即時偵測餵食脂肪酸對粒線體生合成之影響

粒線體可藉由氧化磷酸化作用 (oxidative phosphorylation, OXPHOS) 及電子傳遞鏈產生ATP 作為細胞能量來源，而粒線體的生合成由核 DNA (nuclear DNA, nDNA) 及粒線體DNA (mitochondria DNA, mtDNA) 所共同調控 PGC-1α (Peroxisome proliferator-activated receptor (PPAR) -γ coactivator 1 α) 為一細胞核所轉譯的轉錄因子，其不僅可調節能量平衡，於粒線體生合成亦扮演重要角色。本實驗藉由活體電擊 (in vivo electroporation) 及活體造影系統 (in vivo imaging system, IVIS) 之技術，建立動物之即時偵測模式，再由此即時偵測模式即時且連續觀察餵食油酸 (oleic acid, OA) 、亞麻油酸 (linoleic acid, LA) 及二十碳五烯酸 (eicosapentaenoic acid, EPA) 是否可誘發 PGC-1α 啟動子之活性，此外亦偵測餵食不同脂肪酸對粒線體生合成之影響。 本實驗藉由電穿孔 (electroporation) 技術將 pGL3-PGC-1α 轉型到 ICR (Institute of Cancer Research) 小鼠脛骨前 (tibialis anterior, TA) 肌中，接著餵食 65% OA 、 65% LA 或 45% EPA + 10% OA + 10% LA 之高脂飲食，三組又分為十天短期實驗及九週長期實驗，實驗期間以即時偵測系統連續且即時觀察脛骨前肌 PGC-1α 啟動子於十天及九週之表現狀況，接著犧牲小鼠並分析粒線體生合成相關指標。於十天短期實驗結果顯示， OA 、 LA 及 EPA 皆可穩定且持續增加 PGC-1α啟動子活性。而於九週長期實驗，發現 OA 亦可穩定且持續增加 PGC-1α 啟動子活性，而 LA 則於中後期才有此效果， EPA 則集中於前三週。此外 OA 及 EPA 可增加 NRF-1(nuclear receptor factor-1) 蛋白質表現量，而 EPA 亦會增加 PGC-1α 蛋白質表現量並增加mtDNA 套數。由以上結果推測不同種類脂肪酸對於誘發 PGC-1α 轉錄活性有時間性之差異，且 EPA 可藉由 PGC-1α 共同活化 NRF-1 而促進粒線體生合成作用，而本實驗亦成功於動物模式建立一套即時偵測之系統。

Real-Time Determination of Effect of Fatty Acids Supplementation on Mitochondrial Biogenesis

ATP is synthesized in mitochondria as an energy source of cell though oxidative phosphorylation (OXPHOS) and electron transport chain (ETC). Mitochondrial biogenesis is coordinately mediated by nuclear DNA (nDNA) and mitochondria DNA (mtDNA). Peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1α (PGC-1α), a nuclear encoded transcription factor, is identified as a key regulator on mediated energy homeostasis and mitochondrial biogenesis. In this study, a real-time determination system was created to analyze PGC-1 α promoter activity in skeletal muscle of mice by electroporation and in vivo imaging system (IVIS). ICR mice were fed with oleic acid (OA), linoleic acid (LA), and eicosapentaenoic acid (EPA) respectively, and to determine whether fatty acids induced PGC-1α promoter activities by real-time and continus monitering. The effect of different fatty acids supplement on mitochondrial biogenesis was also determined. In this study, the pGL3-PGC-1α plasmid DNA was transfected into tibialis anterior (TA) muscle of ICR mice by electric pulse-mediated gene transfer, and supplement 65 ％ OA or LA, and 45 ％ EPA+10 ％ OA+10 ％ LA diet for 10 days and 9 weeks. The PGC-1α transcriptional activity also was determined continually by real-time system during the 10 days and 9 weeks. Finally the mice were scarified and analysis the marker of mitochondrial biogenesis. During 10 days, the result represents OA, LA and EPA continually and stably increased PGC-1α promoter activities. During 9 weeks, the OA also continually and stably increased PGC-1α promoter activities, and the LA increased in middle-later period. However, EPA effects were mainly at 1st to 3rd, besides EPA induced mitochondrial biogenesis. OA and EPA increased NRF-1 (nuclear receptor factor-1) protein expression. Besides, EPA also increased PGC-1α protein expression and mtDNA copy number. These results suggest different fatty acids provided verified affects on PGC-1α promoter activity. And EPA increased mitochondrial biogenesis by induced PGC-1α and to coactivatied NRF-1. This study also created a successful real-time imaging model of the animal.