具降血糖活性藥物Quercetin和CAPE在C2C12細胞之分子機制研究

Abstract

背景：糖尿病是影響全世界人口健康的代謝疾病，隨著罹患糖尿病人數日益增加，尋找新的治療方法是必要的。Quercetin是一種flavonoid，存在許多天然物中，臨床研究顯示quercetin除了抗氧化外，還有其他生物活性，更有研究更指出quercetin可以預防糖尿病。CAPE為蜂蠟的主要成分，研究指出CAPE可以促進3T3-L1 adipocytes分泌adiponectin，可能有助於治療第二型糖尿病。本篇論文主要的研究方向是利用培養細胞來探討兩個具有降血糖作用的藥物quercetin與CAPE的作用機轉，以骨骼肌細胞C2C12作為探討帄台，確立quercetin與CAPE作為降血糖藥物的發展潛力。 材料及方法：本篇實驗使用C2C12小鼠骨骼肌細胞株作為細胞模式，看Quercetin與CAPE對AMPK和Akt活性的影響，並進一步測試Quercetin與CAPE對葡萄糖吸收、ATP含量、肝醣合成的改善作用。 結果： 本篇實驗結果發現Quercetin與CAPE可以促進C2C12小鼠骨骼肌細胞對於葡萄糖的吸收，兩藥分別皆在0.03 μM開始顯著增加葡萄糖再吸收的作用，在濃度10 μM作用最強。給予不同濃度的Quercetin 15分鐘，在濃度0.1 μM有明顯的活化AMPK的作用，而在0.3 μM和10 μM時AMPK有最大的磷酸化。在Quercetin 0.3 μM在30分鐘時AMPK才有明顯的磷酸化現象，到120分鐘達到最大磷酸化；而在Quercetin高濃度10 μM作用時，5分鐘時AMPK則已有磷酸化的現象，並在120分鐘時作用最大。CAPE 0.3 μM在30分鐘時AMPK磷酸化開始明顯增加，60分鐘至120分鐘達到最大磷酸化；而在CAPE高濃度10 μM作用下，5分鐘時AMPK即有磷酸化的現象，隨著藥物作用時間增加，AMPK磷酸化程度也逐漸上升，到120分鐘達到最大。然而Quercetin與CAPE活化Akt的模式與活化AMPK不同，給予不同濃度的Quercetin 15分鐘，在濃度0.03 μM下，Akt活化即有明顯增加，而在較高濃度的1 μM、3 μM與10 μM達到最大磷酸化。給予Quercetin 0.3 μM在 30分鐘時Akt磷酸化才明顯增加；給予Quercetin 10 μM，15分鐘時Akt開始被磷酸化，到30分鐘時Akt磷酸化程度最大。另外給予不同濃度的CAPE 15分鐘，Akt磷酸化程度在0.03 μM就增加，亦是在1 μM開始磷酸化的情形變得更明顯。給予CAPE 0.3 μM，Akt磷酸化程度在15分鐘時開始，到30分鐘增加；而給予CAPE 10 μM，Akt磷酸化現象在15分鐘開始被活化，隨著時間增加，Akt活化程度最高，到120分鐘時Akt磷酸化程度最大。 C2C12骨骼肌細胞以不同濃度Quercetin作用 15分鐘，在濃度0.03 μM時ATP的含量就有顯著的下降，濃度為10 μM下降最多；給予細胞不同濃度的CAPE 15分鐘，在濃度0.03 μM時ATP含量顯著的下降，且ATP含量隨著CAPE濃度的升高而降低。Quercetin與CAPE於低濃度(0.3 μM)和高濃度(10 μM)時，5分鐘時ATP的含量顯著的降低，且隨著藥物刺激時間的拉長而ATP含量逐漸回復，但還是比未加藥組來的低。間接證明了Quercetin與CAPE會藉由降低細胞的ATP含量來提高AMP：ATP的比率，進而活化AMPK，而隨著AMPK的活化，ATP的含量又漸漸攀升。 此外，Quercetin與CAPE增加AMPK與Akt的活化分別會被Compound C與Akt inhibitor抑制。而Quercetin與CAPE促進葡萄糖吸收與肝醣合成的作用亦會被Compound C與Akt inhibitor給抑制，但不會抑制ATP含量的降低。可以得知Quercetin與CAPE促進細胞的葡萄糖吸收與肝醣合成是經由AMPK與Akt兩條路徑。 結論：Quercetin與CAPE的降血糖活性的機制可能是藉由AMPK路徑與Akt路徑兩條路徑來調控，而Quercetin與CAPE活化AMPK與Akt是否和在生物體內降血糖作用有關以及彼此如何互相調控仍頇進一步研究。

Background: Diabetes mellitus is one of the most common metabolic disorders that disturbs lots of people’s health all over the world. With the growth of diabetic patients amount, the search for new agent in treatment of diabetes is necessary. Quercetin is a kind of flavonoid, existing in many nature products. Clinical research shows that quercetin possesses many biological activities besides anti-oxidative activity. There are some studies showing that quercetin can prevent diabetes. CAPE is a main component of propolis. There is a study suggesting that CAPE is able to stimulate 3T3-L1 adipocytes to secrete much more adiponectin, referring to assisting to treat Type II diabetes. The research purpose of this study is to use cultured cell figuring out the potential of quercetin and CAPE as antibiabetic drugs. Material and methods: The effects of quercetin and CAPE on activity of AMPK and Akt and the effects of both agents on glucose uptake, ATP level and glycogen synthesis were examined and compared in C2C12 myotube. Results: Quercetin and CAPE both significantly increased the glucose uptake and glycogen synthesis of C2C12 myotube at 0.03 μM and reached the strongest effect at 10 μM. In parallel with the stimulation of glucose uptake and glycogen synthesis, both quercetin and CAPE stimulated AMPK and Akt phosphrylation of C2C12 myotube in concentration and time-dependent manner. Quercetin started to stimulate AMPK phosphorylation at 0.3 μM while CAPE started to stimulate AMPK phosphorylation at 0.03 μM. Increasing concentrations of both agents from 0.3 to 10 μM shortened the onset time of AMPK phosphrylation from 30 to 5 minutes. The stimulation of AMPK phosphorylation by both agents reached highest level in 60 to 120 minutes. With regard to the effects on Akt, both quercetin and CAPE started to stimulate Akt phosphorylation at 0.03 μM. Increasing quercetin and CAPE concentrations from 0.3 to 10 μM did not shorten the onset time of Akt phosphorylation but increase the level of Akt phosphorylation. Treating with quercetin and CAPE resulted in a reduction of ATP level in C2C12 myotube concentration-dependently, ATP level was significantly decreased by both agents at 0.03 μM. Both agents induced initial decrease of ATP level in 5 to 15 minutes, which then recover with time to a level less than control condition. The stimulation of glucose uptake and glycogen synthesis by both agents was reduced by AMPK inhibition (Compound C) and Akt inhibition. The decrease of ATP level by both agents however remained unaffected by Compound C and Akt inhibitor. Conclusion: The present study proved that both quercetin and CAPE can stimulate glucose uptake and glycogen synthesis in C2C12 myotube. Since AMPK and Akt inhibitors could inhibit the stimulation effects of both agents on glucose uptake and glycogen synthesis, the stimulation effects of both agents on glucose utilization could be attributed to the stimulation of AMPK and Akt activity. The stimulation of AMPK activity by both agents may be related to the reduction of ATP level by both agents. The mechanisms of ATP depletion by both agents remain unknown and need further study.