Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

A Study on the Gene Expression of Adipogenic Regulators by an Herbal Composition

생약복합물에 의한 지방세포형성 조절자의 유전자 발현 연구

  • Lee, Hae-Yong (Department of Microbiology, Chung-Ang University College of Medicine) ;
  • Kang, Ryun-Hwa (Department of Microbiology, Chung-Ang University College of Medicine) ;
  • Bae, Sung-Min (Department of Microbiology, Chung-Ang University College of Medicine) ;
  • Chae, Soo-Ahn (Department of Pediatrics, Chung-Ang University College of Medicine) ;
  • Lee, Jung-Ju (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) ;
  • Cho, Soo-Hyun (Department of Family Medicine, Chung-Ang University College of Medicine) ;
  • Shim, Yae-Jie (Department of General Education, Seoul Women's University) ;
  • Yoon, Yoo-Sik (Department of Microbiology, Chung-Ang University College of Medicine)
  • 이해용 (중앙대학교 의과대학 미생물학교실) ;
  • 강련화 (중앙대학교 의과대학 미생물학교실) ;
  • 배성민 (중앙대학교 의과대학 미생물학교실) ;
  • 채수안 (중앙대학교 소아과학교실) ;
  • 이정주 (중앙대학교 소아과학교실) ;
  • 오동진 (중앙대학교 내과학교실) ;
  • 박석원 (중앙대학교 방사선종양학과) ;
  • 조수현 (중앙대학교 가정의학과) ;
  • 심예지 (서울여자대학교 교양학부) ;
  • 윤유식 (중앙대학교 의과대학 미생물학교실)
  • Received : 2010.02.02
  • Accepted : 2010.03.10
  • Published : 2010.05.31
Download PDF
⟨ Previous Next ⟩

Abstract

In our previous study, it was reported that an herbal mixture, SH21B, inhibits fat accumulation and adipogenesis both in vitro and in vivo models of obesity. SH21B is a mixture composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd, and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). The aim of this study was to investigate the detailed molecular mechanisms of the effects of SH21B on various regulators of the adipogenesis pathway. During the adipogenesis of 3T3-L1 cells, SH21B significantly decreased the expression levels of central transcription factors of adipogenesis, such as peroxisome proliferator-activated receptor (PPAR)$\gamma$ and CCAAT/enhancer binding protein (C/EBP)$\alpha$. To elucidate the detailed molecular mechanism of the anti-adipogenic effects of SH21B, we examined the expression levels of the various pro-adipogenic or anti-adipogenic regulators of adipogenesis upstream of $PPAR{\gamma}$ and C/$EBP{\alpha}$. The mRNA levels of Krox20 and Kruppel-like factor (KLF) 15, which are pro-adipogenic regulators, were significantly down-regulated by SH21B treatment, whereas the mRNA levels of C/$EBP{\gamma}$ and KLF5 were not changed. KLF2 and C/EBP homologous protein (CHOP), which are anti-adipogenic regulators, were significantly up-regulated by SH21B treatment. These results suggest that the molecular mechanism of the anti-adipogenic effect of SH21B involves both the down-regulations of pro-adipogenic regulators, such as Krox20 and KLF15, and the up-regulations of anti-adipogenic regulators, such as KLF2 and CHOP, which results in the suppression of central transcription factors of adipogenesis including $PPAR{\gamma}$ and C/$EBP{\alpha}$.

본 연구의 목적은 생약복합제제인 SH21B의 adipogenesis 억제 효능에 대한 상세한 분자적 메커니즘을 3T3-L1 지방세포를 이용하여 밝히는 데 있다. 실험에 사용된 SH21B는 7가지 생약성 천연물질인, 황금, 행인, 마황, 석창포, 포황, 원지 및 하엽으로 이루어졌다. 최근, 본 연구진에 의해 3T3-L1을 이용한 in vitro 연구와 마우스를 이용한 in vivo 연구에서 SH21B의 adipogenesis 억제효능이 밝혀진 바 있다. 본 연구에서는 3T3-L1 지방세포가 분화될 때 작용하는 다양한 지방세포형성 조절자들의 유전자 발현이 SH21B에 의해 어떻게 변하는지 살펴보고자 하였다. 실시간중합효소반응(real time PCR) 기술을 이용하여 SH21B를 처리한 지방세포와 그렇지 않은 지방세포를 비교한 결과, 최종마커인 ADIPOQ와 SLC2A4의 유전자 발현이 SH21B에 의해 급격하게 감소함을 알 수 있었다. 최종마커의 발현을 유도하는 핵심전사인자인 $PPAR{\gamma}$와 C/$EBP{\alpha}$의 유전자 발현 역시 SH21B의 처리 시 유의하게 억제되었다. 좀 더 상세한 분자적 메커니즘을 규명하기 위해, 핵심전사인자의 상위에 위치한 다양한 조절자들의 유전자 발현을 분석하였다. 그 결과, 여러 지방세포형성 유도조절자 중, Krox20과 KLF15의 유전자 발현이 SH21B 처리에 의해 유의하게 감소된 반면, C/$EBP{\beta}$와 KLF5의 유전자 발현은 SH21B 처리에 영향을 받지 않았다. 그리고 지방세포형성 억제조절자인 KLF2와 CHOP의 유전자 발현은 SH21B 처리에 의해 유의하게 증가되었다. 이러한 결과들은 SH21B의 지방세포형성 억제효능이 지방세포의 분화에 작용하는 다양한 상위조절자 중 지방세포형성 유도조절자인 Krox20과 KLF15 그리고 지방세포형성 억제조절자인 KLF2와 CHOP 등의 유전자 발현이 변화되면서 일어나는 복합적인 반응의 결과임을 제시한다.

Keywords

References

  1. Ahmed, W., O. Ziouzenkova, J. Brown, P. Devchand, S. Francis, M. Kadakia, T. Kanda, G. Orasanu, M. Sharlach, F. Zandbergen, and J. Plutzky. 2007. PPARs and their metabolic modulation: new mechanisms for transcriptional regulation? J. Intern. Med. 262, 184-198. https://doi.org/10.1111/j.1365-2796.2007.01825.x
  2. Chen, Z., J. I. Torrens, A. Anand, B. M. Spiegelman, and J. M. Friedman. 2005. Krox20 stimulates adipogenesis via C/EBPbeta-dependent and -independent mechanisms. Cell Metab. 1, 93-106. https://doi.org/10.1016/j.cmet.2004.12.009
  3. Darlington, G. J., S. E. Ross, and O. A. MacDougald. 1998. The role of C/EBP genes in adipocyte differentiation. J. Biol. Chem. 273, 30057-30060. https://doi.org/10.1074/jbc.273.46.30057
  4. Freytag, S. O., D. L. Paielli, and J. D. Gilbert. 1994. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 8, 1654-1663. https://doi.org/10.1101/gad.8.14.1654
  5. Gray, S., M. W. Feinberg, S. Hull, C. T. Kuo, M. Watanabe, S. Sen-Banerjee, A. DePina, R. Haspel, and M. K. Jain. 2002. The Kruppel-like factor KLF15 regulates the insulin-sensitive glucose transporter GLUT4. J. Biol. Chem. 277, 34322-34328. https://doi.org/10.1074/jbc.M201304200
  6. 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
  7. Hamm, J. K., A. K. el Jack, P. F. Pilch, and S. R. Farmer. 1999. Role of PPAR gamma in regulating adipocyte differentiation and insulin-responsive glucose uptake. Ann. N Y Acad. Sci. 892, 134-145. https://doi.org/10.1111/j.1749-6632.1999.tb07792.x
  8. Kaczynski, J., T. Cook, and R. Urrutia. 2003. Sp1- and Kruppel-like transcription factors. Genome Biol. 4, 206. https://doi.org/10.1186/gb-2003-4-2-206
  9. Kadowaki, T., T. Yamauchi, N. Kubota, K. Hara, K. Ueki, and K. Tobe. 2006. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J. Clin. Invest 116, 1784-1792. https://doi.org/10.1172/JCI29126
  10. Koerner, A., J. Kratzsch, and W. Kiess. 2005. Adipocytokines: leptin--the classical, resistin--the controversical, adiponectin-- the promising, and more to come. Best Pract. Res. Clin. Endocrinol. Metab. 19, 525-546. https://doi.org/10.1016/j.beem.2005.07.008
  11. Lee, H., R. Kang, and Y. Yoon. 2009. SH21B, an anti-obesity herbal composition, inhibits fat accumulation in 3T3-L1 adipocytes and high fat diet-induced obese mice through the modulation of the adipogenesis pathway. J. Ethnopharmacol. DOI:10.1016/j.jep.2009.12.002
  12. Lin, F. T. and M. D. Lane. 1994. CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc. Natl. Acad. Sci. USA 91, 8757-8761. https://doi.org/10.1073/pnas.91.19.8757
  13. 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
  14. Medina-Gomez, G., S. Virtue, C. Lelliott, R. Boiani, M. Campbell, C. Christodoulides, C. Perrin, M. Jimenez-Linan, M. Blount, J. Dixon, D. Zahn, R. R. Thresher, S. Aparicio, M. Carlton, W. H. Colledge, M. I. Kettunen, T. Seppanen-Laakso, J. K. Sethi, S. O'Rahilly, K. Brindle, S. Cinti, M. Oresic, R. Burcelin, and A. Vidal-Puig. 2005. The link between nutritional status and insulin sensitivity is dependent on the adipocyte-specific peroxisome proliferatoractivated receptor-gamma2 isoform. Diabetes 54, 1706-1716. https://doi.org/10.2337/diabetes.54.6.1706
  15. Mori, T., H. Sakaue, H. Iguchi, H. Gomi, Y. Okada, Y. Takashima, K. Nakamura, T. Nakamura, T. Yamauchi, N. Kubota, T. Kadowaki, Y. Matsuki, W. Ogawa, R. Hiramatsu, and M. Kasuga. 2005. Role of Kruppel-like factor 15 (KLF15) in transcriptional regulation of adipogenesis. J. Biol. Chem. 280, 12867-12875. https://doi.org/10.1074/jbc.M410515200
  16. Morrison, R. F. and S. R. Farmer. 2000. Hormonal signaling and transcriptional control of adipocyte differentiation. J. Nutr. 130, 3116S-3121S.
  17. 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
  18. Nawrocki, A. R. and P. E. Scherer. 2005. Keynote review: the adipocyte as a drug discovery target. Drug Discov. Today 10, 1219-1230. https://doi.org/10.1016/S1359-6446(05)03569-5
  19. Oishi, Y., I. Manabe, K. Tobe, K. Tsushima, T. Shindo, K. Fujiu, G. Nishimura, K. Maemura, T. Yamauchi, N. Kubota, R. Suzuki, T. Kitamura, S. Akira, T. Kadowaki, and R. Nagai. 2005. Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation. Cell Metab. 1, 27-39. https://doi.org/10.1016/j.cmet.2004.11.005
  20. Rosen, E. D. and O. A. MacDougald. 2006. Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 7, 885-896. https://doi.org/10.1038/nrm2066
  21. 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
  22. Shepherd, P. R. and B. B. Kahn. 1999. Glucose transporters and insulin action-implications for insulin resistance and diabetes mellitus. N. Engl. J. Med. 341, 248-257. https://doi.org/10.1056/NEJM199907223410406
  23. Spiegelman, B. M. and J. S. Flier. 2001. Obesity and the regulation of energy balance. Cell 104, 531-543. https://doi.org/10.1016/S0092-8674(01)00240-9
  24. Takano, H. and I. Komuro. 2009. Peroxisome proliferatoractivated receptor gamma and cardiovascular diseases. Circ. J. 73, 214-220. https://doi.org/10.1253/circj.CJ-08-1071
  25. Tanaka, T., N. Yoshida, T. Kishimoto, and S. Akira. 1997. Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO. J. 16, 7432-7443. https://doi.org/10.1093/emboj/16.24.7432
  26. Tontonoz, P., E. Hu, and B. M. Spiegelman. 1994. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79, 1147-1156. https://doi.org/10.1016/0092-8674(94)90006-X
  27. Zhang, J., M. Fu, T. Cui, C. Xiong, K. Xu, W. Zhong, Y. Xiao, D. Floyd, J. Liang, E. Li, Q. Song, and Y. E. Chen. 2004. Selective disruption of PPARgamma 2 impairs the development of adipose tissue and insulin sensitivity. Proc. Natl. Acad. Sci. USA 101, 10703-10708. https://doi.org/10.1073/pnas.0403652101