Comparison between Single and Co-culture of Adipocyte and Muscle Cell Lines in Cell Morphology and Cytosolic Substances

지방과 근육 세포주의 단독 및 공동배양을 통한 세포형태학 및 세포물질 비교 연구

  • Received : 2012.02.27
  • Accepted : 2012.04.24
  • Published : 2012.04.30


Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1 (adipocyte) and L6 (muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco's modified eagle's medium (DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a $0.4{\mu}m$ insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase (GPDH; for 3T3-L1) and creatine kinase (CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically (P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa (P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.


Cell line;Co-culture;Adipocyte;Muscle cells


  1. Ailhaud, G., Grimaldi, P. and Negrel, R. 1992. Cellular and molecular aspects of adipose tissue development. Ann. Rev. Nutr. 12:207-233.
  2. Baek, K. H. 2003. Studies on the production of lean meats and the identification of Hanwoo (Korean Cattle) brand beef using immunological techniques. Ph.D. thesis. Yeungnam Univ., Gyeongsan, Korea.
  3. Bernt, E. and Bergmeyer, H. U. 1974. Hexokinase. In : H. U. Bergmeyer and K. Gawehn (Ed.). Methods of enzyme analysis. Academic Press, New York. 473-474.
  4. Brindle, P. K. and Montiminy, M. R. 1992. The CREB family of transcription activators. Curr. Opin. Genet. Dev. 2:199-204.
  5. Calvo, J. C., Rodbard, D., Katki, A., Chernick, S. and Yanagishita, M. 1991. Differentiation of 3T3-L1 preadipocytes with 3- isobutyl-1-methyl-xanthine and dexamethasone stimulates cellassociated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266:11237-11244.
  6. Cao, Z., West, C., Norton-Wenzel, C. S., Rej, R., Davis, F. B., Davis, P. J. and Rej., R. 2009. Effects of resin or charcoal treatment on fetal bovine serum and bovine calf serum. Endo. Res. 34:101-108.
  7. Choi, C. W. 2011. Sera taken from aged Korean native steers increase adipocyte differentiation. J. Agr. Life Sci. 45(2):85-92.
  8. Choi, C. B., Shin, H. W., Lee, S. W., Kim, S. I., Jung, K. K., Choi, C. W., Baek, K. H., Lunt, D. K. and Smith, S. B. 2008. Comparison of cholesterol contents and fatty acid composition in M. longissimus of Hanwoo, Angus and Wagyu crossbred steers. J. Anim. Sci. & Technol. (Kor.) 50:519-526.
  9. Choi, C. W., Baek, K. H., Smith, S. B., Kim, Y. H., Ford, L. A., Kim, S. J., Oh, Y. K., Kim, K. H., Kang, S. W., Nam, I. S. and Lee, B. S. 2007. Screening of media conditions to establish optimum conditions for 3T3-L1 cells co-cultured with L6 cells. Proceedings of Animal Science & Technology. 2007. 6. 28. Chungang University, Anseong, Gyeonggi.
  10. Chung, K. Y., Park, S. K., Chung, H. J. and Choi, C. B. 2001. Screening of media components to establish optimum conditions for the differentiation of Hanwoo adipocytes. Korean J. Anim. Sci. & Technol. 43(1):65-74.
  11. Darimont, C., Gaillard, D., Ailhaud, G. and Negrel, R. 1993. Terminal differentiation of mouse preadipocyte cell : adipogenic and antimitogenic role of triiodothyronine. Mol. cell. Endocrinol. 98:67-73.
  12. Dietze, D., Koenen, M., Rȍhrig, K., Horikoshi, H., Hauner, H. and Eckel, J. 2002. Impairment of insulin signaling in human skeletal muscle cells by co-culture with human adipocytes. Diabetes. 51: 2369-2376.
  13. Dieudonne, M. N., Pecquery, R., Leneveu, M. C. and Giudicelli, Y. 2000. Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: Evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor $\gamma$. Endo. 141:649-656.
  14. Diez, J. and Iglesias, P. 2003. The role of the novel adipocyte-derived hormone adiponectin in human disease. European J. Endo. 148:293-300.
  15. Duncan, D. B. 1955. Multiple range and multiple F test. Biometrics. 11:1-42.
  16. Gaben-Cogneville, A., Quignard-Boulange, A., Aron, Y., Brignant, L., Jahchan, T., Pello, J. and Swierczewski, E. 1984. Development under the control of insulin of lipogenic enzymes, lipoprotein lipase, isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture. Biochem. Biophys. Acta. Nov. 13:252-260.
  17. Greene, E. A. and Allen, R. E. 1991. Growth factor regulation of bovine satellite cell growth in vitro. J. Anim. Sci. 69:146-152.
  18. Gregoire, F. M., Smas, C. M. and Sul, H. S. 1998. Understanding adipocyte differentiation. Physiol. Rev. 78:783-809.
  19. Hausman, G. J. and Poulos, S. P. 2005. A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles. J. Anim. Sci. 83: 1010-1016.
  20. Hemmer, W. and Wallimann, T. 1994. Creatine kinase in non-muscle tissues and cells. Mol. Cell. Biochem. 133:193-220.
  21. Isgaard, J., Nilsson, A., Vikman, K. and Isaksson, O. G. P. 1989. Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle. J. Endo. 120:107-112.
  22. Ishida, Y., Taniguchi, H. and Baba, S. 1988. Possible involvement of $1{\alpha}$, 25-dihydroxyvitamin D3 in proliferation and differentiation of 3T3-L1 cells. Biochem. Biophys. Res. commun. 151:1122-1127.
  23. Jarett, L., Wong, E., Macaulay, S. and Smith, J. 1985. Insulin mediators from rat skeletal muscle have differential effects on insulin-sensitive pathways of intact adipocytes. Science. 227: 533-535.
  24. Kawada, T., Aoki, N., Kamei, Y., Maeshige, K., Nishiu, S. and Sugimoto, E. 1990. Comparative investigation of vitamins and their analogues on terminal differentiation from preadipocyte to adipocyte, of 3T3-L1 cell. Comp. Biochem. Physiol. 96A: 323-326.
  25. Kozak, L. P. and Jensen, J. T. 1974. Genetic and Developmental Control of Multiple Forms of L-Glycerol 3-Phosphate Dehydrogenase. J. Bio. Chem. 249(24):7775-7781.
  26. Lee, S. C., Kim, D. W., Lee, H. J., Kim, J. W., Hong, S. G. and Chung, Y. H. 1997. Differentiation of adipose stromal-vascular cells from Korean native steers in culture. Korean J. Anim. Sci. & Technol. 39(4):415-422.
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
  28. Oh, Y. S., Cho, S. B., Baek, K. H. and Choi, C. B. 2005. Effects of testosterone, $17{\beta}$-estradiol, and progesterone on the differentiaton of bovine intramuscular adipocytes. Asian-Aust. J. Anim. Sci. 18(11):1589-1593.
  29. Oyama. K., Matsuda, K., Torii, S., Matsui, T., Yano, H., Kawada, T. and Ishihara, T. 1998. The interaction between vitamin A and thiazolidmedione on bovine adipocyte differentiation in primary culture. J. Anim. Sci. 76:61-65.
  30. Ramsay, T. G., White, M. E. and Wolverton, C. K. 1989. Insulin-like growth factor 1 induction of differentiation of porcine preadipocytes. J. Anim. Sci. 67:2452-2459.
  31. RDA. 2008. Livestock Research Report. National Instititute of Animal Science, Rural Development Administration. Reg. No. 11- 1390271-000032-10.
  32. Safonova, I., Darimont, C., Amri, E. Z., Grimaid, P., Ailhaud, G., Reichert, U. and Shroot, B. 1994. Retinoids are positive effectors of adipose cell differentiation. Mol. cell. Endocrinol. 104: 201-211.
  33. Skottman, H. and Hovatta, O. 2006. Culture conditions for human embryonic stem cells. Reproduction. 132:691-698.
  34. Sottile, V. and Seuwen, K. 2001. A high-capacity screen for adipogenic differentiation. Anal. Biochem. 293:124-128.
  35. Sztalryd, C., Levacher, C. and Picon, L. 1989. Acceleration by triiodothyronine of adipose conversion of rat preadipocytes from two adipose localizations. Cell. Mol. Biol. 35:81-88.
  36. Wise, L. S. and Green, H. 1979. Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. The J. Biol. Chem. 254:273-275.
  37. Yeh, W. C., Cao, Z., Classon, M. and Mcknight, S. 1995. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Gene & Dev. 9:168-181.

Cited by

  1. Suppression of adipogenic differentiation by muscle cell-induced decrease in genes related to lipogenesis in muscle and fat co-culture system vol.37, pp.9, 2013,