Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate's protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity. The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate's protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity. IntroductionIt has long been recognized that increased plasma free fatty acids are associated with insulin resistance in humans (1Schalch D.S. Kipnis D.M. J. Clin. Invest. 1965; 44: 2010-2020Crossref PubMed Scopus (123) Google Scholar). Indeed, plasma free fatty acid concentrations are increased in obese subjects (2Adams 2nd, J.M. Pratipanawatr T. Berria R. Wang E. DeFronzo R.A. Sullards M.C. Mandarino L.J. Diabetes. 2004; 53: 25-31Crossref PubMed Scopus (511) Google Scholar) as well as in genetically obese or high fat diet-induced insulin-resistant mice (3Dentin R. Benhamed F. Hainault I. Fauveau V. Foufelle F. Dyck J.R. Girard J. Postic C. Diabetes. 2006; 55: 2159-2170Crossref PubMed Scopus (315) Google Scholar, 4Bonnard C. Durand A. Peyrol S. Chanseaume E. Chauvin M.A. Morio B. Vidal H. Rieusset J. J. Clin. Invest. 2008; 118: 789-800Crossref PubMed Scopus (639) Google Scholar). In such conditions, circulating free fatty acid concentrations are elevated, and FA 4The abbreviations used are: FAfatty acid(s)ASPacid-soluble product(s)CCCPcarbonyl cyanide m-chlorophenyl hydrazoneCPT1mtmutant CPT1A M593SDAGdiacylglycerol(s)LacZβ-galactosidaseLCFAlong-chain fatty acidMUFAmonounsaturated fatty acid(s)OApreincubation with oleatePLphospholipid(s)SFAsaturated fatty acid(s)SMsphingomyelin(s)TGtriglyceride(s)G55 mm glucoseG20+I20 mm glucose plus 100 nm insulin. metabolism is altered (5McGarry J.D. Diabetes. 2002; 51: 7-18Crossref PubMed Scopus (1205) Google Scholar, 6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar, 7Kelley D.E. Goodpaster B. Wing R.R. Simoneau J.A. Am. J. Physiol. 1999; 277: E1130-E1141Crossref PubMed Google Scholar), leading to ectopic accumulation of FA in the liver, pancreatic β-cells, or skeletal muscle, where they interfere with normal cell function. For instance, FA overload induces skeletal muscle insulin resistance (6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar), inflammation (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), and cell death via apoptosis (4Bonnard C. Durand A. Peyrol S. Chanseaume E. Chauvin M.A. Morio B. Vidal H. Rieusset J. J. Clin. Invest. 2008; 118: 789-800Crossref PubMed Scopus (639) Google Scholar, 6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar), a phenomenon commonly referred as “lipotoxicity.” The toxic effects of FA are known to depend on their chain length and degree of saturation. Long-chain saturated FA (SFA), such as palmitate (C16:0) and stearate (C18:0), are the most lipotoxic. Consistently, palmitate induces apoptosis in many cell types (9Sparagna G.C. Hickson-Bick D.L. Buja L.M. McMillin J.B. Antioxid. Redox Signal. 2001; 3: 71-79Crossref PubMed Scopus (36) Google Scholar, 10Shimabukuro M. Zhou Y.T. Levi M. Unger R.H. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 2498-2502Crossref PubMed Scopus (1006) Google Scholar, 11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar, 12Paumen M.B. Ishida Y. Muramatsu M. Yamamoto M. Honjo T. J. Biol. Chem. 1997; 272: 3324-3329Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). In muscle cells, palmitate's cytotoxic effect is mediated by increased intracellular concentrations of diacylglycerols (DAG) and ceramides (11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar). In contrast, monounsaturated FA (MUFA), such as oleate (C18:1), protect against SFA-induced toxicity (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 13Miller T.A. LeBrasseur N.K. Cote G.M. Trucillo M.P. Pimentel D.R. Ido Y. Ruderman N.B. Sawyer D.B. Biochem. Biophys. Res. Commun. 2005; 336: 309-315Crossref PubMed Scopus (125) Google Scholar, 14Chavez J.A. Summers S.A. Arch. Biochem. Biophys. 2003; 419: 101-109Crossref PubMed Scopus (380) Google Scholar). Whether oleate exerts such a protective effect on palmitate-induced apoptosis in skeletal muscle cells has not been reported.The mechanisms by which oleate protects cells from palmitate toxicity are not well understood. SFA, which are reported to be less efficiently incorporated into triglycerides (TG) than MUFA, lead to increased accumulation of DAG (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 14Chavez J.A. Summers S.A. Arch. Biochem. Biophys. 2003; 419: 101-109Crossref PubMed Scopus (380) Google Scholar, 15Montell E. Turini M. Marotta M. Roberts M. Noé V. Ciudad C.J. Macé K. Gómez-Foix A.M. Am. J. Physiol. Endocrinol. Metab. 2001; 280: E229-E237Crossref PubMed Google Scholar). Oleate has been proposed to protect cells from palmitate-induced lipotoxicity by promoting its esterification into TG, a neutral form of FA storage (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 15Montell E. Turini M. Marotta M. Roberts M. Noé V. Ciudad C.J. Macé K. Gómez-Foix A.M. Am. J. Physiol. Endocrinol. Metab. 2001; 280: E229-E237Crossref PubMed Google Scholar, 16Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3077-3082Crossref PubMed Scopus (1366) Google Scholar). However, it was recently hypothesized that oleate protects from palmitate-induced insulin resistance and inflammation by increasing its mitochondrial oxidation (as shown by increased CPT1 (carnitine palmitoyltransferase 1) gene expression) (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). CPT1 is a transmembrane enzyme of the mitochondrial outer membrane, which converts long-chain acyl-CoA to acylcarnitine, which enters the mitochondrial matrix and undergoes β-oxidation. Because of its inhibition by malonyl-CoA, an intermediate of lipogenesis synthesized by acetyl-CoA carboxylase (ACC), CPT1 is the key regulatory enzyme of long-chain fatty acid (LCFA) β-oxidation (17McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar). CPT1 exists in at least two isoforms, CPT1A (liver isoform) and CPT1B (muscle isoform), each of which is found in several tissue and cell types (17McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 18Perdomo G. Commerford S.R. Richard A.M. Adams S.H. Corkey B.E. O'Doherty R.M. Brown N.F. J. Biol. Chem. 2004; 279: 27177-27186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).Whether defects in muscle mitochondrial metabolism are a cause or a consequence of insulin resistance has been extensively investigated but remains controversial. In rodents, some argue against the concept that insulin resistance is mediated by muscle mitochondrial dysfunction (19Turner N. Bruce C.R. Beale S.M. Hoehn K.L. So T. Rolph M.S. Cooney G.J. Diabetes. 2007; 56: 2085-2092Crossref PubMed Scopus (417) Google Scholar, 20Hancock C.R. Han D.H. Chen M. Terada S. Yasuda T. Wright D.C. Holloszy J.O. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7815-7820Crossref PubMed Scopus (396) Google Scholar). Others have demonstrated that skeletal muscles from obese and insulin-resistant patients exhibit diminished rates of palmitate oxidation, in association with a decrease in CPT1 activity (21Kim J.Y. Hickner R.C. Cortright R.L. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2000; 279: PubMed Google Scholar). and insulin-resistant skeletal muscles have and which decrease FA D.E. J. Diabetes. 2002; 51: PubMed Scopus Google Scholar, J. S. D.E. Diabetes. 2001; PubMed Scopus Google Scholar). Because accumulation of muscle cells is associated with mitochondrial FA increasing LCFA oxidation a protective have been reported the of a of mitochondrial LCFA oxidation on palmitate-induced apoptosis in (11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar, D.E. J. Diabetes. 2002; 51: PubMed Scopus Google Scholar) and pancreatic T.A. LeBrasseur N.K. Cote G.M. Trucillo M.P. Pimentel D.R. Ido Y. Ruderman N.B. Sawyer D.B. Biochem. Biophys. Res. Commun. 2005; 336: 309-315Crossref PubMed Scopus (125) Google Scholar, J.Y. Am. J. Physiol. Physiol. 2002; PubMed Scopus Google Scholar, E. G. Biochem. Biophys. Res. Commun. 2008; PubMed Scopus Google Scholar). This has been in skeletal muscle the we aimed to oleate protects skeletal muscle cells from palmitate-induced apoptosis and to examine the role of mitochondrial LCFA in mediating such effect of we a mutant form of CPT1A which is active but insensitive to malonyl-CoA inhibition M. A. E. N. A. G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), in C2C12 The and of an increased LCFA oxidation through CPT1mt expression with the effects of preincubation with oleate to the mechanisms underlying oleate's protective effect and to of mitochondrial LCFA oxidation lead to the same protective effect as that oleate preincubation protects skeletal muscle cells from palmitate-induced apoptosis by on its metabolism through two the of palmitate toward and palmitate targeting mitochondrial LCFA oxidation via the expression of a CPT1 (CPT1mt), has a protective of palmitate metabolism toward oxidation to be sufficient against its lipotoxic effect with (11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar, G.L. 2007; PubMed Scopus Google Scholar), we showed that palmitate apoptosis in C2C12 of myotubes to palmitate promoted intracellular accumulation of with at least two and DAG and and ceramides (11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar), deleterious result from enhanced than from content was by palmitate palmitate increased and MUFA, leading to a have that palmitate-induced DAG and accumulation result from a esterification flux palmitate than oleate E. Turini M. Marotta M. Roberts M. Noé V. Ciudad C.J. Macé K. Gómez-Foix A.M. Am. J. Physiol. Endocrinol. Metab. 2001; 280: E229-E237Crossref PubMed Google Scholar, 16Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3077-3082Crossref PubMed Scopus (1366) Google Scholar), as we However, that palmitate is less to oxidation than oleate, which its metabolism toward found that preincubation with oleate palmitate esterification into DAG and of palmitate in a However, oleate preincubation promoted intracellular in at least and a palmitate This from an enhanced expression of is than S. Zhou E. B. T. Farese Jr., R.V. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, oleate preincubation and DAG that (i.e. and DAG that preincubation with oleate DAG to with the that oleate preincubation palmitate content and increased the preincubation with oleate content in cells, which be to gene Because oleate is a of we hypothesized that it a on gene has been reported that oleate preincubation led to an in CPT1 in skeletal muscle cells (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, M. M. Diabetes. PubMed Scopus Google Scholar). We demonstrated that oleate preincubation enhanced mitochondrial oxidation of as a consequence of increased CPT1 gene and protein that preincubation with oleate mitochondrial LCFA oxidation through a of which leads to inhibition to increased CPT1 investigated on mitochondrial oxidation, by leads to the same protective effect as oleate In with the of G. Am. J. Physiol. Endocrinol. Metab. 2007; PubMed Scopus Google Scholar), we showed that CPT1mt expression an in LCFA oxidation at the of their oleate preincubation, DAG and are by CPT1mt In contrast to preincubation with oleate, CPT1mt expression the of esterification of palmitate into the two by palmitate) and on the or content of FA in contrast with of G. Commerford S.R. Richard A.M. Adams S.H. Corkey B.E. O'Doherty R.M. Brown N.F. J. Biol. Chem. 2004; 279: 27177-27186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), reported that expression of the CPT1 not TG, or This the of malonyl-CoA in the of CPT1 has been that increased mitochondrial LCFA oxidation lead to an accumulation of D.M. Biochem. 2006; PubMed Scopus Google Scholar). However, in the palmitate was not CPT1mt expression, in the presence of glucose and high This that CPT1mt expression the of oxidation of accumulation of is well that skeletal muscle insulin resistance is associated with of CPT1 by an increased in skeletal muscle and insulin resistance in high R.L. B. V. J. J.D. Diabetes. 2001; PubMed Scopus Google Scholar), that in LCFA flux into is the deleterious effect of on insulin Furthermore, it has been recently reported that in CPT1 in skeletal muscle enhanced mitochondrial LCFA oxidation and high fat diet-induced insulin resistance C.R. C. N. Cooney G.J. Am. J. Physiol. Endocrinol. Metab. 2007; PubMed Scopus Google Scholar, C.R. N. Watt M.J. K. Cooney G.J. Febbraio M.A. Diabetes. PubMed Scopus Google Scholar). effects associated with a decrease in and palmitate into We that effects be to the expression of which is to malonyl-CoA Indeed, it has been shown in muscle from obese and subjects that malonyl-CoA is leading to a decrease in mitochondrial LCFA oxidation C.R. C. N. Cooney G.J. Am. J. Physiol. Endocrinol. Metab. 2007; PubMed Scopus Google Scholar, C.R. N. Watt M.J. K. Cooney G.J. Febbraio M.A. Diabetes. PubMed Scopus Google Scholar). we demonstrated that targeting mitochondrial LCFA oxidation, via the expression of a led to the same protective effect as preincubation with oleate, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced apoptosis and insulin In conclusion, we that increased CPT1 activity with malonyl-CoA be a the of the deleterious effects of accumulation by insulin or IntroductionIt has long been recognized that increased plasma free fatty acids are associated with insulin resistance in humans (1Schalch D.S. Kipnis D.M. J. Clin. Invest. 1965; 44: 2010-2020Crossref PubMed Scopus (123) Google Scholar). Indeed, plasma free fatty acid concentrations are increased in obese subjects (2Adams 2nd, J.M. Pratipanawatr T. Berria R. Wang E. DeFronzo R.A. Sullards M.C. Mandarino L.J. Diabetes. 2004; 53: 25-31Crossref PubMed Scopus (511) Google Scholar) as well as in genetically obese or high fat diet-induced insulin-resistant mice (3Dentin R. Benhamed F. Hainault I. Fauveau V. Foufelle F. Dyck J.R. Girard J. Postic C. Diabetes. 2006; 55: 2159-2170Crossref PubMed Scopus (315) Google Scholar, 4Bonnard C. Durand A. Peyrol S. Chanseaume E. Chauvin M.A. Morio B. Vidal H. Rieusset J. J. Clin. Invest. 2008; 118: 789-800Crossref PubMed Scopus (639) Google Scholar). In such conditions, circulating free fatty acid concentrations are elevated, and FA 4The abbreviations used are: FAfatty acid(s)ASPacid-soluble product(s)CCCPcarbonyl cyanide m-chlorophenyl hydrazoneCPT1mtmutant CPT1A M593SDAGdiacylglycerol(s)LacZβ-galactosidaseLCFAlong-chain fatty acidMUFAmonounsaturated fatty acid(s)OApreincubation with oleatePLphospholipid(s)SFAsaturated fatty acid(s)SMsphingomyelin(s)TGtriglyceride(s)G55 mm glucoseG20+I20 mm glucose plus 100 nm insulin. metabolism is altered (5McGarry J.D. Diabetes. 2002; 51: 7-18Crossref PubMed Scopus (1205) Google Scholar, 6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar, 7Kelley D.E. Goodpaster B. Wing R.R. Simoneau J.A. Am. J. Physiol. 1999; 277: E1130-E1141Crossref PubMed Google Scholar), leading to ectopic accumulation of FA in the liver, pancreatic β-cells, or skeletal muscle, where they interfere with normal cell function. For instance, FA overload induces skeletal muscle insulin resistance (6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar), inflammation (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), and cell death via apoptosis (4Bonnard C. Durand A. Peyrol S. Chanseaume E. Chauvin M.A. Morio B. Vidal H. Rieusset J. J. Clin. Invest. 2008; 118: 789-800Crossref PubMed Scopus (639) Google Scholar, 6Hulver M.W. Berggren J.R. Cortright R.N. Dudek R.W. Thompson R.P. Pories W.J. MacDonald K.G. Cline G.W. Shulman G.I. Dohm G.L. Houmard J.A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E741-E747Crossref PubMed Scopus (276) Google Scholar), a phenomenon commonly referred as “lipotoxicity.” The toxic effects of FA are known to depend on their chain length and degree of saturation. Long-chain saturated FA (SFA), such as palmitate (C16:0) and stearate (C18:0), are the most lipotoxic. Consistently, palmitate induces apoptosis in many cell types (9Sparagna G.C. Hickson-Bick D.L. Buja L.M. McMillin J.B. Antioxid. Redox Signal. 2001; 3: 71-79Crossref PubMed Scopus (36) Google Scholar, 10Shimabukuro M. Zhou Y.T. Levi M. Unger R.H. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 2498-2502Crossref PubMed Scopus (1006) Google Scholar, 11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar, 12Paumen M.B. Ishida Y. Muramatsu M. Yamamoto M. Honjo T. J. Biol. Chem. 1997; 272: 3324-3329Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). In muscle cells, palmitate's cytotoxic effect is mediated by increased intracellular concentrations of diacylglycerols (DAG) and ceramides (11Turpin S.M. Lancaster G.I. Darby I. Febbraio M.A. Watt M.J. Am. J. Physiol. Endocrinol. Metab. 2006; 291: E1341-E1350Crossref PubMed Scopus (133) Google Scholar). In contrast, monounsaturated FA (MUFA), such as oleate (C18:1), protect against SFA-induced toxicity (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 13Miller T.A. LeBrasseur N.K. Cote G.M. Trucillo M.P. Pimentel D.R. Ido Y. Ruderman N.B. Sawyer D.B. Biochem. Biophys. Res. Commun. 2005; 336: 309-315Crossref PubMed Scopus (125) Google Scholar, 14Chavez J.A. Summers S.A. Arch. Biochem. Biophys. 2003; 419: 101-109Crossref PubMed Scopus (380) Google Scholar). Whether oleate exerts such a protective effect on palmitate-induced apoptosis in skeletal muscle cells has not been reported.The mechanisms by which oleate protects cells from palmitate toxicity are not well understood. SFA, which are reported to be less efficiently incorporated into triglycerides (TG) than MUFA, lead to increased accumulation of DAG (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 14Chavez J.A. Summers S.A. Arch. Biochem. Biophys. 2003; 419: 101-109Crossref PubMed Scopus (380) Google Scholar, 15Montell E. Turini M. Marotta M. Roberts M. Noé V. Ciudad C.J. Macé K. Gómez-Foix A.M. Am. J. Physiol. Endocrinol. Metab. 2001; 280: E229-E237Crossref PubMed Google Scholar). Oleate has been proposed to protect cells from palmitate-induced lipotoxicity by promoting its esterification into TG, a neutral form of FA storage (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 15Montell E. Turini M. Marotta M. Roberts M. Noé V. Ciudad C.J. Macé K. Gómez-Foix A.M. Am. J. Physiol. Endocrinol. Metab. 2001; 280: E229-E237Crossref PubMed Google Scholar, 16Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3077-3082Crossref PubMed Scopus (1366) Google Scholar). However, it was recently hypothesized that oleate protects from palmitate-induced insulin resistance and inflammation by increasing its mitochondrial oxidation (as shown by increased CPT1 (carnitine palmitoyltransferase 1) gene expression) (8Coll T. Eyre E. Rodríguez-Calvo R. Palomer X. Sánchez R.M. Merlos M. Laguna J.C. Vázquez-Carrera M. J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). CPT1 is a transmembrane enzyme of the mitochondrial outer membrane, which converts long-chain acyl-CoA to acylcarnitine, which enters the mitochondrial matrix and undergoes β-oxidation. Because of its inhibition by malonyl-CoA, an intermediate of lipogenesis synthesized by acetyl-CoA carboxylase (ACC), CPT1 is the key regulatory enzyme of long-chain fatty acid (LCFA) β-oxidation (17McGarry J.D. Brown N.F. Eur. J. 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J. Diabetes. 2002; 51: PubMed Scopus Google Scholar) and pancreatic T.A. LeBrasseur N.K. Cote G.M. Trucillo M.P. Pimentel D.R. Ido Y. Ruderman N.B. Sawyer D.B. Biochem. Biophys. Res. Commun. 2005; 336: 309-315Crossref PubMed Scopus (125) Google Scholar, J.Y. Am. J. Physiol. Physiol. 2002; PubMed Scopus Google Scholar, E. G. Biochem. Biophys. Res. Commun. 2008; PubMed Scopus Google Scholar). This has been in skeletal muscle the we aimed to oleate protects skeletal muscle cells from palmitate-induced apoptosis and to examine the role of mitochondrial LCFA in mediating such effect of we a mutant form of CPT1A which is active but insensitive to malonyl-CoA inhibition M. A. E. N. A. G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), in C2C12 The and of an increased LCFA oxidation through CPT1mt expression with the effects of preincubation with oleate to the mechanisms underlying oleate's protective effect and to of mitochondrial LCFA oxidation lead to the same protective effect as