臺灣博碩士論文加值系統

近年來隨著人們生活飲食的改變，高血壓、高血脂、高血糖等代謝病越益增高，因此WHO (World Health Organization)為此定義了代謝症候群，代謝症候群是一種綜合型的內分泌疾病，會增加發展中的心血管疾病以及糖尿病的風險。而第二型糖尿病症狀與高濃度的游離脂肪酸有關，會降低肌肉細胞對葡萄糖的攝取，增加血液中葡萄糖含量和對胰島素抗性。AMPK-activate protein kinase (AMPK)是感應細胞內能量代謝的關鍵調控者，磷酸化AMPK可減少脂肪的合成和增加脂肪酸代謝，此外，AMPK也被廣泛運用為第二型糖尿病的標靶分子。近年來研究發現，甜菊可以改善第二型糖尿病的症狀。在本篇研究當中透過Glucose uptake實驗，對數十種甜菊修飾後的化合物進行篩選，並從中選出效果最佳的兩個化合物CTUT2和CTUT3進行機轉的探討。CTUT2和CTUT3增加細胞對葡萄糖攝取量與胰島素依賴性路徑無關。CTUT2和CTUT3可增加AMPK及下游ACC蛋白磷酸化，也可促進PKC δ蛋白的磷酸化；當本篇研究將C2C12細胞以AMPK抑制劑(Compound C) 預處理後，發現可以抑制CTUT3誘發的PKC δ蛋白質磷酸化，且抑制CTUT2對葡萄糖的攝取；最後，透過免疫螢光染色發現CTUT2和CTUT3可促進GLUT4從細胞質轉移到細胞膜上，而增進葡萄糖的攝取。綜合以上結果本篇研究認為CTUT2和CTUT3可以透過AMPK調控PKCδ並促進GLUT4轉移到細胞膜上而增加葡萄糖的攝取。

Metabolic syndrome is a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes. The World Health Organization (WHO) defines Metabolic syndrome with the characetistics of elevated blood fatty-acid concentrations, reduced glucose uptake in muscle, and increased insulin resistance and high blood sugar, which is highly associated with diabetes mellitus type II. AMP-activated protein kinase (AMPK) is a key sensor regulating intracellular and whole body energy metabolism. Phosphorylation of AMPK suppresses lipid synthesis and increase fatty acid β-oxidation, so AMPK has been a therapeutic molecular target for diabetes. Recent studies suggested that stevioside may exert therapeutic beneficial effects for type-2 diabetes. In our studies, screening of compounds derived from stevioside for glucose uptake was performed using C2C12 myotube. We found that CTUT2 and CTUT3 have significantly effects and we further investigated the mechanism. CTUT2 and CTUT3 stimulates glucose uptake in insulin independent pathway. CTUST2 and CTUT3 increased AMPK and the downstream ACC phosphorylation in a concentration-dependent manner in C2C12 myotubes. CTUST2 and CTUT3 also increased PKCδ phosphorylation. When we pretreated the cells by compound C (AMPK inhibitor), the increases of PKCδ phosphorylation and glucose uptake by CTUT2 and CTUT3 were reduced. Finally, CTUT2 and CTUT3 promoted GLUT4 transporting to membrane from cytoplasm and increased glucose uptake. These results indicate that CTUT2 and CTUT3 promoted the transportation of GLUT4 from cytoplasm to membrane and glucose uptake via increasing AMPK and the downstream PKCδ phosphorylation.

中文摘要 5
Abstract 6
縮寫表 12
背景 14
代謝性症候群（Metabolic syndrome） 14
糖尿病（Diabetes mellitus） 14
胰島素抗性 (Insulin Resistance) 16
胰島素訊息傳遞路徑 (Insulin signaling pathway) 16
AMP-activated protein kinase (AMPK) 17
Acetyl CoA carboxylase (ACC) 18
Protein Kinase C δ (PKC δ) 19
Glucose transporter (GLUT) 20
Glucose transporter 4 (GLUT4) 20
甜菊(stevia) 21
研究動機 22
實驗方法與材料 23
一、 實驗材料 23
1. 藥品試劑 23
2. 常用儀器 25
3. 常用溶液 26
二、 實驗方法 28
1. C2C12細胞的培養 28
2. 細胞蛋白質的測定 28
3. 西方墨漬法 29
4. 葡萄糖攝取試驗 29
5. 免疫螢光染色 30
6. MTT細胞存活率實驗 31
7. 細胞膜及細胞質蛋白分離 31
實驗結果 33
一、 甜菊糖衍生物CTUT2及CTUT3可增加AMPK及下游ACC蛋白磷酸化表現 33
二、 甜菊糖衍生物CTUT2和CTUT3可增加葡萄糖攝取量 34
三、 CTUT2和CTUT3對於細胞存活率的影響 34
四、 CTUT2和CTUT3增加葡萄糖攝取與胰島素訊息路徑無關 35
五、 CTUT2和CTUT3不會增加AMPK下游的p38 MAPK蛋白磷酸化 36
六、 CTUT2和CTUT3透過AMPK調控下游的PKCδ蛋白的磷酸化 36
七、 CTUT2和CTUT3是透過AMPK來調控葡萄糖攝取量 37
八、 CTUT2和CTUT3增加GLUT4的蛋白表現 38
九、 CTUT2&3是透過AMPK使GLUT4從細胞質轉移到細胞膜上 39
討論 41
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