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: 3233567
• 负责人: PHILIPPE A HALBAN
• 金额: $ 8.39万
• 依托单位: UNIVERSITY OF GENEVA
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233567
• 关键词: Golgi apparatus; HTC cell; antibiotics; cell population study; cyproheptadine; diabetes mellitus; endoplasmic reticulum; flow cytometry; genetically modified animals; high performance liquid chromatography; hormone biosynthesis; hormone metabolism; immunocytochemistry; insulin; insulin receptor; laboratory mouse; laboratory rat; maleimides; pancreatic islet function; proinsulin; protein structure function; protein transport; receptor binding; species difference; transfection

Insulin production consists of a series of interdependent events involving movement of precursors from one organelle to the next and their proteolytic conversion. The underlying hypothesis of this Project is that structural domains of insulin and its precursors are implicated in each of these steps. Other domains will be equally important once insulin is released from the B-cell, being responsible for biological activity and, ultimately, degradation by target tissues. The longterm goals of this study are to: a) characterize each step in insulin production and pinpoint defects responsible for abnormal insulin production in given diabetic states; b) study the interplay between human and mouse insulins in transgenic mice in order to understand better the impact of bioartificial insulin delivery on B-cell function. The specific aims for the next 3 Year Period are: 1. How is proinsulin transported from the RER to the cis-Golgi? The effects of inhibitors on localization (immunocytochemistry) and chemistry of proinsulin in rat islet B-cells will be evaluated. Brefeldin A inhibits RER-Golgi transfer but allows the reverse movement of molecules (regurgitation from the Golgi). NEM (N-ethylmaleimide) prevents fusion of transport vesicles with their target organelle. Cyproheptadine leads to accumulation of material in dilated cisternae of the RER. 2. Which proinsulin domains are involved in targeting from the trans-Golgi to granules and in recognition by the conversion endoproteases? Proinsulin conversion/release will be studied in transfected AtT20 (pituitary corticotroph) cells. Studying expression of mutant genes will show whether the altered domain is involved in targeting or conversion. 3. Can proinsulin be converted (or partially converted) if released via the constitution pathway? FAO (hepatoma) cells will be transfected with the proinsulin gene. Since these cells do not express the regulated pathway, all proinsulin must be handled by the constitutive pathway. HPLC analysis of products synthesized and released by transfected FAO cells will show whether any conversion arises in this pathway. 4. Why is rat proinsulin I converted to insulin more rapidly than proinsulin II, and is there any difference in the biological activity of the two rat insulins? The rate of conversion of proinsulin to conversion intermediates (split proinsulins) and of intermediates to insulin will be studied in rat islets. The affinity of the rat insulin receptor for the two insulins will be measured, and the kinetics of receptor-mediated degradation followed. 5. Is newly synthesized proinsulin/insulin really released in preference to older, stored, insulin, or is the phenomenon merely a reflection of B- cell heterogeneity? B-cells will be separated from non-B-cells by flow cytometry and further sorted into metabolically active and inactive subpopulations based upon NAD(P)H autofluorescence. Rates of release of new (labeled) and old insulin will then be followed from the two subpopulations. 6. How is human insulin synthesis regulated in transgenic mouse B-cells, and what is its impact on endogenous insulin production and metabolism? Rates of synthesis and release of human and mouse insulins, as well as their biological activity, will be compared in transgenic mice expressing human insulin in their B-cells.

胰岛素的产生包括一系列相互依赖的事件， 前体从一个细胞器到下一个细胞器的运动及其蛋白水解 转换. 该项目的基本假设是， 胰岛素及其前体的结构域涉及这些中的每一个 步 一旦胰岛素被释放，其他领域将同样重要 从B细胞，负责生物活性，最终， 通过靶组织降解。 本研究的长期目标是：a） 描述胰岛素生产的每个步骤并查明缺陷 在给定的糖尿病状态下负责异常胰岛素产生; B） 在转基因小鼠中研究人类和小鼠胰岛素之间的相互作用， 为了更好地了解生物人工胰岛素输送对 B细胞功能 下一个三年期的具体目标是： 1. 胰岛素原是如何从粗面内质网转运到高尔基体的？的 抑制剂对定位（免疫细胞化学）和化学的影响 将评估大鼠胰岛B细胞中胰岛素原的水平。 布雷菲德菌素A抑制 RER-Golgi转移，但允许分子反向运动 （来自高尔基体的反流）。 NEM（N-乙基马来酰亚胺）可防止 运输囊泡和它们的目标细胞器。 赛庚啶导致 粗面内质网扩张池中物质的积累。 2. 哪些胰岛素原结构域参与了反式高尔基体的靶向 到颗粒和识别的转换内切蛋白酶？ 胰岛素原 将在转染的AtT 20（垂体）中研究转化/释放 促肾上腺皮质激素细胞）。 研究突变基因的表达将表明 改变的结构域参与靶向或转化。 3. 胰岛素原是否可以转化（或部分转化）， 体质途径？ FAO（肝细胞瘤）细胞将用以下转染： 胰岛素原基因 由于这些细胞不表达受调节的 在胰岛素原通路中，所有胰岛素原必须由组成性通路处理。 HPLC 对转染的FAO细胞合成和释放的产物的分析将 显示在该途径中是否发生任何转化。 4. 为什么大鼠胰岛素原I转化为胰岛素的速度比 胰岛素原II，以及是否有任何差异的生物活性， 两种老鼠胰岛素 胰岛素原转化率 中间体（裂解胰岛素原）和胰岛素中间体的生产将 在大鼠胰岛中进行了研究。 大鼠胰岛素受体对胰岛素的亲和力 将测量两种胰岛素，并测定受体介导的 退化随之而来。 5. 新合成的胰岛素原/胰岛素真的会优先释放吗 或者这种现象仅仅是B的反映- 细胞异质性？B细胞将通过流动与非B细胞分离 流式细胞术，并进一步分选成代谢活性和非活性 基于NAD（P）H自发荧光的亚群。 释放率 新的（标记的）和旧的胰岛素将随后从两个 亚群 6. 在转基因小鼠B细胞中人胰岛素合成是如何调节的， 它对内源性胰岛素的产生和代谢有什么影响？ 人和小鼠胰岛素的合成和释放速率，以及 它们的生物学活性，将在转基因小鼠中比较， B细胞中的人类胰岛素