Metabolic ROS Signaling: To Immunity and Beyond.

Metabolism is a critical determinant of immune cell functionality. Immunometabolism, by definition, is a multidisciplinary area of immunology research that integrates the knowledge of energy transduction mechanisms and biochemical pathways. An important concept in the field is metabolic switch, a transition of immune cells upon activation to preferential utilization of select catabolic pathways for their energy needs. Mitochondria are not inert in this process and contribute to the metabolic adaptation by different mechanisms which include increasing ATP production to match dynamic bioenergetic demands and serving as a signaling platform. The latter involves generation of reactive oxygen species (ROS), one of the most intensively studied mitochondrial processes. While the role of mitochondrial ROS in the context of oxidative stress is well established, ROS signaling in immunity is an emerging and quickly changing field. In this review, we discuss ROS signaling and immunometabolism concepts from the standpoint of bioenergetics. We also provide a critical insight into the methodology for ROS assessment, outlining current challenges in the field. Finally, based on our analysis of the literature data, we hypothesize that regulatory ROS production, as opposed to oxidative stress, is controlled by mitochondrial biogenesis rather than metabolic switches.

[The effect of fenofibrate on expression of genes involved in fatty acids beta-oxidation and associated free-radical processes]. / Vliianie fenofibrata na ékspressiiu genov beta-okisleniia zhirnykh kislot i sviazannye s nim svobodnoradikal'nye protsessy.

Fenofibrate is a synthetic ligand for peroxisome proliferator-activated receptors subtype alpha (PPARa); it is used for the treatment of a wide range of metabolic diseases such as hypertriglyceridemia, dyslipidemia, diabetes and various neurodegenerative diseases. We have studied the effect of fenofibrate on b-oxidation of fatty acids and related free-radical processes. The most effective concentration of fenofibrate (0.3%) added to the chow caused a significant decrease of the body weight of mice. The data obtained by quantitative PCR demonstrated increased hepatic gene expression responsible for b-oxidation of fatty acids in peroxisomes and mitochondria. Enhancement of oxidative processes caused a 2-fold increase in the rate of reactive oxygen species (ROS) production, as evidenced by determination of the level of lipid peroxidation (LPO) products in the liver. Mitochondrial antioxidant systems are more sensitive to elevated ROS production, as they respond by increased expression of SOD2 and PRDX3 genes, than cytoplasmic and peroxisomal antioxidant systems, where expression of CAT1, SOD1, PRDX5 genes remained unaltered.

Mitochondrial ROS Metabolism: 10 Years Later.

The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66(shc) protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field.

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.).

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.

Impaired brain creatine kinase activity in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is associated with impaired energy metabolism in the brain. Creatine kinase (CK) catalyzes ATP-dependent phosphorylation of creatine (Cr) into phosphocreatine (PCr), thereby serving as readily available high-capacity spatial and temporal ATP buffering. OBJECTIVE: Substantial evidence supports a specific role of the Cr/PCr system in neurodegenerative diseases. In the brain, the Cr/PCr ATP-buffering system is established by a concerted operation of the brain-specific cytosolic enzyme BB-CK and ubiquitous mitochondrial uMt-CK. It is not yet established whether the activity of these CK isoenzymes is impaired in HD. METHODS: We measured PCr, Cr, ATP and ADP in brain extracts of 3 mouse models of HD - R6/2 mice, N171-82Q and HdhQ(111) mice - and the activity of CK in cytosolic and mitochondrial brain fractions from the same mice. RESULTS: The PCr was significantly increased in mouse HD brain extracts as compared to nontransgenic littermates. We also found an approximately 27% decrease in CK activity in both cytosolic and mitochondrial fractions of R6/2 and N171-82Q mice, and an approximately 25% decrease in the mitochondria from HdhQ(111) mice. Moreover, uMt-CK and BB-CK activities were approximately 63% lower in HD human brain samples as compared to nondiseased controls. CONCLUSION: Our findings lend strong support to the role of impaired energy metabolism in HD, and point out the potential importance of impairment of the CK-catalyzed ATP-buffering system in the etiology of HD.

Mitochondrial metabolism of reactive oxygen species.

Oxidative stress is considered a major contributor to etiology of both "normal" senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.

Effect of electron-transport inhibitors on the generation of reactive oxygen species by pea mitochondria during succinate oxidation.

The effect of inhibitors of the cytochrome pathway and alternative oxidase on the rate of respiration and generation of reactive oxygen species by pea mitochondria was studied. Respiration of mitochondria from pea cotyledons was inhibited by 70-80% by salicylhydroxamate (SHAM). The rate of hydrogen peroxide production by pea cotyledon mitochondria during succinate oxidation was 0.15 nmol/min per mg protein. SHAM considerably accelerated the hydrogen peroxide production. The SHAM-dependent H2O2 production was stimulated by 2 micro M antimycin A and inhibited by 5 mM KCN and 1 micro M myxothiazol. The study of the rate of O2*- generation by pea mitochondria using EPR spin traps and epinephrine oxidation showed that H2O2 accumulation can be accounted for by a significant increase in the rate of O2*- production.

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+.

At low Ca2+ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca2+ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential (DeltaPsi) across the inner mitochondrial membrane caused by 10 micro M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the "classic" state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of "classic pore" opening in Ca2+-loaded mitochondria myristic acid induces the cyclosporin A-sensitive DeltaPsi decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P(i) and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped DeltaPsi decrease induced by 5 micro M myristic acid and 5 micro M Ca2+. In the last case nigericin, when added after cyclosporin A, reversed the DeltaPsi decrease, and the following addition of EGTA produced only a weak (if any) DeltaPsi increase. In P(i)-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the DeltaPsi decrease. These data show that the cyclosporin A-sensitive decrease in DeltaPsi by low concentrations of fatty acids and Ca2+ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca2+ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive DeltaPsi decrease results from a conversion of DeltaPsi to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca2+-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.

Thyroxine reversibly inhibits the uncoupling action of protonophores on energy production in rat thymus lymphocytes.

Earlier we reported that some thyroid and steroid hormones and also 6-ketocholestanol used in micromolar concentrations modulated the effects of protonophoric uncouplers on isolated mitochondria (Starkov et al. (1997) Biochim. Biophys. Acta, 1318, 173-183). In the present study we investigated the effects of a thyroid hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and mitochondria isolated from these cells. The resting (oligomycin-inhibited) respiration of the isolated intact lymphocytes was stimulated by the addition of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of micromolar concentrations of thyroxin decreased the rate of uncoupler-stimulated respiration and partially reversed uncoupler-induced decrease of membrane potential (DeltaPsi). In experiments with mitochondria isolated from thymus lymphocytes the re-coupling effect of thyroxine was not observed. In this case thyroxine did not influence mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation of respiration and DeltaPsi decrease induced with another uncoupler, SF6847. The data are discussed in terms of a hypothesis that aromatic uncouplers are transported into the cell by the thyroxine carrier of the plasma membrane.

Structural determinants of fluorochemical-induced mitochondrial dysfunction.

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria.

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO4 and ascorbate (vitamin C) was studied. FeSO4 (1-4 microM) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca2+. All the effects of FeSO4 + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca2+-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO4 + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO4 and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca2+-dependent pore in the inner mitochondrial membrane.

Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain.

Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex III is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking. We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H(2)O(2) in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H(2)O(2) production by mitochondria oxidizing malate, succinate, or alpha-glycerophosphate. Stigmatellin inhibited H(2)O(2) production induced by myxothiazol. Myxothiazol-induced H(2)O(2) production was dependent on the succinate/fumarate ratio but in a manner different from H(2)O(2) generation induced by antimycin A. We conclude that myxothiazol-induced H(2)O(2) originates from a site located in the complex III Q(o) center but different from the site of H(2)O(2) production inducible by antimycin A.

Mitochondrial targets of drug toxicity.

Mitochondria have long been recognized as the generators of energy for the cell. Like any other power source, however, mitochondria are highly vulnerable to inhibition or uncoupling of the energy harnessing process and run a high risk for catastrophic damage to the cell. The exquisite structural and functional characteristics of mitochondria provide a number of primary targets for xenobiotic-induced bioenergetic failure. They also provide opportunities for selective delivery of drugs to the mitochondrion. In light of the large number of natural, commercial, pharmaceutical, and environmental chemicals that manifest their toxicity by interfering with mitochondrial bioenergetics, it is important to understand the underlying mechanisms. The significance is further underscored by the recent identification of bioenergetic control points for cell replication and differentiation and the realization that mitochondria play a determinant role in cell signaling and apoptotic modes of cell death.

Fatty acids as natural uncouplers preventing generation of O2.- and H2O2 by mitochondria in the resting state.

Both natural (laurate) and artificial (m-chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O2.- and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate, an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the releasing effect of carboxyatractylate. GDP is much less effective than carboxyatractylate in releasing the laurate inhibition of reactive oxygen species (ROS) formation. Micromolar laurate concentrations arresting the ROS formation cause strong inhibition of reverse electron transfer from succinate to NAD+, whereas State 4 respiration and the transmembrane electric potential difference (delta psi) level are affected only slightly. It is suggested that (i) free fatty acids operate as natural 'mild uncouplers' preventing the transmembrane electrochemical H+ potential difference (delta muH+) from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of the respiratory chain, and (ii) it is the ATP/ADP-antiporter, rather than uncoupling protein 2, that is mainly involved in this antioxidant mechanism of heart muscle mitochondria.

Inhibition of the alternative oxidase stimulates H2O2 production in plant mitochondria.

The hypothesis that a non-coupled alternative oxidase of plant mitochondria operates as an antioxygen defence mechanism [Purvis, A.C. and Shewfelt, R.L., Physiol. Plant. 88 (1993) 712-718; Skulachev, V.P., Biochemistry (Moscow) 59 (1994) 1433-1434] has been confirmed in experiments on isolated soybean and pea cotyledon mitochondria. It is shown that inhibitors of the alternative oxidase, salicyl hydroxamate and propyl gallate strongly stimulate H2O2 production by these mitochondria oxidizing succinate. Effective concentrations of the inhibitors proved to be the same as those decreasing the cyanide-resistant respiration. The inhibitors proved to be ineffective in stimulating H2O2 formation in rat liver mitochondria lacking the alternative oxidase.

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria.

Formation of H2O2 has been studied in rat heart mitochondria, pretreated with H2O2 and aminotriazole to lower their antioxidant capacity. It is shown that the rate of H2O2 formation by mitochondria oxidizing 6 mM succinate is inhibited by a protonophorous uncoupler, ADP and phosphate, malonate, rotenone and myxothiazol, and is stimulated by antimycin A. The effect of ADP is abolished by carboxyatractylate and oligomycin. Addition of uncoupler after rotenone induces further inhibition of H2O2 production. Inhibition of H2O2 formation by uncoupler, malonate and ADP+Pi is shown to be proportional to the delta psi decrease by these compounds. A threshold delta psi value is found, above which a very strong increase in H2O2 production takes place. This threshold slightly exceeds the state 3 delta psi level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169-2021 that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation.

Thyroxine induces cyclosporin A-insensitive, Ca2+-dependent reversible permeability transition pore in rat liver mitochondria.

The effect of thyroxine on Ca2+-dependent mitochondrial permeability transition has been examined. It is shown that 40 microM thyroxine induces high amplitude swelling and decrease in membrane potential in Ca2+-loaded rat liver mitochondria, both in the presence and absence of cyclosporin A. Thyroxine-induced decrease in membrane potential is partially or completely reversed by addition of EGTA into the incubation medium. Nigericin and ADP are shown to prevent, or significantly delay, the effects of thyroxine on both mitochondrial swelling and membrane potential, whereas nicotinamide potentiates the permeabilisation of mitochondria. It is suggested that thyroxine induced reversible, cyclosporin A-insensitive permeability transition pore (PTP) opening in the inner mitochondrial membrane.

"Mild" uncoupling of mitochondria.

Recently, it was proposed that the thyroid hormone-mediated uncoupling in mitochondria is involved in the cellular defence system against free radicals (Skulachev V.P. (1996) Quart. Rev. Biophys. 29:169-202). This phenomenon was named "mild" uncoupling. It was postulated to be a protein-mediated process controlled by several factors. The data reported during the past 40 years, pointing to the protein-mediated uncoupling mechanism in mitochondria, are reviewed in a context of hypothetical properties of "mild" uncoupling. The mechanism of "mild" uncoupling is suggested to be the following: (a) mitochondria possess protein(s) that regulate the proton permeability of inner mitochondrial membrane; (b) these proteins are regulated by binding of an unidentified low-molecular-weight endogenous compound with properties resembling those of the most active artificial uncouplers like FCCP and SF6847; (c) the interaction of this compound with its target protein(s) is modulated by a thyroid hormone in a positive (i.e. enhancing the proton permeability) way and by sex steroid hormones in a negative way; (e) endogenous fatty acids can attenuate the influence of both thyroid and steroid hormones.