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 glucolunase 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.Glucokinase, a key enzyme of the glycolytic pathway, catalyses the ATP-dependent phosphorylation of glucose into glucose 6-phosphate. It is the predominant isoform of hexokinase expressed in the liver and the only one present in parenchymal cells [l-31. It has a higher K, for glucose (=lo mh4 versus =20-130 pM) and a higher specificity for glucose [4] than other hexokinases and it is not inhibited by its reaction product glucose 6-phosphate. These properties allow for rapid adjustements in the rate of liver glucose phosphorylation in response to changes in plasma glucose concentration [5].In adult rat liver, glucokinase activity is subject to longterm changes depe...

PTP1B deficiency increases glucose uptake in neonatal hepatocytes: involvement of IRA/GLUT2 complexes

“…Figure 2A shows that the expression of GK was very low in neonatal livers and neonatal hepatocytes. These results agree with those previously reported (6,34). Conversely, adult hepatocytes expressed high levels of GK, with no differences found between both genotypes.…”

“…Net hepatic glucose flux is the balance between the rate of glucose phosphorylation catalyzed by GK and the rate of dephosphorylation of G6P catalyzed by G6Pase. Importantly, in hepatocytes from suckling mice, constitutively expressed HK is the main enzyme responsible for glucose phosphorylation (6,34). Next, we investigated whether changes in the activities of HK and G6Pase could account for the net Fig.…”

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes

“…Glucokinase is expressed for the first time after transition from a milk diet to a high-carbohydrate diet after weaning, which is concomitant with an increase in insulin level. Glucokinase expression in suckling rat liver can also be induced earlier in vivo by giving a carbohydrate diet or in vitro by adding insulin to cultured hepatocytes (20,24). If our hypothesis relating glucokinase expression to SREBP-1c is true, the expression of SREBP-1c should be absent from the suckling rat liver because of the low insulin and high glucagon concentrations.…”

Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes

“…For instance, GK gene transcription, a SREBP-1c-dependent process [3], is activated by the hormone in less than 1 h in adult rat hepatocytes [15,16]. If one of the main actions of insulin on gene transcription is through an enhanced amount of the mature Figure 3 Time course of the disappearance of SREBP-1c precursor and mature forms after cycloheximide treatment in cultured hepatocytes (A) In order to test the efficiency of the cycloheximide treatment, hepatocytes cultured overnight in the presence of 5 mM glucose and in the absence of insulin were then cultured for 6 h in the presence of insulin without or with 5 µM cycloheximide.…”