Stimulation by insulin of glucokinase gene transcription in liver of diabetic rats.

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.