BBRC Biochemical and Biophysical Research Communications 312 (2003) 983–988 www.elsevier.com/locate/ybbrc

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus) Darren A. Talbot,a Nicolas Hanuise,b Benjamin Rey,b Jean-Louis Rouanet,b Claude Duchamp,b and Martin D. Branda,* b

a Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY, UK Laboratoire Physiologie Int egrative, Cellulaire et Mol eculaire, CNRS-Universit e Lyon 1, 43 Bld 11 Novembre 1918, F-69622, Villeurbanne Cedex, France

Received 6 November 2003

Abstract We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88–92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Superoxide; Uncoupling protein; avUCP; Proton leak; Mitochondria

In mammals, brown adipose tissue is specialised for adaptive non-shivering thermogenesis. It responds to noradrenergic stimulation by activating a regulated uncoupling of mitochondrial oxidative phosphorylation through an uncoupling protein, UCP1. Two homologues (UCP2 and UCP3) of UCP1 have been identified in mammals, and several others have been found in a variety of other organisms, including fish, birds, and plants [1–5]. The ability of UCP1 to catalyse the transport of protons across the mitochondrial inner membrane and partially dissipate the mitochondrial membrane potential is well established [6]. However, despite the fact that the predicted amino-acid sequences of the UCP1 homologues closely resemble that of UCP1, evidence that they are able to function as true uncouplers to catalyse a proton conductance under physiological conditions in vivo is much debated. It is clear that neither UCP2 nor UCP3 contributes to the * Corresponding author. Fax : +44-1223-252805. E-mail address: [email protected] (M.D. Brand).

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.022

basal proton leak that is a feature of all mitochondria [7]. Also, of all the mammalian UCPs, only UCP1 is involved in non-shivering thermogenesis [8]. Therefore, in spite of a large literature on the area, UCP1 remains as the only protein of the UCP family to have a wellestablished function. Despite the probable differences in the physiological functions of the mammal UCPs, it has recently been demonstrated that UCP1, UCP2, and UCP3 all share the common property of a superoxide-activated proton conductance [9]. The superoxide activation of these UCPs only occurs in the presence of free fatty acids and is potently inhibited by nucleoside di- and tri-phosphates. A similar observation has also been reported for the plant UCP homologue, StUCP, expressed in mitochondria from the tubers of potato (Solanum tuberosum) [10]. While the plant UCP protein was reported to be expressed in response to cold when it was first identified [5], its function, like UCP2 and UCP3, is still unclear. The superoxide activation of these UCPs suggests a role in antioxidant defence [11].

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Birds lack brown adipose tissue, yet show adaptive thermogenesis, probably in muscle [12]. However, chicken (Gallus gallus) [4] and a hummingbird (Eupetomena macroura) [13] do possess a UCP homologue, avUCP. When the avUCP gene was first identified in 2001, the expression of avUCP mRNA in muscle was shown to be sensitive to cold and glucagon [4,13]. Recent evidence has demonstrated further hormonal control of the avUCP mRNA by thyroid hormone but not insulin [14]. The mRNA is primarily expressed in skeletal muscle [4,13] and in this respect it closely mirrors the mammalian UCP3. However, little progress has been made towards understanding the function of avUCP. Its contribution to the basal proton conductance of bird mitochondria, its ability to catalyse an inducible proton conductance in a UCP1-like fashion and its potential activation by superoxide have not yet been examined. Due to their natural environment, penguins provide a good model to investigate potential thermogenic mechanisms in birds. This study aimed to examine the presence of an avUCP homologue in penguin skeletal muscle and to investigate its contribution, if any, to the basal proton leak of mitochondria from these birds. Furthermore, we aimed to study its bioenergetic properties in relation to its potential ability to act as a true uncoupler and whether such activity required superoxide activation. Materials and methods The study was conducted on the Crozet archipelago (Possession Island) at the French Alfred Faure Station (46°250 S, 52°450 E). According to the Agreed Measures for the Preservation of Antarctic Fauna, the project received the agreement of the French Committee for Antarctic Research (Programme 131). Animals. Eight juvenile king penguins (Aptenodytes patagonicus) of both sexes, 12–13 months old, that had completed moulting, were naturally adapted to marine life and weighed 9–11 kg, were captured and kept in an outside enclosure (average temperature 7.7 °C) for up to 14 days. All birds were weighed every second day and force-fed ice fish (Champsocephalus gunnari) up to 500 g per day, to maintain a constant body weight. On completion of the study the penguins were released at the site of their capture. Isolation and sequencing of penguin avUCP cDNA. Total RNA was isolated from frozen pectoralis muscle using the TriReagent procedure (Sigma, St. Quentin Fallavier, France) and the integrity of extracted RNA was assessed by electrophoresis on agarose gels. Reverse transcription (RT) of 1 lg of total RNA was performed using 200 U of M-MLV-RTase (Promega, France) according to the manufacturer’s instructions and as described previously [15]. The resulting cDNAs were submitted to polymerase chain reaction (PCR) amplification in a total volume of 50 ll containing 1 lM of forward (50 -GTGGATGCCTACA GGACCAT-30 ) and reverse (50 -ATGAACATCACCACGTTCCA-30 ) primers (Invitrogen, Cergy Pontoise, France), 2.5 U of Taq DNA polymerase (Eurobio, Les Ullis, France), reaction buffer, 200 lM dNTPs, and 1.5 mM MgCl2 . Primers were selected according to the chicken avUCP sequence [4]. After an initial denaturation step at 94 °C for 2 min, PCR cycles were run with the following parameters: denaturation 94 °C for 45 s, annealing at 62 °C for 1 min, and elongation at

72 °C for 60 s using a Thermo Hybaid thermocycler (Ashforf, UK). The amplified product (389 nt) was separated on agarose gel and recovered using a Maestro gelex DNA purification kit (Eurobio). The purified amplicon was cloned directly into pGEM-T vector (Promega) and sequenced (Genoscreen, Lille, France). Mitochondrial isolation. Skeletal muscle intermyofibrillar mitochondria were isolated as previously described for ducklings [16]. Penguins were anaesthetised with a mixture of air and 2.5% (v/v) halothane in air. Approximately 6 g of pectoralis muscle was removed under sterile conditions and placed in ice-cold isolation buffer containing: 100 mM sucrose; 50 mM Tris base; 50 mM KCl; and 5 mM EDTA, adjusted to pH 7.4 with HCl at 4 °C. Muscle tissue was shredded with a razor blade on a pre-cooled tile, minced with scissors and then homogenised with 10 passes of the plunger of a Potter homogeniser before being centrifuged at 800g for 10 min. The pellet was diluted in 40 ml of isolation buffer containing nagarse (1 mg/g of muscle) and stirred on ice for 5 min. Forty millilitres of isolation buffer was added to stop the protease digestion followed by a further ten passes of the plunger of the homogeniser. Mitochondria were then isolated using differential centrifugation. The final suspension medium contained 250 mM sucrose, 1 mM EGTA, and 20 mM Tris base, pH 7.4. Protein concentration was determined using the Biuret method with bovine serum albumin as standard [17]. Yield was approximately 5–10 mg mitochondrial protein per gram muscle. Penguin muscle mitochondria were well coupled, with respiratory control ratios of 2.6–3.7 with succinate as substrate. After surgery, birds were monitored for a few days and then released back into the colony. No adverse postsurgical outcomes were noted. Measurement of oxygen consumption. Oxygen consumption was measured using a 2.5 ml Clark-type oxygen electrode (Hansatech, King’s Lynn, Norfolk, UK) maintained at 38 °C and calibrated with air-saturated assay medium (120 mM KCl, 5 mM KH2 PO4 , 3 mM Hepes, 1 mM EGTA, 1 mM MgCl2 , and 0.3% (w/v) BSA, pH 7.2), which was assumed to contain 402 nmol O/ml [18]. Electrode linearity was checked routinely by following the uncoupled respiration rate in the presence of 0.4 lM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) from 100% to 0% air saturation. For all experiments, mitochondria (0.35 mg protein/ml) were incubated in assay medium with 4 mM succinate, 5 lM rotenone, 1 lg/ml oligomycin, and 65 ng/ml nigericin. Measurement of proton conductance. The respiration rate of mitochondria, in the presence of oligomycin to inhibit ATP synthesis, is proportional to the rate at which protons leak across the mitochondrial inner membrane. The kinetic response of the proton conductance to its driving force (proton motive force) can therefore be measured as the relationship between respiration rate and mitochondrial membrane potential when the potential is varied with titration with electron transport chain inhibitors [19–21]. Respiration rate and membrane potential were determined simultaneously using electrodes sensitive to oxygen and to the potential-dependent probe triphenylmethylphosphonium cation (TPMPþ ) [22]. Mitochondria (0.35 mg protein/ml) were incubated at 38 °C in assay media containing 5 lM rotenone, 1 lg/ml oligomycin, and 65 ng/ml nigericin (to collapse the difference in pH across the mitochondrial inner membrane). The electrode was calibrated with sequential additions up to 2 lM TPMPþ and 4 mM succinate was added to start the reaction. Respiration and potential were inhibited progressively through successive steady states by additions of malonate up to 3 mM. At the end of each run 0.4 lM FCCP was added to dissipate the membrane potential and release all TPMPþ back into the medium, allowing correction for any small electrode drift. The TPMPþ binding correction factor was taken as 0.45 ll/mg of protein [23]. Where indicated, exogenous superoxide was generated using xanthine (50 lM) and xanthine oxidase (0.01 U per 2.5 ml). Stocks of xanthine at 0.35 mM and xanthine oxidase at 2 U/ml were prepared in assay medium. Xanthine and xanthine oxidase (XXO) were added before the TPMPþ calibration. Where XXO stimulated basal oxygen

D.A. Talbot et al. / Biochemical and Biophysical Research Communications 312 (2003) 983–988 consumption before the addition of succinate, this was averaged over the period of the TPMPþ calibration and subtracted from all subsequent steady state respiration rates of that run. The maximum size of this correction was 10% of the state 4 rate. All experiments were conducted in the presence of 0.3% BSA and 150 lM palmitate. Where indicated, 12 U/ml superoxide dismutase (SOD) or 1 mM guanosine diphosphate (GDP) was added.

Results and discussion Partial sequencing of penguin avUCP As shown in Fig. 1, the selected primers led to the PCR amplification of a DNA fragment of the expected size with cDNAs from penguin pectoralis muscle. The sequence of the amplified fragment (Table 1) was closely related to those of other avian UCPs. Overlapping sequence was 91.5%, 91.8%, and 88.4% homologous to chicken, turkey, and hummingbird avUCPs, respectively. Homology was 64% with rat UCP1 (GenBank Accession No. NM012682), 71% with rat UCP2 (AF039033) and 73% with rat UCP3 (AF035943). These results indicate that a UCP homologue is expressed, at least at the mRNA level, in skeletal muscle from king penguins. Mitochondrial proton conductance Fig. 2 shows the kinetic response of the proton leak rate to its driving force, membrane potential, in mitochondria isolated from penguin pectoralis muscle. Under control conditions (circles, Figs. 2A and D),

Fig. 1. RT-PCR detection of an mRNA encoding a UCP-like protein in the pectoralis muscle of a juvenile king penguin. L: 100 bp ladder; H2 O: PCR negative control; 24 cycles of PCR were used and hybridisation temperature was 62 °C.

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penguin muscle mitochondria exhibited the same characteristic non-linear response of proton leak to membrane potential as liver mitochondria from other birds [24,25] and liver and skeletal muscle mitochondria from mammals [23]. Fig. 2A shows that in the presence of the superoxide generating system, xanthine and xanthine oxidase (XXO; squares), the proton conductance was greater than in controls. This is demonstrated by the upward displacement of the kinetic curves: the mitochondria required approximately 60% higher respiration rate to maintain any given membrane potential when incubated with XXO. The increase in proton conductance was prevented by addition of exogenous superoxide dismutase (Fig. 2B), indicating that it was dependent on extramitochondrial superoxide generated by the xanthine and xanthine oxidase. A superoxide stimulated increase in proton conductance has previously been observed in mammalian and plant mitochondria and shown to be mediated by UCPs [9,10]. A distinguishing property of superoxide activation of UCPs is its potent inhibition by purine nucleoside di- and tri-phosphates. Fig. 2C shows that the superoxide-stimulated proton conductance of penguin skeletal muscle mitochondria was similarly completely inhibited by 1 mM GDP, strongly suggesting that much like the mammalian and plant UCPs, superoxide was acting through the avUCP. In the absence of XXO, GDP did not inhibit proton conductance (Fig. 2D), which suggests that avUCP is not involved in the basal proton conductance of penguin mitochondria. This confirms previous results from mammalian mitochondria that UCPs are not in any way regulators of the ubiquitous and energetically expensive mitochondrial proton leak [7] at least under unstimulated conditions. Overall, our findings demonstrate that an avUCP, very similar to that in other birds [4] is present in penguins. They show that avUCP does not contribute to the basal proton conductance of mitochondria isolated from penguin skeletal muscle. They strongly suggest that avUCP, like the mammalian and plant UCPs, requires activation by superoxide before its proton conductance can be seen, and that this superoxide-activated proton conductance is fully inhibitable by GDP. Furthermore, this study complements the growing body of evidence that GDP-sensitive superoxide activation can be used as a diagnostic test for the presence of functional UCP protein in isolated mitochondria [9,10,26]. The function of avUCP remains to be established. It may act to cause mild uncoupling and diminish endogenous superoxide production by mitochondria, as proposed for mammalian UCP2 and UCP3 [11]. Such a role might be of utmost importance in diving penguins likely to be subjected to oxidative stress during recovery from episodes of apnea. Indeed, the resumption of breathing when

71 71 71 71

141 141 141 141

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281 281 281 281

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Chicken Turkey Hummingbird King penguin

Chicken Turkey Hummingbird King penguin

Chicken Turkey Hummingbird King penguin

Chicken Turkey Hummingbird King penguin

Chicken Turkey Hummingbird King penguin

GTGGATGCCT GTGGATGCCT CTGGATGCCT GTGGATGCCT ********* TCGCCCGCAA TCGCCCGCAA TCGCCCGCAA TCGCCCGCAA ********** GGCACAGCTG GGCGCAGCTG GGAGCACCTG GGCACAGCTG ** ****** GTGGTGGCGT GTGGTGGCGT GTGGTGGCGT ATGGTGGCGT ********* TGCCGAGCTG TGCCCAGCTG CCCTGAGCTG TGCTCAGCTG * ***** CTTCCTGCGC CTTCCTGCGT TTTCCTTCGC CTTCCTGCGG ***** **

ACAGGACCAT ACAGGACCAT ACAGAACCAT ACAGGACCAT ********** CTCCATCATT TGCCATCATT CGCCGTCATC CGCCATCGTC ** ** * ATGACAGACA ATGACAGACA ATGGCAGACG ATGACAGACA *** ***** CGCCGGTGGA CACCGGTGGA CGCCGGTGGA CACCGGTGGA * ******** CCTGCTGGCC CCTGCTGGCC CCTCCTGGCT CCTCATCGCC *** * ** CTCGGCTCCT CTCGGCTCCT CTTGGCTCCT CTCGGCTCCT ** *******

CGCCAGGGAG CGCCAGGGAG CGCCAGGGAG CGCCAGGGAG ********** AACTGCGGCG AACTGCGGTG AACTGCGGGG AACTGCGGGG ******** * ACGTCCCCTG ACGTCCCCTG ATGTCCCCTG ACGTCCCCTG * ******** TGTGGTGAAG TGTGGTGAAG CGTGGTGAAG CGTGGTGAAG ********* CTGCTGCTGC CTGCTGATGC CTCCTCATGC CTGCTCATGC ** ** *** GGAACGTGGT GGAACGTGGT GGAATGTGGT GGAACGTGGT **** *****

GAGGGAGTGC GAGGGAGTGC GAGGGAGTCC GAGGGCGTCC ***** ** * AGCTCGTCAC AGCTCGTCAC AGCTCGTCAC AGCTCGTCAC ********** TCACTTCGTG TCACTTCGTG TCACTTCGTG CCACTTCGTA ******** ACGCGGTACA ACGCGGTACA ACCCGGTACA ACGCGGTACA ** ******* AGGATGGCAT AGGACGGCAT AGGATGGGAT AGGACGGCCT **** ** * GATGTTCAT GATGTTCAT GATGTTCAT GATGTTCAT *********

GTGGGCTGTG GTGGGCTGTG GGGGGCTCTG GCGGGCTCTG * ***** ** CTACGACCTC CTACGACCTC CTACGACCTC CTACGACCTC ********** GCTGCCTTCG GCTGCTTTTG GCCGCTTTCG GCTGCCTTCG ** ** ** * TGAACGCCAG TGAATGCCAG TGAACGCTGG TGAACGCCGG **** ** * TGCTGGCCTC CTCTGGCCTC CACCGGGTTC CGCCGGCTTC * ** **

GAGAGGGACG GAGAGGGACG GAGAGGGACT GAGAGGGACG ********* ATCAAGGACA ATCAAGGACA ATTAAGGACG ATTAAGGACG ** ****** GGGCCGGATT GGGCCGGATT GGGCCGGGTT GGGCCGGCTT ******* ** CCCCGGGCAG CCCTGGGCAG GCCTGGGCAG CCCCGGGCAG ** ****** TACAAGGGGT TACAAGGGGT TACAAGGGGT TACAAGGGGT **********

CTGCCCAACA CTGCCCAACA TTGCCCAACA CTGCCCAACA ********* CACTGCTGCG CGCTGCTGCG CTCTGCTGCG CGCTGCTGCG * ******** CTGCGCCACG CTGCGCCACG CTGCGCCACG CTGCGCCACG ********** TACCGCAATG TACCGGAATG TACAGGAATG TACCGGAACG *** **** * TCGTCCCCTC TCGTCCCCTC TTGTCCCCTC TCGTCCCCTC * ********

GenBank Accession No. of reported sequences were AF287144 for chicken (Gallus gallus), AF436811 for turkey (Meleagris gallopavo), and AF255729 for hummingbird (Eupetomena macroura) avUCPs. * indicates fully conserved.

1 1 1 1

Chicken Turkey Hummingbird King penguin

Table 1 Sequence alignment of king penguin UCP partial cDNA sequence with other related proteins in birds

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Fig. 2. Effect of superoxide on the proton conductance of penguin skeletal muscle mitochondria. For full details see “Materials and methods.” Penguin skeletal muscle mitochondria (0.35 mg/ml) were incubated at 38 °C in assay buffer containing 120 mM KCl, 5 mM KH2 PO4 , 3 mM Hepes, 1 mM EGTA, 1 mM MgCl2 , 0.3% (w/v) BSA, 150 lM palmitate, 5 lM rotenone, 1 lg/ml oligomycin, and 65 ng/ml nigericin, pH 7.2. The kinetics of the mitochondrial proton leak were obtained by simultaneous measurement of membrane potential and oxygen consumption, using succinate as a substrate and varying the potential with sequential additions of malonate up to 3 mM. (A) Effect of superoxide; (B) prevention of activation by addition of exogenous superoxide dismutase; (C) prevention of activation by GDP; and (D) lack of inhibition by GDP in the absence of superoxide. (s) Control; () plus 50 lM xanthine and 0.01 U per 2.5 ml xanthine oxidase, n ¼ 8; (n) plus xanthine, xanthine oxidase, and 12 U/ml superoxide dismutase, n ¼ 4; (d) plus xanthine, xanthine oxidase, and 1 mM GDP, n ¼ 8; and (j) plus 1 mM GDP, n ¼ 4. All data are means
SEM.

penguins resurface after prolonged diving is likely to create a potentially dangerous situation of overgeneration of endogenous superoxide by analogy with the oxidative stress created by ischemia and reperfusion. On the other hand, it may have a thermogenic role in bird muscle, akin to UCP1 in mammalian brown adipose tissue. Superoxide activation of thermogenesis could therefore contribute to the rapid rewarming of penguins after diving-induced hypothermia [27] and play a part in the adaptive non-shivering thermogenesis demonstrated in king penguins [28]. Whether these speculations are accurate and if so, whether such physiological functions are unique to penguins due to the extremes of their natural environment needs to be investigated in future studies investigating the activity of avUCP in vivo.

Acknowledgments This work was supported by grants from the Institut Polaire Francßais Paul Emile Victor (programme 131), which also provided the logistic support, the Universite Claude Bernard Lyon 1, the Centre National de la Recherche Scientifique, and the Medical Research Council.

References [1] C. Fleury, M. Neverova, S. Collins, S. Raimbault, O. Champigny, C. Levi-Meyrueis, F. Bouillaud, M.F. Seldin, R.S. Surwit, D. Ricquier, C.H. Warden, Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia, Nat. Genet. 15 (1997) 269–272.

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[2] A. Vidal-Puig, G. Solanes, D. Grujic, J.S. Flier, B.B. Lowell, UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue, Biochem. Biophys. Res. Commun. 235 (1997) 79–82. [3] J.A. Stuart, J.A. Harper, K.M. Brindle, M.D. Brand, Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates, Biochim. Biophys. Acta 1413 (1999) 50–54. [4] S. Raimbault, S. Dridi, F. Denjean, J. Lachuer, E. Couplan, F. Bouillaud, A. Bordas, C. Duchamp, M. Taouis, D. Ricquier, An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds, Biochem. J. 353 (2001) 441–444. [5] M. Laloi, M. Klein, J.W. Riesmeier, B. Muller-Rober, C. Fleury, F. Bouillaud, D. Ricquier, A plant cold-induced uncoupling protein [letter], Nature 389 (1997) 135–136. [6] D.G. Nicholls, E. Rial, A history of the first uncoupling protein, UCP1, J. Bioenerg. Biomembr. 31 (1999) 399–406. [7] S. Cadenas, J.A. Buckingham, S. Samec, J. Seydoux, N. Din, A.G. Dulloo, M.D. Brand, UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged, FEBS Lett. 462 (1999) 257–260. [8] J. Nedergaard, V. Golozoubova, A. Matthias, A. Asadi, A. Jacobsson, B. Cannon, UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency, Biochim. Biophys. Acta 1504 (2001) 82–106. [9] K.S. Echtay, D. Roussel, J. St-Pierre, M.B. Jekabsons, S. Cadenas, J.A. Stuart, J.A. Harper, S.J. Roebuck, A. Morrison, S. Pickering, J.C. Clapham, M.D. Brand, Superoxide activates mitochondrial uncoupling proteins, Nature 415 (2002) 96–99. [10] M.J. Considine, M. Goodman, K.E. Echtay, M. Laloi, J. Whelan, M.D. Brand, L.J. Sweetlove, Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein, J. Biol. Chem. 278 (2003) 22298–22302. [11] M.D. Brand, J.B. Buckingham, T.C. Esteves, K. Green, A.J. Lambert, S. Miwa, M.P. Murphy, J.L. Pakay, D.A. Talbot, K.S. Echtay, Mitochondrial superoxide and ageing, Biochem. Soc. Symp. (2003), in press. [12] C. Duchamp, H. Barre, Skeletal muscle as the major site of nonshivering thermogenesis in cold-acclimated ducklings, Am. J. Physiol. 265 (1993) R1076–R1083. [13] C.R. Vianna, T. Hagen, C.Y. Zhang, E. Bachman, O. Boss, B. Gereben, A.S. Moriscot, B.B. Lowell, J.E. Bicudo, A.C. Bianco, Cloning and functional characterization of an uncoupling protein homolog in hummingbirds, Physiol. Genom. 5 (2001) 137–145. [14] A. Collin, M. Taouis, J. Buyse, N.B. Ifuta, V.M. Darras, P. Van As, R.D. Malheiros, V.M. Moraes, E. Decuypere, Thyroid status, but not insulin status, affects expression of avian uncoupling protein mRNA in chicken, Am. J. Physiol. 284 (2003) E771–E777. [15] F. Denjean, J. Lachuer, A. Geloen, F. Cohen-Adad, C. Moulin, H. Barre, C. Duchamp, Differential regulation of uncoupling

[16]

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats, FEBS Lett. 444 (1999) 181–185. H. Barre, G. Berne, P. Brebion, F. Cohen-Adad, J.L. Rouanet, Loose-coupled mitochondria in chronic glucagon-treated hyperthermic ducklings, Am. J. Physiol. 256 (1989) R1192–R1199. A.G. Gornall, C.J. Bardawill, M.M. David, Determination of serum proteins by means of the biuret reaction, J. Biol. Chem. 177 (1949) 751–766. B. Reynafarje, L.E. Costa, A.L. Lehninger, O2 solubility in aqueous media determined by a kinetic method, Anal. Biochem. 145 (1985) 406–418. M.D. Brand, The proton leak across the mitochondrial inner membrane, Biochim. Biophys. Acta 1018 (1990) 128–133. D.G. Nicholls, The influence of respiration and ATP hydrolysis on the proton- electrochemical gradient across the inner membrane of rat-liver mitochondria as determined by ion distribution, Eur. J. Biochem. 50 (1974) 305–315. S. Cadenas, J.A. Buckingham, J. St-Pierre, K. Dickinson, R.B. Jones, M.D. Brand, AMP decreases the efficiency of skeletalmuscle mitochondria, Biochem. J. 351 (2000) 307–311. M.D. Brand, Measurement of mitochondrial proton motive force, in: G.C. Brown, C.E. Cooper (Eds.), Bioenergetics—a Practical Approach, IRL Press, Oxford, 1995, pp. 39–62. D.F.S. Rolfe, A.J. Hulbert, M.D. Brand, Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat, Biochim. Biophys. Acta 1188 (1994) 405–416. P.S. Brookes, J.A. Buckingham, A.M. Tenreiro, A.J. Hulbert, M.D. Brand, The proton permeability of the inner membrane of liver mitochondria from ectothermic and endothermic vertebrates and from obese rats: correlations with standard metabolic rate and phospholipid fatty acid composition, Comp. Biochem. Physiol. B 119 (1998) 325–334. M.D. Brand, N. Turner, A. Ocloo, P.L. Else, A.J. Hulbert, Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds, Biochem. J. (2003), in press. S. Cadenas, K.S. Echtay, J.A. Harper, M.B. Jekabsons, J.A. Buckingham, E. Grau, A. Abuin, H. Chapman, J.C. Clapham, M.D. Brand, The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3, J. Biol. Chem. 277 (2002) 2773–2778. Y. Handrich, R.M. Bevan, J.-B. Charassin, P.J. Butler, K. Ptz, A.J. Woakes, J. Lage, Y. Le Maho, Hypothermia in foraging king penguins, Nature 388 (1997) 64–67. C. Duchamp, H. Barre, D. Delage, J.L. Rouanet, F. Cohen-Adad, Y. Minaire, Nonshivering thermogenesis and adaptation to fasting in king penguin chicks, Am. J. Physiol. 257 (1989) R744– R751.