Baldwin D, Prince M, Tsai P, Johnson C, Lotan R, Rubenstein A H, Olefsky J M
Am J Physiol. 1981 Sep;241(3):E251-60. doi: 10.1152/ajpendo.1981.241.3.E251.
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.
使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。结合迅速且与孵育温度呈负相关；在所有研究温度下均实现了长时间的稳态结合，且细胞外胰岛素的降解量极少。竞争曲线表明，在未标记胰岛素浓度为7 ng/ml时，可使125I - 胰岛素结合抑制一半。结合数据的Scatchard图呈曲线状，显示成纤维细胞每个细胞约含7000个受体位点。在30℃时，结合的125I - 胰岛素从成纤维细胞解离的半衰期为10分钟，在16℃时为35分钟。在30℃解离60分钟后，45%的解离放射性由125I - 胰岛素降解产物组成，而在16℃解离60分钟后，只有8%的解离物质呈降解产物形式。这表明成纤维细胞具有温度敏感的受体介导的胰岛素降解过程。用胰岛素预孵育单层细胞会导致激素诱导的胰岛素受体丧失。因此，在37℃用25 ng/ml胰岛素孵育细胞6小时会使随后测量的125I - 胰岛素结合减少50%。这种激素诱导的受体丧失对生理胰岛素水平敏感，约5 ng/ml可导致一半最大受体丧失。当用溶酶体促渗剂氯喹处理单层细胞，随后用5×10(-11) M 125I - 胰岛素孵育时，在3℃孵育若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，其动力学特性和特异性与其他胰岛素靶细胞相同；2）用生理浓度的胰岛素孵育成纤维细胞会导致细胞表面胰岛素受体显著丧失；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。
