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系統識別號 U0026-0409201221061600
論文名稱(中文) 探討白藜蘆醇與轉譯後修飾對於調控肌肉細胞粒線體形態的角色
論文名稱(英文) The role of resveratrol and posttranslational modifications in mitochondrial morphology in muscle cells
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 黃詩涵
研究生(英文) Shih-Han Huang
學號 S96994049
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-蔡曜聲
口試委員-黃朝慶
口試委員-呂佩融
口試委員-蔡佩珍
中文關鍵字 AMPK  粒線體斷裂  Opa1  白藜蘆醇 
英文關鍵字 AMPK  Mitochondrial fission  Opa1  RSV 
學科別分類
中文摘要 在代謝疾病中,粒線體功能異常與胰島素抗性被認為是粒線體動態不平衡所導致。粒線體動態平衡,藉由融合 (fusion) 與分裂 (fission) 的機制來調控動態平衡。粒線體融合,藉由融合分子調控—mitofusins (Mfn1與Mfn2),而粒線體分裂,藉由分裂分子調控—Drp1與Fis1。已知粒線體動態蛋白能藉由轉譯後修飾快速調控粒線體形態。先前文獻指出蛋白激酶A會對Drp1 S637進行磷酸化,而使Drp1離開粒線體。白藜蘆醇 (RSV) 為天然多酚類,對於粒線體功能具有保護作用。白藜蘆醇活化Sirt1與AMPK藉由去乙醯化和磷酸化調控PGC-1α活性,進而改善粒線體功能。因此我們探討白藜蘆醇與轉譯後修飾對於調控粒線體形態的角色。游離脂肪酸誘使粒線體斷裂,伴隨著Drp1轉位至粒線體。然而此轉位之現象與Drp1上之乙醯化或絲胺酸的磷酸化無關。在我們系統之下,給予蛋白激酶A之活化物,forskolin對Drp1S637位點進行磷酸化,但此現象並不改變Drp1在粒線體的分佈或粒線體形態。我們亦發現到在過多游離脂肪酸下,不影響p53蛋白上的乙醯化、Sirt1基因與蛋白之表現。顯示在過多游離脂肪酸下,Sirt1活性不受影響。在過多游離脂肪酸下同時給予白藜蘆醇,能改善粒線體斷裂,而此現象是由於AMPK活化所導致。白藜蘆醇改善高脂所引起之粒線體斷裂,且伴隨著增加融合蛋白Opa1之表現量與轉位至粒線體上,但此現象在給予AMPK抑制劑或shAMPKα1後會被抑制。白藜蘆醇亦會降低高脂下所引起之Drp1轉位至粒線體。在同時給予白藜蘆醇後,會降低活性氧自由基之產生,但抑制AMPK之活化,並不影響白藜蘆醇降低活性氧自由基產生。顯示,白藜蘆醇改善高脂所引起之粒線體斷裂不經由AMPK活化所導致。在此研究,我們發現到白藜蘆醇活化AMPK在高脂肪酸下所導致粒線體斷裂中扮演重要角色。因此白藜蘆醇活化AMPK可以作為一治療方針,預防代謝疾病之產生。
英文摘要 In metabolic diseases, mitochondrial dysfunction and insulin resistance are related to unbalanced mitochondrial dynamics, which is controlled by mitochondrial fusion and fission. Mitochondrial fusion is regulated by fusion proteins, Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is regulated by fission proteins, Drp1 and Fis1. It has been known that posttranslational modifications on mitochondrial dynamic proteins rapidly changes mitochondrial morphology. Previous studies showed that cyclic-AMP-dependent protein kinase (PKA) phosphorylated Drp1 at S637 and induced translocation of Drp1 away from mitochondria. Resveratrol (RSV), a natural polyphenol, has the beneficial effects on mitochondrial functions. RSV activates both sirtuin1 (Sirt1) and AMP-activated protein kinase (AMPK) that regulate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) activity by deacetylation and phosphorylation, and improves mitochondrial functions. Therefore, we hypothesized that RSV and posttranslational modifications of mitochondrial dynamic proteins play important roles in regulation of mitochondrial morphology. We found that free fatty acids (FFA)-induced mitochondrial fission was related to Drp1 translocation to mitochondria. However, Drp1 translocation to mitochondria in response to FFA was not associated changes in the acetylation level and S637 phosphorylation of Drp1. Phosphorylation of Drp1 at S637per se by forskolin did not affect Drp1 translocation and mitochondrial morphology. Moreover, FFA-induced mitochondrial fission was not associated with
changes in acetylation level of p53, the downstream target of Sirt1, and mRNA or protein level of Sirt1. These data suggest that FFA-induced mitochondrial fission is not related to changes in Sirt1 activity. Interestingly, we found that RSV co-treatment attenuated FFA-induced mitochondrial fission. The reversal of FFA-induced mitochondrial fission by RSV co-treatment was dependent on activation of AMPK, but not Sirt1. Inhibition of AMPK activation by compound C, and shAMPKα1 blocked the reversal of FFA-induced mitochondrial fission by RSV co-treatment. Moreover, RSV co-treatment increased Opa1 protein level and translocation of Opa1 to mitochondria, which were attenuated by inhibition of AMPK activation. Taken together, although we did not find the changes of posttranslational modifications on mitochondrial dynamic proteins in response to FFA treatment, our findings showed that RSV and AMPK activation play important roles in the reversal of FFA-induced mitochondrial fission. Thus, the effect of RSV on alteration of mitochondrial dynamics may be used a therapeutic strategy to prevent the metabolic diseases.
論文目次 INTRODUCTION ...................................................................................................... 1
Mitochondria and metabolic diseases ................................................................... 1
Mitochondrial dynamics ....................................................................................... 2
Physiology of dynamic proteins ........................................................................... 3
Posttranslational modifications on dynamic proteins ........................................... 4
Resveratrol in mitochondria and metabolic diseases ............................................ 5
Significance .......................................................................................................... 7
MATERIALS AND METHODS ................................................................................ 8
Cell culture ............................................................................................................ 8
Freezing cells. ....................................................................................................... 8
Mitochondrial morphology ................................................................................... 8
Protein extraction .................................................................................................. 9
Mitochondrial extraction ...................................................................................... 9
RNA extraction ..................................................................................................... 9
Real-time PCR .................................................................................................... 10
Acetylation sites prediction ................................................................................ 11
Immunoprecipitation ........................................................................................... 11
Immunoflouroscence .......................................................................................... 11
ROS production .................................................................................................. 12
Transfection ........................................................................................................ 12
Immunoblotting .................................................................................................. 12
Data analysis ....................................................................................................... 13
RESULTS ................................................................................................................. 14
FFA-induced mitochondrial fission was related to Drp1 translocation to mitochondria ....................................................................................................... 14
Increased Drp1 translocation to mitochondria was not associated with changes in the acetylation level and S637 phosphorylation of Drp1 in response to FFA .. 14
Forskolin increased Drp1 S637 phosphorylation, but did not cause Drp1 translocation and change mitochondrial morphology ........................................ 15
FFA-induced mitochondrial fission was not associated with changes in acetylation level of Drp1 and Sirt1 activity ........................................................ 15
RSV co-treatment attenuated FFA-induced mitochondrial fission .................... 16
The reversal of FFA-induced mitochondrial fission by RSV co-treatment was not related to change in Sirt1 activity ................................................................. 18
The reversal of FFA-induced mitochondrial fission by RSV co-treatment was dependent on activation of AMPK ..................................................................... 18
DISCUSSION ........................................................................................................... 21
REFERENCES ......................................................................................................... 28
TABLE ...................................................................................................................... 34
Appendix ................................................................................................................... 57
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