Olefsky J, Johnson J, Liu F, Edwards P, Baur S
Diabetes. 1975 Sep;24(9):801-10. doi: 10.2337/diab.24.9.801.
We have compared the ability of rat liver plasma membranes and isolated hepatocytes to bind and degrade insulin. Isolated cells were prepared in two different ways: by mechanical separation of cells and by collagenase digestion of extracellular matric. In all studies the binding and degradative characteristics of both types of hepatocyte preparations were identical. Furthermore, with one exception, the binding characteristics of membranes and cells were also quite similar. The only exception concerned the amount of insulin bound by hepatocytes as compared to liver membranes. Thus, at concentrations of cells (1.2 times 10(6) cells per milliliter) and membranes (150 mug. protein per milliliter) that gave equal binding at insulin concentrations less than 100 ng./ml., the amount of insulin specifically bound at insulin concentrations greater than 100 ng./ml. was greater with use of hepatocytes. Additional studies indicated that, in contrast to membranes, at the higher insulin concentration only 75 per cent of the previously bound insulin could be recovered from hepatocytes. Thus, a nondissociable component exists; which probably represents intracellular radioactivity and appears to account for the higher specific insulin binding by cells at higher insulin concentrations. When insulin degradation was studied at the above hepatocyte and plasma membrane concentrations, cells degraded 30 per cent more insulin than did membranes. Kinetic analysis of these data revealed that the Km for insulin degradation (5 times 10(-7) M at 37 degrees) was the same for both systems wereas the Vmax was greater with use of hepatocytes.
(1) Preparation of hepatocytes by collagenase digestion does not appear to alter insulin binding or degradation; (2) studies of liver membranes and isolated hepatocytes obtained from normal rats should yield similar information about insulin-receptor interaction as long as insulin concentrations less than 100 ng./ml. are used; (3) at very high insulin concentrations, some of the radioactivity appears to enter the cells; (4) the kinetics of insulin degradation by hepatocytes and liver membranes are similar; and (5) insulin degradation appears to be primarily a membrane phenomenon.
我们比较了大鼠肝细胞膜和分离的肝细胞结合及降解胰岛素的能力。分离细胞有两种不同方法制备：通过机械分离细胞和通过胶原酶消化细胞外基质。在所有研究中，两种类型肝细胞制剂的结合和降解特性相同。此外，除一个例外，膜和细胞的结合特性也非常相似。唯一的例外涉及与肝细胞膜相比肝细胞结合的胰岛素量。因此，在细胞浓度（每毫升1.2×10⁶个细胞）和膜浓度（每毫升150μg蛋白质）下，在胰岛素浓度低于100ng/ml时结合量相等，但在胰岛素浓度高于100ng/ml时，使用肝细胞时特异性结合的胰岛素量更大。进一步研究表明，与膜相反，在较高胰岛素浓度下，仅75%先前结合的胰岛素可从肝细胞中回收。因此，存在一种不可解离成分；这可能代表细胞内放射性，似乎解释了在较高胰岛素浓度下细胞对胰岛素的更高特异性结合。当在上述肝细胞和质膜浓度下研究胰岛素降解时，细胞比膜多降解30%的胰岛素。对这些数据的动力学分析表明，胰岛素降解的Km（37℃时为5×10⁻⁷M）对两个系统相同，而使用肝细胞时Vmax更大。
（1）通过胶原酶消化制备肝细胞似乎不会改变胰岛素结合或降解；（2）只要使用胰岛素浓度低于100ng/ml，对从正常大鼠获得的肝细胞膜和分离的肝细胞的研究应能产生关于胰岛素 - 受体相互作用的类似信息；（3）在非常高的胰岛素浓度下，一些放射性似乎进入细胞；（4）肝细胞和肝细胞膜对胰岛素降解的动力学相似；（5）胰岛素降解似乎主要是一种膜现象。
