Long-term maintenance of low concentrations of fructose for the study of hepatic glucose phosphorylation

The stimulation of glucose phosphorylation in isolated hepatocytes by low f ructose concentrations is transient due to the rapid metabolism of fructose . To prolong this stimulatory effect fructose was enzymically generated in the incubation medium from either sucrose with invertase or inulin with inu linase. A maximal rate of glucose phosphorylation was achieved when fructos e was formed at at least 0.01 mu mol/min, which maintained a concentration of 70 mu M fructose in the medium. In the presence of a fructose concentrat ion of 70 mu M, the rate of phosphorylation with 5 mM glucose was doubled a nd remained constant over a 2.5 h period. Under these conditions the rate o f glycolysis was increased more than 3-fold. The stimulation of flux throug h glucokinase by low concentrations of fructose decreased the proportion of glucose phosphorylated, which was cycled between glucose and glucose 6-pho sphate, and increased the proportion that was glycolysed. The method descri bed for maintaining the stimulation of glucose phosphorylation by isolated hepatocytes over prolonged incubation periods is especially suited to the f urther study of the control of glucokinase activity, in particular how the variation of flux through glucokinase affects the flux through all the path ways that utilize the product, glucose 6-phosphate.