Glycolytic regulation in rat liver and hepatomas

Abstract

The recent development of a series of transplantable liver tumors that display a wide range of glycolytic and respiratory activity, correlated roughly with growth rate and degree of differentiation, provides a useful tool for studies of control mechanisms in glycolysis and respiration. Experiments here described point to major sites of regulation at pyruvate kinase and/or P glycerate kinase. This assumption is based on experiments with a model system in which whole, fortified homogenates were incubated with fructose-diP as substrate and 2-deoxyglucose, P i , and yeast hexokinase were added to regenerate ADP from ATP formed by glycolysis and respiration. Measurement of O 2 uptake, lactate formation and 2-DG disappearance allowed estimation of glycolysis, respiration, and glycolytic and respiratory phosphorylation. Endogenous respiration was high in well-differentiated and low in poorly-differentiated tumors, and in both types P/O ratios were 1 to 2. Addition of FDP to homogenates of well-differentiated tumors caused only low lactate formation and low glycolytic phosphorylation and no change in respiration or respiratory ATP production. In contrast, FDP addition to poorly-differentiated tumor homogenates caused high glycolysis; and though respiration was not lowered, oxidative phosphorylation was negligible, whereas glycolytic phosphorylation was very high. These findings point to the transphosphorylating enzymes as a major site of glycolytic control, presumably owing to competition for ADP with the respiratory phosphorylation system. Additional support for this hypothesis was obtained by intermixing mitochondrial and supernatant fractions. Replacement of particles from a low-respiring, poorly-differentiated tumor by particles from a high-respiring, well-differentiated tumor resulted in a pronounced Pasteur effect; respiration was increased, together with respiratory ATP production, while glycolysis was markedly decreased. However, when particles of a high-respiring tumor were replaced with particles of a low-respiring tumor, respiration and respiratory phosphorylation were decreased, and glycolysis was markedly increased. Further evidence for glycolytic control at pyruvate kinase was obtained from enzyme assays. Pyruvate kinase levels in the poorly-differentiated tumors were up to 20-fold higher in poorly-differentiated than in well-differentiated tumors. Rat liver has two pyruvate kinase isozymes differing in adsorption on DEAE-cellulose. The low levels in liver and well-differentiated tumor predominate in the adsorbable form whereas the high levels in the poorly-differentiated tumors consist primarily of the non-adsorbed form. If competition for ADP plays a part in regulating glycolysis in liver tumors, the low levels of pyruvate kinase in the well-differentiated tumors should favor uptake of ADP by the respiratory acceptor systems and thus lower glycolysis further. On the other hand, the very high pyruvate kinase levels of the poorly-differentiated tumors should favor preferential uptake of ADP by this enzyme, to enhance glycolysis. The characteristically high glycolytic activity of tumors may in general be attributable to low levels of respiratory ADP acceptor systems in combination with high pyruvate kinase activity.