过SDS-聚丙烯酰胺平板凝胶分离蛋白质一维或二维电泳。游离钙将通过Borle和Snowdowne的HOST-水母发光蛋白方法测量。