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Quantification of lipid in cultured 3T3-L1 adipocytes

Published online by Cambridge University Press:  18 August 2016

P. S. Mir ,
J. L. Vierck ,
Z. Mir  and
M. V. Dodson
P. S. Mir
Affiliation:
Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada
J. L. Vierck
Affiliation:
Department of Animal Sciences, Washington State University, Pullman, WA, USA
Z. Mir
Affiliation:
Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada
M. V. Dodson
Affiliation:
Department of Animal Sciences, Washington State University, Pullman, WA, USA
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Abstract

A preliminary study was conducted to quantify the lipid produced by differentiated 3T3-L1 cells after incubation in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS), supplemented with or without dimethyl-sulphoxide (DMSO; 9•6 g/l) and acetone (1•2 g/l). The two media treatments were applied to 3T3-L1 cells, plated at either 15K or 30K cells per well in 24-well plates. Cells were grown to confluence (96 h) and then treated with dexamethasone, methyl-isobutylxanthine and insulin for 48 h and later maintained in their respective media treatments for another 144 h. Cells from each treatment were recovered after two, 5-min incubations with trypsin, washed and resuspended in DMEM and counted on a haemocytometer. The lipid in the cells was extracted with hexane derivatized with tetramethyl-guanidine and analysed by gas chromatography. Final mean cell density was 6•8 (s.e. 0•18) ✕ 105 and 4•6 (s.e. 0•19) ✕ 105 when initially plated at 30K and 15K cells per well, respectively. Inclusion of DMSO and acetone in the medium did not affect final cell numbers. Plating density did not affect concentration of lipid (0•55 (s.e. 0•08) mg per 1 ✕ 105 cells) but inclusion of DMSO and acetone led to overall decreases in total lipid concentration. Results indicate that initial plating density influenced final cell number in treatment cultures, but that DMSO and acetone treatments only had an effect on final lipid concentration. Collectively, these data suggest that the application of treatments to cell cultures may be influenced by the carrier vehicle that the treatment is contained in and this should be considered when developing an in vitro system to evaluate growth and development of adipocytes.

Keywords

adipocytesgas chromatographyhaemocytometerplating densitylipids
Type
Research Article
Information
Animal Science , Volume 71 , Issue 3 , December 2000 , pp. 521 - 526
Copyright
Copyright © British Society of Animal Science 2000

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References

Ip, C. 1997. Review of the effect of trans-fatty acids, oleic acid, n-3 polyunsaturated fatty acids and conjugated linoleic acid on mammary carcinogenesis in animals. American Journal of Clinical Nutrition 66: 1523S-1529S.CrossRefGoogle ScholarPubMed
Middleton, C. K., Kazala, E. C., Lozeman, F. J., Hurly, T. A., Mir, P. S., Bailey, D. R. C., Jones, S. D. M. and Weselake, R. J. 1998. Evaluation of diacylglycerol acyltransferase as an indicator of intramuscular fat content in beef cattle. Canadian Journal of Animal Science 78: 265—270.CrossRefGoogle Scholar
Mir, Z., Goonewardene, L. A., Okine, E. K., Jaegar, S. and Scheer, H. D. 1999. Effect of canola oil on constituents, conjugated linoleic acid (CLA) and long chain fatty acids in goats milk. Small Ruminant Research 33: 137—143.Google Scholar
Mir, Z., Rushfeldt, M. L., Mir, P. S., Paterson, L. J. and Weselake, R. J. 2000. Effect of dietary supplementation with either conjugated linoleic acid (CLA) or linoleic acid rich oil on the CLA content of lamb tissues. Small Ruminant Research 36: 25—31.CrossRefGoogle Scholar
Ohyama, M., Matsuda, K., Torii, S., Matsui, T., Yano, H., Kawada, T. and Ishihara, T. 1998. The interaction between vitamin A and thiazolidinedione on bovine adipocyte differentiation in primary culture. Journal of Animal Science 76: 61—65.Google Scholar
Sadowski, H. B., Wheeler, T. T. and Young, D. A. 1992. Gene expression during 3T3-L1 adipocyte differenetiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation. Journal of Biological Chemistry 266: 4722—4731.Google Scholar
Satory, D. L. and Smith, S. B. 1999. Conjugated linoleic acid inhibits proliferation but stimulates lipid filling of murine 3T3-L1 preadipocytes. Journal of Nutrition 129: 92—97.CrossRefGoogle ScholarPubMed
Smith, S. B. and Crouse, J. D. 1984. Relative contributions of acetate, lactate and glucose to lipogenesis in bovine intramuscular and subcutaneous adipose tissue. Journal of Nutrition 114: 792—800.Google Scholar
Suryawan, A., Swanson, L. V. and Hu, C. Y. 1997. Insulin and hydro cortisone, but not triiodothyronine are required for the differentiation of pig preadipocytes in primary culture. Journal of Animal Science 75: 105—111.Google Scholar
Vierck, J. L., McNamara, J. P. and Dodson, M. V. 1996. Proliferation and differentiation of progeny of ovine unilocular fat cells adipofibroblasts. In Vitro Cellular and Developmental Biology — Animal 32: 564—572.Google Scholar