Insulin Decreases H4IIE Cell PEPCK mRNA by a Mechanism That Does Not Involve cAMP

Insulin is thought to influence some metabolic events by decreasing the intracellular concentration of cyclic AMP (cAMP). To test whether this explains the repression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) by insulin we measured intracellular cAMP, cAMP-dependent protein kinase; mRNA^PEPCK, and PEPCK gene transcription in cultured Reuber H4IIE hepatoma cells treated with forskolin with and without insulin. In untreated cells, the concentration of cAMP was 2.9 pmol/mg of protein. Forskolin at 1,10, and 50 μM increased the level of cAMP to 9.2, 35.8, and 115 pmol/mg of protein, respectively; 5 nM insulin had no significant effect on these cAMP concentrations. In untreated cells, the activity ratio of cAMP-dependent protein kinase was 0.43, and 50 μM forskolin increased this to 0.96; insulin had no effect on this ratio at times from 15–180 min. In untreated cells mRNA^PEPCK bound 15 cpm of a ³²P-labeled cDNA probe per microgram of total cellular RNA. Forskolin, at 1, 10, and 50 μM, increased this to 48, 96, and 115 cpm/μg RNA. Insulin (5 nM), in combination with 0, 1, 10, and 50 μM forskolin, decreased the concentration of mRNA^PEPCK to 5, 8, 23, and 29 cpm/μg RNA, respectively. Finally, the rate of transcription of the PEPCK gene was 85, 168, 630, 823, and 884 parts per million (ppm) in H4IIE cells treated for 30 min with 0, 1, 5, 10, and 50 μM forskolin, respectively, while the corresponding rates in the presence of 5 nM insulin were 49, 45, 84, 85, and 136 ppm. These results demonstrate that insulin represses mRNA^PEPCK and PEPCK gene transcription in H4IIE cells by a mechanism that is independent of the regulation of intracellular cAMP concentration and cAMP-dependent protein kinase activity.