Effect of endurance training on mRNA expression of uncoupling proteins 1, 2, and 3 in the rat

^a Department of Medical Biochemistry, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland
^b Department of Physiology, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland
^c UMR 5578 CNRS University Lyon 1, 69373 Lyon Cedex 08, France

	ABSTRACT

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Endurance exercise training has been shown to decrease diet-induced thermogenesis (DIT) in rats and humans. In rodents, most thermogenesis is thought to occur in brown adipose tissue via activation of the uncoupling protein-1 (UCP1) and in skeletal muscle. Since the level of UCP1 mRNA in rat BAT was reported to be unmodified by exercise training, the newly described uncoupling proteins UCP2 and UCP3 could be responsible for the decreased DIT in trained rats. UCP3 mRNA levels in endurance-trained rats were found to be reduced by 76% and 59% in tibialis anterior and soleus muscles, respectively. UCP2 mRNA levels were also decreased in tibialis anterior and in heart by 54% and 41%, respectively. Neither white adipose tissue UCP2 nor brown adipose tissue UCP1, UCP2, and UCP3 mRNA levels were modified. The results of this study show that a need for a higher metabolic efficiency is associated with decreased mRNA expression of the uncoupling proteins in skeletal and heart muscles, which would decrease energy dissipation in these tissues. The down-regulation of UCP3 and UCP2 expressions might also contribute to the rapid weight gain known to occur when exercise training ceased.—Boss, O. Samec, S., Desplanches, D., Mayet, M.-H., Seydoux, J., Muzzin, P., Giacobino, J.-P. Effect of endurance training on mRNA expression of uncoupling proteins 1, 2, and 3 in the rat. FASEB J. 12, 335–339 (1998)

Key Words: exercise training • uncoupling protein • skeletal muscle • heart • adipose tissue

	INTRODUCTION

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THE UNCOUPLING PROTEIN-1 (UCP1)² gene encodes a mitochondrial protein carrier that stimulates heat production by uncoupling respiration from ATP synthesis in brown adipose tissue (BAT), and thus plays an important role in nonshivering thermogenesis in rodents (1). Recently, new proteins sharing 55% and 56% amino acid identities with UCP1 have been identified in humans and called UCP2 and UCP3 (2, 3). Tissue distribution of UCP2 and UCP3 is very different from that of UCP1, which is BAT-specific and expressed either ubiquitously for UCP2 or specifically in skeletal muscle for UCP3 (2, 3). In rodents, UCP3 was also expressed in BAT (3). UCP2 was shown to function as an uncoupling protein in transfected yeast, where it partially uncoupled mitochondrial respiration (2). Preliminary results obtained in transfected C₂C₁₂ myoblasts indicate that UCP3 also uncouples mitochondrial respiration (unpublished results). In rat BAT, the mRNA expression of UCP2 was found to be increased by cold exposure and not modified by fasting, whereas in skeletal muscle it was markedly increased by fasting (4).

Rat UCP3 cDNA was first cloned from a rat skeletal muscle cDNA library and sequenced to determine the amino acid sequence of its corresponding protein. Skeletal muscle is an important site of catecholamine and diet-induced thermogenesis (DIT) in rats (5) and humans (6, 7). If UCP3 plays a major role in whole body thermogenesis, down-regulation of its expression is to be expected in endurance-trained rats, where it has been shown that DIT is reduced (8). Similarly, a decreased meal-induced thermogenesis was reported in endurance athletes (9).

The aim of the present study was therefore to investigate the effects of an 8-wk endurance training program in rats on mRNA expression of UCP3 and UCP2 in two types of skeletal muscles—tibialis anterior (TA), which is composed of types IIa and IIb fast-twitch fibers, and soleus (SO), which is composed mainly of type I slow-twitch fibers—and in the heart. We also studied mRNA expression of UCP1, UCP2, and UCP3 in BAT and of UCP2 in white adipose tissue (WAT), which, as a lipid storage tissue, might be strongly solicited during endurance exercise.

The present study describes the sequence of rat UCP3 cDNA, the amino acid sequence of the corresponding protein, as well as the effects of an 8-wk endurance training program in rats on mRNA expression of UCP3 and UCP2 in skeletal muscle and heart, of UCP1, UCP2, and UCP3 in BAT, and of UCP2 in WAT.

	MATERIALS AND METHODS

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Materials
Chemicals were purchased from Merck (Darmstadt, Germany), Sigma (St. Louis, Mo.), or Fluka (Buchs, Switzerland). Hybond N Nylon membranes, [-³²P]dCTP (3000 Ci/mmol), [-³²P]ATP (3000 Ci/mmol), Megaprime DNA labeling system, and Hyperfilm ECL films were from Amersham (Bucks, U.K.). Reverse transcription-polymerase chain reaction (RT-PCR) reagents and 0.24–9.5 kb RNA Ladder were from Gibco BRL (New York, N.Y.). SeaKem GTG agarose was from FMC BioProducts (Rockland, Maine). QuikHyb Rapid Hybridization Solution and sonicated salmon sperm DNA were from Stratagene (La Jolla, Calif.). Sephadex G-50 NICK Columns were from Pharmacia (Uppsala, Sweden).

Library screening
The rat atypical UCP cDNA probe used was obtained from rat TA muscle as described previously (3). Briefly, total RNA was amplified by RT-PCR, using oligonucleotide primers corresponding to nucleotides 279–298 (UCPRF) and 1021–1044 (UCPRR) on rat UCP1 cDNA (GenBank Accession M11814). This rat atypical UCP cDNA was used as a probe to screen a rat skeletal muscle cDNA library (Stratagene, #937510, La Jolla, Calif.).

Clone sequencing
cDNA clones were purified by Qiagen plasmid kit according to the manufacturer's instructions (Quiagen, Santa Clarita, Calif.) and sequenced by using standard protocols for the ABI373A automated sequencer with M13–20 and triiodothyronine (T3) primers and gene-specific primers until the sequence was determined on both strands.

Animals and training
Twenty-four male pathogen-free OFA rats (110 g) were housed in a temperature-controlled room (22±2°C) with a light-dark cycle (12:12 h) and fed ad libitum Purina laboratory chow. After 1 wk, animals were randomly assigned to either a sedentary group (n=12) or a training group (n=12). During the treadmill training program, rats initially ran for 10 min at 30 m/min on a 10% incline. The duration of exercise was then progressively increased by 5 min per day so that, by the end of the fourth week the animals were running for 90 min at 30 m/min up a 10% slope, 5 days/wk, as described previously (10). The rats were maintained at this level of training for another 4 wk. At the end of the experiment, the mean body weight was 414 ± 5 g for the trained and 393 ± 14 g for the sedentary rats.

RNA isolation and Northern blot analysis
The rats were killed by decapitation 24 to 30 h after the last exercise bout. Tissues were excised, immediately frozen in liquid nitrogen, and stored at -80°C. TA, SO, heart, interscapsular BAT, and epididymal WAT of four or five randomly chosen rats from each group were used for RNA preparation. Tissues were homogenized in 4 M guanidium thiocyanate and total RNA was isolated by the method of Chomczynski and Sacci (11). Total RNA (20 µg) was electrophoresed in a 1.2% agarose gel containing formaldehyde, as described by Lehrach et al. (12), and Northern blots were performed as described previously (4). Size estimates for the RNA species were established by comparison with an RNA ladder. The amount of RNA in the signals on the autoradiograms was quantified by scanning photodensitometry. Only signals obtained on the same Northern blot were compared. Hybridizations of the total RNA blots with a [-³²P]ATP-labeled synthetic oligonucleotide specific for the 18S rRNA subunit were performed to ensure that equivalent amounts of RNA were loaded onto the gel.

Enzyme activities
Two skeletal muscles involved in running (TA and plantaris) were chosen. Muscle samples (10 mg) were homogenized at 4°C in 0.3 M phosphate buffer containing 0.05% bovine serum albumin (pH 7.7) with a glass Potter-Elvehjem homogenizer. The samples were frozen at -80°C and thawed three times to disrupt the mitochondrial membranes. 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activity was determined fluorometrically as described by Lowry and Passonneau (13). Citrate synthase (EC 4.1.3.7) was measured spectrophotometrically according to Srere (14).

cDNA probes
The rat UCP1 cDNA probe of 766 bp was obtained by RT-PCR on rat BAT total RNA using oligonucleotide primers corresponding to nucleotides 279–298 (UCPRF) and 1021–1044 (UCPRR) on rat UCP1 cDNA (GenBank Accession M11814). The human UCP2 cDNA probe of 772 bp was obtained by PCR from cloned human UCP2 cDNA (GenBank Accession U82819) (3), using oligonucleotide primers corresponding to nucleotides 188–207 and 936–959. The rat UCP3 cDNA probe of 769 bp was obtained by PCR from cloned rat UCP3 cDNA (GenBank Accession U92069), using oligonucleotide primers UCPRF and UCPRR.

Statistics
The individual mRNA levels were expressed as a ratio to the mean of the control group. Student's unpaired t test was used to determine statistical significance, which was accepted at the P < 0.05 level.

	RESULTS

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Screening of ca. 1 million phages from the rat skeletal muscle cDNA library with a rat atypical UCP cDNA probe (3) resulted in the isolation of three positive clones, all with the same sequence. The predicted polypeptide sequence contains 308 amino acids and is illustrated in Fig. 1. It has amino acid identities of 57% and 86% to rat UCP1 (15) and to the recently isolated human UCP3 (3), respectively. This protein is therefore the rat homologue of human UCP3, and thus was called rat UCP3 (GenBank Accession U92069). Like other mitochondrial carriers, UCP3 contains six predicted transmembrane domains and three mitochondrial energy–transfer–protein signature domains (16). The potential purine nucleotide binding domain extends between amino acid residues 275 and 297 by homology with rat UCP1 (SWISS-PROT P04633) ( Fig. 1).

View larger version (37K): [in this window] [in a new window]   Figure 1. Amino acid sequence alignment of human UCP3 (GenBank Accession U84763) and rat UCP3 (GenBank Accession U92069) obtained with the ClustalW Multiple Sequence Alignment program (K. C. Worley, Human Genome Center, Baylor College of Medicine). The sequences are presented in single-letter code. Gaps introduced into the sequences to optimize alignments are illustrated by a dash. Identical amino acids are shaded. Potential transmembrane -helices are underlined and numbered in Roman numerals (I–VI). The potential purine-nucleotide binding domain (PNBD) is underlined twice. The three mitochondrial energy–transfer–protein signature domains (16) are boxed.

The endurance training program used in this study has previously been shown to increase the activity of skeletal muscle oxidative enzymes such as citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) (10). Similar results were obtained in this study: in TA and in plantaris, two muscles involved in running, the activity of citrate synthase was increased by 39% (P<0.05) and 31% (P<0.05), respectively, and that of 3-hydroxyacyl-CoA dehydrogenase was increased by 92% (P<0.001) and 34% (P<0.05), respectively. These results confirm that the training program used here was effective.

The effects of training on mRNA expression of UCP3 and UCP2 in TA and SO muscles as well as in the heart were studied. As shown in Fig. 2, in exercise-trained rats killed 24–30 h after the last exercise bout, UCP3 mRNA expression was decreased by 76% and 59% in TA and SO, respectively, as compared to control sedentary animals. UCP2 mRNA expression in exercise-trained rats was decreased in TA and the heart by 54% and 41%, respectively, but not significantly changed in SO muscle. Due to its very low abundance in the heart (3), UCP3 mRNA levels in trained rats could not be quantified accurately. The effects of training on WAT and BAT were also investigated. As shown in Fig. 3, in exercise-trained rats neither UCP2 mRNA expression in WAT nor UCP1, UCP2, and UCP3 mRNA expressions in BAT were changed.

View larger version (36K): [in this window] [in a new window]   Figure 2. Endurance training decreases UCP3 and UCP2 expression in skeletal muscle and heart. A) Expression levels of UCP2 and UCP3 mRNA in tibialis anterior (TA) and soleus muscles and the heart of control (empty columns) and endurance-trained (hatched columns) rats. The three tissues were obtained from the same rats. Photodensitometric comparison of signals obtained from total RNA hybridized with a [32P]-labeled human UCP2 or rat UCP3 probe, as described in Materials and Methods. The results are expressed as percentages ± SE of the mean respective control value taken as 100%. The number of experiments was four or five. The main signals (1.7 kb for UCP2 and 2.5 kb for UCP3) were quantified by scanning photodensitometry and normalized using the corresponding 18S rRNA values. *P < 0.03, ***P < 0.005 vs. control. B) Representative UCP2, UCP3 mRNA, and 18S rRNA signals under the various conditions studied. Duration of exposure of the Northern blots hybridized with UCP probes was between 4 and 50 h.

View larger version (41K): [in this window] [in a new window]   Figure 3. Endurance training does not change the expressions of UCP1, UCP2, and UCP3 in adipose tissues. A) Expression levels of UCP2 mRNA in white adipose tissue (WAT) and of UCP1, UCP2, and UCP3 mRNA in brown adipose tissue (BAT) of control (empty columns) and endurance-trained (hatched columns) rats. Photodensitometric comparison of signals obtained from total RNA hybridized with a [32P]-labeled rat UCP1, human UCP2, or rat UCP3 probe, as described in Materials and Methods. The results are expressed as percentages ±SE of the mean respective control value taken as 100%. Four or five experiments were performed. The main signals (1.4 kb for UCP1, 1.7 kb for UCP2, and 2.5 kb for UCP3) were quantified by scanning photodensitometry and normalized using the corresponding 18S rRNA values. All differences are nonsignificant. B) Representative UCP1, UCP2, UCP3 mRNA, and 18S rRNA signals under the various conditions studied. Duration of exposure of the Northern blots hybridized with the UCP probes was between 4 and 50 h.

	DISCUSSION

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The UCP3 sequence is very conserved between species, as shown by the 86% amino acid identity between rat and human UCP3 sequences. Furthermore, three main UCP features—six potential transmembrane domains, three mitochondrial energy-transfer-protein signature domains, and a potential purine nucleotide binding domain—are shared by both rat and human UCP3.

This study describes a new effect of endurance training on skeletal muscle. It has been shown that after a prolonged endurance exercise bout, skeletal muscle glycogen returns to its pre-exercise level within 24 h (17). Levels of UCP3 and UCP2 mRNA were measured in 8-wk endurance-trained rat TA and SO muscles after recovery from the last exercise bout. The results show that endurance training reduces considerably UCP3 and UCP2 mRNA equilibrium levels in both TA and SO muscles. A decreased expression of uncoupling proteins in skeletal muscle would allow for a higher level of metabolic efficiency of this tissue, which in turn would favor energy storage during the recovery phase and allow a higher capacity to perform mechanical work during subsequent exercise.

The greater effect of training observed on TA (types IIa and IIb, fast-twitch fibers) than on SO (type I, slow-twitch fibers) UCP2 and UCP3 mRNA suggests that muscles depending more on glycolysis than on fatty acid oxidation for their ATP supply gain more energetic efficiency upon training. The effect of training on heart UCP2 mRNA expression suggests a role for this protein in heart metabolic efficiency.

Neither WAT nor BAT UCP mRNA expression was modified by endurance training. The latter finding agrees with a previous study that shows a lack of effect of training on the level of UCP1 mRNA in BAT (18). The effect of exercise training on mRNA expression of UCPs is specific to skeletal muscle and heart, tissues in which the metabolic rate is chronically increased by repeated exercise bouts. In these tissues, an increased metabolic efficiency should be beneficial to physical performance. The absence of effect in BAT suggests that endurance exercise training does not lead to chronic sympathetic stimulation in this tissue. The absence of effect in WAT suggests that the increased turnover of triglycerides induced by endurance training in this tissue does not affect UCP2 mRNA expression.

We have previously shown that skeletal muscle UCP2 mRNA expression is increased in fasted rats, which was unexpected for a protein possibly involved in DIT (4). The present results suggest, however, that a decrease in the expression of both UCP2 and UCP3 could contribute to the decreased DIT levels in trained animals.

Endurance exercise training has been shown to result in decreased levels of plasma T3 in rats (9) and in humans (19). Very recently it has been shown that decreased levels of T3 in hypothyroid rats are associated with a decreased expression of UCP3 mRNA in skeletal muscle, but not in BAT (20). In our trained rats, plasma leptin levels as well as leptin mRNA expression in WAT were found to be significantly decreased by 25% (P<0.01) and 29% (P<0.001), respectively (unpublished results). It was also reported recently that leptin increases UCP3 mRNA expression in skeletal muscle of obese ob/ob mice (20).

These results suggest that the effects of exercise training observed in our study could be mediated by (among others) changes in circulating T3 and leptin levels. Down-regulation of skeletal muscle uncoupling proteins might contribute to the decrease observed in diet-induced thermogenesis (8) and to the apparent greater food efficiency (21, 22) induced by endurance training in rats and in human athletes.

	ACKNOWLEDGMENTS

 
O.B. was supported by a grant from the Swiss Institute of Sport Sciences. We acknowledge financial support from the Swiss National Science Foundation (grants No. 31–43405.95 and 31–47211.96). We thank Françoise Kuhne, Frédéric Preitner, and Martine Vollenweider for help in dissection, Colette Rossier for the sequencing, Frédéric Levasseur for precious help, and Françoise Assimacopoulos-Jeannet for fruitful discussions.

	FOOTNOTES

 
¹ Correspondence: Medical Biochemistry, Faculty of Medicine, 1 Michel Servet, 1211 Geneva 4, Switzerland. E-mail: Olivier.Boss{at}medecine.unige.ch

² Abbreviations: DIT, diet-induced thermogenesis; UCP, uncoupling protein; BAT, brown adipose tissue; WAT, white adipose tissue; TA, tibialis anterior; SO, soleus; RT-PCR, reverse transcription-polymerase chain reaction; T3, triiodothyronine.

Received for publication August 14, 1997. Accepted for publication October 29, 1997.

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