Interrelation between mitochondrial respiration, substrate supply and redox ratio in perifused permeabilized rat...

A one-step perifusion technique is described for studying the regulation of energy metabolism in intact hepatocytes and in mitochondria of the same cells after their permeabilization by digitonin. Cell count and activities of glutamate dehydrogenase, the latter being used as an indicator of mitochondrial integrity, were found to be nearly unchanged after permeabilization and perifusion for at least 40 min at 37 degrees C. The residual activity of lactate dehydrogenase after permeabilized indicated that permeabilized cells were almost depleted of soluble cytosolic components. The composition of the perifusion medium was chosen so that various metabolic states could be adjusted of both intact and permeabilized hepatocytes without the need to change the perfusion medium. Oxidative phosphorylation of mitochondria within permeabilized hepatocytes remained intact throughout the perifusion as indicated by the response of respiration to the addition of ADP, carboxyatractyloside and uncoupler. The application of the perifusion technique allows us to sample indicator metabolites in the effluent medium like acetoacetate (AcAc) and 3-hydroxybutyrate (HB) for calculating the mitochondrial redox ratios and rates of ketogenesis. In the presence of octanoate and ADP, an improvement of substrate supply by glutamate and malate led to increases in the intramitochondrial HB/AcAc ratio and the respiration rate. Glutamate/malate concentrations of 1 mM resulted in maximal respiration rates, whereas concentrations of 5 mM further enhanced the HB/AcAc ratio. Mitochondria responded to increasing ATP/ADP ratios in the perifusion medium by decreased respiration rates at higher HB/AcAc ratios. By comparing respiration rates and redox ratios of mitochondria in permeabilized cells with those before permeabilization (gluconeogenic conditions of hepatocytes), it is concluded that in the intact cell oxidative phosphorylation is limited with respect to substrate supply as well as by the ATP demand.