Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

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.     

Alkylsulfonates activate the uncoupling protein UCP1: implications for the transport mechanism

Fatty acids activate the uncoupling protein UCP1 by a still controversial mechanism. Two models have been put forward where the fatty acid operates as either substrate ("fatty acid cycling hypothesis") or prosthetic group ("proton buffering model"). Two sets of experiments that should help to discriminate between the two hypothetical mechanisms are presented. We show that undecanosulfonate activates UCP1 in respiring mitochondria under conditions identical to those required for the activation by fatty acids. Since alkylsulfonates cannot cross the lipid bilayer, these experiments rule out the fatty acid cycling hypothesis as the mechanism of uncoupling. We also demonstrate that without added nucleotides and upon careful removal of endogenous fatty acids, brown adipose tissue (BAT) mitochondria from cold-adapted hamsters respire at the full uncoupled rate. Addition of nucleotides lower the respiratory rate tenfold. The high activity observed in the absence of the two regulatory ligands is an indication that UCP1 displays an intrinsic proton conductance that is fatty acid-independent. We propose that the fatty acid uncoupling mediated by other members of the mitochondrial transporter family probably involves a carrier to pore transition and therefore has little in common with the activation of UCP1.     

Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis

Background  

Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.     

棕色脂肪的产热及其调控机制

棕色脂肪组织是小型哺乳动物重要的兼性产热器官,位于棕色脂肪细胞线粒体内膜的解偶联蛋白是此种产热机制的关键物质.产热刺激激活解偶联蛋白构成的质子通道,在线粒体内膜形成质子短路,从而解除ATP合成偶联对氧化呼吸速率的控制,使产热高速进行.去甲肾上腺素、甲状腺激素、胰岛素、pH值以及食物、环境温度等因素综合调控着棕色脂肪组织的结构与功能状态.     

Fatty Acid Interaction with Mitochondrial Uncoupling Proteins

The phenomena of fatty acid interaction with mitochondrial integral membrane proteins, namelyuncoupling proteins (UCPs), are reviewed to emphasize the fatty acid cycling mechanism thathas been suggested to explain the UCP function. Fatty acid-induced uncoupling is suggestedto serve in bioenergetic systems, to set the optimum efficiency, and to tune the degree ofcoupling of oxidative phosphorylation. Fatty acid interaction with the classic uncouplingprotein (UCP1) from mitochondria of thermogenic brown adipose tissue (BAT) is well known.UCP1 is considered to mediate purine nucleotide-sensitive uniport of monovalent unipolaranions, including anionic fatty acids. The return of protonated fatty acid leads to H⁺ uniportand uncoupling. Experiments supporting this mechanism are also reviewed for plant uncouplingmitochondrial protein (PUMP) and ADP/ATP carrier. The fatty acid cycling mechanism ispredicted, as well for the recently discovered uncoupling proteins, UCP2 and UCP3.     

Induction of fatty acid-binding protein 3 in brown adipose tissue correlates with increased demand for adaptive thermogenesis in rodents

We investigated the contribution of fatty acid-binding protein 3 (FABP3) to adaptive thermogenesis in brown adipose tissue (BAT) in rodents. The expression of FABP3 mRNA in BAT was regulated discriminatively in response to alteration of the ambient temperature, which regulation was similar and reciprocal to the regulation of uncoupling protein 1 (UCP1) and leptin, respectively. FABP3 expression in the BAT was significantly higher in the UCP1-knockout (KO) mice than in the wild-type ones, and these KO mice showed a higher clearance rate of free fatty acid from the plasma. In addition, FABP3 expression in the BAT was increased greatly with the development of diet-induced obesity in mice. These results indicate that the induction of FABP3 in BAT correlates with an increased demand for adaptive thermogenesis in rodents. FABP3 appears to be essential for accelerating fatty acid flux and its oxidation through UCP1 activity for non-shivering thermogenesis in BAT.     

Uncoupling proteins and thermoregulation.

Energy balance in animals is a metabolic state that exists when total body energy expenditure equals dietary energy intake. Energy expenditure, or thermogenesis, can be subcategorized into groups of obligatory and facultative metabolic processes. Brown adipose tissue (BAT), through the activity of uncoupling protein 1 (UCP1), is responsible for nonshivering thermogenesis, a major component of facultative thermogenesis in newborn humans and in small mammals. UCP1, found in the mitochondrial inner membrane in BAT, uncouples energy substrate oxidation from mitochondrial ATP production and hence results in the loss of potential energy as heat. Mice that do not express UCP1 (UCP1 knockouts) are markedly cold sensitive. The recent identification of four new homologs to UCP1 expressed in BAT, muscle, white adipose tissue, brain, and other tissues has been met by tremendous scientific interest. The hypothesis that the novel UCPs may regulate thermogenesis and/or fatty acid metabolism guides investigations worldwide. Despite several hundred publications on the new UCPs, there are a number of significant controversies, and only a limited understanding of their physiological and biochemical properties has emerged. The discovery of UCP orthologs in fish, birds, insects, and even plants suggests the widespread importance of their metabolic functions. Answers to fundamental questions regarding the metabolic functions of the new UCPs are thus pending and more research is needed to elucidate their physiological functions. In this review, we discuss recent findings from mammalian studies in an effort to identify potential patterns of function for the UCPs.     

Energy metabolism in uncoupling protein 3 gene knockout mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.     

Fusion of the Endoplasmic Reticulum and Mitochondrial Outer Membrane in Rats Brown Adipose Tissue: Activation of Thermogenesis by Ca2+

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca²⁺-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca²⁺ effect in BAT mitochondria thermogenesis. We found that Ca²⁺ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca²⁺ concentration needed for half-maximal activation varied between 0.08 and 0.11 µM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN.Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca²⁺ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca²⁺ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.     

Crucial yet divergent roles of mitochondrial redox state in skeletal muscle vs. brown adipose tissue energetics

Reduced glutathione (GSH) is the major determinant of redox balance in mitochondria and as such is fundamental in the control of cellular bioenergetics. GSH is also the most important nonprotein antioxidant molecule in cells. Surprisingly, the effect of redox environment has never been examined in skeletal muscle and brown adipose tissue (BAT), two tissues that have exceptional dynamic range and that are relevant to the development of obesity and related diseases. Here, we show that the redox environment plays crucial, yet divergent, roles in modulating mitochondrial bioenergetics in skeletal muscle and BAT. Skeletal muscle mitochondria were found to naturally have a highly reduced environment (GSH/GSSG≈46), and this was associated with fairly high (～40%) rates of state 4 (nonphosphorylating) respiration and decreased reactive oxygen species (ROS) emission. The deglutathionylation of uncoupling protein 3 (UCP3) following an increase in the reductive potential of mitochondria results in a further increase in nonphosphorylating respiration (～20% in situ). BAT mitochondria were found to have a much more oxidized status (GSH/GSSG≈13) and had basal reactive oxygen species emission that was higher (～250% increase in ROS generation) than that in skeletal muscle mitochondria. When redox status was subsequently increased (i.e., more reduced), UCP1-mediated uncoupling was more sensitive to GDP inhibition. Surprisingly, BAT was found to be devoid of glutaredoxin-2 (Grx2) expression, while there was abundant expression in skeletal muscle. Taken together, these findings reveal the importance of redox environment in controlling bioenergetic functions in both tissues, and the highly unique characteristics of BAT in this regard.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Lack of activation of UCP1 in isolated brown adipose tissue mitochondria by glucose-O-ω-modified saturated fatty acids of various chain lengths

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.     

Mitoproteome plasticity of rat brown adipocytes in response to cold acclimation

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.     

Functional characterisation of UCP1 in the common carp: uncoupling activity in liver mitochondria and cold-induced expression in the brain

Mammalian uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis in brown adipose tissue. We previously reported on the presence of a UCP1 orthologue in ectothermic fish and observed downregulation of UCP1 gene expression in the liver of the common carp. Neither the function of UCP1, nor the mode of UCP1 activation is known in carp liver mitochondria. Here, we compared the proton conductance at 25°C of liver mitochondria isolated from carp either maintained at 20°C (warm-acclimated, WA) or exposed to 8°C (cold-acclimated, CA) water temperature for 7–10 days. Liver mitochondria from WA carp had higher state four rates of oxygen consumption and greater proton conductance at high membrane potential. Liver mitochondria from WA, but not from CA, carp showed a strong increase in proton conductance when palmitate (or 4-hydroxy-trans-2-nonenal, HNE) was added, and this inducible proton conductance was prevented by addition of GDP. This fatty acid sensitive proton leak is likely due to the expression of UCP1 in the liver of WA carp. The observed biochemical properties of proton leak strongly suggest that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as mammalian UCP1. Significant UCP1 expression was also detected in our previous study in whole brain of the carp. We here observed a twofold increase of UCP1 mRNA in carp brain following cold exposure, suggesting a role of UCP1 in the thermal adaptation of brain metabolism. In situ hybridization located the UCP1 gene expression to the optic tectum responsible for visual system control, the descending trigeminal tract and the solitary tract. Taken together, this study characterises uncoupling protein activity in an ectotherm for the first time. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.