Hostname: page-component-cd9895bd7-q99xh Total loading time: 0 Render date: 2024-12-21T23:43:40.609Z Has data issue: false hasContentIssue false
  • English
  • Français

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model

Published online by Cambridge University Press:  01 May 2009

S. M. Waters ,
D. A. Kenny ,
A. P. Killeen ,
S. A. Spellman ,
A. Fitzgerald ,
A. A. Hennessy  and
A. C. Hynes
S. M. Waters*
Affiliation:
Animal Bioscience Centre, Teagasc Grange, Dunsany, Co., Meath, Ireland
D. A. Kenny
Affiliation:
School of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
A. P. Killeen
Affiliation:
Animal Bioscience Centre, Teagasc Grange, Dunsany, Co., Meath, Ireland
S. A. Spellman
Affiliation:
Department of Physiology, National University of Ireland, Galway, Ireland
A. Fitzgerald
Affiliation:
Department of Physiology, National University of Ireland, Galway, Ireland
A. A. Hennessy
Affiliation:
Teagasc, Moorepark Food Research Centre, Fermoy, Co., Cork, Ireland
A. C. Hynes
Affiliation:
Department of Physiology, National University of Ireland, Galway, Ireland
*
E-mail: [email protected]
Get access

Abstract

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal’s diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of Δ-9 desaturasein vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, Δ-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 μM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration (P < 0.01). Expression of Δ-9 desaturase mRNA was decreased five- and seven-fold, respectively, following 50 and 100 μM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6- and 18-fold in cells supplemented with 50 and 100 μM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both Δ-9 desaturase (P < 0.001) and SREBP-1c (P < 0.001), while a significant positive relationship was observed between Δ-9 desaturase and SREBP-1c gene expression (P < 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both Δ-9 desaturase and SREBP-1cin vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

Keywords

adipocytebovinein vitroEPAΔ-9 desaturase
Type
Full Paper
Information
animal , Volume 3 , Issue 5 , May 2009 , pp. 718 - 727
Copyright
Copyright © The Animal Consortium 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Asano, M, Nakajima, T, Hazama, H, Iwasawa, K, Tomaru, T, Omata, M, Soma, M, Asakura, Y, Mizutani, M, Suzuki, S, Yamashita, K, Okuda, Y 1998. Influence of cellular incorporation of n-3 eicosapentaenoic acid on intracellular Ca2+ concentration and membrane potential in vascular smooth muscle cells. Atherosclerosis 138, 117–127.CrossRefGoogle ScholarPubMed
Aso, H, Abe, H, Nakajima, I, Ozutsumi, K, Yamaguchi, T, Takamori, Y, Kodama, A, Hoshino, FB, Takano, S 1995. A preadipocyte clonal line from bovine intramuscular adipose tissue: nonexpression of GLUT-4 protein during adipocyte differentiation. Biochemical and Biophysical Research Communications 213, 369–375.CrossRefGoogle ScholarPubMed
Bahar, B 2006. Development of isotopic and molecular techniques for the authentication of dietary background information in bovine tissues. PhD, National University of Ireland, UCD, Dublin.Google Scholar
Bate, C, Tayebi, M, Diomede, L, Salmona, M, Williams, A 2008. Docosahexaenoic and eicosapentaenoic acids increase prion formation in neuronal cells. BMC Biology 12, 6–39.Google Scholar
Cabrero, A, Alegret, M, Sánchez, RM, Adzet, T, Laguna, JC, Vázquez, M 2001. Bezafibrate reduces mRNA levels of adipocyte markers and increases fatty acid oxidation in primary culture of adipocytes. Diabetes 50, 1883–1890.CrossRefGoogle Scholar
Darin, N, Kadhom, N, Brière, JJ, Chretien, D, Bébéar, CM, Rötig, A, Munnich, A, Rustin, P 2003. Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis. BMC Biochemistry 4, 15.Google Scholar
De La Torre, A, Gruffat, D, Chardigny, JM, Sebedio, JL, Durand, D, Loreau, O, Bauchart, D 2005. In vitro metabolism of rumenic acid in bovine liver slices. Reproduction Nutrition and Development 45, 441–451.Google Scholar
De Simone, V, Cortese, R 1992. Transcription factors and liver-specific genes. Biochimica et Biophysica Acta 1132, 119–126.CrossRefGoogle ScholarPubMed
Dodson, MV, Fernyhough, ME, Vierck, JL, Hausman, GJ 2005. Adipocytes may not be a terminally differentiated cell type: implications for animal production. Animal Science 80, 239–240.Google Scholar
Dorroch, U, Goldammer, T, Brunner, RM, Kata, SR, Kühn, C, Womack, JE, Schwerin, M 2001. Isolation and characterization of hepatic and intestinal expressed sequence tags potentially involved in trait differentiation between cows of different metabolic type. Mammalian Genome 12, 528–537.Google Scholar
Dux, L, Mesaros, MG, Rohan, J, Gajdos, L, Jakab, G, Guba, F 1981. The value of simple lipid stains for typing skeletal muscle fibers. Histochemical Journal 13, 63–71.Google Scholar
Faulconnier, Y, Thévenet, M, Fléchet, J, Chilliard, Y 1994. Lipoprotein lipase and metabolic activities in incubated bovine adipose tissue explants: effects of insulin, dexamethasone, and fetal bovine serum. Journal of Animal Science 72, 184–191.Google Scholar
Fernyhough, ME, Vierck, Jl, Hausman, GJ, Mir, PS, Okine, EK, Dodson, MV 2005. Primary adipocyte culture: adipocyte purification methods may lead to a new understanding of adipose tissue growth and development. Cytotechnology 46, 163–172.CrossRefGoogle Scholar
Folch, J, Lees, M, Sloane Stanley, GH 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509.Google Scholar
Forest, C, Grimaldi, P, Czerucka, D, Negrel, R, Alihaud, G 1983. Establishment of a preadipocyte cell line from the epididymal fat pad of the lean C57 BL/6J mouse – Long term effects of insulin and triiodothyronine on adipose conversion. In Vitro 19, 344–354.Google Scholar
Grant, AC, Ortiz-Colòn, G, Doumit, ME, Buskirk, DD 2008. Optimization of in vitro conditions for bovine subcutaneous and intramuscular preadipocyte differentiation. Journal of Animal Science 86, 73–82.Google Scholar
Gregoire, FM, Smas, CM, Sul, HS 1998. Understanding adipocyte differentiation. Physiological Reviews 78, 783–809.CrossRefGoogle ScholarPubMed
Hausman, GJ, Poulos, SP, Pringle, TD, Azain, MJ 2008. The influence of thiazolidinediones on adipogenesis in vitro and in vivo: potential modifiers of intramuscular adipose tissue deposition in meat animals. Journal of Animal Science 86, 236–243.CrossRefGoogle Scholar
Hoashi, S, Ashida, N, Ohsaki, H, Utsugi, T, Sasazaki, S, Taniguchi, M, Oyama, K, Mukai, F, Mannen, H 2007. Genotype of bovine sterol regulatory element binding protein-1 (SREBP-1) is associated with fatty acid composition in Japanese Black cattle. Mammalian Genome 18, 880–886.Google Scholar
Kershaw, EE, Schupp, M, Guan, HP, Gardner, NP, Lazar, MA, Flier, JS 2007. PPAR-γ regulates adipose triglyceride lipase in adipocytes in vitro and in vivo. American Journal of Physiology, Endocrinology and Metabolism 293, 1736–1745.CrossRefGoogle ScholarPubMed
Kuhajda, FP, Jenner, K, Wood, FD, Hennigar, RA, Jacobs, LB, Dick, JD, Pasternack, GR 1994. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proceedings of the National Academy of Sciences 91, 6379–6383.CrossRefGoogle ScholarPubMed
Lehnert, SA, Byrne, KA, Reverter, A, Nattrass, GS, Greenwood, PL, Wang, YH, Hudson, NJ, Harper, GS 2006. Gene expression profiling of bovine skeletal muscle in response to and during recovery from chronic and severe undernutrition. Journal of Animal Science 84, 3239–3250.CrossRefGoogle ScholarPubMed
Li, J, Adams, L, Schwartz, SM, Bumgarner, RE 2003. RNA amplification, fidelity and reproducibility of expression profiling. Comptes Rendus Biologies 326, 1021–1030.CrossRefGoogle ScholarPubMed
Livak, KJ, Schmittgen, TD 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2(-delta delta CT)) method. Methods 25, 402–408.Google Scholar
Matfield, M 1996. The ethics of animal research. A special lecture to the Japanese Association of Laboratory Animal Science 6th June 1995. Experimental Animals 45, 209–215.CrossRefGoogle Scholar
Moloney, AP, Mooney, MT, Kerry, JP, Troy, DJ 2001. Producing tender and flavoursome beef with enhanced nutritional characteristics. Proceedings of the Nutrition Society 60, 221–229.Google Scholar
Morganstein, DL, Christian, M, Turner, JJ, Parker, MG, White, R 2008. Conditionally immortalized white preadipocytes: a novel adipocyte model. Journal of Lipid Research 49, 679–685.Google Scholar
Morris, CA 2007. A review of genetic resistance to disease in Bos taurus cattle. Veterinary Journal 174, 481–491.CrossRefGoogle ScholarPubMed
Natal, C, Fortuño, MA, Restituto, P, Bazán, A, Colina, I, Díez, J, Varo, N 2008. Cardiotrophin-1 is expressed in adipose tissue and upregulated in the metabolic syndrome. American Journal of Physiology. Endocrinology and Metabolism 294, 52–60.CrossRefGoogle ScholarPubMed
Nkrumah, JD, Sherman, EL, Li, C, Marques, E, JrCrews, DH, Bartusiak, R, Murdoch, B, Wang, Z, Basarab, JA, Moore, SS 2007. Primary genome scan to identify putative quantitative trait loci for feedlot growth rate, feed intake, and feed efficiency of beef cattle. Journal of Animal Science 85, 3170–3181.CrossRefGoogle ScholarPubMed
Park, PW, Goins, RE 1994. In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. Journal of Food Science 59, 1262–1266.CrossRefGoogle Scholar
Pitchford, WS, Deland, MP, Siebert, BD, Malau-Aduli, AE, Bottema, CD 2002. Genetic variation in fatness and fatty acid composition of crossbred cattle. Journal of Animal Science 80, 2825–2832.Google Scholar
Radzikowski, C 2006. Protection of animal research subjects. Science and Engineering Ethics 12, 103–110.CrossRefGoogle ScholarPubMed
Renaville, B, Mullen, A, Moloney, F, Larondelle, Y, Schneider, YJ, Roche, HM 2006. Eicosapentaenoic acid and 3,10 dithia stearic acid inhibit the desaturation of trans-vaccenic acid into cis-9, trans-11-conjugated linoleic acid through different pathways in Caco-2 and T84 cells. British Journal of Nutrition 95, 688–695.Google Scholar
SAS 2001. Statistical Analysis Systems. Version 8.2. SAS Institute, Inc., North Carolina State University, USA.Google Scholar
Scollan, N, Hocquette, JF, Nuernberg, K, Dannenberger, D, Richardson, I, Moloney, A 2006. Innovations in beef production systems that enhance the nutritional and health value of beef lipids and their relationship with meat quality. Meat Science 74, 17–33.CrossRefGoogle ScholarPubMed
Seo, JB, Moon, HM, Noh, MJ, Lee, YS, Jeong, HW, Yoo, EJ, Kim, WS, Park, J, Youn, BS, Kim, JW, Park, SD, Kim, JB 2004. Adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression. Journal of Biological Chemistry 279, 22108–22117.Google Scholar
Sladek, FM 1993. Orphan receptor HNF-4 and liver-specific gene expression. Receptor 3, 223–232.Google Scholar
Stalenhoef, AF, Armstrong, VW, Steinmetz, A, Taskinen, MR, Zechner, R 1995. The 17th annual meeting of the European Lipoprotein Club. Arteriosclerosis, Thrombosis, and Vascular Biology 15, 543–549.CrossRefGoogle ScholarPubMed
Su, X, Han, X, Yang, J, Mancuso, DJ, Chen, J, Bickel, PE, Gross, RW 2004. Sequential ordered fatty acid alpha oxidation and Delta9 desaturation are major determinants of lipid storage and utilization in differentiating adipocytes. Biochemistry 43, 5033–5044.Google Scholar
Sun, YR, Yang, Z 2003. Advances of nuclear translation factor: peroxisome proliferator-actived receptor gamma2. Yi Chuan 25, 713–717.Google Scholar
Szekely, A, Kitajka, K, Panyi, G, Marian, T, Gaspar, R, Krasznai, Z 2007. Nutrition and immune system: certain fatty acids differently modify membrane composition and consequently kinetics of KV1.3 channels of human peripheral lymphocytes. Immunobiology 212, 213–227.CrossRefGoogle ScholarPubMed
Vernon, RG, Taylor, E 1988. Insulin, dexamethasone and their interactions in the control of glucose metabolism in adipose tissue from lactating and nonlactating sheep. Biochemical Journal 256, 509–514.Google Scholar
Viravaidya, K, Shuler, ML 2002. The effect of various substrates on cell attachment and differentiation of 3T3-F442A preadipocytes. Biotechnology and Bioengineering 78, 454–458.Google Scholar
Waters, SM, Kelly, JP, O’Boyle, P, Moloney, AP, Kenny, DA 2009. Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of Δ-9 desaturase in muscle of beef cattle. Journal of Animal Science, in press.CrossRefGoogle Scholar
Wolnicka-Glubisz, A, King, W, Noonan, FP 2005. SCA-1+ cells with an adipocyte phenotype in neonatal mouse skin. Journal of Investigative Dermatology 125, 383–385.Google Scholar
Zhang, S, Knight, TJ, Reecy, JM, Beitz, DC 2008. DNA polymorphisms in bovine fatty acid synthase are associated with beef fatty acid composition. Animal Genetics 39, 62–70.CrossRefGoogle ScholarPubMed
Zhou, QL, Jiang, ZY, Holik, J, Chawla, A, Hagan, GN, Leszyk, J, Czech, MP 2008. Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes. Biochemistry Journal 411, 647–655.Google Scholar