臺灣博碩士論文加值系統

本實驗室過去研究在肝癌細胞株Hep3B/T2中發現胰島素確實能夠抑制 B型肝炎病毒顆粒的產生。有趣的是，調控核心啟動子的活性和參與糖質新生基因的表現的轉錄因子有相似之處。此外已知胰島素與Metformin治療第II類型糖尿病是透過抑制肝臟進行糖質新生作用(gluconeogenesis)，有鑑於此，提出了一個假說為Metformin是否能成為一種新穎的藥物，藉由調控宿主的糖質新生作用而影響了 B 型肝炎病毒基因的表現。首先，利用Luciferase reporter assay，發現 cAMP 及葡萄糖皮質素dexamethasone 促進核心啟動子活性及PEPCK 和 G6Pase mRNA具有協同的效果 (synergistic effect) ，再利用B 型肝炎病毒核心啟動子不同長度序列剔除 (serial deletion) 的核心啟動子以及不同區域的點突變 (point mutation)，鑑定出 nt1656-1675 為核心啟動子上Metformin的反應序列 (response element)。在過去有研究報告指出，利用帶有 B 型肝炎病毒基因的轉殖鼠進行短時間禁食時，發現體內 B 型肝炎病毒及PGC-1α表現量上升，PGC-1α在維持能量的穩定上扮演著重要的角色，它可以和 HNF-4α結合去活化糖質新生 (gluconeogenesis)的相關基因。
接著進一步發現在Hep3B/T2細胞，Metformin會透過活化AMPK路徑影響PGC-1α蛋白質的表現，進而影響了B 型肝炎病毒核心啟動子與G6Pase啟動子的活性，為了更加了解Metformin活化AMPK路徑和B 型肝炎病毒基因的關聯，透過特定的抑制劑以及活化劑處理細胞做進一步探討，發現處理A769662 (AMPK活化劑)會抑制cAMP/dexamethasone 促進核心啟動子及G6Pase啟動子的活性以及抑制PGC-1α蛋白質的表現；另外處理AMPK抑制劑Compound C，當AMPK受到抑制時，Metformin 就無法抑制8-bromo-cAMP/dexamethasone 所活化G6Pase mRNA的表現，同樣地，Metformin 就無法抑制核心啟動子的活性，另外發現大量表現PGC-1α蛋白質同樣也可以回復Metformin 抑制核心啟動子的活性，有趣的是核心啟動子上nt1656-1675 的構築質體p19nt*3會受到cAMP/dexamethasone以及大量表現PGC-1α蛋白質的活化。在本論文的研究中，發現治療第II類型糖尿病Metformin具有抗HBV的效果，進一步發現Metformin抑制核心啟動子及糖質新生基因的表現是透過活化AMPK路徑，而轉錄輔因子PGC-1α也參與在其中，希望本論文的研究對於慢性肝炎以及糖尿病患者的治療能有所貢獻。

Our laboratory has found that insulin inhibited the hepatitis B virus particles generated in the hepatoma cell line Hep3B/T2. Interestingly, the regulation of core promoter activity and transcription factors of gluconeogenesis gene expression is similar. In addition, insulin and Metformin treatment of type II diabetes through inhibition of liver gluconeogenesis. In light of this, it is therefore hypothesize that anti-diabetic drug, metformin, as novel class of anti-HBV therapeutics, by regulation of host gluconeogenesis gene expression. First, it was shown that combination of cAMP and glucocorticoids had a synergistic effect on the induction of the HBV core promoter activity and PEPCK and G6Pase mRNA. Then, it was identified nt1656-1675 which as a response element of Metformin in the HBV core promoter using serial deletion as well as point mutation at different regions of the core promoter.
Previous studies have shown that peroxisome proliferators-activated receptor-γ coactivator 1 (PGC-1) is strongly induced to coactivate HBV gene expression in liver of fasting mice. PGC-1 is a major metabolic regulator of key gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) , it is associated with HNF-4α binding to activated gluconeogenesis.
In Hep3B/T2 cells, Metformin suppress hepatitis B virus core promoter and G6Pase promoter activity through inhibits PGC-1α protein expression by activated AMPK, and the inhibition was resembled with specific activator A769662 (AMPK activator) ; another treated the AMPK inhibitor compound C, when AMPK was inhibited, Metformin was not be able to inhibit the cAMP/dexamethasone activated the G6Pase mRNA. Similarly, Metformin was not be able to inhibit the activity of the core promoter when treated with compound C. On the other hand, overexpression of PGC-1α protein can reverse Metformin inhibition of the core promoter activity, it is interesting that nt1656-1675 on the core promoter construct plasmid p19nt 3 was stimulated by cAMP/dexamethasone, and overexpression of PGC-1α protein activation.
This study has revealed that treatment of cells the first type II diabetes Metformin has anti-HBV effect, and further found that Metformin inhibition of the core promoter and gluconeogenic gene expression via activation of AMPK pathway, and the transcription cofactor (co-activator) of PGC -1α is also involved in this pathway, Then it is hope of this research can contribute to the treatment of chronic hepatitis B and patients with diabetes.

中文摘要…………………………………………………….…...……………4
Abstract…………………………………………………….….………………5
壹. 緒論(Introduction) …………………………………….………………….7
1-1 B 型肝炎病毒簡介……………………………….…………..……7
1-2 B 型肝炎病毒結構……………………………….…………..……8
1-3 B 型肝炎病毒基因之表現與調控…………...……………………8
1-4 慢性B 型肝炎病毒治療之藥物及其優缺點……………………10
1-5 糖質新生作用 (gluconeogenesis) …..……………………………11
1-6 Metformin…………………………………………………………12
1-7 研究動機…………………………….………………….…………13
貳. 材料與方法 (Materials and Methods)…….…..…………..…………….14
2-1 使用儀器及設備……………………………………………………14
2-2 材料 (Materials) ……………………………………………………15
2-3細胞株 (cell line) …………………………………...………………17
2-4 細胞培養 (cell culture) ……………………..……...………………17
2-5 細胞生長分析 (Analysis of cell growth) …………..………………17
2-6 B 型肝炎病毒表面抗原量分析 (HBsAg test) ……..……………17
2-7 質體轉染 (transfection) ……………………………………………18
2-8 啟動子活性分析 (promoter activity assay) ……………….………18
2-9 β-galactosidase activity assay……………………………..………18
2-10 製備全細胞萃取物 (whole cell lysate extraction)…….…………18
2-11 蛋白質濃度測量………………………………………..…………19
2-12 西方墨點法 (Western blotting) ………….………………….……19
2-13 核醣核酸萃取 (RNA extraction) ……….………………..………19
2-14 反轉錄聚合酶鏈鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) ………………………………………………………20
2-15 即時定量聚合酶鏈鎖反應 (quantitative PCR, Q-PCR) .…..……20
2-16 病毒DNA純化 (Virus DNA Purification) ………………....……21
2-17 統計分析…………………………………………………….….…21
參. 結果 (Results) ………………………………………………..………22
3-1. Synergistic effects of 8-bromo-cAMP, and dexamethasone on HBV core promoter in Hep3B/T2 cells………………………………………22
3-2. Synergistic effects of cAMP, and dexamethasone on gluconeogenesis in Hep3B/T2 cells…………………………………………………………23
3-3. Metformin and insulin suppress gluconeogenesis and HBV core promoter activity in Hep3B/T2 cells……………………………………24
3-4. Metformin suppress PGC-1α expression through activate AMPK in Hep3B/T2 cells…………………………………………………………26
3-5. AMPK inhibitor Compound C reverses metformin suppressed core promoter activity in Hep3B/T2 cells……………………………………28
3-6. Metformin suppresses HBV replication and expression. ….………29
肆. 討論 (Discussion) ……………………………………………...………30
4-1. Synergistic effects of 8-bromo-cAMP, and dexamethasone on HBV core promoter in Hep3B/T2 cells………………………………….……30
4-2. Synergistic effects of cAMP, and dexamethasone on gluconeogenesis in Hep3B/T2 cells…………………………………………….……………31
4-3. Metformin and insulin suppress gluconeogenesis and HBV core promoter activity in Hep3B/T2 cells…………………………………….32
4-4. Metformin suppress PGC-1α expression through activate AMPK in Hep3B/T2 cells……………………………………………………….….33
4-5. AMPK inhibitor Compound C reverses metformin suppressed core promoter activity in Hep3B/T2 cells……………………………………..34
4-6. Metformin suppresses HBV replication and expression…………….35
伍.參考文獻 (References) ………………………………………….………36
陸.圖表 (Figures) …………………………………………………...………41
柒.附錄 (Appendices) ………………………………………………………55

[1] Allen MI, Deslauriers M, Andrews CW, Tripples GA, Walters KA, Tyrrell DLJ, Brown N and Condreay LD (1998) Identification and characterization of mutation in hepatitis B virus resistant to lamivudine. Hepatology 27: 1670-1677.
[2] Chakravarty K, Cassuto H, Reshef L and Hanson RW (2005) Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C. Critical Reviews in Biochemistry and Molecular Biology 40: 129-154.
[3] Chanda D, Kim SJ, Lee IK, Shong M and Choi HS (2008) Sodium arsenite induces orphan nuclear receptor SHP gene expression via AMP-activated protein kinase to inhibit gluconeogenic enzyme gene expression. AM. J. Physiol. Endocrinol. Metab. 295: E368-E379.
[4] Chen CL, Yang HI, Yang WS, Chen PJ, You SL, Wang LY, Sun CA, Lu SN, Chen DS, and Chen CJ (2008) Metabolic factors and risk of hepatocellular carcinoma by chronic hepatitis B/C infection: A Follow-up Study in Taiwan.Gastroenterology. 135:111-21.
[5] Chou CK, Su TS, Chan CC, Hu CP, Huang MY, Suen CS, Chou NW and Ting LP (1989) Insulin suppresses hepatitis B virus surface antigen expression in human hepatoma cells. J. Biol. Chem. 264:15304-15308.
[6] Chou CK, Wang LH, Lin HM and Chi CW (1992) Glucocorticoid stimulates hepatitis B viral gene expression in cultured human hepatoma cells. Hepatology 16:13-18.
[7] Collier CA, Bruce CR, Smith AC, Lopaschuk G and Dyck DJ (2006) Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle. Am J Physiol Endocrinol Metab. 291:E182-E189.
[8] Dane DS, Cameron CH and Briggs M (1970) Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet. 1:695-698.
[9] Dankel SN, Hoang T, Flågeng MH, Sagen JV and Mellgren G (2010) cAMP-mediated regulation of HNF-4α depends on the level of coactivator PGC-1α. Biochim Biophys Acta. 1803:1013-9
[10] Ding XR, Yang J, Sun DC, Lou SK and Wang SQ (2008) Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the poteneial targets of anti-HBV drugs. Pharmacogenomics J. 8:61-70.
[11] Doitsh G and Shaul Y (2004) Enhancer I predominance in hepatitis B virus gene expression. Mol Cell Biol 24:1799-1808.
[12] Edgington TS and Chisari FV (1975) Immunological aspects of hepatitis B virus infection. Am J Med Sci 270:212-227.
[13] Ganem D and Prince AM (2004) Hepatitis Bvirus infection-natural history and clinical consequences. N Engl J Med 350:1118-1129.
[14] Geller SA (2002) Hepatitis B and hepatitis C. Clin Liver Dis 6:317-334.
[15] Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B and Montminy M (2001) CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413:179-183.
[16] Horton ES, Foley JE and Shen SG (2004) Efficacy and tolerability of initial combination therapy with nateglinide and metformin in treatment-naïve patients with type 2 diabetes. Curr Med Res Opin 20:883-889.
[17] Ishizaki T, Sasaki F, Ameshima S, Shiozaki K, Takahashi H, Abe Y, Ito S, Kuriyama M, Nakai T and Kitagawa M (1996) Pneumonitis during interferon and/or herbal drug therapy in patients with chronic active hepatitis. J Eur Resp
9:2691-2696.
[18] Jammeh S, Tavner F, Waston R, Thomas HC, Karayiannis P (2008) Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 89 : 901-909.
[19] Jared Diamond (2011) Diabetes in India Nature. Nature. 469:478-479.
[20] Johnson AB, Webster JM, Sum CF, Heseltine L, Argyraki M, Cooper BG and Taylor R (1993) The impact of metformin therapy on hepatic glucose production and skeletal muscle glycogen synthase activity in overweight type II diabetic patients. Metabolism 42:1217-22.
[21] Kim YD, Park KG, Lee YS, Park YY, Kim DK, Nedumaran B, Jang WG, Cho WJ, Ha J, Lee IK, Lee CH and Choi HS (2008) Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP. Diabetes 57:306-14.
[22] Lalau JD and Race JM (2001) Lactic acidosis in metformin therapy: searching for a link with metformin in reports of metformin-associated lactic acidosis. Diabetes Obes Metab 3: 195-201.
[23] Lee MJ, Seo WY, Song KH, Chanda D, Kim YD, Kim DK, Lee MW, Ryu D, Kim YH, Noh JR, Lee CH, John YL, Chiang, Koo SH, and Choi HS (2010) AMPK-dependent repression of hepatic gluconeogenesis via disruption of CREB-CRTC2 complex by orphan nuclear receptor small heterodimer partner. The Journal of Biological Chemistry 285:42, 32182-32191.
[24] Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, Tanwandee T, Tao QM, Shue K, Keene ON, Dixon JS, Gray DF and Sabbat J (2004) Lamivudine for patients with chronic hepatitis B and advanced liver disease.
N Engl J Med. 351:1521-31.
[25] Lin YL, Chou CK, Chen HC and Yeh SF (1995) Differential pathways of insulin action upon the hepatitis B surface antigen gene expression and cell proliferation in human hepatoma cells. Endocrinology 136: 2922-2927.
[26] Ling H, Amin S, Stephen D, Ryan M, Xiaojian S, Mehboob Hussain A, Sally R and Fredric EW (2009) Metformin and Insulin Suppress Hepatic Gluconeogenesis through Phosphorylation of CREB Binding Protein. Cell 137:4, 635-646.
[27] Maddrey WC (2000) Hepatitis B: an important public health issue. J Med Virol 61:362-366.
[28] Marcellin P (2009) Hepatitis B and hepatitis C in 2009. Liver Int. 29:1-8
[29] Melchior WR and Jaber LA (1996) Metformin: an antihyperglycemic agent for treatment of type II diabetes. Ann Pharmacother 30:158-64.
[30] Moolla N, Kew M and Arbuthnot P (2002) Regulatory elements of hepatitis B virus transcription. J Viral Hepat 9:323-331.
[31] Mutel E (2011) Control of blood glucose in the absence of hepatic glucose production during prolonged fasting in mice: induction of renal and intestinal gluconeogenesis by glucagon. Diabetes 60:3121-3131.
[32] Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J, Dong H. Accili D and Spiegelman BM (2003) Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1α interaction. Nature 423: 550-555.
[33] Puigserver P and Spiegelman BM (2003) Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 24: 78-90.
[34] Raney AK, Johnson JL, Palmer CN and McLachlan A (1997) Members of the nuclear receptor superfamily regulate transcription from the hepatitis B virus nucleocapsid promoter. J Virol 71:1058-1071.
[35] Risérus U, Willett WC and Hu FB (2009) Dietary fats and prevention of type 2 diabetes. Progress in Lipid Research 48:44-51.
[36] Rother KI (2007) Diabetes treatment bridging the divide. The New England Journal of Medicine 356:1499-501.
[37] Seeger C and Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64:51-68.
[38] Shlomai A, Paran N and Shaul Y (2006) PGC-1 controls hepatitis B virus through nutritional signals. Proc Nat1 Acad Sci USA 103:16003- 16008.
[39] Tan A, Yeh SH, Liu CJ, Cheung C and Chen PJ (2008) Viral hepatocarcinogenesis:from infection to cancer. Liver Int 28:175-188.
[40] Tang H and McLachlan A (2001) Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism. Proc Natl Acad Sci USA 98:1841-1846.
[41] Towler MC and Hardie DG (2007) AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res 100:328-41.
[42] Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB and Spiegelman BM (2001) Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131-138.
[43] Yuh CH and Ting LP (1990) The genome of hepatitis B virus contains a second enhancer: cooperation of two elements within this enhancer is required for its function. J Virol 64:4281-4287.
[44] Yuh CH, Chang YL and Ting LP (1992) Transcriptional regulation of precore and pregenomic RNAs of hepatitis B virus. J Virol 66:4073-4084.
[45] Zhang L, He H and Balschi JA (2007) Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration. Am J Physiol Heart Circ Physiol. 293:H457-66.
[46] Zuckerman AJ (1999) More than third of world's population has been infected with hepatitis B virus. BMJ 318:1213.