Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting Maria Jimenez, Cedric Yvon, Lorenz Lehr, Bertrand Le ´ ger, Patrick Keller, Aaron Russell, Franc¸oise Kuhne, Pierre Flandin, Jean-Paul Giacobino and Patrick Muzzin Department of Medical Biochemistry, Faculty of Medicine, University of Geneva, Switzerland Uncoupling protein-3 (UCP3) is a mitochondrial inner- membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differen- tially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully valid- ated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxi- dative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mito- chondria. Taken together, these results suggest that oxida- tive muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modu- lation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes. Keywords: fasting; intermyofibrillar mitochondria; muscle type; subsacorlemmal; uncoupling protein-3 (UCP3). The first uncoupling protein described, uncoupling protein-1 (UCP1), is an inner-mitochondrial membrane protein, which, by dissipating the mitochondrial proton gradient driven by the respiratory chain, uncouples oxidation from phosphorylation and therefore produces heat instead of ATP. UCP1 was found to be exclusively expressed in brown adipose tissue (for review see [1]). The novel UCP3, discovered in 1997, is abundantly expressed in rodent and human skeletal muscle. Its high sequence homology with UCP1 suggested that it had similar uncoupling activities [2]. In fact, using heterologous yeast and mammalian cell expression systems, UCP3 was shown to decrease the mitochondrial membrane potential, as measured by uptake of potential sensitive fluorescent dyes (reviewed in [3–5]). Also recent data obtained using muscle mitochondria of UCP3 knockout (UCP3KO) mice [6,7] and of transgenic mice overexpressing UCP3 in their skeletal muscle [8] confirmed the uncoupling activity of UCP3. A more recent study clearly established that UCP3 is, like UCP1, a H + transporter sensitive to nucleotides and fatty acids [9]. Many studies have been performed on the metabolic regulation of brown adipose tissue and muscle UCP3 mRNA expression in both rodents and humans (for review see [3–5]). Very few of the control mechanisms of UCP3 observed in muscle at the mRNA level have so far been studied at the protein level. The reasons for this are the difficulty in obtaining specific antibodies and validation tests and also good Western blot conditions. Skeletal muscle mitochondria consist of two distinct subfractions, the subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, located beneath the sarcolemma and between the myofibrils, respectively. These two mitochond- rial populations possess different characteristics, such as higher cardiolipin content and more elevated state-3 respir- ation rate in IMF mitochondria [10]. In this study, we validated an antibody to UCP3 using a fully controlled Western blot technique and present a comparative study in mice of UCP3 protein expression in SS and IMF mitochondria of various muscle types. We also examined UCP3 protein expression in the two mitochon- dria populations of fed and fasted mouse gastrocnemius muscle. Correspondence to P. Muzzin, De ´ partement de Biochimie Me ´ dicale, Centre Me ´ dical Universitaire, 1 rue Michel Servet, CH-1211 Gene ` ve 4 Switzerland. Fax: 41 22 702 5502, Tel.: 41 22 702 5492, E-mail: Patrick.Muzzin@medecine.unige.ch. Abbreviations: UCP, uncoupling protein; SS, subsarcolemmal; IMF, intermyofibrillar; COX, cytochrome oxidase; UCP3KO, UCP3 knockout. (Received 17 January 2002, revised 12 April 2002, accepted 23 April 2002) Eur. J. Biochem. 269, 2878–2884 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02953.x MATERIALS AND METHODS Bio-Rad Protein Assay and nonfat dry milk were purchased from Bio-Rad Laboratories (Hercules, CA, USA). ECL kit Hyperfilm ECL and Coomassie blue (PhastGelÒ BlueR) were obtained from Amersham International Biotech (Amersham, Bucks, UK), the antibody to human UCP3 C-terminus (CabrX) from Research Diagnostics, Inc (San Antonio, LA, USA) and the monoclonal antibody to the cytochrome oxidase (COX) subunit IV were from Molecu- lar Probes (Eugene, OR, USA). Goat anti-rabbit and anti- mouse immunoglobulins were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Trizol reagent was from Life Technologies (Basel, Switzerland). The protease inhibitor cocktail was purchased from Sigma (St Louis, MO, USA). Animals Three-month-old female C57BL/6J mice fed ad libitum a standard laboratory chow and maintained under a 12-h light/dark cycle at 23 °C were used. All animals were caged individually during the experimental periods. Mice were either fed ad libitum orfastedforaperiodof24hwithfree access to water. The animals were killed by cervical dislocation, and tibialis anterior, gastrocnemius and soleus muscles were carefully dissected and kept on ice. In fasted animals and the respective controls, one gastrocnemius muscle was used for the mitochondria preparation and the other for RNA isolation. All experiments were performed in accordance with the Office Ve ´ te ´ rinaire de Gene ` ve author- ization covering animal experiments. Preparation of muscle mitochondria SS and IMF mitochondria were prepared from skeletal muscle by the following procedure. Muscle (50–250 mg) was minced with scissors in 5 mL ice-cold homogenization buffer containing 100 m M sucrose, 180 m M KCl, 10 m M EDTA, 5 m M MgCl 2 ,1m M ATP, 50 m M Tris/HCl, pH 7.4, and 0.06% protease inhibitor cocktail. They were then homogenized using a Teflon pestle in an ice-cold glass Potter–Elvehjem homogenizer (clearance 0.37 mm, 10 up and down strokes, 1800 r.p.m.; clearance 0.12 mm, 2 up and down strokes, 1800 r.p.m.). The homogenate was centri- fuged at 1600 g for 10 min at 4 °C. The pellet was kept at 4 °C for the extraction of IMF mitochondria. The super- natant was filtered through two layers of surgical gauze and then centrifuged at 9200 g for 10 min at 4 °C. The resulting SS mitochondria pellet was suspended in the appropriate volume of distilled water. Being located just beneath the sarcolemmal membrane, the SS mitochondria are easier to extract from the muscle by homogenization than IMF mitochondria. In a first attempt to prepare IMF mitochon- dria, we used the original technique of Krieger et al. [11], which involves digestion by the Nagarse protease. The latter, even when tightly controlled, was found by Western blot analysis to degrade UCP3. Therefore we adopted another technique in which we broke down myofibrils by strong mechanical disruption with a tight-fitting homoge- nizer. The 1600 g pellet was resuspended in 4 mL ice-cold homogenization buffer containing 100 m M KCl, 1 m M EDTA, 5 m M MgSO 4 ,1m M ATP, 50 m M Tris/HCl, pH 7.4, and 0.06% protease inhibitor cocktail using a Teflon pestle in an ice-cold glass Elvehjem homogenizer (clearance 0.12 mm for 3 min, 1800 rpm). The resulting homogenate was centrifuged at 1600 g for 10 min at 4 °C. After filtration through two layers of surgical gauze, the supernatant was centrifuged at 15 000 g for 45 min at 4 °C, and the resulting IMF mitochondrial pellet was resuspended in an appropriate volume of distilled water. Mitochondrial protein concentrations were determined as described by Bradford [12] using the Bio-Rad Protein Assay, with BSA as a standard. Isolated mitochondria were stored at )20 °Cas 15-lg mitochondrial protein aliquots. Western blotting Purified muscle mitochondria (15 lg) were dried under vacuum and resuspended in 10 lL loading buffer contain- ing 50% glycerol, 5% SDS, 2.5% bromophenol blue and 0.5 M Tris/HCl, pH 6.8. The samples were electrophoresed on a 12% polyacrylamide/0.1% SDS gel, and transferred to a poly(vinylidene difluoride) membrane by electroblotting transfer with a buffer containing 10% methanol, 25 m M Tris/HCl, pH 6.8, and 190 m M glycine. The transfer was performed for 15 h at 30 V, 70 mA and 4 °C. After blotting, the gel was stained with 0.1% Coomassie blue to check transfer efficiency. No band was visible below 50 kDa on the gel. The membrane was blocked with a NaCl/P i buffer containing 0.1% Tween and 2% nonfat dry milk. This same buffer was used for all subsequent hybridizations. UCP3 protein was detected using CabrX at a concentration of 1 lgÆmL )1 . The membrane was washed twice in NaCl/P i containing 0.1% Tween and hybridized with a 1 : 1000 diluted goat anti-rabbit peroxidase-labeled secondary anti- body. The signals were detected by chemiluminescence using a standard ECL kit and developed Hyperfilm ECL film. They were quantified by scanning photodensitometry using ImageQuant Software 3.3 (Molecular Dynamics, Sunnyvale, CA, USA). COX protein was detected as above using a 1 : 1000 diluted monoclonal antibody specific for COX subunit IV and a 1 : 1000 diluted goat anti-mouse peroxidase-labeled secondary antibody. To compare the UCP3 signals, linear standard curves were constructed using increasing concentrations of the human and mouse UCP3 recombinant proteins provided by Dr Michele Chiesi at Novartis (Basel, Switzerland) and Stratagene (La Jolla, CA, USA), respectively. The specificity of the antibody to UCP3 was tested using UCP3KO muscle mitochondria, which were described by Gong et al. [7] and generously provided by Dr Mary Ellen Harper (University of Ottawa, Ottawa, Ontario, Canada). The replication-defective recombinant adenoviral vector that contains the human UCP3 cDNA under the transcriptional control of the cytomegalovirus promoter was constructed as previously described [13]. Real-time quantitative RT-PCR Total muscle RNA was isolated using the Trizol reagent technique according to the manufacturer’s instructions. Oligo-dT first-strand cDNA was synthesized from 2 lg total RNA using Superscript II reverse transcriptase. Real-time PCR was performed using a Lightcycler rapid thermal cycler system and designated software (Roche Diagnostics Ltd, Rotkreuz, Switzerland) according to the Ó FEBS 2002 UCP3 protein quantitation (Eur. J. Biochem. 269) 2879 manufacturer’s instructions. Reactions were performed in a 20-lL reaction mixture containing 50 ngÆlL )1 of first- strand cDNA, 0.5 l M primers and 2.4 m M MgCl 2 .Nucleo- tides, Taq DNA polymerase, and buffer were included in the Lightcycler-DNA Master SYB Green I mix (Roche Diag- nostics). The PCR protocol consisted of 2 min of denatur- ing at 95 °C, followed by 30 cycles with 95 °Cdenaturing for 1 s, 56 °C annealing for 5 s and 72 °C extension for 16 s. The fluorescent product was detected at the end of the 72 °C extension period. To confirm the amplification specificity, the PCR product was subjected to a melting curve analysis and then agarose gel electrophoresis. A linear standard curve was constructed using known concentra- tions of a mouse UCP3 plasmid. The 10-fold serial dilutions ranged between 3.0 ngÆlL )1 and 3.0 pgÆlL )1 . The concen- trations of the experimental samples were calculated by comparison with the standard curves. Background fluores- cence was removed by setting a noise band. The number of cycles at which the best-fit line through the log-linear portion of each amplified curve intersected the noise band was inversely proportional to the log copy number [14]. The samples were also normalized against b-actin using the same conditions as described above. Northern blot analysis Total RNA from gastrocnemius muscle was isolated using the Trizol reagent technique. Total RNA (20 lg) was separated on a 1.2% agarose/formaldehyde gel and trans- ferred to nylon membrane as described by Boss et al.[16]. To detect UCP3 mRNA, we used a probe derived from a full-length rat UCP3 cDNA [15]. The probe was labeled by random priming with [a- 32 P]dCTP (Amersham). Hybridization and washing were carried out as previously reported [16]. Blots were exposed to Hyperfilm ECL films (Amersham) at )80 °C with intensifying screens. The signals on the autoradiograms were quantified by scanning photodensitometry using ImageQuant Software version 3.3. Hybridization of the blots with a [c- 32 P]ATP-labeled synthetic oligonucleotide specific for the 18S rRNA subunit was used to correct for differences in the amounts of RNA loaded on to the gel. Student’s unpaired t test was used to determine statistical significance. RESULTS Validation of antibodies to UCP3 Table 1 shows a list of antibodies that have been used for the Western blot analysis of UCP3 protein expression in rodents. The studies performed on UCP3KO or transgenic mice overexpressing UCP3 in their skeletal muscle provide convincing validation of the antibodies used. The Lilly antibody to mouse and rat UCP3 [6] and the Chemicon antibody to human UCP3 (AB3046) [7] were found to react specifically with mouse UCP3, as the signal observed in the wild-type animal was found to be abolished in UCP3KO mouse muscle. The a-Diagnostic antibody to human UCP3 (UCP32-A) was found to cross-react specifically with human UCP3 as validated in a UCP3 transgenic mouse model [8]. In the other studies in Table 1, in which the possible modulations of UCP3 at the protein level were analysed, validated Lilly, Chemicon and a-Diagnostic antibodies were used [17–21]. Pedraza et al.[22]usedan antibody to human UCP3 from a-Diagnostic (UCP31-A) which was not validated in UCP3 transgenic mice but using human UCP3 transfected cells. In this study, we used a new antibody to human UCP3 C-terminus from Research Diagnostics (CabrX) and devel- oped a Western-blot technique that optimizes protein transfer. We obtained a 34-kDa UCP3 signal that was validated using a UCP3KO mouse model, adenovirus human UCP3-transfected cells and recombinant UCP3. In our hands, the CabrX antibody showed higher sensitivity than the other commercial antibodies listed in Table 1. Table 1. Western blot analysis of rodent muscle mitochondria with UCP3 antibodies. TM3, 3rd transmembrane domain; TM4, 4th transmembrane domain; h, human. Reference Antibody name (supplier) Transgenic animals Transfected cells Species (muscle mitochondria) Vidal-Puig et al. [6] Peptide sequence between TM3 and TM4 of mouse and rat UCP3 (Lilly) UCP3KO – Mouse muscle Gong et al. [7] C-Terminus of human UCP3, AB3046 (Chemicon) UCP3KO – Mouse muscle Cadenas et al. [17] C-Terminus of human UCP3, AB3046 (Chemicon) – hUCP3/HEK293 Rat muscle (starvation) Zhou et al. [19] C-Terminus of human UCP3, AB3046 (Chemicon) – – Rat muscle (exercise, hypoxia, AMPkinase activation) Clapham et al. [8] C-Terminus of human UCP3, UCP32-A (a-Diagnostic) hUCP3Tg – Mouse muscle Sivitz et al. [18] C-Terminus of human UCP3, UCP32-A (a-Diagnostic) – – Rat muscle (fasting, leptin) Jucker et al. [20,21] C-Terminus of human UCP3, UCP32-A (a-Diagnostic) – – Rat muscle (fasting, T3) Pedraza et al. [22] Peptide sequence between TM2 and TM3 of human UCP3, UCP31-A (a-Diagnostic) – hUCP3/HEK293 Mouse muscle (lactation) 2880 M. Jimenez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Muscle mitochondria prepared from UCP3KO mice [7] were compared with wild-type mitochondria. As shown in Fig. 1A lanes 1 and 2, the strong signal observed in wild- type mouse mitochondria was absent from those of UCP3KO mice. Figure 1A (lane 3) shows that the CabrX antibody reacts with the mouse recombinant protein. The size of the signal is higher than 34 kDa because of the presence of a His 6 tag in the mouse recombinant protein. C 2 C 12 cells, which do not express UCP3, were infected with an adenovirus containing the human UCP3 gene. As shown in Fig. 1B, no signal was observed in the wild-type C 2 C 12 cells whereas the human UCP3 was detected, at the expected size of 34 kDa, in the infected cells and in a sample of the human UCP3 recombinant protein. Taken together these results demonstrate that the 34-kDa signal observed in our Western blots is due to a specific interaction between the CabrX antibody and mouse or human UCP3. Figure 2A shows that the UCP3 signal of human recombinant protein interacting with the antibody to human UCP3 increases linearly as a function of increasing amounts of the protein over the relatively large range 5–30 ng. Figure 2B shows data from a similar experiment performed with mouse recombinant protein. Representative signals obtained at different concentrations of recombinant protein are shown under the figures. The times of exposure werethesameforFig2AandB. The reproducibility of the Western blot quantification was analyzed using mouse gastrocnemius and tibialis muscle mitochondria. The mean variation between quadruplicates for four different samples was 27 ± 7% and 20 ± 6% for UCP3 and COX, respectively. It should be stressed that larger and unpredictable variations were observed when values obtained with a given sample on two different gels were compared. Therefore we only compared values obtained on the same gel for all our subsequent quantitative studies. Preparation of SS and IMF mitochondria We developed a technique using selective conditions of mechanical disruption to prepare SS and IMF mitochon- dria. As shown in Table 2, the quantity of IMF mitochon- dria recovered from 1 g gastrocnemius muscle was 1.7-fold higher than SS mitochondria. The specific and total levels of COX protein were not significantly different in IMF and SS mitochondria. The yield in mitochondria, which was determined by comparing the level of COX protein in the sum of the two mitochondria populations with that in the homogenate, was 88%. Expression of UCP3 protein in SS and IMF mitochondria of various muscles Figure 3A illustrates the distribution of UCP3 in SS and IMF mitochondria obtained from different types of mouse muscle, i.e. tibialis anterior (two-thirds fast oxidative glycolytic, one-third glycolytic), gastrocnemius (one-third slow oxidative, one-third fast oxidative glycolytic, one-third fast glycolytic) and soleus (90% slow oxidative). It can be seen in Fig. 3A that the UCP3 protein levels in SS mitochondria (expressed as arbitrary units per mg mitoch- ondrial protein) are higher in the tibialis anterior and gastrocnemius than in the soleus (1.4-fold and 1.7-fold, respectively). The levels of UCP3 in IMF mitochondria are also higher in the tibialis anterior and gastrocnemius muscles than in the soleus muscle (2.2-fold and 1.8-fold, respectively). UCP3 is expressed at a significantly lower level in IMF than SS mitochondria in the three types of muscle (by 37%, 58% and 46% in tibialis anterior, gastrocnemius and soleus muscle, respectively). No difference was observed in the level of COX per mg of mitochondrial protein in the three muscle types and in IMF vs. SS mitochondria, except Fig. 2. Increase in the UCP3 signal as a function of increasing amounts of (A) human recombinant protein and (B) mouse recombinant protein. Representative signals are shown under the figures. Fig. 1. Western blot analysis. (A) Western-blot signals obtained with 20 lg mitochondria isolated from wild-type (lane 1) or UCP3KO (lane 2) mouse gastrocnemius. Lane 3, 20 ng mouse recombinant UCP3. (B) 20 lg homogenate from C 2 C 12 wild-type myoblasts (lane 1) and C 2 C 12 myoblasts infected with adenoviruses containing the human UCP3 gene (lane 2). Lane 3, 40 ng human recombinant UCP3. The immu- noblots were hybridized with CabrX (Anti-hUCP3) antibody. They were also hybridized with antibodies to COX (Anti-COX) and pro- hibitin. Representative signals are shown under the figures. Table 2. Recovery of SS and IMF mitochondria from gastrocnemius muscle. The results are expressed as means ± SEM from the number of experiments in parentheses. SS IMF Protein yield (mg protein per g muscle) 1.9 ± 0.2 (4) 3.2 ± 0.2 (3) a COX protein specific level (arbitrary units per mg protein) 131 ± 12 (4) 86 ± 26 (3) COX protein total level (arbitrary units per g muscle) 239 ± 14 (4) 265 ± 69 (3) a P < 0.01 in IMF vs. SS mitochondria. Ó FEBS 2002 UCP3 protein quantitation (Eur. J. Biochem. 269) 2881 for the gastrocnemius, where the level of COX is lower in IMF than SS mitochondria by 43% (Fig. 3B). As shown in Fig. 3C, the UCP3/COX ratio in SS mitochondria is higher in the gastrocnemius than in the soleus (1.5-fold) and in IMF mitochondria in the tibialis anterior and in the gastrocnemius than in the soleus (2.0-fold and 1.9-fold, respectively). In the gastrocnemius and soleus muscles, the UCP3/COX ratio is 37% and 41% lower, respectively, in IMF than SS mitochondria. This indicates that the compo- sitions of the two mitochondrial populations are different. UCP3 mRNA was determined by quantitative RT-PCR in the same muscles to allow a comparison between the respective UCP3 mRNA and protein levels. As shown in Fig. 4, UCP3 mRNA levels were higher in the tibialis anterior and gastrocnemius muscles than in the soleus muscle (1.7-fold and 2.1-fold, respectively). Therefore the relative amounts of UCP3 protein and mRNA in the three muscles are comparable. Effect of fasting on UCP3 protein expression We and others have shown that fasting induces upregula- tion of UCP3 mRNA expression in skeletal muscle of mice and rats (for review see [5]). To study further the regulation of UCP3 in fasting, we measured the protein levels in SS and IMF mitochondria of gastrocnemius muscle in 24 h-fasted mice. Figure 5 illustrates the effects of 24-h fasting on the expression of muscle UCP3 and COX per mg of protein in both mitochondria populations. After a 24-h fast, the UCP3 protein level was increased 1.5-fold (P < 0.01) in SS mitochondria, whereas it was unaffected in IMF mitochon- dria. COX protein level was found to be unchanged by fasting. In five animals from each group of mice, we also Fig. 3. UCP3 protein levels (A) and COX protein levels (B) in mouse tibialis anterior (TA), gastrocnemius (Gn) and soleus (So) muscle SS (empty columns) and IMF (shaded columns) mitochondria (20 lg). (A) The immunoblots were hybridized with CabrX antibody. The signals were quantified by scanning photodensitometry and are presented as means ± SEM of absolute values, n ¼ 4–6. **P <0.02 and ***P < 0.001 vs. SS mitochondria; #P < 0.05 vs. tibialis anterior values; °P <0.05 and°°°P < 0.005 vs. gastrocnemius values. (B) Same as in (A) except that the immunoblots were hybridized with a COX antibody. ***P < 0.005 vs. SS mitochondria. (C) UCP3 values normalized using the corresponding COX values. *P <0.05 and ***P < 0.005 vs. SS mitochondria; #P < 0.05 vs. tibialis anterior values; °°°P < 0.005 vs. gastrocnemius values. Fig. 4. UCP3 mRNA levels in mouse tibialis anterior (TA), gastroc- nemius (Gn) and soleus (So) muscle. The results, obtained by real-time quantitative RT-PCR as described in Materials and Methods, are presented as means ± SEM of values normalized using actin. *P < 0.05 vs. gastrocnemius values. 2882 M. Jimenez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 determined the UCP3 mRNA levels in gastrocnemius muscle. We observed that fasting induced a 16.4-fold (P < 0.001) increase in UCP3 mRNA expression, whereas it augmented the total amount of UCP3 protein by 1.5-fold in the gastrocnemius from the same animals. DISCUSSION This study first validates a Western blot technique for the detection and quantitation of UCP3 using a specific and sensitive antibody raised against 14 amino acids located at the C-terminus of human UCP3 protein (CabrX). The use of both mitochondria from knockout mouse gastrocnemius and homogenates of C 2 C 12 myoblasts infected with an adenovirus encoding for human UCP3 allowed clear validation of our Western blot signal. Good cross-reactivity of the CabrX antibody with mouse recombinant UCP3 was also demonstrated. The signals can be quantitated and compared with a reasonable degree of accuracy, but only if obtained on the same gel. The density of UCP3 protein (i.e. the intensity of the signal expressed per mg of proteins) was similar in the SS mitochondria of the two glycolytic muscles, tibialis anterior and gastrocnemius, and higher than that in the oxidative muscle soleus. The same was observed with IMF mito- chondria. It is noteworthy that identical results were obtained in this study when the levels of UCP3 mRNA were measured in the tibialis anterior, gastrocnemius and soleus muscles. Hesselink et al. [23], who studied the distribution of UCP3 protein in various human muscle types by immunofluorescence, showed a higher expression of UCP3 protein in glycolytic than oxidative fibers of vastus lateralis muscle. Our results show that these findings cannot be extended to rodent muscles. If indeed the UCP3 protein was more abundantly expressed in glycolytic than oxidative muscles, its density would be higher in the tibialis anterior than in the gastrocnemius. The density of COX showed a tendency to be lower in IMF than SS mitochondria, the difference being significant only in the gastrocnemius muscle. In rats, muscle IMF mitochondria have been shown to display higher state-III respiration [10,11] and slightly higher COX and lower succinate dehydrogenase activities [10] than SS mitochon- dria. The results of our study, showing that the COX protein level tended to be lower in IMF than SS mitochon- dria is not in total agreement with these results. When the density of UCP3 was compared in SS and IMF mitochondria, it was found to be higher in SS mitochondria of all three muscle types studied. Our UCP3 data show that (a) the muscle type that relies most on oxidative phosphorylation for ATP synthesis, i.e. the soleus muscle, contains less UCP3 per mg of mitoch- ondrial protein than glycolytic muscle types and (b) in all muscle types the mitochondria most closely associated with the myofibrills, i.e. the IMF mitochondria, have a lower UCP3 density. If UCP3 is an uncoupling protein, the oxidative muscle (soleus) and the IMF mitochondria (which should be most involved in muscle contraction) would be less prone to uncoupling and therefore more efficient at producing ATP. In a recent paper on pig muscle SS and IMF mitochondria, Lombardi et al. [24] showed that IMF mitochondria had a higher capacity for ATP production than SS. The possible role of the lower level of UCP3 in this difference would be interesting to study. Most studies on regulation of UCP3 expression have investigated changes in UCP3 mRNA levels. Fasting has repeatedly been shown to dramatically increase muscle UCP3 mRNA in rats and mice [5]. This is surprising because UCP3, which has been shown to exhibit uncoupling activity, would be expected to be turned off in muscle under conditions that dictate energy sparing such as starvation. In this study, a 24-h fasting period, which was shown to increase gastrocnemius muscle UCP3 mRNA level 16.4- fold, was found to induce an increase of 1.5-fold in the UCP3 protein level of gastrocnemius muscle from the same animal. Consistent with these observations, Cadenas et al. [17] and Sivitz et al. [18] reported increases in rat UCP3 protein induced by fasting that were less than half those of UCP3 mRNA level in experiments carried out in parallel. These results are in agreement with studies reporting no change in mitochondrial proton conductance [17] and energy coupling [21] in muscle of starved rats. Thus, UCP3 protein level rather than mRNA expression would be appear to be an indication of UCP3 activity in rodent muscle. The observation of a marked increase in UCP3 Fig. 5. Effect of a 24-h fast on (A) UCP3 protein levels and (B) COX protein levels in mouse gastrocnemius SS (empty columns) and IMF (shaded columns) mitochondria (20 lg). The methods for the detection and quantitation of UCP3 and COX are described in the legend of Fig. 3. Data are presented as means ± SEM, n ¼ 9–11. (A) ***P < 0.005 vs. SS mitochondria; ##P < 0.01 vs. respective mitochondrial population in muscle of fed mice. (B) **P <0.01and*P < 0.05 vs. SS mitochondria. Ó FEBS 2002 UCP3 protein quantitation (Eur. J. Biochem. 269) 2883 mRNA associated with only a minor change at the protein level during fasting suggests that UCP3 expression is regulated at the post-transcriptional level and therefore that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level. We can speculate that the increase in UCP3 mRNA produces an RNA pool ready to be rapidly translated after the initiation of refeeding. This point will be addressed in future studies. The present findings put into perspective the so-called Ôfasting paradoxÕ, suggesting that UCP3 activity might not be modified in the muscle during fasting. Furthermore our data show that the two mitochondria populations are differently affected by metabolic changes. Further studies on the regulation of UCP3 protein expres- sion may have important implications for the understanding of its physiological role. In conclusion, in this study, using a carefully validated antibody to UCP3 and a new protocol to prepare SS and IMF mitochondria, UCP3 was found at higher density in mitochondria of glycolytic muscle than in those of oxidative muscle. Our data also indicate that SS mitochondria contain more UCP3 than IMF mitochondria, raising the possibility that these organelles have different capacities for oxidative ATP production and a moderate increase in UCP3 protein content in SS mitochondria of fasted mice. ACKNOWLEDGEMENTS This work was supported by grants from the Swiss National Science Foundation no. 31-53707.98 and 31-54306.98. We are indebted to the Office Fe ´ de ´ ral du Sport Macolin, the Fonds Euge ` ne Rapin, the Fondation du Centenaire de la socie ´ te ´ Suisse d’Assurances ge ´ ne ´ rales sur la vie humaine pour la sante ´ publique et les recherches me ´ dicales and the Roche Research Foundation. REFERENCES 1. Cannon, B. & Nedergaard, J. 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Keywords: fasting; intermyofibrillar mitochondria; muscle type; subsacorlemmal; uncoupling protein-3 (UCP3). The first uncoupling protein described, uncoupling