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• W2070789450 abstract "Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1–/– mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1–/– mice, irrespective of whether they were adapted to thermoneutral temperature (30 °C) or to the cold (4 °C). The absence of oxidative damage in UCP1–/– mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage. Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1–/– mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1–/– mice, irrespective of whether they were adapted to thermoneutral temperature (30 °C) or to the cold (4 °C). The absence of oxidative damage in UCP1–/– mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage. A generally accepted view of the molecular mechanism of UCP1 function and regulation has not been achieved (reviewed in Refs. 1Ricquier D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (745) Google Scholar, 2Nedergaard J. Golozoubova V. Matthias A. Asadi A. Jacobsson A. Cannon B. Biochim. Biophys. Acta. 2001; 1504: 82-106Crossref PubMed Scopus (452) Google Scholar, 3Brand M.D. Affourtit C. Esteves T.C. Green K. Lambert A.J. Miwa S. Pakay J.L. Parker N. Free Radic. Biol. Med. 2004; 37: 755-767Crossref PubMed Scopus (807) Google Scholar, 4Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4354) Google Scholar). Roles for purine nucleotides as inhibitors and for fatty acids as (re)activators are generally accepted, but the issue has been raised as to whether alternative physiological activators exist and/or whether certain cofactors are necessary for UCP1 activity. These discussions may also be relevant concerning the phylogenetically related mitochondrial membrane proteins UCP2 and UCP3 (for review, see Refs. 5Nedergaard J. Cannon B. Exp. Physiol. 2003; 88: 65-84Crossref PubMed Scopus (189) Google Scholar, 6Esteves T.C. Brand M.D. Biochim. Biophys. Acta. 2005; 1709: 35-44Crossref PubMed Scopus (126) Google Scholar, 7Krauss S. Zhang C.Y. Lowell B.B. Nat. Rev. Mol. Cell Biol. 2005; 6: 248-261Crossref PubMed Scopus (548) Google Scholar, 8Nedergaard J. Ricquier D. Kozak L.P. EMBO Rep. 2005; 6: 917-921Crossref PubMed Scopus (103) Google Scholar). Particularly, to explain the lack of constitutive uncoupling activity of uncoupling proteins reconstituted in liposomes, or the lack of basal uncoupling (inhibitable by GDP) in skeletal muscle mitochondria expressing UCP3 (9Cadenas S. Brand M.D. Biochem. J. 2000; 348: 209-213Crossref PubMed Scopus (43) Google Scholar), it has been suggested that non-fatty acid cofactors are required for activation, ubiquinone (10Echtay K.S. Winkler E. Klingenberg M. Nature. 2000; 408: 609-613Crossref PubMed Scopus (287) Google Scholar) and superoxide (11Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1124) Google Scholar) being those originally suggested. Although the necessity for additional activators has been questioned in reconstituted systems (12Jaburek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), in isolated mitochondria (13Couplan E. del Mar Gonzalez-Barroso M. Alves-Guerra M.C. Ricquier D. Goubern M. Bouillaud F. J. Biol. Chem. 2002; 277: 26268-26275Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and in animals (14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar), several other cofactor candidates have been discussed. A series of compounds that are formed downstream of superoxide interaction with the mitochondrial membrane have been proposed to be the true activators of UCPs (or to be directly involved in the uncoupling mechanism). These include carbon-centered radicals (15Murphy M.P. Echtay K.S. Blaikie F.H. Asin-Cayuela J. Cocheme H.M. Green K. Buckingham J.A. Taylor E.R. Hurrell F. Hughes G. Miwa S. Cooper C.E. Svistunenko D.A. Smith R.A. Brand M.D. J. Biol. Chem. 2003; 278: 48534-48545Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), hydroperoxy fatty acids (16Goglia F. Skulachev V.P. FASEB J. 2003; 17: 1585-1591Crossref PubMed Scopus (205) Google Scholar, 17Jaburek M. Miyamoto S. Di Mascio P. Garlid K.D. Jezek P. J. Biol. Chem. 2004; 279: 53097-53102Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and lipid peroxidation products, e.g. 4-hydroxy-2-nonenal (HNE) 3The abbreviations used are: HNE, 4-hydroxy-2-nonenal; UCP, uncoupling protein; ROS, reactive oxygen species; BSA, bovine serum albumin; DHE, dihydroethidium; SOD, superoxide dismutase; MDA, malonyl dialdehyde; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3The abbreviations used are: HNE, 4-hydroxy-2-nonenal; UCP, uncoupling protein; ROS, reactive oxygen species; BSA, bovine serum albumin; DHE, dihydroethidium; SOD, superoxide dismutase; MDA, malonyl dialdehyde; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. (3Brand M.D. Affourtit C. Esteves T.C. Green K. Lambert A.J. Miwa S. Pakay J.L. Parker N. Free Radic. Biol. Med. 2004; 37: 755-767Crossref PubMed Scopus (807) Google Scholar, 6Esteves T.C. Brand M.D. Biochim. Biophys. Acta. 2005; 1709: 35-44Crossref PubMed Scopus (126) Google Scholar, 24Echtay K.S. Esteves T.C. Pakay J.L. Jekabsons M.B. Lambert A.J. Portero-Otin M. Pamplona R. Vidal-Puig A.J. Wang S. Roebuck S.J. Brand M.D. EMBO J. 2003; 22: 4103-4110Crossref PubMed Scopus (503) Google Scholar). The question of a cofactor necessity for UCP function has thus been linked to the earlier suggestion that UCP activity can control the formation of reactive oxygen species (ROS) (18Negre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Penicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (676) Google Scholar). A much discussed (8Nedergaard J. Ricquier D. Kozak L.P. EMBO Rep. 2005; 6: 917-921Crossref PubMed Scopus (103) Google Scholar) scheme has been proposed where conditions leading to increased oxygen stress lead to superoxide production, which results in the formation of compounds such as HNE that could activate UCPs. This should lower the mitochondrial membrane potential and, consequently, diminish the risk of oxidative damage (3Brand M.D. Affourtit C. Esteves T.C. Green K. Lambert A.J. Miwa S. Pakay J.L. Parker N. Free Radic. Biol. Med. 2004; 37: 755-767Crossref PubMed Scopus (807) Google Scholar, 6Esteves T.C. Brand M.D. Biochim. Biophys. Acta. 2005; 1709: 35-44Crossref PubMed Scopus (126) Google Scholar). Due to the broad implications of this hypothesis for pathological processes, we have here studied the proposed interaction between HNE and the originally identified uncoupling protein UCP1. The choice of UCP1 was made because it is uncontroversial that UCP1 functions as an uncoupling protein; the proton leak associated with UCP1 is thus easily observable. We have used the availability of UCP1-ablated mice (19Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar) to enable us to dissociate the UCP1-dependent effects of HNE from UCP1-independent effects, which may occur in the mitochondria. We conclude that the reported HNE effects on brown fat mitochondria are UCP1-independent. In vivo studies confirm that no protective effect of UCP1 can be identified physiologically. Our results may have significance for the understanding of the regulation not only of UCP1 but also of the other uncoupling proteins and may also contribute to the identification of physiological conditions associated with the increased risk of oxidative damage. Animals—UCP1-ablated mice (progeny of those described in Ref. 19Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1060) Google Scholar) were backcrossed to C57Bl/6 mice for 10 generations and after intercrossing were maintained as UCP1–/– and UCP1+/+ strains. The mice were fed ad libitum (R70 Standard Diet, Lactamin), had free access to water, and were kept on a 12:12-h light:dark cycle, routinely at normal (24 °C) animal house temperature. Adult (8–12-week-old) male mice were routinely used for the experiments. For experiments on warm-acclimated animals, UCP1–/– and UCP1+/+ adult male mice were divided into age- (7–8-week-old) and body weight (23–24 g)-matched groups, one per cage, and acclimated at 30 °C (i.e. thermoneutral temperature for both wild-type and UCP1-ablated mice) 4H. Feldmann, B. Cannon, and J. Nedergaard, unpublished observations. for at least 1 month before the start of the experiment. For experiments on cold-acclimated animals, UCP1+/+ and UCP1–/– adult male mice were similarly either acclimated (one per cage) at 24 °C or successively acclimated to cold by first placing them at 18 °C for 4 weeks with the following 4 weeks at 4 °C (the intermediate 18 °C step was required to allow for survival of the UCP1–/– animals at 4 °C (20Golozoubova V. Hohtola E. Matthias A. Jacobsson A. Cannon B. Nedergaard J. FASEB J. 2001; 15: 2048-2050Crossref PubMed Scopus (350) Google Scholar)). The experiments were approved by the Animal Ethics Committee of the North Stockholm region. Mitochondrial Preparation—Brown fat mitochondria were prepared principally as described (21Cannon B. Nedergaard J. Methods Mol. Biol. 2001; 155: 295-303PubMed Google Scholar) with some modifications (22Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Mitochondrial protein concentration was measured using the fluorescamine method (23Udenfriend S. Stein S. Bohlen P. Dairman W. Leimgruber W. Weigele M. Science. 1972; 178: 871-872Crossref PubMed Scopus (2185) Google Scholar), and the suspensions were diluted to stock concentrations of 25 mg of mitochondrial protein/ml of 125 mm sucrose with 0.2% (1% in experiments with warm-acclimated mice) fatty acid-free bovine serum albumin (BSA). Oxygen Consumption—Isolated mitochondria, at final concentrations of 0.5 or 0.3 mg (indicated in figure legends) of mitochondrial protein/ml, were added to 1.1 ml of a continuously stirred incubation medium consisting of 125 mm sucrose, 20 mm K+-Tes (pH 7.2), 2 mm MgCl2, 1 mm EDTA, 4 mm potassium Pi, and 1.3 μg of oligomycin/ml. The substrates were 5 mm pyruvate plus 3 mm malate or 5 mm glycerol 3-phosphate in the presence of 2 μg of rotenone/ml. UCP1–/– and UCP1+/+ brown fat mitochondria exhibit identical oxidative capacities, estimated as maximal rates of FCCP-stimulated respiration (22Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The final concentration of fatty acid-free BSA was adjusted to 0.1% (w/v) for oxygen consumption measurements performed in mitochondria respiring on pyruvate plus malate. This was increased to 1% (w/v) in experiments where glycerol 3-phosphate was used as substrate (as indicated) (i.e. similarly to Ref. 24Echtay K.S. Esteves T.C. Pakay J.L. Jekabsons M.B. Lambert A.J. Portero-Otin M. Pamplona R. Vidal-Puig A.J. Wang S. Roebuck S.J. Brand M.D. EMBO J. 2003; 22: 4103-4110Crossref PubMed Scopus (503) Google Scholar). Oxygen consumption rates were monitored with a Clark-type oxygen electrode (Yellow Springs Instrument Co.) in a sealed chamber at 37 °C, as described (22Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Data for GDP concentration-response curves were analyzed with the general fit option of the KaleidaGraph application for Macintosh for adherence to simple Michaelis-Menten kinetics, V(x) = Vmax – ΔVmax · (x/(Km + x)), where x is the concentration of GDP. Mitochondrial Membrane Potential—Measurements were performed in brown fat mitochondria with the dye safranin O (25Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (661) Google Scholar). Mitochondria were incubated with 2 μg/ml oligomycin, 5 μm safranin, 0.1% (w/v) fatty acid-free BSA, and 5 mm glycerol 3-phosphate in the presence of 2 μg of rotenone/ml. The changes in absorbance of safranin O were followed at 37 °C in an Aminco DW-2 dual-wavelength spectrophotometer at 511–533 nm with a 3-nm slit. Signals were recorded every 0.5 s via a PowerLab/ADInstrument. The data were stored and analyzed using the Chart version 5.1.1 program. Calibration curves were made for each mitochondrial preparation in K+-free medium and were obtained from traces in which the extramitochondrial K+,[K+]out, was altered by addition of KCl in a 0.1–20 mm final concentration range. The change in absorbance then caused by the addition of 3 μm valinomycin was plotted against [K+]out. The intramitochondrial K+, [K+]in, was estimated by extrapolation of the line to the zero uptake point, as described in Ref. 26Nedergaard J. Eur. J. Biochem. 1983; 133: 185-191Crossref PubMed Scopus (33) Google Scholar. The absorbance readings were used to calculate the membrane potential (mV) by the Nernst equation according to: Δψm = 61 mV·log ([K+]in/[K+]out). To determine the basal proton leak, mitochondrial membrane potential and oxygen consumption measurements were performed in parallel using the same media and conditions, but in the presence of increasing amounts of antimycin A, as indicated. Western Blotting—For HNE-adduct detection, aliquots of freshly isolated mitochondrial suspension were stored under nitrogen at –80 °C after supplementation with protease inhibitor mixture (Complete Mini, Roche). Protein concentrations of the thawed mitochondrial samples were requantified using the Lowry method. Equal amounts of mitochondrial protein were loaded on SDS-polyacrylamide gel. After electrophoresis, proteins were transferred by electroblotting to a polyvinylidene difluoride membrane. HNE protein adducts were detected with polyclonal antibodies from Alpha Diagnostics (HNE12-S, dilution 1:1000). After incubation with horseradish peroxidase-conjugated secondary antibodies, the membrane was incubated with detection reagent (ECL, Amersham Biosciences) and the chemiluminescence signal was detected with a CCD camera (Fuji). Quantifications were performed with the Image Gauge 3 software. For UCP1 determination, the membrane used for detection of the HNE adducts was stripped and blotted with UCP1 polyclonal antibodies (prepared in rabbit from the C-terminal decapeptide of mouse UCP1), dilution 1:3000. For cytochrome oxidase subunit 1 determination, the membrane used for detection of HNE adducts and UCP1 was blotted, after stripping, with cytochrome oxidase subunit 1-monoclonal antibodies (Molecular Probes), diluted 1:2000. Superoxide Measurement—Net superoxide release rates were assessed directly in isolated brown fat mitochondria by fluorescence measurements with the dye dihydroethidium (DHE) (Molecular Probes), the conversion of which to ethidium is superoxide-induced (14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar, 27Benov L. Sztejnberg L. Fridovich I. Free Radic. Biol. Med. 1998; 25: 826-831Crossref PubMed Scopus (423) Google Scholar). The fluorescence emitted by the ethidium formed was followed on a spectrophotometer (Sigma ZES II) at 37 °C using an excitation wavelength of 495 nm and collecting the emission via a cutoff filter at 580 nm. The data were acquired, stored, and analyzed using the Chart 4.1.1 program (PowerLab/ADInstrument). Chemical and biological validations of this method were reported in Ref. 14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar: generation of superoxide by the xanthine plus xanthine oxidase system or by mitochondria in the presence of DHE resulted in a significant increase in ethidium-emitted fluorescence, which was blocked by addition of recombinant SOD (14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar). When estimated by this method, superoxide generation was diminished in brown fat mitochondria from hSOD2+ mice (i.e. mitochondrial superoxide dismutase-overexpressing mice) as compared with wild-type mice (14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar). The assay thus detected mitochondrial superoxide release. Aconitase and Citrate Synthase Activities—Aconitase activity was measured spectrophotometrically as NADPH formation, monitored at 340 nm using a coupled assay (28Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (244) Google Scholar). The frozen mitochondrial samples (the same as those used for Western blotting) were rapidly thawed immediately prior to assay, and 2 μl of 10 times diluted sample were added to 500 μl of assay buffer (50 mm Tris-HCl, pH 7.4, 0.6 mm MnCl2, 5 mm sodium citrate, 0.2 mm NADP+, 0.1% (v/v) Triton X-100, and 0.4 units/ml isocitrate dehydrogenase (Sigma)) pre-equilibrated to 30 °C. Each sample was assayed in duplicate; readings were taken at 15-s intervals over 7 min, and the resulting linear slopes were averaged to give a measurement of aconitase activity for that sample. Superoxide inactivates the Krebs cycle enzyme aconitase, whereas citrate synthase, another Krebs cycle enzyme, is insensitive to superoxide; therefore, the aconitase/citrate synthase ratio is a convenient measure of oxidative damage in mitochondria (28Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (244) Google Scholar). Citrate synthase activity was determined as in Ref. 29Alp P.R. Newsholme E.A. Zammit V.A. Biochem. J. 1976; 154: 689-700Crossref PubMed Scopus (281) Google Scholar. To validate the assay and to induce extensive oxidative damage, brown fat mitochondria were also exposed to a superoxide-generating system (370 μm xanthine plus 23 μg/ml xanthine oxidase) for 15 min at 30 °C. Confirmation that this system provides a high level of superoxide was obtained in Ref. 14Silva J.P. Shabalina I.G. Dufour E. Petrovic N. Backlund E.C. Hultenby K. Wibom R. Nedergaard J. Cannon B. Larsson N.-G. EMBO J. 2005; 24: 4061-4070Crossref PubMed Scopus (92) Google Scholar. Lipid Peroxidation—Interscapular brown adipose tissue was rapidly dissected from UCP1–/– and UCP1+/+ mice and immediately frozen in liquid nitrogen. The wet weight of brown adipose tissue was measured and the left lobe of tissue was used for the lipid peroxidation assay and the right lobe for the determination of antioxidant enzyme activities. The amount of peroxidative reactants was estimated as the formation of thiobarbituric acid-reactive substances (mainly the lipid peroxidation product, malonyl dialdehyde (MDA) (30Yagi K. Methods Mol. Biol. 1998; 108: 101-106PubMed Google Scholar)). Brown adipose tissue homogenates were prepared in 50 mm Tris-HCl buffer (pH 7.4) in a ratio of 1:50 (w/v). Basal MDA levels were measured in freshly homogenized samples. Inducible peroxidative reactions were stimulated by addition of 312.5 μm ascorbic acid and 6.25 μm FeSO4 (final concentrations) and incubation at 37 °C for 1 h. Spontaneous peroxidative reactions were analyzed in samples incubated in parallel without addition of ascorbic acid and FeSO4. Antioxidant Enzyme Assays—The right lobe of interscapular brown adipose tissue was homogenized in buffer containing 250 mm sucrose, 50 mm Tris-HCl, and 1 mm EDTA (pH 7.2), in a 1:10 ratio (w/v) and sonicated three times at 100 watts for 20 s with 10-s pauses in a Bronson model B-12 sonicator. The samples were then centrifuged at 20,000 × g in an Eppendorf centrifuge for 60 min. Supernatants were used for determination of manganese-containing mitochondrial superoxide dismutase 2 (Mn-SOD), CuZn-containing cytosolic superoxide dismutase 1 (CuZn-SOD), and catalase. SOD activity was measured by the adrenaline method (31Misra H.P. Fridovich I. J. Biol. Chem. 1972; 247: 3170-3175Abstract Full Text PDF PubMed Google Scholar). 100 μl of acidified adrenaline solution was added to 3 ml of alkaline carbonate buffer (50 mm Na2CO3, 0.1 mm EDTA, pH 10.2), and absorbance of adrenochrome at 480 nm was monitored for 4 min. The decrease in the rate of change of the absorbance caused by the samples was followed. One unit of SOD was defined as the amount of enzyme reducing the rate of autoxidation of adrenaline by 50%. Mn-SOD-specific activity was obtained by inhibiting CuZn-SOD for 20 min at room temperature with 4 mm KCN (final concentration) before the dismutase assay. CuZn-SOD activity was calculated by subtracting Mn-SOD activity from the total SOD activity. Catalase activity was measured spectrophotometrically by a method (32Beutler E. Beutler E. Red Cell Metabolism, a Manual of Biochemical Methods. Grune and Stratton, New York1982: 105-106Google Scholar) based on the rate of hydrogen peroxide degradation by the catalase contained in the examined samples. Chemicals—Fatty acid-free bovine serum albumin, fraction V, was from Roche Diagnostics GmbH (Germany). HNE was from Cayman Chemical (Ann Arbor, MI). Nonanoic acid and fluorescamine (4-phenyl spiro-[furan-2(3H),1-phthalan]-3,3′-dione) were from Fluka Chemie Gmbh. 2-Nonenoic acid was from CHEMOS Gmbh (Regenstauf). Other chemicals were all from Sigma. GDP was dissolved in 20 mm Tes (pH 7.2) and the pH of the solution readjusted. FCCP was dissolved in 95% ethanol and diluted in 50% ethanol; oligomycin and rotenone were dissolved in 95% ethanol. Nonanoic acid, 2-nonenoic acid, 2-nonenal, all-trans-retinal, and HNE were dissolved in 95% ethanol, divided into small aliquots, and stored under nitrogen at –80 °C. Ethanol in a final concentration of 0.1% did not in itself have any effects on the parameters measured. Statistics—All data are expressed as mean ± S.E. Statistical analysis for the comparison of two groups was performed using Student's t test. The Presence of a Carboxyl Group Is an Absolute Requirement for Compounds (Re)activating UCP1—HNE, 2-nonenal, and all-trans-retinal have been suggested as general UCP activators (24Echtay K.S. Esteves T.C. Pakay J.L. Jekabsons M.B. Lambert A.J. Portero-Otin M. Pamplona R. Vidal-Puig A.J. Wang S. Roebuck S.J. Brand M.D. EMBO J. 2003; 22: 4103-4110Crossref PubMed Scopus (503) Google Scholar). We have examined their effects on UCP1, i.e. the uncoupling protein found in brown fat mitochondria and the only uncoupling protein with a verified physiological uncoupling ability. We recently demonstrated a role for fatty acids as kinetically competitive (re)activators of UCP1 in GDP-inhibited brown fat mitochondria (22Shabalina I.G. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 2004; 279: 38236-38248Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Our interpretation of those experiments was that fatty acids only function to overcome the GDP inhibition, in our hands in a kinetically simple competitive manner, and that fatty acids do not participate in the uncoupling function of UCP1 as such. We therefore initially examined the ability of HNE to reactivate GDP-inhibited UCP1, i.e. to have a mechanism of action similar to that of fatty acids. To identify the UCP1-dependent effects, we examined the effect of HNE in brown fat mitochondria isolated from both UCP1–/– and UCP1+/+ mice. For comparison of activating efficiency, we first determined the effects of the corresponding fatty acids 2-nonenoic and nonanoic acid. In brown fat mitochondria without UCP1, small uncoupling effects of these fatty acids were observed (Fig. 1, A–C, thin lines), indicating a minor UCP1-independent uncoupling mediated by these fatty acids. Both fatty acids were able to (re)stimulate oxygen consumption (preinhibited by GDP) in UCP1+/+ mitochondria (Fig. 1, B and C, thin lines). The UCP1-dependent effect was estimated as the difference between the fatty acid effects in the two mitochondrial preparations (UCP1+/+ and UCP1–/–) (Fig. 1D). As seen, the efficiency and apparent affinity were essentially identical for 2-nonenoic and nonanoic acid. The (re)activation ability was thus independent of the presence or absence of a double bond in the fatty acid. The activator candidate, the aldehyde HNE, had practically no effect in UCP1–/– mitochondria (Fig. 1, A–C, heavy lines). Similarly but notably, no ability to stimulate oxygen consumption was found in UCP1+/+ mitochondria (Fig. 1, B and C, heavy lines). Thus, there was no UCP1-dependent effect of HNE at all (Fig. 1D), i.e. HNE cannot (re)activate GDP-inhibited UCP1. Consequently, in contrast to the corresponding fatty acids, aldehydes were not able to reactive GDP-inhibited UCP1 in brown fat mitochondria. Because this inhibited state of UCP1 probably mimics the UCP1 state in unstimulated brown fat cells, it is not evident how HNE could activate UCP1 in situ. Oxidized Aldehyde Can Activate UCP1—Mammalian mitochondria possess several highly active pathways for HNE metabolism, and one, aldehyde dehydrogenase, has as its product hydroxy-nonenoic acid (33Siems W. Grune T. Mol. Aspects Med. 2003; 24: 167-175Crossref PubMed Scopus (173) Google Scholar). Because a fatty acid is thus a major product of HNE metabolism, we investigated the effect of supplying isolated UCP1+/+ mitochondria with aldehyde dehydrogenase (plus NAD+) when studying the effect of the aldehyde 2-nonenal (Fig. 2). Nonenal did not in itself reactivate GDP-inhibited UCP1 (Fig. 2A). The presence of aldehyde dehydrogenase plus NAD+ dramatically altered the response of the mitochondria to the aldehyde. Addition of 2-nonenal now stimulated UCP1-dependent oxygen consumption to the same extent as did 2-nonenoic acid (Fig. 2, B and C). This demonstrated that the aldehyde has a stimulatory effect only if converted to a fatty acid. Thus, HNE (Fig. 1), 2-nonenal (Fig. 2), or all-trans-retinal (data not shown) could not stimulate oxygen consumption in an UCP1-dependent manner in brown fat mitochondria. Conversion of the aldehyde into a fatty acid led to stimulation of oxygen consumption in UCP1+/+ mitochondria. The presence of a carboxyl group is thus an absolute requirement for compounds (re)activating UCP1. No HNE Activation of Uninhibited UCP1—Although the above experiments demonstrate that HNE cannot (re)activate GDP-inhibited UCP1, it would be possible that HNE could function as an activator for (or enhance the activity of) UCP1 when UCP1 is in its uninhibited state. Indeed, it has been reported by Echtay et al. (24Echtay K.S. Esteves T.C. Pakay J.L. Jekabsons M.B. Lambert A.J. Portero-Otin M. Pamplona R. Vidal-Puig A.J. Wang S. Roebuck S.J. Brand M.D. EMBO J. 2003; 22: 4103-4110Crossref PubMed" @default.
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• W2070789450 title "UCP1 and Defense against Oxidative Stress" @default.
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