以脂肪與肌肉細胞模式評估山苦瓜水萃物暨其區分物對細胞汲取葡萄糖之影響與其機制探討

Effect and Mechanisms of Momordica charantia Extract on Glucose Uptake in 3T3-L1 Adipocytes and L6 Myotubes

10.6342/NTU.2010.01226

周怡君

血糖調控異常 ； 苦瓜 ； 脂肪細胞 ； 肌肉細胞 ； 細胞葡萄糖汲取 ； blood glucose homeostasis ； bitter gourd ； adipocytes ； myotube ； glucose uptake

臺灣大學生化科技學系學位論文

2010年

碩士

黃青真

繁體中文

苦瓜是熱帶地區常見蔬菜，印度傳統醫療則用來治療糖尿病。近代研究報告亦有證據支持苦瓜具有降血糖功能。早期研究曾自苦瓜分離出類胰島素胜肽，近年也有研究自苦瓜分離出含三萜類物質，可促進細胞攝入葡萄糖，顯示苦瓜降血糖的活性成分與機制不只一種。本實驗以山苦瓜作為試驗食材，以肌肉及脂肪細胞模式探討山苦瓜水萃物及區分物對細胞汲取葡萄糖之影響及改善胰島素抗性上的潛力及其作用的相關機制。 本研究採用之細胞包括 L6 肌肉細胞株及 3T3-L1 脂肪細胞株，其分化後進行實驗。實驗分為正常及以棕櫚酸誘發細胞胰島素阻抗等兩種模式中，山苦瓜水萃物及各種區分物分別處理 30 分鐘或 12 小時後，對此兩株細胞分別於添加或不添加胰島素狀態下，以偵測 2-Deoxy-D-[1-3H]glucose 進行葡萄糖汲取試驗。試驗樣品包括：山苦瓜水萃物（WE）、水萃物酵素水解後乙酸乙酯萃物（We-E）、水萃物酵素水解後正丁醇萃物（We-B）、水萃物中 3Kd 以下小分子（WES）、小分子酵素水解後之乙酸乙酯萃物（Se-E）、小分子酵素水解後正丁醇萃物（Se-B）與植物類胰島素（Pf）。結果顯示，在脂肪細胞試驗中， WE、WES、Se-E 與 Pf 在有無誘發胰島素抗性、各種處理時間皆增加脂肪細胞汲取葡萄糖，並增加細胞對胰島素之反應。經由前述結果進一步分析以樣品處理 12 小時的脂肪細胞中之訊息傳遞，蛋白質表現及其磷酸化由西方墨點分析。結果顯示，山苦瓜樣品經酵素處理後之萃取物：We-B、Se-E、Se-B 可增加脂肪細胞中 Akt 磷酸化。而在肌肉細胞實驗中，經酵素水解後之樣品 We-E、Se-E 及 Pf 處理 30 分鐘、Se-E 處理肌肉細胞 12 小時可增加細胞汲取葡萄糖。所有樣品與棕櫚酸共處理 12 小時皆可改善肌肉細胞胰島素抗性。本研究結果，花蓮四號山苦瓜水萃物中含有增加脂肪與肌肉細胞汲取葡萄糖的活性成分，其中又以 Se-E 於多種試驗模式中皆有促進之效果，其增加 Akt 磷酸化、增加脂肪與肌肉細胞汲取葡萄糖，而具調控血糖的潛力。

Bitter gourd (Momordica Charantia, BG) is a common tropical vegetable that has also been used in traditional medicine for treating diabetes. Numerous research in past decades have provided evidences supporint the hypoglycemic effect of BG. Insulin like polypeptide-p and triterpenoids from BG have been demonstrated for their hypoglycemic effect in vivo and in vitro. This study aims at examing the effects of BG water extract and its feactions on the glucose uptake of 3T3-L1 adipocytes and L6 myotute. Lyophilized powder of wild bitter gourd (Hualian NO.4) was extracted with water (WE), treated with enzyme and extracted with ethyl acetate(We-E), and butanol (We-B). Small molecular weight fraction of WE (WES) was treated similarly and give rise to fractions Se-E and Se-B. Insulin-like peptide fraction (Pf) was isolated from WE. Cells, treated with various BG samples for 30min or 12 hr, were examined for their 3H-labeled 2-deoxyglucose uptake at basal or palmitate-induced insulin resistant state and in the presence or presence of insulin. In 3T3-L1 adipocytes, WE, WES, Se-E and Pf were increased glucose uptake in both basal and insulin resistant state (p<0.05), and also improved insulin resistance and amplified insulin action. Associated with these effects, 3T3-L1 adipocytes treated with BG WE and fractions for 12hr showed enhanced Akt phosphorylation, detected by western-blot analysis. In L6 myotubes, We-E, Se-E, and Pf increased glucose uptake after 30min treatment. Myotubes treated with Se-E for 12 hrs also showed higher glucose uptake. Most importantly, all BG samples prevented palmitate-induced inhibition of glucose uptake. These datas suggested that the wild bitter gourd contains hypoglycemic components wihich increased adipocytes and myptubes uptake glucose. Among our BG fraction, Se-E might be the most effective fraction.

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