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

胰岛素减少细胞表面胰岛素受体的数量 导致胰岛素抵抗。 受体调节的当前模型 提出胰岛素改变了表面受体的分布， 内部位点，增加某些细胞中的受体降解。 的 降血糖药物甲苯磺丁脲通过未知的方式抵消这一过程 增加细胞表达的表面受体水平的机制。 该提案将继续评估接受国的步骤， 在3 T3-L1脂肪细胞中，胰岛素和甲苯磺丁脲调节的加工。 的 表面受体运动的动力学将通过定量 受体与分离的网格蛋白包被囊泡（包被小凹）的结合 与抗胞苷抗体，及其之间的转运表面和内部 由其各自的敏感性或耐药性表示的池 蛋白水解 甲苯磺丁脲和胰岛素对内分泌功能的影响 将测量标记受体的浓度和降解速率 他们的关系建立起来了。 这些研究和进一步分析 光标记的受体代谢应确定这些的作用位点， 试剂和确定受体占有和解离在 正常受体调节。 受体定位与 磷酸盐和唾液酸含量，以及影响pI和 规模将受到监控。 与受体相互作用的蛋白质也将被 通过将标记的细胞提取物吸附到纯化的受体结合物上 硝化纤维素或亲和支持物，并通过原位交联 标记的细胞蛋白质与完整或透化细胞中的受体结合。 与胰岛素或甲苯磺丁脲中受体相互作用的蛋白质 调节的方式将被纯化并进一步表征。 无论是 通过免疫沉淀分离和定量相受体， 凝胶电泳 这些研究应该更直接地确定药物的动力学和途径。 受体处理，识别重要的受体相互作用或 涉及的修饰，以及它们在介导胰岛素作用中的作用 和甲苯磺丁脲对受体加工的影响。 这些研究将提供 更好地了解一些胰岛素抵抗性疾病的影响 状态及其治疗对胰岛素受体代谢和功能的影响。

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.