Role for mitochondrial oxidants as regulators of cellular metabolism

Abstract

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.

FIG. 1

Pyruvate stimulates an increase in cytosolic JNK1 activity. (A) Time course of JNK1 activity in HeLa cells following pyruvate (100 mM) stimulation. Activity was assessed in 30 μg of protein extract by immunocomplex kinase assays using GST-ATF2 as a substrate. Lysates were also probed by Western blot analysis for JNK1 protein levels. (B) Levels of JNK1 activity 1 h after pyruvate addition in various cell types. The normal concentration of pyruvate in basal medium was 1 mM.

FIG. 2

Role of the mitochondria in pyruvate-stimulated JNK1 activity. (A) Peak levels of pyruvate (100 mM) and IL-1β (10 ng/ml) stimulated JNK1 activity in cells pretreated (1 h, 25 μM) with rotenone, a specific mitochondrial complex I inhibitor. (B) Lack of pyruvate-induced JNK1 activity but preservation of IL-1β-stimulated JNK1 activity in HeLa cells depleted of mitochondria by treatment with ethidium bromide (400 ng/ml) for 7 days.

FIG. 3

Pyruvate addition stimulates the release of mitochondrial oxidants. Confocal image of DHR123 fluorescence (A) under basal conditions and (B) 2 h following 100 mM pyruvate addition. Dose-response of (C) pyruvate-induced DHR123 fluorescence and (D) DCFDA fluorescence. (E) Quantitation of DHR123 fluorescence under basal conditions and 2 h following pyruvate addition in the presence (25 μM for 1 h) and absence of rotenone.

FIG. 3

Pyruvate addition stimulates the release of mitochondrial oxidants. Confocal image of DHR123 fluorescence (A) under basal conditions and (B) 2 h following 100 mM pyruvate addition. Dose-response of (C) pyruvate-induced DHR123 fluorescence and (D) DCFDA fluorescence. (E) Quantitation of DHR123 fluorescence under basal conditions and 2 h following pyruvate addition in the presence (25 μM for 1 h) and absence of rotenone.

FIG. 4

Role of mitochondrial oxidants in the activation of JNK1. (A) Inhibitory effect of the peroxide-scavenging agent NAC on JNK1 activity. Where indicated, cells were incubated with NAC for 15 h prior to pyruvate stimulation. (B) Effects of increasing amounts of GSTpi expression on pyruvate-stimulated JNK1 activity. (C) Corresponding level of GSTpi protein expression in total cellular lysate in control transfected (−) or GSTpi-transfected (2 μg) cells. The amount of GSTpi in transfected cells is underestimated in this blot since transfection efficiency was approximately 20%. (D) Exogenous catalase blocks pyruvate-induced JNK1 activation. (E) Levels of DHR123 fluorescence 15 min after treatment with various mitochondrial respiratory inhibitors and (F) the corresponding levels of JNK1 activity.

FIG. 5

Redox-dependent activation of GSK-3β. (A) Levels of GSK-3β activity following pyruvate addition in the presence and absence of the peroxide scavenger NAC (circles, pyruvate only; triangles, pyruvate with 2 mM NAC; squares, pyruvate with 10 mM NAC). (B) GSK-3β activity following treatment with 25 μM antimycin A (triangles), 25 μM rotenone (squares), or vehicle only (circles).

FIG. 6

Role for JNK1 in cytosolic metabolism. (A) Levels of GSK-3β activity in a HeLa cell line expressing a tetracycline-inducible dominant negative SEK1 isoform. Activity following pyruvate addition was assessed in the presence (solid circles) or absence (open circles) of doxycycline (DOX, 1 μg/ml). (Inset) Levels of expression of the dominant negative SEK1 isoform and the corresponding JNK1 activity in the presence and absence of doxycycline. (B) Glycogen synthase activity following pyruvate treatment (stippled bar), antimycin treatment (shaded bar), or under basal conditions (open bar). Activity was assessed in cells expressing a doxycycline (DOX)-inducible form of a dominant negative SEK1 isoform.

FIG. 7

In vivo assessment of JNK1 activity. (A) JNK1 activity in rat liver homogenates 1 h following injection of the indicated amount of pyruvate. (B) In vivo dose-response of JNK1 activity following pyruvate. b.w., body weight.

FIG. 8

Activation of RSK3 by mitochondrial oxidants. (A) Assessment of RSK3, p70S6K, and AKT1 activity under basal conditions or following pyruvate stimulation or antimycin treatment. (Inset) In vitro phosphorylation of a GSK fusion protein using immunoprecipitated RSK3 from control (C), pyruvate (P)-, or antimycin (A)-stimulated lysate. (B) Basal and stimulated levels of RSK3 activity in cells stably expressing an approximately twofold increase in GSTpi or in control (neo)-transfected cells. (C) Levels of RSK3 activity with or without overnight pretreatment with 20 mM NAC. RSK3 activity was assessed under basal conditions (open bar) or 90 min after 100 mM pyruvate (stippled bar) or 25 μM antimycin (shaded bar) treatment. (D) Levels of RSK2 or RSK3 activity under basal conditions or after increased mitochondrial oxidant release induced by antimycin treatment. Activity was assessed in the absence (−) or presence (+) of a dominant negative SEK1 isoform.

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 ROS level is sensed by the cytosol, resulting in the activation of JNK1. In turn, JNK1 inhibits the activity of GSK-3β, most likely through a pathway involving RSK3. The resulting change in GSK-3 activity leads to augmented glycogen synthase (GS) activity. Increased glycogen synthase activity results in an increased conversion of glucose to glycogen, with a subsequent reduction in metabolic substrate and hence a fall in mitochondrial oxidants. In addition to the effects of peroxide on JNK activity, peroxide can also result in the decarboxylation of pyruvate and thereby reduce mitochondrial metabolism, represented as the flow of electrons (e⁻) through complexes I through IV. CoA, coenzyme A; CoQ, coenzyme Q; MnSOD, manganese superoxide dismutase.