Uncoupling protein-3 mRNA up-regulation in C2C12 myotubes after etomoxir treatment

Biochimica et Biophysica Acta 1532 (2001) 195^202 www.bba-direct.com

Uncoupling protein-3 mRNA up-regulation in C2C12 myotubes after etomoxir treatment é gatha Cabrero, Marta Alegret, Rosa Sa¨nchez, Toma¨s Adzet, Juan C. Laguna, A Manuel Va¨zquez * Pharmacology Unit, School of Pharmacy, University of Barcelona, Diagonal 643, 08028 Barcelona, Spain Received 12 February 2001; received in revised form 9 April 2001; accepted 8 May 2001

Abstract Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of C2C12 myotubes for 24 h with 40 WM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated uncoupling protein 3 (UCP3) mRNA levels (2-fold induction), whereas UCP-2 mRNA levels were not modified. Etomoxir treatment also caused a 2.5fold induction in M-CPT-I (muscle-type CPT-I) mRNA levels. In contrast, other well-known peroxisome proliferatoractivated receptor K (PPARK) target genes, such as acyl-CoA oxidase and medium-chain acyl-CoA dehydrogenase, were not affected, suggesting that this transcription factor was not involved in the effects of etomoxir. Since it has been reported that CPT-I inhibition by etomoxir leads to a further increase in ceramide synthesis, we test the possibility that ceramides were involved in the changes reported. Similarly to etomoxir, addition of 20 WM C2 -ceramide to C2C12 myotubes for 3, 6 and 9 h resulted in increased UCP-3 and M-CPT-I mRNA levels. These results indicate that the effects on UCP-3 mRNA levels could be mediated by increased ceramide synthesis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Uncoupling protein; Etomoxir; C2C12; Ceramide; Carnitine palmitoyltransferase I; Peroxisome proliferator-activated receptor

1. Introduction Carnitine palmitoyltransferase I is located on the mitochondrial outer membrane and catalyses the entry of long-chain fatty acids into the mitochondrial matrix [1]. This enzyme converts fatty acyl-CoA to acyl-carnitine, which are then transported into the mitochondria by a speci¢c translocase (carnitineacyl-carnitine carrier) and re-esteri¢ed to acyl-thioesters by carnitine palmitoyltransferase II (CPT-II).

* Corresponding author. Fax: +34-93-403-5982. E-mail address: [email protected] (Manuel Va¨zquez).

Once into the mitochondria, fatty acyl-thioesters can undergo L-oxidation, generating reducing equivalents used to produce ATP via oxidative phosphorylation. CPT-I exists as two isoforms: liver-type (L-CPT-I), which is expressed in liver and ¢broblasts [2], and muscle-type (M-CPT-I), abundantly expressed in heart, skeletal muscle and brown and white adipose tissue [3^5]. Both isoforms of CPT-I are inhibited by malonyl-CoA, the product of acetyl-CoA carboxylase, but with di¡erent sensitivity [6,7]. Thus, the IC50 of M-CPT-I for malonyl-CoA is approximately 100-fold lower than that of L-CPT-I [8]. Pharmacological inhibition of CPT-I is also achieved by oxirane carboxylates, such as etomoxir, which is an irre-

1388-1981 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 1 ) 0 0 1 3 1 - 7

BBAMCB 55796 9-7-01

196

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

versible inhibitor of CPT-I. The inhibition by this drug of the transport of long-chain acyl-CoA compounds into the mitochondria increases the longchain fatty acyl-CoA/long-chain fatty acyl-carnitine ratio in the cytoplasm and inhibits L-oxidation. Since increased fatty acid L-oxidation, as a result of elevated plasma FFA, may contribute to insulin resistance [9] these drugs were developed for the treatment of non-insulin-dependent diabetes mellitus. In fact, CPT-I activity and the availability of fatty acids are the main factors that determine the £ux of mitochondrial L-oxidation. As stated above, this process provides reducing equivalents (NADH) which are oxidized by the respiratory chain, resulting in the generation of an electrochemical gradient of protons [10]. Normally, protons descend this gradient via ATP synthase, coupling synthesis of ATP to fuel oxidation. However, under some circumstances, dissipation of the proton electrochemical gradient is also possible, at the expense of ATP synthesis, through inner mitochondrial proton transporters called uncoupling proteins (UCPs) [11]. Three UCP subtypes have now been identi¢ed. UCP-1, the ¢rst uncoupling protein discovered is expressed exclusively in brown adipose tissue [11]. Recently, two new uncoupling proteins, UCP-2 and UCP-3, have been discovered [12^15]. Like UCP-1, these new UCPs seem to function as uncouplers of oxidative phosphorylation in mitochondria [12,14^16], although their expression pattern is di¡erent. UCP-2 mRNA is widely expressed in many human and rat tissues, including WAT and skeletal muscle. UCP-3 mRNA is expressed in rat skeletal muscle and brown adipose tissue, and to a lesser extent in white adipose tissue and heart, whereas in humans its expression is restricted to skeletal muscle, an important site of energy expenditure [13^15]. The increase in plasma FFA observed after fasting up-regulates UCP-3 mRNA levels in skeletal muscle [17,18]. The molecular mechanism by which FFA activate the expression of UCP-3 mRNA levels seems to involve PPARs. Ligands of these receptors, which are also activated by fatty acids, increase UCP-3 mRNA levels in muscle and WAT [19^21]. Furthermore, three putative peroxisome proliferator response elements (PPREs) have been found in the 5P £anking region of the human UCP-3 gene, pointing to the involvement of PPARs in the expression of

this gene [22]. In addition, increased glucose in£ux up-regulates UCP-3 mRNA expression in muscle and WAT [23], although the molecular mechanism responsible has not been elucidated. Here we examine the e¡ects of etomoxir on UCP-3 and UCP-2 mRNA levels in C2C12 myotubes. We report that treatment with etomoxir for 24 h upregulated UCP-3 mRNA expression. In contrast, UCP-2 mRNA levels were not signi¢cantly modi¢ed. Etomoxir treatment also increased M-CPT-I mRNA levels, but other well-known PPARK target genes, such as acyl-CoA oxidase (ACO) and medium-chain acyl-CoA dehydrogenase (MCAD) were not a¡ected by the treatment. Since it has been reported that etomoxir treatment increases ceramide synthesis [24], we studied the e¡ects of C2 -ceramide in C2C12 myotubes. C2 -Ceramide treatment led to increased UCP-3 and M-CPT-I mRNA levels, suggesting they were involved in the e¡ects mediated by etomoxir. 2. Materials and methods 2.1. Cell culture Mouse C2C12 myoblasts (ATCC) were maintained in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 50 units/ml penicillin and 50 Wg/ml streptomycin. As cells reached con£uence, the medium was switched to the di¡erentiation medium containing DMEM and 2% horse serum. Medium was changed every other day. After 4 additional days, the di¡erentiated C2C12 cells had fused into myotubes, which were then treated in serum-free DMEM with either vehicle (0.1% DMSO or ethanol), 40 WM etomoxir for 24 h or 20 WM C2 -ceramide (Sigma) for 3, 6 or 9 h. After the incubation, RNA was extracted from myotubes as described below. 2.2. RNA preparation and analysis Total RNA was isolated using the Ultraspec reagent (Biotecx). Relative levels of speci¢c mRNAs were assessed by reverse transcriptase^polymerase chain reaction (RT^PCR). Complementary DNA was synthesized from RNA samples by mixing 0.5

BBAMCB 55796 9-7-01

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

Wg of total RNA, 125 ng of random hexamers as primers in the presence of 50 mM Tris^HCl bu¡er (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies), 20 U RNAsin (Life Technologies) and 0.5 mM of each dNTP (Sigma) in a total volume of 20 Wl. Samples were incubated at 37³C for 60 min. A 5-Wl aliquot of the RT reaction was then used for subsequent PCR ampli¢cation with speci¢c primers. Each 25-Wl PCR reaction contained 5Wl of the RT reaction, 1.2 mM MgCl2 , 200 WM dNTPs, 1.25 WCi [32 P]-dATP (3000 Ci/mmol, Amersham), 1 U Taq polymerase (Ecogen, Barcelona, Spain), 0.5 Wg of each primer and 20 mM Tris^HCl, pH 8.5. To avoid unspeci¢c annealing, cDNA and Taq polymerase were separated from primers and dNTPs by using a layer of para¤n (reaction components contact only when para¤n fuses, at 60³C). The sequences of the sense and antisense primers used for ampli¢cation were: UCP-3, 5P-GGAGCCATGGCAGTGACCTGT-3P and 5P-TGTGATGTTGGGCCAAGTCCC3P; UCP-2, 5P-AACAGTTCTACACCAAGGGC-3P and 5P-AGCATGGTAAGGGCACAGTG-3P; MCPT-I, 5P-TTCACTGTGACCCCAGACGGG-3P and 5P-AATGGACCAGCCCCATGGAGA; ACO, 5P-ACTATATTTGGCCAATTTTGTG-3P and 5PTGTGGCAGTGGTTTCCAAGCC-3P; MCAD, 5PTCGAAAGCGGCTCACAAGCAG-3P and 5P-CACCGCAGCTTTCCGGAATGT-3P; adenosyl phosphoribosyl transferase (APRT), 5P-AGCTTCCCGGACTTCCCCATC-3P and 5P-GACCACTTTCTGCCCCGGTTC-3P. PCR was performed in an MJ Research Thermocycler equipped with a peltier system and temperature probe. After an initial denaturation for 1 min at 94³C, PCR was performed for 20 (MCAD), 21 (UCP-2), 26 (UCP-3), 28 (APRT), 30 (ACO) or 33 (M-CPT-I) cycles. Each cycle consisted of denaturation at 92³C for 1 min, primer annealing at 60³C (except 58³C for ACO), and primer extension at 72³C for 1 min and 50 s. A ¢nal 5-min extension step at 72³C was performed. Five microliters of each PCR sample was electrophoresed on a 1-mmthick 5% polyacrylamide gel. The gels were dried and subjected to autoradiography using Kodak X-ray ¢lms to show the ampli¢ed DNA products. Ampli¢cation of each gene yielded a unique band of the expected size (UCP-3, 198 bp; UCP-2, 471 bp; M-

197

CPT-I, 222 bp; ACO, 195 bp; MCAD, 216 bp; APRT, 329 bp). Preliminary experiments were carried out with various amounts of cDNA to determine non-saturating conditions of PCR ampli¢cation for all the genes studied. Therefore, under these conditions, relative quanti¢cation of mRNA was assessed by the RT^PCR method used in this study [25]. Radioactive bands were quanti¢ed by video-densitometric scanning (Vilbert Lourmat Imaging). The results for the expression of speci¢c mRNAs are always presented relative to the expression of the control gene (aprt).

Fig. 1. E¡ect of etomoxir on the expression of UCP-3 (A) and UCP-2 (B) mRNA in C2C12 myotubes. Cells were incubated for 24 h with or without 40 WM etomoxir. All cells were exposed to 0.1% DMSO. 0.5 Wg of total RNA was analyzed by RT^PCR. A representative autoradiogram and the quanti¢cation of the aprt-normalized UCPs mRNA levels are shown. Data are expressed as mean þ S.D. of three experiments. *P = 0.007 compared with control experiments. F.I., fold induction.

BBAMCB 55796 9-7-01

198

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

2.3. Statistical analyses Results are expressed as mean þ S.D. Statistical signi¢cance was evaluated using Student's t-test. 3. Results When cultured in the presence of 2% horse serum for 4 days after reaching con£uence, C2C12 myoblasts di¡erentiated morphologically to fuse into myotubes. In these culture conditions the e¡ects of etomoxir, and irreversible inhibitor of CPT-I, was assessed on UCP-3 and UCP-2 mRNA levels in C2C12 myotubes for 24 h. Treatment of C2C12 myotubes with 40 WM etomoxir, an irreversible inhibitor of CPT-I, caused a 2-fold induction in UCP-3 mRNA levels (P = 0.007) (Fig. 1A). In contrast, UCP-2 mRNA levels were not modi¢ed by the addition of this drug (Fig. 1B). Drug treatment caused a 2.5-fold increase in the levels of M-CPT-I transcripts (P = 0.006) (Fig. 2). On the other hand, it has been reported that irreversible inhibition of CPT-I by etomoxir, which leads to an accumulation of intracellular lipids, induces a PPARK-mediated feedback activation of target genes, such as ACO, involved in alternate oxidation pathways. Thus, mice receiving

Fig. 2. E¡ect of etomoxir on the expression of M-CPT-I mRNA in C2C12 myotubes. Cells were incubated for 24 h with or without 40 WM etomoxir. All cells were exposed to 0.1% DMSO. 0.5 Wg of total RNA was analyzed by RT^PCR. A representative autoradiogram and the quanti¢cation of the aprtnormalized mRNA levels are shown. Data are expressed as mean þ S.D. of three experiments. *P = 0.006 compared with control experiments. F.I., fold induction.

Fig. 3. E¡ect of etomoxir on the expression of ACO (A) and MCAD (B) mRNA in C2C12 myotubes. Cells were incubated for 24 h with or without 40 WM etomoxir. All cells were exposed to 0.1% DMSO. 0.5 Wg of total RNA was analyzed by RT^PCR. A representative autoradiogram and the quanti¢cation of the aprt-normalized mRNA levels are shown. Data are expressed as mean þ S.D. of three experiments.

a 5-day course of etomoxir treatment showed an induction of the mRNA expression of ACO in heart and liver [26]. In order to explore a similar response in C2C12 myotubes we studied the e¡ect of etomoxir treatment on ACO, the rate-limiting enzyme of peroxisomal fatty acid L-oxidation, whose transcription is controlled by PPARK [27]. Treatment with etomoxir caused no increase in ACO mRNA levels (Fig. 3A). Similarly, MCAD, an enzyme catalyzing a rate-limiting step in the mitochondrial oxidation of medium-chain fatty acyl-thioesters produced by peroxisomal L-oxidation of long-chain fatty acids has been reported to be induced by a 5 days treatment of etomoxir in liver and heart. However, C2C12 myotubes showed no increase in MCAD mRNA levels after 24 h of etomoxir treatment (Fig. 3B). The lack

BBAMCB 55796 9-7-01

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

199

Fig. 4. E¡ect of C2 -ceramide on the expression of M-CPT-I (A) and UCP-3 (B) mRNA in C2C12 myotubes. Cells were incubated for 3, 6 and 9 h with or without 20 WM C2 -ceramide. All cells were exposed to 0.1% ethanol. 0.5 Wg of total RNA was analyzed by RT^ PCR. A representative autoradiogram and the quanti¢cation of the apart-normalized mRNA levels are shown. Data are the mean of two experiments.

of induction of these PPARK target genes suggest that PPARK was not involved in the e¡ects of etomoxir on UCP-3 and M-CPT-I mRNA levels. On the other hand, CPT-I inhibition by etomoxir prevents the entrance of palmitoyl-CoA into mitochondria, leading to its accumulation in the cytoplasm. Since palmitoyl-CoA is a precursor of sphingolipid synthesis, etomoxir treatment may result in enhanced ceramide synthesis as previously reported [24]. Thus, to gain further insight into the mechanism by which etomoxir up-regulates UCP-3 and M-CPT-I mRNA levels we treated C2C12 cells with 20 WM C2 -ceramide, a cell-permeable ceramide analog, for 3, 6 and 9 h. Addition of C2 -ceramide to C2C12 myotubes resulted in up-regulation of M-CPT-I (1.8-, 1.5and 3.2-fold induction after 3, 6 and 9 h of treatment, respectively) (Fig. 4A) and UCP-3 (2.6-, 2.1and 3.1-fold induction after 3, 6 and 9 h of treatment, respectively) mRNA levels (Fig. 4B). These results suggest that enhanced ceramide synthesis is the mechanism underlying the e¡ects caused by etomoxir treatment on UCP-3 and M-CPT-I mRNA levels in C2C12 myotubes. 4. Discussion In the present study we show that etomoxir, an irreversible inhibitor of CPT-I, and therefore, of mi-

tochondrial L-oxidation of fatty acids, increases the expression of UCP-3 mRNA in C2C12 myotubes. UCP-2 mRNA expression, however, was not modi¢ed by the treatment. Acute administration of etomoxir and other 2-oxiranecarboxylates, such as clomoxir or palmoxirate, to animals and man increases plasma FFA [28,29]. This makes it di¤cult to interpret the direct e¡ects of etomoxir on UCP-3 expression in vivo [30], since FFA are potent inducers of this UCP subtype [20,21]. Further, we have previously reported that etomoxir treatment increased UCP-3 mRNA levels in a rat primary culture of undi¡erentiated preadipocytes [31]. However, it remained to study whether muscle cells, which express higher levels of UCP-3, had a similar behavior after etomoxir treatment. In addition, the e¡ects of etomoxir in muscle cells may be di¡erent when compared to the e¡ects on preadipocytes due to the different pattern of expression of CPT-I isoforms [2,3]. Moreover, etomoxir administration leads to cardiac hypertrophy [32], suggesting that this drug especially a¡ects muscle cells. At the level of gene regulation it has been shown that etomoxir inhibition of mitochondrial fatty acid import, causes a PPARK-mediated activation of target genes involved in alternate oxidation pathways. As a consequence, mRNA levels of peroxisomal ACO and mitochondrial MCAD, known PPARK target genes, were induced in heart and liver of

BBAMCB 55796 9-7-01

200

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

mice receiving etomoxir (50 Wg/g body weight, for 5 days) [26]. Likewise, the mRNA levels of M-CPT-I, which expression is controlled by PPARK [33], is also induced in the heart of mice treated with etomoxir [34]. The direct involvement of PPARK in the e¡ects caused by etomoxir on ACO and MCAD [26] and M-CPT-I [34] was demonstrated by using mice lacking PPARK, in which the response of these PPARK target genes to etomoxir was abolished. According to Forman et al. [35], the mechanism by which etomoxir activates PPARK includes direct binding to this receptor and, indirectly, as a metabolic inhibitor, may lead to the accumulation of endogenous ligands. Further, we have previously reported that C2C12 myotubes express PPARK at the mRNA level, and that PPARK activators, such as Wy-14,643 and beza¢brate, induce M-CPT-I mRNA levels [36]. Thus, initially all these data pointed to the potential involvement of PPARK in the e¡ects of etomoxir on M-CTP-I and UCP-3 mRNA levels in C2C12 myotubes. However, in contrast to the e¡ects reported in preadipocytes, where etomoxir treatment led to the induction of ACO mRNA levels [31], in C2C12 myotubes etomoxir treatment for 24 h did not modify the mRNA levels of the PPARK target genes ACO and MCAD. These data suggest that other mechanisms, not related to PPARK, may be involved in the acute e¡ects of etomoxir in C2C12 myotubes. However PPARK mediated mechanisms could not be completely discarded. It is likely that PPARK activation by etomoxir requires a higher accumulation of fatty acids, which can be attained after more prolonged treatments, similarly to what is observed in vivo, where MCAD mRNA levels are induced in heart of etomoxir-treated mice after 5 days but not after 1 day of treatment [26]. Among the potential mechanisms involved in the e¡ects of etomoxir, it is interesting to note that inhibition of CPT-I by this drug prevents the entrance of palmitoyl-CoA into mitochondria, resulting in its accumulation in the cytoplasm [24]. Given that palmitoyl-CoA is a precursor of sphingolipid synthesis, etomoxir treatment may result in enhanced ceramide synthesis as previously reported [24]. Treatment of C2C12 cells with 20 WM C2 -ceramide, a cell-permeable ceramide analog, increased UCP-3 mRNA levels, suggesting the involvement of ceramides in the

UCP-3 mRNA up-regulation caused by etomoxir. Besides, C2-ceramide increased M-CPT-I mRNA levels, which is consistent with the notion that CPT-I is a ceramide-activated enzyme [37]. Taking into account the results here presented, MCPT-I and UCP-3 induction by ceramides is interesting in view of the possible implication of these genes in ceramide-mediated apoptosis. Indeed, expression of high CPT-I activity a¡orded protection from apoptosis [24]. Although the functions of UCPs are yet unknown, a role for muscle UCP-3 has been proposed in the limitation of the level of reactive oxygen species [11]. Therefore, it is likely that simultaneous activation of M-CPT-I and UCP-3 by ceramide may form part of a common mechanism. On the other hand, inhibition of CPT-I by etomoxir could explain recently published results, in which overexpression of glucose transporter 4 (GLUT4) in skeletal muscle and WAT of transgenic mice harboring a GLUT4 minigene led to up-regulation of UCP3 in these tissues [23]. Increased glucose entry to skeletal myocytes and white adipocytes, due to high GLUT4 levels, increases the concentration of malonyl-CoA, a known inhibitor of CPT-I [6,7]. Therefore, forced glucose £ux into the cells may lead to CPT-I inhibition by malonyl-CoA, causing an accumulation of palmitoyl-CoA, which in turn would increase ceramide synthesis, leading to up-regulation of UCP-3 mRNA levels. In summary, we show an up-regulation of UCP-3 mRNA levels after etomoxir treatment in C2C12 myotubes. The results here presented suggest that the e¡ects produced by etomoxir were mediated by increased ceramide synthesis. Acknowledgements We thank Mr. Rycroft (Language Advice Service of the University of Barcelona) for his helpful assistance. We are grateful to Dr. G. Asins (Unidad de Bioqu|¨mica, Facultad de Farmacia, Universidad de Barcelona) for generously providing us with etomoxir. This study was partly supported by grants from the FPCNL, Ministerio de Ciencia y Tecnolog|¨a (SAF97-0215, SAF98-0105 and SAF2000-0201) and FISS (00/1124). We also thank the Generalitat de

BBAMCB 55796 9-7-01

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202

Catalunya for grants SGR96-84 and 1998SGR-33. é gatha Cabrero was supported by a grant from the A Ministerio de Educacio¨n of Spain. References [1] J.D. McGarry, N.F. Brown, The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis, Eur. J. Biochem. 244 (1997) 1^14. [2] E.A. Park, M.L. Ste¡en, S. Song, V.M. Park, G.A. Cook, Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene, Biochem. J. 330 (1998) 217^224. [3] N. Yamazaki, Y. Shinohara, A. Shima, H. Terada, High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone, FEBS Lett. 363 (1995) 41^45. [4] N. Yamazaki, Y. Shinohara, A. Shima, Y. Yamanaka, H. Terada, Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I, Biochim. Biophys. Acta 1307 (1996) 157^ 161. [5] V. Esser, N.F. Brown, A.T. Cowan, D.W. Foster, J.D. McGarry, Expression of a cDNA isolated from rat brown adipose tissue and heart identi¢es the product as the muscle isoform of carnitine palmitoyltransferase (M-CPT I): MCPT I is the predominant CPTI isoform expressed in both white (epididymal) and brown adipocytes, J. Biol. Chem. 271 (1996) 6972^6977. [6] J.D. McGarry, K.F. Woeltje, M. Kuwajima, D.W. Foster, Regulation of ketogenesis and the renaissance of carnitine palmitoyltransferase, Diabetes Metab. Rev. 5 (1989) 271^ 284. [7] J.D. McGarry, The mitochondrial carnitine palmitoyltransferase system: its broadening role in fuel homeostasis and new insights into its molecular features, Biochem. Soc. Trans. 23 (1995) 321^324. [8] J.D. McGarry, S.E. Mills, C.S. Long, D.W. Foster, Observations on the a¤nity of carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non hepatic tissues of the rat, Biochem. J. 214 (1983) 21^ 28. [9] P.J. Randle, P.B. Garland, C.N. Hales, E.A. Newsholme, The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus, Lancet 1 (1963) 785^789. [10] G.C. Brown, Control of respiration and ATP synthesis in mammalian mitochondria and cells, Biochem. J. 284 (1992) 1^13. [11] D. Ricquer, F. Bouillad, The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP, Biochem. J. 345 (2000) 161^179.

201

[12] C. Fleury, M. Neverova, S. Collins, S. Raimbault, O. Champigny, M.C. Levi, F. Bouillaud, M.F. Seldin, R.S. Surwitt, D. Ricquier, C.H. Warden, Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia, Nat. Genet. 15 (1997) 269^272. [13] A. Vidal-Puig, G. Solanes, D. Grujic, J.S. Flier, B.B. Lowell, UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue, Biochem. Biophys. Res. Commun. 235 (1997) 79^82. [14] O. Boss, S. Samec, G.A. Paoloni, C. Rossier, A. Dulloo, J. Seydoux, P. Muzzin, J.P. Giacobino, Uncoupling protein-3 : a new member of the mitochondrial carrier family with tissue-speci¢c expression, FEBS Lett. 408 (1997) 39^42. [15] D. Gong, Y. He, M. Karas, M. Reitman, Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, L3-adrenergic agonists, and leptin, J. Biol. Chem. 272 (1997) 24129^24132. [16] R.E. Gimeno, M. Dembski, X. Weng, N. Deng, A.W. Shyjan, C.J. Gimeno, F. Iris, S.J. Ellis, E.A. Woolf, L.A. Tartaglia, Cloning and characterization of an uncoupling protein homolog: a potential mediator of human thermogenesis, Diabetes 46 (1997) 900^906. [17] L. Millet, H. Vidal, F. Andreeli, D. Larrouy, J.P. Riou, D. Ricquier, M. Laville, D. Langin, Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans, J. Clin. Invest. 100 (1997) 2665^2670. [18] D.S. Weigle, L.E. Selfridge, M.W. Schwartz, R.J. Seely, D.E. Cummings, P.J. Havel, J.L. Kuipjer, H. BeltrandelRio, Elevated free fatty acids induce uncoupling protein 3 expression in muscle, Diabetes 47 (1998) 298^302. [19] J. Matsuda, K. Hosoda, H. Itoh, C. Son, K. Doi, Y. Hanaoka, G. Inoue, H. Nishimura, Y. Yoshimasa, Y. Yamori, H. Odaka, K. Nakao, Increased adipose expression of the uncoupling protein-3 gene by thiazolidinediones in Wistar fatty rats and in culture adipocytes, Diabetes 47 (1998) 1809^1814. [20] S. Brun, M. Carmona, T. Mampel, O. Vin¬as, M. Giralt, R. Iglesias, F. Villarroya, Activators of peroxisome proliferatoractivated receptor-alpha induce the expression of the uncoupling protein-3 gene in skeletal muscle. A potential mechanism for the lipid-intake dependent activation of uncoupling protein-3 gene expression at birth, Diabetes 48 (1999) 1217^ 1222. [21] A. Cabrero, G. Llaver|¨as, N. Roglans, M. Alegret, R. Sa¨nchez, T. Adzet, J.C. Laguna, M. Va¨zquez, Uncoupling Protein-3 mRNA Levels are increased in white adipose tissue and skeletal muscle of beza¢brate-treated rats, Biochem. Biophys. Res. Commun. 260 (1999) 547^556. [22] A. Ac|¨n, M. Rodr|¨guez, H. Rique, E. Canet, J.A. Boutin, J.-P. Galizzi, Cloning and characterization of the 5P £anking region of the human uncoupling protein 3 (UCP3) gene, Biochem. Biophys. Res. Commun. 258 (1999) 278^283. [23] N. Tsuboyama-Kasoaka, N. Tsunoda, K. Maruyama, M. Takahashi, H. Kim, D.W. Cooke, D. Lane, O. Ezaki, Overexpression of GLUT4 in mice causes up-regulation of UCP3

BBAMCB 55796 9-7-01

202

[24]

[25]

[26]

[27]

[28]

[29]

[30]

A. Cabrero et al. / Biochimica et Biophysica Acta 1532 (2001) 195^202 mRNA in skeletal muscle, Biochem. Biophys. Res. Commun. 258 (1999) 187^193. M.B. Paumen, Y. Ishida, M. Muramatsu, M. Yamamoto, T. Honjo, Inhibition of carnitine palmitoyltransferase I augments sphingolipid synthesis and palmitate-induced apoptosis, J. Biol. Chem. 272 (1997) 3324^3329. W.M. Freeman, S.J. Walker, E.V. Vrana, Quantitative RT^ PCR: pitfalls and potential, BioTechniques 26 (1999) 112^ 125. F. Djouadi, C.J. Weinheimer, J.E. Sa¤tz, C. Pitchford, J. Bastin, F.J. Gonzalez, D.P. Kelly, A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator-activated receptor alpha-de¢cient mice, J. Clin. Invest. 102 (1998) 1083^1091. W. Wahli, O. Braissant, B. Desvergne, Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more, Chem. Biol. 2 (1995) 261^266. P.P. Koundakjian, D.M. Turnbull, A.J. Bone, S.I.M. Younen, H.S.A. Sherrat, Metabolic changes in fed rats caused by chronic administration of ethyl2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate, a new hypoglycaemic compound, Biochem. Pharmacol. 33 (1984) 465^473. G.M. Reaven, H. Chang, B.B. Ho¡man, Additive hypoglycaemic e¡ects of drugs that modify free-fatty-acid metabolism by di¡erent mechanism in rats with streptozocin-induced diabetes, Diabetes 37 (1998) 28^32. S. Samec, J. Seydoux, A.G. Dulloo, Skeletal muscle UCP3 and UCP2 gene expression in response to inhibition of free fatty acid £ux through mitochondrial beta-oxidation, Eur. J. Physiol. 438 (1999) 452^457.

[31] A. Cabrero, M. Alegret, R. Sa¨nchez, T. Adzet, J.C. Laguna, M. Va¨zquez, Sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2carboxylate, up-regulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes, Biochem. Biophys. Res. Commun. 263 (1999) 87^93. [32] R. Bressler, R. Gay, J.G. Copeland, J.J. Bahl, J. Bedotto, S. Goldman, Chronic inhibition of fatty acid oxidation: new model of diastolic dysfunction, Life Sci. 44 (1989) 1897^ 1906. [33] C. Mascaro¨, E. Acosta, J.A. Ortiz, P.F. Marrero, F.G. Hegardt, D. Haro, Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor, J. Biol. Chem. 273 (1998) 8560^ 8563. [34] J.M. Brandt, F. Djouadi, D.P. Kelly, Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferatoractivated receptor K, J. Biol. Chem. 273 (1998) 23786^ 23792. [35] B.A. Forman, J. Chen, R.M. Evans, Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta, Proc. Natl. Acad. Sci. USA 94 (1997) 4312^4317. [36] A. Cabrero, M. Alegret, R. Sa¨nchez, T. Adzet, J.C. Laguna, M. Va¨zquez, Down-regulation of uncoupling protein-3 and -2 by thiazolidinediones in C2C12 myotubes, FEBS Lett. 484 (2000) 37^42. [37] R.N. Kolesnick, M. Kro«nke, Regulation of ceramide production and apoptosis, Annu. Rev. Physiol. 60 (1998) 643^ 665.

BBAMCB 55796 9-7-01