Studies on hepatic fructose metabolism

Abstract

The hepatic metabolism of fructose in fed rats was studied using perfused liver and isolated hepatocytes. Fru-l-P has been shown by many workers to be the major initial product of fructose metabolism. However, the present study shows the simultaneous accumulation of a higher molecular-weight compound. This product was found to predominate at low fructose concentrations, whereas Fru-l-P was the major product on incubation with more than 4mM-fructose. The unknown metabolite was isolated and purified by a variety of techniques. Gel-filtration studies indicated a molecular weight of 583 and on acid hydrolysis the compound was shown to contain carbohydrate, phosphate and amino acid positive material. The unknown was unstable to acid, alkaline phosphatase and barium acetate precipitation, yielding a variety of different products in each case. Fructokinase, purified 100-fold from the livers of adult male rats, was shown to utilise GTP as well as ATP as nucleotide triphosphate. In both cases Fru-l-P was the sole labelled product of the fructokinase re-action with [u14c] fructose. The purified enzyme was found, with ATP as substrate, to have K values of 0.83mM for fructose and 1.43mM for MgATP.m With GTP as substrate, constants of 0.56mM for fructose and 1.65mM for MgGTP were determined. Both reactions were inhibited by ADP and GDP and inhibition constants calculated. The utilisation of both nucleotide triphosphates following a fructose load is discussed. Phosphofructokinase activity, assayed in crude extracts of isolated hepatocytes, was shown to be increased on incubation of the cells with low concentrations (l-8mM) of fructose, dihydroxyacetone or galactose or with high glucose (20mM) concentrations. The activation, proposed to be due to Fru-2,6-P accumulation, was abolished by filtration of the extract down a Sephadex G-25 column. Glycerol (2-8mM) and glucose (1-8mM) were without significant effect and 20mM-fructose caused an inhibition of enzyme activity relative to controls. The latter effect is thought to be due to the accumulation of Fru-l-P, an inhibitor of hepatic fructokinase.