4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H(+) leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H(+) leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenal-induced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed.     

解偶联蛋白2与衰老

解偶联蛋白（uncoupling protein,UCP）属于内膜上的一类载体蛋白,其生理作用是消除线粒体膜电位,使氧化磷酸化解偶联,从而抑制酸腺苷（adenosine triphosphate,ATP）合成,能量以热能形式散发。研究发现UCP2具有一种质子漏功能,表现对线粒体活性氧（reactive oxygen species,ROS）产生的调控和降低ROS的功能：在不同组织器官,不同代谢状态下UCP2的生理功能对细胞的影响不完全相同。特别是近年来的研究发现,UCP2参与了能量代谢、ROS的产生、子宫内膜退化、衰老等过程,并且与非酒精性脂肪肝、抗肥胖、动脉粥样硬化、局部缺血以及缺血再灌注损伤和2型糖尿病等有一定的相关性,倍受人们的关注。     

Evidence for Involvement of Uncoupling Proteins in Cerebral Mitochondrial Oxidative Phosphorylation Deficiency of Rats Exposed to 5,000 m High Altitude

The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.     

Avian UCP: The Killjoy in the Evolution of the Mitochondrial Uncoupling Proteins

The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs' activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.     

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.     

UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism

Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.     

Use the Protonmotive Force: Mitochondrial Uncoupling and Reactive Oxygen Species

Mitochondrial respiration results in an electrochemical proton gradient, or protonmotive force (pmf), across the mitochondrial inner membrane. The pmf is a form of potential energy consisting of charge (?ψ_m) and chemical (?pH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Although capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review, we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models—mitochondria and cells—only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo.     

Mitochondria and the regulation of free radical damage in the eye

Neuronal cell death can be determined by the overall level of reactive oxygen species (ROS) resulting from the combination of extrinsic sources and intrinsic production as a byproduct of oxidative phosphorylation. Key controllers of the intrinsic production of ROS are the mitochondrial uncoupling proteins (UCPs). By allowing a controlled leak of protons across the inner mitochondrial membrane activation of these proteins can decrease ROS and promote cell survival. In both primate models of Parkinson’s disease and mouse models of seizures, increased activity of UCP2 significantly increased neuronal cells survival. In the retina UCP2 is expressed in many neurons and glial cells, but was not detected in rod photoreceptors. Retinal ganglion cell survival following excitotoxic damage was much greater in animals overexpressing UCP2. Traditional Chinese medicines, such as an extract of Cistanche tubulosa, may provide benefit by altering mitochondrial metabolism.     

棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.     

Antioxidant properties of UCP1 are evolutionarily conserved in mammals and buffer mitochondrial reactive oxygen species

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of “mild uncoupling”. Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.     

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.     

Mitochondrial uncoupling proteins--facts and fantasies

Instead of a comprehensive review, we describe the basic undisputed facts and a modest contribution of our group to the fascinating area of the research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, protein-mediated uncoupling, we discuss the assumption that due to their low abundance the novel mitochondrial uncoupling proteins (UCP2 to UCP5) can provide only a mild uncoupling, i.e. can decrease the proton motive force by several mV only. Contrary to this, the highly thermogenic role of UCP1 in brown adipose tissue is not given only by its high content (approximately 5 % of mitochondrial proteins) but also by the low ATP synthase content and high capacity respiratory chain. Fatty acid cycling mechanism as a plausible explanation for the protonophoretic function of all UCPs and some other mitochondrial carriers is described together with the experiments supporting it. The phylogenesis of all UCPs, estimated UCP2 content in several tissues, and details of UCP2 activation are described on the basis of our experiments. Functional activation of UCP2 is proposed to decrease reactive oxygen species (ROS) production. Moreover, reaction products of lipoperoxidation such as cleaved hydroperoxy-fatty acids and hydroxy-fatty acid can activate UCP2 and promote feedback down-regulation of mitochondrial ROS production.     

Control of Mitochondrial pH by Uncoupling Protein 4 in Astrocytes Promotes Neuronal Survival

Brain activity is energetically costly and requires a steady and highly regulated flow of energy equivalents between neural cells. It is believed that a substantial share of cerebral glucose, the major source of energy of the brain, will preferentially be metabolized in astrocytes via aerobic glycolysis. The aim of this study was to evaluate whether uncoupling proteins (UCPs), located in the inner membrane of mitochondria, play a role in setting up the metabolic response pattern of astrocytes. UCPs are believed to mediate the transmembrane transfer of protons, resulting in the uncoupling of oxidative phosphorylation from ATP production. UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms expressed in the brain are UCP2, UCP4, and UCP5. We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling and measured a range of functional metabolic parameters including mitochondrial electrical potential and pH, reactive oxygen species production, NAD/NADH ratio, ATP/ADP ratio, CO₂ and lactate production, and oxygen consumption rate. In brief, we found that UCP4 regulates the intramitochondrial pH of astrocytes, which acidifies as a consequence of glutamate uptake, with the main consequence of reducing efficiency of mitochondrial ATP production. The diminished ATP production is effectively compensated by enhancement of glycolysis. This nonoxidative production of energy is not associated with deleterious H₂O₂ production. We show that astrocytes expressing more UCP4 produced more lactate, which is used as an energy source by neurons, and had the ability to enhance neuronal survival.     

Autoregulation of Free Radicals via Uncoupling Protein Control in Pancreatic β-Cell Mitochondria

Pancreatic β-cells sense the ambient blood-glucose concentration and secrete insulin to signal other tissues to take up glucose. Mitochondria play a key role in this response as they metabolize nutrients to produce ATP and reactive oxygen species (ROS), both of which are involved in insulin secretion signaling. Based on data available in the literature and previously developed mathematical models, we present a model of glucose-stimulated mitochondrial respiration, ATP synthesis, and ROS production and control in β-cells. The model is consistent with a number of experimental observations reported in the literature. Most notably, it captures the nonlinear rise in the proton leak rate at high membrane potential and the increase in this leak due to uncoupling protein (UCP) activation by ROS. The functional forms used to model ROS production and UCP regulation yield insight into these mechanisms, as many details have not yet been unraveled in the experimental literature. We examine short- and long-term effects of UCP activation inhibition and changes in the mitochondrial density on mitochondrial responses to glucose. Results suggest increasing mitochondrial density while decreasing UCP activity may be an effective way to increase glucose-stimulated insulin secretion while decreasing oxidative stress.     

Uncoupling protein 2 mediates resistance to gemcitabine-induced apoptosis in hepatocellular carcinoma cell lines

Oxidative stress induction is a common effector pathway for commonly used chemotherapeutic agents like gemcitabine (GEM) in hepatocellular carcinoma (HCC) patients. However, GEM alone or in combination with oxiplatin hardly renders any survival benefits to HCC patients. Mitochondrial uncoupling protein 2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) generation, thus mitigating oxidative stress-induced apoptosis. We demonstrate in the present study, using a panel of HCC cell lines that sensitivity to GEM in HCC well correlate with the endogenous level of UCP2 protein expression. Moreover, ectopic overexpression of UCP2 in a HCC cell line with low endogenous UCP2 expression, HLE, significantly decreased mitochondrial superoxide induction by the anti-cancer drug GEM. Conversely, UCP2 mRNA silencing by RNA interference in HCC cell lines with high endogenous UCP2 expression significantly enhanced GEM-induced mitochondrial superoxide generation and apoptosis. Cumulatively, our results suggest a critical role for mitochondrial uncoupling in GEM resistance in HCC cell lines. Hence, synergistic targeting of UCP2 in combination with other chemotherapeutic agents might be more potent in HCC patients.     

Mitochondrial uncoupling proteins in unicellular eukaryotes

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH₂) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.