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

• 批准号: 3233571
• 负责人: PHILIPPE A HALBAN
• 金额: $ 8.71万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 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/3233571
• 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

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细胞产生胰岛素涉及许多密切相关的过程。 综合步骤。 了解胰岛素产生的唯一方法是 整体，从而识别与之相关的潜在缺陷，或 负责，糖尿病，是为了更好地了解 这些步骤的分子机制。 这是我们的目标。 本研究。 为此，有必要研究B细胞亚细胞 部分（高尔基体亚部分，颗粒亚群和溶酶体）。 由于亚细胞分离需要的组织比 使用分离的胰岛容易获得，大鼠胰岛素瘤细胞将被 采用 胰岛素瘤细胞将首先在以下方面进行表征： 胰岛素原生物合成和转化以及胰岛素释放，以验证 作为B细胞功能的模型。 为了改变密度或 在亚分级分离之前感兴趣的细胞器的其它性质， 从而提高了细分过程的分辨率， 产品（包括胰岛素原）通过高尔基复合体的运输将 被莫能菌素或三羟甲基氨基甲烷阻断 胰岛素原转化为胰岛素将是 被精氨酸和赖氨酸类似物的掺入所阻断;这导致 包衣颗粒成熟至未包衣颗粒成熟过程中的相关阻滞 (the网格蛋白包被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）高尔基复合体对胰岛素原的加工： 胰岛素原必须以某种方式被高尔基复合体识别， 集中在选择的高尔基体区域， 包装成分泌颗粒。 识别过程可能是 受体介导的 高尔基体膜上的胰岛素原受体 被研究。 2)胰岛素原向胰岛素的转化： 高尔基复合体中的转化起始将被定位， 负责将胰岛素原和转化酶 接触研究。 3)颗粒异质性： 将研究包衣和未包衣颗粒的重要性， 特别提到负责优先执行的机制， 释放新合成的胰岛素。 4)胰岛素储备的降解： 将研究该途径的机制和调节。 食鼻癖 是最好的途径，但尚未得到生物化学证实。 的影响 胰岛素晶体对胰岛素降解的敏感性 将评价溶酶体。