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

Next Section Abstract 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.

Previous Section Next Section Introduction It has long been recognized that increased plasma free fatty acids are associated with insulin resistance in humans (1). Indeed, plasma free fatty acid concentrations are increased in obese subjects (2) as well as in genetically obese or high fat diet-induced insulin-resistant mice (3, 4). In such conditions, circulating free fatty acid concentrations are elevated, and FA4 metabolism is altered (5,–,7), 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 (6), inflammation (8), and cell death via apoptosis (4, 6), 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 (9,–,12). In muscle cells, palmitate's cytotoxic effect is mediated by increased intracellular concentrations of diacylglycerols (DAG) and ceramides (11). In contrast, monounsaturated FA (MUFA), such as oleate (C18:1), protect against SFA-induced toxicity (8, 13, 14). 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 (8, 14, 15). Oleate has been proposed to protect cells from palmitate-induced lipotoxicity by promoting its esterification into TG, a neutral form of FA storage (8, 15, 16). 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) (8). 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 (17). 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 (17, 18). 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 (19, 20). 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 (21). Obese and insulin-resistant skeletal muscles also have fewer and dysmorphic mitochondria, which may decrease FA oxidative capacity (22, 23). Because accumulation of palmitate-derived metabolites within muscle cells is associated with decreased mitochondrial FA oxidative capacity, increasing LCFA oxidation may exert a protective effect. Additionally, contradictory results have been reported concerning the impact of a modulation of mitochondrial LCFA oxidation on palmitate-induced apoptosis in cardiomyocytes (11, 22) and pancreatic β-cells (13, 24, 25). This question has never been addressed in skeletal muscle cells. In the present study, we aimed to determine whether oleate protects skeletal muscle cells from palmitate-induced apoptosis and to examine the role of mitochondrial LCFA in mediating any such effect of oleate. To this end, we expressed a mutant form of CPT1A (CPT1 M593S, CPT1mt), which is active but insensitive to malonyl-CoA inhibition (26), in C2C12 myotubes. The metabolic and cellular consequences of an increased LCFA oxidation through CPT1mt expression were compared with the effects of preincubation with oleate (OA) to decipher the mechanisms underlying oleate's protective effect and to determine if direct manipulation of mitochondrial LCFA oxidation would lead to the same protective effect as OA.

Previous Section Next Section EXPERIMENTAL PROCEDURES Cell Culture Mouse C2C12 myoblasts (ATCC) were cultured in controlled humidified atmosphere (5% CO 2 , 37 °C) in Dulbecco's modified Eagle's medium (DMEM, PAA Laboratories) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 1% (v/v) amphotericin B, gentamicin (Invitrogen). Confluent myoblasts were induced to differentiate in DMEM containing 2% (v/v) horse serum (Invitrogen) for 4 days prior to all experiments. Adenoviruses (Ad) encoding either β-galactosidase (Ad-LacZ) or rat CPT1A mutated at methionine 593 (Ad-CPT1mt) were constructed and produced as previously described (27). Adenoviral infection was performed in myotubes in serum-free medium containing 5 infectious particles/cell of Ad-LacZ or Ad-CPT1mt for 16 h. Sixteen h following the removal of the infection medium, cells were cultured for 24 h in a medium containing either 5 mm glucose (G5) or 20 mm glucose plus 100 nm insulin (G20+I), as indicated. For insulin treatment, cells were incubated for 10 min in fresh medium in the absence or presence of 100 nm insulin (Novo Nordisk). FA Treatment Sodium salts of palmitic acid and oleic acid (Sigma) were conjugated with FA- and endotoxin-free BSA (PAA Laboratories), and 4 mm stock solution was made in serum-free DMEM containing 5% (w/v) BSA. Myotubes were incubated with carnitine (1 mm), with or without (1% (w/v) BSA) various concentrations of palmitate for the time periods indicated. Preincubation with 0.3 mm oleate was done for 16 h before exposure to palmitate. Western Blot Antibodies against CPT1A (28), CPT2 (27), ACC, phospho-ACC, Akt, phospho-Akt, AMP-activated protein kinase (AMPK), phospho-AMPK, caspase-3 (Cell Signaling Technology), cytochrome c, tubulin α (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and mitochondrial respiratory chain subunit B6 of Complex 1 and subunit Fe/S of Complex 3 (gift from Dr. Anne Lombès, INSERM U582, Paris, France) were used. Subcellular Fractionation C2C12 myotubes were homogenized in an isolation buffer (0.3 m sucrose, 5 mm Tris-HCl, 1 mm EGTA, pH 7.4) and centrifuged at 1200 × g for 4 min. The supernatant containing mitochondria was then centrifuged at 4900 × g for 10 min. The pellet and the supernatant correspond to the mitochondrial and cytosolic fractions, respectively. CPT1 Activity Assay CPT1 activity was determined in mitochondria-enriched fractions (50 μg of protein, 1 mg/ml) using l-[methyl-3H]carnitine (400 μm; 10 Ci/mol) and palmitoyl-CoA (150 μm) as substrates (27). Activity assays were performed with different concentrations of malonyl-CoA to determine the malonyl-CoA concentration required to inhibit CPT1 activity by 50% (IC 50 ). Determining Cellular Oxygen Consumption Cells were trypsinized and resuspended in their medium. Measurement of respiration was performed by a polarographic oxygen sensor in a 2-ml glass chamber of an Oxygraph 2K respirometer (Oroboros Instruments). The amplified signal from the oxygen sensor was recorded on a computer at sampling intervals of 1 s. Data were acquired using DatLab3 software and processed with the DatLab2 program, which permits subtraction of oxygen consumption due to the electrode. The respiration medium was equilibrated with air in the oxygraph chambers at 37 °C and stirred until a stable signal was obtained for calibration at air saturation. After calibration, the medium was replaced by 2 ml of aerated cell suspensions, and the chambers were closed with stoppers. Basal respiration was determined after 5–10 min of stabilization. Oligomycin (0.5 μg/ml) was then added to determine the State-4 respiratory rate. Finally, increasing concentrations (40 nm) of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were used to estimate the maximum (uncoupled) respiratory rate. FA Metabolism FA oxidation and esterification were determined during the last 3 h of culture in the presence of either 0.3 mm [1-14C]oleate (0.5 Ci/mol) or 0.8 mm [1-14C]palmitate (2 Ci/mol) bound to 1% (w/v) BSA and 1 mm carnitine. Then medium was collected to determine [14C]CO 2 and [14C]acid-soluble products (ASP) (27). ASP consist mainly of shortened acyl-carnitines, Krebs cycle intermediates, and acetyl-CoA. Cells were washed and scraped in PBS to determine [14C]TG, [14C]phospholipids (PL), [14C]DAG, and [14C]non-esterified fatty acid. Lipids were extracted with chloroform/methanol (2:1, v/v) and separated by thin layer chromatography on silica gel plates (Merck) (27). Immunofluorescence Assay C2C12 myotubes cultured on coverslips were fixed as described previously (27). Cells were stained with anti-cytochrome c (BD Biosciences) and anti-cleaved caspase-3 (Cell Signaling Technology) antibodies, which were detected by an anti-mouse IgG conjugated with Alexa fluor 594 and an anti-rabbit IgG conjugated with Alexa fluor 488 (Molecular Probes), respectively. After incubation with Hoechst 33342 (2 μm; Molecular Probes) for DNA staining, coverslips were mounted on glass slides with Fluoromount G (Clinisciences). Caspase-3 Activity Assay Caspase-3 activity was measured fluorometrically in extracts (100 μg of protein) using the Caspase-3 Cellular Assay Kit Plus with Ac-DEVD-p-nitroanilide as a substrate (BIOMOL International), according to the manufacturer's protocol. Lipidomic Analysis Myotubes were homogenized in methanol, 5 mm EGTA (2:1 v/v) with FAST-PREP (MP Biochemicals) and evaporated. The dry pellets were dissolved overnight in 0.1 m NaOH. Lipids were extracted from homogenates according to the method of Bligh and Dyer (29) and analyzed by gas-liquid chromatography for determination of neutral lipid molecular species, ceramides, sphingomyelins (SM) (30) and FA methyl esters (31). Data were normalized to protein concentration. RT-PCR Total RNA was extracted using the RNeasy® minikit (Qiagen), following the manufacturer's instructions. RT-PCR was performed with SuperScriptTM II reverse transcriptase (Invitrogen) using 1 μg of total RNA. The resulting cDNA was mixed with a buffer containing 25 mm MgCl 2 , 15 μm sense and antisense primers, and 0.75 μl of FastStart DNA-MasterSYBR Green I (Roche Applied Science) and analyzed with online quantitative PCR (LightCycler® system), as follows: denaturing at 95 °C for 15 min, annealing at 58 °C for 7 s, elongation at 72 °C for 15 s, and denaturing at 95 °C. Primers (Eurofins, MWG Operon) used are listed in supplemental Table 1. The relative mRNA abundance was calculated using LightCycler® software and normalized to 18 S mRNA. Statistical Analysis Data represent at least three independent experiments, are reported as means ± S.E., and were analyzed with analysis of variance using GraphPad Prism 5 software. The Student-Newman-Keuls test was used for post hoc analyses. Differences were considered significant at p < 0.05.

Previous Section Next Section DISCUSSION Our findings indicate that oleate preincubation protects skeletal muscle cells from palmitate-induced apoptosis by acting on its metabolism through two mechanisms. It promotes the clearance of DAG-containing palmitate moieties toward TG and palmitate oxidation. Second, because targeting mitochondrial LCFA oxidation directly, via the expression of a malonyl-CoA-insensitive CPT1 (CPT1mt), also has a protective effect, redirection of palmitate metabolism toward oxidation appears to be sufficient for protection against its lipotoxic effect (Fig. 7). View larger version: Download as PowerPoint Slide FIGURE 7. CPT1mt expression and OA reach similar final effects via different mechanisms. Control, the SFA palmitate induces apoptosis in skeletal muscle cells by promoting intracellular TG, DAG, and ceramide accumulation. CPT1mt, CPT1mt expression reduces palmitate-induced apoptosis by increasing mitochondrial oxidation of palmitate and decreasing the production of TG, DAG, and ceramides. OA, preincubation with oleate prevents palmitate-induced apoptosis by promoting mitochondrial LCFA oxidation, secondary to increased CPT1mt expression and phospho-ACC/ACC ratio, and by channeling palmitate toward TG as a result of an enhanced DGAT2 expression. These two mechanisms reduce the availability of palmitate for incorporation into DAG and de novo ceramide synthesis. In conclusion, OA induces palmitate oxidation and storage into TG, whereas CPT1mt expression increases mitochondrial oxidation of palmitate. Consistent with previous studies (11, 35), we showed that palmitate induced apoptosis in C2C12 myotubes. First, prolonged exposure of myotubes to palmitate promoted intracellular accumulation of TG (specifically TG species with at least two C16: C51 and C53), DAG (specifically 16-16 and 16-18), and ceramides (specifically Cer16:0). As previously suggested (11), these deleterious metabolites result from enhanced de novo ceramide synthesis rather than from SM degradation because cellular SM content was unaffected by palmitate treatment. Second, palmitate exposure increased SFA and decreased MUFA, leading to a decreased MUFA/SFA ratio. Previous studies have suggested that palmitate-induced DAG and TG accumulation might result from a higher esterification flux for palmitate than for oleate (15, 16), as we also observed. However, our metabolic investigations also revealed that palmitate is much less prone to oxidation than oleate, which could favor directing its metabolism toward esterification. We found that preincubation with oleate decreased palmitate esterification into DAG and TG after 3 h of palmitate exposure, resulting in a decreased DAG/TG ratio. However, oleate preincubation promoted intracellular TG accumulation, in particular TG species containing at least one C18 (C53, C55, and C57) after a 24-h palmitate exposure. This difference probably results from an enhanced expression of DGAT2, whose affinity is 50% higher for oleyl-CoA than for palmitoyl-CoA (36, 37). Indeed, oleate preincubation decreased TG and DAG species that contain solely C16 (i.e. C51 and DAG 16-16), suggesting that preincubation with oleate diverts DAG 16-16 to TG enriched with C18. Additionally, the lipidomic analysis revealed that oleate preincubation reduced palmitate content and increased the MUFA/SFA ratio. Interestingly, preincubation with oleate also reduced palmitoleate content in palmitate-exposed cells, which might be due to decreased SCD1 gene expression. Because oleate is a product of SCD1, we hypothesized that it can induce a negative feedback on SCD1 gene expression. It has been reported that oleate preincubation led to an increase in CPT1 mRNA level in skeletal muscle cells (8, 38). We demonstrated that oleate preincubation enhanced mitochondrial oxidation of palmitate, as a consequence of increased CPT1 gene and protein expressions. Our findings also indicate that preincubation with oleate increases mitochondrial LCFA oxidation through a second mechanism, namely activation of AMPK, which leads to ACC inhibition and, thus, to increased CPT1 activity. We investigated whether acting directly on mitochondrial oxidation, by expressing CPT1mt, leads to the same protective effect as oleate preincubation. In agreement with the work of Sebastián et al. (34), we showed that CPT1mt expression induced an increase in LCFA oxidation at the expense of their esterification. Like oleate preincubation, DAG and ceramide contents are reduced by CPT1mt expression. In contrast to preincubation with oleate, CPT1mt expression decreased the rate of esterification of palmitate into TG (regarding the two species raised by palmitate) and had no impact on the nature or content of FA in myotubes. These results contrast with those of Perdomo et al. (18), who reported that expression of the wild-type CPT1 did not modify TG, DAG, or ceramide contents. This difference highlights the importance of malonyl-CoA in the regulation of CPT1 activity. It has been suggested that increased mitochondrial LCFA oxidation can lead to an accumulation of ASP (39). However, in our study, the CO 2 /ASP ratio for palmitate was increased, not decreased, following CPT1mt expression, both in the presence of low glucose and high glucose/insulin concentrations. This indicates that CPT1mt expression permits the maintenance of complete oxidation of palmitate, thus avoiding accumulation of incompletely oxidized molecules. It is well established that skeletal muscle insulin resistance is associated with intramuscular lipid accumulation. Inhibition of CPT1 by etomoxir, an irreversible inhibitor, increased lipid deposition in skeletal muscle and exacerbated insulin resistance in high fat-fed animals (40), indicating that alteration in LCFA flux into mitochondria is critical for regulating the deleterious effect of lipids on insulin sensitivity. Furthermore, it has been recently reported that in vivo wild-type CPT1 overexpression in rat skeletal muscle enhanced mitochondrial LCFA oxidation and decreased high fat diet-induced insulin resistance (41, 42). These effects were associated with a moderate decrease in both TG level and palmitate incorporation into DAG. We hypothesize that these mild effects may be due to the expression of wild-type CPT1, which is sensitive to malonyl-CoA inhibition. Indeed, it has been shown in muscle from obese and diabetic subjects that malonyl-CoA level is increased, leading to a decrease in mitochondrial LCFA oxidation (41, 42). Here we demonstrated that targeting mitochondrial LCFA oxidation, via the expression of a malonyl-CoA-insensitive CPT1, 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 resistance. In conclusion, we propose that increased CPT1 activity together with decreased malonyl-CoA sensitivity may be a promising strategy for the treatment of the deleterious effects of lipid accumulation caused by obesity, insulin resistance, or aging.

Previous Section Next Section Acknowledgments The C2C12 mouse skeletal muscle cell line was kindly provided by Dr. Chantal Wrutniak (INRA, Montpellier, France). We thank Dr. Anne Lombes (INSERM U582, Paris, France) for antibodies against complexes 1 and 3 and Dr. Catherine Postic (INSERM U1016, Paris, France) for critical reading of the manuscript and for selected primers. We thank Justine Bertrand Michel (Lipidomic Core, IFR 150, Toulouse, France) for help with and expertise about lipid content analysis. We appreciate the assistance of the Flow Cytometry Core of the Cochin Institute, and we thank the Vector Core of the University Hospital of Nantes supported by the Association Francaise contre les Myopathies for providing the adenovirus vectors.

Previous Section Next Section Footnotes ↵1 Supported by a graduate fellowship from the Ministère de l'Education Nationale, de la Recherche, et de la Technologie.

↵2 Supported by a Postdoctoral Award from the Association de Langue Française pour l'Etude du Diabète et des Maladies Métaboliques (ALFEDIAM).

↵* This research was funded by the Agence Nationale de la Recherche, ANR Mithycal and by Grant no. 13714 from the Association Française contre les Myopathies (AFM).

↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. S1 and S2.

↵4 The abbreviations used are: FA fatty acid(s) ASP acid-soluble product(s) CCCP carbonyl cyanide m-chlorophenyl hydrazone CPT1mt mutant CPT1A M593S DAG diacylglycerol(s) LacZ β-galactosidase LCFA long-chain fatty acid MUFA monounsaturated fatty acid(s) OA preincubation with oleate PL phospholipid(s) SFA saturated fatty acid(s) SM sphingomyelin(s) TG triglyceride(s) G5 5 m m glucose G20+I 20 m m glucose plus 100 n m insulin.