Peavy D E, DeSante D C
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120.
Biochem J. 1990 May 1;267(3):671-7. doi: 10.1042/bj2670671.
The role of insulin to regulate protein turnover in fetal liver was investigated using primary cultures of fetal-rat hepatocytes. The basal rate of protein degradation (in the presence of insulin and amino acids) was the same in cultured fetal and adult hepatocytes (2.48 +/- 0.16 versus 2.46 +/- 0.06% of total protein degraded/h respectively). Incubation of cells in an unsupplemented media (without insulin or amino acids) resulted in a deprivation-induced increase in degradation in cells from both groups (P less than 0.05). Rates of proteolysis could be returned to their respective basal values by the addition of amino acids at 5 times their normal plasma concentrations. In adult cells, addition of insulin alone significantly inhibited protein degradation (P less than 0.05), whereas, in contrast, insulin was without effect on protein degradation in fetal hepatocytes. Both fetal and adults cells responded to dibutyryl cyclic AMP with an increase in protein degradation above that seen in the no-additions group. Results of experiments in which the effect of inhibitors of protein degradation (chloroquine, NH4Cl, amino acids and dinitrophenol) were tested suggested that lysosomes were responsible for 20-30% of total protein degradation in fetal hepatocytes. Impaired insulin processing in fetal hepatocytes was examined as a possible cause of the insulin-resistance in these cells. As determined by h.p.l.c. analysis, the same pattern of initial degradation products of insulin was found in fetal hepatocytes as had previously been found in adult hepatocytes. Incubation of cells with various doses of chloroquine resulted in an increase in cell-associated 125I-insulin and a decrease in insulin degradation in both fetal and adult cells. At the highest dose of chloroquine tested (500 microM), a slightly greater increase in insulin binding and a decrease in insulin degradation were observed in fetal cells as compared with adult cells. Rates of insulin internalization were also compared between fetal and adult cells. A 30% slower rate of insulin internalization was observed in fetal cells, as compared with adult cells. It was concluded that the absence of an effect of insulin on protein degradation in fetal hepatocytes is not the result of a major difference in insulin internalization and processing between fetal and adult hepatocytes.
利用原代培养的胎鼠肝细胞研究了胰岛素在调节胎儿肝脏蛋白质周转中的作用。在培养的胎儿和成年肝细胞中，蛋白质降解的基础速率（在存在胰岛素和氨基酸的情况下）相同（分别为总蛋白质降解量的2.48±0.16%/小时和2.46±0.06%/小时）。将细胞置于无补充培养基（无胰岛素或氨基酸）中孵育，导致两组细胞中因营养剥夺引起的降解增加（P<0.05）。通过添加正常血浆浓度5倍的氨基酸，蛋白水解速率可恢复到各自的基础值。在成年细胞中，单独添加胰岛素可显著抑制蛋白质降解（P<0.05），而相比之下，胰岛素对胎儿肝细胞中的蛋白质降解无影响。胎儿和成年细胞对二丁酰环磷腺苷的反应均表现为蛋白质降解比未添加组增加。测试蛋白质降解抑制剂（氯喹、氯化铵、氨基酸和二硝基苯酚）作用的实验结果表明，溶酶体负责胎儿肝细胞中20 - 30%的总蛋白质降解。研究了胎儿肝细胞中胰岛素加工受损作为这些细胞胰岛素抵抗可能原因的情况。通过高效液相色谱分析确定，胎儿肝细胞中胰岛素初始降解产物的模式与先前在成年肝细胞中发现的相同。用不同剂量的氯喹孵育细胞导致胎儿和成年细胞中细胞相关的125I - 胰岛素增加，胰岛素降解减少。在测试的最高氯喹剂量（500 microM）下，与成年细胞相比，胎儿细胞中胰岛素结合的增加和胰岛素降解的减少略大。还比较了胎儿和成年细胞之间胰岛素内化的速率。与成年细胞相比，胎儿细胞中胰岛素内化速率慢30%。得出的结论是，胰岛素对胎儿肝细胞中蛋白质降解无影响并非胎儿和成年肝细胞之间胰岛素内化和加工存在重大差异的结果。
