Skip to main content

A semi-quantitative RT-PCR method to measure thein vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression

Abstract

Conjugated linoleic acid (CLA) can activate (in vitro) the nuclear transcription factors known as the peroxisome proliferators activated receptors (PPAR). CLA was fed at 11 g CLA/kg of feed for 45d to castrated male pigs (barrows) to better understand long term effects of PPAR activationin vivo. The barrows fed CLA had lean muscle increased by 3.5% and overall fat reduced by 9.2% but intramuscular fat (IMF %) was increased by 14% (P<0.05). To measure the effect of long term feeding of CLA on porcine muscle gene expression, a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping genes cyclophilin and β-actin. This method does not require radioactivity or expensive PCR instruments with real-time fluorescent detection. PPAR and the PPAR responsive gene AFABP but not PPARα were significantly increased (P<0.05) in the CLA fed pig’s muscle. PPARα and PPARγ were also quantitatively tested for large differences in gene expression by western blot analysis but no significant difference was detected at this level. Although large differences in gene expression of the PPAR transcriptional factors could not be confirmed by western blotting techniques. The increased expression of AFABP gene, which is responsive to PPAR transcriptional factors, confirmed that dietary CLA can induce a detectable increase in basal PPAR transcriptional activity in the live animal.

References

  1. 1.

    Dugan MER, Aalhus JL, Schaefer AL, Kramer JKG. The effect of conjugated linoleic acid on fat to lean repartitioning and feed conversion in pigs.Canadian Journal of Animal Science 1997; 77:723–725.

    CAS  Google Scholar 

  2. 2.

    Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature 1990; 347: 645–650.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-deoxy-δ12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARγ.Cell 1995; 83: 803–812.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Oritz JA, Mallolas J, Nicot C, Bofarull J, Rodriguez JC, Hegardt FG, Haro D, Marrero PF. Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element.Biochemistry Journal 1999; 337: 329–335.

    Article  Google Scholar 

  5. 5.

    Mukherjee R, Jow L, Croston GE, Paterniti JR. Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPARγ2 verses PPARγ1 and activation with retinoid X receptor agonists and antagonists.Journal of Biological Chemistry 1997; 272: 8071–8076.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Mandrup S, Lane MD. Regulating adipogenesis.Journal of Biological Chemistry 1997; 272: 5367–5370.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Braissant O, Wahli W. Differential expression of peroxisome proliferator-activated receptor -α, -β, and -γ during rat embryonic development.Endocrinology 1998; 139: 2748–2754.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Michalik L, Wahli W. Peroxisome proliferator-activated receptors: three isotypes for a multitude of functions.Current Opinion in Biotechnology 1999; 10: 564–570.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Bustin, SA. Invited review: Quanitification of mRNA using real-time reverese transcription PCR (RT-PCR): trends and problems.Journal of Molecular Endocrinology 2002; 29:23–39.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Stangl GI, Muller H, Kirchgessner M. Conjugated linoleic acid effects on circulating hormones, metabolites and lipoproteins, and its proportion in fasting serum and erythrocyte membranes of swine.European Journal of Nutrition 1999; 38: 271–277.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Wang J, Liu R, Hawkins M, Barzilai N, Rossetti L. A nutrient sensing pathway regulates leptin gene expression in muscle and fat.Nature 1998; 393: 684–688.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Hawkins M, Angelov I, Liu R, Barzilai N, Rossetti L. The tissue concentration of UDP-N-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake.Journal of Biological Chemistry 1997; 272: 4889–4895.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Kramer JKG, Sehat N, Dugan MER, Mossoba MM, Yurawecz MP, Roach JAG, Eulitz K, Aalhus JL, Schaefer AL, Ku Y. Distribution of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography.Lipids 1998; 33: 549–558.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    National Research Council. Nutrient requirements of swine. 9th ed. National Academy Press, Washington, DC. 1998.

    Google Scholar 

  15. 15.

    Canadian Council on Animal Care. Guide to the care and use of experimental animals. 1993. Vol.1 Ed. Olfert ED, Cross BM, McWilliam AA. CCAC, Ottawa, ON.

    Google Scholar 

  16. 16.

    Martin AH, Fredeen HT, Weiss GM, Fortin A, Sim D. Yield of trimmed pork product in relation to weight and backfat thickness of the carcass.Canadian Journal of Animal Science 1981; 61: 299–310.

    Article  Google Scholar 

  17. 17.

    Association of Official Analytical Chemists. Official methods of AOAC international. 16th ed. AOAC, Washington DC. 1995. Method 39.1.05.

    Google Scholar 

  18. 18.

    Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Analytical Biochemistry 1987; 161:156–1599.

    Article  Google Scholar 

  19. 19.

    Meadus WJ, Macinnis R. Testing for the RN-gene in retail pork chops.Meat Science 2000; 54:231–237.

    Article  CAS  Google Scholar 

  20. 20.

    Spencer WE, Christensen MJ. Multiplex relative RT-PCR method for verification of differential gene expression.BioTechniques 1999; 27: 1044–1052.

    PubMed  CAS  Google Scholar 

  21. 21.

    Statistical Analysis System Institute. SAS user’s guide: statistics, 5th ed. SAS Institute Inc., Cary, NC. 1985.

    Google Scholar 

  22. 22.

    Meadus WJ, MacInnis R, Dugan MER. Prolonged dietary treatment with conjugated linoleic acid stimuates porcine muscle peroxisome proliferator activated receptor g and glutamine fructose aminotransferase gene expressionin vivo. Journal of Molecular Endocrinology 2002; 28: 79–86.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Thoennes SR, Tate PL, Price TM, Kilgore MW. Differential transcriptional activation of peroxisome proliferator-activated receptor gamma by omega-3 and omega-6 fatty acids in MCF-7 cells.Molecular and Cellular Endocrinology 2000; 160: 67–73.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Gerbens F, de Koning DJ, Harders FL, Meuwissen THE, Janss LGG, Groenen MAM, Veerkamp JH, Van Arendonk JAM, te Pas MFW. The effect of adipocyte and heart fatty acid-binding protein genes on intramuscular fat and backfat content in Meishan crossbred pigs.Journal of Animal Science 2000; 78: 552–559.

    PubMed  CAS  Google Scholar 

  25. 25.

    Moya-Camarena SY, Belury MA. Species differences in the metabolism and regulation of gene expression by conjugated linoleic acid.Nutrition reviews 1999; 57: 336–340.

    PubMed  CAS  Google Scholar 

  26. 26.

    Muller HL, Kirchgessner M, Roth FX, Stangl GI. Effect of conjugated linoleic acid on energy metabolism in growing-finishing pigs.Journal of Animal Physiology and Animal Nutrition 2000; 83: 85–94.

    Article  CAS  Google Scholar 

  27. 27.

    Rooyackers OE, Nair KS.Hormonal regulation of human muscle protein metabolism.Annual Review of Nutrition 1997; 17: 457–485.

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Ezaki O. Regulatory elements in the insulin-responsive glucose transporter (GLUT4) gene.Biochemical and Biophysical Research Communication 1997; 241: 1–6.

    Article  CAS  Google Scholar 

  29. 29.

    Huang J, Forsberg NE. Role of calpain in skeletal-muscle protein degredation.Proceedings of the National Academy of Sciences USA 1998; 95: 12100–12105.

    Article  CAS  Google Scholar 

  30. 30.

    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Research 1997; 25:3389–3402.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to W. J. Meadus.

Additional information

Published: February 17, 2003

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Meadus, W.J. A semi-quantitative RT-PCR method to measure thein vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression. Biol. Proced. Online 5, 20–28 (2003). https://doi.org/10.1251/bpo43

Download citation

Indexing terms

  • RT-PCR
  • PPAR
  • gene expression
  • in vivo
  • pigs
  • muscle