Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells
2009; Elsevier BV; Volume: 50; Issue: 9 Linguagem: Inglês
10.1194/jlr.m800535-jlr200
ISSN1539-7262
AutoresDavid Sebastián, María Guitart, Cèlia Garcia‐Martínez, Caroline Mauvezin, Josep Maria Orellana‐Gavaldà, Dolors Serra, Anna M. Gómèz‐Foix, Fausto G. Hegardt, Guillermina Asins,
Tópico(s)Adipose Tissue and Metabolism
ResumoCarnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for β-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids. Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for β-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids. Fatty acids are a major energy source for skeletal muscle. They are transported into the cell via a protein-mediated mechanism, involving fatty acid translocase/CD36 (FAT/CD36), plasma-membrane-associated fatty acid binding protein, and fatty acid transport protein (FATP1-6) (1Bonen A. Chabowski A. Luiken J.J. Glatz J.F. Is membrane transport of FFA mediated by lipid, protein, or both? Mechanisms and regulation of protein-mediated cellular fatty acid uptake: molecular, biochemical, and physiological evidence.Physiology (Bethesda). 2007; 22: 15-29Crossref PubMed Scopus (196) Google Scholar). Once inside the cell, fatty acids can be esterified, metabolized to lipid second messengers, such as leukotrienes and eicosanoids, or β-oxidized in the mitochondrion. The first step of the oxidative pathway is the transport of fatty acids into the mitochondrial matrix. This step is controlled by the carnitine palmitoyltransferase (CPT) system, consisting of CPT1, acylcarnitine translocase, and CPT2 (2McGarry J.D. Brown N.F. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1342) Google Scholar). CPT1 catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine and is the rate-limiting step in the transport of long-chain fatty acids (LCFAs) from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. CPT1 is tightly regulated by its physiological inhibitor malonyl-CoA. This regulation allows CPT1 to signal the availability of lipid and carbohydrate fuels to the cell (2McGarry J.D. Brown N.F. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1342) Google Scholar). Mammalian tissues express three isoforms: CPT1A (liver), CPT1B (muscle and heart), and CPT1C (brain) (3Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function.J. 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Thus, malonyl-CoA is not responsible for the increase in fatty acid oxidation during the transition from low to moderate aerobic exercise or for the decrease in fatty acid oxidation during more strenuous exercise (6Odland L.M. Heigenhauser G.J. Lopaschuk G.D. Spriet L.L. Human skeletal muscle malonyl-CoA at rest and during prolonged submaximal exercise.Am. J. Physiol. 1996; 270: E541-E544PubMed Google Scholar, 7Odland L.M. Heigenhauser G.J. Wong D. Hollidge-Horvat M.G. Spriet L.L. Effects of increased fat availability on fat-carbohydrate interaction during prolonged exercise in men.Am. J. Physiol. 1998; 274: R894-R902PubMed Google Scholar, 8Dean D. Daugaard J.R. Young M.E. Saha A. Vavvas D. Asp S. Kiens B. Kim K.H. Witters L. Richter E.A. et al.Exercise diminishes the activity of acetyl-CoA carboxylase in human muscle.Diabetes. 2000; 49: 1295-1300Crossref PubMed Scopus (102) Google Scholar). Moreover, concentrations of malonyl-CoA measured in muscle (1–4 µM) (9Chien D. Dean D. Saha A.K. Flatt J.P. Ruderman N.B. Malonyl-CoA content and fatty acid oxidation in rat muscle and liver in vivo.Am. J. Physiol. Endocrinol. Metab. 2000; 279: E259-E265Crossref PubMed Google Scholar, 10McGarry J.D. Mills S.E. Long C.S. Foster D.W. Observations on the affinity for 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. 1983; 214: 21-28Crossref PubMed Scopus (466) Google Scholar) should inhibit CPT1 activity at any time, since the IC50 of CPT1B for malonyl-CoA is much lower than this concentration (∼0.03 µM). Several hypotheses have been advanced to explain this discordance between malonyl-CoA levels, CPT1 regulation, and LCFA oxidation in muscle. Thus, alteration in compartmentalization/cellular distribution of malonyl-CoA may be involved in the control of CPT1 activity. It has been proposed that the malonyl-CoA involved in CPT1 regulation is that synthesized by acetyl-CoA carboxylase 2, which resides in the vicinity of CPT1 in the outer mitochondrial membrane and modulates CPT1 activity (11McGarry J.D. Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes.Diabetes. 2002; 51: 7-18Crossref PubMed Scopus (1218) Google Scholar). Moreover, other proteins could also be involved in the transport of fatty acids into mitochondria. Thus, Bonen and coworkers demonstrated that exercise induces the translocation of FAT/CD36 to mitochondria, thus increasing muscle fatty acid oxidation (12Campbell S.E. Tandon N.N. Woldegiorgis G. Luiken J.J. Glatz J.F. Bonen A. A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria.J. Biol. Chem. 2004; 279: 36235-36241Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). FATPs are a family of membrane-bound proteins that catalyze the ATP-dependent esterification of LCFA and very-LCFA to their acyl-CoA derivatives (13Hall A.M. Smith A.J. Bernlohr D.A. Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1.J. Biol. Chem. 2003; 278: 43008-43013Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Stahl A. A current review of fatty acid transport proteins (SLC27).Pflugers Arch. 2004; 447: 722-727Crossref PubMed Scopus (235) Google Scholar, 15Bonen A. Miskovic D. Kiens B. Fatty acid transporters (FABPpm, FAT, FATP) in human muscle.Can. J. Appl. Physiol. 1999; 24: 515-523Crossref PubMed Scopus (54) Google Scholar). Mammals express six different forms of FATP (FATP1-6) with tissue-specific expression patterns (14Stahl A. A current review of fatty acid transport proteins (SLC27).Pflugers Arch. 2004; 447: 722-727Crossref PubMed Scopus (235) Google Scholar). FATP1 is a 63 kDa protein that is expressed in cells and tissues with high levels of fatty acid uptake for metabolism or storage, such as skeletal muscle and adipose tissue (15Bonen A. Miskovic D. Kiens B. Fatty acid transporters (FABPpm, FAT, FATP) in human muscle.Can. J. Appl. Physiol. 1999; 24: 515-523Crossref PubMed Scopus (54) Google Scholar). The regulatory role of FATP1 in metabolism has been partially elucidated by gene manipulation. Overexpression of FATP1 in cultured fibroblasts increases fatty acid uptake (16Schaffer J.E. Lodish H.F. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein.Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (743) Google Scholar). Cardiac-specific overexpression of FATP1 in transgenic mice increases myocardial fatty acid uptake and free FA accumulation, but not triacylglyceride (TG) levels, and enhances palmitate oxidation leading to a lipotoxic cardiomyopathy (17Chiu H.C. Kovacs A. Blanton R.M. Han X. Courtois M. Weinheimer C.J. Yamada K.A. Brunet S. Xu H. Nerbonne J.M. et al.Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy.Circ. Res. 2005; 96: 225-233Crossref PubMed Scopus (350) Google Scholar). In contrast, when overexpressed in cultured human skeletal myotubes, FATP1 stimulates FA uptake and storage as TG, but not oxidation (18Garcia-Martinez C. Marotta M. Moore-Carrasco R. Guitart M. Camps M. Busquets S. Montell E. Gomez-Foix A.M. Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells.Am. J. Physiol. Cell Physiol. 2005; 288: C1264-C1272Crossref PubMed Scopus (62) Google Scholar). Deletion of FATP1 protects mice from fatty-acid-induced insulin resistance and intramuscular accumulation of fatty acyl-CoAs (19Kim J.K. Gimeno R.E. Higashimori T. Kim H.J. Choi H. Punreddy S. Mozell R.L. Tan G. Stricker-Krongrad A. Hirsch D.J. et al.Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle.J. Clin. Invest. 2004; 113: 756-763Crossref PubMed Scopus (206) Google Scholar). Furthermore, FATP1 is involved in hormonal regulation of fatty acid uptake, translocating to the plasma membrane in response to insulin in adipocytes and primary skeletal muscle cells (20Stahl A. Evans J.G. Pattel S. Hirsch D. Lodish H.F. Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes.Dev. Cell. 2002; 2: 477-488Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 21Wu Q. Ortegon A.M. Tsang B. Doege H. Feingold K.R. Stahl A. FATP1 is an insulin-sensitive fatty acid transporter involved in diet-induced obesity.Mol. Cell. Biol. 2006; 26: 3455-3467Crossref PubMed Scopus (214) Google Scholar). We have recently shown that overexpression of CPT1A in L6E9 myotubes enhanced CPT1 activity up to 15-fold, whereas fatty acid oxidation did not increase more than 2-fold, even when a mutant form of CPT1 insensitive to malonyl-CoA was overexpressed (22Sebastian D. Herrero L. Serra D. Asins G. Hegardt F.G. CPT I overexpression protects L6E9 muscle cells from fatty acid-induced insulin resistance.Am. J. Physiol. Endocrinol. Metab. 2007; 292: E677-E686Crossref PubMed Scopus (58) Google Scholar). These data indicate that other factors independent of CPT1/malonyl-CoA interaction could be involved in the control of fatty acid oxidation in these cells. In the search for a novel regulator of fatty acid oxidation in skeletal muscle, we examined the effect of FATP1 overexpression on fatty acid oxidation in L6E9 muscle cells. We show that FATP1 localized in mitochondria and coimmunoprecipitated with CPT1. Moreover, FATP1 overexpression enhanced mitochondrial oxidation of fatty acids. We also observed all these effects in cells overexpressing FAT/CD36, which may have a role in this process in muscle (12Campbell S.E. Tandon N.N. Woldegiorgis G. Luiken J.J. Glatz J.F. Bonen A. A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria.J. Biol. Chem. 2004; 279: 36235-36241Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These data reveal a new protein involved in β-oxidation and could help us to understand its complex regulation in skeletal muscle. Protran® nitrocellulose membranes for protein analysis were from Scheicher and Schuell (Keene, NH). The enhanced chemifluorescence detection kit from Amersham Biosciences was used for Western blot analysis. The Bradford solution for protein assay was from Bio-Rad Laboratories (Hercules, CA). FBS, DMEM, and antibiotics were from Gibco-Invitrogen. Defatted BSA (BSA), palmitate, and other chemicals were purchased from Sigma-Aldrich. [1-14C]Palmitic acid and [1-14C]palmitoyl-CoA were from GE Healthcare. Antibodies used included rabbit polyclonal anti-FATP1 (sc-14497) and rabbit polyclonal anti-FAT/CD36 (sc-9154) from Santa Cruz Biotechnologies, anti-green fluorescent protein (GFP) (ref. 8360-1) from Clontech, anti-COI of complex IV from Molecular Probes, and anti-porin from Calbiochem. Silica gel 60 TLC plates were from Merck (Rahway, NJ). Etomoxir was provided by H. P. O. Wolf (GMBH, Allensbach, Germany). Samples of gastrocnemius and soleus muscle were taken from male Sprague-Dawley rats (180–200 g) bred in our laboratory. Animals were maintained under a 12 h dark/light cycle at 23°C with free access to food and water. All experimental protocols were approved by the Animal Ethics Committee at the University of Barcelona. The L6E9 rat skeletal muscle cell line was cultured in a humidified atmosphere containing 5% CO2 in DMEM medium containing 10% FBS, 100 units/ml penicillin G, and 100 μg/ml streptomycin and 25 mmol/l HEPES (pH 7.4) (growth medium). Preconfluent myoblasts (80–90%) were induced to differentiate by lowering FBS to a final concentration of 2% (v/v) (differentiation medium). Cells were completely differentiated after 4 days in this medium. Ad-CPT1A encoding rat CPT1A, Ad-FATP1 encoding mouse FATP1, Ad-FAT/CD36 encoding rat FAT/CD36, and Ad-LacZ, which expresses bacterial β-galactosidase, were constructed as previously described (18Garcia-Martinez C. Marotta M. Moore-Carrasco R. Guitart M. Camps M. Busquets S. Montell E. Gomez-Foix A.M. Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells.Am. J. Physiol. Cell Physiol. 2005; 288: C1264-C1272Crossref PubMed Scopus (62) Google Scholar, 23Rubi B. Antinozzi P.A. Herrero L. Ishihara H. Asins G. Serra D. Wollheim C.B. Maechler P. Hegardt F.G. Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion.Biochem. J. 2002; 364: 219-226Crossref PubMed Scopus (66) Google Scholar). Adenoviruses were amplified using the human embryonic kidney cell line (HEK 293) as host. Lysates obtained were titrated using the Adeno-XTM Rapid Titer kit (Clontech) and used directly for cell transduction. The titers of the lysates were 2.3 × 109 pfu/ml for Ad-LacZ, 1 × 1010 pfu/ml for Ad-CPT1A, 1 × 1010 pfu/ml for Ad-FAT/CD36, and 3 × 1010 pfu/ml for Ad-FATP1. Myotubes were transduced at day 4 of differentiation with 20 pfu/cell of Ad-LacZ, Ad-FATP1, or Ad-CPT1A and 5 pfu/cell of Ad-FAT/CD36 in serum-free medium for 30 h. After this time, the infection medium was removed and cells were incubated with serum-free medium for a further 16 h. The plasmids pFATP1-GFP and pFAT/CD36-GFP, which include an NH2-terminal fusion protein construct with the GFP (20Stahl A. Evans J.G. Pattel S. Hirsch D. Lodish H.F. Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes.Dev. Cell. 2002; 2: 477-488Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), were transfected in L6E9 myoblasts using FuGENE 6 (Roche) following the manufacturer's guidelines. After transfection, cells were differentiated and used for the experiments. Palmitate oxidation to CO2 and acid-soluble products (ASPs), essentially acyl-carnitine, Krebs cycle intermediates, and acetyl-CoA (24Veerkamp J.H. van Moerkerk T.B. Glatz J.F. Zuurveld J.G. Jacobs A.E. Wagenmakers A.J. 14CO2 production is no adequate measure of [14C]fatty acid oxidation.Biochem. Med. Metab. Biol. 1986; 35: 248-259Crossref PubMed Scopus (72) Google Scholar), were measured in L6E9 cells grown in 6-well plates, differentiated, and transduced as described above. On the day of the assay, cells were washed in Krebs-Ringer bicarbonate HEPES buffer (KRBH buffer: 135 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, and 10 mM HEPES, pH 7.4) that contained 0.1% BSA, preincubated at 37°C for 30 min in KRBH 1% BSA and washed again in KRBH 0.1% BSA. Cells were then incubated for 3 h at 37°C with fresh KRBH containing 2.5 mM glucose and 0.8 mM carnitine plus 0.25 mM palmitate and 1 μCi/ml [1-14C]palmitate bound to 1% BSA. Oxidation measurements were performed by trapping the radioactive CO2 in a parafilm-sealed system. The reaction was stopped by the addition of 40% perchloric acid through a syringe that pierced the parafilm. Mitochondria-enriched cell fractions from L6E9 myotubes were obtained as previously described (23Rubi B. Antinozzi P.A. Herrero L. Ishihara H. Asins G. Serra D. Wollheim C.B. Maechler P. Hegardt F.G. Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion.Biochem. J. 2002; 364: 219-226Crossref PubMed Scopus (66) Google Scholar) with some modifications. Briefly, L6E9 cells were grown in 150 mm dishes, left to differentiate, and transduced as described above. Cells were scraped out and resuspended in 1 ml of solution A (100 mM KCl, 5 mM MgSO4, 5 mM EDTA, 1 mM ATP, and 50 mM Tris-HCl, pH 7.4). Cells were homogenized using a glass homogenizer (20 strokes with both the loose and the tight pestle) and centrifuged at 2,000 g for 3 min at 4°C. The supernatant was centrifuged again at 16,000 g for 30 min at 4°C. The mitochondria-enriched pellet was resuspended in 50–100 µl of solution B (220 mM sucrose, 70 mM mannitol, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4) and used for immunoprecipitation or palmitate oxidation assays. The quality of mitochondria was assessed measuring the malonyl-CoA-resistant CPT1 activity, attributable basically to CPT2 activity inside the mitochondrion, allowing us to quantify broken mitochondria. According to this method, the integrity of mitochondria was higher than 80% (data not shown). Mitochondria-enriched fractions from rat muscle were obtained by differential centrifugation. Two soleus muscle samples or one gastrocnemius muscle sample from each animal were homogenized separately in 9 volumes of solution A using an omnimixer and then centrifuged at 1,000 g for 15 min. The pellet was homogenized and centrifuged at 600 g for 10 min. The resulting supernatant was centrifuged at 15,000 g for 15 min, and the pellet was washed twice in solution A and resuspended at 1 µl/mg tissue in solution B. L6E9 cells were cultured in 150 mm dishes, differentiated, and transduced as described above. Mitochondria-enriched fractions were obtained and resuspended in solution B. Fatty acid oxidation was measured as described elsewhere (12Campbell S.E. Tandon N.N. Woldegiorgis G. Luiken J.J. Glatz J.F. Bonen A. A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria.J. Biol. Chem. 2004; 279: 36235-36241Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) with minor modifications. For palmitate oxidation assays, 50 µl (150 µg) of mitochondria and 50 µl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 µCi/ml [1-14C]palmitate (final concentration: 0.25 mM palmitate and 1 µCi/ml [1-14C]palmitate) were incubated for 1 h with agitation in 400 µl of pregassed (37°C for 15 min with 5% CO2-95% O2) modified Krebs Ringer HEPES (MKRH) buffer (115 mM NaCl, 2.6 mM KCl, 1.2 mM KH2PO4, 10 mM NaHCO3, and 10 mM HEPES, pH 7.4) supplemented with 5 mM ATP, 1 mM NAD+, 0.5 mM l-carnitine, 0.1 mM CoA, 0.5 mM malate, and 25 μM cytochrome C (complete MKRH buffer). For palmitoyl-CoA oxidation assays 50 µl (400 µg) of mitochondria and 50 µl of a 2.5 mM 5:1 palmitoyl-CoA-BSA complex containing 1 µCi/ml [1-14C]palmitoyl-CoA was added to the reaction mixture (final concentration: 0.25 mM palmitoyl-CoA and 0.1 µCi/ml [1-14C]palmitoyl-CoA) were incubated for 0.5 h with agitation in 400 µl of pregassed complete MKRH buffer. Oxidation measurements were performed by trapping the radioactive CO2 and ASPs in a parafilm-sealed system in a 6-well plate with agitation. The reaction was stopped by the addition of 40% perchloric acid through a syringe that pierced the parafilm. Palmitate incorporation into complex lipids was measured in L6E9 cells that were cultured on 6-well plates and pretreated as described above. Cells were incubated for 16 h at 37°C in serum-free medium containing 0.25 mM palmitate and 1 μCi/ml [1-14C]palmitate bound to 1% BSA. On the day of the assay cells were washed in PBS, and lipids were extracted as described previously (23Rubi B. Antinozzi P.A. Herrero L. Ishihara H. Asins G. Serra D. Wollheim C.B. Maechler P. Hegardt F.G. Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion.Biochem. J. 2002; 364: 219-226Crossref PubMed Scopus (66) Google Scholar). Total lipids were dissolved in 30 μl of chloroform and separated by TLC to measure the incorporation of labeled fatty acid into phospholipids (PLs), diacylglycerol (DAG), TGs, and nonesterified labeled palmitate (NE palm), as described (22Sebastian D. Herrero L. Serra D. Asins G. Hegardt F.G. CPT I overexpression protects L6E9 muscle cells from fatty acid-induced insulin resistance.Am. J. Physiol. Endocrinol. Metab. 2007; 292: E677-E686Crossref PubMed Scopus (58) Google Scholar). Samples were assayed for acyl-CoA synthetase activity by the conversion of [1-14C]palmitate to its CoA derivative (13Hall A.M. Smith A.J. Bernlohr D.A. Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1.J. Biol. Chem. 2003; 278: 43008-43013Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The assay mixture contained, in a total volume of 200 µl: 100 mM Tris-HCl buffer, pH 7.5, 50 µM [1-14C]palmitate (0.2 µCi/ml), 10 mM ATP, 5 mM MgCl2, 200 µM CoA, 200 µM DTT, and 0.4% Triton X-100. The assay was initiated by addition of 5–10 µl of enzyme suspension (30–50 µg) from total or mitochondrial extracts from L6E9 myotubes. The reaction was carried out at 30°C for 5 min. Reactions were terminated with the addition of 1.25 ml of isopropyl alcohol:heptane:H2SO4 (40:10:1, v/v/v), 0.5 ml of water, and 0.75 ml of heptane to facilitate organic phase separation. The aqueous phase was extracted three times with 0.75 ml of heptane to remove unreacted fatty acids, and the radioactivity was determined by liquid phase scintillation counting. Immunoprecipitation studies were performed in L6E9 myotubes and rat skeletal muscle. L6E9 cells were grown on 150 mm plates, differentiated, and transduced with the adenoviruses. After transduction, cells were collected in PBS and homogenized with a douncer in 500 μl of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM EGTA, 1 μM PMSF, 25 μg/ml leupeptin, and 25 μg/ml aprotinin) and centrifuged at 700 g for 10 min to remove nuclei, cell debris, and floating cells. For muscle studies, two soleus samples or one gastrocnemius sample were homogenized in 9 volumes of lysis buffer using a polytron. Homogenates were rotated for 1 h at 4°C in an orbital shaker and centrifuged at 5,000 g for 10 min at 4°C. After that, 1,000 or 2,000 μg of protein was immunoprecipitated with 5 μg/ml of either anti-FATP1 or anti-FAT/CD36 antibody. For immunoprecipitation studies in mitochondria, mitochondrial fractions were obtained as described above, 1% Triton X-100 was added to each preparation, and 150 or 300 μg of each fraction was used. Immunoprecipitates were collected on protein A-Sepharose G beads, washed five times in lysis buffer, resuspended, and incubated for 5 min at 95°C in 1× SDS-PAGE sample buffer. Samples were resolved in 8% SDS-PAGE. A CPT1A-specific polyclonal antibody against amino acids 317–430 of the rat CPT1A (25Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae.FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar) (1/6,000 dilution) was used for the detection of CPT1A protein in the immunoprecipitates from L6E9 myotubes. A CPT1B-specific antibody against amino acids 259–760 of the rat CPT1B (26van der Leij F.R. Cox K.B. Jackson V.N. Huijkman N.C. Bartelds B. Kuipers J.R. Dijkhuizen T. Terpstra P. Wood P.A. Zammit V.A. et al.Structural and functional genomics of the CPT1B gene for muscle-type carnitine palmitoyltransferase I in mammals.J. Biol. Chem. 2002; 277: 26994-27005Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) was used (1/1,000 dilution) for detection of CPT1B protein in the immunoprecipitates from rat skeletal muscle. Cells grown on cover slips were fixed for 15 min with 3% paraformaldehyde in PBS and then rinsed three times with PBS and subsequently incubated for 10 min in PBS containing 50 mM NH4Cl, for 10 min with PBS containing 20 mM glycine, and for 10 min in PBS containing 0.1% triton X-100 and 0.05% colic acid. Cells were then washed three times in PBS 1× and for 30 min in PBS containing 10% goat serum. Thereafter, cover slips were incubated with primary antibodies at 0.5–1.0 µg/ml anti-COI of complex IV (Molecular Probes) and revealed with fluorochrom-conjugated goat secondary antibodies (Texas Red). Cells were rinsed three times with PBS prior to mounting in medium for fluorescence microscopy (Vectashield). Fluorescence images were obtained with a Leica TCS 4D confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope and 63× (numerical aperture 1.4 oil) Leitz Plan-Apo objectives. The light source was an argon/krypton laser (75 mW), with which optical sections (0.1 µm) were obtained. Qualitative analyses of colocalization were assessed using the plugin RGB Profiler from the WCIF ImageJ software. A line was drawn arbitrarily over an area of interest and the plot presented the overlap of the staining of each laser (from distinct labeled proteins). Quantitative analyses were performed by calculating Pearson's correlation coefficient and Mander's overlap coefficient, which are both described in the online manual for the WCIF ImageJ collection. In many forms of correlation analysis, the value for Pearson's range from 1 to −1. A value close to 1 does indicates reliable colocalization. The Mander's overlap coefficient ranges from 1 to 0, with 1 being high colocalization and 0 being low. Having defined and eliminated the background noise from the original picture, we used the plugin Intensity Correlation Analysis from WCIF ImageJ software. Mitochondrial fractions were obtained by differential centrifugation of muscle cell homogenates. Extracts were prepared by scraping L6E9 cell monolayers from 6 cm dishes into 700 µl of homogenization buffer consisting of 250 mM sucrose, 10 mM HEPES (pH 7.4), 1 mM EDTA, 1 μM PMSF, 25 μg/ml leupeptin, 25 μg/ml pepstatin A, and 25 μg/ml aprotinin. Cells were homogenized with the aid of a 22G-syringe (15 times). The homogenates were centrifuged at 1,500 g for 10 min, and the supernatant was then centrifuged at 10,000 g for 10 min to obtain a pellet enriched in mitochondria and other organelles (27Lanni A. Moreno M. Lombardi A. Goglia F. Biochemical and functional differences in rat liver mitochondrial subpopulations obtained at different gravitational forces.Int. J. Biochem. Cell Biol. 1996; 28: 337-343Crossref PubMed Scopus (48) Google Scholar). All fractions were stored at −80°C, and an aliquot of the cell extracts was used for the measurement of protein concentration. Fifty micrograms of protein was resolved in 10% SDS-PAGE, and immunoblotting was performed with the corresponding antibody. Data are expressed as means ± SE. The significance of differences was assessed by a one-way or two-w
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