Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers.

Previous estimates of flux through the pyruvate-dehydrogenase complex were made by measuring 14CO2 generated from oxidation of [1-14C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between 14CO2 produced from pyruvate dehydrogenase and 14CO2 generated from phospho-enolpyruvate carboxykinase and citric-acid-cycle dehydrogenases. While some previous reports have attempted to correct for the additional 14CO2 production by comparing 14CO2 generated by [1-14C]pyruvate with [2-14C]pyruvate or [3-14C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the 14C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4-14C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate-dehydrogenase complex is negligible compared to the other reactions which generate 14CO2 from [1-14C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, 14CO2 production via the pyruvate-dehydrogenase complex is large (approximately 11.5 nmol.min-1.mg mitochondrial protein-1) compared to 14CO2 production via phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases (approximately 6.4 nmol.min-1.mg-1) when the pyruvate-dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.

The mechanism of Ca2(+)-related control of gluconeogenesis in perfused liver.

A kinetic expression for rat-liver mitochondrial aspartate formation in situ was developed in order to determine whether hormonally induced decreases in 2-oxoglutarate levels can regulate hepatic gluconeogenesis from lactate via control of aspartate formation. Previous studies from this laboratory showed that 2-oxoglutarate can inhibit aspartate production by isolated mitochondria. These present studies were designed to probe the physiological significance of the decrease in 2-oxoglutarate levels observed when Ca2(+)-mobilizing gluconeogenic hormones are administered to isolate perfused rat livers. First, estimates were made of the kinetic constants which determine the rate of aspartate formation in isolated mitochondria. The concentrations of the substrates and products of this process were then measured in perfused livers. From these values, it was possible to estimate aspartate efflux from mitochondria in situ. The calculated rates of aspartate production were increased by decreases in 2-oxoglutarate levels which occurred when glucagon or phenylephrine was added to the perfused livers. Glucagon also effected an inhibition of pyruvate kinase, evidenced by the fact that the calculated rate of aspartate efflux equalled the rate of gluconeogenesis (the difference between the two is equivalent to the pyruvate-kinase flux). By contrast, in control livers and with phenylephrine stimulation, aspartate formation was higher than gluconeogenesis suggesting significant pyruvate-kinase flux in this condition. The calculations also show a correlating increase in flux through pyruvate carboxylase (30% with phenylephrine, 15% with glucagon, compared with approximately 50% increases in gluconeogenic flux). The mechanism of this increase is discussed.

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin.

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes.

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.