MORPHOLOGICAL STUDIES IN DIABETES AND METABOLISM

这项提案的目标是利用形态学和生物化学 分析胰岛素受体调节的方法。 超微结构研究 对胰岛素敏感细胞的作用表现出显著的细胞特异性 胰岛素组织、分布和流动性的异质性 受体。 这些形态学观察结果与细胞特异性 在相同细胞的生化结合研究中发现的差异。 最近 有证据表明，细胞与细胞之间的变异性可能存在于这些过程中， 参与胰岛素受体的内化和再循环。 这项建议的具体目标是： (1)定性和定量测定和比较胰岛素 受体组织和分布在不同的细胞类型。 将在占用胰岛素之间建立关系（如果有的话） 受体和特定的细胞表面结构和成分。 (2)分析细胞上胰岛素受体的后占据迁移率， 表明占位诱导的受体组织变化，或 分布 各种化学试剂将被用来试图阻止 受体微聚集，以确定所涉及的机制， 与胰岛素作用的关系。 (3)以确定参与的途径和亚细胞结构， 胰岛素的内化、降解或加工以及再循环， 胰岛素受体 除了使用单体铁蛋白-胰岛素进行形态学研究外， 使用抗胰岛素受体抗体和 生物素基标记的胰岛素与胰岛素受体结合，随后是抗体 或抗生物素蛋白缀合的胶体金颗粒和/或过氧化物酶将被 用于证明胰岛素受体的完整性以及 透化细胞 这个实验室处于一个独特的位置， 这一建议，因为我们已经证明有能力进行定量分析， 形态学和相关生化研究。 这些研究将 有助于我们理解胰岛素的作用和可能的位点， 胰岛素抵抗状态中的后结合缺陷。

The goal of this proposal is to use morphological and biochemical approaches to analyze insulin receptor regulation. Ultrastructural studies on insulin-sensitive cells have shown significant cell-specific heterogeneity in the organization, distribution and mobility of insulin receptors. These morphological observations correlate with cell-specific differences found in biochemical binding studies on the same cells. Recent evidence suggests that cell-to-cell variability may exist in the processes involved in insulin receptor internalization and recycling. The specific aims of this proposal are: (1) to qualitatively and quantitatively determine and compare insulin receptor organization and distribution on different cell types. Relationships, if any, will be established between occupied insulin receptor and specific cell surface structures and components. (2) to analyze the post-occupancy mobility of insulin receptors on cells that demonstrate occupancy-induced changes in receptor organization or distribution. Various chemical reagents will be used in attempts to block receptor microaggregation in order to determine the mechanisms involved and relationships to insulin action. (3) to determine the routes and subcellular structures involved in the internalization, degradation or processing, and recycling of insulin and insulin receptors. In addition to using monomeric ferritin-insulin for morphological studies, immunocytochemistry using both anti-insulin receptor antibody and biotinyl-labeled insulin binding to insulin receptors followed by antibody or avidin conjugated colloidal gold particles and/or peroxidase will be used to demonstrate the insulin receptors in intact as well as permeabilized cells. This laboratory is in a unique position to accomplish this proposal because of our proven capability to perform both quantitative morphological and correlative biochemical studies. These studies will contribute to our understanding of insulin action and of possible sites for post-binding defects in insulin resistant states.