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研究生:郭宣霆
研究生(外文):Kuo, Hsuan-Ting
論文名稱:探討一種新穎AMPK活化劑對3T3-L1脂肪前驅細胞分化之影響
論文名稱(外文):Investigation of The Effect of A Novel AMPK Activator on The Differentiation of 3T3-L1 Preadipocytes
指導教授:陳翰民
指導教授(外文):Chen, Han-Min
口試委員:賴金美高紹軒
口試委員(外文):Lai, Jin-MeiKao, Shao-Hsuan
口試日期:2016-07-25
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:67
中文關鍵詞:脂肪分化
外文關鍵詞:3T3-l1AMPKAdipocyte differentiation
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:0
肥胖是造成代謝症候群的主要因素之一,當熱量的攝取超過所需熱量時,多餘的熱量會促使脂肪細胞的生長,肥胖可能引起許多代謝疾病的產生,像是動脈粥狀硬化症、高血糖、高血壓以及血脂異常等。AMPK (AMP-activated Protein Kinase)為一種在細胞內維持能量平衡的酵素,有文獻指出,活化AMPK可以抑制脂肪細胞生成並促進脂肪細胞分解,也有文獻的結果與其相反,因此AMPK的活化對於脂肪細胞的分化與糖類攝取所扮演的角色尚未完全了解。本篇擬以常用的AMPK活化劑「AICAR」與一種新型的AMPK活化劑「ENERGI」,共同探討其對脂肪細胞形成的影響。實驗結果顯示,在3T3-L1之脂肪分化時期投以ENERGI處理可以促進脂肪細胞生成,其機制為透過調控脂肪細胞之轉錄因子,包括PPAR與C/EBP等,處理AICAR的結果則與其相反。在脂肪細胞分化晚期使其處於低葡萄糖濃度可促進脂肪細胞中三酸甘油脂分解成游離脂肪酸,並輔以ENERGI、AICAR與腎上腺素的處理,結果顯示處於低糖環境對於脂肪細胞,使細胞中三酸甘油脂分解成游離脂肪酸是主要的關鍵。綜上所述,證實ENERGI可能透過其他途徑,並非是以活化AMPK來調控脂肪細胞在分化時期促進脂肪細胞生成。
Obesity is one of causative factors involved in the development of metabolic syndrome. Obesity is the consequence of an imbalance between energy intake and expenditure, so obesity is not only a serious health, but also predisposes a person to a variety of metabolic diseases like atherosclerosis, hyperglycemia, dyslipidemia, and hypertension. The AMPK (AMP-activated Protein Kinase) is a sensor of cellular energy status, and it plays a critical role in systemic energy balance in response to metabolic stress. Recently, many studies indicated that activate AMPK can inhibit adipogenesis but promote lipolysis, also there hold opposite. Therefore, the role of AMPK in adipocyte differentiation is not completely understood. In this study, we aim to clarify the effect of ENERGI, a novel AMPK activator discovered by our lab, compare with AICAR, on adipocyte differentiation in fibroblast, 3T3-L1. The results showed that 3T3-L1 cells display a significant adipogenesis with ENERGI treatment, through regulation of transcription factors, including PPAR and C/EBPhowever, the result of AICAR treatment was opposite. In the late adipocyte differentiation stage, while treated in low glucose concentration can promote cellular triglycerides transformation to free fatty acid, addition of ENERGI, AICAR, or epinephrine contribute to adipocyte lipolysis. In summary, it was confirmed ENERGI possible through other pathways regulates adipocyte differentiation facilitate adipogenesis without activating AMPK.
目錄
中文摘要 1
英文摘要 2
第一章 概論 3
 1.1 肥胖與代謝症候群 3
 1.2 脂肪細胞分化 (adipocyte differentiation)與轉錄因子之調控機
制 3
 1.3 AMP-activated protein kinase (AMPK) 4
 1.4 AMPK於脂肪細胞分化之相關性 5
 1.5 實驗動機與目的 6
第二章 材料與方法 7
 2.1 細胞培養 7
 2.1.1 貼附型細胞之培養與繼代 7
 2.2 細胞保存 8
 2.3 細胞存活分析 9
 2.4 細胞破裂法 10
 2.5 蛋白質定量 11
 2.5.1 BCA method 12
 2.6 電泳檢定法 13
 2.7 蛋白質轉印法 15
 2.8 酵素免疫染色 17
 2.9 脂肪細胞分化 18
 2.10 細胞內ATP測定 19
 2.11 油紅染色 (Oil Red O staining) 20
 2.12 細胞內Triglyceride測定 21
 2.13 RNA萃取 22
 2.14 定量即時聚合酶連鎖反應 (qPCR) 23
 2.15 細胞外Free Fatty Acid測定 24
第三章 實驗結果 26
3.1 ENERGI於3T3-L1 cell line脂肪細胞分化之影響 26
 3.1.1 ENERGI於3T3-L1生長之影響 26
 3.1.2 ENERGI與AICAR於3T3-L1之AMPK與ACC活化 26
 3.1.3 細胞密度於3T3-L1脂肪細胞分化之影響 26
 3.1.4 3T3-L1脂肪細胞分化於各個時期處理ENERGI與AICAR
之細胞內ATP含量 27
 3.1.5 3T3-L1脂肪細胞分化處理ENERGI、AICAR與
Rosiglitazone之影響 27
 3.1.6 3T3-L1脂肪細胞分化處理ENERGI與AICAR於細胞內
三酸甘油脂含量之影響 27
 3.1.7 3T3-L1脂肪細胞分化處理ENERGI、AICAR與
Rosiglitazone於細胞內Δ三酸甘油脂之影響 28
 3.1.8 3T3-L1脂肪細胞分化處理ENERGI與AICAR之脂肪細
胞相關mRNA表現量 28
 3.1.9 3T3-L1脂肪細胞分化處理ENERGI與AICAR之脂肪細
胞相關蛋白質表現 29
 3.1.10 3T3-L1脂肪細胞分化於A1時期處理ENERGI與AICAR
基因表現變化之分析 29
3.2 ENERGI於3T3-L1 cell line脂肪細胞脂肪分解之影響 30
 3.2.1 不同葡萄糖濃度於3T3-L1脂肪細胞之影響 30
 3.2.2 不同葡萄糖濃度佐以ENERGI、AICAR與Epinephrine
於3T3-L1脂肪細胞之影響 30
3.2 圖表集 31
 圖一. ENERGI與AICAR於3T3-L1生長之影響 31
 圖二. ENERGI與ACIAR於3T3-L1之AMPK與ACC活化 32
 圖三. 細胞密度於3T3-L1脂肪細胞分化之影響 33
 圖四. 3T3-L1脂肪細胞分化於A1、A2及A3時期處理ENERGI
與AICAR之細胞內ATP含量 34
 圖五. 3T3-L1脂肪細胞分化處理ENERGI、AICAR與
Rosiglitazone之Oil Red O染色 35
 圖六. 3T3-L1脂肪細胞分化A1或A3處理ENERGI與AICAR
於A3後測量細胞內TG含量 36
 圖七. 3T3-L1脂肪細胞分化A1時期處理ENERGI、AICAR與
Rosiglitazone於A1時期之細胞內ΔTG含量 37
 圖八. 3T3-L1脂肪細胞分化A1時期處理ENERGI、AICAR與
Rosiglitazone於A2時期之細胞內ΔTG含量 38
 圖九. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI、
AICAR與Rosiglitazone於A3時期之細胞內ΔTG含量 39
 表一. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI、
AICAR與Rosiglitazone於各個時期之細胞內蛋白質總量 41
 圖十. 3T3-L1脂肪細胞分化A1時期處理ENERGI與AICAR
於A1時期之mRNA表現量 42
 圖十一. 3T3-L1脂肪細胞分化A1時期處理ENERGI與AICAR
於A2時期之mRNA表現量 43
 圖十二. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI與
AICAR於A3時期之mRNA表現量 44
 圖十三. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI與
AICAR之不同時期ACC活化 45
 圖十四. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI與
AICAR之不同時期AMPK活化 46
 圖十五. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI與
AICAR之不同時期脂肪細胞分化之相關蛋白質表現量 47
 圖十六. 3T3-L1脂肪細胞分化A1或A3時期處理ENERGI與
AICAR之不同時期脂肪細胞分化之相關蛋白質表現量 48
 圖十七. 3T3-L1脂肪細胞分化A1時期處理ENERGI與AICAR
之mRNA microarray熱點圖 49
 圖十八. 以GeneGo分析ENERGI處理3T3-L1之mRNA assay
實驗數據 50
 圖十九. 不同葡萄糖濃度於3T3-L1脂肪細胞分化完全後細胞
外FFA之含量 51
 圖二十. 不同葡萄糖濃度、ENERGI、AICAR與Epinephrine
於3T3-L1脂肪細胞分化完全後細胞外FFA之含量 52
第四章 討論 53
第五章 文獻參考 55
第六章 答問錄 58


1.Y.F. Cheng, G.H. Young, J.T. Lin, H.H. Jang, C.C. Chen, J.Y. Nong, P.K. Chen, C.Y. Kuo, S.H. Kao, Y.J. Liang, H.M. Chen. 2015. Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells. Plos One. 10(11): e0142283. doi:10.1371.
2.C.C. Chen, J.T. Lin, Y.F. Cheng, C.F. Huang, S.H. Kao, Y.J. Liang, C.Y. Cheng, H.M. Chen. 2014. Amelioration of LPS-Induced Inflammation Response in Microglia by AMPK Activation. Hindawi Publishing Corporation. Article ID 692061.
3.J.H. Qin, J.Z. Ma, X.W. Yang, Y.J. Hu, Juan Zhou, L.C. Fu, R.H. Tian, Shan Liu, Gang Xu, X.L. Shen. 2015. A Triterpenoid Inhibited Hormone-Induced Adipocyte Differentiation and Alleviated Dexamethasone-Induced Insulin Resistance in 3T3-L1 adipocytes. Nat. Prod. Bioprospect.5:159-166.
4.S.W. Hong, J. Lee, S.E. Park, E.J. Rhee, C.Y. Park, K.W. Oh, S.W. Park, W.Y. Lee. 2014. Activation of AMP-Activated Protein Kinase Attenuates Tumor Necrosis Factor-α-Induced Lipolysis via Protection of Perilipin in 3T3-L1 Adipocytes. Endocrinology and Metabolism. 29:553-560.
5.Shailendra Giri, Ramandeep Rattan, Ehtishamul Haq, Mushfiquddin Khan, Rifat Yasmin, Je-song Won, Lyndon Key, Avtar K Singh and Inderjit Singh. 2006. AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model. Nutrition&Metabolism. 3:31.
6.M.H. Yang, Y.W. Chin, H.S. Chae, K.D. Yoon, J. Kim. 2014. Anti-adipogenic Constituents from Dioscorea opposite in 3T3-L1 Cells. Biol. Pharm. Bull. 37(10) 1683-1688.
7.Byulchorong Min, Heejin Lee, J.H. Song, M.J. Han and Jayong Chung. 2014. Arctiin inhibits adipogenesis in 3T3-L1 cells and decreases adiposity and body weight in mice fed a high-fat diet. Nutrition Research and Practice. 8(6):655-661.
8.Q.X. Meng, Long Wen, X.Y. Chen and H.J. Zhong. 2013. Association of serum angiopoietin-like protein 2 and epinephrine levels in metabolically healthy but obese individuals: In vitro and in vivo evidence. 5:1631-1636.
9.H.J. Kim, B.K. Yoon, H. Park, J. W. Seok, H. Choi, J.H. Yu, Y. Choi, S.J. Song, A. Kim, J.W. Kim. 2016. Caffeine inhibits adipogenesis through modulation of mitotic clonal expansion and the AKT/GSK3 pathway in 3T3-L1 adipocytes. BMB Rep. Feb;49(2):111-5.
10.Sunmin Park, Suna Kang, D.Y. Jeong, S.Y. Jeong, J.J. Park, H.S. Yun. 2015. Cyanidin and malvidin in aqueous extracts of black carrots fermented with Aspergillus oryzae prevent the impairment of energy, lipid and glucose metabolism in estrogen-deficient rats by AMPK activation. Genes Nutr. 10:6.
11.E. Trevellin, M. Scorzeto, M. Olivieri, M. Granzotto, A. Valerio, L. Tedesco, R. Fabris, R. Serra, M. Quarta, C. Reggiani, E. Nisoli, and R. Vettor. 2014. Exercise Training Induces Mitochondrial Biogenesis and Glucose Uptake in Subcutaneous Adipocyte Tissue Through eNOS-Dependent Mechanisms. Diabetes. 63:2800-2811.
12.Y. Ohsaka, H. Nishino, Y. Nomura. 2014. Adipose Cells Induce Phospho-Thr-172 AMPK Production by Epinephrine or CL316243 in Mouse 3T3-L1 Adipocytes or MAPK Activation and G Protein-Associated PI3K Responses Induced by CL316243 or Aluminum Fluoride in Rat White Adipocytes. Folia Biologica (Praha). 60,168-179.
13.J. Zhang, H. Tang, Y. Zhang, R. Deng, L. Shao, Y. Liu, F. Li, X. Wang and L. Zhou. 2014. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation. International Journal of Molecular Medicine. 33:1209-1219.
14.Muthuraman Pandurangan, Sambandam Ravikumar. 2014. Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes. Cytotechnology. 66:619-624.
15.G. Wang, X. Xu, X. Yao, Z. Zhu, L. Yu, L. Chen, J. Chen, X. Shen. 2013. Latanoprost effectively ameliorates glucose and lipid disorders in db/db and ob/ob mice. Diabetologia. 56:2702-2712.
16.Gormand, C. Berggreen, L. Amar, E. Henriksson, I. Lund, S. Albinsson and O. Goransson. 2014. LKB1 signalling attenuates early events of adipogenesis and responds to adipogenic cues. Molecular Endocrinology. 53:1,117-130.
17.X. Yu, L. Ye, H. Zhang, J. Zhao, G. Wang, C. Guo and W. Shang. 2014. Ginsenoside Rb1 ameliorates liver fat accumulation by upregulating perilipin expression in adipose tissue of db/db obese mice. Ginseng Research. 39:119-205.
18.S. Ilavenil, D.H. Kim, M. V. Arasu, S. Srigopalram, R. Sivanesan and K.C. Choi. 2015. Phenyllactiv Acid from Lactobacillus plantarum Promotes Adipogenic Activity in 3T3-L1 Adipocyte via Up-Regulation of PPAR-2. Molecules. 20:15359-15379.
19.Bolsoni-Lopes, W.T. Festuccis, P. Chinmin, T.S. Farias, F. Torres-Leal, M. Cruz, P. Andrade, S. Hirabara, F. Lima and M.I. Alonso-Vale. 2014. Palmitoleic acid (n-7) increases white adipocytes GLUT4 content and glucose uptake in association with AMPK activation. Lipids in Health and Disease. 13:119
20.J. Rhyu, M.S. Kim, M.K. You, M.A. Bang, H.A. Kim. 2014. Pear pomace water extract inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes. Nutrition Research and Practice. 8(1):33-39.
21.J. Love, T. Suzuki, D. Robison, C. Harris, J. Johnson, P. Mohler, W. Jerome, L. Swift. 2015. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes. PLOS ONE. DOI:10.1371
22.W.W. Winder and D.G. Hardie. 1999. AMP-activated protein kinase, a metabolic master switch:possible roles in Type 2 diabetes. Am J Physiol. Jul;277:E1-10.
23.J. Han, N. Yang, F. Zhang, C. Zhang, F.Y. Liang, W.F. Xie, W.S. Chen. 2015. Rhizoma Anemarrhenae extract ameliorates hyperglycemia and insulin resistance via activation of AMP-activated protein kinase in diabetic rodents. Ethnopharmacology. 172:368-376.
24.K.H. Lee, U.I. Ju, J.Y. Song and Y.S. Chun. 2014. The Histone Demethylase PHF2 Promotes Fat Cell Differentiaiton as an Epigenetic Activator of Both C/EBP and C/EBP. Molecules and Cells. 37(10):734-741.
25.S.R. Yoo, C.S. Seo, H.K. Shin and S.J. Jeong. 2015. Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes. Evidence-Based Complementary and Alternative Medicine. Article ID 949461, 10 pages.
26.J. Han, J. Yi, F.Y. Liang, B. Jiang, Y. Xiao, S.H. Gao, N. Yang, H. Hu, W.F. Xie and W.S. Chen. 2015. X-3, a mangiferin derivative, stimulates AMP-activated protein kinase and reduces hyperglycemia and obesity in db/db mice. Molecular and Cellular Endocrinology. 405:63-73.
27.G.R. Burton, R. Nagarajan, C.A. Peterson and R.E. Mcgehee Jr. 2004. Microarray analysis of differentiation-specific gene expression during 3T3-L1 adipogenesis. Gene. 329:167-185
28.Maris M. Mihaylova and Reuben J. Shaw. 2012. The AMP-activated protein kinase (AMPK) signaling pathway coordinates cell growth, autophagy, &metabolism. Nat Cell Biol. 13(9):1016-1023.
29.Evan D. Rosen and Bruce M. Spiegelman. 2006. Adipocytes as regulators of energy balance and glucose homeostasis. Nature. 444(7121):847-853.
30.K.K. Li, C.L. Liu, H.T. Shiu, H.L. Wong, W.S. Siu, C. Zhang, X.Q. Han, C.X. Ye, P.C. Leung and C.H. Ko. 2016. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes. SCIENTIFIC REPORTS. 6:20172.
31.Jonathan E. Campbell, Ashley J. Peckett, Anna M. D’souza, Thomas J. Hawke, and Michael C. Riddell. 2011. Adipogenic and lipolytic effects of chronic glucocorticoid exposure. Cell Physiol. 300:C198-C209.

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