Mitochondrial-Cytosolic Interactions in Perfused Rat Heart: ROLE OF COUPLED TRANSAMINATION IN REPLETION OF CITRIC ACID CYCLE INTERMEDIATES

Abstract

Abstract The kinetics of changes in the tissue content of citric acid cycle intermediates and free amino acids have been investigated in isolated perfused hearts during the transition from substrate-free perfusion to perfusion with medium containing 5 mm glucose and 5 x 10-3 units of insulin per ml. Glycolytic flux increased within 1 min to about 300 µmoles of glucose equivalents per g (dry weight) per hour, and oxygen uptake increased by 18 to 28% from a basal rate of 3000 µatoms per g (dry weight) per hour. The ratio of lactate to pyruvate in the perfusion fluid showed a transient rise from a value of 0.4 to a peak of 18 after 2 min and reached a final steady state value of 4. The tissue contents of oxalacetate and aspartate decreased by 76 and 59%, respectively, within the first 3 min, whereas the glutamate content increased reciprocally by 230%. The tissue contents of malate, citrate, and alanine increased more gradually over a 12-min period by 4.3-, 8.6-, and 4.4-fold, respectively. The α-ketoglutarate content initially increased rapidly and then fell before rising again to final values about 2-fold greater than the initial. The changes in the tissue contents of amino acids other than glutamate, aspartate, and alanine were minor. Net accumulation of citric acid cycle intermediates in the heart following addition of glucose can be accounted for by double transamination between alanine and aspartate aminotransferases. Creatine-P levels increased by 14 µmoles per g (dry weight), whereas tissue inorganic phosphate levels decreased by 12 µmoles per g (dry weight). These changes together with the observed increase of the ATP:ADP ratio from 10.8 to 14.2 indicate a higher energy state of the adenine nucleotide system after glucose addition. From the measured rates of glucose metabolism, oxygen uptake, and changes of the aspartate and alanine contents, together with the previously verified assumption that glycolytically generated NADH is transported to the mitochondria via the malate-aspartate cycle, calculations were made of flux through steps of the citric acid cycle and the anion exchange reactions across the mitochondrial membrane. The data indicate a rapid increase of α-ketoglutarate-malate exchange upon addition of glucose and insulin, but a 1- to 2-min delay in the onset of increased glutamate-aspartate exchange and flux through mitochondrial aspartate aminotransferase. This delay of intramitochondrial glutamate transamination was attributed to a low affinity of the glutamate carrier for extramitochondrial glutamate in the presence of high extramitochondrial aspartate concentrations. The calculations showed that flux through citrate synthase initially increased, whereas flux through α-ketoglutarate dehydrogenase decreased. It is postulated that these changes are a direct consequence of the regulation of glutamate influx across the mitochondrial membrane. A uniform flux through the citric acid cycle except for malate dehydrogenase was achieved by a fall of flux through citrate synthase and increase of flux through α-ketoglutarate dehydrogenase to common steady state values. Increased flux through citrate synthase is attributed to the observed rise of acetyl-CoA levels, whereas decreased flux through α-ketoglutarate dehydrogenase is attributed to inhibition of the enzyme by an increase of succinyl-CoA and NADH levels. The reciprocal reversals of flux after 1.5 min are attributed to competition for oxalacetate between citrate synthase and mitochondrial aspartate aminotransferase, and an increased rate of generation of intramitochondrial α-ketoglutarate which overcomes product inhibition of α-ketoglutarate dehydrogenase by increasing the intramitochondrial α-ketoglutarate concentration.