臺灣博碩士論文加值系統

研究顯示中性脂質的油滴在調節細胞內的脂質代謝扮演重要的角色。靈芝是中國傳統上品中草藥，具有多種藥理功能，亦有影響脂肪細胞分化和生成的可能性。然而，靈芝對脂質代謝和細胞內的油滴形成、融合和分解的相關機制尚未完全了解。本研究使用松杉靈芝做為實驗材料。初步結果顯示松杉靈芝乙醇萃取物(GTEE)會影響在分化中及成熟後的3T3-L1細胞中油滴的形成；因此本研究目的即在探討松杉靈芝對分化中和成熟後的3T3-L1前驅脂肪細胞油滴的形成和脂質代謝及其相關的分子機制。結果顯示，GTEE會使脂肪細胞內的油滴變小，同時也發現與分化中相關蛋白如DGAT1和FSP27表現會減少，而SREBP-1c, FAS, aP2表現會增加，在脂質分解的蛋白中如p-HSL和ATGL會增加，導致脂質分解的甘油釋放量也增加。但GTEE對成熟後的3T3-L1脂肪細胞中脂質分解相關的蛋白表現沒有明顯的影響，儘管如此，GTEE在成熟後的3T3-L1脂肪細胞中短時間內的甘油釋放量也會增加。綜合以上結果GTEE 在調節細胞內的脂質紊亂中扮演重要的角色。

Lipid droplets (LDs) play a critical role in regulating cellular lipid metabolism, especially in adipocytes. It is known that Ganoderma (Lingzhi; Reishi), which is the top grade of Traditional Chinese Medicine, has a variety of pharmacological functions, including effect on adipogenesis. However, the relevant mechanisms mediated by Ganoderma on cellular lipid metabolism and intracellular LDs formation, fusion, and mobilization are yet to be investigated. Our previous results showed that the ethanolic extract of Ganoderma tsugae (GTEE) might affect the LDs formation size. Therefore, we aimed to investigate the potential effects of GTEE on intracellular LDs formation and related lipid metabolism in differentiating and differentiated 3T3-L1 cells. GTEE was found to contribute to adipocyte differentiation and the reduction of LDs size in differentiating and differentiated 3T3-L1 cells. During adipogenesis, GTEE up-regulated adipogenic-related protein expression (SREBP-1c, FAS, Perilipin, and aP2) and down-regulated lipogenic- and fusion-related protein expression (DGAT1 and Fsp27). Interestingly, GTEE also up-regulated lipolytic-related protein expression (ATGL, p-HSL). In addition, GTEE increased glycerol release during differentiation of 3T3-L1 cells. However, GTEE down-regulated lipogenic- and fusion-related protein expression clearly, but didn’t affect lipolytic-related protein expression obviously in differentiated 3T3-L1 adipocytes. Nevertheless, GTEE also led to the increase of glycerol release temporarily in differentiated 3T3-L1 adipocytes. Taken together, these findings reveal that GTEE may play an important role in regulating intracellular lipid disturbances of adipocytes.

致謝 I
圖目錄 III
縮寫目錄 IV
中文摘要 V
Abrstract VI
第一章 研究緣起與目的 1
第二章 文獻回顧 3
第一節、靈芝(Ganoderma) 3
第二節、脂肪細胞(adipocyte) 4
第三節、脂肪油滴形成與調控 5
第三章 材料與方法 9
第一節、實驗藥材及儀器 9
第二節、實驗方法 14
第四章 實驗結果 18
第一節、松杉靈芝乙醇萃取物對分化中3T3-L1脂肪細胞的影響 18
第二節 松杉靈芝乙醇萃取物對成熟後3T3-L1脂肪細胞的影響 20
第五章 討論 23
第六章 結論 28
第七章 參考文獻 29
第八章 圖表 35
第九章 附錄 47

Anthonsen, M.W., Ronnstrand, L., Wernstedt, C., Degerman, E., and Holm, C. (1998). Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. J Biol Chem 273, 215-221.

Anthony, N.M., Gaidhu, M.P., and Ceddia, R.B. (2009). Regulation of visceral and subcutaneous adipocyte lipolysis by acute AICAR-induced AMPK activation. Obesity 17, 1312-1317.

Batra, P., Sharma, A.K., and Khajuria, R. (2013). Probing Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes): a bitter mushroom with amazing health benefits. International journal of medicinal mushrooms 15, 127-143.

Beller, M., Thiel, K., Thul, P.J., and Jackle, H. (2010). Lipid droplets: a dynamic organelle moves into focus. FEBS letters 584, 2176-2182.

Boh, B. (2013). Ganoderma lucidum: a potential for biotechnological production of anti-cancer and immunomodulatory drugs. Recent patents on anti-cancer drug discovery 8, 255-287.

Bunnell, B.A., Flaat, M., Gagliardi, C., Patel, B., and Ripoll, C. (2008). Adipose-derived stem cells: Isolation, expansion and differentiation. Methods 45, 115-120.

Cameron, A.J., Shaw, J.E., and Zimmet, P.Z. (2004). The metabolic syndrome: prevalence in worldwide populations. Endocrinology and metabolism clinics of North America 33, 351-375, table of contents.

Choi, S.M., Tucker, D.F., Gross, D.N., Easton, R.M., DiPilato, L.M., Dean, A.S., Monks, B.R., and Birnbaum, M.J. (2010). Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway. Molecular and cellular biology 30, 5009-5020.

Chu, T.T., Benzie, I.F., Lam, C.W., Fok, B.S., Lee, K.K., and Tomlinson, B. (2012). Study of potential cardioprotective effects of Ganoderma lucidum (Lingzhi): results of a controlled human intervention trial. The British journal of nutrition 107, 1017-1027.

Farmer, S.R. (2006). Transcriptional control of adipocyte formation. Cell metabolism 4, 263-273.

Gong, J., Sun, Z., Wu, L., Xu, W., Schieber, N., Xu, D., Shui, G., Yang, H., Parton, R.G., and Li, P. (2011). Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites. The Journal of cell biology 195, 953-963.

Green, H., and Kehinde, O. (1976). Spontaneous Heritable Changes Leading to Increased Adipose Conversion in 3t3-Cells. Cell 7, 105-113.

Gregoire, F.M., Smas, C.M., and Sul, H.S. (1998). Understanding adipocyte differentiation. Physiological reviews 78, 783-809.

Harris, C.A., Haas, J.T., Streeper, R.S., Stone, S.J., Kumari, M., Yang, K., Han, X., Brownell, N., Gross, R.W., Zechner, R., et al. (2011). DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes. Journal of lipid research 52, 657-667.

Hsu, S.C., Ou, C.C., Chuang, T.C., Li, J.W., Lee, Y.J., Wang, V., Liu, J.Y., Chen, C.S., Lin, S.C., and Kao, M.C. (2009). Ganoderma tsugae extract inhibits expression of epidermal growth factor receptor and angiogenesis in human epidermoid carcinoma cells: In vitro and in vivo. Cancer letters 281, 108-116.

Hsu, S.C., Ou, C.C., Li, J.W., Chuang, T.C., Kuo, H.P., Liu, J.Y., Chen, C.S., Lin, S.C., Su, C.H., and Kao, M.C. (2008). Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G(2)/M cell cycle arrest. Journal of ethnopharmacology 120, 394-401.

Jambunathan, S., Yin, J., Khan, W., Tamori, Y., and Puri, V. (2011). FSP27 promotes lipid droplet clustering and then fusion to regulate triglyceride accumulation. PloS one 6, e28614.

Jaworski, K., Sarkadi-Nagy, E., Duncan, R.E., Ahmadian, M., and Sul, H.S. (2007). Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue. American journal of physiology Gastrointestinal and liver physiology 293, G1-4.

Ji, Z., Tang, Q., Zhang, J., Yang, Y., Jia, W., and Pan, Y. (2007). Immunomodulation of RAW264.7 macrophages by GLIS, a proteopolysaccharide from Ganoderma lucidum. Journal of ethnopharmacology 112, 445-450.

Jiang, Y.J., and Feingold, K.R. (2011). The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis. Dermato-endocrinology 3, 70-76.

Jin, X., Ruiz Beguerie, J., Sze, D.M., and Chan, G.C. (2012). Ganoderma lucidum (Reishi mushroom) for cancer treatment. The Cochrane database of systematic reviews 6, CD007731.

Kan, Y., Chen, T., Wu, Y., Wu, J., and Wu, J. (2014). Antioxidant activity of polysaccharide extracted from Ganoderma lucidum using response surface methodology. International journal of biological macromolecules 72C, 151-157.

Ko, H.H., Hung, C.F., Wang, J.P., and Lin, C.N. (2008). Antiinflammatory triterpenoids and steroids from Ganoderma lucidum and G. tsugae. Phytochemistry 69, 234-239.

Krahmer, N., Guo, Y., Farese, R.V., Jr., and Walther, T.C. (2009). SnapShot: Lipid Droplets. Cell 139, 1024-1024 e1021.

Kuo, H.P., Hsu, S.C., Ou, C.C., Li, J.W., Tseng, H.H., Chuang, T.C., Liu, J.Y., Chen, S.J., Su, M.H., Cheng, Y.C., et al. (2013). Ganoderma tsugae Extract Inhibits Growth of HER2-Overexpressing Cancer Cells via Modulation of HER2/PI3K/Akt Signaling Pathway. Evidence-based complementary and alternative medicine : eCAM 2013, 219472.

Langin, D. (2006). Control of fatty acid and glycerol release in adipose tissue lipolysis. Comptes rendus biologies 329, 598-607; discussion 653-595.

Lee, I., Kim, H., Youn, U., Kim, J., Min, B., Jung, H., Na, M., Hattori, M., and Bae, K. (2010a). Effect of lanostane triterpenes from the fruiting bodies of Ganoderma lucidum on adipocyte differentiation in 3T3-L1 cells. Planta medica 76, 1558-1563.

Lee, I., Seo, J., Kim, J., Kim, H., Youn, U., Lee, J., Jung, H., Na, M., Hattori, M., Min, B., et al. (2010b). Lanostane triterpenes from the fruiting bodies of Ganoderma lucidum and their inhibitory effects on adipocyte differentiation in 3T3-L1 Cells. Journal of natural products 73, 172-176.

Letexier, D., Pinteur, C., Large, V., Frering, V., and Beylot, M. (2003). Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue. Journal of lipid research 44, 2127-2134.

Lin, J.Y., Chen, M.L., Chiang, B.L., and Lin, B.F. (2006). Ganoderma tsugae supplementation alleviates bronchoalveolar inflammation in an airway sensitization and challenge mouse model. International immunopharmacology 6, 241-251.

Lv, G.P., Zhao, J., Duan, J.A., Tang, Y.P., and Li, S.P. (2012). Comparison of sterols and fatty acids in two species of Ganoderma. Chemistry Central journal 6, 10.

MacDougald, O.A., and Lane, M.D. (1995). Transcriptional regulation of gene expression during adipocyte differentiation. Annual review of biochemistry 64, 345-373.

Meng, L.Z., Xie, J., Lv, G.P., Hu, D.J., Zhao, J., Duan, J.A., and Li, S.P. (2014). A comparative study on immunomodulatory activity of polysaccharides from two official species of ganoderma (lingzhi). Nutrition and cancer 66, 1124-1131.

Moro, C., Bajpeyi, S., and Smith, S.R. (2008). Determinants of intramyocellular triglyceride turnover: implications for insulin sensitivity. American journal of physiology Endocrinology and metabolism 294, E203-213.

Ntambi, J.M., and Young-Cheul, K. (2000). Adipocyte differentiation and gene expression. The Journal of nutrition 130, 3122S-3126S.

Oluba, O.M., Adebisi, K.E., Eidangbe, G.O., Odutuga, A.A., and Onyeneke, E.C. (2014). Modulatory Effect of Crude Aqueous Extract of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes), on Hematological and Antioxidant Indices in Plasmodium berghei-infected Mice. International journal of medicinal mushrooms 16, 499-506.

Schweiger, M., Schreiber, R., Haemmerle, G., Lass, A., Fledelius, C., Jacobsen, P., Tornqvist, H., Zechner, R., and Zimmermann, R. (2006). Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism. J Biol Chem 281, 40236-40241.

Shimojo, Y., Kosaka, K., and Shirasawa, T. (2011). Effect of Ganoderma lucidum extract on adipocyte differentiation and adiponectin gene expression in the murine pre-adipocyte cell line, 3T3-L1. Phytotherapy research : PTR 25, 202-207.

Sliva, D. (2004). Cellular and physiological effects of Ganoderma lucidum (Reishi). Mini reviews in medicinal chemistry 4, 873-879.

Smith, P.J., Wise, L.S., Berkowitz, R., Wan, C., and Rubin, C.S. (1988). Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes. J Biol Chem 263, 9402-9408.

Thyagarajan-Sahu, A., Lane, B., and Sliva, D. (2011). ReishiMax, mushroom based dietary supplement, inhibits adipocyte differentiation, stimulates glucose uptake and activates AMPK. BMC complementary and alternative medicine 11, 74.

Wang, C.D., Teng, B.S., He, Y.M., Wu, J.S., Pan, D., Pan, L.F., Zhang, D., Fan, Z.H., Yang, H.J., and Zhou, P. (2012). Effect of a novel proteoglycan PTP1B inhibitor from Ganoderma lucidum on the amelioration of hyperglycaemia and dyslipidaemia in db/db mice. The British journal of nutrition 108, 2014-2025.

Yang, H., Galea, A., Sytnyk, V., and Crossley, M. (2012). Controlling the size of lipid droplets: lipid and protein factors. Current opinion in cell biology 24, 509-516.

Yu, Y.H., Kuo, H.P., Hsieh, H.H., Li, J.W., Hsu, W.H., Chen, S.J., Su, M.H., Liu, S.H., Cheng, Y.C., Chen, C.Y., et al. (2012). Ganoderma tsugae Induces S Phase Arrest and Apoptosis in Doxorubicin-Resistant Lung Adenocarcinoma H23/0.3 Cells via Modulation of the PI3K/Akt Signaling Pathway. Evidence-based complementary and alternative medicine : eCAM 2012, 371286.

Zhang, X., Heckmann, B.L., Xie, X., Saarinen, A.M., and Liu, J. (2014). Regulation of FSP27 protein stability by AMPK and HSC70. American journal of physiology Endocrinology and metabolism, ajpendo 00125 02014.

Zhu, K., Nie, S., Li, C., Lin, S., Xing, M., Li, W., Gong, D., and Xie, M. (2013). A newly identified polysaccharide from Ganoderma atrum attenuates hyperglycemia and hyperlipidemia. International journal of biological macromolecules 57, 142-150.