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標題: | KS-C370G在培養細胞及ICR小鼠降血糖作用及機制研究 Antihyperglycemic Effect and Mechanism of KS-C370G on ICR Mice and Cultured Cell Lines |
作者: | Yen-Chu Lin 林彥竹 |
指導教授: | 蘇銘嘉(Ming-Jai Su) |
關鍵字: | 蜂膠,AMP活化蛋白激酶,降血糖, CAPE,AMPK,Akt,diabetes,antihyperglycemic effects, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | KS-C370G是caffeic acid phenethyl ester (CAPE)的相似物,CAPE是蜂膠裡的成分,KS-C370G具有許多生化及藥理作用類似CAPE甚至更好。我們實驗室之前研究發現,KS-C370G在STZ-induced的type 1糖尿病鼠和high fat-induced的type 2糖尿病鼠單次給藥有降空腹血糖作用,長期治療type 2糖尿病鼠則有降血糖作用及治療血中高胰島素(hyperinsulinemia)等糖尿病症狀,惟詳細機轉仍不清楚。
本實驗使用C2C12小鼠骨骼肌細胞株及Hep 3B人類肝癌細胞株來作為細胞模式探討KS-C370G降血糖作用及機制。本實驗發現,KS-C370G可以促進C2C12小鼠骨骼肌細胞株對葡萄糖的吸收及增加Hep 3B肝臟細胞肝醣含量。在C2C12細胞給予不同濃度藥物刺激30分鐘,從0.03 µM開始可以顯著活化AMPK,濃度增加到1 µM AMPK活化有下降的趨勢,到了3 µM來到了最低點,但是10 µM又有開始活化的現象。使用高濃度10 µM在不同時間測C2C12細胞AMPK活化狀態,發現15分鐘AMPK有最大的活化,一直到30分鐘還會活化AMPK,60分鐘後AMPK活化消失,但給藥120分鐘後又開始活化AMPK,而使用低濃度0.03 µM做time course實驗,發現KS-C370G從5分鐘開始活化C2C12細胞的AMPK,一直持續到120分鐘AMPK都有很強的活化,然而KS-C370G活化Akt模式與其活化AMPK有所不同,在低濃度和高濃度下KS-C370G從5分鐘開始活化Akt,此作用持續到120分鐘。 在Hep 3B細胞中,給予不同濃度藥物1小時,發現KS-C370G在濃度0.3和1 µM 可使AMPK活化,但是此條件下Akt並沒有被活化,而在濃度10 µM刺激下,我們發現給予藥物15分鐘AMPK活性增至最大,但是在30分鐘活化狀態下降持續到60分鐘,在120分鐘後AMPK活化漸增加,而Akt則是在15分鐘開始被活化,60分鐘活化消失,但是給予藥物120分鐘Akt又被活化。 我們進一步探討KS-C370G對細胞ATP含量之影響,結果發現在C2C12細胞給予藥物不同濃度30分鐘,ATP隨著濃度提高而下降,高濃度10 µM作用下,則發現ATP隨著給藥時間越久有漸恢復的趨勢,但是仍然低於未給藥組別。此外,KS-C370G促進肝臟細胞肝醣合成的現象會被Compound C和prazosin抑制。在IL-6引起之胰島素阻抗細胞模式中,KS-C370G可以些微提升C2C12細胞對胰島素阻抗的反應,增加葡萄糖吸收,其作用機制可能是透過改善insulin receptor substrate對胰島素的反應。 在健康的ICR小鼠,給予KS-C370G藥物有降血糖、促進胰島素釋放、增加對於葡萄糖的耐受性,同時KS-C370G可以促進ICR小鼠肝臟和骨骼肌肝醣的含量,其效果比單純使用胰島素或同時併用胰島素和KS-C370G的作用來的好。 總結本篇實驗,我們發現KS-C370G降血糖作用的有效劑量與其活化AMPK及Akt訊息傳遞的有效濃度一致,但是活化AMPK和Akt是否和降血糖作用有關以及彼此如何互相調控仍須進一步研究。 KS-C370G is a derivative of caffeic acid phenethyl ester (CAPE), an active component of propolis, which has several biological and pharmacological properties similar to CAPE or even better. Our previous research has shown that KS-C370G has an antidiabetic effects in streptozotocin -induced diabetic mice and high-fat-induced type 2 diabetic mice. In acute treatment, KS-C370G can reduce fasting blood glucose in both STZ-induced diabetic mice and high-fat induced type 2 diabetic mice. In chronic treatment, KS-C370G can also lower the fasting blood glucose and reverse hyperinsulinemia in high-fat induced type 2 diabetic mice. In this study, we used C2C12 mouse skeletal myoblast cell lines and Hep 3B human hepatocellular carcinoma cell lines as cell models to investigate the mechanism of KS-C370G hypoglycemic effectss. KS-C370G enhanced C2C12 cells glucose uptake and Hep 3B cells glycogen synthesis. In C2C12 cells, KS-C370G incubation for 30 minute induced AMPK activation at concentrations of 0.03 µM and 1 µM but activation disappeared at 3 µM, and AMPK activation reappeared at 10 µM. In study of the time course of cellular response, KS-C370G at 10 µM induced C2C12 cells AMPK activation at 15 minutes and sustained to 30 minutes. AMPK activation diminished after 60 minutes and reappeared at 120 minutes which seemed like biphasic effectss. In a time course of response to 0.03 µM KS-C370G, C2C12 cells AMPK activation started from 5 minutes and sustained to 120 minutes in a highly activation manner. However, KS-C370G showed different activating patterns on Akt signals. KS-C370G induced Akt activation in C2C12 cells both at 10 µM and 0.03 µM which sustained from 5 minutes to 120 minutes after the drug incubation. In Hep 3B cells, KS-C370G incubation for one hour showed that AMPK was activated at 0.3 and 10 µM but Akt was not activated under this condition. In study of the time course of cellular response, KS-C370G at 10 µM induced peak AMPK activation at 15 minutes which declined from 30 minutes to 60 minutes, and reactivated at 120 minutes. Akt was activated from 15 minutes to 30 minutes but declined at 60 minutes, and reactivated at 120 minutes. We further investigated whether stimulation by KS-C370G affected cell ATP contents. We found that KS-C370G induced initial depletion of C2C12 cells ATP contents concentration-dependently in 30 minutes. The ATP content decreased to a minimal level in 5 minutes which then recovered time-dependently to a level lower than control value. The KS-C370G-stimulated Hep 3B glycogen synthesis effectss could be blocked by Compound C and prazosin. The IL-6 induced inhibition of insulin-stimulated glucose uptake was reduced and insulin receptor signaling was partly recovered by KS-C370G via improving insulin substrate-1 responding to insulin stimulation. In healthy ICR mice, we found that KS-C370G had hypoglycemic effectss, stimulating insulin release, improving glucose tolerance. Also, KS-C370G increased liver and skeletal muscle glycogen content in ICR mice even better than insulin-stimulated groups. In conclusion, KS-C370G was found to have hypoglycemic activity to a dose ranges comparable to the effectsive concentrations required for the activation of AMPK and Akt signaling in cultured cells.The relationship between AMPK and Akt regulation and the hypoglycemic effects of KS-C370G require for further investigation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43276 |
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