Mechanism of the inhibition by insulin of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in rat hepatocyte cultures. Action on gene transcription, mRNA level and -stability as well as hysteresis effect

Abstract

The mechanism of the antagonistic action of insulin on the glucagon-dependent stimulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied in primary cultures of rat hepatocytes. Gene expression was monitored by the transcriptional activity of the PEPCK gene and the accumulation and degradation of PEPCK mRNA. 1) Insulin in concentrations from 0.1 to 100nM shifted the dose-response curve of the glucagon-dependent accumulation of PEPCK mRNA to the right, increasing the half-maximally effective glucagon concentration gradually from 0.1 to 0.7nM. At saturating 10nM glucagon concentrations insulin was not antagonistic. 2) Glucagon at 0.1nM concentrations increased PEPCK gene transcription and PEPCK mRNA to a transient maximum at 0.5 and 2 h, respectively. Insulin, added at 10nM concentrations simultaneously with glucagon, reduced the maximal increase in PEPCK gene transcription by 70% and in PEPCK mRNA by 45%, respectively. 3) Following the maximal glucagon-induced increase after 2 h PEPCK mRNA declined to half-maximal levels after another 2.3 h. Insulin, added at 2 h at the PEPCK mRNA maximum, accelerated the disappearance of PEPCK mRNA, which reached half-maximal values already after another 1.2 h. 4) The transcriptional inhibitor cordycepin, added at 2 h at the PEPCK mRNA maximum, clearly retarded the normal and the insulin-accelerated decay of PEPCK mRNA so that half-maximal levels were reached only after another 5 h and 3 h, respectively. However, cordycepin did not retard the decay of PEPCK mRNA, when insulin was present from the beginning of induction by glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)