N6,O2'-dibutyryl Cycle AMP and Glucose Regulate the Amount of Messenger RNA Coding for Hepatic Phosphoenolpyruvate Carboxykinase (GTP)

Rat liver phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA was purified to 25% of total mRNA activity (greater than 50-fold enrichment) by polysome immunoprecipitation. This preparation was used as template for the synthesis of cDNA that was subsequently cloned in Escherichia coli. The resulting clones were screened by in situ hybridization and by hybrid-selected translation of phosphoenolpyruvate carboxykinase mRNA. The cDNA insert of one plasmid, pPC2, was complementary to phosphoenolpyruvate carboxykinase mRNA as determined by these screening procedures. pPC2 cDNA was 760 base pairs in length and a partial restriction enzyme map was constructed. pPC2 was labeled with 32P by nick translation and was used as a hybridization probe to quantitate phosphoenolpyruvate carboxykinase mRNA following N6,O2'-dibutyryl cAMP (Bt2cAMP) injection or glucose feeding. Bt2cAMP increased whereas glucose decreased the level of hybridizable phosphoenolpyruvate carboxykinase mRNA and in all cases the changes were proportional to the in vitro translational activities measured in a reticulocyte lysate system. The half-life of phosphoenolpyruvate carboxykinase mRNA sequences was measured by an indirect procedure involving their quantitation, by hybridization assay, during deinduction and induction. The half-life was approximately 10-40 min during deinduction by glucose or during induction stimulated by Bt2cAMP. Our data indicate that cAMP enhances some step in the generation of phosphoenolpyruvate carboxykinase mRNA.