INSULIN RECEPTOR METABOLISM IN 3T3-L1 AND 3T3-C2 CELLS

胰岛素对细胞表面胰岛素受体数量的影响 会导致胰岛素抵抗。受体调节的现行模型 建议胰岛素将表面受体的分布转移到 内部部位，增加某些细胞中受体的降解。这个 降血糖药物甲苯磺丁胺可抵消这一过程，但未知 提高细胞表面受体表达水平的机制。 该提案将继续对受体中的步骤进行评估 胰岛素和甲苯磺丁胺对3T3-L1脂肪细胞加工的调节。这个 表面受体运动的动力学将通过对两者进行量化来评估 分离的笼状蛋白包被小泡(包被小凹)与受体的结合 抗网织蛋白抗体，及其在表面和内部之间的转运 以其各自的敏感度或抵抗力表示的池 蛋白质分解。甲苯磺丁胺和胰岛素对心脏功能的影响 将测量标记受体的浓度和降解率 他们的关系也建立起来了。这些研究和进一步的分析 光标记受体新陈代谢应该确定这些受体的作用部位。 受体的占据和解离在体内的作用 正常的受体调节。受体位置与基因表达的关系 磷酸盐和唾液酸含量，以及影响等电点和 大小将受到监控。与受体相互作用的蛋白质也将是 通过吸附标记的细胞提取物与纯化的受体结合来鉴定 硝化纤维素或亲和载体，并通过原位交联化 在完整或通透性的细胞中标记的细胞蛋白与受体。 胰岛素或甲苯丁胺中与受体相互作用的蛋白质 受调控的方式将被提纯并进一步表征。在这两个地方 相受体的分离和定量将通过免疫沉淀和 凝胶电泳法。 这些研究应该更直接地定义动力学和路线 受体处理，确定重要的受体相互作用或 所涉及的修饰及其在调节胰岛素效应中的作用 和甲苯磺丁胺对受体的作用。这些研究应该提供 更好地了解某些胰岛素抵抗疾病的影响 胰岛素受体代谢和功能的状态及其治疗。

The reduction of the number of cell-surface insulin receptors by insulin contributes to insulin resistance. A current model for receptor regulation proposes that insulin shifts the distribution of surface receptor to internal sites, increasing receptor degradation in some cells. The hypoglycemic drug tolbutamide counteracts this process by unknown mechanisms increasing the level of surface receptor expressed by the cell. The proposal will continue an evaluation of the steps in receptor processing regulated by insulin and tolbutamide in 3T3-L1 adipocytes. The kinetics of surface receptor movement will be assessed by quantitating both receptor association with clathrin coated vesicles (coated pits) isolated with anticlathrin antibody, and its transit between surface and internal pools represented by its respective sensitivity or resistance to proteolysis. The effects of tolbutamide and insulin on the internal concentration and rate of degradation of labelled receptor will be measured and their relationship established. These studies and further analysis of photolabelled receptor metabolism should define the site of action of these agents and determine the role of receptor occupancy and dissociation in normal receptor regulation. The relationship of receptor location to phosphate and sialic acid content, and to modifications affecting pI and size will be monitored. Protein interacting with the receptor also will be identified by adsorbing labelled cell extracts to purified receptor bound to nitrocellulose or affinity supports, and by in situ crosslinkling of labelled cellular proteins to receptor in intact or permeabilized cells. Proteins interacting with the receptor in an insulin or tolbutamide regulated manner will be purified and further characterized. In both phases receptor will be isolated and quantitated by immunoprecipitation and gel electrophoresis. These studies should more directly define the kinetics and routes of receptor processing, identify the important receptor interactions or modifications involved, and their role in mediating the effects of insulin and tolbutamide on receptor processing. These studies should provide better understanding of the effects of some insulin resistant disease states and their treatment on insulin receptor metabolism and function.