In a continuation of studies on glycolytic and respiratory interrelationships in a series of rat hepatomas ranging widely in growth rate and degree of differentiation, whole, fortified homogenates of these tissues were incubated at 28°C in the presence of fructose-1,6-diphosphate (FDP), 2-deoxyglucose (2-DG), and exogenous hexokinase; respiration, lactate formation, and uptake of 2-DG were measured. Preliminary studies with this model system established that uptake of 2-DG was a valid measure of ATP formation. On the assumption that each mole of lactate formed from FDP leads to production of 2 moles of ATP via phosphoglycerate kinase and pyruvate kinase, glycolytic phosphorylation was estimated as twice that of lactate, and respiratory phosphorylation was calculated as the difference between total ATP and glycolytic ATP formation.
Without exogenous substrate, respiration was high in homogenates of well-differentiated tumors, and low in those of poorly differentiated tumors. In both tumor types respiration was coupled with ATP formation, yielding P/O ratios of 1 to 2. Addition of FDP to liver homogenates resulted in moderate lactate and glycolytic ATP formation, the latter being formed largely at the expense of respiratory phosphorylation. In homogenates of well-differentiated tumors, lactate formation and glycolytic phosphorylation were low, and neither respiration nor respiratory phosphorylation was decreased. In contrast, homogenates of the poorly differentiated hepatomas exhibited high lactate formation, and though respiration was increased somewhat by FDP addition, essentially all of the ATP was formed via glycolysis. It thus appears that, in this system, transphosphorylating enzymes of glycolysis are a major site of glycolytic control, presumably through competition with the respiratory ADP acceptors for the available ADP. Further evidence for such competition was obtained by intermixing the supernatant and particulate 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.
These findings provide evidence for the suggestion offered many years ago by Johnson and by Lynen that the Pasteur effect may reflect competition for ADP and inorganic phosphate (P1) at the transphosphorylating sites of glycolysis and respiration. They also suggest that the high aerobic glycolysis which is, in general, characteristic of highly dedifferentiated tumors may be, in part, a resultant of their low respiratory activity and high levels of glycolytic transphosphorylating enzymes.
This study was aided by Grants AM 05487 and CA-07174 of the National Institutes of Health, USPHS, and Grant P-202 from the American Cancer Society.