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Butyric acid induces spontaneous adipocytic differentiation of porcine bone marrow–derived mesenchymal stem cells

  • Benedetta Tugnoli
  • Chiara Bernardini
  • Monica Forni
  • Andrea Piva
  • Chad H. Stahl
  • Ester GrilliEmail author
Article
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Abstract

Butyric acid (BA) affects the differentiation of mesenchymal stem cells (MSC) through the activation of different transcriptional pathways. The aim of this study was to determine the effects of BA on proliferation and spontaneous differentiation of porcine bone marrow–derived MSC. Second passage MSC (n = 6) were cultured in either a basal medium (BM, DMEM + 10% FBS), or BM + 2.5 mmol/L BA (BA-2.5) or BM + 5 mmol/L BA (BA-5). Cell proliferation was significantly decreased by both BA-2.5 and BA-5 after 48 h and 72 h (− 55% and − 63%, respectively). To assess the impact of BA on spontaneous differentiation, MSC were cultured for 27 d, with complete media changes every 3 d. At day 27, cells were stained for osteocytic, chondrocytic, and adipocytic differentiation. No terminal differentiation was detected in control MSC, while accumulated small drops of lipids were stained by Oil-Red-O in BA-treated cells. The phenotypic changes were associated with changes in gene expression, determined by qPCR. Treatment with BA modulated the expression of adipocytic differentiation markers: peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α were significantly increased by both BA-2.5 and BA-5 throughout the study, while lipoprotein lipase and fatty acid-binding protein 4 were increased by BA-5 at day 3, and decreased by both BA-5 and BA-2.5 later throughout the study. Osteocalcin and aggrecan mRNA was reduced throughout the experiment by both doses of BA (P < 0.05). In conclusion, our data support that BA promotes the spontaneous differentiation of porcine bone marrow–derived MSC toward an adipocytic lineage in the absence of inducing cocktail media.

Keywords

Butyric acid Mesenchymal stem cells Pig Adipocytic differentiation In vitro 

Abbreviations

BA

butyric acid

MSC

mesenchymal stem cells

BM

basal medium

DPBS

Dulbecco’s phosphate-buffered solution

RPL35

ribosomal protein L35

GOI

gene of interest

HK

housekeeping gene

PPARγ

peroxisome proliferator-activated receptor γ

FABP4

fatty acid-binding protein 4

LPL

lipoprotein lipase

C/EBPα

CCAAT/enhancer binding protein α

ACAN

aggrecan

BGLAP

bone gamma-carboxyglutamate (gla) protein (osteocalcin)

Notes

Acknowledgements

We thank Dr. Francesca Bianchi for her technical assistance in the immunophenotyping of cells.

References

  1. Alex S, Lange K, Amolo T, Grinstead JS, Haakonsson AK, Szalowska E, Koppen A, Mudde K, Haenen D, Al-Lahham S, Roelofsen H, Houtman R, van der Burg B, Mandrup S, Bonvin AMJJ, Kalkhoven E, Müller M, Hooiveld GJ, Kersten S (2013) Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor. Mol Cell Biol 33:1303–1316CrossRefGoogle Scholar
  2. Alexander LS, Seabolt BS, Rhoads RP, Stahl CH (2012) Neonatal phosphate nutrition alters in vivo and in vitro satellite cell activity in pigs. Nutrients 4:436–448CrossRefGoogle Scholar
  3. Berni Canani R, Di Costanzo M, Leone L (2012) The epigenetic effects of butyrate: potential therapeutic implications for clinical practice. Clin Epigenetics 4:4CrossRefGoogle Scholar
  4. Canani RB, Costanzo MD, Leone L, Pedata M, Meli R, Calignano A (2011) Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World J Gastroenterol 17:1519–1528CrossRefGoogle Scholar
  5. Chen T-H, Chen W-M, Hsu K-H, Kuo C-D, Hung S-C (2007) Sodium butyrate activates ERK to regulate differentiation of mesenchymal stem cells. Biochem Biophys Res Commun 355:913–918CrossRefGoogle Scholar
  6. Cristancho AG, Lazar MA (2011) Forming functional fat: a growing understanding of adipocyte differentiation. Nat Rev Mol Cell Biol 12:722–734CrossRefGoogle Scholar
  7. Di Benedetto A, Brunetti G, Posa F, Ballini A, Grassi FR, Colaianni G, Colucci S, Rossi E, Cavalcanti-Adam EA, Lo Muzio L, Grano M, Mori G (2015) Osteogenic differentiation of mesenchymal stem cells from dental bud: role of integrins and cadherins. Stem Cell Res 15:618–628CrossRefGoogle Scholar
  8. Frenette PS, Pinho S, Lucas D, Scheiermann C (2013) Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol 31:285–316CrossRefGoogle Scholar
  9. Furuhashi M, Hotamisligil GS (2008) Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov 7:489–503CrossRefGoogle Scholar
  10. Gregoire FM, Smas CM, Sul HS (1998) Understanding adipocyte differentiation. Physiol Rev 78:783–809CrossRefGoogle Scholar
  11. Guilloteau P, Martin L, Eeckhaut V, Ducatelle R, Zabielski R, Van Immerseel F (2010) From the gut to the peripheral tissues: the multiple effects of butyrate. Nutr Res Rev 23:366–384CrossRefGoogle Scholar
  12. Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer R-J (2008) Review article: the role of butyrate on colonic function. Aliment Pharmacol Ther 27:104–119CrossRefGoogle Scholar
  13. Heimann E, Nyman M, Degerman E (2015) Propionic acid and butyric acid inhibit lipolysis and de novo lipogenesis and increase insulin-stimulated glucose uptake in primary rat adipocytes. Adipocyte 4:81–88CrossRefGoogle Scholar
  14. Heinritz SN, Mosenthin R, Weiss E (2013) Use of pigs as a potential model for research into dietary modulation of the human gut microbiota. Nutr Res Rev 26:191–209CrossRefGoogle Scholar
  15. Klemmt PA, Vafaizadeh V, Groner B (2011) The potential of amniotic fluid stem cells for cellular therapy and tissue engineering. Expert Opin Biol Ther 11:1297–1314CrossRefGoogle Scholar
  16. Lee S, Park J-R, Seo M-S, Roh K-H, Park S-B, Hwang J-W, Sun B, Seo K, Lee YS, Kang SK, Jung JW, Kang KS (2009) Histone deacetylase inhibitors decrease proliferation potential and multilineage differentiation capability of human mesenchymal stem cells: HDAC inhibitors regulate stemness of MSCs. Cell Prolif 42:711–720CrossRefGoogle Scholar
  17. Li G, Yao W, Jiang H (2014) Short-chain fatty acids enhance adipocyte differentiation in the stromal vascular fraction of porcine adipose tissue. J Nutr 144:1887–1895CrossRefGoogle Scholar
  18. Liang G, Taranova O, Xia K, Zhang Y (2010) Butyrate promotes induced pluripotent stem cell generation. J Biol Chem 285:25516–25521CrossRefGoogle Scholar
  19. Liu J, Wang Y, Wu Y, Ni B, Liang Z (2014) Sodium butyrate promotes the differentiation of rat bone marrow mesenchymal stem cells to smooth muscle cells through histone acetylation. PLoS One 9:e116183CrossRefGoogle Scholar
  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  21. Mahajan A, Stahl CH (2009) Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells. J Nutr Biochem 20:512–520CrossRefGoogle Scholar
  22. Mali P, Chou B-K, Yen J, Ye Z, Zou J, Dowey S, Brodsky RA, Ohm JE, Yu W, Baylin SB, Yusa K, Bradley A, Meyers DJ, Mukherjee C, Cole PA, Cheng L (2010) Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 28:713–720CrossRefGoogle Scholar
  23. Ntambi JM, Young-Cheul K (2000) Adipocyte differentiation and gene expression. J Nutr 130:3122S–3126SCrossRefGoogle Scholar
  24. Pappa KI, Anagnou NP (2009) Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regen Med 4:423–433CrossRefGoogle Scholar
  25. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefGoogle Scholar
  26. Reese-Wagoner A, Thompson J, Banaszak L (1999) Structural properties of the adipocyte lipid binding protein. Biochim Biophys Acta 1441:106–116CrossRefGoogle Scholar
  27. Rosen ED, Hsu C-H, Wang X, Sakai S, Freeman MW, Gonzalez FJ, Spiegelman BM (2002) C/EBPα induces adipogenesis through PPARγ: a unified pathway. Genes Dev 16:22–26CrossRefGoogle Scholar
  28. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM (2000) Transcriptional regulation of adipogenesis. Genes Dev 14:1293–1307Google Scholar
  29. Russo R, De Caro C, Avagliano C, Cristiano C, La Rana G, Mattace Raso G, Berni Canani R, Meli R, Calignano A (2015) Sodium butyrate and its synthetic amide derivative modulate nociceptive behaviors in mice. Pharmacol Res 103:279–291CrossRefGoogle Scholar
  30. Schwab M, Reynders V, Loitsch S, Steinhilber D, Stein J, Schröder O (2007) Involvement of different nuclear hormone receptors in butyrate-mediated inhibition of inducible NFκB signalling. Mol Immunol 44:3625–3632CrossRefGoogle Scholar
  31. Sun W, Wang H, Li Y, Zhou X, Teng Y, Chen J (2013) Acquisition of pig intramuscular preadipocytes through dedifferentiation of mature adipocytes and establishment of optimal induction conditions. Genet Mol Res 12:5926–5936CrossRefGoogle Scholar
  32. Tontonoz P, Spiegelman BM (2008) Fat and beyond: the diverse biology of PPARγ. Annu Rev Biochem 77:289–312CrossRefGoogle Scholar
  33. Toscani A, Soprano DR, Soprano KJ (1990) Sodium butyrate in combination with insulin or dexamethasone can terminally differentiate actively proliferating Swiss 3T3 cells into adipocytes. J Biol Chem 265:5722–5730Google Scholar
  34. Wang L, Waltenberger B, Pferschy-Wenzig E-M, Blunder M, Liu X, Malainer C, Blazevic T, Schwaiger S, Rollinger JM, Heiss EH, Schuster D, Kopp B, Bauer R, Stuppner H, Dirsch VM, Atanasov AG (2014) Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review. Biochem Pharmacol 92:73–89CrossRefGoogle Scholar
  35. Yan H, Ajuwon KM (2015) Mechanism of butyrate stimulation of triglyceride storage and Adipokine expression during adipogenic differentiation of porcine stromovascular cells. PLoS One 10:e0145940CrossRefGoogle Scholar
  36. Zaniboni A, Bernardini C, Bertocchi M, Zannoni A, Bianchi F, Avallone G, Mangano C, Sarli G, Calzà L, Bacci ML, Forni M (2015) In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells. Am J Physiol Cell Physiol 309:C320–C331CrossRefGoogle Scholar
  37. Zuk PA (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279–4295CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Benedetta Tugnoli
    • 1
  • Chiara Bernardini
    • 1
  • Monica Forni
    • 1
  • Andrea Piva
    • 1
  • Chad H. Stahl
    • 2
  • Ester Grilli
    • 1
    Email author
  1. 1.Department of Veterinary Medical SciencesUniversity of BolognaBolognaItaly
  2. 2.Department of Animal and Avian Sciences, College of Agriculture and Natural ResourcesUniversity of MarylandCollege ParkUSA

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