S J Pilkis

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-P2) 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-P2 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-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km 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-P2 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 that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 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.

Expression of rat liver fructose-1,6-bisphosphatase in Escherichia coli

Rat liver fructose-1,6-bisphosphatase was expressed in Escherichia coli using a T7 RNA polymerase-transcribed expression system. Maximum yields of soluble active enzyme were obtained when the bacterial host cell, BL21(DE3), carrying the expression plasmid was grown and transcription induced in LB medium at 37 degrees C. Approximately 20mg of fructose-1,6-bisphosphatase are synthesized per liter of culture after 4hr, of which about 10mg are soluble and enzymatically active. Expressed fructose-1,6-bisphosphatase, purified to homogeneity by substrate elution from a carboxymethyl Sephadex column, was indistinguishable from that purified from rat liver in terms of subunit size and kinetic properties. The in vitro expression of fructose-1,6-bisphosphatase in an heterologous system is a necessary preliminary step for future studies on site-directed mutant enzyme forms.

Multihormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in primary cultures of rat hepatocytes.

In the absence of hormone, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA rapidly declined to undetectable levels in primary cultures of rat hepatocytes. Dexamethasone increased mRNA levels and this effect was blocked by actinomycin D or cycloheximide. Insulin or thyroxine had no effect but potentiated the dexamethasone induction. Dibutyryl cyclic AMP decreased the stimulatory effect of dexamethasone at early times but after 24 hours the cyclic nucleotide increased mRNA levels compared with dexamethasone alone. It was concluded that expression of the bifunctional enzyme gene in hepatocytes is under a complex multihormonal control. Glucocorticoids are the most important transcriptional factor and their effect requires ongoing protein synthesis.