Hatada E N, McClain D A, Potter E, Ullrich A, Olefsky J M
Department of Medicine, School of Medicine, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1989 Apr 25;264(12):6741-7.
We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.
我们研究了（Hep G2）细胞中胰岛素受体数量和胰岛素受体mRNA水平随生长及胰岛素处理的变化情况。生长中的细胞胰岛素受体水平相对较低。培养约5天后，若细胞不再换液，它们就会停止分裂，每个细胞的受体数量增加，到第9天时达到初始值的4倍。给细胞重新换液可完全防止生长停滞和胰岛素受体数量增加。每天向细胞添加0.33微摩尔的胰岛素会导致受体下调，但并不能阻止结合增加3倍以及生长停滞。对用代谢标记（[35S]甲硫氨酸）的细胞进行脉冲追踪研究表明，快速生长的细胞与生长停滞的细胞相比，受体降解率（表观半衰期为18 - 20小时）相当。未换液细胞中受体水平的增加并非汇合细胞产生可溶性因子所致，也不是由于培养基中胰岛素、葡萄糖或胰岛素样生长因子I的消耗引起的。通过Northern印迹法测定的胰岛素受体mRNA水平在生长停滞的细胞中与受体数量的增加平行升高。mRNA值从培养第3天开始增加，到第9天时达到第3天水平的6.0倍。慢性胰岛素诱导的受体下调在任何研究的时间点都未改变胰岛素受体mRNA水平。这些数据表明，生长停滞细胞中每个细胞胰岛素受体数量的增加与胰岛素受体mRNA含量的增加平行，而受体降解率没有变化。这表明胰岛素受体数量的增加是由于受体mRNA含量增加导致受体合成增强。相反，胰岛素受体的下调并不影响胰岛素受体mRNA水平，因此一定是由于受体降解增加所致。
