NQ-1 經由活化 AMPK 路徑增加肌肉細胞對葡萄糖的攝取

NQ-1 increases glucose uptake through activation of AMP-activated protein kinase in muscle cells

陳鈺蒨(Yu-Chien Chen)

指導教授 ： 梁有志

臺北醫學大學/醫學院/醫學科學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00136

摘要

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。

關鍵字

糖尿病 ； 腺苷單磷酸活化蛋白激酶葡萄糖的攝取

並列摘要

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.

並列關鍵字

Diabetes mellitus ； AMP-activated protein kinase ； Glucose uptake

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