Adenovirus-mediated overexpression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in gluconeogenic rat hepatoma cells. Paradoxical effect on Fru-2,6-P2 levels.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated to 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 S0.5 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.

Duke Scholars

Author Thomas Charles Becker Medicine, Endocrinology, Metabolism, and Nutrition

Author Christopher Bang Newgard Pharmacology & Cancer Biology