PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle
2015; Elsevier BV; Volume: 56; Issue: 12 Linguagem: Inglês
10.1194/jlr.m060533
ISSN1539-7262
AutoresNanami Senoo, Noriyuki Miyoshi, Naoko Goto‐Inoue, Kimiko Minami, Ryoji Yoshimura, Akihito Morita, Naoki Sawada, Junichiro Matsuda, Yoshihiro Ogawa, Mitsutoshi Setou, Yasutomi Kamei, Shinji Miura,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoExercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle. Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle. Phospholipids are important structural components of membranes, and they influence a number of physical properties related to membrane function, including fluidity, permeability, and the anchoring of membrane-related proteins. Because altering dietary fatty acids (1.Andersson A. Nalsen C. Tengblad S. Vessby B. Fatty acid composition of skeletal muscle reflects dietary fat composition in humans.Am. J. Clin. Nutr. 2002; 76: 1222-1229Crossref PubMed Scopus (168) Google Scholar, 2.Ayre K.J. Hulbert A.J. Dietary fatty acid profile influences the composition of skeletal muscle phospholipids in rats.J. Nutr. 1996; 126: 653-662Crossref PubMed Scopus (75) Google Scholar, 3.Pan D.A. Storlien L.H. 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Physiol. (1985). 2001; 90: 670-677Crossref PubMed Scopus (4) Google Scholar) can both influence the composition of skeletal muscle membrane fatty acids, changes in phospholipid fatty acids may be involved in diet-induced or exercise training-induced physiological adaptation of the skeletal muscle. This effect on skeletal muscle adaptation may ultimately influence its function. Previous studies examined the effect of endurance training on the molecular species of skeletal muscle phospholipids. Exercise training increases phosphatidylcholine (PC) in muscle (8.Morgan T.E. Short F.A. Cobb L.A. Effect of long-term exercise on skeletal muscle lipid composition.Am. J. Physiol. 1969; 216: 82-86Crossref PubMed Scopus (58) Google Scholar), and the effects of exercise on numerous phospholipid species in skeletal muscle have been examined in rats fed a standard chow diet (9.Mitchell T.W. Turner N. Hulbert A.J. Else P.L. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.J. Appl. Physiol. (1985). 2004; 97: 1823-1829Crossref PubMed Scopus (36) Google Scholar) or a high-fat diet (10.Mitchell T.W. Turner N. Else P.L. Hulbert A.J. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet.Int. J. Mol. Sci. 2010; 11: 3954-3964Crossref PubMed Scopus (9) Google Scholar). With chow feeding, exercise training increased the abundance of two PC species (9.Mitchell T.W. Turner N. Hulbert A.J. Else P.L. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.J. Appl. Physiol. (1985). 2004; 97: 1823-1829Crossref PubMed Scopus (36) Google Scholar): PC (16:0/18:1), the species also identified as an endogenous PPARα ligand in liver, and PC (16:0/18:2). In addition, exercise training with the normal chow diet enhanced the abundance of phosphatidic acid (PA) (16:0/18:2), PA (18:1/18:2), plasmenyl-phosphatidylethanolamine (PE) (p-16:0/18:2), and phosphatidylinositol (PI) (18:0/22:5), and decreased the abundance of PE (18:0/22:6), PC (16:0/20:4), and PC (16:0/22:6) (9.Mitchell T.W. Turner N. Hulbert A.J. Else P.L. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.J. Appl. Physiol. (1985). 2004; 97: 1823-1829Crossref PubMed Scopus (36) Google Scholar). With high-fat diet (10.Mitchell T.W. Turner N. Else P.L. Hulbert A.J. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet.Int. J. Mol. 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They found that PC (16:0/18:2), PC (18:0/22:6), and SM (d18:1/16:0) were chronic exercise training-induced lipids and, in contrast, PC (18:0/20:4) and SM (d18:1/24:1) were high-fat diet-induced lipids. The largest differences were also observed when comparing oxidative and glycolytic muscles, such as a decrease in plasmenyl-PE (16:0/20:4) and an increase in PE (18:0/22:6) in the oxidative muscle (9.Mitchell T.W. Turner N. Hulbert A.J. Else P.L. Hawley J.A. Lee J.S. Bruce C.R. Blanksby S.J. Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.J. Appl. Physiol. (1985). 2004; 97: 1823-1829Crossref PubMed Scopus (36) Google Scholar). Although evidence has shown that exercise training induces changes in skeletal muscle phospholipid species, it is not fully understood how exercise training induces these changes, or what roles these phospholipids play in the functional changes of exercise-trained skeletal muscle. 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Isoform-specific increases in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) mRNA in response to beta2-adrenergic receptor activation and exercise.Endocrinology. 2008; 149: 4527-4533Crossref PubMed Scopus (117) Google Scholar). PGC-1α-b, whose N terminus is different from that of PGC-1α-a protein, is the predominant PGC-1α isoform in skeletal muscles that is expressed in response to exercise (26.Miura S. Kai Y. Kamei Y. Ezaki O. Isoform-specific increases in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) mRNA in response to beta2-adrenergic receptor activation and exercise.Endocrinology. 2008; 149: 4527-4533Crossref PubMed Scopus (117) Google Scholar). Overexpression of PGC-1α-b promoted fiber-type switch, mitochondrial biogenesis, and exercise capacity, increased the expression of fatty acid transporters, and enhanced angiogenesis and oxygen utilization kinetics in skeletal muscle (25.Tadaishi M. Miura S. Kai Y. Kano Y. Oishi Y. Ezaki O. Skeletal muscle-specific expression of PGC-1alpha-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake.PLoS One. 2011; 6: e28290Crossref PubMed Scopus (119) Google Scholar, 27.Kano Y. Miura S. Eshima H. Ezaki O. Poole D.C. The effects of PGC-1alpha on control of microvascular P(O2) kinetics following onset of muscle contractions.J. Appl. Physiol. (1985). 2014; 117: 163-170Crossref PubMed Scopus (10) Google Scholar). Recently, we also found that PGC-1α-b induced branched chain amino acid metabolism, which might also be involved in endurance capacity (28.Hatazawa Y. Tadaishi M. Nagaike Y. Morita A. Ogawa Y. Ezaki O. Takai-Igarashi T. Kitaura Y. Shimomura Y. Kamei Y. et al.PGC-1alpha-mediated branched-chain amino acid metabolism in the skeletal muscle.PLoS One. 2014; 9: e91006Crossref PubMed Scopus (60) Google Scholar). Because exercise training induces changes in skeletal muscle phospholipid species, it is possible that PGC-1α-b may be the underlying mechanism of induction. In this study, to understand how exercise training induces changes in phospholipid species, we performed lipidomics of skeletal muscle from genetically modified mice that overexpressed PGC-1α-b and mice that carry KO alleles of PGC-1α in skeletal muscle. We found that PGC-1α is involved in exercise training-induced changes in skeletal muscle phospholipid species. The methods for generating transgenic mice overexpressing PGC-1α-b in skeletal muscle (PGC-1α-Tg mice) were described previously (26.Miura S. Kai Y. Kamei Y. Ezaki O. Isoform-specific increases in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) mRNA in response to beta2-adrenergic receptor activation and exercise.Endocrinology. 2008; 149: 4527-4533Crossref PubMed Scopus (117) Google Scholar). The promoter for human α-skeletal actin, provided by Drs. E. C. Hardeman and K. L. Guven (Children's Medical Research Institute, Australia) was used to express PGC-1α-b in skeletal muscle. The transgenic mice (heterozygotes, BDF 1 background) and WT C57BL6 mice were crossed and female 10–13-week-old offspring (heterozygote and WT, from the same litter) were used for the experiments. To generate skeletal muscle-specific PGC-1α KO mice (muscle PGC-1α-KO mice), we inactivated PGC-1α expression in skeletal muscles by crossing mice carrying a floxed PGC-1α allele with mice transgenic for the human α-skeletal actin promoter driven-Cre transgenic. PGC-1αflox/flox mice were obtained from the Jackson Laboratory (Bar Harbor, ME) (29.Handschin C. Choi C.S. Chin S. Kim S. Kawamori D. Kurpad A.J. Neubauer N. Hu J. Mootha V.K. Kim Y.B. et al.Abnormal glucose homeostasis in skeletal muscle-specific PGC-1alpha knockout mice reveals skeletal muscle-pancreatic beta cell crosstalk.J. Clin. Invest. 2007; 117: 3463-3474Crossref PubMed Scopus (286) Google Scholar, 30.Sawada N. Jiang A. Takizawa F. Safdar A. Manika A. Tesmenitsky Y. Kang K.T. Bischoff J. Kalwa H. Sartoretto J.L. et al.Endothelial PGC-1alpha mediates vascular dysfunction in diabetes.Cell Metab. 2014; 19: 246-258Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Mice were maintained in a 12 h light/dark cycle at 22°C and were fed a normal chow diet ad libitum (CE-2; CLEA Japan, Tokyo, Japan). Mice were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and our institutional guidelines. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the University of Shizuoka (number 135024). Male (9-week-old) muscle PGC-1α-KO mice and control PGC-1αflox/flox mice were randomly assigned to one of two experimental groups: the sedentary control group or the training group. Mice assigned to training were housed individually in cages (22 × 9 × 8 cm) equipped with a running wheel (20 cm diameter; Shinano Co., Tokyo, Japan) for 5 weeks. The running wheel was equipped with a tachometer to determine the total running distance. Sedentary mice were housed in cages without a running wheel. Lipids were extracted from the extensor digitorum longus (EDL) and the soleus. Frozen muscle was homogenized and powdered in liquid nitrogen. Total lipids were extracted from homogenates with 1 ml chloroform/methanol (2:1, v/v with 0.2 mg/ml butyl hydroxyl toluene) overnight. For targeted analysis of phospholipids, 0.05 mg/ml PC (17:0/17:0) was added to the chloroform/methanol for use as an internal standard. The lipid fractions were evaporated to dryness under vacuum. Samples were reconstituted in an equal volume of acetonitrile/isopropanol/water (65:30:5, v/v/v). Ten microliters of samples were injected onto the LC/MS system. Lipidomic analysis was performed using a Q-ExactiveTM benchtop orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with an electrospray source ionization probe and an autosampler, Accela quaternary HPLC pump (Thermo Fisher Scientific). For LC analysis, an Acquity UPLC CSH C18 column (1.7 μm, 2.1 × 150 mm; Waters, Milford, MA) was used. Mobile phase A consisted of water/acetonitrile (60:40, v/v) and mobile phase B consisted of isopropanol/acetonitrile (90:10, v/v). Both mobile phases, A and B, were supplemented with 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.4 ml/min. The gradient was as follows: 10% B at 0 min, 10% B at 2 min, 50% B at 8 min, 75% B at 20 min, 90% B at 55 min, and 10% B at 60 min. For MS analysis, the spray voltage was set to 3.5 kV, the capillary temperature was set to 350°C, the S-lens radio frequency (RF) level was set to 50, and heater temperature was set to 300°C. The sheath gas flow rate was set to 40, and the auxiliary gas flow rate was set to 10. These conditions were applied to both positive and negative ionization modes. All samples were analyzed by both positive and negative ionization mode acquiring full scan MS, and the scan range was between m/z 120 and 1,200. To identify lipid species, m/z and retention time were used to obtain MS/MS data of the target peak by tandem mass spectrometric analysis. Using the LIPID MAPS online MS tool, the MS/MS data obtained were searched against a database of glycerophospholipid (http://www.lipidmaps.org/tools/ms/GP_prod_search.html) and glycerolipid (http://www.lipidmaps.org/tools/ms/GL_prod_search.html) precursor/product ions. Targeted LC/MS/MS analysis was performed using an LCMS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) in positive ionization mode equipped with an electrospray source ionization probe, LC-30AD binary pump (Shimadzu), SIL-30AC auto sampler (Shimadzu), and CTO-20AC column oven (Shimadzu). For HPLC analysis, an Accucore RP-MS C18 column (2.6 μm, 2.1 × 50 mm, Thermo Fisher Scientific) was used. The composition of the mobile phase and the flow rate were the same as above. The gradient was as follows: 40% B at 0 min, 40% B at 2 min, 52% B at 8 min, 60% B at 20 min, 100% B at 25 min, and 40% B at 30 min. For MS analysis, the nebulizer gas flow was set to 3.0 l/min, the drying gas flow was set to 15.0 l/min, the desolvation line temperature was set to 250°C, the heat block temperature was set to 400°C, and the collision-induced dissociation gas was set to 17 kPa. Target analysis of phospholipid species was operated in selected reaction monitoring (SRM) mode. The details of the conditions for SRM for each phospholipid are shown in supplementary Table 1. The relative peak area for each species was normalized by the peak area of internal standard and muscle weight. For quantitative analysis of each lipid, we used conventional TLC. Briefly, total lipid extracts from each gastrocnemius dissolved in chloroform/methanol (2:1, v/v) were manually applied as 5 mm wide spots to silica gel 60 HPTLC plates (Merck, Darmstadt, Germany). The plates were developed with a solvent system consisting of methyl acetate/1-propanol/chloroform/methanol/0.25% aqueous potassium chloride (25:25:25:10:9, v/v/v/v/v) for phospholipids, whereas for neutral lipid separation, the developing solvent consisted of n-hexane/diethyl ether/acetic acid (80:30:1, v/v/v). These chromatograms were sprayed with primuline reagent and lipid bands were visualized under UV light at 366 nm. The relative density of each band was quantified by Image J software (http://imagej.nih.gov/ij/). The localization of each lipid was analyzed by IMS. The developed TLC plates were transferred to a polyvinylidene difluoride (PVDF) membrane by the TLC-blot method, as described previously (31.Goto-Inoue N. Hayasaka T. Taki T. Gonzalez T.V. Setou M. A new lipidomics approach by thin-layer chromatography-blot-matrix-assisted laser desorption/ionization imaging mass spectrometry for analyzing detailed patterns of phospholipid molecular species.J. Chromatogr. A. 2009; 1216: 7096-7101Crossref PubMed Scopus (44) Google Scholar), and transferred PVDF membranes were attached to the MALDI target plate for IMS analyses. For TLC-blot-imaging analyses, we used a QSTAR Elite high-performance hybrid quadrupole TOF mass spectrometer (Applied Biosystems, Foster City, CA). The laser irradiated 500 times per position on the PVDF membrane. We set the spatial resolution to 400 μm. All analyses were performed in the positive ion mode within the mass ranges of m/z 400–1,200, with 2,5-dihydroxybenzoic acid at 50 mg/ml as a matrix. The ion images were constructed using BioMap software (Novartis, Basel, Switzerland). The tissue blocks (gastrocnemius) were rapidly frozen in isopentane cooled by liquid nitrogen. Transverse cross-sections of 10 μm were made with a cryostat (Leica, CM1510; Germany) at −20°C. For positive-ion mode, 2,5-dihydroxybenzoic acid of 50 mg/ml in methanol/water (7:3, v/v) was uniformly sprayed over the muscle tissue sections with a 0.2 mm nozzle caliber airbrush (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan). We used a MALDI TOF/TOF-type instrument, the Ultraflex II (Bruker Daltonics, Billerica, MA). The laser irradiated 200 times per position. All pixel sizes of imaging were 100 μm. The MS parameters were set to obtain the highest sensitivity with m/z values in the range of 400–1,000. The ion images were constructed using FlexImaging software (Bruker Daltonics). Normalization by total ion current was performed using the same software. RNA preparation methods and quantitative (q)RT-PCR were performed as described previously (26.Miura S. Kai Y. Kamei Y. Ezaki O. Isoform-specific increases in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) mRNA in response to beta2-adrenergic receptor activation and exercise.Endocrinology. 2008; 149: 4527-4533Crossref PubMed Scopus (117) Google Scholar). The mouse-specific primer pairs used are shown in supplementary Table 2. For lipidomic analyses, the detected peaks were aligned according to the m/z value and normalized retention time using Signpost MS (Reifycs, Tokyo, Japan). After applying autoscaling, mean-centering, and scaling by standard deviation on a per-peak basis as pretreatment, a hierarchical clustering analysis and a principal component analysis (PCA) were conducted using JMP version 11 (SAS Institute, Cary, NC). In hierarchical clustering analysis, the resulting data sets of each genotype were clustered using Euclidean distance with Ward's method (32.Ward J.H. Hierarchical grouping to optimize an objective function.J. Am. Stat. Assoc. 1963; 58: 236-244Crossref Scopus (13317) Google Scholar). The relative area value of each peak was calculated and used for the comparison between the PGC-1α-Tg and WT groups. In PCA, a score plot of the first and second principal components was generated. Statistical hypothesis testing of factor loading in PCA was performed to select species that had a statistically significant correlation to the principal component score. The P value was calculated as reported previously (33.Yamamoto H. Fujimori T. Sato H. Ishikawa G. Kami K. Ohashi Y. Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis.BMC Bioinformatics. 2014; 15: 51Crossref PubMed Scopus (62) Google Scholar). A change trend was defined at P < 0.05. Furthermore, a Bonferroni adjustment was applied to determine the level of significance for multiple testing (the adjusted α = 0.05/97 = 0.000515 for retention time 14–29 min peaks and α = 0.05/80 = 0.000625 for retention time 30–41 min peaks). Other data were analyzed by one-way ANOVA. In case of significant differences, each group was compared with the other groups by a Student's t-test (JMP, version 11). Values are shown as the mean ± SE. To examine the differences between the lipid profiles of glycolytic and oxidative muscle fibers such as EDL and soleus, and to determine the impact of PGC-1α on these profiles, lipidomic analyses were performed using high-resolution LC/MS that allows for accurate identification of lipid species. Figure 1A shows PCA scatter plots of the samples. The first principal component effectively and distinctly separated the mice based on muscle fiber type (x axis), and the second principal component separated the mice based on the genotype (y axis). The results suggested that overexpression of PGC-1α in the skeletal muscle caused a significant change in the overall lipid profiles of the muscle. However, the lipid profile of EDL from PGC-1α-Tg mice, which showed oxidative characteristics, was different from the profile of the originally oxidative muscle, such as the soleus. In the loading plot of this PCA (Fig. 1B), lipid species with chromatographic retention times between 14 and 29 min contributed to PGC-1α-driven alterations in the lipid profile. On the other hand, lipid species having chromatographic retention times between 30 and 41 min contributed to the differences in lipid profiles between the EDL and soleus. Because our preliminary study showed that the fractions having chromatographic retention times between 14 and 29 min contained many phospholipid species, this fraction was termed the phospholipid fraction. The fraction having chromatographic retention times between 30 and 41 min contained many TG species and was termed the TG fraction. To identify the lipid species that contribute to PGC-1α-driven changes in lipid profile, PCA was performed using lipid species in the phospholipid and TG fractions. In PCA of the phospholipid fraction, the first principal component separated the WT and PGC-1α-Tg mice (x axis), and the differences were obvious in EDL (Fig. 1C). Statistical hypothesis testing for factor loading in the first and second principal components was performed, and the lipid species that showed statistically significant differences (P < 0.05) are shown in Fig. 1D and Table 1. The phospholipids, 12 PC and 6 PE, were identified as significant in the first principal component, and 3 PC and 1 SM were identified in the second principal component. In PCA of the TG fraction, the first principal component separated EDL and soleus, and the second principal component separated soleus from WT and PGC-1α-
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