Multisite control of the Crabtree effect in ascites hepatoma cells

Abstract

AS-30D hepatoma cells, a highly oxidative and fastgrowing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mm glucose or 10 mm fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular 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 Ca 21 and Mg 21 were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D 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 Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.Keywords: Crabtree effect; fast-growth tumor cells; multisite control; oxidative phosphorylation.Fast-growing tumor cells are characterized by a high glycolytic activity, even in the presence of saturating oxygen. In these cells it is also known that cellular oxidative phosphorylation is strongly inhibited by exogenous glucose [1]. A diminished or absent Pasteur effect would explain the former phenomenon. However, for the glucose-induced inhibition of oxidative phosphorylation, known as the Crabtree effect [2], there is no satisfactory explanation. The Crabtree effect is not restricted to malignant cells as it is also observed in normal proliferative cells [3]. In fact, in several nontumoral tissues (pig platelets, hamster embryos, proliferating thymocytes, retina, intestinal mucosa) [4±7] and in bacteria and yeast [8,9], it has been reported that oxidative phosphorylation is inhibited by exogenous hexoses.Eigenbrodt et al. proposed that this process could be a selective advantage for tumor cells, as the consequence of a glucose-dependent accumulation of essential metabolic intermediaries, such as serine, phosphoribosyl-pyrophosphate, fructose 1,6-bisphosphate, and glycerol 3-phosphate, which can trigger the mitogenic events In this work, we have evaluated some of these hypotheses, using the oxidative ascites tumor AS-30D cell line as the experimental model. We show that several factors are involved in the onset of the Crabtree effect. M A T E R I A L S A N D M E T H O D SCell preparation AS-30D ascites tumor cells were propagated by inoculation of 2 Â 10 8 cells into the peritoneal cavity of female Wistar rats of 200±250 g weight. The cells were harvested, and washed as described previously [17]. They were stored in ice at a density of 2±3 Â 10 8 cells´mL 21 until use. Isolation of mitochondriaAS-30D mitochondria were isolated by the digitonin permeabilization procedure...

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