Initial expression of glucokinase gene in cultured hepatocytes from suckling rats is linked to the synthesis of an insulin-dependent protein

Abstract

The initial accumulation of glucokinase mRNA in response to insulin in cultured hepatocytes from 10-day-old suckling rats was characterized by a delay of 18-24 h with a maximal level reached after 48 h. This delay is not observed in cultured adult rat hepatocytes. When hepatocytes from 10-day-old suckling rats were cultured for 48 h in the presence of insulin (to obtain a maximal accumulation of glucokinase mRNA) and then deprived of insulin for 18 h, glucokinase mRNA returned to very low levels. Reexposure of these cultured hepatocytes to insulin allowed a rapid accumulation of glucokinase mRNA, with a maximal level reached after 8 h, as in adult rat hepatocytes. The aim of the present study was to investigate the factors responsible for the delay in insulin action during first exposure to insulin. The difference in the kinetics of glucokinase mRNA accumulation after the first and secondary exposure to insulin was due to differences in the rate of transcriptional activity of the glucokinase gene, as shown by a run-on assay on isolated nuclei. The half-life of glucokinase mRNA was similar after the first and second exposure to insulin. The delay in the initial accumulation of glucokinase mRNA in response to the first exposure to insulin was not due to elevated levels of cAMP (a potent inhibitor of glucokinase gene expression) or to a defect in insulin signalling (insulin inhibited without delay phosphoenolpyruvate carboxykinase gene expression). In contrast, it was markedly dependent upon whether glucokinase has been already expressed in vivo. Hepatocytes from rats that had already expressed glucokinase in vivo (suckling rats force-fed with glucose or rats weaned to a high-carbohydrate diet) showed no delay in their response to insulin in culture, whereas hepatocytes from rats that have never expressed glucokinase in vivo (suckling rats or rats weaned to a high-fat diet) showed a delay of 24 h. Two different inhibitors of protein synthesis (cycloheximide and puromycin) prevented the initial accumulation of glucokinase mRNA in response to the first exposure to insulin but not to the secondary accumulation of glucokinase mRNA in response to reexposure to insulin. This suggests that the synthesis of one or several insulin-dependent proteins is necessary for the first activation of glucokinase gene transcription in response to the first exposure to insulin.