Iynedjian P B, Gjinovci A, Renold A E
Institut de Biochimie Clinique, University of Geneva School of Medicine, Switzerland.
J Biol Chem. 1988 Jan 15;263(2):740-4.
The purpose of this work was to investigate the molecular mechanism responsible for the induction of hepatic glucokinase in diabetic rats acutely treated with insulin. Experimental diabetes was provoked by injection of streptozotocin 8-10 days before the experiments. Regular insulin was given by three intraperitoneal injections at 8-h intervals, and the time course of glucokinase induction was followed over a time period of 24 h. The amount of glucokinase in liver was estimated by Western blotting of total cytosol protein with affinity-purified antibodies, as well as by conventional enzyme activity assay. Both measurements showed that glucokinase was reduced by more than 90% in the livers of diabetic rats as compared to normal controls. Following insulin administration, the amount (and activity) of glucokinase increased in a time-dependent fashion, after an initial lag of 4 h, to reach 65% of the nondiabetic control level 24 h after the initial dose of insulin. Northern blot analysis with a cloned cDNA probe was used to quantitate glucokinase mRNA. In contrast with the slow onset of enzyme accumulation, the amount of glucokinase mRNA was shown to be increased dramatically as early as 1 h after insulin administration. The abundance of specific mRNA increased until 8 h after the initial dose of insulin. Subsequently, the level of the mRNA decayed rapidly so that little message was left after 16 h and virtually none after 24 h. Run-on transcription experiments with isolated nuclei showed that the rate of transcription of the glucokinase gene was increased about 20-fold within 45 min of insulin administration and returned to the prestimulation level after 8 h. From these data, it was concluded that the induction of glucokinase resulted primarily from a burst in the transcriptional activity of the gene, leading to a short-term accumulation of glucokinase mRNA. The more sustained elevation of the enzyme level can be accounted for by the long half-life of the enzyme (greater than 30 h). The virtually immediate activation of glucokinase gene transcription suggests a direct effect of insulin on the liver cell.
本研究旨在探讨胰岛素急性处理糖尿病大鼠后肝葡萄糖激酶诱导产生的分子机制。实验性糖尿病在实验前8 - 10天通过注射链脲佐菌素诱发。普通胰岛素以8小时间隔进行三次腹腔注射，在24小时内跟踪葡萄糖激酶诱导的时间进程。通过用亲和纯化抗体对全细胞溶质蛋白进行蛋白质免疫印迹法以及传统酶活性测定来估计肝脏中葡萄糖激酶的量。两种测量方法均显示，与正常对照组相比，糖尿病大鼠肝脏中葡萄糖激酶减少了90%以上。给予胰岛素后，葡萄糖激酶的量（和活性）在最初4小时的滞后时间后以时间依赖性方式增加，在首次注射胰岛素后24小时达到非糖尿病对照水平的65%。用克隆的cDNA探针进行Northern印迹分析以定量葡萄糖激酶mRNA。与酶积累的缓慢起始相反，早在胰岛素给药后1小时，葡萄糖激酶mRNA的量就显著增加。特异性mRNA的丰度在首次注射胰岛素后8小时之前一直增加。随后，mRNA水平迅速下降，以至于在16小时后几乎没有剩余信息，24小时后几乎没有。用分离的细胞核进行的核转录实验表明，胰岛素给药后45分钟内葡萄糖激酶基因的转录速率增加了约20倍，并在8小时后恢复到刺激前水平。从这些数据得出结论，葡萄糖激酶的诱导主要源于该基因转录活性的爆发，导致葡萄糖激酶mRNA的短期积累。酶水平更持续的升高可以由酶的长半衰期（大于30小时）来解释。葡萄糖激酶基因转录几乎立即被激活表明胰岛素对肝细胞有直接作用。
