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.     

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.     

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.     

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.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

Mitochondrial proton leak: a role for uncoupling proteins 2 and 3?

In mitochondria ATP synthesis is not perfectly coupled to oxygen consumption due to proton leak across the mitochondrial inner membrane. Quantitative studies have shown that proton leak contributes to approximately 25% of the resting oxygen consumption of mammals. Proton leak plays a role in accounting for differences in basal metabolic rate. Thyroid studies, body mass studies, phylogenic studies and obesity studies have all shown that increased mass-specific metabolic rate is linked to increased mitochondrial proton leak. The mechanism of the proton leak is unclear. Evidence suggests that proton leak occurs by a non-specific diffusion process across the mitochondrial inner membrane. However, the high degree of sequence homology of the recently cloned uncoupling proteins UCP 2 and UCP 3 to brown adipose tissue UCP 1, and their extensive tissue distribution, suggest that these novel uncoupling proteins play a role in proton leak. Early indications from reconstitution experiments and several in vitro expression studies suggest that the novel uncoupling proteins uncouple mitochondria. Furthermore, mice overexpressing UCP 3 certainly show a phenotype consistent with increased metabolism. The evidence for a role for these novel UCPs in mitochondrial proton leak is reviewed.     

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.     

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.     

Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2

The production of reactive oxygen species (ROS) in mitochondria is very sensitive to the proton motive force and may be decreased by mild uncoupling, mediated e.g. by mitochondrial uncoupling proteins (UCPs). UCPs were conversely hypothesized to be activated by ROS. Conclusions from experiments studying the reactive product of lipid peroxidation 4-hydroxy-2-nonenal (HNE) in isolated mitochondria and UCP knock-out mice are highly controversial. Here we investigated the molecular mechanism of HNE action by evaluating the separate contributions of lipid and protein phases of the membrane and by comparing UCP1 and UCP2, which were reconstituted in planar lipid bilayers. We demonstrated that aldehyde does not directly activate either UCP1 or UCP2. However, HNE strongly potentiated the membrane conductance increase (G_m) mediated by different long-chain fatty acids in UCP-containing and in UCP-free membranes and this suggest the involvement of both lipid-mediated and protein-mediated mechanisms with FA playing the central role. G_m increase was concentration-dependent and exhibited a typical saturation kinetic with the binding constant 0.3 mM. By using Electron Paramagnetic Resonance, membrane fluidity change could be excluded as a cause for the HNE-mediated increase in the presence of FA. The impact of the HNE binding to definite positively charged UCP amino acid residues is discussed as a possible protein-mediated mechanism of the UCP activation.     

Uncoupling proteins and their role in the regulation of brain and heart tolerance to impact of ischemia and reperfusion

Experimental data indicate that moderate uncoupling oxidative phosphorylation induces reduction in production of reactive oxygen species (ROS) and promotes an increase in survival of neurons and cardiomyocytes under hypoxia and re-oxygenation conditions. Uncoupling proteins (UCP) are expressed by cardiomyocytes and neurons. These proteins are involved in the thermogenesis, inhibit ROS generation by mitochondria, reduce deltaphi, elevate respiration rate of these organelles. It was established that UCP contributed to the elevation of cardiomyocyte and neuron tolerance of an impact of hypoxia and re-oxygenation. They also promote cell resistance to oxidative stress. Experimental data indicate the important role of the UCP in the neuroprotective and cardioprotective effects of ischemic preconditioning. At the same time, real contribution of the UCP in preconditioning is still to be verified.     

Mitochondrial uncoupling proteins--what is their physiological role?

The physiological functions of the mitochondrial uncoupling proteins (UCP2 and UCP3) are still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, they are not crucial for nonshivering thermogenesis and do not catalyze the basal proton conductance of mitochondria. Rather, there is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. This review critically examines the evidence of the different proposed mechanisms for UCPs functions, namely (a) to export fatty acid anions from mitochondria, (b) to regulate insulin secretion in pancreatic beta-cells, and (c) to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against oxidative damage. Beside, available scientific data on UCP4 and UCP5/BMCP1 will be reviewed. However, their physiological function has not yet been established.     

解偶联蛋白与肥胖及2型糖尿病发病的关系

解偶联蛋白（UCPs）是线粒体内膜上的一种转运蛋白,它能够降低线粒体内膜上的质子梯度,使底物氧化和ADP磷酸化解偶联,减少ATP的产生。基于其功能,解偶联蛋白基因被视为肥胖病及2型糖尿病的重要候选基因。UCP同系物过表达的遗传工程小鼠表现出对饮食导致的肥胖具有耐受性,同时UCP2基因3＇非翻译区的45bp插入／缺失以及UCP3基因C-55T多态与肥胖表型的相关性等研究结论支持这一假说。本文对UCP基因与多基因控制的肥胖病及2型糖尿病发病的相关研究进行综述和讨论。 Abstract:Uncoupling proteins (UCPs) are mitochondria carrier proteins,which are able to dissipate the proton gradient of the inner mitochondria membrane.The uncoupling procedure reduces the amount of ATP generated through an oxidation of fuels.Therefore,UCPs are suggested as candidate genes for human obesity or type II diabetes mellitus.Experimental evidences,that genetically engineered mice over expressing different UCP homologues were resistant to diet-induced obesity and 45bp insertion polymorphism in the UCP2 3＇untranslated region and C-55T in UCP3 promoter region were associated with obesity related phenotype,supported the hypothesis.The roles of UCP genes in polygenic obesity and type II diabetes are evaluated and discussed in this paper.     

Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.     

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.     

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.     

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.     

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.     

Respiration under control of uncoupling proteins: Clinical perspective

The term 'uncoupling protein' was originally used for the mitochondrial membrane protein UCP1, which is uniquely present in mitochondria of brown adipocytes, thermogenic cells that regulate body temperature in small rodents, hibernators and mammalian newborns. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP-synthase resulting in a futile proton cycling and dissipation of oxidation energy as heat. Recent identification of new homologues to UCP1 expressed in brown and white adipose tissue, muscle, brain and other tissues together with the hypothesis that these novel uncoupling proteins (UCPs) may regulate thermogenesis and/or fatty acid metabolism and furthermore may protect against free radical oxygen species production have generated considerable optimism for rapid advances in the identification of new targets for pharmacological management of complex pathological syndromes such as obesity, type 2 diabetes or chronic inflammatory diseases. However, since the physiological and biochemical roles of the novel UCPs are not yet clear, the main challenge today consists first of all in providing mechanistic explanation for their functions in cellular physiology. This lively awaited information may be the basis for potential pharmacological targeting of the UCPs in future.     

线粒体解偶联蛋白UCP2的研究进展

本文综述了线粒体解偶联蛋白2（uncoupling protein2,UCP2）研究方面的进展。UCP2定位于线粒体内膜上,通过消散线粒体内膜的质子梯度调节线粒体的功能,包括线粒体内膜电位、ATP合成、呼吸链ROS产生、线粒体钙库的存储和释放等。目前,UCP2的质子漏机理并不清楚,但体内实验表明UCP2活性可被过氧化物激活。特别是近年来UCP2调控胰岛素分泌方面的研究取得了重要进展。     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.