Regulation of fructose-2,6-bisphosphate content in rat hepatocytes, perfused hearts, and perfused hindlimbs.

Abstract

A method has been developed to specifically measure fructose-2,6-bisphosphate in tissues.After elimination of the hexose-6-phosphate present in the extract, fructose-2,6-bisphosphate is hydrolyzed and the acid-revealed fructose-6-phosphate is measured in a coupled enzymatic assay with bacterial NADH-linked luciferase.Fructose-2,6-bisphosphate was found in all tissues studied; and, in order of increasing concentration, epididymal fat, kidney, skeletal muscle, lung, heart, liver, and brain contain 0.1 to 2.3 nmol/g of tissue.Values lower than 0.1 nmol/g cannot be accurately measured and represent the limit of detection of the method.Incubation of isolated hepatocytes from fed rats in the presence of 20 m~ alanine, pyruvate, glycerol, Lsorbose, lactate, tagatose, fructose, and glyceraldehyde caused, in order of increasing effect, a 2-to 10-fold decrease in fructose-2,6-bisphosphate content.Incubation in the presence of 20 m~ dihydroxyacetone, galactose, mannose, xylitol, arabinose, or ribose had little or no effect on the concentration of fructose-2,6-bisphosphate. When hepatocytes were incubated under anoxic conditions or in the presence of carbonyl cyanide mchlorophenylhydrazone, a mitochondrial uncoupler, the fructose-2,6-bisphosphate content was decreased by 40 and 608, respectively.In perfused rat hindlimb muscle, insulin and epinephrine, respectively, caused a 2-and 4-fold increase in fructose-2,6-bisphosphate and a stimulation of glycolysis.Electrical stimulation caused muscular contraction and a 2.5-fold increase in lactate production, but decreased fructose-2,6-bisphosphate. It also abolished the increase in this metabolite induced by epinephrine.In perfused rat hearts, epinephrine caused the activation of protein kinase and phosphorylase but did not alter the fructose-2,6-bisphosphate content.Fructose-2,6-bisphosphate is a very potent stimulator of liver and muscle phosphofructokinase (1, 2), and changes in its concentration in the liver have been related to changes in glycolytic fluxes in this tissue (3-5).In hepatocytes, glucose,