Mechanism of the Inhibition by Insulin of the Glucagon-Dependent Activation of the Phosphoenolpyruvate Carhoxykinase Gene in Rat Hepatocyte Cultures. Action on Gene Transcription, mRNA Level and -Stability as well as Hysteresis Effect

Publisher Website
Google Scholar
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 100 mM 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 0.7 nM. At saturating 10 nM glucagon concentrations insulin was not antagonistic. 2) Glucagon at 0.1 nM concentrations increased PEPCK gene transcription and PEPCK mRNA to a transient maximum at 0.5 and 2 h, respectively. Insulin, added at 10 nM 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 transcription inhibitor cordyncepin, 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 glycagon. 5) Insulin, added at 10 nM concentrations for only 1 h before initiation of induction by 0.1 nM glucagon and then omitted during induction, lowered the maximal increase in PEPCK gene transcription and in PEPCK mRNA by 60%. Thus insulin showed a hysteresis effect remaining effective although no longer present. The study permits the following conclusions: Insulin inhibited the glucagon-dependent induction of the PEPCK gene by reduction of the transcriptional activity of the gene and by acceleration of the PEPCK mRNA decay. The degradation of PEPCK mRNA was in part dependent on ongoing gene transcription. It is proposed that PEPCK mRNA-destabilizing activities were induced de novo by insulin rapidly as an antagonistic principle and by glucagon slowly as a self-regulated shut-off mechanism.