Insulin binding, internalization, and receptor regulation in cultured human fibroblasts.
Insulin binding, internalization, and receptor regulation in cultured human fibroblasts.
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培养的人成纤维细胞中的胰岛素结合、内化和受体调节。
DOI：
10.1152/ajpendo.1981.241.3.e251
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Olefsky,JM
中科院分区：
文献类型：
--
作者：
BaldwinJr,D;Prince,M;Tsai,P;Johnson,C;Lotan,R;Rubenstein,AH;Olefsky,JM
利用培养的正常人成纤维细胞单层研究胰岛素与受体的结合。结合速度快，且与孵育温度成反比；在所研究的所有温度下，都实现了长时间的稳态结合，并且细胞外胰岛素的降解量最小。竞争曲线显示，当未标记的胰岛素浓度为7 ng/ml时，125i -胰岛素结合受到半抑制。结合数据的Scatchard图呈曲线状，显示成纤维细胞每个细胞含有约7000个受体位点。绑定125 i-insulin分离成纤维细胞与t 1/2的10分钟30摄氏度,35分钟16度C . 60分钟后离解在30摄氏度,45%的分离放射性由125 i-insulin降解产物,而只有8%的分离材料的降解产品后60分钟的离解16度C。这表明成纤维细胞具有温度敏感受体介导胰岛素降解的过程。单分子层与胰岛素的预孵育导致激素诱导的胰岛素受体的丢失。因此，将细胞与25 ng/ml胰岛素在37℃下孵育6小时，导致随后测量的125i -胰岛素结合减少50%。这种激素诱导的受体损失对生理胰岛素水平很敏感，大约5 ng/ml会导致一半的最大受体损失。当单层细胞用促溶体剂氯喹处理后，随后用5 X 10(-11) M 125i -胰岛素孵育，在30℃下120分钟后，观察到细胞相关放射性增加130%。总之，1)培养的正常人成纤维细胞具有胰岛素受体，其表现出与其他胰岛素靶细胞相同的动力学特性和特异性；2)具有生理胰岛素浓度的成纤维细胞孵育导致细胞表面胰岛素受体的显著丧失；3)受体结合的125i -胰岛素通过能量依赖的内吞途径内化，随后通过氯喹敏感反应降解。
Insulin binding to receptors was studied using monolayers of cultured normal human fibroblasts. Binding was rapid and inversely related to the incubation temperature; prolonged periods of steady-state binding were achieved at all temperatures studied and the amount of degradation of extracellular insulin was minimal. Competition curves demonstrated half-maximal inhibition of 125I-insulin binding at an unlabeled insulin concentration of 125I-insulin binding at an unlabeled insulin concentration of 7 ng/ml. Scatchard plots of the binding data were curvilinear and revealed that fibroblasts contained about 7,000 receptor sites per cell. Bound 125I-insulin dissociated from fibroblasts with a t 1/2 of 10 min at 30 degrees C and 35 min at 16 degrees C. After 60 min dissociation at 30 degrees C, 45% of the dissociated radioactivity consisted of 125I-insulin degradation products, whereas only 8% of the dissociated material was in the form of degraded products after 60 min of dissociation at 16 degrees C. This indicates that fibroblasts possess a temperature-sensitive receptor-mediated process for insulin degradation. Preincubation of the monolayers with insulin led to a hormone-induced loss of insulin receptors. Thus, incubating cells with 25 ng/ml insulin for 6 h at 37 degrees C caused a 50% reduction in subsequently measured 125I-insulin binding. This hormone-induced receptor loss was sensitive to physiologic insulin levels, with approximately 5 ng/ml causing a half-maximal receptor loss. When monolayers were treated with the lysosomotropic agent chloroquine and subsequently incubated with 5 X 10(-11) M 125I-insulin, a 130% increase in cell-associated radioactivity was observed after 120 min at 30 degrees C. In summary, 1) cultured normal human fibroblasts possess insulin receptors that exhibit kinetic properties and specificity identical to that of other insulin target cells; 2) incubation of fibroblasts with physiologic concentrations of insulin causes a marked loss of cell-surface insulin receptors; and 3) receptor-bound 125I-insulin is internalized through an energy-dependent endocytotic pathway and subsequently degraded by a chloroquine-sensitive reaction.