Multisite control of the Crabtree effect in ascites hepatoma cells

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, show ed glucose-induced and fructose-induced inhibition of oxidative phosphoryla tion (the Crabtree effect) of 54% and 34%, respectively. To advance the und erstanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several met abolites was determined. The addition of glucose or fructose lowered intrac ellular P-i (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%) , glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate ( 15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ an d Mg2+ were not modified. The addition of galactose or glycerol did not mod ify the concentrations of the metabolites. Mitochondria isolated from AS-30 D cells, incubated in media with low P-i (0.6 mM) at pH 6.8, exhibited a 40 % inhibition of oxidative phosphorylation. The data suggest that the Crabtr ee effect is the result of several small metabolic changes promoted by addi tion of exogenous glucose or fructose.