The Bioenergetics of Brown Fat Mitochondria from UCP1-ablated Mice
1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês
10.1074/jbc.274.40.28150
ISSN1083-351X
AutoresA. Matthias, Anders Jacobsson, Barbara Cannon, Jan Nedergaard,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe 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, Ucp2mRNA levels in brown adipose tissue were 14-fold higher andUcp3 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. 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, Ucp2mRNA levels in brown adipose tissue were 14-fold higher andUcp3 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. The thermogenic function of brown adipose tissue is generally believed to result from the expression in the mitochondria of this tissue of the uncoupling protein, UCP1 (for reviews, see Refs. 1Klaus S. Casteilla L. Bouillaud F. Ricquier D. Int. J. Biochem. 1991; 23: 791-801Crossref PubMed Scopus (241) Google Scholar, 2Nedergaard J. Cannon B. New Comprehensive Biochemistry: Molecular Mechanisms in Bioenergetics. 23. Elsevier, Amsterdam1992: 385-420Google Scholar, 3Klingenberg M. J. Bioenerg. Biomembr. 1993; 25: 447-457Crossref PubMed Scopus (93) Google Scholar, 4Ricquier D. Bouillaud F. Prog. Nucleic Acids Res. Mol. Biol. 1997; 56: 83-108Crossref PubMed Google Scholar, 5Jezek P. Engstova H. Zackova M. Vercesi A.E. Costa A.D. Arruda P. Garlid K.D. Biochim. Biophys. Acta. 1998; 1365: 319-327Crossref PubMed Scopus (185) Google Scholar). However, until now, analysis of the functional significance of UCP1 has had to be indirect, and there has been no way to identify the properties of brown fat mitochondria that are directly due to the presence of UCP1. Only through the recent development of UCP1-ablated mice in Leslie P. Kozak's laboratory (6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar) have investigations dedicated to this issue, which is central not only in thermoregulatory research, but also in obesity research, become possible. We present here a study examining the bioenergetics of brown fat mitochondria from UCP1-ablated mice, enabling a delineation of UCP1 function in its native environment and a distinction between UCP1-related and non-UCP1-related properties of brown fat mitochondria (meeting reports of studies of these mitochondria have been published previously (Refs. 7Monemdjou S. Kozak L.P. Harper M.-E. Obesity Res. 1997; 5: 26SGoogle Scholar, 8Matthias A. Jacobsson A. Cannon B. Nedergaard J. EBEC Short Rep. 1998; 10 (155): 155Google Scholar, 9Cannon B. Matthias A. Golozoubova V. Ohlson K.B.E. Andersson U. Jacobsson A. Nedergaard J. Prog. Obesity Res. 1999; 8: 13-26Google Scholar and 67Monemdjou S. Kozak L.P. Harper M.-E. Am. J. Physiol. 1999; 276: E1073-E1082PubMed Google Scholar). In most respects, ablation of UCP1 altered the characteristics of the mitochondria in ways predicted from earlier studies of wild-type brown fat mitochondria or from studies of UCP1 ectopically expressed in yeast or reconstituted into liposomes; there were, however, notable exceptions. Most remarkably, we observed that the ability of free fatty acids to (re)induce de-energization (uncoupling) in brown fat mitochondria, generally thought to result from an activation of UCP1, was notUCP1-dependent. Additionally, as brown adipose tissue from UCP1-ablated mice exhibit high expression levels of Ucp2 and Ucp3, the present study allowed for observations of the bioenergetic consequences in isolated mitochondria of such high expression levels. The results indicate that a high expression level of these members of the uncoupling protein family is not intrinsically associated with the corresponding isolated mitochondria being in a de-energized state. The UCP1-ablated mice were progeny of those described by Enerbäck et al. (6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar), in which UCP1 was inactivated by homologous recombination with a deletion vector in which exon 2 and parts of exon 3 had been replaced with a neomycin resistance gene; in the brown fat of these mice, no UCP1 can be detected with polyclonal antibodies (6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar). The mice were bred within the institute and were phenotypically (hair color, body weight, growth rate, etc.) identical to mice of the C57BL/6 strain that were the donors of the blastocysts (genetically they are infiltrated with 129/SvJ (from the embryonic stem cells) and 129/SvPas (to which the chimeras were bred)). The wild-type mice were thus of the C57BL/6 strain; these mice (of the same age) were obtained from B & K Universal, Stockholm, Sweden. Before the experiments, adult male mice of either strain were acclimated (one per cage) to 24 °C (12 h light, 12 h dark) for 3 weeks with free access to food and water. Brown adipose tissue and liver (and other tissues, to be detailed elsewhere) were rapidly dissected out and pieces thereof frozen in liquid nitrogen and stored at −80 °C until use. Total RNA was isolated in 1.2 ml of Ultraspec (Biotecx), as described in the manufacturer's protocol. The RNA was separated on an agarose gel (1.25%) containing 20 mm MOPS 1The abbreviations used are:MOPS4-morpholinepropanesulfonic acidFCCPcarbonyl cyanidep-(trifluoromethoxy) phenylhydrazone (pH 7.0), 6.7% formaldehyde, 50 mm NaOAc, and 10 mm EDTA. Ethidium bromide (0.075 μg/ml) was added to the gel for routine examination under UV light of RNA distribution and equal loading. The RNA was transferred to a Hybond-N membrane (Amersham Pharmacia Biotech) by capillary blotting overnight. The membrane was prehybridized at 42 °C for 2 h in 10 ml/membrane of prehybridizing solution (5× SSC (pH 7.0), 5× Denhardt's, 0.5% SDS, 50 mm sodium phosphate (pH 6.5), 50% formamide, and 100 μg/ml herring sperm DNA (Sigma)). The membranes were then hybridized overnight at 42 °C in the same solution with the addition of [32P]CTP-labeled cDNA, labeled by random priming (Roche Molecular Biochemicals). The cDNA clone corresponding to theUcp1 mRNA was that earlier characterized (10Jacobsson A. Stadler U. Glotzer M.A. Kozak L.P. J. Biol. Chem. 1985; 260: 16250-16254Abstract Full Text PDF PubMed Google Scholar). The cDNA clones corresponding to the Ucp2 andUcp3 mRNAs were obtained from Genome Systems Inc. as EST clones 1040737 and 482847, respectively. The identity of these clones was confirmed by sequencing. After hybridization, the solution was removed and the membranes were washed twice in 2× SSC and 0.2% SDS for 20 min at 30 °C, followed by another two washes in 0.1× SSC and 0.2% SDS for 40 min at 50 °C. The membranes were then exposed to a PhosphorImager screen and scanned in a Molecular Dynamics PhosphorImager and analyzed with ImageQuant software. The same membranes were analyzed with all three clones; between hybridizations, the membranes were stripped of previous hybridizations by incubating them for 30 min in 0.1× SSC with 0.1% SDS at 95 °C. 4-morpholinepropanesulfonic acid carbonyl cyanidep-(trifluoromethoxy) phenylhydrazone Both brown fat and liver mitochondria were prepared principally as described by Cannon and Lindberg (11Cannon B. Lindberg O. Methods Enzymol. 1979; 55: 65-78Crossref PubMed Scopus (190) Google Scholar). For brown fat mitochondria, the interscapular, periaortic, axillary, and cervical deposits from 10 animals were dissected out and pooled. Livers from two of these animals were also dissected out and pooled. Tissues were then minced with scissors, homogenized in 40 ml of 250 mm sucrose solution, filtered through gauze, and centrifuged at 8500 × g for 10 min. The pellets were resuspended in sucrose and centrifuged at 800 ×g for 10 min. The resulting supernatants were then centrifuged at 8500 × g for 10 min, and the pellets resuspended in 100 mm KCl, 20 mm Tris (pH 7.2), 0.2% fatty-acid-free bovine serum albumin. After recentrifugation at 8500 × g for 10 min, the mitochondria were further washed in and finally resuspended in KCl/Tris (without albumin). Protein was measured with the fluorescamine method (Fluram from Fluka) and the suspensions diluted to stock concentrations of 20 mg/ml. The GDP-binding capacity of the mitochondria was estimated essentially as described previously (12Nedergaard J. Cannon B. Eur. J. Biochem. 1987; 164: 681-686Crossref PubMed Scopus (23) Google Scholar). Briefly, mitochondria were incubated for 10 min at room temperature in glass vials at a concentration of 1 mg/ml mitochondrial protein in a medium consisting of 125 mmsucrose, 20 mm Tris (pH 7.2), 2 mmMgCl2, 1 mm EDTA, 0.1% fatty-acid-free bovine serum albumin, 4 mm potassium phosphate, and 5 μm rotenone. (A hypotonic medium is necessary to avoid matrix condensation (13Nicholls D.G. Grav H.J. Lindberg O. Eur. J. Biochem. 1972; 31: 526-533Crossref PubMed Scopus (76) Google Scholar).) 10 μm GDP (Sigma) labeled with 800,000 cpm/ml [3H]GDP (Amersham Pharmacia Biotech) was added for the binding, and [14C]sucrose (Amersham Pharmacia Biotech) was added to about 300,000 cpm/ml, as a marker for the extramitochondrial volume. 0.4 ml of the incubation mixture was filtered under vacuum through a 0.45-μm cellulose-nitrate filter (Sartorius GmbH, Götingen, Germany). The filters were then fully dissolved in 5 ml of scintillation fluid for 1 h. The amount of [3H]GDP found on the filter in excess of that predicted from the [14C]sucrose data was defined as specific binding. All assays were performed in quadruplicate for each mitochondrial preparation. Mitochondria, at a final concentration of 0.2 mg/ml mitochondrial protein, were added to 1.1 ml of a continuously stirred incubation medium of the same composition as for the GDP-binding experiments, with the further addition of 0.6 μmrhodamine 123 (Sigma) and either 5 mm glycerol 3-phosphate for brown fat mitochondria or 5 mm succinate for liver mitochondria (brown fat mitochondria may exhibit low permeability for succinate and similar substrates (14Cannon B. Bernson V.S.M. Nedergaard J. Biochim. Biophys. Acta. 1984; 766: 483-491Crossref PubMed Scopus (12) Google Scholar), and the use of a substrate oxidizable without membrane permeation (i.e.glycerol-3-phosphate; Ref. 15Nicholls D.G. Bernson V.S.M. Eur. J. Biochem. 1977; 75: 601-612Crossref PubMed Scopus (85) Google Scholar) was therefore preferred in the brown fat preparations). All incubations were carried out at 37 °C. Membrane potential was monitored with the cationic fluorescent dye rhodamine 123, on an Aminco DW-2 spectrophotometer in the dual-wavelength mode (516–495 nm) in a manner similar to that described by Emaus et al. (16Emaus R.K. Grunwald R. Lemasters J.J. Biochim. Biophys. Acta. 1986; 850: 436-448Crossref PubMed Scopus (732) Google Scholar). The absorbance readings were transferred to mV membrane potential based on calibration curves constructed (principally as described previously (Ref. 17Nedergaard J. Eur. J. Biochem. 1983; 133: 185-191Crossref PubMed Scopus (33) Google Scholar)) for each of the three types of mitochondrial preparations. The calibration curves were based on the Nernst equation: Δψ = 61 mV · log ([K+]in/[K+]out). Calibration values were obtained from traces in which the extramitochondrial K+ level ([K+]out) was altered in the 1–5 mm range. The change in absorbance caused by the addition of 9 μm valinomycin (mean from two to three independent preparations) was plotted against [K+]out. [K+]in was then estimated by extrapolation of the line to the zero uptake point. The [K+]invalue obtained was 19 mm for brown fat mitochondria from wild-type mice (in good agreement with earlier estimates; Ref. 17Nedergaard J. Eur. J. Biochem. 1983; 133: 185-191Crossref PubMed Scopus (33) Google Scholar). It was nearly the same for brown fat mitochondria from UCP1-ablated mice (17 mm); the value for liver mitochondria from wild-type mice was 26 mm (when calculated as means from each preparation, the values were 18 ± 3, 17 ± 2, and 30 ± 12 mm, respectively). These [K+]in values and the [K+]out values were then entered in the Nernst equation for the calibration. Due to the presence of phosphate in the incubation system used here, the ΔpH is expected to be minimal, and the Δψ and the proton motive force (ΔμH+) should therefore be practically identical (16Emaus R.K. Grunwald R. Lemasters J.J. Biochim. Biophys. Acta. 1986; 850: 436-448Crossref PubMed Scopus (732) Google Scholar). Additions were made as indicated in the legends to the figures. FCCP and oleate were dissolved in 50% ethanol; the ethanol did not in itself have any effects on the parameters measured and is used as zero concentration of agent. As the mitochondrial protein/albumin ratio was low (1/5), albumin is the dominant protein and the level of free fatty acids in the incubation can therefore be considered to be buffered by the albumin. The free concentration of oleate was therefore calculated using the equation from Richieri et al. (18Richieri G.V. Anel A. Kleinfeld A.M. Biochemistry. 1993; 32: 7574-7580Crossref PubMed Scopus (337) Google Scholar) for the binding of oleate to bovine serum albumin at 37 °C: [FFA] = 6.5ν − 0.19 + 0.13e1.54ν, where ν is the molar ratio of oleate to albumin. The molecular weight of albumin was taken as 60,000. Nominal levels of oleate added were 10, 20, 40, 60, 80, 100, and 120 μm; these thus transfer to free levels of 4, 8, 21, 56, 239, 1354, and 8365 nm. To facilitate comparisons with other studies, the free values of oleate were those used in graphs. As corresponding binding affinity data do not exist for all fatty acids, comparative data on different fatty acids are given as nominal additions (nmol/ml). Oxygen consumption rates were monitored with a Clark-type oxygen electrode in a medium and under other conditions identical to those used for the membrane potential determinations, including the presence of rhodamine. The UCP1-ablated mice had the same body weight as wild-type mice (as earlier observed (Ref. 6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar)). The total amount of brown adipose tissue that could be dissected out was somewhat larger in the UCP1-ablated mice than in the wild-type, but the tissue was lighter brown, in agreement with it being more fat-filled (6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar) (data not shown). To confirm the validity and examine the consequences of the UCP1 ablation, mRNA levels for the three members of the uncoupling protein family were determined in brown adipose tissue and in liver (Table I). In wild-type mice, mRNA coding for UCP1, UCP2, and UCP3 was found in brown adipose tissue, in agreement with earlier observations (19Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (998) Google Scholar, 20Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1562) Google Scholar, 21Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar). As expected, no full-length Ucp1 mRNA was observable in the brown adipose tissue of the UCP1-ablated mice (but a short transcript was observable (data not shown), as mentioned previously (Ref. 6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar)). Correspondingly, no UCP1 protein was detectable with polyclonal UCP1-antibodies (22Cannon B. Hedin A. Nedergaard J. FEBS Lett. 1982; 150: 129-132Crossref PubMed Scopus (120) Google Scholar) in immunoblots in these mice (data not shown). The ablation of UCP1 led to a 14-fold increase in Ucp2 mRNA level in the brown adipose tissue (principally in accordance with earlier observations (Ref. 6Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1098) Google Scholar)) leading to a level of Ucp2mRNA about half that found in spleen (which has the highest reported Ucp2 mRNA level (Refs. 23Negre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Penicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (686) Google Scholar and24Hidaka S. Kakuma T. Yoshimatsu H. Yasunaga S. Kurokawa M. Sakata T. Biochim. Biophys. Acta. 1998; 1389: 178-186Crossref PubMed Scopus (40) Google Scholar)). 2A. Matthias, V. Golozoubova, A. Jacobsson, B. Cannon, and J. Nedergaard, unpublished observation. The ablation of UCP1 also led to some decrease in Ucp3 mRNA level in the brown adipose tissue (Table I), but the level remained close to that observed in skeletal muscle (data not shown), which, together with brown and white adipose tissue, has the highest reported levels (19Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (998) Google Scholar,25Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (682) Google Scholar).2Table IEffect of UCP1 ablation on mRNA levels of uncoupling proteinsMouse strainTissuemRNA levelsUCP1 (n = 3)UCP2 (n = 5)UCP3 (n = 3)arbitrary unitsUCP1+/+BAT34 ± 22 ± 120 ± 2UCP1−/−BAT0 ± 0**28 ± 6**12 ± 1*UCP1+/+Liver0 ± 04 ± 00 ± 0The indicated tissues were prepared from UCP1-ablated (UCP−/−) and wild-type (UCP+/+) mice and the levels of mRNA coding for UCP1, UCP2, and UCP3 were determined as described under "Materials and Methods." The values are arbitrary units for each UCP and cannot be compared. It may be noted that basal levels of gene expression of any of the three UCPs may differ between different mouse strains (see,e.g., Ref. 66Surwit R.S. Wang S. Petro A.E. Sanchis D. Raimbault S. Ricquier D. Collins S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4061-4065Crossref PubMed Scopus (295) Google Scholar), and the differences given below can therefore not be generalized to all strains; they are, however, those relevant for the present functional study. ** and * indicate significant differences in mRNA levels between wild-type and UCP1-ablated mice (p < 0.01 and p < 0.05, respectively; Student's unpaired t test). Open table in a new tab The indicated tissues were prepared from UCP1-ablated (UCP−/−) and wild-type (UCP+/+) mice and the levels of mRNA coding for UCP1, UCP2, and UCP3 were determined as described under "Materials and Methods." The values are arbitrary units for each UCP and cannot be compared. It may be noted that basal levels of gene expression of any of the three UCPs may differ between different mouse strains (see,e.g., Ref. 66Surwit R.S. Wang S. Petro A.E. Sanchis D. Raimbault S. Ricquier D. Collins S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4061-4065Crossref PubMed Scopus (295) Google Scholar), and the differences given below can therefore not be generalized to all strains; they are, however, those relevant for the present functional study. ** and * indicate significant differences in mRNA levels between wild-type and UCP1-ablated mice (p < 0.01 and p < 0.05, respectively; Student's unpaired t test). In the liver of wild-type mice, there was, as expected, noUcp1 expression, low Ucp2 expression (reported to be only from Kupffer cells under these circumstances (Ref. 26Larrouy D. Laharrague P. Carrera G. Viguerie-Bascands N. Levi-Meyrueis C. Fleury C. Pecqueur C. Nibbelink M. Andre M. Casteilla L. Ricquier D. Biochem. Biophys. Res. Commun. 1997; 235: 760-764Crossref PubMed Scopus (129) Google Scholar)), and noUcp3 expression (Table I) (19Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (998) Google Scholar, 25Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (682) Google Scholar). Thus, the isolated liver mitochondria cannot be expected to contain any UCP1 or UCP3. There may be trace amounts of UCP2 in the preparation, but as Ucp2 is only expressed in the Kupffer cells (26Larrouy D. Laharrague P. Carrera G. Viguerie-Bascands N. Levi-Meyrueis C. Fleury C. Pecqueur C. Nibbelink M. Andre M. Casteilla L. Ricquier D. Biochem. Biophys. Res. Commun. 1997; 235: 760-764Crossref PubMed Scopus (129) Google Scholar) and not in the parenchymal liver cells (27Carretero M.V. Torres L. Latasa U. Garcia-Trevijano E.R. Prieto J. Mato J.M. Avila M.A. FEBS Lett. 1998; 439: 55-58Crossref PubMed Scopus (53) Google Scholar) from which the bulk of the mitochondria in the preparation originates, there is no reason to suspect that this UCP2 will affect the collective properties of the isolated mitochondria to any appreciable extent. The brown fat mitochondria from wild-type mice will be expected to have high UCP1 levels. Based on the expression levels shown above, the brown fat mitochondria from the UCP1-ablated mice could be expected to contain rather high levels of UCP2 and some UCP3. Thus, comparison of the properties of these three types of mitochondria may be helpful in furthering the understanding not only of the effects of UCP1 on mitochondrial bioenergetics, but perhaps also of those of UCP2 and UCP3. For analysis of the effect of UCP1 ablation on mitochondrial bioenergetics, mitochondria were isolated from the brown adipose tissue of wild-type and of UCP1-ablated mice, and from the liver of wild-type mice. The amount of brown fat mitochondria obtained from wild-type mice was slightly higher than from the UCP1-ablated mice. The presence of UCP mRNA in the brown adipose tissue of the wild-type mice (which were housed at 24 °C, i.e. at an ambient temperature significantly below their thermoneutral zone of 30–32 °C) would be expected to lead to the presence of UCP1 associated with a specific GDP-binding capacity. In accordance with this, brown fat mitochondria from wild-type mice had a [3H]GDP-binding capacity of 0.15 nmol/mg (TableII), a value in good agreement with earlier observations made on mice at this acclimation temperature (28Jennings G. Richard D. Trayhurn P. Comp. Biochem. Physiol. A. 1986; 85: 583-586Crossref PubMed Scopus (13) Google Scholar). In UCP1-ablated mice, the [3H]GDP-binding capacity was practically eliminated and was reduced to the level observed in the UCP1,2,3-free liver mitochondria (Table II). Thus, indeed, the presence of UCP1 is associated with the ability to bind [3H]GDP.Table IIGDP-binding capacity and effects of GDP on membrane potential of mitochondria isolated from brown adipose tissue of UCP1-ablated and wild-type mice and from liver of wild-type miceMitochondriaGDP bindingMembrane potentialStrainTissue− GDP+ GDPnmol/mg proteinmVUCP1+/+BAT0.15 ± 0.02−33 ± 8−141 ± 2***UCP1−/−BAT0.05 ± 0.02*−195 ± 6**−190 ± 8**UCP1+/+Liver0.05 ± 0.01*−184 ± 3**−177 ± 3**,***[3H]GDP-binding capacities were measured as described under "Materials and Methods," in three independent preparations of each type, with four replicates for each preparation. In control experiments with brown fat mitochondria from wild-type mouse, we have verified that 10 μm GDP is a saturating concentration in these preparations (data not shown). In some experiments, 10 μm[3H]GDP was also competed with 100-fold excess (1 mm) unlabeled GDP, leading to practically full elimination of [3H]GDP binding in all three types of mitochondria; thus, the residual binding observed in brown-fat mitochondria from UCP1-ablated mice and that observed in liver mitochondria does not represent unspecific (unsaturable) binding but binding to other site(s) than that of UCP1. The membrane potential determinations were performed as those exemplified in Fig. 1, with 1 mm GDP. Results are means from five preparations. * and ** indicate a value statistically different from the corresponding value for mitochondria from brown adipose tissue of wild-type mice (p < 0.05 and <0.001, respectively; Student's unpaired t test), and *** a statistically significant effect of GDP addition (p< 0.001; Student's paired t test). Open table in a new tab [3H]GDP-binding capacities were measured as described under "Materials and Methods," in three independent preparations of each type, with four replicates for each preparation. In control experiments with brown fat mitochondria from wild-type mouse, we have verified that 10 μm GDP is a saturating concentration in these preparations (data not shown). In some experiments, 10 μm[3H]GDP was also competed with 100-fold excess (1 mm) unlabeled GDP, leading to practically full elimination of [3H]GDP binding in all three types of mitochondria; thus, the residual binding observed in brown-fat mitochondria from UCP1-ablated mice and that observed in liver mitochondria does not represent unspecific (unsaturable) binding but binding to other site(s) than that of UCP1. The membrane potential determinations were performed as those exemplified in Fig. 1, with 1 mm GDP. Results are means from five preparations. * and ** indicate a value statistically different from the corresponding value for mitochondria from brown adipose tissue of wild-type mice (p < 0.05 and <0.001, respectively; Student's unpaired t test), and *** a statistically significant effect of GDP addition (p< 0.001; Student's paired t test). UCP2 and UCP3 possess amino acid sequences similar to that thought to be responsible for nucleotide binding in UCP1. Therefore, these members of the mitochondrial carrier family have been suggested to also be able to bind purine nucleotides (23Negre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Penicaud L. Casteilla L. FASEB J. 1997; 11: 809-815Crossref PubMed Scopus (686) Google Scholar, 29Boss O. Muzzin P. Giacobino J.-P. Eur. J. Endocrinol. 1998; 139: 1-9Crossref PubMed Scopus (227) Google Scholar). As both of these genes are highly expressed in brown adipose tissue of UCP1-ablated mice (Table I), it may be suggested that some of the residual GDP-binding capacity (TableII) could represent binding to UCP2 or UCP3. However, since the GDP-binding capacities of mitochondria from the UCP1,2,3-free liver and from the highly Ucp2- and Ucp3-expressing brown adipose tissue from UCP1-ablated mice were equal, it could be concluded that high expression levels of Ucp2 or Ucp3 are not predictive of the presence of a high capacity for purine nucleotide binding to mitochondria from that tissue, and perhaps even that UCP2 and UCP3 do not carry a purine nucleotide site with properties similar to that of UCP1. The possibility cannot, of course, be excluded that a purine nucleotide binding site exists on UCP2 or UCP3 with a purine nucleotide selectivity or affinity that is markedly different from that of UCP1 and would thus not be detected with 10 μm[3H]GDP. To examine the effect of the absence of UCP1 on the bioenergetics of brown fat mitochond
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