Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10477
RESEARCH ARTICLE
Palmitate exerts opposite effects on proliferation and differentiation of skeletal myoblasts Kamil Grabiec, Marta Milewska, Maciej Błaszczyk, Małgorzata Gajewska and Katarzyna Grzelkowska-Kowalczyk* Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Nowoursynowska 159 02-776, Warsaw, Poland
Abstract The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (a-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth. Keywords: G1 cyclins; long-chain fatty acids; myogenesis; myogenic regulatory factors
Introduction Skeletal muscle, making up approximately 40% of the total body mass, provides structural support, enables the body to maintain posture and controls motor movements. It is a key insulin-responsive tissue, important in maintaining homeostasis, due to its relatively large mass and energy needs (Lowell and Shulman, 2005; Houmard 2008). Fetal stage is crucial for skeletal muscle development, especially in humans and farm animals, because there is no increase in muscle fiber number after birth (Zhu et al., 2006). Early stages of muscle fiber formation involve two excluding processes (Floyd et al., 2001). Myoblast proliferation occurs through series of events arranged in precise order, i.e. cell cycle, whereas differentiation concerns the cells, which withdraw the cell cycle in G1 phase (Blomen and Boonstra, 2007). Proper myogenesis depends on the expression of primary (MyoD and Myf5) and secondary (myogenin and MRF-4) myogenic transcription factors
(Ferri et al., 2009), required for subsequent muscle-specific gene expression and morphological changes (Le Grand and Rudnicki, 2007). There is increasing evidence supporting on effects of fatty acids on muscle metabolic function. Excess of dietary lipids and acute exposure to palmitate cause insulin resistance and relevant mechanisms, i.e. impairment of insulin signaling (Hulver and Dohm, 2004; Delarue and Magnan, 2007), GLUT transporter activity (Alkhateeb et al., 2007) and mitochondrial uncoupling (Hirabara et al., 2006) have been extensively investigated. According to recent studies on human and animal models, maternal high-fat feeding and obesity increase fat-to-lean-mass ratio and cause an impaired glucose tolerance in offspring (reviewed in Warner and Ozanne, 2010). Using the obese pregnant sheep model, Tong et al. (2009) reported down-regulation of myogenesis and Wnt/b-catenin signaling, probably resulting from activation of inflammatory NF-kB pathway in fetal skeletal muscle. Such observations implicate an impairment of fetal
Corresponding author: e-mail: [email protected] Abbreviations: 7-AAD, 7-aminoactinomycin D; cdk, cyclin-dependent kinase; CV, crystal violet; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HS, horse serum; IOD, integrated optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; MyHC, myosin heavy chain; MyoD, Myogenic determination factor; PA, palmitate; RIPA, RadioImmunoPrecipitation Assay; SDS–PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis
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skeletal muscle growth under exposure to humoral factors associated with maternal obesity. The present study was designed to explore whether palmitate, one of the most prevalent fatty acids in skeletal muscle (Chavez and Summers, 2003), can affect mechanisms controlling cell cycle progression and arrest, as well as differentiation of skeletal myoblasts. We examined cellular levels and localizations of cyclins promoting cell cycle progression (cyclin A, D1), proteins essential for cell cycle arrest and the onset of myogenesis (p21, cyclin D3), the distribution of p21 in the cdk4 complexes as well as markers of myogenic differentiation (myogenin, integrinb1, a-actinin and myosin heavy chain) in mouse C2C12 myoblasts exposed to palmitate in vitro. Materials and methods
Preparing fatty acid-containing media The solution of palmitate for the experimental treatment was prepared by conjugation with fatty acid-free bovine serum albumin, as described by Coll et al. (2008). First, palmitate (the sodium salt, obtained from Sigma-Aldrich), was dissolved in ethanol and then diluted 1:100 in DMEM (Dulbecco modified Eagle medium) supplemented with 2% (w/v) fatty-acid free bovine serum albumin.
Cell culture Research work was carried out on murine myogenic C2C12 cell line (satellite cells from thigh muscle), purchased from the European Collection of Animal Cell Culture (ECACC). This cell line undergoes proliferation and differentiation in response to growth factors present in the extracellular environment and serves as a model to study mechanisms controlling myogenesis (Yaffe and Saxel, 1977). Mouse C2C12 myoblasts were maintained free of contamination at the exponential phase of growth in DMEM supplemented with 10% FBS (Foetal Bovine Serum) and an antibiotic-antimycotic mixture (Life Technologies), in controlled humidified air supplemented with 5% CO2, at 37 C. After reaching 40% confluence, proliferating myoblasts were subjected to 24-h exposure to palmitate (final concentration 0.1 mmol/l) added to 5%FBS/DMEM, according to experimental protocol described previously (Lee et al., 2009). When the cells reached 80% confluence, myogenic differentiation was induced after switching to a medium containing 2% horse serum (HS) supplemented with palmitate (final concentration 0.1 mmol/l) and used during 3-day period of observation. Proliferating and differentiating control cultures were maintained in 5% FBS/DMEM or 2% HS/DMEM medium, respectively, containing the same amounts of ethanol and FFA-free
Palmitate affects skeletal myogenesis
bovine serum albumin as experimental media. To preserve the characteristics of C2C12 cell line, the cells were split up to a maximum of 7 times.
Assessment of DNA content and cell viability A crystal violet (CV) assay was performed to determine the total amount of nuclear DNA. Viability of proliferating and differentiating cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (Grabiec et al., 2014).
Analysis of myoblast number and myotube size C2C12 myoblasts were seeded onto Petri dishes and cultured as described above. Changes in cell number were monitored using phase contrast microscope (IX 70, Olympus Optical Co., Hamburg, Germany) and the Kodak DC 290 zoom digital camera (Eastman Kodak Co., Rochester, NY, USA). The images of 10 independent visual fields for each dish were captured before (0 h) and after 24-h-treatment and myoblast number in control and palmitate-treated cultures were compared using the images related to the same visual field. The results were presented as the percentage of initial cell number. Differentiating C2C12 myogenic cells were used for experiments at day 3. To visualize morphological changes in cell cultures, ten non-overlapping visual fields for control and experimental treatment were photographed. Myotubes were identified as clearly bigger and longer morphology than undifferentiated myoblasts. All photographed myotubes (i.e. 39 for control and 41 for palmitate treatment) were measured and used for calculation and comparison of mean myotube length and diameter for each group. Myotube length was measured as the longest distance between two myotube ends, and the myotube diameter was measured in the biggest part of the entire tube, using MicroImage analysis system (Olympus Optical, Poland), and presented in arbitrary units.
Immunoblotting Whole cell lysates were obtained using RIPA (RadioImmunoPrecipitation Assay) buffer supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Aliquots of cell extracts corresponding to 100 mg of protein were resolved through SDS-PAGE as described previously (Grabiec et al., 2014). The membranes were incubated with appropriate primary antibody (Santa Cruz Biotechnology): anti-cyclin A (rabbit polyclonal, sc596), anti-cyclin D1 (rabbit polyclonal, sc-718), anti-p21 (goat polyclonal, sc-397-G), anti-cdk4 (goat polyclonal, sc260-G), anti-cyclin D3 (goat polyclonal, sc-182-G), anti-
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Figure 1 The effect of palmitate supplementation on C2C12 myoblast number. A) Representative phase-contrast images (magnification 100) of control (Ctrl) and palmitate-treated (PA, 0.1 mmol/l) cultures. B) The results are shown as percentage of initial cell number. *Significantly different versus control value, n ¼ 3.
MyoD (rabbit polyclonal, sc-760), anti-myogenin (mouse monoclonal, sc-12732), anti-integrin b1 (rabbit polyclonal, sc-8978), anti-a-actinin (rabbit polyclonal, sc-15335), and anti-MyHC (rabbit polyclonal, sc-20641). Quantification of the integrated optical density (IOD ¼ optical density area) was performed using the analysis software provided with the Odyssey scanner (LI-COR Biosciences). Control probing with anti-actin antibody (goat polyclonal, sc-1616, Santa Cruz Biotechnology) was also performed, to ensure that all lanes contained equal amounts of total protein.
Immunoprecipitation The p-21-cdk4 interaction was evaluated on the basis of presence of p21 protein in the material after immunoprecipitation with anti-cdk4 antibody, as was described previously (Grabiec et al., 2014).
Immunofluorescence staining and confocal microscopy Cell cultures were carried out directly on glass Lab-tec coverslips (Nunc Inc., USA), fixed with 3.7% paraformaldehyde and permeabilized with 0.05% Triton X-100 in PBS, as described previously (Grabiec et al., 2014). The cells were incubated overnight in darkness at 4 C with primary antibody (Santa Cruz Biotechnology) and with Alexa Fluor 488 secondary antibody (Eugene, USA). For nuclear visualization, the cells were stained with 7-aminoactinomycin D (7-AAD). The cells were visualized by confocal laser scanning microscope FV-500 system (Olympus Optical Co, Hamburg, Germany). The combination of excitation/ 1046
emission were: Argon 488 nm laser with 505–525 nm filter for Alexa Fluor 488 and HeNe 543 nm laser with 610 nm filter for 7AAD nucleus staining. Stack of cross-sections were gathered separately for each fluorescence channel. Ten independent fields from each repetition of control and experimentally treated cell cultures were photographed and the IOD values were measured using MicroImage analysis system (Olympus Optical Poland). To estimate the nuclear localization of examined proteins, circle was drawn around every nucleus with red channel and then pixels inside the circles were counted from green channel. Total fluorescence of fields with similar cell density reflected the green fluorescence measured both in green and red channel.
Statistical analyses The results of MTT and CV tests are representative of four separate experiments performed in triplicate (n ¼ 12). The assessment of myoblast number was performed in triplicate. The individual data (n ¼ 3) used for statistical analysis represent average increments of cell number calculated for each dish from 10 independent visual fields. The data obtained from immunoblotting analysis represent three separate experiments performed in triplicate (n ¼ 9). The experiments visualizing the cellular localization of examined proteins or the morphological changes were performed three times with 3 wells/treatment. Ten randomly selected nonoverlapping fields with similar cell density were photographed. The individual data (n ¼ 10) used for the statistical analysis represent the total fluorescence and calculated nucleus/cytoplasm fluorescence ratio. All results were
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Figure 2 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin A (CycA) in C2C12 myoblasts. A) The cellular content and localization of cyclin A assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of CycA and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
presented as means SE. For each assay, the Student t-test was used for the comparison of two means (control vs. experimental treatment) and the criterion for statistical significance was P < 0.05. The analyses were performed using GraphPad Prism 5 (GraphPad Software, USA). Results Addition of palmitate significantly decreased the number of myoblast in proliferating cultures after 24-h-treatment (by 42% in comparison to control value, P ¼ 0.019, Figure 1). This mitoinhibitory effect of palmitate was confirmed in crystal violet test (drop by 51.6% in comparison to control value, P < 0.0001, Table 1). Proliferating C2C12 myoblast cultures exposed to palmitate for 24 h exhibited lower cell viability (drop by 56.5% below control value in MTT assay, P < 0.0001), which probably resulted from decreased myoblast number. Palmitate slightly diminished the number and viability of myogenic cells subjected to 3-day
differentiation (by 15.3% and 33% in comparison to control value, respectively, P < 0.0001). Presence of palmitate markedly decreased the intracellular level of cyclin A during myoblast proliferation (Figure 2).
Table 1 The effect of palmitate supplementation (PA, 0.1 mmol/l) on DNA content assessed in crystal violet test, and cell viability assessed in MTT test in proliferating and differentiating C2C12 myogenic cells.
Proliferating myoblasts DNA content Cell viability Differentiation, 3 days DNA content Cell viability
Ctrl
PA
0.43 0.03 0.26 0.01
0.21 0.02* 0.12 0.002*
1.71 0.04 0.93 0.02
1.45 0.03* 0.62 0.02*
*Significantly different versus control value (Ctrl), n ¼ 12.
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Figure 3 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin D1 (CycD1) in C2C12 myoblasts. A) The cellular content and localization of cyclin D1 assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of CycD1 and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
In control myoblasts cyclin A appeared both in cytoplasm and in nuclei. Supplementation of cell culture medium with the fatty acid salt did not alter markedly the localization of cyclin A in myoblasts. Both in control and in palmitate-treated cells cyclin A-related green fluorescence clearly overlapped with nuclear red 7-AAD fluorescence, manifested by yellow fluorescence (resulting from simultaneous excitation of green and red fluorochromes). In myoblasts treated with palmitate, a decreased level of cyclin D1 was observed (Figure 3). Moreover, palmitate markedly decreased the level of cyclin D1 in myoblast nuclei. Exposure to palmitate caused an increase in cellular content of the cell cycle inhibitor p21 as well as in nuclear localization of this protein (Figure 4). In control myoblasts p21 bound to cdk4 was hardly detected, and palmitate treatment significantly increase the fraction of p21 associated with cdk4. In differentiating C2C12 cells supplementation with palmitate significantly stimulated the expression of cyclin 1048
D3, associated with cell cycle exit (Figure 5). The cellular content of MyoD was not modified by palmitate; however, myogenin was significantly augmented. Integrin-b1 was not affected by palmitate, whereas increases in a-actinin and in myosin heavy chain (MyHC) levels were observed on the 3rd day of myogenesis (Figure 6). C2C12 myogenic cells placed in differentiation medium began to fuse after 2 days of culturin, both under control and experimental conditions; moreover, a similar numbers of myotubes were found in control and palmitate-treated cultures (39 and 41 myotubes, respectively). All these myotubes were measured and presented in Figure 6D. Addition of palmitate had no effect on myotube length; however, it caused a significant increase in myotube diameter (by 67% in comparison to the control value, P < 0.0001). Discussion Maternal obesity can affect fetal skeletal muscle formation, leading to predisposition to metabolic disturbances in
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Figure 4 The effect of palmitate supplementation (PA, 0.1 mmol/l) on p21 in C2C12 myoblasts. A) The cellular content and localization of p21 assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of total p21, cyclin-dependent kinase 4 (Cdk4)-bound p21, cdk-4 and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
offspring (Du et al., 2010). Therefore, mechanisms controlling processes that determine skeletal muscle mass, i.e. myoblast proliferation, cell cycle exit, differentiation, and their potential modifications by humoral factors acting during fetal and postnatal period merit interest.
There are studies concerning the effect of fatty acids on mechanisms controlling muscle cell growth and differentiation; however, reported data differ depending on experimental protocol. According to Lee et al. (2009), palmitate (0.1–10 mmol/l) did not affect either proliferation or
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Figure 5 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin D3 (A), MyoD (B) and myogenin (Myog, C) content in C2C12 cells on the 3rd day of differentiation. The densitometric quantitation of the specific bands (IOD, integrated optical density) is presented in arbitrary units, with the value obtained in control (Ctrl) cells set as 100%. Blots are representative for three separate experiments. *Significantly different versus control value.
Figure 6 The effect of palmitate supplementation (PA, 0.1 mmol/l) on integrin-b1 (A), a-actinin (B) and myosin heavy chain (MyHC, C) content in C2C12 cells on the 3rd day of differentiation. The densitometric quantitation of the specific bands (IOD, integrated optical density) is presented in arbitrary units, with the value obtained in control (Ctrl) cells set as 100%. Blots are representative for three separate experiments. D) Representative phase-contrast images (magnification 100) of control and palmitate-treated cultures on the 3rd day of differentiation. The points on the scatter charts represent individual myotubes. The length and diameter of myotubes are presented in arbitrary units. *Significantly different versus control value.
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differentiation of C2C12 myoblasts during 48-h-exposure. In our experiments, the higher concentration of fatty acid was used (0.1 mmol/l) and palmitate decreased myoblasts proliferation (Figure 1 and Table 1). In fact, at the beginning of our experiments, we used higher concentration of palmitate, i.e. 0.5 mmol/l, which is known to cause insulin resistance in muscle cells (Alkheteeb et al., 2007; Coll et al., 2008; Bakhtiyari et al., 2010; Feng et al., 2012). However, this concentration appeared highly toxic for proliferating myoblasts (not shown); therefore, finally, we decided to treat cell cultures with fatty acid salt at concentration of 0.1 mmol/l. Palmitate present in extracellular environment of proliferating myoblasts diminished the level of cyclins that play an essential role in cell cycle progression (Figures 2 and 3). Drop in cyclin A, which is known to promote cell cycle in S and G2 phases (Cheng et al., 2007), in cultures treated with palmitate was revealed; however, the localization of cyclin A in myoblast nuclei was not altered, suggesting cyclin activity. Decreased level of cyclin D1 and drop in its nuclear localization were observed in myoblasts subjected to proliferation in the presence of palmitate. Moreover, the cdk4-bound abundance of p21 increased under palmitate treatment (Figure 4), which can result in cdk4 inhibition. Thus, the inhibitory effect of palmitate on myoblast number (Figure 1 and Table 1) was, at least partly, attributed to the inhibition of cyclin D1 expression and cdk-4 activity, whereas the role of CycA is less evident. In contrast to its mitoinhibitory effect, the presence of palmitate seems to promote myogenic differentiation. This was manifested by increases of examined markers of cell cycle withdrawal and myogenesis, i.e. cyclin D3, myogenin, a-actinin and myosin heavy chain in C2C12 cells subjected to 3-day differentiation with palmitate (Figures 5 and 6). The onset of myocyte fusion and myotube number were similar in control and fatty acid-treated cultures (Figure 6), indicating proper dynamics of early myogenesis, similar to that described previously (Ferri et al., 2009). Interestingly, Yang et al. (2013) recently found negative effects of high palmitate (0.2–0.6 mmol/l) on C2C12 myotubes, manifested by drop in expression of late myogenic markers (myosin heavy chain 2b and muscle creatine kinase), health benefit myokines (FGF21, FnDC5, CTRP15) and an increase in proteolytic system expression. Fatty acid-induced stimulation of protein degradation through the ubiquitin-proteasome pathway, as well as an inhibition of protein synthesis through the increased phosphorylation of eukaryotic initiation factor 2 (eIF2) in skeletal muscle cells have already been described (Zhou et al., 2007). Thus, effects of palmitate on myogenic cells are clearly dose-dependent, as in our experiments, the myotubes exposed to fatty acid did not manifest the symptoms of wasting assessed on the basis of molecular markers of myogenic differentiation and at the
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microscopic level (Figure 6). Moreover, such observations indicated that effects of palmitate present in extracellular environment of skeletal myoblasts may be secondary, due to modulation of cell response to environmental stimuli. In fact, several disturbances in hormone signaling, i.e. enhanced phosphorylation of protein kinase C-theta (Wang et al., 2010), increased protein tyrosine phosphatase-1B (Bakhtiyari et al., 2010), decreased phosphorylation of Akt (Feng et al., 2012) or activation of NF-kB (Barma et al., 2009; Zhang et al., 2010; Salvad o et al., 2013), usually attributed to palmitate-induced insulin resistance, could also affect myoblast differentiation. Clarification of this issue requires further investigation, involving transcriptional profiling or proteomic studies. Tong et al. (2009) reported reduction of key myogenic markers, MyoD, myogenin and desmin, in fetal skeletal muscle in an obese sheep model. Our present study investigating the effect of individual fatty acid on myoblast proliferation and differentiation in vitro is complementary to those results, indicating that inhibition of fetal skeletal muscle growth associated with maternal obesity can result from disturbances of mechanisms controlling proliferation of muscle precursor cells, independent of effects on myogenic differentiation. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth. Acknowledgment and funding This study was supported by Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW). References Alkhateeb H, Chabowski A, Glatz JF, Luiken JF, Bonen A (2007) Two phases of palmitate-induced insulin resistance in skeletal muscle: impaired GLUT4 translocation is followed by a reduced GLUT4 intrinsic activity. Am J Physiol Endocrinol Metab 293: E783–93. Bakhtiyari S, Meshkani R, Taghikhani M, Larijani B, Adeli K (2010) Protein tyrosine phosphatase-1B (PTP-1B) knockdown improves palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Lipids 45: 237–44. Barma P, Bhattacharya S, Bhattacharya A, Kundu R, Dasgupta S, Biswas A, Bhattacharya S, Roy SS, Bhattacharya S (2009) Lipid induced overexpression of NF-kappaB in skeletal muscle cells is linked to insulin resistance. Biochim Biophys Acta 1792: 190–20. Blomen VA, Boonstra J (2007) Cell fate determination during G1 phase progression. Cell Mol Life Sci 64 3084–104.
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Chavez JA, Summers SA (2003) Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 419: 101–9. Cheng RK, Asai T, Tang H, Dashoush NH, Kara RJ, Costa KD, Naka Y, Wu EX, Wolgemuth DJ, Chaudhry HW (2007) Cyclin A2 induces cardiac regeneration after myocardial infarction and prevents heart failure. Circ Res 100: 1741–8. Coll T, Eyre E, Rodriguez-Calvo R, Palomer X, Sanchez RM, Merlos M, Laguna JC, Vazquez-Carrera M (2008) Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem 283: 11107–16. Delarue J, Magnan C (2007) Free fatty acids and insulin resistance. Curr Opin Clin Nutr Metab Care 10: 142–8. Du M, Yan X, Tong JF, Zhao J, Zhu MJ (2010) Maternal obesity, inflammation, and fetal skeletal muscle development. Biol Reprod 82: 4–12. Feng XT, Wang TZ, Leng J, Chen Y, Liu JB, Liu Y, Wang WJ (2012) Palmitate contributes to insulin resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes. Biosci Biotechnol Biochem 76: 1356–61. Ferri P, Barbieri E, Burattini S, Guescini M, D’Emilio A, Biagiotti L, Del Grande P, De Luca A, Stocchi V, Falcieri E (2009) Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem 108: 1302–17. Floyd ZE, Trausch-Azar JS, Reinstein E, Ciechanover A, Schwartz AL (2001) The nuclear ubiquitin-proteasome system degrades MyoD. J Biol Chem 276: 22468–75. Grabiec K, Gajewska M, Milewska M, Błaszczyk M, GrzelkowskaKowalczyk K (2014) The influence of high glucose and high insulin on mechanisms controlling cell cycle progression and arrest in mouse C2C12 myoblasts: the comparison with IGF-I effect. J Endocrinol Invest 37: 233–45. Hirabara SM, Silveira LR, Alberici LC, Leandro CV, Lambertucci RH, Polimeno GC, Cury Boaventura MF, Procopio J, Vercesi AE, Curi R (2006) Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle. Biochim Biophys Acta 1757: 57–66. Houmard JA (2008) Insulin resistance and cardiometabolic syndrome: adipose tissue and skeletal muscle factors. Am J Physiol Regul Integr Comp Physiol 294: R1111–6.
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Hulver MW, Dohm GL (2004) The molecular mechanisms linking muscle fat accumulation to insulin resistance. Proc Nutr Soc 63: 375–80. Lee J-H, Tachibana H, Morinaga Y, Fujimura Y, Yamada K (2009) Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids. Life Sci 84: 415–20. Le Grand F, Rudnicki MA (2007) Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 19: 628–33. Lowell BB, Shulman GI (2005) Mitochondrial dysfunction and type 2 diabetes. Science 307: 384–7. Salvad o L, Coll T, G omez-Foix AM, Salmer on E, Barroso E, Palomer X, Vazquez-Carrera M (2013) Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPKdependent mechanism. Diabetologia 56: 1372–82. Tong JF, Xu Y, Zhu MJ, Ford SP, Nathanielsz PW, Du M (2009) Maternal obesity downregulates myogenesis and b-catenin signaling in fetal skeletal muscle. Am J Physiol Endocrinol Metab 296: E917–24. Wang X, Yu W, Nawaz A, Guan F, Sun S, Wang C (2010) Palmitate induced insulin resistance by PKCtheta-dependent activation of mTOR/S6K pathway in C2C12 myotubes. Exp Clin Endocrinol Diabetes 118: 657–61. Warner MJ, Ozanne SE (2010) Mechanisms involved in the developmental programming of adulthood disease. Biochem J 427: 333–47. Yang M, Wei D, Mo C, Zhang J, Wang X, Han X, Wang Z, Xiao H (2013) Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes. Lipids Health Dis 12: 104. Zhang J, Wu W, Li D, Guo Y, Ding H (2010) Overactivation of NF-kB impairs insulin sensitivity and mediates palmitateinduced insulin resistance in C2C12 skeletal muscle cells. Endocrine 37: 157–66. Zhou Q, Du J, Hu Z, Walsh K, Wang XH (2007) Evidence for adipose-muscle cross-talk: opposing regulation of muscle proteolysis by adiponectin and fatty acids. Endocrinology 148: 5696–705. Received 3 November 2014; accepted 31 March 2015. Final version published online 20 May 2015.
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RESEARCH ARTICLE
Palmitate exerts opposite effects on proliferation and differentiation of skeletal myoblasts Kamil Grabiec, Marta Milewska, Maciej Błaszczyk, Małgorzata Gajewska and Katarzyna Grzelkowska-Kowalczyk* Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Nowoursynowska 159 02-776, Warsaw, Poland
Abstract The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (a-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth. Keywords: G1 cyclins; long-chain fatty acids; myogenesis; myogenic regulatory factors
Introduction Skeletal muscle, making up approximately 40% of the total body mass, provides structural support, enables the body to maintain posture and controls motor movements. It is a key insulin-responsive tissue, important in maintaining homeostasis, due to its relatively large mass and energy needs (Lowell and Shulman, 2005; Houmard 2008). Fetal stage is crucial for skeletal muscle development, especially in humans and farm animals, because there is no increase in muscle fiber number after birth (Zhu et al., 2006). Early stages of muscle fiber formation involve two excluding processes (Floyd et al., 2001). Myoblast proliferation occurs through series of events arranged in precise order, i.e. cell cycle, whereas differentiation concerns the cells, which withdraw the cell cycle in G1 phase (Blomen and Boonstra, 2007). Proper myogenesis depends on the expression of primary (MyoD and Myf5) and secondary (myogenin and MRF-4) myogenic transcription factors
(Ferri et al., 2009), required for subsequent muscle-specific gene expression and morphological changes (Le Grand and Rudnicki, 2007). There is increasing evidence supporting on effects of fatty acids on muscle metabolic function. Excess of dietary lipids and acute exposure to palmitate cause insulin resistance and relevant mechanisms, i.e. impairment of insulin signaling (Hulver and Dohm, 2004; Delarue and Magnan, 2007), GLUT transporter activity (Alkhateeb et al., 2007) and mitochondrial uncoupling (Hirabara et al., 2006) have been extensively investigated. According to recent studies on human and animal models, maternal high-fat feeding and obesity increase fat-to-lean-mass ratio and cause an impaired glucose tolerance in offspring (reviewed in Warner and Ozanne, 2010). Using the obese pregnant sheep model, Tong et al. (2009) reported down-regulation of myogenesis and Wnt/b-catenin signaling, probably resulting from activation of inflammatory NF-kB pathway in fetal skeletal muscle. Such observations implicate an impairment of fetal
Corresponding author: e-mail: [email protected] Abbreviations: 7-AAD, 7-aminoactinomycin D; cdk, cyclin-dependent kinase; CV, crystal violet; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HS, horse serum; IOD, integrated optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; MyHC, myosin heavy chain; MyoD, Myogenic determination factor; PA, palmitate; RIPA, RadioImmunoPrecipitation Assay; SDS–PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis
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skeletal muscle growth under exposure to humoral factors associated with maternal obesity. The present study was designed to explore whether palmitate, one of the most prevalent fatty acids in skeletal muscle (Chavez and Summers, 2003), can affect mechanisms controlling cell cycle progression and arrest, as well as differentiation of skeletal myoblasts. We examined cellular levels and localizations of cyclins promoting cell cycle progression (cyclin A, D1), proteins essential for cell cycle arrest and the onset of myogenesis (p21, cyclin D3), the distribution of p21 in the cdk4 complexes as well as markers of myogenic differentiation (myogenin, integrinb1, a-actinin and myosin heavy chain) in mouse C2C12 myoblasts exposed to palmitate in vitro. Materials and methods
Preparing fatty acid-containing media The solution of palmitate for the experimental treatment was prepared by conjugation with fatty acid-free bovine serum albumin, as described by Coll et al. (2008). First, palmitate (the sodium salt, obtained from Sigma-Aldrich), was dissolved in ethanol and then diluted 1:100 in DMEM (Dulbecco modified Eagle medium) supplemented with 2% (w/v) fatty-acid free bovine serum albumin.
Cell culture Research work was carried out on murine myogenic C2C12 cell line (satellite cells from thigh muscle), purchased from the European Collection of Animal Cell Culture (ECACC). This cell line undergoes proliferation and differentiation in response to growth factors present in the extracellular environment and serves as a model to study mechanisms controlling myogenesis (Yaffe and Saxel, 1977). Mouse C2C12 myoblasts were maintained free of contamination at the exponential phase of growth in DMEM supplemented with 10% FBS (Foetal Bovine Serum) and an antibiotic-antimycotic mixture (Life Technologies), in controlled humidified air supplemented with 5% CO2, at 37 C. After reaching 40% confluence, proliferating myoblasts were subjected to 24-h exposure to palmitate (final concentration 0.1 mmol/l) added to 5%FBS/DMEM, according to experimental protocol described previously (Lee et al., 2009). When the cells reached 80% confluence, myogenic differentiation was induced after switching to a medium containing 2% horse serum (HS) supplemented with palmitate (final concentration 0.1 mmol/l) and used during 3-day period of observation. Proliferating and differentiating control cultures were maintained in 5% FBS/DMEM or 2% HS/DMEM medium, respectively, containing the same amounts of ethanol and FFA-free
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bovine serum albumin as experimental media. To preserve the characteristics of C2C12 cell line, the cells were split up to a maximum of 7 times.
Assessment of DNA content and cell viability A crystal violet (CV) assay was performed to determine the total amount of nuclear DNA. Viability of proliferating and differentiating cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (Grabiec et al., 2014).
Analysis of myoblast number and myotube size C2C12 myoblasts were seeded onto Petri dishes and cultured as described above. Changes in cell number were monitored using phase contrast microscope (IX 70, Olympus Optical Co., Hamburg, Germany) and the Kodak DC 290 zoom digital camera (Eastman Kodak Co., Rochester, NY, USA). The images of 10 independent visual fields for each dish were captured before (0 h) and after 24-h-treatment and myoblast number in control and palmitate-treated cultures were compared using the images related to the same visual field. The results were presented as the percentage of initial cell number. Differentiating C2C12 myogenic cells were used for experiments at day 3. To visualize morphological changes in cell cultures, ten non-overlapping visual fields for control and experimental treatment were photographed. Myotubes were identified as clearly bigger and longer morphology than undifferentiated myoblasts. All photographed myotubes (i.e. 39 for control and 41 for palmitate treatment) were measured and used for calculation and comparison of mean myotube length and diameter for each group. Myotube length was measured as the longest distance between two myotube ends, and the myotube diameter was measured in the biggest part of the entire tube, using MicroImage analysis system (Olympus Optical, Poland), and presented in arbitrary units.
Immunoblotting Whole cell lysates were obtained using RIPA (RadioImmunoPrecipitation Assay) buffer supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Aliquots of cell extracts corresponding to 100 mg of protein were resolved through SDS-PAGE as described previously (Grabiec et al., 2014). The membranes were incubated with appropriate primary antibody (Santa Cruz Biotechnology): anti-cyclin A (rabbit polyclonal, sc596), anti-cyclin D1 (rabbit polyclonal, sc-718), anti-p21 (goat polyclonal, sc-397-G), anti-cdk4 (goat polyclonal, sc260-G), anti-cyclin D3 (goat polyclonal, sc-182-G), anti-
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Figure 1 The effect of palmitate supplementation on C2C12 myoblast number. A) Representative phase-contrast images (magnification 100) of control (Ctrl) and palmitate-treated (PA, 0.1 mmol/l) cultures. B) The results are shown as percentage of initial cell number. *Significantly different versus control value, n ¼ 3.
MyoD (rabbit polyclonal, sc-760), anti-myogenin (mouse monoclonal, sc-12732), anti-integrin b1 (rabbit polyclonal, sc-8978), anti-a-actinin (rabbit polyclonal, sc-15335), and anti-MyHC (rabbit polyclonal, sc-20641). Quantification of the integrated optical density (IOD ¼ optical density area) was performed using the analysis software provided with the Odyssey scanner (LI-COR Biosciences). Control probing with anti-actin antibody (goat polyclonal, sc-1616, Santa Cruz Biotechnology) was also performed, to ensure that all lanes contained equal amounts of total protein.
Immunoprecipitation The p-21-cdk4 interaction was evaluated on the basis of presence of p21 protein in the material after immunoprecipitation with anti-cdk4 antibody, as was described previously (Grabiec et al., 2014).
Immunofluorescence staining and confocal microscopy Cell cultures were carried out directly on glass Lab-tec coverslips (Nunc Inc., USA), fixed with 3.7% paraformaldehyde and permeabilized with 0.05% Triton X-100 in PBS, as described previously (Grabiec et al., 2014). The cells were incubated overnight in darkness at 4 C with primary antibody (Santa Cruz Biotechnology) and with Alexa Fluor 488 secondary antibody (Eugene, USA). For nuclear visualization, the cells were stained with 7-aminoactinomycin D (7-AAD). The cells were visualized by confocal laser scanning microscope FV-500 system (Olympus Optical Co, Hamburg, Germany). The combination of excitation/ 1046
emission were: Argon 488 nm laser with 505–525 nm filter for Alexa Fluor 488 and HeNe 543 nm laser with 610 nm filter for 7AAD nucleus staining. Stack of cross-sections were gathered separately for each fluorescence channel. Ten independent fields from each repetition of control and experimentally treated cell cultures were photographed and the IOD values were measured using MicroImage analysis system (Olympus Optical Poland). To estimate the nuclear localization of examined proteins, circle was drawn around every nucleus with red channel and then pixels inside the circles were counted from green channel. Total fluorescence of fields with similar cell density reflected the green fluorescence measured both in green and red channel.
Statistical analyses The results of MTT and CV tests are representative of four separate experiments performed in triplicate (n ¼ 12). The assessment of myoblast number was performed in triplicate. The individual data (n ¼ 3) used for statistical analysis represent average increments of cell number calculated for each dish from 10 independent visual fields. The data obtained from immunoblotting analysis represent three separate experiments performed in triplicate (n ¼ 9). The experiments visualizing the cellular localization of examined proteins or the morphological changes were performed three times with 3 wells/treatment. Ten randomly selected nonoverlapping fields with similar cell density were photographed. The individual data (n ¼ 10) used for the statistical analysis represent the total fluorescence and calculated nucleus/cytoplasm fluorescence ratio. All results were
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Figure 2 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin A (CycA) in C2C12 myoblasts. A) The cellular content and localization of cyclin A assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of CycA and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
presented as means SE. For each assay, the Student t-test was used for the comparison of two means (control vs. experimental treatment) and the criterion for statistical significance was P < 0.05. The analyses were performed using GraphPad Prism 5 (GraphPad Software, USA). Results Addition of palmitate significantly decreased the number of myoblast in proliferating cultures after 24-h-treatment (by 42% in comparison to control value, P ¼ 0.019, Figure 1). This mitoinhibitory effect of palmitate was confirmed in crystal violet test (drop by 51.6% in comparison to control value, P < 0.0001, Table 1). Proliferating C2C12 myoblast cultures exposed to palmitate for 24 h exhibited lower cell viability (drop by 56.5% below control value in MTT assay, P < 0.0001), which probably resulted from decreased myoblast number. Palmitate slightly diminished the number and viability of myogenic cells subjected to 3-day
differentiation (by 15.3% and 33% in comparison to control value, respectively, P < 0.0001). Presence of palmitate markedly decreased the intracellular level of cyclin A during myoblast proliferation (Figure 2).
Table 1 The effect of palmitate supplementation (PA, 0.1 mmol/l) on DNA content assessed in crystal violet test, and cell viability assessed in MTT test in proliferating and differentiating C2C12 myogenic cells.
Proliferating myoblasts DNA content Cell viability Differentiation, 3 days DNA content Cell viability
Ctrl
PA
0.43 0.03 0.26 0.01
0.21 0.02* 0.12 0.002*
1.71 0.04 0.93 0.02
1.45 0.03* 0.62 0.02*
*Significantly different versus control value (Ctrl), n ¼ 12.
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Figure 3 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin D1 (CycD1) in C2C12 myoblasts. A) The cellular content and localization of cyclin D1 assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of CycD1 and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
In control myoblasts cyclin A appeared both in cytoplasm and in nuclei. Supplementation of cell culture medium with the fatty acid salt did not alter markedly the localization of cyclin A in myoblasts. Both in control and in palmitate-treated cells cyclin A-related green fluorescence clearly overlapped with nuclear red 7-AAD fluorescence, manifested by yellow fluorescence (resulting from simultaneous excitation of green and red fluorochromes). In myoblasts treated with palmitate, a decreased level of cyclin D1 was observed (Figure 3). Moreover, palmitate markedly decreased the level of cyclin D1 in myoblast nuclei. Exposure to palmitate caused an increase in cellular content of the cell cycle inhibitor p21 as well as in nuclear localization of this protein (Figure 4). In control myoblasts p21 bound to cdk4 was hardly detected, and palmitate treatment significantly increase the fraction of p21 associated with cdk4. In differentiating C2C12 cells supplementation with palmitate significantly stimulated the expression of cyclin 1048
D3, associated with cell cycle exit (Figure 5). The cellular content of MyoD was not modified by palmitate; however, myogenin was significantly augmented. Integrin-b1 was not affected by palmitate, whereas increases in a-actinin and in myosin heavy chain (MyHC) levels were observed on the 3rd day of myogenesis (Figure 6). C2C12 myogenic cells placed in differentiation medium began to fuse after 2 days of culturin, both under control and experimental conditions; moreover, a similar numbers of myotubes were found in control and palmitate-treated cultures (39 and 41 myotubes, respectively). All these myotubes were measured and presented in Figure 6D. Addition of palmitate had no effect on myotube length; however, it caused a significant increase in myotube diameter (by 67% in comparison to the control value, P < 0.0001). Discussion Maternal obesity can affect fetal skeletal muscle formation, leading to predisposition to metabolic disturbances in
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Figure 4 The effect of palmitate supplementation (PA, 0.1 mmol/l) on p21 in C2C12 myoblasts. A) The cellular content and localization of p21 assessed by confocal microscopy. The images are representative for three separate experiments. Bar, 20 mm. The integrated optical density (IOD) obtained in control (Ctrl) cells was set as 100%. B) Representative blots of total p21, cyclin-dependent kinase 4 (Cdk4)-bound p21, cdk-4 and actin (as a loading control) in myoblasts proliferating for 24 h are shown. *Significantly different versus control value.
offspring (Du et al., 2010). Therefore, mechanisms controlling processes that determine skeletal muscle mass, i.e. myoblast proliferation, cell cycle exit, differentiation, and their potential modifications by humoral factors acting during fetal and postnatal period merit interest.
There are studies concerning the effect of fatty acids on mechanisms controlling muscle cell growth and differentiation; however, reported data differ depending on experimental protocol. According to Lee et al. (2009), palmitate (0.1–10 mmol/l) did not affect either proliferation or
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Figure 5 The effect of palmitate supplementation (PA, 0.1 mmol/l) on cyclin D3 (A), MyoD (B) and myogenin (Myog, C) content in C2C12 cells on the 3rd day of differentiation. The densitometric quantitation of the specific bands (IOD, integrated optical density) is presented in arbitrary units, with the value obtained in control (Ctrl) cells set as 100%. Blots are representative for three separate experiments. *Significantly different versus control value.
Figure 6 The effect of palmitate supplementation (PA, 0.1 mmol/l) on integrin-b1 (A), a-actinin (B) and myosin heavy chain (MyHC, C) content in C2C12 cells on the 3rd day of differentiation. The densitometric quantitation of the specific bands (IOD, integrated optical density) is presented in arbitrary units, with the value obtained in control (Ctrl) cells set as 100%. Blots are representative for three separate experiments. D) Representative phase-contrast images (magnification 100) of control and palmitate-treated cultures on the 3rd day of differentiation. The points on the scatter charts represent individual myotubes. The length and diameter of myotubes are presented in arbitrary units. *Significantly different versus control value.
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differentiation of C2C12 myoblasts during 48-h-exposure. In our experiments, the higher concentration of fatty acid was used (0.1 mmol/l) and palmitate decreased myoblasts proliferation (Figure 1 and Table 1). In fact, at the beginning of our experiments, we used higher concentration of palmitate, i.e. 0.5 mmol/l, which is known to cause insulin resistance in muscle cells (Alkheteeb et al., 2007; Coll et al., 2008; Bakhtiyari et al., 2010; Feng et al., 2012). However, this concentration appeared highly toxic for proliferating myoblasts (not shown); therefore, finally, we decided to treat cell cultures with fatty acid salt at concentration of 0.1 mmol/l. Palmitate present in extracellular environment of proliferating myoblasts diminished the level of cyclins that play an essential role in cell cycle progression (Figures 2 and 3). Drop in cyclin A, which is known to promote cell cycle in S and G2 phases (Cheng et al., 2007), in cultures treated with palmitate was revealed; however, the localization of cyclin A in myoblast nuclei was not altered, suggesting cyclin activity. Decreased level of cyclin D1 and drop in its nuclear localization were observed in myoblasts subjected to proliferation in the presence of palmitate. Moreover, the cdk4-bound abundance of p21 increased under palmitate treatment (Figure 4), which can result in cdk4 inhibition. Thus, the inhibitory effect of palmitate on myoblast number (Figure 1 and Table 1) was, at least partly, attributed to the inhibition of cyclin D1 expression and cdk-4 activity, whereas the role of CycA is less evident. In contrast to its mitoinhibitory effect, the presence of palmitate seems to promote myogenic differentiation. This was manifested by increases of examined markers of cell cycle withdrawal and myogenesis, i.e. cyclin D3, myogenin, a-actinin and myosin heavy chain in C2C12 cells subjected to 3-day differentiation with palmitate (Figures 5 and 6). The onset of myocyte fusion and myotube number were similar in control and fatty acid-treated cultures (Figure 6), indicating proper dynamics of early myogenesis, similar to that described previously (Ferri et al., 2009). Interestingly, Yang et al. (2013) recently found negative effects of high palmitate (0.2–0.6 mmol/l) on C2C12 myotubes, manifested by drop in expression of late myogenic markers (myosin heavy chain 2b and muscle creatine kinase), health benefit myokines (FGF21, FnDC5, CTRP15) and an increase in proteolytic system expression. Fatty acid-induced stimulation of protein degradation through the ubiquitin-proteasome pathway, as well as an inhibition of protein synthesis through the increased phosphorylation of eukaryotic initiation factor 2 (eIF2) in skeletal muscle cells have already been described (Zhou et al., 2007). Thus, effects of palmitate on myogenic cells are clearly dose-dependent, as in our experiments, the myotubes exposed to fatty acid did not manifest the symptoms of wasting assessed on the basis of molecular markers of myogenic differentiation and at the
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microscopic level (Figure 6). Moreover, such observations indicated that effects of palmitate present in extracellular environment of skeletal myoblasts may be secondary, due to modulation of cell response to environmental stimuli. In fact, several disturbances in hormone signaling, i.e. enhanced phosphorylation of protein kinase C-theta (Wang et al., 2010), increased protein tyrosine phosphatase-1B (Bakhtiyari et al., 2010), decreased phosphorylation of Akt (Feng et al., 2012) or activation of NF-kB (Barma et al., 2009; Zhang et al., 2010; Salvad o et al., 2013), usually attributed to palmitate-induced insulin resistance, could also affect myoblast differentiation. Clarification of this issue requires further investigation, involving transcriptional profiling or proteomic studies. Tong et al. (2009) reported reduction of key myogenic markers, MyoD, myogenin and desmin, in fetal skeletal muscle in an obese sheep model. Our present study investigating the effect of individual fatty acid on myoblast proliferation and differentiation in vitro is complementary to those results, indicating that inhibition of fetal skeletal muscle growth associated with maternal obesity can result from disturbances of mechanisms controlling proliferation of muscle precursor cells, independent of effects on myogenic differentiation. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth. Acknowledgment and funding This study was supported by Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW). References Alkhateeb H, Chabowski A, Glatz JF, Luiken JF, Bonen A (2007) Two phases of palmitate-induced insulin resistance in skeletal muscle: impaired GLUT4 translocation is followed by a reduced GLUT4 intrinsic activity. Am J Physiol Endocrinol Metab 293: E783–93. Bakhtiyari S, Meshkani R, Taghikhani M, Larijani B, Adeli K (2010) Protein tyrosine phosphatase-1B (PTP-1B) knockdown improves palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Lipids 45: 237–44. Barma P, Bhattacharya S, Bhattacharya A, Kundu R, Dasgupta S, Biswas A, Bhattacharya S, Roy SS, Bhattacharya S (2009) Lipid induced overexpression of NF-kappaB in skeletal muscle cells is linked to insulin resistance. Biochim Biophys Acta 1792: 190–20. Blomen VA, Boonstra J (2007) Cell fate determination during G1 phase progression. Cell Mol Life Sci 64 3084–104.
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Chavez JA, Summers SA (2003) Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 419: 101–9. Cheng RK, Asai T, Tang H, Dashoush NH, Kara RJ, Costa KD, Naka Y, Wu EX, Wolgemuth DJ, Chaudhry HW (2007) Cyclin A2 induces cardiac regeneration after myocardial infarction and prevents heart failure. Circ Res 100: 1741–8. Coll T, Eyre E, Rodriguez-Calvo R, Palomer X, Sanchez RM, Merlos M, Laguna JC, Vazquez-Carrera M (2008) Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem 283: 11107–16. Delarue J, Magnan C (2007) Free fatty acids and insulin resistance. Curr Opin Clin Nutr Metab Care 10: 142–8. Du M, Yan X, Tong JF, Zhao J, Zhu MJ (2010) Maternal obesity, inflammation, and fetal skeletal muscle development. Biol Reprod 82: 4–12. Feng XT, Wang TZ, Leng J, Chen Y, Liu JB, Liu Y, Wang WJ (2012) Palmitate contributes to insulin resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes. Biosci Biotechnol Biochem 76: 1356–61. Ferri P, Barbieri E, Burattini S, Guescini M, D’Emilio A, Biagiotti L, Del Grande P, De Luca A, Stocchi V, Falcieri E (2009) Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem 108: 1302–17. Floyd ZE, Trausch-Azar JS, Reinstein E, Ciechanover A, Schwartz AL (2001) The nuclear ubiquitin-proteasome system degrades MyoD. J Biol Chem 276: 22468–75. Grabiec K, Gajewska M, Milewska M, Błaszczyk M, GrzelkowskaKowalczyk K (2014) The influence of high glucose and high insulin on mechanisms controlling cell cycle progression and arrest in mouse C2C12 myoblasts: the comparison with IGF-I effect. J Endocrinol Invest 37: 233–45. Hirabara SM, Silveira LR, Alberici LC, Leandro CV, Lambertucci RH, Polimeno GC, Cury Boaventura MF, Procopio J, Vercesi AE, Curi R (2006) Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle. Biochim Biophys Acta 1757: 57–66. Houmard JA (2008) Insulin resistance and cardiometabolic syndrome: adipose tissue and skeletal muscle factors. Am J Physiol Regul Integr Comp Physiol 294: R1111–6.
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Hulver MW, Dohm GL (2004) The molecular mechanisms linking muscle fat accumulation to insulin resistance. Proc Nutr Soc 63: 375–80. Lee J-H, Tachibana H, Morinaga Y, Fujimura Y, Yamada K (2009) Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids. Life Sci 84: 415–20. Le Grand F, Rudnicki MA (2007) Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 19: 628–33. Lowell BB, Shulman GI (2005) Mitochondrial dysfunction and type 2 diabetes. Science 307: 384–7. Salvad o L, Coll T, G omez-Foix AM, Salmer on E, Barroso E, Palomer X, Vazquez-Carrera M (2013) Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPKdependent mechanism. Diabetologia 56: 1372–82. Tong JF, Xu Y, Zhu MJ, Ford SP, Nathanielsz PW, Du M (2009) Maternal obesity downregulates myogenesis and b-catenin signaling in fetal skeletal muscle. Am J Physiol Endocrinol Metab 296: E917–24. Wang X, Yu W, Nawaz A, Guan F, Sun S, Wang C (2010) Palmitate induced insulin resistance by PKCtheta-dependent activation of mTOR/S6K pathway in C2C12 myotubes. Exp Clin Endocrinol Diabetes 118: 657–61. Warner MJ, Ozanne SE (2010) Mechanisms involved in the developmental programming of adulthood disease. Biochem J 427: 333–47. Yang M, Wei D, Mo C, Zhang J, Wang X, Han X, Wang Z, Xiao H (2013) Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes. Lipids Health Dis 12: 104. Zhang J, Wu W, Li D, Guo Y, Ding H (2010) Overactivation of NF-kB impairs insulin sensitivity and mediates palmitateinduced insulin resistance in C2C12 skeletal muscle cells. Endocrine 37: 157–66. Zhou Q, Du J, Hu Z, Walsh K, Wang XH (2007) Evidence for adipose-muscle cross-talk: opposing regulation of muscle proteolysis by adiponectin and fatty acids. Endocrinology 148: 5696–705. Received 3 November 2014; accepted 31 March 2015. Final version published online 20 May 2015.
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