臺灣博碩士論文加值系統

　　腺苷單磷酸活化蛋白激酶 (AMP-activated protein kinase, AMPK) 是一個感應細胞內能量代謝的關鍵調控者， AMPK 在骨骼肌、肝臟及脂肪組織中活化會減少血糖濃度、血漿中脂質濃度及異常的脂肪累積，並增加胰島素敏感性，因此 AMPK 可作為預防與治療第二型糖尿病的分子標耙。 NQ-1 是由中草藥萃取出來的物質，本實驗室先前的研究發現 NQ-1 具有降血糖的活性，但 NQ-1 影響降血糖的詳細機制目前尚不清楚，因此本論文主要為探討 NQ-1 在動物體內的影響及使用細胞實驗探討降血糖的機轉。本論文所使用的實驗動物為糖尿病小鼠 (db/db mice) 與肥胖小鼠 (ob/ob mice) ，研究結果發現，給予 NQ-1 的組別之血糖相較於控制組有下降的情形。此外，本論文利用老鼠骨骼肌細胞 L6 與人類肝癌細胞 HepG2 作為探討分子機轉的模式，在給予 NQ-1 後利用西方墨點法分析蛋白質的表現及變化，結果發現， NQ-1 會活化 AMPK ，並抑制下游 Acetyl-CoA carboxylase (ACC) 的活性，這兩個蛋白質在葡萄糖攝取及代謝中扮演很重要的角色。此外，由 NQ-1 所引起的 ACC 磷酸化會被 Compound C (AMPK 抑制劑) 抑制，代表 NQ-1 所誘發的訊息傳遞屬於 AMPK 依賴性路徑。接著利用帶有螢光的葡萄糖類似物探討 NQ-1 是否會增加 L6 細胞對於葡萄糖攝取的能力，由結果發現隨著 NQ-1 濃度增加，細胞攝取葡萄糖的能力亦顯著增加。並且在西方墨點法實驗中亦發現 NQ-1 會增加第四型葡萄糖轉運蛋白 (Glucose transporter 4, GLUT4) 表現至細胞膜上，使 GLUT4 發揮運送葡萄糖的功能。接著利用 HPLC 方法探討 NQ-1 對 AMP/ATP 比例的影響，發現 NQ-1 會增加 AMP/ATP 的比例。這些結果顯示 NQ-1 會增加 AMP/ATP 比例引發 AMPK 訊息路徑的活化，並增加細胞膜上 GLUT4 的含量與葡萄糖攝取的能力。因此我們認為 NQ-1 或許具有開發成為第二型糖尿病藥物的潛力。

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscle, liver, and adipose tissue results in a favorable metabolic milieu for the prevention or treatment of type 2 diabetes, i.e., decreased circulating glucose, reduced plasma lipid, and ectopic fat accumulation, as well as enhanced insulin sensitivity. NQ-1 was extracted from Chinese herb medicine, and was found that had a hypoglycemic activity in our previous study. However, the detail hypoglycemic mechanisms of NQ-1 are still unclear. In animal model, male db/db mice and ob/ob mice were used to examine the possible hypoglycemic activity, and showed that NQ-1 significantly decreased plasma blood glucose levels in both animal strains. In vitro study, mouse skeletal muscle cells L6 and human hepatoma cells HepG2 were used to examine the underlying molecular machenisms of NQ-1 on hypoglycemic activity. Western blot analysis revealed that NQ-1 induced the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), which play an important role in glucose uptake and metabolism. In addition, NQ-1-induced phosphorylation of ACC was suppressed by compound C, an AMPK inhibitor, suggesting that NQ-1-induced phosphorylation was mediated through AMPK-dependent manner. Next, we examined whether NQ-1 could increase glucose uptake by a fluorescent glucose analog in L6 cells. We found that NQ-1 significantly increased glucose uptake in a dose-dependent manner. In addition, NQ-1 also increased glucose transporter 4 (GLUT4) translocation to plasma membrane by Western blot analysis. To examine whether NQ-1 activaed AMPK through the change of AMP/ATP ratio, we determined the ATP and AMP levels by HPLC. The results showed that NQ-1 dose-dependently increased AMP/ATP ratio. These results suggest that the increase in the AMP/ATP ratio by NQ-1 triggers the activation of AMPK signaling pathway and leads to increase plasma membrane GLUT4 content and glucose uptake. Therefore, we hypothesize that NQ-1 has potential as an antidiabetes agent for the treatment of type 2 diabetes.

縮寫表 VI
Abstract VIII
中文摘要 IX
第一章 研究動機與目的 (Study aim) 1
第二章 緒論 (Introduction) 2
第一節 糖尿病 (Diabetes mellitus, DM) 2
一、糖尿病的定義 (Definition of diabetes mellitus) 2
二、糖尿病診斷標準 (Diagnoses of diabetes mellitus) 2
三、糖尿病的分類 (The classification of diabetes) 2
四、糖尿病的發病機制 (Diabetes pathogenesis) 4
五、糖尿病併發症 (Complications of diabetes mellitus) 6
六、糖尿病的治療 (The treatment of diabetes mellitus) 6
第二節 血糖恆定 (Glucose homeostasis) 10
第三節 胰島素訊息傳遞路徑 (Insulin signaling pathway) 11
一、 PI3-K 的活化 13
二、胰島素誘導之磷酸化連鎖反應 (Insulin-stimulated phosphorylation cascades) 13
三、 Cbl/CAP 路徑與脂質筏 (Cbl/CAP pathway and lipid rafts) 14
第四節 腺苷單磷酸活化蛋白激酶 (AMP-activated protein kinase, AMPK) 14
一、 AMPK 的結構與調控 (Structure and regulation of AMPK) 15
二、 AMPK 與血糖恆定 (AMPK and Glucose homeostasis) 17
三、 AMPK 與糖尿病 (AMPK and diabetes mellitus) 19
第五節 NQ-1 之簡介 20
第六節 實驗動物 (Laboratory animals) 21
一、糖尿病小鼠 (db/db mice) 21
二、肥胖小鼠 (ob/ob mice) 21
第三章 材料與方法 (Materials and methods) 22
一、細胞培養 (Cell culture) 22
二、西方墨點法 (Western blot) 23
三、膜蛋白的萃取 (Membrane protein extraction) 25
四、結晶紫染色試驗 (Crystal violet assay) 25
五、腺嘌呤核苷酸分析 (Adenine nucleotides analysis) 26
六、葡萄糖攝取試驗 (Glucose uptake assay) 26
七、反轉錄聚合酶酵素連鎖反應 (Reverse transcriptase polymerase chain reaction, RT-PCR) 27
八、動物實驗 ( Animal experiment) 29
九、統計方法 (Statistics) 30
第四章 結果 (Results) 31
一、 NQ-1 在動物體內具有降血糖功效 31
二、老鼠肌纖維母細胞的分化 31
三、 NQ-1 對於細胞的毒性測試 32
四、 NQ-1 具有活化 AMPK 的能力 32
五、 Compound C 可以抑制由 NQ-1 誘發的 AMPK 磷酸化 33
六、 NQ-1具有促進細胞攝取葡萄糖的能力 33
七、 NQ-1 具有增加 AMP/ATP 比例的能力 34
八、 NQ-1 具有活化 PI3-K /Akt signaling pathway 的能力 34
九、 NQ-1 對 AS160 的影響 35
第五章 討論 (Discussion) 36
第六章 參考文獻 (References) 41
第七章 目次圖表 (Figures lists) 49
Figure 1. Anti-diabetic effect of NQ-1 in vivo experiment. 49
Table 1. Clinical and biochemical parameters in the db/db mice study. 50
Table 2. Clinical and biochemical parameters in the ob/ob mice study. 50
Figure 2. Differentiation of mouse skeletal myoblasts, L6 and C2C12. 51
Figure 3. Effect of NQ-1 on cell viability. 52
Figure 4. NQ-1 increased phosphorylation of AMPK and ACC in rat myoblast cells in a dose- and time-dependent manner. 53
Figure 5. NQ-1 increased phosphorylation of AMPK in HepG2, human hepatoma cells, in a dose- and time-dependent manner. 54
Figure 6. The effect of AMPK inhibitor Compound C on phosphorylation of AMPK after NQ-1 treatment in mouse L6 muscle cells. 55
Figure 7. NQ-1 promoted GLUT4 translocation and stimulated glucose uptake in skeletal muscle cells. 56
Figure 8. NQ-1 increased the cellular AMP/ATP ratio. 58
Figure 9. Phosphorylation of Akt serine-473, GSK3??/?? serine-21/9 and AS160 in differentiated L6 cells. 59
第八章 附錄 (Appendixes) 60

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