Backer J M, Kahn C R, White M F
Research Division, Joslin Diabetes Center, Boston, Massachusetts.
J Biol Chem. 1990 Sep 5;265(25):14828-35.
We have studied the intracellular processing of insulin in the rat hepatoma cell line Fao. Fao cells internalized cohorts of surface-bound 125I-insulin or 125I-insulin-like growth factor II within 3-5 min. Degraded 125I-insulin-like growth factor II did not appear in the medium until 20-30 min after uptake, consistent with a time course of lysosomal delivery. In contrast, internalized insulin was completely degraded within 7-10 min. The half-times for dissociation and degradation of internalized insulin were identical at 37 degrees C (3 min), suggesting that the two processes occurred in the same compartment. Subcellular fractionation of Fao cells showed that a pulse of internalized insulin was largely intact after 3 min and associated with a light membrane fraction devoid of lysosomal markers. After an additional 4 min, the amount of insulin in this compartment decreased by 40%, with an increase in degraded insulin in the cytosol; no transfer of intact insulin to lysosomes or the cytosol was detected. The relationship between insulin-receptor dissociation and insulin degradation was further studied with inhibitors of insulin processing. Monensin blocked both dissociation and degradation of internalized insulin, as did incubation of the cells at 20 degrees C, suggesting that both endosomal acidification and endosomal fusion were required for insulin processing. At 25 degrees C, dissociation (+ t 1/2 = 12.9 min) preceded degradation (+ t 1/2 = 15.8 min). Inhibitors of lysosomal proteases were without effect on the half-time for either process. In contrast, bacitracin, an inhibitor of insulin degradation, caused a 2-fold increase in the half-times for both dissociation and degradation. Thus, intracellular insulin dissociation and degradation are tightly coupled endosomal processes in Fao cells, and insulin degradation facilitates the dissociation of insulin from its receptor inside the cell.
我们研究了大鼠肝癌细胞系Fao中胰岛素的细胞内加工过程。Fao细胞在3 - 5分钟内内化表面结合的125I -胰岛素或125I -胰岛素样生长因子II群体。降解的125I -胰岛素样生长因子II直到摄取后20 - 30分钟才出现在培养基中，这与溶酶体递送的时间进程一致。相比之下，内化的胰岛素在7 - 10分钟内完全降解。内化胰岛素解离和降解的半衰期在37℃时相同（3分钟），表明这两个过程发生在同一区室。Fao细胞的亚细胞分级分离显示，内化胰岛素脉冲在3分钟后大部分保持完整，并与不含溶酶体标记物的轻膜部分相关。再过4分钟后，该区室中胰岛素的量减少了40%，同时胞质溶胶中降解胰岛素增加；未检测到完整胰岛素向溶酶体或胞质溶胶的转移。我们用胰岛素加工抑制剂进一步研究了胰岛素 -受体解离与胰岛素降解之间的关系。莫能菌素阻断内化胰岛素的解离和降解，在20℃孵育细胞时也是如此，这表明胰岛素加工需要内体酸化和内体融合。在25℃时，解离（+ t1/2 = 12.9分钟）先于降解（+ t1/2 = 15.8分钟）。溶酶体蛋白酶抑制剂对这两个过程的半衰期均无影响。相比之下，胰岛素降解抑制剂杆菌肽使解离和降解的半衰期均增加了2倍。因此，细胞内胰岛素解离和降解是Fao细胞中紧密偶联的内体过程，胰岛素降解促进胰岛素在细胞内与其受体的解离。
