Our results demonstrate that an increase in metabolite supply, in this case in the form of pyruvate, is capable of activating JNK1. This is not a direct effect of pyruvate, but instead requires the mitochondrion-dependent metabolism of this substrate. An increase in mitochondrial ROS develops as a consequence of the increase in metabolism, and the subsequent generation of H2O2 is essential for the activation of JNK1. Not unexpectedly, pyruvate causes a fall in GSK-3β activity. Interestingly, this reduction in GSK-3β activity requires the redox-dependent activation of JNK1 and appears to proceed through RSK3 activation. As such, these results describe the outline of the novel regulatory loop depicted in Fig. .
FIG. 9 Model for mitochondrial oxidants as regulators of cellular metabolism. As described, an increase in metabolite flow to the mitochondria results in an increase in O2 consumption with the subsequent increased release of ROS into the cytosol. This increased (more ...)
We observed an increase in JNK1 activity in a variety of different cell types over a range of pyruvate concentrations. The largest increase in JNK1 activity came at a high pyruvate concentration, suggesting that the pathways described here may primarily operate in the setting of a sudden, large increase in metabolic supply. Nonetheless, it is important to note that cells in culture, particularly transformed cells, have an increased rate of anaerobic glycolysis. As such, the percentage of pyruvate entering the mitochondria may be significantly reduced in cultured cells. It should also be noted that it has been known for many years that pyruvate can scavenge peroxide directly, resulting in the oxidative decarboxylation of pyruvate (26
). In light of the results presented here, it is tempting to speculate that this property of pyruvate has a homeostatic effect. In particular, small increases in pyruvate would cause increases in mitochondrial peroxide generation that in turn could be scavenged by cytosolic pyruvate. In the process of scavenging hydrogen peroxide, pyruvate is decarboxylated to lactate and hence diverted away from aerobic metabolism and further ROS generation. It is possible that the scavenging and decarboxylation of pyruvate help regulates metabolic flux under basal conditions, while the pathway described in this paper may primarily operate following a large metabolic load. Under such conditions, the ROS generated by pyruvate would exceed the scavenging capacity of pyruvate. This is consistent with our observations that under conditions in which pyruvate stimulated JNK1 activity, both mitochondrial and cytosolic ROS levels rose.
The in vivo activation of JNK1 by pyruvate occurred with an approximately sixfold rise in serum pyruvate levels. Although this represents a significant rise in pyruvate levels, recent evidence suggests that vigorous exercise can produce a similar three- to fivefold elevation in human subjects (12
). Similarly, in preliminary studies in our laboratory, pyruvate levels rose approximately threefold 1 h after eating a large slice of chocolate cake (unpublished observations). As such, the activation of JNK1 by pyruvate occurs at apparently high but physiologically achievable doses of serum pyruvate.
In the process of review of the manuscript, a report appeared suggesting that the increase in mitochondrial ROS induced by elevated glucose levels may contribute to many of the pathological changes observed in diabetes (29
). In particular, elevated mitochondrial ROS appeared to contribute to glucose-stimulated activation of protein kinase C, sorbitol accumulation, and the activation of NF-κB. Inhibition of each of these pathways appears in previous animal studies to provide benefits from some of the complications of diabetes. Given these recent results as well as the data presented here, it is tempting to speculate that the homeostatic regulation provided by mitochondrial oxidants may be perturbed in certain disease states such as diabetes. Under these conditions the normal negative feedback provided by mitochondrial oxidants may not be present or as robust. In this setting, high levels of metabolic substrate such as glucose or pyruvate would not be appropriately channeled into glycogen. Consistent with this notion, glycogen synthesis activity is known to be impaired in type II diabetics (18
). Direct scavenging of mitochondrial oxidants or augmenting the activation of JNK1 may therefore be one strategy to prevent these perturbations. In this regard, the compound RO 31-8220 has been recently shown to activate JNK and increase cellular glycogen synthase activity (34
). Interestingly, preliminary evidence suggests that in vivo administration of this compound appears to reverse some of the metabolic abnormalities seen in the type II diabetic Goto-Kakizaki rats (34
A growing body of literature suggests that oxidants function as signaling molecules (14
). Many ligands appear to stimulate cytosolic oxidant production, and this ligand-activated change in oxidant production appears to affect a variety of downstream pathways. In contrast to the growing appreciation that oxidants generated in the cytosol contribute to signaling pathways, the continuous release of mitochondrial oxidants has been generally regarded solely as a deleterious byproduct of aerobic metabolism. Nonetheless, oxidants are also employed as signaling molecules in plants (2
), suggesting that this represents an evolutionarily conserved method of signal transduction. The ancient incorporation of mitochondria into eukaryotic cells undoubtably required that the cell cytosol coordinate metabolite flow and metabolic rates with these newly acquired energy-producing organelles. Our results suggest that one means by which the mitochondria and the cytoplasm communicate is by the release of the small diffusible molecule hydrogen peroxide.