Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.     

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.     

GDP在体外对大鼠脑线粒体脱耦联蛋白活性和表达的影响

本研究通过GDP体外处理大鼠脑组织块,观察GDP对脑线粒体脱耦联蛋白(uncoupling proteins,UCPs)活性、UCP4和UCP5表达的影响,以探讨嘌呤核苷酸对大鼠脑UCPs的调节作用.取Sprague-Dawley大鼠双侧大脑半球,将脑组织切成约8-10 mm3的脑组织块,与含1 mmol/L GDP的孵育介质共孵育30 min后,匀浆并差速离心分离提取大鼠脑组织线粒体,采用[3H]-GTP结合法测定UCPs活性,并以Scatehard作图法计算两者结合的解离常数(Kd)和最大结合量(Bmax);RT-PCR和Western blot分别检测UCP4和UCP5的mRNA和蛋白表达.结果显示,1 mmol/L GDP可降低体外大鼠脑组织线粒体中UCPs与[3H]-GTP结合的Bmax,提高Kd,但对脑纰织中UCP4和UCP5 mRNA和蛋白表达量的改变无统计学意义.上述结果提示,GDP可直接抑制体外大鼠脑组织中UCPs的活性,但并不影响UCP4和UCP5的表达.     

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.     

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.     

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.     

Functional analysis of skunk cabbage SfUCPB, a unique uncoupling protein lacking the fifth transmembrane domain, in yeast cells

Skunk cabbage, Symplocarpus foetidus, expresses two uncoupling proteins (UCPs), termed SfUCPA and SfUCPB, in the thermogenic organ spadix. SfUCPB exhibits unique structural features characterized by the absence of the putative fifth transmembrane domain (TM5) observed in SfUCPA, which is structurally similar to UCP1, and is abundantly expressed in the thermogenic spadix. Here, we conducted a series of comparative analyses of UCPs with six transmembrane domains, SfUCPA and rat UCP1, and TM5-deficient SfUCPB, using a heterologous yeast expression system. All UCPs were successfully expressed and targeted to the mitochondria, although the expression level of SfUCPB protein was approximately 10% of rat UCP1. The growth rate, mitochondrial membrane potential, and ATP content were significantly lower in cells expressing SfUCPB than in those expressing rat UCP1 and SfUCPA. These results suggest that SfUCPB, a novel TM5-deficient UCP, acts as an uncoupling protein in yeast cells.     

Identification and characterization of uncoupling protein 4 in fat body and muscle mitochondria from the cockroach <Emphasis Type="Italic">Gromphadorhina cocquereliana</Emphasis>

We have identified and characterized an uncoupling protein in mitochondria isolated from leg muscle and from fat body, an insect analogue tissue of mammalian liver and adipose tissue, of the cockroach Gromphadorhina coquereliana (GcUCP). This is the first functional characterization of UCP activity in isolated insect mitochondria. Bioenergetic studies clearly indicate UCP function in both insect tissues. In resting (non-phosphorylating) mitochondria, cockroach GcUCP activity was stimulated by the addition of micromolar concentrations of palmitic acid and inhibited by the purine nucleotide GTP. Moreover, in phosphorylating mitochondria, GcUCP activity was able to divert energy from oxidative phosphorylation. Functional studies indicate a higher activity of GcUCP-mediated uncoupling in cockroach muscle mitochondria compared to fat body mitochondria. GcUCP activation by palmitic acid resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of UCPs in insects. GcUCP protein was immunodetected using antibodies raised against human UCP4 as a single band of around 36 kDa. GcUCP protein expression in cockroach muscle mitochondria was significantly higher compared to mitochondria isolated from fat body. LC-MS/MS analyses revealed 100% sequence identities for peptides obtained from GcUCP to UCP4 isoforms from D. melanogaster (the highest homology), human, rat or other insect mitochondria. Therefore, it can be proposed that cockroach GcUCP corresponds to the UCP4 isoforms of other animals.     

Towards comprehension of the physiological role of UCP3.

Thyroid hormones have long been known to stimulate energy expenditure partly via loss of metabolic efficiency. The mechanism underlying the loss in metabolic efficiency observed, however, is not yet understood. An important candidate gene responsible for thyroid hormone induced thermogenesis was identified in 1997 with the discovery of skeletal muscle-uncoupling protein 3 (UCP3), a protein with approximately 60 % homology to the brown adipose tissue uncoupling protein 1 (UCP1). This short review summarizes our presentation held at the 'Thyroid and Sports' meeting; it does not aim to provide a concise overview of the available literature at this topic. Although induction of the UCP3 gene and increased protein expression during hyperthyroidism has been shown, there are no convincing data that increased UCP3 levels account for the increase in thermogenesis in the hyperthyroid state in humans. In contrast to cell and animal studies using ectopic overexpression of UCP3 as a model, induction of UCP3 in humans does not result in any apparent mitochondrial uncoupling. Hence, the primary physiological role of UCP3 may not be mitochondrial uncoupling, but uncoupling may occur as a side effect of a more pivotal role played by UCP3. Recently, UCP3 has been hypothesized to export fatty acid anions and/or lipid peroxides away from the mitochondrial matrix to prevent mitochondria from the harmful effects of peroxidized lipids. The present review aims to provide an overview of studies testing the feasibility of this unconventional function of UCP3.     

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.     

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.     

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.     

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.     

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.     

Regulation of mitochondrial uncoupling respiration during exercise in rat heart: role of reactive oxygen species (ROS) and uncoupling protein 2

The physiological significance of cardiac mitochondrial uncoupling protein 2 (UCP2)-mediated uncoupling respiration in exercise is unknown. In the current study, mitochondrial respiratory function, UCP2 mRNA level, UCP2-mediated respiration (UCR), and reactive oxygen species (ROS) generation, as well as manganese superoxide dismutase (MnSOD) activity were determined in rat heart with or without endurance training after an acute bout of exercise of different duration. In the untrained rats, state 4 respiration and UCR-independent respiration rates were progressively increased with exercise time and were 64 and 70% higher, respectively, than resting rate at 150 min, whereas UCR was elevated by 86% with no significant change in state 3 respiration. UCP2 mRNA level showed a 5- and 4-fold increase, respectively, after 45 and 90 min of exercise, but returned to resting level at 120 and 150 min. Mitochondrial ROS production and membrane potential (Deltapsi) increased progressively until 120 min, followed by a decrease to the resting level at 150 min. MnSOD mRNA abundance showed a 2-fold increase at 120 min but MnSOD activity did not change with exercise. Training significantly increased mitochondrial ATP synthetase activity, ADP to oxygen consumption (P/O) ratio, respiratory control ratio, and MnSOD activity, whereas exercise-induced state 4 respiration, UCR, ROS production, and Deltapsi were attenuated in the trained rats. We conclude that (1) UCP2 mRNA expression and activity in rat heart can be upregulated during prolonged exercise, which may reduce cross-membrane Deltapsi and thus ROS production; and (2) endurance training can blunt exercise-induced UCP2 and UCR, and improve mitochondrial efficiency of oxidative phosphorylation due to increased removal of ROS.     

Redox State of Endogenous Coenzyme Q Modulates the Inhibition of Linoleic Acid-Induced Uncoupling by Guanosine Triphosphate in Isolated Skeletal Muscle Mitochondria

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.     

Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells.

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.     

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.