Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Association of β-Catenin with Fat Accumulation in 3T3-L1 Adipocytes and Human Population

β-catenin 유전자의 3T3-L1 지방세포 및 인체에서의 지방축적 연관성 연구

  • Bae, Sung-Min (Department of Microbiology, Chung-Ang University College of Medicine) ;
  • Lee, Hae-Yong (Department of Microbiology, Chung-Ang University College of Medicine) ;
  • Chae, Soo-Ahn (Department of Pediatrics, Chung-Ang University College of Medicine) ;
  • Oh, Dong-Jin (Department of Internal Medicine, Chung-Ang University College of Medicine) ;
  • Park, Suk-Won (Department of Radiation Oncology, Chung-Ang University College of Medicine) ;
  • Yoon, Yoo-Sik (Department of Microbiology, Chung-Ang University College of Medicine)
  • 배성민 (중앙대학교 의과대학 미생물학교실) ;
  • 이해용 (중앙대학교 의과대학 미생물학교실) ;
  • 채수안 (중앙대학교 의과대학 소아과학교실) ;
  • 오동진 (중앙대학교 의과대학 내과학교실) ;
  • 박석원 (중앙대학교 의과대학 방사선종양학과) ;
  • 윤유식 (중앙대학교 의과대학 미생물학교실)
  • Received : 2011.07.25
  • Accepted : 2011.08.30
  • Published : 2011.09.30
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Abstract

The major function of adipocytes is to store fat in the form of triglycerides. One of the signaling pathways known to affect adipogenesis, i.e. fat formation, is the WNT/${\beta}$-catenin pathway which inhibits the expression and activity of key regulators of adipogenesis. The purpose of this research is to find genes among the WNT/${\beta}$-catenin pathway which regulate adipogenesis by using small interfering (si) RNA and to find the association of single nucleotide polymorphisms (SNPs) of the gene with serum triglyceride levels in the human population. To elucidate the effects of ${\beta}$-catenin siRNA on adipogenesis key factors, PPAR${\gamma}$ and C/EBP${\alpha}$, we performed real-time PCR and western blotting experiments for the analyses of mRNA and protein levels. It was found that the siRNA-mediated knockdown of ${\beta}$-catenin upregulates adipogenesis key factors. However, upstream regulators of the WNT/${\beta}$-catenin pathway, such as DVL2 and LRP6, had no significant effects compared to ${\beta}$-catenin. These results indicate that ${\beta}$-catenin is a candidate gene for human fat accumulation. In general, serum triglyceride level is a good indicator of fat accumulation in humans. According to statistical analyses of the association between serum triglyceride level and SNPs of ${\beta}$-catenin, -10,288 C>T SNP (rs7630377) in the promoter region was significantly associated with serum triglyceride levels (p<0.05) in 290 Korean subjects. On the other hand, serum cholesterol levels were not significantly associated with SNPs of the ${\beta}$-catenin gene. The results of this study showed that ${\beta}$-catenin is associated with fat accumulation both in vitro and in the human population.

비만은 중성지방이 체내에 과잉으로 축적되어 지방 본래의 에너지 저장과 대사조절의 기능을 정상적으로 하지 못하는 상태를 말한다. 본 연구진은 siRNA 방법을 이용하여 Wingless-type MMTV integration site (WNT)/${\beta}$-catenin pathway에 의한 지방축적 조절에서 중요한 역할을 하는 유전자를 확인하고자 하였다. WNT/${\beta}$-catenin pathway에 속한 유전자 중 ${\beta}$-catenin을 siRNA기법을 통하여 knock down 한 후 adipogenesis의 핵심 조절자인 peroxisome proliferator-activated receptor (PPAR)${\gamma}$, CCAAT/enhancer binding protein (C/EBP)${\alpha}$의 mRNA와 단백질 발현 변화를 확인해 보았다. 그 결과 ${\beta}$-catenin유전자의 knock down에 의하여 PPAR${\gamma}$, CEBP${\alpha}$의 유전자 및 단백질 발현이 유의하게 증가함을 확인하였다. WNT/${\beta}$-catenin pathway에서 ${\beta}$-catenin의 상위 조절자인 LRP6와 DVL2의 knock down에 의한 adipogenesis 조절 유무를 분석하였으나 유의적인 영향을 미치지 못하는 것으로 발견되었다. 이는 ${\beta}$-catenin이 상위 조절자들의 영향을 받기 보다는 독립적인 기작으로 PPAR${\gamma}$, CEBP${\alpha}$의 mRNA, 단백질 발현의 조절함으로써 adipogenesis의 negative regulator의 기능을 하는 것으로 판단된다. 또한 290명의 한국인을 대상으로 비만의 대표적인 표지인자인 혈중 중성지방 농도와 혈중 콜레스테롤 농도에 대한 ${\beta}$-catenin 유전자의 단일염기다형성(SNP)과의 연관성을 통계 분석해보았다. 그 결과 프로모터 부분에 위치한 4종류의 SNP 중에서 transcription개시 지점으로부터 -10,288위치에 존재하는 C>T polymorphism인 rs7630377이 유의하게 혈중 중성지방 농도와 연관성이 있음을 확인할 수 있었다. 본 연구의 결과는 ${\beta}$-catenin이 세포 수준에서 뿐 아니라 인체에서도 지방축적에 유의적인 영향을 미치고 있음을 제시하고 있다.

Keywords

References

  1. Ahmadian, A., B. Gharizadeh, A. C. Gustafsson, F. Sterky, P. Nyren, M. Uhlen, and J. Lundeberg. 2000. Single-nucleotide polymorphism analysis by pyrosequencing. Anal. Biochem. 280, 103-110. https://doi.org/10.1006/abio.2000.4493
  2. Al-Shemari, H., Y. Bosse, T. J. Hudson, M. Cabaluna, M. Duval, M. Lemire, S. Vallee-Smedja, S. Frenkiel, and M. Desrosiers. 2008. Influence of leukotriene gene polymorphisms on chronic rhinosinusitis. BMC Med. Genet. 9, 21.
  3. Albrink, M. J. and J. W. Meigs. 1965. The relationship between serum triglycerides and skinfold thickness in obese subjects. Ann. N. Y. Acad. Sci. 131, 673-683. https://doi.org/10.1111/j.1749-6632.1965.tb34830.x
  4. Behari, J., T. H. Yeh, L. Krauland, W. Otruba, B. Cieply, B. Hauth, U. Apte, T. Wu, R. Evans, and S. P. Monga. 2010. Liver-specific beta-catenin knockout mice exhibit defective bile acid and cholesterol homeostasis and increased susceptibility to diet-induced steatohepatitis. Am. J. Pathol. 176, 744-753. https://doi.org/10.2353/ajpath.2010.090667
  5. Cadigan, K. M. and Y. I. Liu. 2006. Wnt signaling: complexity at the surface. J. Cell Sci. 119, 395-402. https://doi.org/10.1242/jcs.02826
  6. Cha, M. H., I. C. Kim, K. S. Kim, B. K. Kang, S. M. Choi, and Y. Yoon. 2007. Association of UCP2 and UCP3 gene polymorphisms with serum high-density lipoprotein cholesterol among Korean women. Metabolism 56, 806-813. https://doi.org/10.1016/j.metabol.2007.01.023
  7. Dahlman, I. and P. Arner. 2010. Genetics of adipose tissue biology. Prog. Mol. Biol. Transl. Sci. 94, 39-74. https://doi.org/10.1016/B978-0-12-375003-7.00003-0
  8. Gustafson, B., and U. Smith. 2006. Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes. J. Biol. Chem. 281, 9507-9516. https://doi.org/10.1074/jbc.M512077200
  9. Hollister, L. E., J. E. Overall, and H. L. Snow. 1967. Relationship of obesity to serum triglyceride, cholesterol, and uric acid, and to plasma-glucose levels. Am. J. Clin. Nutr. 20, 777-782.
  10. Huelsken, J. and J. Behrens. 2002. The Wnt signalling pathway. J. Cell Sci. 115, 3977-3978. https://doi.org/10.1242/jcs.00089
  11. Kawai, M., S. Mushiake, K. Bessho, M. Murakami, N. Namba, C. Kokubu, T. Michigami, and K. Ozono. 2007. Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha. Biochem. Biophys. Res. Commun. 363, 276-282. https://doi.org/10.1016/j.bbrc.2007.08.088
  12. Lee, H., S. Bae, K. Kim, W. Kim, S. I. Chung, and Y. Yoon. 2010. Beta-Catenin mediates the anti-adipogenic effect of baicalin. Biochem. Biophys. Res. Commun. 398, 741-746. https://doi.org/10.1016/j.bbrc.2010.07.015
  13. Lee, H., R. Kang, S. Bae, and Y. Yoon. 2011. AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/beta-catenin pathway in 3T3-L1 adipocytes. Int. J. Mol. Med. 28, 65-71.
  14. Liu, J. and S. R. Farmer. 2004. Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes. J. Biol. Chem. 279, 45020-45027. https://doi.org/10.1074/jbc.M407050200
  15. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408. https://doi.org/10.1006/meth.2001.1262
  16. Ma, X., X. Ren, P. Han, S. Hu, J. Wang, and J. Yin. 2010. SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction. Mol. Biol. Rep. 37, 4003-4011. https://doi.org/10.1007/s11033-010-0059-5
  17. Moldes, M., Y. Zuo, R. F. Morrison, D. Silva, B. H. Park, J. Liu, and S. R. Farmer. 2003. Peroxisome-proliferator- activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis. Biochem. J. 376, 607-613. https://doi.org/10.1042/BJ20030426
  18. Morrison, R. F. and S. R. Farmer. 1999. Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis. J. Biol. Chem. 274, 17088-17097. https://doi.org/10.1074/jbc.274.24.17088
  19. Ntambi, J. M. and K. Young-Cheul. 2000. Adipocyte differentiation and gene expression. J. Nutr. 130, 3122S-3126S.
  20. Pereira, D. S., D. M. Garcia, F. M. Narciso, M. L. Santos, J. M. Dias, B. Z, Queiroz, E. R. Souza, O. T. Nobrega, and L. S. Pereira. 2011. Effects of 174 G/C polymorphism in the promoter region of the interleukin-6 gene on plasma IL-6 levels and muscle strength in elderly women. Braz. J. Med. Biol. Res. 44, 123-129. https://doi.org/10.1590/S0100-879X2010007500152
  21. Qin, L., Y. Chen, Y. Niu, W. Chen, Q. Wang, S. Xiao, A. Li, Y. Xie, J. Li, X. Zhao, Z. He, and D. Mo. 2010. A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/beta-catenin signaling pathway. BMC Genomics 11, 320. https://doi.org/10.1186/1471-2164-11-320
  22. Ross, S. E., N. Hemati, K. A. Longo, C. N. Bennett, P. C. Lucas, R. L. Erickson, and O. A. MacDougald. 2000. Inhibition of adipogenesis by Wnt signaling. Science 289, 950-953. https://doi.org/10.1126/science.289.5481.950
  23. Sakurai, K., M. Amarzguioui, D. H. Kim, J. Alluin, B. Heale, M. S. Song, A. Gatignol, M. A. Behlke, and J. J. Rossi. 2011. A role for human Dicer in pre-RISC loading of siRNAs. Nucleic Acids Res. 39, 1510-1525. https://doi.org/10.1093/nar/gkq846
  24. Salpea, K. D., D. R. Gable, J. A. Cooper, J. W. Stephens, S. J. Hurel, H. A. Ireland, M. D. Feher, I. F. Godsland, and S. E. Humphries. 2009. The effect of WNT5B IVS3C>G on the susceptibility to type 2 diabetes in UK Caucasian subjects. Nutr. Metab. Cardiovasc. Dis. 19, 140-145. https://doi.org/10.1016/j.numecd.2008.02.009
  25. Shao, D. and M. A. Lazar. 1997. Peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha, and cell cycle status regulate the commitment to adipocyte differentiation. J. Biol. Chem. 272, 21473-21478. https://doi.org/10.1074/jbc.272.34.21473
  26. Van Tienen, F. H., H. Laeremans, C. J. Van der Kallen, and H. J. Smeets. 2009. Wnt5b stimulates adipogenesis by activating PPARgamma, and inhibiting the beta-catenin dependent Wnt signaling pathway together with Wnt5a. Biochem. Biophys. Res. Commun. 387, 207-211. https://doi.org/10.1016/j.bbrc.2009.07.004
  27. Vermeulen, A. 1993. Metabolic effects of obesity in men. Verh. K. Acad. Geneeskd. Belg. 55, 383-393.
  28. Wall, N. R., and Y. Shi. 2003. Small RNA: can RNA interference be exploited for therapy? Lancet 362, 1401-1403. https://doi.org/10.1016/S0140-6736(03)14637-5
  29. Wang, D. G., J. B. Fan, C. J. Siao, A. Berno, P. Young, R. Sapolsky, G. Ghandour, N. Perkins, E. Winchester, J. Spencer, L. Kruglyak, L. Stein, L. Hsie, T. Topaloglou, E. Hubbell, E. Robinson, M. Mittmann, M. S. Morris, N. Shen, D. Kilburn, J. Rioux, C. Nusbaum, S. Rozen, T. J. Hudson, R. Lipshutz, M. Chee, and E. S. Lander. 1998. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280, 1077-1082. https://doi.org/10.1126/science.280.5366.1077
  30. Wang, K., W. D. Li, C. K. Zhang, Z. Wang, J. T. Glessner, S. F. Grant, H. Zhao, H. Hakonarson, and R. A. Price. 2011. A genome-wide association study on obesity and obesity-related traits. PLoS One 6, e18939. https://doi.org/10.1371/journal.pone.0018939
  31. Yamaguchi, Y., M. Moritani, T. Tanahashi, D. Osabe, K. Nomura, Y. Fujita, P. Keshavarz, K. Kunika, N. Nakamura, T. Yoshikawa, E. Ichiishi, H. Shiota, N. Yasui, H. Inoue, and M. Itakura. 2008. Lack of association of genetic variation in chromosome region 15q14-22.1 with type 2 diabetes in a Japanese population. BMC Med. Genet. 9, 22.
  32. Zuckerman, J. E., T. Hsueh, R. C. Koya, M. E. Davis, and A. Ribas. 2011. siRNA knockdown of ribonucleotide reductase inhibits melanoma cell line proliferation alone or synergistically with temozolomide. J. Invest. Dermatol. 131, 453-460. https://doi.org/10.1038/jid.2010.310