Proteomic Analysis of the Palmitate-induced Myotube Secretome Reveals Involvement of the Annexin A1-Formyl Peptide Receptor 2 (FPR2) Pathway in Insulin Resistance*
2015; Elsevier BV; Volume: 14; Issue: 4 Linguagem: Inglês
10.1074/mcp.m114.039651
ISSN1535-9484
AutoresJong Hyuk Yoon, Dayea Kim, Jin-Hyeok Jang, Jaewang Ghim, Soyeon Park, Parkyong Song, Yonghoon Kwon, Jaeyoon Kim, Daehee Hwang, Yoe‐Sik Bae, Pann‐Ghill Suh, Per‐Olof Berggren, Sung Ho Ryu,
Tópico(s)Vitamin D Research Studies
ResumoElevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity. Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity. The obesity epidemic has been linked to the development of metabolic complications such as hyperlipidemia, insulin resistance, and hypertension (1Moller D.E. Kaufman K.D. Metabolic syndrome: a clinical and molecular perspective.Annu. Rev. Med. 2005; 56: 45-62Crossref PubMed Scopus (501) Google Scholar, 2Coenen K.R. Hasty A.H. 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In molecular studies, palmitate has been found to induce inflammation and insulin resistance in skeletal muscle cells by promoting diacylglycerol accumulation, which in turn activates protein kinase C (PKC)-θ 1The abbreviations used are:APEXabsolute protein expression profilingERKextracellular signal-regulated kinaseESIelectrospray ionizationFPR2formyl peptide receptor 2GLUT4glucose transporter type 4GOBPgene ontology biological processGOCCgene ontology cellular componentGTTglucose tolerance testHFDhigh-fat dietIRS1insulin receptor substrate 1LCliquid chromatographyMS/MStandem mass spectrometryNCDnormal chow dietPKCprotein kinase CQ-PCRquantitative polymerase chain reactionWATwhite adipose tissue. 1The abbreviations used are:APEXabsolute protein expression profilingERKextracellular signal-regulated kinaseESIelectrospray ionizationFPR2formyl peptide receptor 2GLUT4glucose transporter type 4GOBPgene ontology biological processGOCCgene ontology cellular componentGTTglucose tolerance testHFDhigh-fat dietIRS1insulin receptor substrate 1LCliquid chromatographyMS/MStandem mass spectrometryNCDnormal chow dietPKCprotein kinase CQ-PCRquantitative polymerase chain reactionWATwhite adipose tissue. and NF-κB, leading to the inhibition of insulin-stimulated Akt phosphorylation through insulin receptor substrate 1 (IRS1) (S307) phosphorylation and IL-6 secretion (9Coll T. Eyre E. Rodriguez-Calvo R. Palomer X. Sanchez R.M. Merlos M. Laguna J.C. Vazquez-Carrera M. Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells.J. Biol. Chem. 2008; 283: 11107-11116Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Sortilin was recently identified as a mediator of palmitate-dependent insulin resistance, which regulates insulin-induced glucose transporter type 4 (GLUT4) trafficking (10Tsuchiya Y. Hatakeyama H. Emoto N. Wagatsuma F. Matsushita S. Kanzaki M. Palmitate-induced down-regulation of sortilin and impaired GLUT4 trafficking in C2C12 myotubes.J. Biol. Chem. 2010; 285: 34371-34381Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Therefore, palmitate is an important hyperlipidemic/dyslipidemic component that induces insulin resistance in skeletal muscle cells. absolute protein expression profiling extracellular signal-regulated kinase electrospray ionization formyl peptide receptor 2 glucose transporter type 4 gene ontology biological process gene ontology cellular component glucose tolerance test high-fat diet insulin receptor substrate 1 liquid chromatography tandem mass spectrometry normal chow diet protein kinase C quantitative polymerase chain reaction white adipose tissue. absolute protein expression profiling extracellular signal-regulated kinase electrospray ionization formyl peptide receptor 2 glucose transporter type 4 gene ontology biological process gene ontology cellular component glucose tolerance test high-fat diet insulin receptor substrate 1 liquid chromatography tandem mass spectrometry normal chow diet protein kinase C quantitative polymerase chain reaction white adipose tissue. Skeletal muscle is thought to function as a tissue that produces and releases cytokines called myokines (11Pedersen B.K. Febbraio M.A. Muscle as an endocrine organ: focus on muscle-derived interleukin-6.Physiol. Rev. 2008; 88: 1379-1406Crossref PubMed Scopus (1420) Google Scholar). As part of its extracellular signaling pathway, skeletal muscle secretes myokines that participate in myogenesis, angiogenesis, and nutrient generation in response to factors such as metabolic disorders, including insulin resistance, and exercise (11Pedersen B.K. Febbraio M.A. Muscle as an endocrine organ: focus on muscle-derived interleukin-6.Physiol. Rev. 2008; 88: 1379-1406Crossref PubMed Scopus (1420) Google Scholar, 12Pedersen B.K. Akerstrom T.C. Nielsen A.R. Fischer C.P. Role of myokines in exercise and metabolism.J. Appl. Physiol. 2007; 103: 1093-1098Crossref PubMed Scopus (520) Google Scholar, 13Chan C.Y. Masui O. Krakovska O. Belozerov V.E. Voisin S. Ghanny S. Chen J. Moyez D. Zhu P. Evans K.R. McDermott J.C. Siu K.W. Identification of differentially regulated secretome components during skeletal myogenesis.Mol. Cell. Proteomics. 2011; 10 (M110 004804)Abstract Full Text Full Text PDF Scopus (60) Google Scholar). Some myokines, including IL-6, IL-8, IL-15, and fibroblast growth factor 21, and brain-derived neurotrophic factor (14Pedersen B.K. Edward F. Adolph distinguished lecture: muscle as an endocrine organ: IL-6 and other myokines.J. Appl. Physiol. 2009; 107: 1006-1014Crossref PubMed Scopus (169) Google Scholar), are induced by exercise. Although myokines are thought to play a critical role in the regulation of (patho)physiological processes, few studies have investigated the role of myokine in metabolism. Because skeletal muscle has a major role in the regulation of glucose metabolism, it is important to identify putative crucial regulators, secreted from skeletal muscle, that modulate glucose metabolism by acting as autocrine/paracrine mediators as well as endocrine mediators (15Yoon J.H. Kim J. Song P. Lee T.G. Suh P.G. Ryu S.H. Secretomics for skeletal muscle cells: a discovery of novel regulators?.Adv. Biol. Regul. 2012; 52: 340-350Crossref PubMed Scopus (34) Google Scholar). Here, using an optimized secretomics approach, we performed a proteomic analysis of proteins in conditioned media from myotube cultures that were either untreated or treated with palmitate to induce insulin resistance (16Yoon J.H. Yea K. Kim J. Choi Y.S. Park S. Lee H. Lee C.S. Suh P.G. Ryu S.H. Comparative proteomic analysis of the insulin-induced L6 myotube secretome.Proteomics. 2009; 9: 51-60Crossref PubMed Scopus (62) Google Scholar, 17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar). Using a label-free quantitative analysis method, our aim was to characterize the skeletal muscle secretome and to identify skeletal muscle-derived proteins whose secretion is modulated by palmitate-induced insulin resistance. We found 36 putative secretory proteins modulated by palmitate-induced insulin resistance. The secretion of annexin A1 was down-regulated after palmitate treatment, and the annexin A1-formyl peptide receptor 2 (FPR2) pathway played a role in palmitate-induced insulin resistance in skeletal muscle by modulating the PKC-θ pathway. Rat L6 GLUT4myc skeletal myoblast cells were used to model skeletal muscle as previously described (17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar, 18Bi Y. Wu W. Shi J. Liang H. Yin W. Chen Y. Tang S. Cao S. Cai M. Shen S. Gao Q. Weng J. Zhu D. Role for sterol regulatory element binding protein-1c activation in mediating skeletal muscle insulin resistance via repression of rat insulin receptor substrate-1 transcription.Diabetologia. 2014; 57: 592-602Crossref PubMed Scopus (18) Google Scholar, 19Zhang W. Liu J. Tian L. Liu Q. Fu Y. Garvey W.T. TRIB3 mediates glucose-induced insulin resistance via a mechanism that requires the hexosamine biosynthetic pathway.Diabetes. 2013; 62: 4192-4200Crossref PubMed Scopus (32) Google Scholar, 20Frangioudakis G. Diakanastasis B. Liao B.Q. Saville J.T. Hoffman N.J. Mitchell T.W. Schmitz-Peiffer C. Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment.Diabetologia. 2013; 56: 2697-2701Crossref PubMed Scopus (20) Google Scholar, 21Dalla-Riva J. Stenkula K.G. Petrlova J. Lagerstedt J.O. Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle.J. Lipid Res. 2013; 54: 1275-1282Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Cells were cultured in the alpha formulation of minimum essential medium (α-MEM; Welgene, Daegu, Korea) supplemented with 10% FBS, 2 mm glutamine, 50 μg/ml streptomycin, and 50 μg/ml penicillin. Cells were provided by Dr. Amira Klip (Hospital for Sick Children, Toronto, Canada). The cells were passaged every second day, and confluency was maintained at less than 80% to prevent spontaneous differentiation. To induce differentiation, cells (4 × 104 cells/ml) were placed into α-MEM supplemented with 2% FBS for 5 days, with the medium refreshed every 48 h. The differentiation status was routinely monitored under a microscope (LSM 510 Meta; Zeiss, Germany). Sodium-palmitate (Sigma-Aldrich, St. Louis, MO) was dissolved in ethanol, heated to 50–60 °C for 30 min, and diluted in medium containing 2% serum (2% FBS in alpha-MEM) (22Huang S. Rutkowsky J.M. Snodgrass R.G. Ono-Moore K.D. Schneider D.A. Newman J.W. Adams S.H. Hwang D.H. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.J. Lipid Res. 2012; 53: 2002-2013Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 23Pu J. Peng G. Li L. Na H. Liu Y. Liu P. Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells.J. Lipid Res. 2011; 52: 1319-1327Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To induce palmitate-mediated insulin resistance, L6 myotubes were incubated with 150 μm of palmitate solution for 18 h in α-MEM supplemented with 2% FBS. Insulin resistance in primary mouse skeletal muscle cells (Cell Biologics Inc., Chicago, IL) was induced in the same way as described above for L6 cells. After the L6 myotubes were treated with palmitate, they were washed three times with completely unsupplemented α-MEM (no serum, phenol red, or antibiotics). Conditioned medium was prepared by incubated the myotubes with unsupplemented α-MEM for 5 h in a 37 °C CO2 incubator. The conditioned medium was collected and centrifuged at 3500 rpm for 10 min (Combi-514R; Hanil Science Industry, Incheon, Korea). The resulting supernatant was collected and filtered using centrifugal filter units (cat# UFC900324; Millipore, MA) to eliminate contaminants. Dried samples filtered from the centrifugal filter units were reduced using 10 mm DTT in 50 mm ammonium bicarbonate and then alkylated with 100 mm iodoacetamide in 50 mm ammonium bicarbonate for tryptic digestion. Finally, each sample was treated with trypsin (Promega, Madison, WI) for 12 h at 37 °C and dried. Tryptic digests (secretome peptides) were analyzed using a linear ion trap LTQ XL mass spectrometer (MS; Thermo, San Jose, CA) interfaced with a nano-electrospray ion source. Chromatographic separation of peptides was achieved on a NanoLC·2D system (Eksigent, Dublin, CA) equipped with a 10-cm PicTip™ emitter with a 75-μm inner diameter (PF360–75-15-N-5; New Objective), packed in-house with a Magic C18AQ 5-μm/200-Å resin (Michrom Bioresources, Auburn, CA). Peptides were loaded from a cooled (4 °C) Eksigent auto-sampler and separated with a linear gradient of ACN/water, containing 0.1% formic acid, at a flow rate of 260 nl/min. Peptide mixtures were separated with a gradient of 2% to 40% ACN in 60 min. The analysis method consisted of a full MS scan with a range of 300–2000 m/z and data-dependent tandem mass spectrometry (MS/MS) on the five most intense ions from the full MS scan. The mass spectrometer was calibrated with the proposed calibration solution according to the manufacturer's instructions. This study used three biological replicates for each condition and two technical replicates for each biological replicate in the proteomic analysis. The peak lists from the MS/MS data (RAW files) of the LTQ were generated using the Trans-Proteomic Pipeline (TPP, ver. 4.6.1) (24Pedrioli P.G. Trans-proteomic pipeline: a pipeline for proteomic analysis.Methods Mol. Biol. 2010; 604: 213-238Crossref PubMed Scopus (115) Google Scholar) and searched using a concatenated database that combined the International Protein Index (IPI) Rat FASTA database (ver. 3.85, 39,924 sequences; 21,245,823 residues) and Bovine FASTA database (ver. 3.73: 30,403 sequences; 16,412,134 residues) with the MASCOT search engine ver. 2.2.2 (Matrix Science, London, UK), selecting the Decoy database analysis. The MASCOT parameters allowed for tryptic specificity of up to two missed cleavages, with methylthio-modifications of cysteine as a fixed modification and oxidation of methionine as a variable modification. The LTQ search parameters for +1, +2, and +3 ions included mass error tolerances of ±2 Da for peptide ions and ±0.8 Da for fragment ions. In the MASCOT search results, when the significance threshold was set at 0.01, the individual ions score was more than 48 (supplemental Table S1). For the raw MASCOT search results, peptide-level (>95% probability) and protein-level (>99% probability) statistical validation was performed with PeptideProphet and ProteinProphet (25Keller A. Eng J. Zhang N. Li X.J. Aebersold R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats.Mol. Syst. Biol. 2005; 1 (2005 0017)Crossref PubMed Scopus (598) Google Scholar) in TPP. We removed frequently observed contaminants such as porcine trypsin, bovine proteins, and human keratins. Proteins supported by a single unique peptide were removed. APEX quantitation was performed using the APEX Quantitative Proteomics Tool ver. 1.1.0 (26Braisted J.C. Kuntumalla S. Vogel C. Marcotte E.M. Rodrigues A.R. Wang R. Huang S.T. Ferlanti E.S. Saeed A.I. Fleischmann R.D. Peterson S.N. Pieper R. The APEX Quantitative Proteomics Tool: generating protein quantitation estimates from LC-MS/MS proteomics results.BMC Bioinformatics. 2008; 9: 529Crossref PubMed Scopus (129) Google Scholar). The APEX abundances of the proteins observed with liquid chromatography (LC)-MS/MS were calculated using the protXML files generated from the PeptideProphet and ProteinProphet analyses. An FDR of 0.43 were then selected (16Yoon J.H. Yea K. Kim J. Choi Y.S. Park S. Lee H. Lee C.S. Suh P.G. Ryu S.H. Comparative proteomic analysis of the insulin-induced L6 myotube secretome.Proteomics. 2009; 9: 51-60Crossref PubMed Scopus (62) Google Scholar, 17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar). Third, secretory proteins were also identified based on nonclassical secretion prediction using SecretomeP 2.0 (28Bendtsen J.D. Jensen L.J. Blom N. Von Heijne G. Brunak S. Feature-based prediction of non-classical and leaderless protein secretion.Protein Eng. Des. Sel. 2004; 17: 349-356Crossref PubMed Scopus (936) Google Scholar) as those with with an NN-score of >0.5 (16Yoon J.H. Yea K. Kim J. Choi Y.S. Park S. Lee H. Lee C.S. Suh P.G. Ryu S.H. Comparative proteomic analysis of the insulin-induced L6 myotube secretome.Proteomics. 2009; 9: 51-60Crossref PubMed Scopus (62) Google Scholar, 17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar). The DAVID software was used for functional enrichment analysis (http://david.abcc.ncifcrf.gov/) for Gene Ontology biological process (GOBP) and GOCC) A list of identified proteins was submitted and analyzed using the following parameters: the count threshold was set at 2, and the p value was set at 0.1 (a default cutoff) (16Yoon J.H. Yea K. Kim J. Choi Y.S. Park S. Lee H. Lee C.S. Suh P.G. Ryu S.H. Comparative proteomic analysis of the insulin-induced L6 myotube secretome.Proteomics. 2009; 9: 51-60Crossref PubMed Scopus (62) Google Scholar, 17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar). Putative exosomal proteins were screened using the ExoCarta exosome database (29Mathivanan S. Simpson R.J. ExoCarta: a compendium of exosomal proteins and RNA.Proteomics. 2009; 9: 4997-5000Crossref PubMed Scopus (662) Google Scholar). Following differentiation, L6 myotubes and primary mouse skeletal muscle cells were treated with 150 μm palmitate for 18 h to induce insulin resistance as described in the section detailing the "Cell culture and establishment of insulin resistance." The cells were cotreated with Ac2–26 (Anygen, Jangseong-gun, Jeonnam, Korea), WKYMVm (Trp-Lys-Tyr-Met-Val-d-Met; Anygen, Korea), WRW4 (Trp-Arg-Trp-Trp-Trp-Trp; Anygen), and palmitate for 18 h, depending on the experimental conditions. Then the cells were treated with 100 nm insulin for 20 min, and the cells were harvested. All experimental procedures involving animals were approved by the POSTECH (Pohang University of Science and Technology) Animal Use and Care Committee. C57BL/6J mice (male, 6 weeks old) were kept under a 12-hour light/dark cycle with free access to water. The mice received a 60% high-fat diet (HFD) for 10 weeks to induce insulin resistance, and the body weight of each animal was monitored. WKYMVm was dissolved in a vehicle solution consisting of 20% water and 80% saline. After 10 weeks of a HFD, mice received intravenous injections of WKYMVm (200 μg/head) or vehicle daily for 5 days. For the glucose tolerance test (GTT), mice subjected to an overnight fast were injected intraperitoneally with glucose (1 g/kg). Blood was collected from the tail vein at 15, 30, 60, and 120 min, and glucose levels were measured. The fasting and feeding insulin levels were measured with an insulin ELISA kit (ALPCO). For lipid profiling, mouse serum was obtained by clotting the blood for 1 h, followed by centrifugation for 1 h at 2000 rpm. Serum triglyceride, cholesterol, LDL cholesterol, and high-density lipoprotein cholesterol were measured using an automatic chemistry analyzer (BS-380; Mindray). To observe insulin signaling in metabolic tissues, mice were fasted overnight; insulin was then injected intraperitoneally (5 units/kg) for 10 min. Phosphorylation of Akt in the skeletal muscles, liver, and epididymal white adipose tissue (WAT) was evaluated with Western blotting. All data were expressed as the mean ± S.E. All statistical analyses were performed using Student's t test (two groups) or one-way ANOVA (>two groups). Tukey's test or Tamhane's T2 test was used for multiple comparisons. A p value of <0.05 was considered significant. (Experimental procedures for the GLUT4 translocation assay, the MTT assay, RNA extraction, quantitative PCR analysis, PCR analysis, and Western blot are presented in the supplemental Data). To find appropriate conditions for palmitate-induced insulin resistance, we performed the GLUT4 translocation assay using differentiated L6 rat skeletal muscle cells treated with different concentrations of palmitate for 18 h. We found that 150–300 μm of palmitate inhibited insulin-induced GLUT4 translocation with statistical confidence (supplemental Fig. S1A). After measuring the cell viability (supplemental Fig. S1B), we selected 150 μm as the optimal concentration for insulin resistance. Treatment with 150 μm palmitate did not affect the morphology of L6 myotubes (Fig. 1A). After stimulating the cells with palmitate, we assessed the expression of pro-inflammatory cytokine genes, such as TNF-α and IL-6. The expression of TNF-α and IL-6 increased under palmitate-treated conditions (Fig. 1B). To examine the possible impairment of downstream insulin-induced signaling after palmitate treatment, we used Western blotting to monitor the phosphorylation of Akt, an important signaling molecule in insulin stimulation. Insulin-induced phosphorylation of Akt (S473) was markedly inhibited after palmitate treatment (Fig. 1C). For the secretome analysis, we collected and processed conditioned media from palmitate-treated and nontreated (NT) cells. The trypsin-digested peptides from the conditioned medium were subjected to LC- electrospray ionization (ESI)-MS/MS. We searched the combined rat/bovine IPI database using the MASCOT search engine and identified 189 proteins from NT and palmitate-treated conditions (Fig. 1D and supplemental Table S1). We used APEX for label-free protein quantification (26Braisted J.C. Kuntumalla S. Vogel C. Marcotte E.M. Rodrigues A.R. Wang R. Huang S.T. Ferlanti E.S. Saeed A.I. Fleischmann R.D. Peterson S.N. Pieper R. The APEX Quantitative Proteomics Tool: generating protein quantitation estimates from LC-MS/MS proteomics results.BMC Bioinformatics. 2008; 9: 529Crossref PubMed Scopus (129) Google Scholar) and found that the abundances of 36 proteins had changed (p value < 0.05 and log2 change > 0.58): the levels of eight proteins were higher in the palmitate-treated group than in the NT control, whereas those of 28 proteins were lower (supplemental Table S1). To select putative secretory proteins from among these proteins, we used a previously reported approach that utilizes three separate methods (16Yoon J.H. Yea K. Kim J. Choi Y.S. Park S. Lee H. Lee C.S. Suh P.G. Ryu S.H. Comparative proteomic analysis of the insulin-induced L6 myotube secretome.Proteomics. 2009; 9: 51-60Crossref PubMed Scopus (62) Google Scholar, 17Yoon J.H. Song P. Jang J.H. Kim D.K. Choi S. Kim J. Ghim J. Kim D. Park S. Lee H. Kwak D. Yea K. Hwang D. Suh P.G. Ryu S.H. Proteomic analysis of tumor necrosis factor-alpha (TNF-alpha)-induced L6 myotube secretome reveals novel TNF-alpha-dependent myokines in diabetic skeletal muscle.J. Proteome Res. 2011; 10: 5315-5325Crossref PubMed Scopus (42) Google Scholar). Among the 36 proteins modulated by palmitate-induced insulin resistance, Gene Ontology selected 21 proteins, signal sequence prediction selected 25 proteins, and nonclassical secretion prediction selected six proteins as secretory proteins. Twelve exosomal proteins were screened with the ExoCarta database. Thus, 36 proteins were selected as secretory proteins modulated by palmitate-induced insulin resistance (Table I).Table IList of secretory proteins modulated by Palmitate-induced insulin resistance. For "Filtering methods," letters indicate how proteins wer
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