PROCESSING OF PROINSULIN/INSULIN BY B-CELL ORGANELLES

B 细胞细胞器对胰岛素原/胰岛素的加工

基本信息

批准号：
3153833
负责人：
PHILIPPE A HALBAN
金额：
$ 11.48万
依托单位：
JOSLIN DIABETES CENTER
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-04-01 至 1989-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3153833
关键词：
Golgi apparatus exocytosis gel electrophoresis granule hormone biosynthesis hormone metabolism hyperinsulinism insulin lysosomes molecular pathology neoplastic cell culture for noncancer research pancreatic islet function proinsulin proteolysis receptor mediated endocytosis secretion vesicle /vacuole

项目摘要

Insulin production by the pancreatic B-cell involves a number of closely integrated steps. The only way to understand insulin production as a whole, and thus identify the potential defects associated with, or responsible for, diabetes, is to gain a better understanding of the molecular mechanism of these individual steps. This is the goal of the present study. For this it will be necessary to study B-cell subcellular fractions (Golgi subfractions, granule subpopulations, and lysosomes). Since subcellular fractionation demands larger amounts of tissue than are readily available using isolated islets, rat insulinoma cells will be used. The insulinoma cells will first be characterized in terms of proinsulin biosynthesis and conversion, and insulin release, to validate them as a model for B-cell function. In order to modify the density or other properties of the organelles of interest before subfractionation, thereby improving the resolution of the subfractionation procedure, the transport of products (including proinsulin) across the Golgi complex will be blocked with monensin or Tris. Proinsulin conversion to insulin will be blocked by incorporation of arginine and lysine analogs; this results in an associated block in the maturation of coated to mature uncoated granules (the clathrin-coated granule being the earliest immature form). The biochemical/ultrastructural transformations selected for detailed study and the issues raised are: 1) Proinsulin processing by the Golgi comlex: proinsulin must in some way be recognized by the Golgi complex and thereby concentrated in select Golgi regions for channeling towards, and ultimate packaging into, secretory granules. The recognition process could be receptor mediated. Putative proinsulin receptors on Golgi membranes will be studied. 2) Proinsulin conversion to isulin: the precise site of initiation of conversion in the Golgi complex will be localized, and the mechanism responsible for bringing proinsulin and the conversion enzyme into contact studied. 3) Granule heterogeneity: the functional significance of coated and uncoated granules will be studied, with particular reference to the mechanism responsible for the preferential release of newly synthesized insulin. 4) Degradation of insulin stores: the mechanism and regulation of this pathway will be studied. Crinophagy is the favored path but has not been confirmed biochemically. The impact of the insulin crystal on sensitivity of insulin to degradation within lysosomes will be evaluated.

胰腺B细胞产生的胰岛素涉及许多密切集成步骤。理解胰岛素产生的唯一方法整个，从而确定与或负责糖尿病，是为了更好地理解这些各个步骤的分子机制。这是目前的研究。为此，有必要研究B细胞亚细胞馏分（高尔基体亚构件，颗粒亚群和溶酶体）。由于亚细胞分馏需要比使用分离的小岛随时可用，大鼠胰岛素瘤细胞将是用过的。胰岛素瘤细胞将首先以促生物素生物合成和转化率以及胰岛素释放，以验证它们是B细胞函数的模型。为了修改密度或分流之前，感兴趣的细胞器的其他特性，从而改善了分数的分辨率，产品（包括促硫素）在高尔基体综合体中的运输（包括促硫素）用Monensin或Tris阻塞。促胰岛素转化为胰岛素将是通过掺入精氨酸和赖氨酸类似物而阻塞；这导致相关块在涂层到成熟的未涂层颗粒的成熟中（涂有网状蛋白的颗粒是最早的未成熟形式）。选择用于详细研究的生化/超微结构转化提出的问题是：1）Golgi Comlex的Proinsulin加工：高尔基综合体必须以某种方式认识到proinsulin，从而集中在选择的高尔基区域，用于引导和最终包装成分泌的颗粒。识别过程可能是受体介导。高尔基膜上的假定素受体将被研究。 2）Proinsulin转化为Isulin：高尔基体综合体中转换的启动将被定位，并且负责带来促硫素和转化酶的机制进入联系人。 3）颗粒异质性：功能将研究涂层和未涂层颗粒的意义，并研究特别提及负责优先的机制释放新合成的胰岛素。 4）胰岛素商店的降解：将研究该途径的机制和调节。 Crinophagy 是受欢迎的路径，但尚未在生化上得到证实。影响胰岛素晶体对胰岛素对降解的敏感性将评估溶酶体。