GLUCOSE-STIMULATED SYNTHESIS OF FRUCTOSE 2,6-BISPHOSPHATE IN RAT-LIVER - DEPHOSPHORYLATION OF FRUCTOSE 6-PHOSPHATE,2-KINASE-FRUCTOSE 2,6-BISPHOSPHATASE AND ACTIVATION BY A SUGAR-PHOSPHATE

The effect of glucose on hepatic fructose (Fru) 2,6-P-2 in starved rat s was investigated. When livers were perfused with high glucose (40 mM ), hexose-P in the liver increased immediately reaching the maximum wi thin in 2 min, but Fru 2,6-P-2 after a lag period of 4 min increased l inearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2, 6-Pase also showed a similar lag period. Determination of the phosphat e contents of the bifunctional enzyme after 10 min of glucose perfusio n revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of c rude extracts of liver perfused with either high glucose or normal glu cose (5.6 mM) showed that high glucose livers contained 50% higher pro tein phosphatase activity, which dephosphorylated the bifunctional enz yme. Subcellular fractionation of the ex tract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the c ytosolic fraction resulted in a 50% loss of the protein phosphatase ac tivity. The low molecular weight activator in the cytosol was isolated , and by various chemical and enzymatic methods it was identified as x ylulose 5-P. The activation of protein phosphatase by xylulose 5-P sho wed a highly sigmoidal saturation curve. The rate of formation of xylu lose 5-P in the perfused liver showed a lag period of approximately 2 min, and after 4 min its concentration reached 10 mu M, the minimum co ncentration necessary for the activation of the protein phosphatase. W e conclude that the mechanism of glucose-induced Fru 2,6-P-2 synthesis was not due to increased Fru 6-P as generally thought but occurred as a result of dephosphorylation of Fru 6-P,2-kinase:Fru 2,6-Pase. Moreo ver, the dephosphorylation was enhanced by increased xylulose 5-P, whi ch activated a specific protein phosphatase. The results suggest a mec hanism for coordinated regulation of glycolysis and the pentose shunt pathway that is mediated by xylulose 5-P.