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研究生:林昱君
研究生(外文):Yu-Chun Lin
論文名稱:胰島素調控粒線體中丙酮酸去氫酶機制之探討
論文名稱(外文):Mechanism by which insulin regulates pyruvate dehydrogenase activity in mitochondria
指導教授:李宏謨李宏謨引用關係
指導教授(外文):Horng-Mo Lee
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:細胞及分子生物研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:肝醣合成酶激脢3b丙酮酸去氫酶
外文關鍵詞:Glycogen synthase kinase 3bpyruvate dehydrogenase
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胰島素是人體中主要的同化激素,可以促進肌肉、脂肪組織、及肝臟中的蛋白質和脂質合成,並促進肝醣合成。經由胰島素受器傳導的訊息傳遞路徑主要有二,為phosphatidylinositol 3 kinase(PI-3K)/ PDK-1/ Akt傳遞路徑,以及MAP kinase傳遞路徑,然而關於胰島素調控粒線體中丙酮酸脫氫酶複合體的機制,目前則尚未釐清。本實驗室先前研究發現,粒線體中肝醣合成激酶3b會與丙酮酸脫氫酶複合體的E3次單元體形成蛋白質複合體。在本篇研究中發現,以胰島素刺激HepG2細胞,Akt受到磷酸化而活化,並轉位到粒線體內,進而磷酸化肝醣合成激酶3b的第9個絲氨酸,抑制其活性,此時丙酮酸脫氫酶的酵素活性上升;在使用doxorubicin刺激肝醣合成激酶3b的活性增加後,則丙酮酸脫氫酶的活性下降;然而若施予肝醣合成激酶3b的抑制劑TDZD-8於細胞中,則可以回復肝醣合成激酶3b的影響,使丙酮酸脫氫酶的活性受胰島素刺激而上升。此外利用二維電泳和西方墨點法,發現在doxorubicin刺激增加肝醣合成激酶3b的活性下,丙酮酸脫氫酶的E2或是E3 binding-protein磷酸化增加,而TDZD-8抑制肝醣合成激酶3b活性後,則會降低此磷酸化現象。綜和以上研究結果可以推論,胰島素刺激Akt磷酸化並轉位到粒線體內,進一步磷酸化肝醣合成激酶3b並抑制其活性,使丙酮酸脫氫酶的磷酸化減少,進而調控並增加丙酮酸脫氫酶的酵素活性。這些發現提供了線索,以進一步了解胰島素藉由肝醣合成激酶3b調控丙酮酸脫氫酶活性的訊息傳遞路徑。
Insulin is a major anabolic hormone that stimulates synthesis of protein, lipid and glycogen in liver, adipose tissue and muscles. Two main signal transduction pathways downstream of insulin receptor are the phosphatidylinositol 3 kinase/ PDK-1/ Akt pathway and the MAP kinase pathway. How insulin might regulate pyruvate dehydrogenase (PDH) activity in mitochondria is not completely known. Our laboratory has previously found that mitochondrial GSK3b was associated with PDH E3 subunit as a complex. In the present study, we demonstrated that?nAkt was translocated to mitochondria upon insulin stimulation, and the mitochondrial Akt was in its phosphorylated and active state. Activation of Akt is known to phosphorylate and inhibit its downstream enzyme, GSK3b?nat Ser9. Inhibitory phosphorylation of GSK3b maintains PDH at its non-phosphorylated and active state. Consistently, treatment of Hep G2 cells with insulin increased phosphorylation of mitochondrial GSK-3b, which was associated with an increase of PDH activity. Activation of GSK3b by doxorubicin suppressed the PDH activity, and this effect was reversed by pretreatment of cells with TDZD-8, a GSK3b-specific inhibitor?|?n Furthermore, treatment of Hep G2 cells with doxorubicin increased phosphorylation of PDH E3 binding-protein as revealed by 2D-immunoblotting, and the inhibition of GSK3b?n?nby TDZD-8 abolished this phosphorylation. Taken together, our results suggest that translocation of Akt to mitochondria and subsequent GSK3b?nphosphorylation may regulate PDH activity in mitochondria by phosphorylating the PDH E2 or E3 binding-protein?|?n These findings might provide clues to understand the mechanism by which insulin regulates mitochondrial pyruvate dehydrogenase activity through GSK3b?|
目 錄
頁數
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅷ
縮寫詞彙說明 ⅩⅠ

壹、緒論
ㄧ、胰島素的作用 2
二、Phosphatidylinositol 3-kinase(PI-3K)
訊息傳遞路徑 4
三、肝醣合成酶激脢3b 5
四、丙酮酸去氫酶 8
五、實驗目的 12

貳、研究材料與方法
ㄧ、藥品試劑 14
二、儀器設備 19
三、實驗方法
(一) HepG2細胞株培養 21
(二) 細胞蛋白質的測定 21
(三) 西方墨點法 22
(四) 蛋白質沉澱 23
(五) 二維電泳 24
(六) 雷射共軛焦顯微鏡 26
(七) 粒線體分離 27
(八) 丙酮酸去氫酶的活性測定 28
(九) 質體DNA的製備 28
(十) HepG2細胞株轉染 29
(十一) 分析轉染後細胞的基因表現 30
(十二) 細胞內ATP含量測定 31

参、實驗結果
ㄧ、胰島素增加HepG2細胞中Akt和GSK3b磷酸化現象 33
二、胰島素增加HepG2細胞中Akt和GSK3b的磷酸化為
短時效應 33
三、胰島素刺激Akt轉位到粒線體內 34
四、胰島素的刺激增加粒線體內Akt和GSK3b的磷酸化 35
五、胰島素可能藉由GSK3b調控丙酮酸去氫酶活性 36
六、GSK3b的活化可能增加丙酮酸去氫酶的磷酸化 36
七、GSK3b質體於轉染後HepG2細胞中的表現 37
八、利用GSK3b shRNA質體轉染細胞探討其對於丙酮酸
去氫酶活性的影響 38
九、GSK3b影響細胞中ATP的含量 39
十、 GSK3b影響細胞中粒線體的型態 39

肆、討論與分析 40

伍、參考文獻 46

陸、實驗結果圖表 55



圖 目 錄
Fig.1. Insulin increased Akt and GSK3b phosphorylation in HepG2 cells. 56

Fig.2. Insulin increased Akt and GSK3b phosphorylation in HepG2 cells. 57

Fig.3. Insulin increased Akt accumulation in mitochondria. 58

Fig.4. GSK3b was partially localized in mitochondria. 59

Fig.5. Mitochondria isolation control. 60

Fig.6. Insulin increased Akt and GSK3b phosphorylation in mitochondria. 61

Fig.7. Insulin might regulates pyruvate dehydrogenase (PDH) activity through GSK3b in mitochondria?| 62
Fig.8. Activated GSK3b increased phosphorylation of pyruvate dehydrogenase (PDH). 63

Fig.9. Map of the GSK3b wild type construct. 64

Fig.10. Map of the GSK3b S9A construct. 65

Fig.11. Map of the GSK3b K85R construct. 66

Fig.12. Sequencing result of GSK3b wild type construct. 67

Fig.13. Sequencing result of GSK3b S9A construct. 68

Fig.14. Sequencing result of GSK3b K85R construct. 69

Fig.15. Map of the GSK3b shRNA construct. 70

Fig.16. Expression of GSK3b mRNA in transfected HepG2 cells. 71
Fig.17. Expression of GSK3b protein in transfected HepG2 cells. 72

Fig.18. Expression of GSK3b in HepG2 cells transfected with GSK3b shRNA construct. 73

Fig.19. Mitochondrial pyruvate dehydrogenase (PDH) activity was up-regulated in HepG2 cells transfected with GSK3b shRNA construct. 74

Fig.20. Cellular ATP content of HepG2 cells transfected with GSK3b constructs. 75

Fig.21. Mitochondrial morphology of HepG2 cells transfected with GSK3b constructs. 76
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