CAMP-INDEPENDENT SYNERGISTIC EFFECTS OF INSULIN AND DEXAMETHASONE ON FRUCTOSE 2,6-BISPHOSPHATE METABOLISM IN H4IIE CELLS

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P-2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P-2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P-2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the K-m for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased Liver 6PF-2-K/Fru-2,6-P-2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude thatthe synergistic effects of insulin and dexamethasone on Fru-2,6-P-2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.