Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.     

Mitochondrial production of reactive oxygen species

Numerous biochemical studies are aimed at elucidating the sources and mechanisms of formation of reactive oxygen species (ROS) because they are involved in cellular, organ-, and tissue-specific physiology. Mitochondria along with other cellular organelles of eukaryotes contribute significantly to ROS formation and utilization. This review is a critical account of the mitochondrial ROS production and methods for their registration. The physiological and pathophysiological significance of the mitochondrially produced ROS are discussed.     

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.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Mitochondrial metabolism in hibernation and daily torpor: a review

Hibernation and daily torpor involve substantial decreases in body temperature and metabolic rate, allowing birds and mammals to cope with cold environments and/or limited food. Regulated suppression of mitochondrial metabolism probably contributes to energy savings: state 3 (phosphorylating) respiration is lower in liver mitochondria isolated from mammals in hibernation or daily torpor compared to normothermic controls, although data on state 4 (non-phosphorylating) respiration are equivocal. However, no suppression is seen in skeletal muscle, and there is little reliable data from other tissues. In both daily torpor and hibernation, liver state 3 substrate oxidation is suppressed, especially upstream of electron transport chain complex IV. In hibernation respiratory suppression is reversed quickly in arousal even when body temperature is very low, implying acute regulatory mechanisms, such as oxaloacetate inhibition of succinate dehydrogenase. Respiratory suppression depends on in vitro assay temperature (no suppression is evident below ~30 degrees C) and (at least in hibernation) dietary polyunsaturated fats, suggesting effects on inner mitochondrial membrane phospholipids. Proton leakiness of the inner mitochondrial membrane does not change in hibernation, but this also depends on dietary polyunsaturates. In contrast proton leak increases in daily torpor, perhaps limiting reactive oxygen species production.     

Mitochondrial potassium channels and reactive oxygen species

Pretreatment of tissues with potassium channel openers (KCO’s) has been observed to be cytoprotective in a broad variety of insults. This phenomenon has been proposed to be intimately linked to activation of mitochondrial potassium channels which apparently modulate the mitochondrial production of reactive oxygen species (ROS). This critical review summarizes literature findings about the mitochondrial production of ROS, the action of KCO’s on mitochondrial ROS production and the putative link to the cytoprotective action of these drugs.     

线粒体呼吸链与活性氧

已知有氧真核生物细胞吸收的氧分子绝大部分都是在线粒体呼吸链末端细胞色素氧化酶上通过四步单电子还原生成水。但同时也有1％-2％的氧可在呼吸链中途接受单电子或双电子被部分还原生成超氧（O2·^-和过氧化氢（H2O2）作为呼吸作用的正常代谢产物。此种来源于线粒体呼吸链的O2·^-和H2O2不但在多种病理的氧化损伤中起关键作用,同样它们也是正常生理条件下对多种细胞过程具有基本调控意义的氧还信号。基于Chance实验室约自20世纪70到90年代的早期研究贡献以及20世纪90年代后其他各实验室的研究新进展,我们聚焦于下述四个相关问题的评述和讨论：（1）由于线粒体内膜面积及其含有的呼吸链复合体酶活力远远高出细胞中所有膜系数量和相关酶活力之总和,因而线粒体呼吸链产生的O2·^-和H2O2构成生物体内最大数量ROS的恒定来源;（2）线粒体呼吸链复合体III的Q循环中Qo位点中半醌自由基（UQH·）已明确是O2·^-的单电子来源;还原细胞色素C-P66^SHC是生成H2O2的双电子供体。虽然复合体I也是产生线粒体基质内O2·^-的主要来源,但由于其确切生成位点尚未明确,在invivo条件下能否产生大量O2·^-也尚有争议;（3）线粒体呼吸链产生O2·^-后的分配和跨膜转移涉及其生理病理作用机制和作用靶点等复杂而重要的问题,直到目前尚未意见一致。“质子和O2·^-循环双回路解偶联模型”整合了目前提出的几种假说的联系点,指出H^＋和O2·^-相互作用生成HO2·及其跨膜很可能是这一复杂问题的中心环节,并与O2·^-对“脂肪酸shuttling model”或O2·^-对“UCPS激活”模型形成了内在的联系;（4）线粒体呼吸形成的△P（△ψ和△pH）能直接控制呼吸链的ROS生成,并以非线性（非欧姆）相关方式通过影响Q循环中的Qo半醌的氧还态和寿命来调节O2·^-生成的急速?     

Mitochondrial production of reactive oxygen species mediate dicumarol-induced cytotoxicity in cancer cells

Dicumarol is a naturally occurring anticoagulant derived from coumarin that induces cytotoxicity and oxidative stress in human pancreatic cancer cells (Cullen, J. J., Hinkhouse, M. M., Grady, M., Gaut, A. W., Liu, J., Zhang, Y., Weydert, C. J. D., Domann, F. E., and Oberley, L. W. (2003) Cancer Res. 63, 5513-5520). Although dicumarol has been used as an inhibitor of the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1), dicumarol is also thought to affect quinone-mediated electron transfer reactions in the mitochondria, leading to the production of superoxide (O2*-) and hydrogen peroxide (H(2)O(2)). We hypothesized that mitochondrial production of reactive oxygen species mediates the increased susceptibility of pancreatic cancer cells to dicumarol-induced metabolic oxidative stress. Dicumarol decreased clonogenic survival equally in both MDA-MB-468 NQO1(-) and MDA-MB-468 NQO1+ breast cancer cells. Dicumarol decreased clonogenic survival in the transformed fibroblast cell line IMRSV-90 compared with the IMR-90 cell line. Dicumarol, with the addition of mitochondrial electron transport chain blockers, decreased clonogenic cell survival in human pancreatic cancer cells and increased superoxide levels. Dicumarol with the mitochondrial electron transport chain blocker antimycin A decreased clonogenic survival and increased superoxide levels in cells with functional mitochondria but had little effect on cancer cells without functional mitochondria. Overexpression of manganese superoxide dismutase and mitochondrial-targeted catalase with adenoviral vectors reversed the dicumarol-induced cytotoxicity and reversed fluorescence of the oxidation-sensitive probe. We conclude mitochondrial production of reactive oxygen species mediates the increased susceptibility of cancer cells to dicumarol-induced cytotoxicity.     

Mitochondrial metabolism during fasting-induced daily torpor in mice

During fasting, mice (Mus musculus) undergo daily bouts of torpor, considerably reducing body temperature (T_b) and metabolic rate (MR). We examined females of different laboratory strains (Balb/c, C57/6N, and CD1) to determine whether liver mitochondrial metabolism is actively reduced during torpor. In all strains, we found that state 3 (phosphorylating) respiration rate measured at 37 °C was reduced up to 35% during torpor for at least one of the substrates (glutamate and succinate) used to fuel respiration. The extent of this suppression varied and was correlated with T_b at sampling. This suggests that, at the biochemical level, the transition to and from a hypometabolic torpid state is gradual. In fasted non-torpid animals, T_b and MR still fluctuated greatly: T_b dropped by as much as 4 °C and MR was reduced up to 25% compared to fed controls. Changes in T_b and MR in fasted, non-torpid animals were correlated with changes in mitochondrial state 3 respiration rate measured at 37 °C. This suggests that fasting mice may conserve energy even when not torpid by occasionally reducing T_b and mitochondrial oxidative capacity to reduce MR. Furthermore, proton conductance was higher in torpid compared to non-torpid animals when measured at 15 °C (the lower limit of torpid T_b). This pattern is similar to that reported previously for daily torpor in Phodopus sungorus.     

Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

Mitochondrial reactive oxygen species and Ca2+ signaling

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca²⁺ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca²⁺]_i) signals. Several Ca²⁺ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca²⁺ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca²⁺]_i signals by Ca²⁺ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca²⁺ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca²⁺ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca²⁺]_i signals. Together, the data reviewed here indicate that Ca²⁺-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca²⁺]_i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production

Pharmacological mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening protects against ischemic damage and mimics ischemic preconditioning. However, physiological and pathological signaling events that open this channel are still not fully understood. We found that catalase, which removes H(2)O(2), is capable of reversing the beneficial effects of ischemic preconditioning but not of mitoK(ATP) agonist diazoxide. On the other hand, 2-mercaptopropionylglycine prevented cardioprotection in both cases, suggesting that this compound may present effects other than scavenging of reactive oxygen species. Indeed, 2-mercaptopropionylglycine and a second thiol-reducing agent, dithiothreitol, impair diazoxide-mediated activation of mitoK(ATP) in isolated heart mitochondria. This demonstrates that mitoK(ATP) activity is regulated by thiol redox status. Furthermore, stimulating the generation of endogenous mitochondrial reactive oxygen species or treating samples with H(2)O(2) strongly enhances mitoK(ATP) activity, in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor. We also demonstrate that mitoK(ATP) channel activity effectively prevents mitochondrial reactive oxygen release. Collectively, our results suggest that mitoK(ATP) acts as a reactive oxygen sensor that decreases mitochondrial free radical generation in response to enhanced local levels of oxidants. As a result, these channels regulate mitochondrial redox state under physiological conditions and prevent oxidative stress under pathological conditions such as ischemia/reperfusion.     

Reduction of metabolic rate and thermoregulation during daily torpor

Physiological mechanisms causing reduction of metabolic rate during torpor in heterothermic endotherms are controversial. The original view that metabolic rate is reduced below the basal metabolic rate because the lowered body temperature reduces tissue metabolism has been challenged by a recent hypothesis which claims that metabolic rate during torpor is actively downregulated and is a function of the differential between body temperature and ambient temperature, rather than body temperature per se. In the present study, both the steady-state metabolic rate and body temperature of torpid stripe-faced dunnarts, Sminthopsis macroura (Dasyuridae: Marsupialia), showed two clearly different phases in response to change of air temperature. At air temperatures between 14 and 30°C, metabolic rate and body temperature decreased with air temperature, and metabolic rate showed an exponential relationship with body temperature (r ²=0.74). The Q ₁₀ for metabolic rate was between 2 and 3 over the body temperature range of 16 to 32°C. The difference between body temperature and air temperature over this temperature range did not change significantly, and the metabolic rate was not related to the difference between body temperature and air temperature (P=0.35). However, the apparent conductance decreased with air temperature. At air temperatures below 14°C, metabolic rate increased linearly with the decrease of air temperature (r ²=0.58) and body temperature was maintained above 16°C, largely independent of air temperature. Over this air temperature range, metabolic rate was positively correlated with the difference between body temperature and air temperature (r ²=0.61). Nevertheless, the Q ₁₀ for metabolic rate between normothermic and torpid thermoregulating animals at the same air temperature was also in the range of 2–3. These results suggest that over the air temperature range in which body temperature of S. macroura was not metabolically defended, metabolic rate during daily torpor was largely a function of body temperature. At air temperatures below 14°C, at which the torpid animals showed an increase of metabolic rate to regulate body temperature, the negative relationship between metabolic rate and air temperature was a function of the differential between body temperature and air temperature as during normothermia. However, even in thermoregulating animals, the reduction of metabolic rate from normothermia to torpor at a given air temperature can also be explained by temperature effects.Abbreviations BM body mass - BMR basal metabolic rate - C apparent conductance - MR metabolic rate - RMR resting metabolic rate - RQ respiratory quotient - T _a air temperature - T _b body temperature - T _lc lower critical temperature - T _tc critical air temperature during torpor - TMR metabolic rate during torpor - TNZ thermoneutral zone - T difference between body temperature and air temperature - VO₂ rate of oxygen consumption     

Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes

Mitochondria are the main source of reactive oxygen species (ROS). The aim of this work was to verify the ROS generation in situ in HeLa cells exposed to prooxidants and antioxidants (menadione, tert-butyl hydroperoxide, antimycin A, vitamin E, N-acetyl-l-cysteine, and butylated hydroxytoluene) using the ROS-sensitive probes 6-carboxy-2,7-dichlorodihydrofluorescein diacetate di-acetomethyl ester (DCDHF) and dihydrofluorescein diacetate (DHF). Mitochondria were counterstained with the potential-sensitive probe tetramethylrhodamine methyl ester perchlorate (TMRM). Both DCDHF and DHF were able to detect the presence of ROS in mitochondria, though with distinct morphological features. DCDHF fluorescence was invariably blurred, smudged, and spread over the cytoplasm surrounding the major mitochondrial clusters. On the contrary, DHF fluorescence was sharp and delineated thin filaments which corresponded in all details to TMRM-stained mitochondria. These data suggest that DCDHF does not reach the mitochondrial matrix but is oxidized by ROS released by mitochondria in the cytosol. On the other hand, DHF enters mitochondria and reacts with ROS released in the matrix. Cytosolic (DCDHF+) ROS but not matrix (DHF+) ROS, were significantly decreased by vitamin E. N-acetyl-l-cysteine was effective in reducing DCDHF and DHF photooxidation in the medium, but was unable to reduce intracellular ROS. ROS generation was accompanied by partial mitochondrial depolarization.     

Mitochondrial reactive oxygen species affect sensitivity to curcumin-induced apoptosis

Curcumin exhibits anticancer activity in vivo and triggers tumor cell apoptosis in vivo and in vitro. Several in vitro studies suggest that curcumin-induced apoptosis is associated with reactive oxygen species (ROS) production and/or oxidative stress in transformed cells. This study compared and contrasted the effects of curcumin on human skin cancer cells and their respiration-deficient (rho0) clones to characterize the prospective oxidative stress signaling responsible for initiating apoptosis. Curcumin promoted a dose-and time-dependent G2/M cell cycle arrest and/or apoptosis in COLO 16 cells. Apoptosis induction in COLO 16 cells was associated with DNA fragmentation, cell shrinkage, the externalization of cell membrane phosphatidylserine, and mitochondrial disruption, which were preceded by an increase in intracellular ROS production. Pharmacologically lowering the mitochondrial bioenergetic capacity, as well as the constitutive ROS levels, in COLO 16 cells suppressed the cytotoxic effects of curcumin. Correspondingly, the rho0 counterparts of COLO 16 cells were markedly resistant to ROS production, mitochondrial disruption, and DNA fragmentation following curcumin exposure. These observations implied that the diminution of mitochondrial ROS production protected cells against the cytotoxic effects of curcumin, and support the notion that mitochondrial respiration and redox tone are pivotal determinants in apoptosis signaling by curcumin in human skin cancer cells.