ADENOVIRUS-MEDIATED OVEREXPRESSION OF LIVER 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE IN GLUCONEOGENIC RAT HEPATOMA-CELLS - PARADOXICAL EFFECT ON FRU-2,6-P(2) LEVELS

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated 60 be a metabolic signaling enzyme, which acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of fructose 2,6-bisphosphate, The effect of overexpressing the bifunctional enzyme was studied in FAO cells transduced with recombinant adenoviral constructs of either the wild-type enzyme or a double mutant that has no bisphosphatase activity or protein kinase phosphorylation site, With both constructs, the mRNA and protein were overexpressed by 150- and 40-fold, respectively, Addition of cAMP to cells overexpressing the wild-type enzyme increased the S-0.5 for fructose 6-phosphate of the kinase by 1.5 fold but had no effect on the overexpressed double mutant, When the wild-type enzyme was overexpressed, there was a decrease in fructose 2,6-bisphosphate levels, even though 6-phosphofructo-2-kinase maximal activity increased more than 22 fold and was in excess of fructose 2,6-bisphosphatase maximal activity, The kinase:bisphosphatase maximal activity ratio was decreased, indicating that the overexpressed enzyme was phosphorylated by cAMP-dependent protein kinase, Overexpression of the double mutant resulted in a 28-fold increase in kinase maximal activity and a 3-4 fold increase in fructose 2,6-bisphosphate levels, Overexpression of this form inhibited the rate of glucose production from dihydroxyacetone by 90% and stimulated the rate of lactate plus pyruvate production by 200%, In contrast, overexpression of the wild-type enzyme enhanced glucose production and inhibited lactate plus pyruvate production, These results provide direct support for fructose 2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway flux and suggest that regulation of bifunctional enzyme activities by covalent modification is more important than the amount of the protein.