Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zuckerfa/farats

Abstract

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P 2) and is a glucokinasebinding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P2 content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P2 levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P2. Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats. liver; glucose metabolism; fructose-2,6-bisphosphate THE BIFUNCTIONAL ENZYME 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) catalyzes both the formation of fructose-2,6-bisphosphate (fructose-2,6-P 2 ) and its degradation (26,27,35). Fructose-2,6-P 2 is a regulator of glycolysis because it is a potent activator of phosphofructokinase-1 and inhibitor of fructose-1,6-bisphosphatase-1. Various tissue-specific isoforms of PFK2 encoded by four genes are expressed in mammals. They differ in their relative kinase and bisphosphatase activities and also in their regulatory mechanisms (36). The liver isoform is regulated by phosphorylation of a serine residue at the NH 2 terminus (Ser-32) by cAMP-dependent protein kinase, which leads to an increase in the bisphosphatase-to-kinase activity ratio. This mechanism accounts for the lowering of fructose-2,6-P 2 and inhibition of glycolysis caused by glucagon (26).Recent studies by Baltrusch et al. (8) showed that PFK2 binds to glucokinase through the bisphosphatase domain. Putative roles for this heterodimeric interaction have been proposed for both the hepatocyte and pancreatic ␤-cells, which express different isoforms of PFK2 (8,31,34). In pancreatic ␤-cells, binding of PFK2 activates gluc...

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