- 學位論文
中國橄欖甲醇萃取-乙酸乙酯區分層之促葡萄糖攝取功效探討
The study of Chinese olive (Canarium album) extract mediated glucose uptake in L6 myotube
摘要
糖尿病名列國人前十大死因之一，其中，第二型糖尿病之成因仍待研究，目前已知慢性發炎和脂質代謝紊亂會造成肌肉、肝臟組織對胰島素的調控失去敏感性(insulin insensitivity)，因而導致第二型糖尿病。Metformin、Thiazolidinedione為目前主要的降血糖藥物，此外，許多植化素(phytochemicals)亦被發現具有調控血糖與增加胰島素敏感的能力，其作用機制各自不同，但有許多植化素可透過活化AMPK進而促進葡萄糖攝取與增加胰島素敏感性。中國橄欖(Canarium album)的療效散見於中國古典藥綱中，含有豐富的多酚類、黃酮類之衍生物，具有抗發炎、抗癌細胞增生、抗氧化等功效，具有減緩糖尿病症狀之功效，然而其降低血糖、增加代謝之功效仍不清楚。本研究以L6大鼠肌管細胞(Myotube)模式進行葡萄糖攝取現象之討論。結果顯示，中國橄欖甲醇萃取 - 乙酸乙酯區分層(OE-MeOH-Ea)可以刺激L6肌管細胞的2-NBDG攝取量增加，並在濃度400 μg/mL處理下達到顯著差異，而在胰島素誘發之胰島素阻抗模式下，仍可以顯著增加2-NBDG攝取。此外，透過利用西方墨點法確認，OE-MeOH-Ea具有刺激AMPK Thr-172磷酸化的能力，使其活性上升。研究發現，OE-MeOH-Ea可促使細胞內活性氧的基值(baseline)下降，粒線體的膜電位上升，以及ATP生成量增加、NADH的量下降，此可能與活化AMPK有關。然而，並未發現OE-MeOH-Ea會增加GLUT4蛋白轉運至膜上的量，其mRNA與蛋白質量亦未增加，推測其增加葡萄糖攝取可能並不是來自於調控GLUT4蛋白。此外，利用抑制劑實驗結果顯示，PI3K、GLUT等抑制劑並無法抑制OE-MeOH-Ea所促進的葡萄糖攝取，說明OE-MeOH-Ea對於葡萄糖攝取的刺激可能透過不同於與胰島素的路徑。蛋白質體學的結果顯示，OE-MeOH-Ea會促使細胞膜分層中與鈣離子調控運輸相關以及適應逆境的蛋白增多，推測OE-MeOH-Ea具有活化粒線體運作、增加細胞代謝速率及葡萄糖攝取功效，可能是來自於影響鈣離子平衡以及造成逆境促使AMPK以及其他的路徑活化，然而詳細的機制仍需要更一步的研究與探討。
並列摘要
Diabetes is the fifth leading cause of death of 2012 in Taiwan. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, however, the pathological mechanism remains unclear. Insulin resistance, a key etiology of T2DM, has been reported to associate with chronic inflammation and dysregulation of lipid metabolism. In addition to Metformin and Thiazolidinediones, the common interventions for lowering blood glucose in T2DM patients, several phytochemicals have been demonstrated to have function in lowing blood glucose and sensitizing insulin. The mechanisms of known phytochemical mediated insulin sensitizing are largely through the activation of AMPK pathway. In this study, we employed Chinese olive, which is rich in polyphenolic compounds, has been used as medicine in traditional Chinese medical science and shows anti-inflammation, anti-oxidation and anti-proliferation effects. Although Chinese olive is considered as a potential supplementary food for preventing diabetes, however, its molecular mechanism is largely unknown. Here we used L6 myotube as a cell model for measuring glucose uptake, and demonstrated that Chinese olive methanol extract – ethyl acetate partition layer (OE-MeOH-Ea) enhanced 2-NBDG uptake and sensitized the function of insulin. Additionally, OE-MeOH-Ea increases the phosphorylation of AMPK at Thr-172 in L6 myotube. We further investigated the mechanism of AMPK activation upon OE-MeOH-Ea, and found that OE-MeOH-Ea could potentiate mitochondria function, such as increased ATP production, higher mitochondrial membrane potential along with lower NADH amount. We also conclude that the OE-MeOH-Ea mediated glucose uptake is not through GLUT4, the major glucose transporter regulated by insulin in muscle, because the membrane translocation, mRNA expression and protein expression of GLUT4 are not affected after OE-MeOH-Ea treatment. Furthermore LY294002, wortmannin (PI3K inhibitors), cytochalasin B (Class I GLUT inhibitor) failed to suppress OE-MeOH-Ea-enhansed 2-NBDG uptake, indicating other glucose transporter might be involved in regulating glucose uptake. To answer this, we used proteomic analysis to analyze the differential protein expression in plasma membrane among control, OE-MeOH-Ea treated L6 myotube. The results showed that Ca2+ regulation proteins and proteins for adaptation of metabolic stress are increased after OE-MeOH-Ea treatment. In summary, OE-MeOH-Ea promotes mitochondrial function and activates AMPK to enhance glucose uptake through regulating Ca2+ signaling pathway.