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Up-Regulation of Uncoupling Protein 3 Gene Expression by Fatty Acids and Agonists for PPARs in L6 Myotubes

Department of Medicine and Clinical Science (C.S., K.H., J.M., J.F., S.Y., H.I., H.M., Y.O., T.H., H.I., H.N., G.I., Y.Yo., K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; and Department of Life Science (K.H., Y.Ya.), Kyoto University Graduate School of Human and Environmental Studies, Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Dr. Kiminori Hosoda, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: kh{at}kuhp.kyoto-u.ac.jp

	Abstract

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Uncoupling protein 3 (UCP3), which uncouples electron transport from ATP synthesis, is expressed at high levels in the skeletal muscle, an important organ in glucose and lipid metabolism. Because several reports proposed that fatty acids induced UCP3 gene expression in skeletal muscle in vivo, in the present study we examined the regulation of UCP3 gene expression by various fatty acids using L6 myotubes. UCP3 gene expression was increased in L6 myotubes by various fatty acids or by -bromopalmitate, a nonmetabolized derivative of palmitic acid. Because fatty acids are also known as agonists for PPARs, we examined the involvement of PPARs in the regulation of the UCP3 gene expression. L-165041, a PPAR agonist, increased UCP3 gene expression in L6 myotubes, whereas neither Wy 14,643, a PPAR agonist, nor Pioglitazone, a PPAR agonist, increased it. Therefore, we conclude that UCP3 gene expression is increased by the activation of PPAR in L6 myotubes and postulate that PPAR mediates at least some part of the increased UCP3 gene expression by fatty acids in skeletal muscle in vivo.

	Introduction

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UNCOUPLING PROTEIN 3 (UCP3) has been identified by several groups, including ours (1, 2, 3). UCP3, which is considered to be involved in energy metabolism by uncoupling electron transport from ATP synthesis in mitochondria, is expressed at high levels in skeletal muscle (2), an important organ in glucose and lipid metabolism (4). UCP3 mRNA is expressed at much higher levels than UCP2 mRNA in skeletal muscle in vivo, although accurate comparison is difficult (1, 2, 3). Because UCP1 gene expression is almost undetectable in skeletal muscle (5), UCP3 is considered to be the most relevant UCP in skeletal muscle.

Recently, two groups have reported on UCP3 knockout mice (6, 7). There was no significant difference between the UCP3 knockout mice and wild-type mice concerning obesity, body temperature, and serum levels of insulin, glucose, triglyceride, and fatty acids. No specific phenotype of the UCP3 knockout mice was observed in response to fasting, cold exposure, or treatment with thyroid hormones compared with those of control mice. These data suggest that lack of UCP3 is not a major determinant of obesity or of metabolic disorders such as diabetes and hyperlipidemia.

Although loss of the UCP3 function appears not to be involved in the pathogenesis of obesity or type 2 diabetes, there still remains the possibility that the up-regulation of the UCP3 function is implicated in the improvement of these disorders. Clapham et al. (8) reported that transgenic mice overexpressing human UCP3 in skeletal muscle weighed approximately 30% less than their wild-type littermates and that these transgenic mice were insulin sensitive, with lower fasting plasma glucose and insulin levels. Although the levels of overexpression of UCP3 were superphysiological (approximately 70-fold), the data indicated a possible beneficial effect of UCP3 against obesity and glucose intolerance.

Several reports have proposed that fatty acids induce UCP3 gene expression in skeletal muscle in vivo. Increased blood levels of FFA by intralipid plus heparin infusion increased UCP3 mRNA levels in rat skeletal muscle (9). UCP3 mRNA levels were positively correlated with circulating levels of FFA in obese human subjects (10). Starvation, in which blood levels of FFA are increased (11), is one of the most potent stimuli of UCP3 gene expression in skeletal muscle (12, 13). As for the effect of fatty acids on the function of the UCP3 protein, Juburek et al. (14) reported that fatty acids may be obligatory for proton flux mediated by UCP3. All of these previous reports indicate that fatty acids are important regulators of gene expression and the function of UCP3. Furthermore, fatty acids have been implicated in glucose metabolism and insulin resistance (15). To elucidate the pathophysiological roles of UCP3, we examined the regulation of UCP3 gene expression by various fatty acids using L6 myotubes.

PPARs are nuclear hormone receptors controlling lipid and glucose metabolism and adipocyte differentiation (16, 17). There has been accumulating evidence that fatty acids are agonists for all three subtypes of PPARs (18, 19). Several groups, including ours, reported that UCP3 gene expression was increased in white and brown adipose tissues by agonists for PPAR or PPAR (20, 21). Therefore, it is hypothesized that PPARs may be involved in the regulation of UCP3 gene expression by fatty acids in skeletal muscle. To test this hypothesis in vitro, we analyzed the gene expression of PPARs in L6 myotubes and examined the effect of various PPAR agonists and of a RXR agonist on UCP3 gene expression.

	Materials and Methods

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Materials
L6 rat skeletal muscle cells were kindly provided by Dr. Amira Klip (Hospital for Sick Children, Toronto, Canada) (22). L-165041 was supplied by Merck Research Laboratories (Rahway, NJ). Pioglitazone was supplied by Takeda Chemical Industries Co., Ltd. (Osaka, Japan). -MEM was purchased from Nikken (Kyoto, Japan). FBS was obtained from Sanko Junyaku (Tokyo, Japan). Penicillin-streptomycin was purchased from Life Technologies, Inc. (Rockville, MD). All other chemicals were purchased from Sigma (St. Louis, MO).

cDNA probes
The preparation of rat UCP3 cDNA probe was described previously (2). The cDNA probes of rat PPAR, PPAR, and PPAR were prepared by RT-PCR using SuperScript (Life Technologies, Inc.) with the following primers (sense, 5'-GCC ATC TTC ACG ATG CTG TCC TCC-3'; antisense, 5'-GTA GAT CTC TTG CAA CAG TGG GTG C-3' for PPAR; sense, 5'-GCT CCA CAC TAT GAA GAC ATC CCG-3'; antisense, 5'-CAG CTG GTC GAT ATC ACT GGA GAT C-3' for PPAR; and sense, 5'-AGT CCA GCC ATA ACG CAC CCT TC-3'; antisense, 5'-TTC CAC CTG TGG CAC GTT CAT G-3' for PPAR). The PCR products were subcloned for sequencing.

Cell culture
L6 myoblasts were maintained in -MEM containing 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin in 10-cm-diameter dishes in an atmosphere of 5% CO₂ at 37 C, as reported by Mitsumoto et al. (22). Cultures were maintained in continuous passages (<10) by trypsinization of subconfluent nonfused cells. When cells reached confluence, the medium was exchanged for -MEM containing 2% FBS. Cells were fed fresh medium every other day. We examined the effects of various reagents on L6 myotubes that had been almost completely differentiated. Cellular differentiation was monitored by counting the percentage of nucleoli present in multinucleated myotubes with phase contrast microscopy. In this study, we used dishes in which more than 85% of myoblasts differentiated into myotubes. By examining dishes with phase contrast microscopy, we confirmed that none of the reagents affected the differentiation of L6 myotubes. Four dishes were used for each experiment.

L6 myotubes were incubated with palmitic acid, oleic acid, linoleic acid, -bromopalmitate, carbaprostacyclin (cPGI), 9-cis RA, cicaprost, Wy 14,643, Pioglitazone, or L-165041. All of the reagents were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1%. As a control experiment, myotubes were incubated with -MEM containing 2% FBS and 0.1% DMSO.

RNA extraction and Northern blot analysis
Total RNA was extracted from cells using Trizol reagent (Life Technologies, Inc.) as previously described (23). Filters containing 30 µg of total RNA were prepared. Northern blot analyses were performed using cDNA probes of rat UCP3, PPAR, PPAR, and PPAR as described previously (24). The density of 18S rRNA stained with ethidium bromide was used to monitor the amount of total RNA in each sample.

RT-PCR analyses
The primer pairs used for preparation of cDNA probes for Northern blot analyses were used for RT-PCR analyses. All primer pairs encompassed an intron. cDNA synthesized from 2.5 µg of total RNA with oligo(dT) primers was subjected to PCR with Ampli Taq Polymerase (Perkin-Elmer Corp., Norwalk, CT) and the following conditions: 20 sec at 94 C, 30 sec at 57 C, and 1 min at 72 C for 30, 35, or 40 cycles. The products of RT-PCR were electrophoresed in 1.0% agarose gel and stained with ethidium bromide. The PCR products with the expected size were verified by direct sequencing. As positive control experiments for RT-PCR, RNA samples from rat brown adipose tissue were used. As negative control experiments, PCR products without RT were also examined. Each experiment was performed in duplicate.

Statistical analysis
The mRNA levels in 30 µg of total RNA from control dishes that were incubated with -MEM containing 2% FBS and 0.1% DMSO were defined as 100 U. Data were expressed as means ± SE. Statistical significance was tested by one-way ANOVA. If F was found to be significant, the t test was used to test individual differences.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
UCP3 gene expression in L6 cells
We examined UCP3 gene expression in L6 cells by Northern blot analysis. UCP3 gene expression was undetectable in myoblasts (Fig. 1). The UCP3 gene was expressed in L6 myotubes, although its mRNA levels were much lower than those in the skeletal muscle in vivo (Fig. 1).

View larger version (57K): [in this window] [in a new window]   Figure 1. Northern blot analyses of UCP3 mRNA in L6 myoblasts and myotubes. Thirty micrograms of total RNA was analyzed. UCP3 mRNA in rat skeletal muscle in vivo is also shown.

View larger version (24K): [in this window] [in a new window]   Figure 2. UCP3 mRNA levels in L6 myotubes incubated with or without various reagents for 24 h. Cells were treated with palmitic acid, oleic acid, and linoleic acid at 10, 30, or 100 µM and with -bromopalmitate at 1, 5, or 10 µM. Thirty micrograms of total RNA was analyzed by Northern blot analyses. All of the reagents were dissolved in DMSO to a final concentration of 0.1%. As a control experiment, myotubes were incubated with -MEM containing 2% FBS and 0.1% DMSO. *, P < 0.05; **, P < 0.001 vs. control. Data are expressed as means ± SE (n = 4).

View larger version (39K): [in this window] [in a new window]   Figure 3. Gene expression of PPARs in L6 myotubes and rat tissues. A, Northern blot analyses of mRNAs for PPARs in L6 myotubes and rat skeletal muscle and brown adipose tissue (BAT). Thirty micrograms of total RNA was analyzed. RNA was obtained from L6 myotubes, rat gastrocnemius muscle, and rat BAT. B, RT-PCR analyses of mRNAs of PPARs in L6 myotubes. The RT-PCR procedure is described in Materials and Methods. Lane a, Product of RT-PCR of L6 myotubes; lane b, product of RT-PCR of brown adipose tissue as a positive control; lane c, product of PCR without reverse transcriptase as a negative control. Each primer pair encompassed an intron. Each band of RT-PCR products was detected at an expected size. Representative results are shown. Each result was confirmed by RT-PCR of another sample.

Regulation of UCP3 gene expression by cPGI and 9-cis RA
We investigated the regulation of the UCP3 gene expression by cPGI and 9-cis RA in L6 myotubes. UCP3 gene expression was increased to 508 ± 14% by 1 µM cPGI, an agonist for all three subtypes of PPARs (P < 0.0001) (Fig. 4). UCP3 gene expression was also increased to 572 ± 26% by 1 µM 9-cis RA, an agonist for RXR (26) (P < 0.0001) (Fig. 4). Coincubated with 1 µM cPGI and 1 µM 9-cis RA, UCP3 gene expression was increased to 1574 ± 105% compared with control experiments (P < 0.0001) (Fig. 4). We also investigated the effect of cicaprost, which is an agonist for PGI₂ receptor but not for PPARs (27), on UCP3 gene expression in L6 myotubes. UCP3 gene expression was not affected by 1 µM cicaprost (91 ± 17%).

View larger version (17K): [in this window] [in a new window]   Figure 4. UCP3 mRNA levels in L6 myotubes incubated with a PPAR agonist and a RXR agonist or with cicaprost or without these agents for 24 h. Cells were treated with 1 µM cPGI, with 1 µM 9-cis RA, with both of these reagents, or with 1 µM cicaprost. Thirty micrograms of total RNA was analyzed by Northern blot analyses. All of the reagents were dissolved in DMSO to a final concentration of 0.1%. As a control experiment, cells were incubated with 0.1% DMSO. **, P < 0.001 vs. control. Data are expressed as means ± SE (n = 4).

View larger version (15K): [in this window] [in a new window]   Figure 5. UCP3 mRNA levels in L6 myotubes incubated with specific agonists for each subtype of PPAR or without these agents for 24 h. Cells were treated with 1, 5, or 10 µM Wy 14,643, with 10, 50, or 100 µM Pioglitazone, or with 0.1, 1, or 10 µM L-165041. Thirty micrograms of total RNA was analyzed by Northern blot analyses. All of the reagents were dissolved in DMSO to a final concentration of 0.1%. As a control experiment, cells were incubated with 0.1% DMSO. **, P < 0.001 vs. control. Data are expressed as means ± SE (n = 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We demonstrate that the UCP3 gene expression was increased by various fatty acids, including palmitic acid, oleic acid, and linoleic acid. The UCP3 gene expression was significantly increased by these fatty acids at the concentration of 100 µM, which is within the physiological range in circulating blood in rats and humans (11). The increased UCP3 mRNA levels by fatty acids in L6 myotubes appeared to be analogous to the induction of UCP3 gene expression in skeletal muscle by increased levels of circulating fatty acids described in previous reports (9). Furthermore, we also demonstrate that UCP3 gene expression was increased by -bromopalmitate, a nonmetabolized derivative of palmitic acid in L6 myotubes, indicating that the increase of UCP3 gene expression was mediated by fatty acids themselves and not by their metabolites. Because there has been accumulating evidence that fatty acids are agonists for all subtypes of PPARs (18), the up-regulation of UCP3 gene expression by fatty acids in skeletal muscle may also be mediated by PPARs. This notion was also supported by the report by Acin et al. (30) about the existence of three sequences similar to consensus PPAR responsive element in the 5' flanking region of the human UCP3 gene.

The present study demonstrates that PPAR mRNA was detected in L6 myotubes by Northern blot analysis, whereas neither PPAR mRNA nor PPAR mRNA was observed. This finding is compatible with the previous report that PPAR was detected by RT-PCR in L6 myotubes (31). In the present study, PPAR mRNA was detected in L6 myotubes by RT-PCR analysis, whereas PPAR mRNA was undetectable. There are reports of detection of very low levels of PPAR mRNA and of PPAR protein in skeletal muscle. Although there is a possibility that a low level of PPAR exists in L6 myotubes, the present findings indicate that PPAR is the major subtype of PPAR in L6 myotubes.

We demonstrate that cPGI, which works as an agonist for all three subtypes of PPARs, and 9-cis RA, an agonist for RXR, increased UCP3 gene expression in L6 myotubes. cPGI is also known to be an agonist for PGI₂ receptor. Because cicaprost, which is an agonist for PGI₂ receptor but not for PPARs (27), failed to affect UCP3 gene expression, cPGI is considered to increase UCP3 gene expression via PPARs. We also demonstrated that cPGI and 9-cis RA synergistically increased UCP3 gene expression. Because it is well known that PPARs transactivate gene expression by forming heterodimers with RXR (32), these data indicate that activation of PPAR-RXR heterodimers induced UCP3 gene expression. All of these findings indicate that the activation of PPARs induces UCP3 gene expression in L6 myotubes.

In the present study, UCP3 mRNA levels were increased neither by Pioglitazone, an agonist for PPAR, nor by Wy 14,643, an agonist for PPAR, at the concentration that is high enough for the activation of each PPAR (33), whereas L-165041, an agonist for PPAR (28), increased UCP3 mRNA levels at the concentration of 0.1 µM, at which PPAR is activated in vitro (28). Together with the expression pattern of PPAR subtypes in the L6 myotubes, the increased UCP3 gene expression in L6 myotubes by fatty acids and cPGI was considered to be mediated by PPAR.

Recently Kersten et al. (34) reported that UCP3 gene expression was induced by fasting in the skeletal muscle of PPAR knockout mice and wild-type mice. We showed that mRNA of PPAR but not PPAR was detected by Northern blot analysis in skeletal muscle in vivo as reported previously (35). Thus, the increase of UCP3 mRNA levels in the skeletal muscle of PPAR knockout mice by fasting is considered to be mediated by PPAR. Although some reports indicated the involvement of PPAR in the regulation of UCP3 gene expression, all of these findings indicate that PPAR mediates at least some part of the increased gene expression of UCP3 by fatty acids in the skeletal muscle in vivo. Because PPAR was detected in rat skeletal muscle by Northern blot analysis, as previously reported (35), the involvement of PPAR is also possible. Further in vivo studies using agonists specific for PPAR or knockout mice of PPAR are required.

Compared with the pathophysiological significance of PPAR and PPAR, that of PPAR is unclear except for its possible roles in adipocyte differentiation (36), implantation in the uterus (37), and tumorigenesis of colorectal cancer (38). The up-regulation of UCP3 gene expression via PPAR in skeletal muscle may give a clue to the role of PPAR in energy metabolism. Recently, Peters et al. (39) reported that PPAR knockout mice did not differ from controls in metabolism, which suggests that the lack of PPAR does not affect energy metabolism at substantial levels. However, there remains the possibility that the PPAR pathway may be involved in energy metabolism.

In conclusion, we demonstrate that fatty acids increased UCP3 gene expression in L6 myotubes, in which we detected mRNA of only the subtype of PPAR by Northern blot analysis. We also demonstrate that UCP3 gene expression was increased by L-165041, whereas neither Wy 14,643 nor Pioglitazone increased it, indicating that UCP3 gene expression is increased by the activation of PPAR in L6 myotubes. PPAR may mediate at least some part of the increased gene expression of UCP3 by fatty acids in skeletal muscle in vivo, although the involvement of PPAR is also possible. The elucidation of the regulatory mechanism of UCP3 gene expression may lead to the development of drugs against obesity and type 2 diabetes.

	Acknowledgments

 
We acknowledge Prof. Amira Klip for providing us L6 rat skeletal muscle cells. We also acknowledge Prof. Joel Berger for providing L-165041. We appreciate Prof. K. Umezono, who died in 1999, Prof. T. Kawata, and Dr. S. Osada for valuable advice and encouragement. We acknowledge Dr. T. Tanaka for help and K. Hiramatsu for her excellent secretarial assistance. J.M. is the recipient of a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists.

	Footnotes

 
This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture, the Japanese Ministry of Health and Welfare, the Yamanouchi Foundation for Research on Metabolic Disorders, the Uehara Memorial Foundation, the Naito Foundation, the Cell Science Research Foundation, the Ono Medical Research Foundation, the Japan Diabetes Foundation, and the Tanabe Medical Frontier Conference.

Abbreviations: cPGI, Carbaprostacyclin; DMSO, dimethyl sulfoxide; UCP, uncoupling protein.

Received March 23, 2001.

Accepted for publication June 27, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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