The effect of lysine on gluconeogenesis from lactate in rat hepatocytes

Abstract

1. In freshly prepared isolated rat liver cells there is a lag in gluconeogenesis from lactate. The magnitude of the lag increases with increasing lactate concentration. 2. The lag is virtually abolished by lysine. 3. A few other amino acids (tyrosine, arginine, asparagine, ornithine) and NH(4)Cl had effects similar to, but less pronounced than, lysine during the early stage of incubation. Lysine was unique in accelerating gluconeogenesis beyond the lag period. 4. The effects of the accelerators are not additive. 5. Glycine, serine, threonine, cysteine, tryptophan and histidine at 2mm markedly inhibit (>20%) gluconeogenesis from lactate. 6. Oleate, which promotes gluconeogenesis from lactate by supplying acetyl-CoA required for the pyruvate carboxylase reaction, had no effect on the lag, yet oleate oxidation showed no lag. 7. Preincubation of cells decreased the lag and decreased the magnitude of the lysine effect. 8. Pyruvate (added at 1mm to give an initial [lactate]/[pyruvate] ratio of 10) also abolished the lag and decreased the lysine effect by about 50%. 9. Lysine reversed the inhibition by ethanol of gluconeogenesis from lactate. 10. All accelerators increased the rate of re-oxidation of cytosolic NADH as shown by a rapid re-adjustment of the [lactate]/[pyruvate] ratio on addition of 10mm-lactate. 11. The accelerated rates of gluconeogenesis are associated with an increased formation of aspartate and glutamate and especially alanine. 12. The existence of the lag period can be explained on the basis of the fact that the accumulation of pyruvate during the lag diverts oxaloacetate from gluconeogenesis to malate formation, i.e. that the re-oxidation of cytosolic NADH takes precedence over gluconeogenesis. This means that much oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred twice from the mitochondria to the cytosol by the aspartate shuttle. Under these conditions the operation of the shuttle limits the rate of gluconeogenesis from lactate. Lysine and other accelerators may increase the effectiveness of the shuttle by providing components of the aspartate aminotransferases involved. The question of why lysine specifically accelerates gluconeogenesis beyond the lag period is discussed.

Alanine and lactate as gluconeogenic substrates during late gestation

“…The rates of gluconeogenesis from lactate in isolated liver cells of fed pregnant rats showed a 3-4-fold increase at days 20, 21 and 22 of gestation when compared to the rates found in fed virgin rats ( fig.lB). It should be pointed out that the basal rates of ~u~oneogenesis observed in virgin rats are lower than those previously reported [ 18,19,22,24]. This difference is most likely due to the use in our experimental protocol of: (i) liver cells isolated from rats fed ad libitum, since starvation is well known to increase the gluconeogenic capacity of the liver 125,261; and (ii) an incubation medium that included glucose and half the concentration of lactate used by others, but that was designed with the purpose of simulating the physiological concentrations of both metabolites to which liver cells are exposed in vivo [ 11,141.…”

“…In our preparations, this effect was not observed; in fact, the rates of gluconeogenesis from L-lactate were linear during 40-50 min of incubation ( fig.lA). The existence of the lag period was explained by Cornell et al [22] by the precedence of the reoxidation of cytosolic NADH over giuconeogenesis, meaning that much of the oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred 'twice' from the mit~hondria to the cytosol by the aspartate shuttle before being converted into phosphoenolpyruvate. The observed discrepancy could be explained on the basis of the previous hypothesis [22]: th?…”

“…The existence of the lag period was explained by Cornell et al [22] by the precedence of the reoxidation of cytosolic NADH over giuconeogenesis, meaning that much of the oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred 'twice' from the mit~hondria to the cytosol by the aspartate shuttle before being converted into phosphoenolpyruvate. The observed discrepancy could be explained on the basis of the previous hypothesis [22]: th? cytosolic redox state of the liver of fed rats is more oxidized than that of the liver from starved rats [23], therefore, under a fed condition there would be no need of oxaloacetate diversfon and the lag period would not be expected to occur.…”

Gluconeogenesis by isolated guinea-pig liver parenchymal cells

“…While these studies were in progress, Cornell et al (1974) reported a similar lag period in glucose synthesis from lactate by isolated rat liver cells which was significantly shortened by the addition of NH4CI, lysine and other amino acids. In their experiments, however, the lag phase was considerably longer (40min) than in our studies but appeared to lead to a similar decrease in the lactate/pyruvate ratio.…”

The capacity of reducing‐equivalent shuttles limits glycolysis during ethanol oxidation

“…Thus, although in this unmodified state they are a useful tool for demonstrating the consequences of limitation of shuttle capacity on metabolism [29], they do not accurately reveal the interactions of glycolysis and ethanol oxidation under physiological conditions where shuttle capacity is intact. During incubation the hepatocytes correct this deficiency in shuttle capacity over a period of 20-30 min [30, 361 but it can be rectified from the commencement of the incubation by inclusion of asparagine in the medium, since this restores shuttle capacity to normal [30]. Thus, if it is desired to examine the interactions of glycolysis and ethanol under physiological conditions, one approach is to employ asparagine-supplemented hepatocytes.…”