Levy J R, Olefsky J M
Department of Medicine, University of California-San Diego.
Endocrinology. 1987 Dec;121(6):2075-86. doi: 10.1210/endo-121-6-2075.
The processing and trafficking of insulin in cultured rat hepatocytes were studied. A time course of binding of radiolabeled insulin to hepatocytes at 37 C revealed a rapid rise in cell-associated radioactivity that reached a steady state by 20 min. Using an acid medium to extract insulin bound to surface receptors, the time courses of receptor binding and internalization of the ligand were characterized. The earliest event in insulin processing was the binding of insulin to surface receptors, reaching steady state by 20 min with a t1/2 of 4 min. The internalization rate of ligand was initially slower than the binding rate, with a t1/2 of 6 min. Similar internalization rates of the insulin receptor were found by measuring the trypsin sensitivity of hepatocyte insulin receptors covalently occupied with a photo-affinity-labeled derivative of insulin [( 125I]B2 (2-nitro-4-azido-phenylacetyl)Des-PheB1-insulin). At steady state, the internalized ligand and receptor comprised approximately 40-45% of the cell-associated radioactivity. The time course of intracellular degradation was assessed by trichloroacetic acid (TCA) precipitability and Sephadex G-50 gel chromatography of solubilized cells containing only internalized radioactivity. Intracellular TCA-soluble and low mol wt degradation products first appeared by 5 min and were released from the cell 3 min later. Chloroquine (100 microM) completely inhibited the formation of intracellular low mol wt degradation products as well as their appearance in the medium. The release of intracellular radioactivity was assessed by first removing surface-bound insulin with acid extraction. Eighty percent of the intracellular radioactivity was released in 45 min with a t1/2 of 8 min. The released radioactivity was assessed by TCA precipitability and gel chromatography. These results demonstrate that after 20 min, 43% of the released intracellular radioactivity is intact insulin. The percentage of intact insulin released increases in a dose-dependent fashion as the amount of insulin bound and internalized increases. In conclusion, the earliest event in insulin processing is binding to surface receptors. After a short delay, insulin and its receptor are internalized and trafficked into either a chloroquine-sensitive degradative pathway or a chloroquine-insensitive retroendocytotic pathway. The amount of insulin that traverses the nondegradative retroendocytotic pathway is proportional to the amount of insulin bound and internalized by the cell.
研究了培养的大鼠肝细胞中胰岛素的加工和运输过程。在37℃下，放射性标记胰岛素与肝细胞结合的时间进程显示，细胞相关放射性迅速上升，20分钟时达到稳定状态。使用酸性介质提取与表面受体结合的胰岛素，对配体的受体结合和内化的时间进程进行了表征。胰岛素加工的最早事件是胰岛素与表面受体结合，20分钟时达到稳定状态，t1/2为4分钟。配体的内化速率最初比结合速率慢，t1/2为6分钟。通过测量与胰岛素的光亲和标记衍生物[（125I]B2（2-硝基-4-叠氮基-苯乙酰基）去苯丙氨酸B1-胰岛素）共价占据的肝细胞胰岛素受体的胰蛋白酶敏感性，发现胰岛素受体的内化速率相似。在稳定状态下，内化的配体和受体约占细胞相关放射性的40-45%。通过三氯乙酸（TCA）沉淀性和仅含有内化放射性的溶解细胞的Sephadex G-50凝胶色谱法评估细胞内降解的时间进程。细胞内TCA可溶性和低分子量降解产物在5分钟时首次出现，并在3分钟后从细胞中释放。氯喹（100 microM）完全抑制细胞内低分子量降解产物的形成及其在培养基中的出现。通过先用酸提取去除表面结合的胰岛素来评估细胞内放射性的释放。80%的细胞内放射性在45分钟内释放，t1/2为8分钟。通过TCA沉淀性和凝胶色谱法评估释放的放射性。这些结果表明，20分钟后，43%的释放的细胞内放射性是完整的胰岛素。随着结合和内化的胰岛素量增加，完整胰岛素释放的百分比以剂量依赖性方式增加。总之，胰岛素加工的最早事件是与表面受体结合。短暂延迟后，胰岛素及其受体被内化并运输到对氯喹敏感的降解途径或对氯喹不敏感的逆向胞吞途径。穿过非降解性逆向胞吞途径的胰岛素量与细胞结合和内化的胰岛素量成正比。
