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第六章、結論
本篇研究的主要目的在探討 HBP1 在脂肪細胞分化過程中的角色，以及 HBP1 參與 quercetin 調控脂質生合成的機制，得到的重要結果如下：
一、HBP1 在 3T3-L1 脂質生合成過程中具階段性的功能 。 (一) MCE 期：
HBP1 的表現在分化初期 2 天的 MCE 期無明顯增加，HBP1 在此期增 加將抑制細胞增生，而造成 MCE 不完全。
(二) Terminal Differentiation 期：
相對地，HBP1 在分化後期的表現增加是最終分化所必須的，尤其，HBP1 可能是PPARγ 調控脂質生合成的下游基因。結論顯示，HBP1 在脂質生合 成過程的 MCE 期和 terminal differentiation 期有不同的生理功能。
二、quercetin 具活化 HBP1，抑制 MCE 期和脂肪細胞分化的作用。
quercetin 活化 MCE 期 HBP1 mRNA 表現，造成 MCE 期細胞週期生長 停滯於 G1，伴隨著轉錄因子 CEBP/ β 和 C/EBPδ mRNA 的表現下降，抑制 脂肪細胞分化。
最終，希望我們的研究探討可以提供HBP1轉錄因子在未來肥胖的研究上作 為一個重要指標或肥胖相關疾病重要標定的可能性。
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參考資料
1. http://www.doh.gov.tw/statistic/index.htm 參考衛生署統計重要指標
2. Rosell M, Hakansson NN, Wolk A. Association between dairy food consumption and weight change over 9 y in 19,352 perimenopausal women. Am J Clin Nutr.
2006 ;84(6):1481-8.
3. Moldes M, Zuo Y, Morrison RF, Silva D, Park BH, Liu J, Farmer, SR, Peroxisome-proliferator-activated receptor c suppresses Wnt/b-catenin signalling during adipogenesis, Biochem. J. 2003;607–613.
4. Park HJ, Yang JY, Ambati S, Della-Fera MA, Hausman DB, Rayalam S, Baile CA.Combined effects of genistein, quercetin, and resveratrol in human and 3T3-L1adipocytes.J Med Food. 2008 ;11(4):773-83.
5. Entenmann G, and Hauner H. Relationship between replication and differentiation in culture human adipocyte precursor cels. Am. J. Physiol. Cell physiol. 1996;
270:c110-c1016.
6. Tang QQ, Otto TC, and Lane MD. CCAAT/enhancer-binding protein β is require for mitotic clonal expansion during adipogenesis. Pnas. 2003; 100: 850-855.
7. Cryer A. Tissue lipoprotein lipase activity and its action in lipoprotein metabolism.
Int J Biochem. 1981;13(5):525-41.
87
8. Brun RP, Kim JB, Hu E, Altiok S, Spiegelman BM. Adipocyte differentiation: A transcriptional regulatory cascade. Curr Opin Cell Biol. 1996 ;8(6):826-32.
9. Zhidan W, Pere P , and Bruce MS. Transcriptional activation of adipogenesis.
Curr. Opin. Cell boil. 1999;11:689-694.
10. Jame MN, and Kim YC. Symposium : Adipocyte function, differentiation and metabolism. J. Nutr. 2000;132:904-910.
11. Green H, and Kehinde O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell. 1976;7(1): 105-113.
12. Green H, and Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell. 1974; 3(2): 127-133.
13. Green H, and Kehinde O. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975;
5(1): 19-27.
14. Green H, and Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J. Cell Physio. 1979;1101:169-171.
15. Wu Z, Bucher NL, Farmer SR. Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids. Mol Cell Biol.
1996 ;16(8):4128-36.
88
16. Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991;5(9):1538-52.
17. Joyner JM, Hutley LJ, Cameron DP. Glucocorticoid receptors in human preadipocytes: Regional and gender differences. J Endocrinol. 2000;166:145-52.
18. Gregoire FM, Smas CM, Sul Hs. Understanding adipocyte differentiation. Physiol Rev. 1998 ;78(3):783-809.
19. Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A, Klip A. An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes. J Biol Chem. 1999 ;274(15):10071-8.
20. Rubin CS, Hirsch A, Fung C, Rosen OM. Development of hormone receptors and hormonal responsiveness in vitro. insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem.1978;253 (20):7570-8.
21. Fajas L, Fruchart JC, and Auwerx J. Transcriptional control of adipogenesis. Curr.
Opin. Cell biology. 1998;10:165-173.
22. Yeh WC, Cao Z, Classon M, and McKnight SL. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev.1995;9 (2): 168-181.
89
23. Wu Z, Xie Y, Morrison RF, Bucher NL, and Farmer SR. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes. J Clin Invest. 1998;
101(1): 22-32.
24. Gregoire FM, Smas CM, Sul Hs. Understanding adipocyte differentiation. Physiol Rev. 1998 ;78(3):783-809.
25. Bruno F. Adipogenesis: cellular and molecular aspects . 2005;19(4):483-499.
26. Darlington GJ, Ross SE, MacDougald OA. The role of C/EBP genes in adipocyte differentiation. J Biol Chem. 1998;273:30057-60.
27. Feve B. Adipogenesis: Cellular and molecular aspects. Best Practice & Research Clinical Endocrinology & Metabolism. 2005;19:483-99.
28. Lee H, Lee YJ, Choi H, Ko EH, and Kim JW. REACTIVE OXYGEN SPECIES FACILITATE ADIPOCYTE DIFFERENTIATION BY ACCELERATING MITOTIC CLONAL EXPANSION. J Biol Chem. 2009;284(16):10601-10609.
29. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990;347:645-50.
30. Werman A, Hollenberg A, Solanes G, Bjorbaek C, Vidal-Puig AJ, Flier JS.
Ligand-independent activation domain in the N terminus of peroxisome proliferator-activated receptor gamma (PPARgamma). differential activity of
90
PPARgamma1 and -2 isoforms and influence of insulin. J Biol Chem.
1997;272:20230-5.
31. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signaling pathways through heterodimer formation of their receptors. Nature. 1992;358:771-4.
32. Schoonjans K, Staels B, and Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res. 1996; 37;907-925.
33. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med.
2002;53:409-35.
34. Gearing KL, Gottlicher M, Teboul M, Widmark E, Gustafsson JA. Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci U S A. 1993;90:1440-4.
35. Guo L, Tabrizchi R. Peroxisome proliferator-activated receptor gamma as a drug target in the pathogenesis of insulin resistance. Pharmacol Ther. 2006;111:145-73.
36. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA. Peroxisome
proliferator-activated receptor (PPAR) gamma: Adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology.
1994;135(2):798-800 .
91
37. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, and Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 1995;270(22): 12953-12956.
38. Staels B, and Fruchart JC. 2005. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes; 54(8): 2460-2470.
39. Kota BP, Huang TH, Roufogalis BD. An overview on biological mechanisms of PPARs. Pharmacological Research. 2005;51:85-94.A
40. Kletzien RF, Clarke SD, and Ulrich RG. Enhancement of adipocyte differentiation by an insulin-sensitizing agent. Mol Pharmacol. 1992;41(2): 393-398.
41. Sandouk T, Reda D, and Hofmann C. Antidiabetic agent pioglitazone enhances adipocyte differentiation of 3T3-F442A cells. Am J Physiol .1993;264(6 Pt 1):
C1600-1608.
42. Tontonoz P, Hu E, and Spiegelman BM. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma. Curr Opin Genet Dev. 1995;5(5): 571-576.
43. Matsusue K, Haluzik M, Lambert G, Yim SH, Gavrilova O, Ward JM, Brewer B Jr, Reitman ML, and Gonzalez FJ Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes. J Clin Invest. 2003; 111(5): 737-747.
92
44. Norris AW, Chen L, Fisher SJ, Szanto I, Ristow M, Jozsi AC, Hirshman MF, Rosen ED, Goodyear LJ, Gonzalez FJ, Spiegelman BM, and Kahn CR.
Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest. 2003;112(4):
608-618.
45. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature.
1994;372:425-32.
46. Lonnqvist F, Arner P, Nordfors L, Schalling M. Overexpression of the obese (ob) gene in adipose tissue of human obese subjects. Nat Med. 1995;1:950-3.
47. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995;269:546-9.
48. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals.
Nature. 1998; 395: 763-70.
49. Monzillo LU, Hamdy O, Horton ES, Ledbury S, Mullooly C, and Jarema C, et al.
Effect of Lifestyle Modification on Adipokine Levels in Obese Subjects with Insulin Resistance, Obesity Reasech. 2003;11(9),1048-1054.
50. MacDougald OA, Hwang CS, Fan H, and Lane MD. Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes. Proc.
93
Natl. Acad. Sci. 1995;92: 9034-9037.
51. Fruhbeck G, Salvador J. Role of adipocytokines in metabolism and disease. Nutr Res. 2004;24:803-26.
52. Yee AS, Paulson EK, McDevitt MA, Rieger-Christ K, Summerhayes I, Berasi SP, Kim J, Huang CY, Zhang X. The HBP1 transcriptional repressor and p38 MAP kinase: Unlikely partners in G1 regulation and tumor suppression. Gene. 2004;
336(1):1-13.
53. Classon M, Salama S, Gorka C, Mulloy R, Braun P, Harlow E. Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control. Proc Natl Acad Sci U S A. 2000 ;97(20): 10820-5.
54. Sekkali B, Szabat E, Ktistaki E, Tolaini M, Roderick K, Harker N, Patel A, Williams K, Norton T, Kioussis D. Human high mobility group box transcription factor 1 affects thymocyte development and transgene variegation. J Immunol, 2005;175(8):5203-12.
55. Shisheva A, Shechter Y. Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes.
Biochemistry. 1992 ;31(34):8059-63.
56. Higgins C, Chatterjee S, and Cherington V. The block of adipocyte differentiation by a C-terminally truncated, but not by full-length, simian virus 40 large tumor antigen is dependent on an intact retinoblastoma susceptibility protein family
94
binding domain. J Virol . 1996;70(2): 745-752.
57. Altiok S, Xu M, and Spiegelman BM. PPARgamma induces cell cycle
withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes 1997 ;11(15): 1987-1998.
58. Morrison R F, and Farmer SR. Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21
(Waf1/Cip1), during adipogenesis. J Biol Chem. 1999b; 274(24):17088-17097.
59. Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, Yee AS. Suppression of wnt signaling by the green tea compound
(-)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells. requirement of the transcriptional repressor HBP1. J Biol Chem. 2006;281(16):10865-75.
60. Logan C.Y.,Nusse R. The Wnt singaling pathway in development and disease.Annu.Rev.Cell Biol.2004;20: 781-810.
61. M. Moldes, Y. Zuo, R.F. Morrison, D. Silva, B.H. Park, J. Liu, S.R.Farmer, Peroxisome-proliferator-activated receptor c suppresses Wnt/b-catenin signalling during adipogenesis, Biochem. J. 2003;607–613.
62. Florian L, Hugnot JP, Amri EZ, Grimaldi P, Barhanin J, Lazdunski M. Expression cloning in K+ transport defective yeast and distribution of HBP1, a new putative HMG transcriptional regulator. Nucleic Acids Research. 1994 ; 22(18):3685-88.
63. Hertog MGL, and Hollman PCH. Potential health effects of the dietary flavonol
95
quercetin. Eur. J. Clin. Nutr. 1996, 50: 63-71.
64. Frankel EN, German JB, Parks E, and Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. The Lancet.
1993; 341: 454-457.
65. Rosen ED,Walkey CJ, Puigserver P,Spiegeiman BM. Transcriptional regulation of adipogenesis. Genes Dev.2000;14:1293-1307.
66. Underhill, Can. J. Biochem. Physiol. 1957, 35:219.
67. Shisheva A, Shechter Y. Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes.
Biochemistry. 1992 ;31(34):8059-63.
68. Hsu CL, Yen GC. Induction of cell apoptosis in 3T3-L1 pre-adipocytes by flavonoids is associated with their antioxidant activity. Mol Nutr Food Res. 2006 ;50(11):1072-9.69.
69. Jiyun A, Lee A, Kim S, Park J, Ha T. The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochemical and Biophysical Research Communications. 2008;373:545-549.
70. Lee Y.T., Don M.J., Hung P.S., Shen Y.C., Lo Y.S., Chang K.W., Chen C.F., Ho L.K. Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells. Cancer letters. 2005;23(1): 19-25.
96
71. Tang Q.Q., Otto T. C., and Lane M.D. CCAAT_enhancer-binding protein is quired for mitotic clonal expansion during adipogenesis. PNAS .
2003 ;100(3)850-855.
72. Chang G.N., Chen E.S., Ho K.C. Molecular Cloning and Expression of a Phase I Flagellin Gene from Salmonella enterica serovar Choleraesuis. Taiwania.
2007;52(3): 201-209.
73. Hollenberg A.N., etal. Functional antagonism between CCAAT/enhancer binding protein-α and Peroxisome Proliferator-activated Receptor-γ on the Leptin Promoter. J Biol Chem. 1997;272(8):5283-5290.
74. Gotoh C., etal. The regulation of adipogenesis through GPR120. Biochemical and Biophysical Research Communications. 2007;354:591-597.
75. He L, Hannon GJ. MicroRNAs: Small RNAs with a big role in gene regulation.
Nature Reviews Genetics. 2004 ;5(7):522-31.
76. Tzameli I., Fang H., Ollero M., Shi H., Hamm JK., Kievit P. Hollenberg A.N., and Flier J.S. Regulated Production of a Peroxisome Proliferator-activated Receptor Ligand during an Early Phase of Adipocyte Differentiation in 3T3-L1 Adipocytes.
J Biol Chem. 2004;279(34):36093-36102.
77. Wittwer C.T.,Herrmann M.G.,Moss A .A.,and Rasmussen R.P., Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques .
97
1997;22(1):130-1,134-8.
78. Constantinos C., etal . The Wnt antagonist Dickkopf-1 and its receptors are
coordinately regulated during early human adipogenesis. Journal of Cell Science.
2006; 119: 2613-2620.
79. Pan C., Yan M., Yao J., Xu J., Long Z., Huang H., Liu Q. Aurora kinase small molecule inhibitor destroys mitotic spindle, suppresses cell growth, and induces apoptosis in oral squamous cancer cells. Oral oncology. 2008;44(7): 639-645.
80. Chang M.C., Chan C.P., Wang Y.J., Lee P.H., Chen L.I., Tsai Y.L., Lin B.R., Wang Y.L., Jeng J.H. Induction of necrosis and apoptosis to KB cancer cells by
sanguinarine is associated with reactive oxygen species production and
mitochondrial membrane depolarization. Toxicology and applied pharmacology.
218(2): 143-151. 2007.
81. Zhidan W, Pere P, and Bruce MS. Transcriptional activation of adipogenesis.
Curr Opin cell boil. 1999;11:689-694.
98