Increased Reactive Oxygen Species Production Down-regulates Peroxisome Proliferator-activated α Pathway in C2C12 Skeletal Muscle Cells

Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor α (PPARα) mRNA expression, achieving an 87% reduction at 80 μm etomoxir. The mRNA levels of most of the PPARα target genes studied were reduced at 100 μm etomoxir. By using several inhibitors of de novo ceramide synthesis and C2-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARα mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-κB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-κB activity, etomoxir did not down-regulate PPARα mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-κB activation are responsible for the down-regulation of PPARα and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells. Generation of reactive oxygen species may contribute to the pathogenesis of diseases involving intracellular lipid accumulation. To explore the mechanisms leading to these pathologies we tested the effects of etomoxir, an inhibitor of carnitine palmitoyltransferase I which contains a fatty acid-derived structure, in C2C12 skeletal muscle cells. Etomoxir treatment for 24 h resulted in a down-regulation of peroxisome proliferator-activated receptor α (PPARα) mRNA expression, achieving an 87% reduction at 80 μm etomoxir. The mRNA levels of most of the PPARα target genes studied were reduced at 100 μm etomoxir. By using several inhibitors of de novo ceramide synthesis and C2-ceramide we showed that they were not involved in the effects of etomoxir. Interestingly, the addition of triacsin C, a potent inhibitor of acyl-CoA synthetase, to etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARα mRNA levels, suggesting that the active form of the drug, etomoxir-CoA, was not involved. Given that saturated fatty acids may generate reactive oxygen species (ROS), we determined whether the addition of etomoxir resulted in ROS generation. Etomoxir increased ROS production and the activity of the well known redox transcription factor NF-κB. In the presence of the pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of NF-κB activity, etomoxir did not down-regulate PPARα mRNA in C2C12 skeletal muscle cells. These results indicate that ROS generation and NF-κB activation are responsible for the down-regulation of PPARα and may provide a new mechanism by which intracellular lipid accumulation occurs in skeletal muscle cells. peroxisome proliferator-activated receptor α carnitine palmitoyltransferase I reactive oxygen species pyrrolidine dithiocarbamate acyl-CoA oxidase muscle-type carnitine palmitoyltransferase I uncoupling protein 3 medium chain acyl-CoA dehydrogenase adenosylphosphoribosyltransferase base pair nuclear factor κB acyl-CoA synthetase 2′,7′-dichlorofluorescein diacetate reverse transcription Skeletal muscle insulin resistance is the major characteristic of non-insulin-dependent diabetes mellitus (1DeFronzo R.A. Gunnarsson R. Bjorkman O. Olsson M. Wahren J. J. Clin. Invest. 1985; 76: 149-155Crossref PubMed Scopus (896) Google Scholar). The mechanisms responsible for the reduced sensitivity of muscle to insulin still remains unclear, but there is a strong correlation between insulin resistance and the presence of increased lipid levels in skeletal muscle (2McGarry J.D. Science. 1992; 258: 766-770Crossref PubMed Scopus (578) Google Scholar). Data from different studies are consistent with this idea. In humans it has been reported that insulin resistance correlates more tightly with intramyocellular lipid levels than with any other factor, including body mass index or percent body fat (3Jacob S. Machann J. Rett K. Brechtel K. Volk A. Renn W. Maerker E. Matthaei S. Schick F. Claussen C-D Häring H-U. Diabetes. 1999; 48: 113-119Google Scholar, 4Krssak M Petersen K.F. Dresner A. Dipietro L. Vogel S.M. Rothman D.L. Shulman G. Roden M. Diabetologia. 1999; 42: 113-116Crossref PubMed Scopus (1028) Google Scholar, 5Perseghin G. De Scifo P. Cobelli F. Pagliato E. Battezzati A. Arcelloni C. Vanzulli A. Testolin G. Pozza G. Del Maschio A. Luzi L. Diabetes. 1999; 48: 1600-1606Crossref PubMed Scopus (755) Google Scholar). Moreover, insulin resistance appears after exposure of rat skeletal muscle to elevated free fatty acids, which is associated with the accumulation of fatty acyl-CoA (6Oakes N.D. Cooney G.J. Camilleri S. Chisholm D.J. Kraegen E.W. Diabetes. 1997; 46: 1768-1774Crossref PubMed Google Scholar) and triglycerides (7Storlien L.H. Jenkins A.B. Chisholm D.J. Pascoe W.S. Khouri S. Kraegen E.W. Diabetes. 1991; 40: 280-289Crossref PubMed Scopus (955) Google Scholar, 8Jucker B. Cline G.W. Barucci N. Shulman G. Diabetes. 1999; 48: 134-140Crossref PubMed Scopus (105) Google Scholar). Similar results have been obtained with cells exposed to increased lipid levels (9Schmitz-Peiffer C. Craig D.L. Dbiden T.J. J. Biol. Chem. 1999; 274: 24202-24210Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Fatty acid catabolism is mainly regulated by the peroxisome proliferator-activated receptor α (PPARα)1 (10Sack M.N. Disch D.L. Rockman H.A. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6438-6443Crossref PubMed Scopus (177) Google Scholar). This PPAR subtype is expressed primarily in tissues with a high level of fatty acid catabolism such as liver, kidney, heart, and skeletal muscle (11Braissant O. Foufelle F. Scotto C. Dauça M. Wahli W. Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1737) Google Scholar,12Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2746) Google Scholar). To be transcriptional active, PPARα needs to heterodimerize with the retinoid X receptor. PPARα-retinoid X receptor heterodimers bind to DNA specific sequences called peroxisome proliferator-response elements consisting of an imperfect direct repeat of the consensus binding site for nuclear hormone receptors (AGGTCA) separated by one nucleotide (DR-1). Recently it has been reported that PPARα activation in skeletal muscle increases expression of enzymes involved in fatty acid β oxidation. These changes lead to prevention of diet-induced obesity and insulin resistance (13Ye J-M. Doyle P.J. Iglesias M.A. Watson D.G. Cooney G.J. Kraegen E.W. Diabetes. 2001; 50: 411-417Crossref PubMed Scopus (327) Google Scholar). On the other hand, hypertrophic growth in cardiac myocytes showed reduced PPARα expression. and its activity is altered at the transcriptional level via the extracellular signal-regulated kinase mitogen-activated protein kinase pathway. These hypertrophied myocytes, with reduced PPARα expression, showed a reduced capacity for cellular lipid homeostasis, resulting in intracellular lipid accumulation in response to fatty acids (14Barger P.M. Brandt J.M. Leone T.C. Weinheimer C.J. Kelly D.P. J. Clin. Invest. 2000; 105: 1723-1730Crossref PubMed Scopus (405) Google Scholar). To gain a better understanding of the mechanism by which exposure of skeletal muscle cells to fatty acids results in lipid accumulation, we have used the myoblast C2C12 cell line, which develops biochemical and morphological properties characteristic of skeletal muscle and has been proven useful for studies of skeletal muscle metabolism (15McMahon D.K. Anderson P.A. Nassar R. Bunting J.B. Saba Z. Oakeley A.E. Malouf N.N. Am. J. Physiol. 1994; 266: C1795-C1802Crossref PubMed Google Scholar, 16Gauthier-Rouviere C. Vandromme M. Tuil D. Lantredou N. Morris M. Soulez M. Kahn A. Fernandez A. Lamb N. Mol. Biol. Cell. 1996; 7: 719-729Crossref PubMed Scopus (73) Google Scholar). To promote fatty acyl-CoA accumulation in these cells, we have taken advantage of the use of etomoxir. Etomoxir is an irreversible inhibitor of CPT-I and. therefore, of fatty acid β-oxidation (17Wolf H.P.O. Horm. Metab. Res. 1992; 26: 62-67Google Scholar). Treatment of C2C12 skeletal muscle cells for 24 h with etomoxir strongly decreased PPARα mRNA levels. Overall, PPARα target genes showed a biphasic response to etomoxir, with small inductions in their expression at low etomoxir concentrations and reductions at higher concentrations. Surprisingly, the effects of etomoxir on PPARα expression were not mediated through CPT-I inhibition, since inhibition of the formation of the active form of the drug, etomoxir-CoA, did not prevent its effects. In contrast, we show that treatment with etomoxir, which contains a saturated fatty acid-derived structure with an oxirane group, resulted in the generation of reactive oxygen species (ROS) and nuclear factor κB (NF-κB) activation. Interestingly, in the presence of the pyrrolidine dithiocarbamate (PDTC), a potent antioxidant and inhibitor of NF-κB activity, etomoxir did not down-regulate PPARα expression in C2C12 skeletal muscle cells. These results indicate that intracellular ROS generation and increased NF-κB activity leads to a down-regulation of the fatty acid oxidation metabolism mediated by PPARα, which in turns may result in intracellular lipid accumulation in skeletal muscle cells. Etomoxir (2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylic acid) was obtained from Dr. Horst Wolf (Germany). C2-ceramide, fumonisin B1, ISP1, and PDTC were from Sigma. PD98059 and triacsin C were fromBiomol Research Labs Inc. (Plymouth Meeting, PA), and 2′,7′-dichlorofluorescein diacetate (DCFH) was purchased from Serva (Heidelberg, Germany). Other chemicals were from Sigma. Mouse C2C12 myoblasts (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. When cells reached confluence, the medium was switched to the differentiation medium containing Dulbecco's modified Eagle's medium and 2% horse serum, which was changed every other day. After 4 additional days, the differentiated C2C12 cells had fused into myotubes, which were then treated in serum-free Dulbecco's modified Eagle's medium with either vehicle (0.1% methanol) or etomoxir. After the incubation, RNA was extracted from myotubes as described below. Total RNA was isolated by using the Ultraspec reagent (Biotecx). Relative levels of specific mRNAs were assessed by reverse transcription (RT)-PCR. Complementary DNA was synthesized from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers as primers in the presence of 50 mm Tris-HCl buffer, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol, 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen), units of (Invitrogen), and in a total of were at for of the reverse transcription was then used for with specific of the reverse transcription mm MgCl2, 200 μm of 1 of μg of and mm pH To and were separated from primers and by using a of at The of the and primers used for acyl-CoA oxidase and uncoupling protein 3 and and medium chain acyl-CoA dehydrogenase and PPARα, and and adenosylphosphoribosyltransferase and was in an M. J. Research with a and After an for 1 at was for or of at for 1 at for and at for 1 and 50 at was of was on a The were and to using to show the DNA of a of the base PPARα, were with of to of for the genes was in and 1999; 26: PubMed Scopus Google Scholar). were by The results for the expression of specific mRNAs are to the expression of the were isolated to and Res. 1991; PubMed Scopus Google Scholar). were into of for 10 and in of mm pH at 4 mm MgCl2, 10 mm KCl, mm dithiothreitol, mm μg/ml and μg/ml by the were to on for 10 and then for 10 samples were for 10 and the was were in 50 of C pH at 4 mm mm MgCl2, mm mm dithiothreitol, mm μg/ml and μg/ml and on for for high was by for at 4 and the DNA binding was at was determined by using the PubMed Scopus Google Scholar). was using for the consensus binding site of the NF-κB nucleotide were with 1 of of kinase buffer, units of and 3 of at 10 at for 1 The was by of mm pH and 1 mm To the from the the was in a to the of nuclear were for 10 on in binding mm pH mm KCl, mm dithiothreitol, mm pH 100 μg/ml and 50 μg/ml in a of was and the was for at specific was the and for 10 on was with the at 4 were by at 4 on a and to was on a cells were with in and with for cells were in and then in a containing and in cells were at for in the and by After 24 h of treatment with either vehicle (0.1% or 80 μm etomoxir with or mm the cells were into and the was in 1 with and for 1 h at in the with 10 μm cells were and in and cell was assessed by using After with cells were in the at 4 and was in a of were and are expressed as of were by or of to the of When were the was the were using the C2C12 myoblasts differentiated to into myotubes in the presence of 2% horse 4 after The effects of different concentrations of etomoxir on PPARα mRNA expression were assessed in C2C12 myotubes for 24 in serum-free these concentrations of etomoxir from 10 to 100 μm strongly decreased PPARα mRNA levels low etomoxir from 10 to the reduction in PPARα mRNA levels was with to the 80 etomoxir reduced PPARα mRNA levels by 87% To whether PPARα reduction resulted in down-regulation of genes regulated by this transcription factor, we the levels of several well known PPARα target and B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2746) Google Scholar). The effects of the different concentrations of etomoxir on expression, which the of β-oxidation of fatty acids, showed a to that reported for PPARα 1 low concentrations of etomoxir changes in mRNA levels were with to the at 80 a reduction was in In to the effects on mRNA levels, etomoxir treatment increased mRNA levels, achieving a at 80 which is consistent with the reported in in after etomoxir treatment J.M. F. Kelly D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (405) Google Scholar). in mRNA levels was at 100 μm etomoxir, with to the cells. The mRNA levels of an a in the were not modified by etomoxir at concentrations from 10 to In contrast, at higher etomoxir a in levels, achieving an and a reduction at 80 and 100 a that may be involved in fatty acid was by low concentrations of etomoxir, a of at 80 μm was with to the 100 μm etomoxir a Etomoxir treatment did not the mRNA expression of that the effects of this etomoxir were specific for the PPARα not CPT-I inhibition by etomoxir the of into leading to its accumulation in the is a of etomoxir treatment may result in ceramide synthesis and M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). to gain into the mechanism by which etomoxir PPARα mRNA levels, we tested the effects of several inhibitors of de novo ceramide The in ceramide synthesis is the formation of from and This is by the at concentrations S. Res. PubMed Scopus Google Scholar). fumonisin ceramide synthetase activity E. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google the in de of fumonisin treatment the down-regulation in PPARα mRNA levels by etomoxir To the of in the down-regulation of PPARα by etomoxir, we treated C2C12 skeletal muscle cells with C2-ceramide, a ceramide for 24 The addition of μm C2-ceramide did not PPARα mRNA levels These that de novo ceramide synthesis is not involved in the effects of etomoxir. In etomoxir treatment did not as by the of nuclear changes were in DNA formation after etomoxir treatment not chain fatty acids be by acyl-CoA synthetase to be as CPT-I Moreover, in cells etomoxir is to etomoxir-CoA, which is the active form of the drug, and it is by this K. Scopus Google Scholar). To whether the effects of etomoxir were mediated through etomoxir-CoA, we tested the effects of a potent inhibitor of activity, triacsin C. The addition of triacsin C to the etomoxir-treated C2C12 skeletal muscle cells did not prevent the down-regulation in PPARα mRNA levels suggesting that etomoxir and not was responsible for the effects by this Interestingly, triacsin C reduced PPARα mRNA levels by the effects of 80 μm etomoxir on and mRNA levels were not altered by triacsin C and Treatment with triacsin C a and in and mRNA levels, which is consistent with the activation of PPARα by free fatty acids R. J. J. PubMed Scopus Google Scholar). has been that cardiac hypertrophic PPARα activity is reduced at the levels of expression as well as by effects involving by the extracellular signal-regulated kinase mitogen-activated protein kinase (14Barger P.M. Brandt J.M. Leone T.C. Weinheimer C.J. Kelly D.P. J. Clin. Invest. 2000; 105: 1723-1730Crossref PubMed Scopus (405) Google Scholar). to whether was involved in the of PPARα by etomoxir, we studied the of a known inhibitor of the extracellular signal-regulated kinase mitogen-activated protein kinase on the mRNA expression of PPARα and several of its target genes after etomoxir treatment In the presence of the reduction in PPARα mRNA levels by etomoxir did not PD98059 did not the reduction in mRNA levels, the 87% reduction in mRNA to a reduction to etomoxir-treated These results that was not involved in the effects by etomoxir in the PPARα it expression. we to the mechanism etomoxir treatment results in a reduction in PPARα mRNA levels. etomoxir contains a saturated fatty acid-derived structure with an oxirane and saturated fatty acids such as generate ROS J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google through protein kinase activation of P. F. M. M. M. Diabetes. 2000; PubMed Scopus Google we determined whether etomoxir addition resulted in ROS generation. To we used which increased with intracellular oxygen which be by of C2C12 skeletal muscle cells with 80 resulted in an in that reactive oxygen were by etomoxir treatment was in of total cell with the 2% in cells. The addition of mm a potent to etomoxir-treated cells resulted in a in to to in cells. These that etomoxir treatment leads to ROS generation. etomoxir treatment of C2C12 skeletal muscle cells results in the generation of ROS and PDTC NF-κB activity N. Am. J. 1997; Google we studied whether this well known transcription factor was involved in the changes by etomoxir. that NF-κB with nuclear I to of the DNA binding was assessed in by an of NF-κB NF-κB binding activity mainly of specific I increased in nuclear from etomoxir-treated cells. The addition of that this to the NF-κB to whether the generation of ROS is for the changes in the PPARα we the of the antioxidant PDTC to PPARα mRNA down-regulation in C2C12 skeletal muscle cells. In the presence of mm etomoxir was to the mRNA expression of PPARα the addition of which ROS generation and NF-κB activation by etomoxir, the reduction in PPARα mRNA expression. The results that PPARα down-regulation after etomoxir treatment in C2C12 skeletal muscle cells is mediated by ROS generation and increased NF-κB The reduction in PPARα expression was by a potent antioxidant and inhibitor of NF-κB. This that ROS generation and NF-κB are involved in the in PPARα expression. studies that reductions in PPARα mRNA levels are mediated through cellular redox and NF-κB activation R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the and have been to a reduction in the expression of PPARα M. L. M. P. 1997; PubMed Scopus Google K. A. D. 1997; PubMed Scopus Google Scholar). These are at high levels in cells from R.A. C. J. Google which reduced mRNA levels of and genes R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). When were to an in the mRNA levels of PPARα and in was to in R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we show that an in the cellular redox in skeletal muscle cells results in a in PPARα mRNA levels. In addition to the effects of NF-κB on PPARα levels, a transcriptional has been reported between PPARα and the of NF-κB P. K. S. W. J.M. A. G. B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). PPARα of a peroxisome response in cells, and it was that between PPARα and occurs mainly via the binding of PPARα P. K. S. W. J.M. A. G. B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). to these mechanisms is that the in NF-κB activity by ROS generation after etomoxir treatment may PPARα mRNA expression and PPARα that ROS generation and NF-κB activation may contribute to the accumulation of intracellular lipid accumulation in skeletal muscle cells. Etomoxir to the of CPT-I which PPARα, and their transcriptional activity with their to bind this nuclear receptor J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). The mechanism by which these PPARα, direct binding to this receptor as a may lead to the accumulation of fatty acid in the C2C12 skeletal muscle cells were fatty acids, accumulation of fatty acyl-CoA these be PPARα activation by etomoxir in C2C12 skeletal muscle cells be to direct binding to this receptor. Interestingly, show a different of etomoxir on the of etomoxir low concentrations from 10 to μm the PPARα reduction in mRNA levels was this reduction was not to the transcriptional of several PPARα target genes such as or other PPARα target genes such as or were not When the concentrations of etomoxir used were higher than the down-regulation in PPARα mRNA levels was of such that a in the mRNA expression of the PPARα target genes studied was the indicate that at low the reduction in PPARα expression is etomoxir PPARα target at higher mechanism leading to a in PPARα expression, and as a the expression of its target genes is has been reported that CPT-I inhibition by etomoxir results in cell and to a in ceramide synthesis in and cells M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). we determined whether the effects of etomoxir on PPARα mRNA levels were the result of cell through ceramide By using inhibitors of the de novo ceramide synthesis and a ceramide we have that were not involved in the effects of etomoxir on PPARα In etomoxir treatment did not result in increased In in the of M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google the addition of etomoxir at a of μm did not cell and 200 μm etomoxir did not cell increased nuclear in cells. In contrast, the addition of etomoxir and resulted in a in DNA formation and nuclear In C2C12 skeletal muscle cells were treated with etomoxir in the of fatty as it is and not contribute to the effects by etomoxir on PPARα expression. On the we have reported A. A. R. M. 2001; PubMed Scopus Google Scholar) that μm etomoxir and mRNA levels in C2C12 skeletal muscle cells. Given that C2-ceramide treatment a in the expression of these we that de synthesis be the mechanism the in and by etomoxir ceramide de novo synthesis is not involved in the down-regulation of PPARα after etomoxir it may be in the of and after treatment with etomoxir. chain fatty acids are not as CPT-I they are by acyl-CoA The of etomoxir to CPT-I activity on this which the active form of the drug, Surprisingly, the effects of etomoxir were not in the presence of the acyl-CoA synthetase triacsin C. These results show that the effects of etomoxir not on acyl-CoA synthetase to gain to the suggesting that CPT-I inhibition is not involved in the effects of this on PPARα expression. remains to whether etomoxir treatment may result in ROS generation in at the of for CPT-I activity in rat is μm M. N. J. 2000; PubMed Scopus Google it is that etomoxir at to ROS generation. is to that generation of of ceramide is for the response and may contribute to the pathogenesis of diseases involving intracellular lipid accumulation J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we a mechanism through which the inhibition of the PPARα by ROS and NF-κB activation may contribute to intracellular lipid accumulation in skeletal muscle cells. cellular with saturated and fatty acids an in ROS J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M.A. J. D. Res. 1999; PubMed Scopus Google Scholar) and binding C. M.A. J. D. Res. 1999; PubMed Scopus Google it remains to whether skeletal muscle to elevated free fatty acids results in PPARα of the of for