Folic acid promotes the myogenic differentiation of C2C12 murine myoblasts through the Akt signaling pathway

Hwang,Seong  Yeon; Kang,Yong  Jung; Sung,Bokyung; Kim,Minjung; Kim,Dong Hwan; Lee,Yujin; Yoo,Mi-Ae; Kim,Cheol  Min; Chung,Hae   Young; Kim,Nam  Deuk

doi:10.3892/ijmm.2015.2311

Folic acid promotes the myogenic differentiation of C2C12 murine myoblasts through the Akt signaling pathway

Authors:
- Seong Yeon Hwang
- Yong Jung Kang
- Bokyung Sung
- Minjung Kim
- Dong Hwan Kim
- Yujin Lee
- Mi-Ae Yoo
- Cheol Min Kim
- Hae Young Chung
- Nam Deuk Kim
View Affiliations

Affiliations: Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea, Department of Molecular Biology, Pusan National University, Busan 609-735, Republic of Korea, Research Center for Anti-Aging Technology Development, Pusan National University, Busan 609-735, Republic of Korea
Published online on: August 12, 2015 https://doi.org/10.3892/ijmm.2015.2311
Pages: 1073-1080

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Folic acid is a water-soluble vitamin in the B-complex group, and an exogenous intake is required for health, growth and development. As a precursor to co-factors, folic acid is required for one-carbon donors in the synthesis of DNA bases and other essential biomolecules. A lack of dietary folic acid can lead to folic acid deficiency and can therefore result in several health problems, including macrocytic anemia, elevated plasma homocysteine levels, cardiovascular disease, birth defects, carcinogenesis, muscle weakness and difficulty in walking. Previous studies have indicated that folic acid exerts a positive effect on skeletal muscle functions. However, the precise role of folic acid in skeletal muscle cell differentiation remains poorly understood. Thus, in the present study, we examined the effects of folic acid on neo-myotube maturation and differentiation using C2C12 murine myoblasts. We found that folic acid promoted the formation of multinucleated myotubes, and increased the fusion index and creatine kinase (CK) activity in a concentration-dependent manner. In addition, western blot analysis revealed that the expression levels of the muscle-specific marker, myosin heavy chain (MyHC), as well as those of the myogenic regulatory factors (MRFs), MyoD and myogenin, were increased in the folic acid-treated myotubes during myogenic differentiation. Folic acid also promoted the activation of the Akt pathway, and this effect was inhibited by treatment of the C2C12 cells with LY294002 (Akt inhibitor). Blocking of the Akt pathway with a specific inhibitor revealed that it was necessary for mediating the stimulatory effects of folic acid on muscle cell differentiation and fusion. Taken together, our data suggest that folic acid promotes the differentiation of C2C12 cells through the activation of the Akt pathway.

View Figures

View References

1	Iyer R and Tomar SK: Folate: a functional food constituent. J Food Sci. 74:R114–R122. 2009. View Article : Google Scholar
2	Lucock M: Is folic acid the ultimate functional food component for disease prevention? BMJ. 328:211–214. 2004. View Article : Google Scholar : PubMed/NCBI
3	Czeizel AE, Dudás I, Vereczkey A and Bánhidy F: Folate deficiency and folic acid supplementation: the prevention of neural-tube defects and congenital heart defects. Nutrients. 5:4760–4775. 2013. View Article : Google Scholar : PubMed/NCBI
4	Hou TC, Lin JJ, Wen HC, Chen LC, Hsu SP and Lee WS: Folic acid inhibits endothelial cell migration through inhibiting the RhoA activity mediated by activating the folic acid receptor/cSrc/p190RhoGAP-signaling pathway. Biochem Pharmacol. 85:376–384. 2013. View Article : Google Scholar
5	Kim YI: Will mandatory folic acid fortification prevent or promote cancer? Am J Clin Nutr. 80:1123–1128. 2004.PubMed/NCBI
6	Li Y, Zhang X, Sun Y, Feng Q, Li G, Wang M, Cui X, Kang L and Jiang Y: Folate deficiency during early-mid pregnancy affects the skeletal muscle transcriptome of piglets from a reciprocal cross. PLoS One. 8:e826162013. View Article : Google Scholar : PubMed/NCBI
7	Swart KM, Enneman AW, van Wijngaarden JP, van Dijk SC, Brouwer-Brolsma EM, Ham AC, Dhonukshe-Rutten RA, van der Velde N, Brug J, van Meurs JB, et al: Homocysteine and the methylenetetrahydrofolate reductase 677C–>T polymorphism in relation to muscle mass and strength, physical performance and postural sway. Eur J Clin Nutr. 67:743–748. 2013. View Article : Google Scholar : PubMed/NCBI
8	Olthof MR and Verhoef P: Effects of betaine intake on plasma homocysteine concentrations and consequences for health. Curr Drug Metab. 6:15–22. 2005. View Article : Google Scholar : PubMed/NCBI
9	Senesi P, Luzi L, Montesano A, Mazzocchi N and Terruzzi I: Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation. J Transl Med. 11:1742013. View Article : Google Scholar : PubMed/NCBI
10	van Meurs JB, Dhonukshe-Rutten RA, Pluijm SM, van der Klift M, de Jonge R, Lindemans J, de Groot LC, Hofman A, Witteman JC, van Leeuwen JP, et al: Homocysteine levels and the risk of osteoporotic fracture. N Engl J Med. 350:2033–2041. 2004. View Article : Google Scholar : PubMed/NCBI
11	Sato Y, Honda Y, Iwamoto J, Kanoko T and Satoh K: Effect of folate and mecobalamin on hip fractures in patients with stroke: a randomized controlled trial. JAMA. 293:1082–1088. 2005. View Article : Google Scholar : PubMed/NCBI
12	Buckingham M, Bajard L, Chang T, Daubas P, Hadchouel J, Meilhac S, Montarras D, Rocancourt D and Relaix F: The formation of skeletal muscle: from somite to limb. J Anat. 202:59–68. 2003. View Article : Google Scholar : PubMed/NCBI
13	Parker MH, Seale P and Rudnicki MA: Looking back to the embryo: defining transcriptional networks in adult myogenesis. Nat Rev Genet. 4:497–507. 2003. View Article : Google Scholar : PubMed/NCBI
14	Weintraub H: The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell. 75:1241–1244. 1993. View Article : Google Scholar : PubMed/NCBI
15	Lassar AB, Skapek SX and Novitch B: Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol. 6:788–794. 1994. View Article : Google Scholar : PubMed/NCBI
16	Olson EN, Perry M and Schulz RA: Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev Biol. 172:2–14. 1995. View Article : Google Scholar : PubMed/NCBI
17	Rudnicki MA, Braun T, Hinuma S and Jaenisch R: Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell. 71:383–390. 1992. View Article : Google Scholar : PubMed/NCBI
18	Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH and Jaenisch R: MyoD or Myf-5 is required for the formation of skeletal muscle. Cell. 75:1351–1359. 1993. View Article : Google Scholar : PubMed/NCBI
19	Perry RL and Rudnick MA: Molecular mechanisms regulating myogenic determination and differentiation. Front Biosci. 5:D750–D767. 2000. View Article : Google Scholar : PubMed/NCBI
20	Moncaut N, Rigby PW, Carvajal JJ and Dial M: Dial M(RF) for myogenesis. FEBS J. 280:3980–3990. 2013. View Article : Google Scholar : PubMed/NCBI
21	Ceci M, Ross J Jr and Condorelli G: Molecular determinants of the physiological adaptation to stress in the cardiomyocyte: a focus on AKT. J Mol Cell Cardiol. 37:905–912. 2004. View Article : Google Scholar : PubMed/NCBI
22	Guttridge DC: Signaling pathways weigh in on decisions to make or break skeletal muscle. Curr Opin Clin Nutr Metab Care. 7:443–450. 2004. View Article : Google Scholar : PubMed/NCBI
23	Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ and Yancopoulos GD: Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 3:1014–1019. 2001. View Article : Google Scholar : PubMed/NCBI
24	Kim M, Sung B, Kang YJ, Kim DH, Lee Y, Hwang SY, Yoon JH, Yoo MA, Kim CM, Chung HY and Kim ND: The combination of ursolic acid and leucine potentiates the differentiation of C2C12 murine myoblasts through the mTOR signaling pathway. Int J Mol Med. 35:755–762. 2015.
25	Erbay E and Chen J: The mammalian target of rapamycin regulates C2C12 myogenesis via a kinase-independent mechanism. J Biol Chem. 276:36079–36082. 2001. View Article : Google Scholar : PubMed/NCBI
26	Gingras AC, Raught B and Sonenberg N: Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15:807–826. 2001. View Article : Google Scholar : PubMed/NCBI
27	Ohanna M, Sobering AK, Lapointe T, Lorenzo L, Praud C, Petroulakis E, Sonenberg N, Kelly PA, Sotiropoulos A and Pende M: Atrophy of S6K1(−/−) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control. Nat Cell Biol. 7:286–294. 2005. View Article : Google Scholar : PubMed/NCBI
28	Lawson MA and Purslow PP: Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific. Cells Tissues Organs. 167:130–137. 2000. View Article : Google Scholar : PubMed/NCBI
29	Nabeshima Y, Hanaoka K, Hayasaka M, Esumi E, Li S, Nonaka I and Nabeshima Y: Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature. 364:532–535. 1993. View Article : Google Scholar : PubMed/NCBI
30	Hasty P, Bradley A, Morris JH, Edmondson DG, Venuti JM, Olson EN and Klein WH: Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature. 364:501–506. 1993. View Article : Google Scholar : PubMed/NCBI
31	Tidball JG and Villalta SA: Regulatory interactions between muscle and the immune system during muscle regeneration. Am J Physiol Regul Integr Comp Physiol. 298:R1173–R1187. 2010. View Article : Google Scholar : PubMed/NCBI
32	Yaffe D and Saxel O: Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 270:725–727. 1977. View Article : Google Scholar : PubMed/NCBI
33	Tapscott SJ: The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development. 132:2685–2695. 2005. View Article : Google Scholar : PubMed/NCBI
34	Hawke TJ and Garry DJ: Myogenic satellite cells: physiology to molecular biology. J Appl Physiol (1985). 91:534–551. 2001.
35	Yoshida N, Yoshida S, Koishi K, Masuda K and Nabeshima Y: Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates 'reserve cells'. J Cell Sci. 111:769–779. 1998.PubMed/NCBI
36	Ferri P, Barbieri E, Burattini S, Guescini M, D'Emilio A, Biagiotti L, Del Grande P, De Luca A, Stocchi V and Falcieri E: Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem. 108:1302–1317. 2009. View Article : Google Scholar : PubMed/NCBI
37	Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ and Fernandez A: The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol. 142:1447–1459. 1998. View Article : Google Scholar : PubMed/NCBI
38	Molkentin JD, Black BL, Martin JF and Olson EN: Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell. 83:1125–1136. 1995. View Article : Google Scholar : PubMed/NCBI
39	Park SY, Yun Y, Kim MJ and Kim IS: Myogenin is a positive regulator of MEGF10 expression in skeletal muscle. Biochem Biophys Res Commun. 450:1631–1637. 2014. View Article : Google Scholar : PubMed/NCBI
40	Ludolph DC and Konieczny SF: Transcription factor families: muscling in on the myogenic program. FASEB J. 9:1595–1604. 1995.PubMed/NCBI
41	Cornelison DD, Olwin BB, Rudnicki MA and Wold BJ: MyoD(−/−) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient. Dev Biol. 224:122–137. 2000. View Article : Google Scholar : PubMed/NCBI
42	Jiang BH, Aoki M, Zheng JZ, Li J and Vogt PK: Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B. Proc Natl Acad Sci USA. 96:2077–2081. 1999. View Article : Google Scholar : PubMed/NCBI
43	Sumitani S, Goya K, Testa JR, Kouhara H and Kasayama S: Akt1 and Akt2 differently regulate muscle creatine kinase and myogenin gene transcription in insulin-induced differentiation of C2C12 myoblasts. Endocrinology. 143:820–828. 2002. View Article : Google Scholar : PubMed/NCBI
44	Peter AK and Crosbie RH: Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway. Exp Cell Res. 312:2580–2591. 2006. View Article : Google Scholar : PubMed/NCBI
45	Izumiya Y, Hopkins T, Morris C, Sato K, Zeng L, Viereck J, Hamilton JA, Ouchi N, LeBrasseur NK and Walsh K: Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice. Cell Metab. 7:159–172. 2008. View Article : Google Scholar : PubMed/NCBI
46	Florini JR, Ewton DZ and Roof SL: Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol. 5:718–724. 1991. View Article : Google Scholar : PubMed/NCBI
47	Xu Q and Wu Z: The insulin-like growth factor-phosphatidylinositol 3-kinase-Akt signaling pathway regulates myogenin expression in normal myogenic cells but not in rhabdomyo-sarcoma-derived RD cells. J Biol Chem. 275:36750–36757. 2000. View Article : Google Scholar : PubMed/NCBI
48	Jagoe RT and Goldberg AL: What do we really know about the ubiquitin-proteasome pathway in muscle atrophy? Curr Opin Clin Nutr Metab Care. 4:183–190. 2001. View Article : Google Scholar : PubMed/NCBI
49	Glass DJ: Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol. 5:87–90. 2003. View Article : Google Scholar : PubMed/NCBI
50	No authors listed. Epidemiologic and methodologic problems in determining nutritional status of older persons. Proceedings of a conference Albuquerque, New Mexico, October 19–21, 1988. Am J Clin Nutr. 50(Suppl): 1121–1235. 1989.
51	Solini A, Santini E and Ferrannini E: Effect of short-term folic acid supplementation on insulin sensitivity and inflammatory markers in overweight subjects. Int J Obes. 30:1197–1202. 2006. View Article : Google Scholar
52	Feng D, Zhou Y, Xia M and Ma J: Folic acid inhibits lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages by suppressing MAPKs and NF-κB activation. Inflamm Res. 60:817–822. 2011. View Article : Google Scholar : PubMed/NCBI
53	Veeranki S, Winchester LJ and Tyagi SC: Hyperhomocysteinemia associated skeletal muscle weakness involves mitochondrial dysfunction and epigenetic modifications. Biochim Biophys Acta. 1852:732–741. 2015. View Article : Google Scholar : PubMed/NCBI
54	Moat SJ, Doshi SN, Lang D, McDowell IF, Lewis MJ and Goodfellow J: Treatment of coronary heart disease with folic acid: is there a future? Am J Physiol Heart Circ Physiol. 287:H1–H7. 2004. View Article : Google Scholar : PubMed/NCBI