Fructose 1‐phosphate and the regulation of glucokinase activity in isolated hepatocytes

Fructose 1‐phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1‐phosphate and fructose. The concentration of fructose 1‐phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05–1 mM) caused a concentration‐dependent and transient increase in the fructose 1‐phosphate content. Glucagon (1 μM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1‐phosphate in cells incubated with fructose, whereas the addition of 0.1 μM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1‐phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1‐phosphate. D‐Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D‐glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon. Both fructose and D‐glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3 H 2 O from [2‐ 3 H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D‐glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1‐phosphate mediates the effects of fructose, D‐glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein [Van Schaftingen, E. (1989) Eur. J. Biochem. 179 , 179–184].