[논문]3T3-L1세포에서 흑효모 SM-2001 추출물(Polycan®)의 항비만 효과

김영숙; 임종민; 구본화; 문승배; 조형래; 이선민; 권정희

doi:10.5352/jls.2020.30.10.835

3T3-L1세포에서 흑효모 SM-2001 추출물(Polycan®)의 항비만 효과
The Anti-obesity Effect of Aureobasidium pullulans SM-2001 Extract (Polycan®) on 3T3-L1 Preadipocytes and Adipocytes 원문보기

생명과학회지 = Journal of life science, v.30 no.10, 2020년, pp.835 - 843

김영숙 ((주)글루칸) , 임종민 ((주)글루칸) , 구본화 ((주)글루칸) , 문승배 ((주)글루칸) , 조형래 ((주)글루칸) , 이선민 (안전성평가연구소 생명자원연구그룹) , 권정희 (주)엔피켐바이오)

초록
AI-Helper

비만은 이제 선진국 뿐만 아니라 개발도상국에서도 가장 문제가 되는 대사성 질환이다. 최근 치료보다는 예방에 중점을 두는 예방의학 시대가 도래함에 따라, 인슐린 저항성 비만과 당뇨병 등의 대사 증후군을 예방하기 위한 천연물들이 큰 관심을 받고 있다. 특히, 베타글루칸은 면역계와 혈중 콜레스테롤 조절에 이로운 효과가 있는 것으로 알려져 있지만 비만에 미치는 영향에 대해서는 완전히 밝혀지지 않았다. 이에 본 연구에서는 β-1,3/1,6-glucan의 함량이 전체 글루칸 함량의 90% 이상 되는 자체 개발 흑효모 균주 SM-2001 추출물(폴리칸)이 3T3-L1 지방전구세포의 지방세포 분화에 미치는 영향을 조사하였다. 폴리칸은 지방전구세포에서 지방축적과 GPDH 효소 활성을 억제하는 것으로 나타났다. 이는 폴리칸이 세포 내에서 지방의 축적을 조절하는 전사인자인 PPARγ와 C/EBPα의 하향조절과 세포 내 에너지 센서 역할을 하는 AMPK 효소의 인산화를 유도함으로써 항비만 효과를 발현하는 것으로 확인되었다. 본 연구를 통해 폴리칸의 항비만 효과를 확인하였으며, 향후 비만과 대사성 질환을 예방할 수 있는 식의약 소재로써 그 활용가치를 더욱 높일 수 있을 것으로 기대한다.

Abstract ▼ AI-Helper

Obesity, the world's leading metabolic disease, is a serious health problem in both industrialized and developing countries. Natural substances are of great interest in preventative medicine, especially in the field of metabolic syndromes-from insulin resistance to obesity and diabetes. In the present study, we investigated the effect of A. pullulans SM-2001 Extract (Polycan®) on the adipocyte differentiation of 3T3-L1 preadipocytes and the anti-obesity effect of 3T3-L1 adipocytes. Although β-glucan has been found to have health benefits in the regulation of the immune system and blood cholesterol levels, its role in obesity has not been fully investigated. Polycan® suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability in 3T3-L1 preadipocytes and adipocytes. Polycan® also inhibited cellular lipid accumulation through down-regulation of transcription factors, such as PPARγ and C/EBPα, and induced dose-dependent phosphorylation of AMP-activated protein kinase (AMPK)-a cellular energy sensor-while the total AMPK protein content remained unchanged. Taken together, this shows that the activation of AMPK by Polycan® in adipocytes plays a critical role in Polycan®-induced inhibition of adipocyte differentiation. Our results show that Polycan® has an anti-obesity action in vitro, suggesting a potential novel preventative agent for obesity and other metabolic diseases.

주제어

표/그림 (6)

그림 Fig. 1. Experimental scheme. For the induction of adipocyte differentiation, 3T3-L1 preadipocytes were seeded. At confluence (day 0), the cultured preadipocytes were induced to differentiate by the addition of differentiating medium containing 0.5mM methylisobutylxanthine (IBMX), 1 μg/ml insulin, and 1 μM dexamethasone (Dex) from day 0 to day 2. At day 2, medium was changed with medium containing 1 μg/ml insulin for an additional 2 days from day 2 to day 6. The medium was refreshed every 2 days. At day 6, differentiated 3T3-L1 cells were subjected to Oil red O solution or used for Western blot analysis. Polycan^® was added to the cell culture medium at concentrations of 0, 100, 200 and 400 ug/ml from day 0 to day 6. DMEM, Dulbecco's modified Eagle's medium.
그림 Fig. 2. Cytotoxic effect of Polycan^® on 3T3-L1 cells. Cell viability was measured using CCK-8 staining. The data represent the mean ± SD of three separate experiments
그림 Fig. 3. Effects of Polycan^® on intracellular lipid accumulation in 3T3-L1 cells. 3T3-L1 cells were treated with polycan (0, 100, 200, 400 μg/ml) for day 6. (A) The mature adipocytes were stained with Oil-red O, and the (B) OD value were measured to quantify intracellular lipid content. Three independent experiments were performed and the data were shown as mean ± SD. Values do not share the same letter are significantly different (p<0.05). ***p<0.001.
그림 Fig. 4. Effects of Polycan^® on glycerol release in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were treated in serum-free medium with Polycan^® (0, 100, 200, 400 ug/ml). The medium was collected and assayed for glycerol content. Three independent experiments were performed and the data were shown as mean ± SD. Values do not share the same letter are significantly different (p<0.05). ***p< 0.001, **p<0.01, *p<0.05.
그림 Fig. 5. Effect of Polycan^® on GPDH activity in cultured 3T3-L1 adipocytes. The 3T3-L1 adipocytes were harvested 6 days after the initiation of differentiation with 0, 100, 200, 400 μg/ml Polycan^®. Three independent experiments were performed and the data were shown as mean ± SD. Values do not share the same letter are significantly different (p<0.05). *** p<0.001.
그림 Fig. 6. Effect of Polycan^® on the protein levels of the PPARγ, C/EBPα, AMPK, and pAMPK in 3T3-L1 adipocytes by Western blotting. 3T3-L1 adipocytes were harvested 6 days after the differentiation with 0, 100, 200, 400 ug/ml Polycan^®. β-actin was used as a housekeeping protein control.

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

대상 데이터

(Korea) and were stored in a refrigerator at 4℃ to protect from light and degradation. 3T3-L1 cells were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). Dulbecco's Modified Eagle Medium/Ham's F-12 nutrient mixture (DMEM/F12), fetal bovine serum (FBS), calf bovine serum (CBS), penicillin-streptomycin, phosphate buffered saline (PBS) and transferrin were obtained from Gibco BRL (Rockville, MD.
). The experiments were performed in triplicate.

데이터처리

). One-way ANOVA was applied to calculate the significance between the groups. Statistical significance was indicated by a p value of <0.

이론/모형

Finally, the solution was centrifuged at 14,000× g for 20 min at 4℃. The supernatant was collected and protein concentrations were determined by the Bradford method. Proteins (30 ug) were loaded onto each lane for 10% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and the separated proteins were blotted to PVDF membrane.

성능/효과

The O.D. absorbance significantly decreased (p<0.05) in cells treated with Polycan® in dose-dependent manner (Fig. 3B), and the decrease rates were 51.2%, 36.03%, and 18.3% at concentrations of 100, 200 and 400 μg/ml of Polycan®, respectively.
These results indicated that Polycan® could inhibit adipocyte differentiation, thus inhibit intracellular lipid accumulation in 3T3-L1 cells.
To the best of our knowledge, the results of the present study demonstrate, for the first time, that Polycan® consisting mainly of β-1,3/1,6-glucan have anti-obesity effects in 3T3-L1 adipocyte.

후속연구

Taken together, Polycan® may have novel preventative potential for obesity and other metabolic diseases, warranting further investigation for the precise mechanism.

참고문헌 (44)

Amri, E. Z., Bonino, F., Ailhaud, G., Abumrad, N. A. and Grimaldi, P. A. 1995. Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors. J. Biol. Chem. 270, 2367-2371.

상세보기
Bell, S., Goldman, V. M., Bistrian, B. R., Arnold, A. H., Ostroff, G. and Forse, R. A. 1999. Effect of beta-glucan from oats and yeast on serum lipids. Crit. Rev. Food Sci. Nutr. 39, 189-202.
Bhathena, S. J. and Velasquez, M. T. 2002. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am. J. Clin. Nutr. 76, 1191-1201.

상세보기
Cani, P. D., Bibiloni, R., Knauf, C., Waget, A., Neyrinck, A. M., Delzenne, N. M. and Burcelin, R. 2008. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 57, 1470-1481.

상세보기
Choi, J., Kim, J., Jung, G., Moon, S., Cho, H., Ku, S. and Sohn, J. 2018. Hypoglycemic and hypolipemic effect of ${\beta}$ -glucan originated from Aureobasidium in STZ-induced diabetic rats. J. Anim. Plant Sci. 28, 9.
Cowherd, R. M., Lyle, R. E. and McGehee, R. E. Jr. 1999. Molecular regulation of adipocyte differentiation. Semin. Cell Dev. Biol. 10, 3-10.

상세보기
Czop, J. K. 1986. The role of beta-glucan receptors on blood and tissue leukocytes in phagocytosis and metabolic activation. Pathol. Immunopathol. Res. 5, 286-296.

상세보기
Daval, M., Foufelle, F. and Ferre, P. 2006. Functions of AMPactivated protein kinase in adipose tissue. J. Physiol. 574, 55-62.
De Paula, A. C., Sousa, R. V., Figueiredo-Ribeiro, R. C. and Buckeridge, M. S. 2005. Hypoglycemic activity of polysaccharide fractions containing beta-glucans from extracts of Rhynchelytrum repens (Willd.) C.E. Hubb., Poaceae. Braz. J. Med. Biol. Res. 38, 885-893.
El-Jack, A. K., Hamm, J. K., Pilch, P. F. and Farmer, S. R. 1999. Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPARgamma and C/EBPalpha. J. Biol. Chem. 274, 7946-7951.
Elberg, G., Gimble, J. M. and Tsai, S. Y. 2000. Modulation of the murine peroxisome proliferator-activated receptor gamma 2 promoter activity by CCAAT/enhancer-binding proteins. J. Biol. Chem. 275, 27815-27822.
Estrada, A., Yun, C. H., Van Kessel, A., Li, B., Hauta, S. and Laarveld, B. 1997. Immunomodulatory activities of oat beta-glucan in vitro and in vivo. Microbiol. Immunol. 41, 991-998.
Finck, B. N. 2018. Targeting metabolism, insulin resistance, and diabetes to treat nonalcoholic steatohepatitis. Diabetes 67, 2485-2493.

상세보기
Freytag, S. O., Paielli, D. L. and Gilbert, J. D. 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.

상세보기
Green, H. and Kehinde, O. 1975. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5, 19-27.

상세보기
Gregoire, F. M., Smas, C. M. and Sul, H. S. 1998. Understanding adipocyte differentiation. Physiol. Rev. 78, 783-809.

상세보기
Haslam, D. W. and James, W. P. 2005. Obesity. Lancet 366, 1197-1209.
Jeon, J. R. and Kim, J. Y. 2006. Effects of pine needle extract on differentiation of 3T3-L1 preadipocytes and obesity in high-fat diet fed rats. Biol. Pharm. Bull. 29, 2111-2115.

상세보기
Jeon, T., Hwang, S. G., Hirai, S., Matsui, T., Yano, H., Kawada, T., Lim, B. O. and Park, D. K. 2004. Red yeast rice extracts suppress adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 cells. Life Sci. 75, 3195-3203.
Johmura, Y. 2007. Characterization of novel genes regulating adipocyte differentiation. Yakugaku Zasshi 127, 135-142.
Kanagasabapathy, G., Chua, K. H., Malek, S. N., Vikineswary, S. and Kuppusamy, U. R. 2014. AMP-activated protein kinase mediates insulin-like and lipo-mobilising effects of beta-glucan-rich polysaccharides isolated from Pleurotus sajor-caju (Fr.), Singer mushroom, in 3T3-L1 cells. Food Chem. 145, 198-204.
Kang, S. A., Jang, K., Hong, K., Choi, W., Jung, K. and Lee, I. 2002. Effects of dietary ${\beta}$ -Glucan on adiposity and serum lipids levels in obese rats induced by high fat diet. J. Kor. Soc. Food Sci. Nutr. 31, 1052-1057.
Kasturi, R. and Joshi, V. C. 1982. Hormonal regulation of stearoyl coenzyme A desaturase activity and lipogenesis during adipose conversion of 3T3-L1 cells. J. Biol. Chem. 257, 12224-12230.
Kersten, S., Desvergne, B. and Wahli, W. 2000. Roles of PPARs in health and disease. Nature 405, 421-424.

상세보기
Kim, M., Kim, O., Chung, C., Jang, K., Kim, C. and Kang, S. A. 2015. ${\beta}$ -Glucan from Aureobasidum species inhibits fat accumulation in 3T3-L1 adipocyte differentiation. Food Sci. Biotechnol. 24, 1147-1150.
Kim, W. K., Lee, C. Y., Kang, M. S., Kim, M. H., Ryu, Y. H., Bae, K. H., Shin, S. J., Lee, S. C. and Ko, Y. 2008. Effects of leptin on lipid metabolism and gene expression of differentiation- associated growth factors and transcription factors during differentiation and maturation of 3T3-L1 preadipocytes. Endocr. J. 55, 827-837.

상세보기
Kim, Y. W., Kim, K. H., Choi, H. J. and Lee, D. S. 2005. Anti-diabetic activity of beta-glucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei. Biotechnol. Lett. 27, 483-487.
Kong, C. S., Kim, J. A. and Kim, S. K. 2009. Anti-obesity effect of sulfated glucosamine by AMPK signal pathway in 3T3-L1 adipocytes. Food Chem. Toxicol. 47, 2401-2406.

상세보기
Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Nakano, R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K., Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Kitamura, T., Fujita, T., Ezaki, O., Aizawa, S. and Kadowaki, T., et al. 1999. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol. Cell 4, 597-609.
Lee, I., Kim, J., Ryoo, I., Kim, Y., Choo, S., Yoo, I., Min, B., Na, M., Hattori, M. and Bae, K. 2010. Lanostane triterpenes from Ganoderma lucidum suppress the adipogenesis in 3T3-L1 cells through down-regulation of SREBP-1c. Bioorg. Med. Chem. Lett. 20, 5577-5581.

상세보기
Lia, A., Hallmans, G., Sandberg, A. S., Sundberg, B., Aman, P. and Andersson, H. 1995. Oat beta-glucan increases bile acid excretion and a fiber-rich barley fraction increases cholesterol excretion in ileostomy subjects. Am. J. Clin. Nutr. 62, 1245-1251.

상세보기
Liu, L. H., Wang, X. K., Hu, Y. D., Kang, J. L., Wang, L. L. and Li, S. 2004. Effects of a fatty acid synthase inhibitor on adipocyte differentiation of mouse 3T3-L1 cells. Acta Pharmacol. Sin. 25, 1052-1057.
MacDougald, O. A. and Lane, M. D. 1995. Transcriptional regulation of gene expression during adipocyte differentiation. Annu. Rev. Biochem. 64, 345-373.

상세보기
Mark, D. H. 2005. Deaths attributable to obesity. JAMA 293, 1918-1919.

상세보기
Masihi, K. N., Madaj, K., Hintelmann, H., Gast, G. and Kaneko, Y. 1997. Down-regulation of tumor necrosis factor-alpha, moderate reduction of interleukin-1beta, but not interleukin-6 or interleukin-10, by glucan immunomodulators curdlan sulfate and lentinan. Int. J. Immunopharmacol. 19, 463-468.
Patsouris, D., Mandard, S., Voshol, P. J., Escher, P., Tan, N. S., Havekes, L. M., Koenig, W., Marz, W., Tafuri, S., Wahli, W., Muller, M. and Kersten, S. 2004. PPARalpha governs glycerol metabolism. J. Clin. Invest. 114, 94-103.
Pick, M. E., Hawrysh, Z. J., Gee, M. I., Toth, E., Garg, M. L. and Hardin, R. T. 1996. Oat bran concentrate bread products improve long-term control of diabetes: a pilot study. J. Am. Diet. Assoc. 96, 1254-1261.

상세보기
Rosen, E. D., Hsu, C. H., Wang, X., Sakai, S., Freeman, M. W., Gonzalez, F. J. and Spiegelman, B. M. 2002. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev. 16, 22-26.

상세보기
Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K., Milstone, D. S., Spiegelman, B. M. and Mortensen, R. M. 1999. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol. Cell 4, 611-617.
Seo, H., Kim, J., Shin, H., Kim, T., Chang, H., Park, B. and Lee, J. 2002. Production of ${\beta}$ -1,3/1,6-glucan by Aureobasidium pullulans SM-2001. Kor. J. Biotechnol. Bioeng. 17, 5.
Sohn, J., Kim, J., Lim, J., Cho, H., Ku, S. and Choi, J. 2018. Effects of ${\beta}$ -glucan from Aureobasidium pullulans in a streptozotocin-induced rat diabetes model. Curr. Nutr. Food Sci. 15, 12.
Soltys, J. and Quinn, M. T. 1999. Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan. Infect. Immun. 67, 244-252.
Takahashi, N., Kawada, T., Goto, T., Yamamoto, T., Taimatsu, A., Matsui, N., Kimura, K., Saito, M., Hosokawa, M., Miyashita, K. and Fushiki, T. 2002. Dual action of isoprenols from herbal medicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes. FEBS Lett. 514, 315-322.
Thompson, G. M., Trainor, D., Biswas, C., LaCerte, C., Berger, J. P. and Kelly, L. J. 2004. A high-capacity assay for PPARgamma ligand regulation of endogenous aP2 expression in 3T3-L1 cells. Anal. Biochem. 330, 21-28.

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증