PROCESSING OF PROINSULIN/INSULIN BY B-CELL ORGANELLES

胰岛素的产生包括一系列相互依赖的事件，包括 前体从一个细胞器到下一个细胞器的运动及其蛋白分解 转换。这个项目的基本假设是结构性的 胰岛素及其前体的结构域与每一个都有关联 台阶。一旦胰岛素释放，其他领域也将同样重要 从B细胞，负责生物活动，并最终， 靶组织的降解。这项研究的长期目标是：a) 描述胰岛素生产的每一步，并找出缺陷 对特定糖尿病状态下胰岛素的异常产生负责；b) 人和小鼠胰岛素在转基因小鼠体内的相互作用 为了更好地了解生物人工胰岛素给药的影响 B细胞功能。 今后三年的具体目标是： 1.胰岛素原是如何从粗面内侧核转运到顺式高尔基体的？这个 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细胞中胰岛素原的含量。灯盏花素A抑制 雷尔-高尔基转移，但允许分子反向运动 (来自高尔基山脉的反流)。NEM(N-乙基马来酰亚胺)阻止 将囊泡与它们的目标细胞器一起运输。赛庚啶导致 大脑皮层扩张脑池内物质积聚。 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，并在识别的转化内切酶？胰岛素原 将在转染腺病毒的AtT20(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 被改变的结构域涉及到靶向或转换。 3.胰岛素原如果通过释放，可以转化(或部分转化)吗 宪法路径？FAO(肝癌)细胞将被转基因 胰岛素原基因。由于这些细胞不表达受调控的 途径，所有的胰岛素原必须由构成途径处理。高效液相色谱仪 转基因粮农组织细胞合成和释放产物的分析 显示在此路径中是否发生了任何转换。 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，对胰岛素原的生物活性有影响吗？ 两只老鼠的胰岛素？胰岛素原转化率 中间体(分裂的胰岛素原)和胰岛素的中间体将是 在大鼠胰岛上进行了研究。大鼠胰岛素受体与胰岛素受体的亲和力 将测量两种胰岛素，以及受体介导的动力学 退化也随之而来。 5.新合成的胰岛素原/胰岛素真的优先释放吗？ 对于较老的、储存的、胰岛素，或者这一现象仅仅是B- 细胞异质性？B细胞和非B细胞将通过流动分离 细胞计数法，并进一步分为代谢活跃和不活跃 基于NAD(P)H自发荧光的亚群。释放率 新的(标记的)和旧的胰岛素将在这两种胰岛素之后 亚群。 6.人胰岛素在转基因小鼠B细胞中的合成是如何调节的， 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度，以及 它们的生物活性，将在表达它们的转基因小鼠中进行比较 B细胞中的人胰岛素。

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.