Role of Pyruvate Kinase in the Regulation of Gluconeogenesis from L-Lactate*

Glucagon and L-epinephrine stimulate gluconeogenesis from 20 mM L-lactate, the effect being about 3 times greater in liver cells from fed rats than in those from fasted rats. The rate of pyruvate kinase flux was estimated to be less than 10% of the rate of gluconeogenesis from lactate in hepatocytes from fasted rats, and neither glucagon nor epinephrine lowered the absolute rate significantly. In hepatocytes from fed rats, however, the rate of pyruvate kinase was nearly one-half that of gluconeogenesis. Glucagon caused a marked depression of pyruvate kinase flux, with 1 muM glucagon lowering the rate to nearly the level found in cells from fasted rats Epinephrine at concentrations from 10(-8) to 10(-6) M actually increased pyruvate kinase flux during gluconeogenesis from lactate in cells from fed rats. These results are in accord with the view that the effects of glucagon and epinephrine on gluconeogenesis are not identical.


Glucagon
and L-epinephrine stimulate gluconeogenesis from 20 mM L-lactate, the effect being about 3 times greater in liver cells from fed rats than in those from fasted rats. The rate of pyruvate kinase flux was estimated to be less than 10% of the rate of gluconeogenesis from lactate in hepatocytes from fasted rats, and neither glucagon nor epinephrine lowered the absolute rate significantly.
In hepatocytes from fed rats, however, the rate of pyruvate kinase was nearly one-half that of gluconeogenesis.
Glucagon caused a marked depression of pyruvate kinase flux, with 1 PM glucagon lowering the rate to nearly the level found in cells from fasted rats. Epinephrine at concentrations from lo-' to lo-" M actually increased pyruvate kinase flux during gluconeogenesis from lactate in cells from fed rats. These results are in accord with the view that the effects of glucagon and epinephrine on gluconeogenesis are not identical.    lactate (20 mM) in isolated liver cells from either fasted or fed rata. The stimulatory effect of the hormones is considerably greater in hepatocytes from fed rata, and the maximal effect of epinephrine we find to be slightly greater than that of glucagon in cells from both fasted and fed rats. We used the high concentration of lactate in order to estimate pyruvate kinase isotopically by a trapping method (5).
The main question which we have attempted to answer in this work is how much of the stimulatory action of the hormones on gluconeogenesis from lactate can be accounted for by an inhibition of a "leak back" reaction, namely the flow from P-enolpyruvate back to pyruvate, catalyzed by pyruvate kinase. In Table I, we show that the estimated absolute rate of pyruvate kinase flux, during gluconeogenesis from lactate in liver cells from fasted rata, is less than 10% of the carbon flux from P-enolpyruvate to glucose, and that neither glucagon nor epinephrine at all concentrations tested had any significant effect on the absolute rate of pyruvate kinase. This shows that the stimulatory effect of the hormones on gluconeogenesis under these conditions is primarily at other sites than pyruvate kinase, presumably at one of the reactions (or transport steps) in the conversion of pyruvate to P-enolpyruvate (12). One might note that the hormones, while causing no net changes in absolute pyruvate kinase flux, did maintain apparent pyruvate kinase flux in the presence of an increased flux through P-enolpyruvate to glucose. One should also point out that the estimations of pyruvate kinase flux become less reliable when the isotopic fluxes are low, when isotopic exchange reactions could make larger contributions to the results (13,14).
In liver cells from fed rata, the rate of pyruvate kinase during gluconeogenesis from lactate is much higher, with the rate of pyruvate kinase approaching one-half that of the conversion of lactate to glucose (Table I). Here a difference in action between glucagon and L-epinephrme becomes very apparent. While both hormones cause a marked stimulation of gluconeogenesis from lactate in liver cells from fed rats, only glucagon causes a marked reduction in absolute pyruvate kinase flux. In fact, epinephrine at concentrations below 1 PM causes a slight increase in absolute pyruvate kinase flux, in spite of which the rate of glucose synthesis is increased. At 10 PM epinephrine, the absolute rate of pyruvate kinase flux is the same as that in the control (no hormone added) cells, but the proportion of P-enolpyruvate returned to pyruvate compared to that converted to glucose is depressed by nearly onehalf. Thus the lack of effect on the absolute rate of pyruvate kinase does not necessarily mean that L-epinephrine at this high concentration has no effect on the pyruvate kinase system, although the effect is certainly much less than that produced by glucagon. At the lower range of concentrations, glucagon produces only small changes in pyruvate kinase flux while causing a substantial increase in gluconeogenesis.
This indicates that glucagon also must act at a forward step in the gluconeogenic pathway. At higher concentrations, glucagon depresses pyruvate kinase flux to a level approaching that in liver cells from fasted rata, and causes an even more striking depression of the proportion of P-enolpyruvate reconverted to pyruvate compared to that converted to glucose. Comparing the decrease in pyruvate kinase flux (about 18 pmol/g/h) caused by 1 PM glucagon, to the net increase in gluconeogenesis (about 23 pmol of glucose/g/h or 46 pmol/g/h of P-enolpyruvate converted to glucose), it is again apparent that depression of pyruvate kinase flux cannot be the sole site of action of glucagon. Under these conditions, however, it is an important site of action. Considering that pyruvate kinase flux could be somewhat underestimated by the isotopic procedure (see "Materials and Methods"), nearly half of the stimulation of gluconeogenesis from lactate could be ascribed to depression of pyruvate kinase in cells from fed animals. On the other hand, the stimulatory effect at the "other site" appears to come into play at lower concentrations of glucagon. Our results show that a marked effect of glucagon on pyruvate kinase flux during gluconeogenesis from lactate can only be seen in hepatocytes from fed rats. However, with other substrates such as pyruvate, fructose, and dihydroxyacetone, we and others have shown that glucagon can cause a definite depression of apparent pyruvate kinase flux even in livers from fasted rats (5,(14)(15)(16). In general, pyruvate kinase flux is lower when more reduced substrates are metabolized (141, suggesting that the NADH/NAD+ ratio may in some manner also regulate pyruvate kinase flux.
It seems highly likely that the glucagon effect on pyruvate kinase is mediated by cyclic AMP, since the actions of glucagon or added cyclic AMP produce very similar effects in the intact liver cell (5,14). The mechanism of the effect is being investigated by several groups (17)(18)(19)(20)(21)(22). Blair and co-workers (17,18) have shown that glucagon treatment of the perfused rat liver decreases the affinity of pyruvate kinase for P-enolpyruvate. Engstrom and co-workers (19,201 have shown that a cyclic AMP-dependent protein kinase caused phosphorylation and inhibition of rat liver pyruvate kinase, and that full activity could be obtained upon dephosphorylation. Epinephrine has much less effect than glucagon on pyruvate kinase flux in hepatocytes from fed rats. The effect of 10 PM epinephrine, on lowering the relative flux through P-enolpyruvate to pyruvate, presumably may be the result of the p-adrenergic effect of epinephrine at this rather high concentration. Our results are in general accord with the views of Fain and coworkers (23) that the mechanisms of action of glucagon and epinephrine on the liver cell are not identical.