不飽和脂肪酸調控小鼠 C2C12 肌纖維細胞粒線體生合成之 探討
近年來之動物及細胞實驗結果皆指出，當體內游離脂肪酸濃度增高時會促進肌肉中粒線
體去偶合蛋白 (uncoupling protein; UCP) 表現，進而促進脂肪酸代謝。本研究旨在探討 不飽和脂肪酸之 n-9 族油酸及 n-3 族亞麻油酸，對於肌肉細胞粒線體生合成相關基因、
脂肪酸代謝相關基因及其上游轉錄因子基因表現之影響。以 C2C12 肌纖維細胞（ C2C1 2 myotube ）作為研究對象，投予 0~1.2 mM 之脂肪酸濃度進行 24 及 48 小時培養，觀 察其細胞型態及檢測細胞存活速率。在測定基因表現方面，給予細胞 0 、 0.3 、 0.6 、 0.9 mM 之油酸及亞麻油酸培養 24 及 48 小時，再以同步定量反轉錄聚合 ? 連鎖反應 (Real-time reverse-transcriptional polymerase chain reaction) 及西方墨點法，測定目標基 因之表現。結果顯示，以 0~1.2 mM 油酸及亞麻油酸處理 C2C12 肌纖維細胞後，細胞型 態於同一濃度及時間點時具一致性，但其細胞存活率則結果不同。經 0.6~1.2 mM 油酸 處理 24 小時後，其細胞存活率為顯著降低之情形，但在 48 小時後則恢復與控制組相同
；經 0.3 mM 亞麻油酸處理 24 小時後則會增加細胞存活率。以 0.9 mM 之油酸及亞麻油 酸處理後皆會增加 PGC-1α (peroxisome proliferators -activated receptor γ coactivator 1α) 之 mRNA 表現，且處理 48 小時後其蛋白質表現量之增加有濃度依賴性之現象。以 0.6 mM 油酸處理 48 小時後會伴隨增加 COX I (cytochrome c oxidase subunit I) 之 mRNA 表 現，而亞麻油酸則是以 0.6~0.9 mM 處理 24 小時即增加其粒線體 flavoprotien 及 COX I mRNA 表現。給予 0.9 mM 油酸及亞麻油酸處理 48 小時，皆會顯著增加其 UCP3 (uncou pling protein 3) 及 CPT-1b (carnitine palmitoyl transferase-1b) 之 mRNA 表現。由上述實 驗數據推論，油酸為經由促進 PGC-1α 之表現，亞麻油酸則並非完全經由 PGC-1α 途徑
，來達到刺激粒線體生合成之效果；兩種脂肪酸皆會促進細胞內 UCP3 及 CPT-1b 之表 現進而達到促進脂肪酸代謝之結果。
Modulation of Mitochondrial Biogenesis on C2C12 Myotube by unaturated Fatty Acids
Recently, uncoupling protein (UCP) located on mitochondrial inner membrane was found that its expression could be induced when plasma free fatty acids level increased to advance fatty a cid metabolism. To evaluate effects of unsaturated fatty acids on mitochondrial biogenesis, mo use C2C12 myotubes were incubated with oleic acid (OA) or linoleic acid (LA). We observed cell morphology and analyzed cell viability after treatment with 0~1.2 mM fatty acid for 24 an d 48 hr using phase contrast microscope and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxy methoxy -phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay. Expression of mitocho ndrial genes and related transcription factor genes were determined after treatment with 0~0.9 mM fatty acid for 24 and 48 hr using real-time RT-PCR and Western blot.
Data showed that C2C12 myotube cell morphology in 0~1.2 mM OA and LA treatment were t he same pattern in various time and concentration, but cell viability of OA and LA treatment were different. Cell viability was decreased in 0.6~1.2 mM OA treatment for 24 h, but it was r ecovered after 48 h treatment. After 0.3 mM LA treatment for 24 h would increase cell viabilit y. Both OA and LA would induce PGC-1α mRNA and protein expression in C2C12 myotube with 0.9 mM treatment, especially after 48 h. 0.6 mM OA treatment would also induce COX I mRNA at 48 h. Besides, 0.6~0.9 mM LA treatment would induce mitochondrial genes includi ng flavoprotein and COX I mRNA expression. Both OA and LA would induce UCP3 and CP T-1b mRNA expression with 0.9 mM treatment at 48 h.
In conclusion, OA would stimulate mitochondrial biogenesis through PGC-1α pathway, but L A might stimulate by other way. Both OA and LA would promote fatty acid metabolism by in creasing UCP3 and CPT-1b gene expression.
