具降血糖活性化合物CAPE和36-25A在HepG2細胞之分子機制研究

Abstract

背景：CAPE是蜂膠裡的主要成分之一，36-25A與CAPE為結構類似物，差別在於36-25A為具amide結構之caffeic acid衍生物。在骨骼肌細胞CAPE已被證實可促進葡萄糖的吸收，本篇實驗則研究在肝臟細胞中CAPE及其結構類似物36-25A是否也會影響能量的調控。 實驗方法與結果：本實驗使用HepG2人類肝癌細胞株作為細胞模式探討具降血糖活性之化合物CAPE及36-25A的作用機制，並以流式細胞儀分析CAPE及36-25A對細胞葡萄糖吸收作用之改變，再進一步以西方點墨法分析相關的訊息傳遞物質，另外也量測細胞內的ATP及肝醣含量以分析CAPE及36-25A對能量調控的影響。實驗結果發現CAPE及36-25A可以促進HepG2細胞對葡萄糖的吸收及增加HepG2細胞的肝醣含量。在HepG2細胞給予不同濃度的CAPE及36-25A刺激15分鐘後，於0.03 μM濃度下CAPE可顯著活化AMPK，而36-25A則從0.3 μM濃度下才可活化AMPK。從給予HepG2細胞不同給藥時間的實驗中則發現CAPE於給藥5分鐘後即可顯著活化AMPK，而36-25A則於給藥15分鐘後才能顯著活化AMPK，顯示與CAPE相比36-25A活化AMPK需較高的濃度及較長的時間。在活化Akt的部分則發現CAPE同樣於0.03 μM濃度下可顯著活化Akt，並於給藥5分鐘後即可活化Akt，而36-25A也於0.03 μM濃度下就明顯活化Akt，且同樣於給藥5分鐘後即可顯著活化Akt，顯示CAPE與36-25A在Akt調控路徑上的作用無明顯差異。進一步探討CAPE與36-25A對細胞ATP含量之影響，由實驗結果發現給予HepG2細胞不同濃度的CAPE及36-25A刺激15分鐘，可顯著降低細胞內ATP含量，且在時間點實驗中發現細胞內ATP的含量會隨著給藥時間拉長而增加，但仍都低於未給藥的對照組，顯示CAPE及36-25A活化AMPK後可能會抑制ATP的消耗並促進ATP的合成。此外CAPE及36-25A促進HepG2細胞對葡萄糖的吸收作用及增加HepG2細胞肝醣合成的作用皆會被compound C及Akt inhibitor所抑制，但compound C及Akt inhibitor不會影響CAPE及36-25A降低細胞內ATP含量的作用，顯示CAPE及36-25A可能是經由降低HepG2細胞內ATP的含量來活化AMPK及影響Akt的調控路徑，促進GLUT4轉移到細胞膜上，增加細胞對葡萄糖的吸收及促進細胞合成肝醣。

結論：總結本篇實驗結果，我們發現具降血糖活性之化合物CAPE及36-25A其增加HepG2細胞對葡萄糖的吸收之有效濃度與其活化AMPK及Akt等訊息傳遞物質之有效濃度相關，也證實CAPE及36-25A會經由降低細胞內ATP的含量來活化AMPK，但CAPE及36-25A如何降低細胞內ATP的含量與如何活化Akt等詳細的作用機制，及活化AMPK與Akt之間彼此如何相互調控仍有待更進一步的研究。

Background: Caffeic aicd phenethyl ester (CAPE) is an active component of propolis, while 36-25A is an analog to CAPE, that 36-25A is a caffeic acid amide derivative. A previous study revealed that CAPE can induce glucose uptake in skeletal muscle cells, so in this study we are interested in whether CAPE and its analog 36-25A are involved in the energy homeostasis in hepatic cells. Methods and Results: In this study, we used HepG2 human liver hepatocellular carcinoma cell line as cell models to investigate the mechanism of CAPE and 36-25A which possesses plasma glucose lowing activities. We use FLOW as a detector for glucose uptake effects induced by CAPE and 36-25A, and we perform the Western blot for identifying the related signaling pathways, furthermore, we measure the cellular ATP level and cellular glycogen content as an indicator for energy balance. The results showed that CAPE and 36-25A can induce glucose uptake in HepG2 cells and increase the cellular glycogen content in HepG2 cells. Giving different concentrations of CAPE and 36-25A for 15 minutes, we find that 0.03 μM CAPE significantly activates AMPK while 0.3 μM 36-25A activates AMPK. In the time course effects experiments we figure out that CAPE activate AMPK after dosing for 5 minutes while 36-25A induces HepG2 cells AMPK activation at 15 minutes. These data means that comparing to CAPE, 36-25A activate AMPK in a higher concentration and with a delayed time in onset of effects. Talking about activation of Akt we can see that CAPE and 36-25A activate Akt in the same concentration (0.03 μM) and in the same time manners (5 minutes after dosing). There is no difference in activating Akt between CAPE and 36-25A. Next, we further examined the influence of cellular ATP level caused by CAPE and 36-25A. The results shows that different concentration of CAPE and 36-25A giving for 15 minutes can significantly lower the cellular ATP level in HepG2 cells. Although there is an elevation in cellular ATP level as the incubation time prolongs, the ATP levels are still lower than control groups. We explain that the elevation is due to the activation of AMPK and which then inhibits the ATP consumption and induces the ATP synthesis. Besides, enhanced glucose uptake and glycogen synthesis caused by CAPE and 36-25A in HepG2 cells can be inhibited by compound C and Akt inhibitor. However, compound C and Akt inhibitors cannot influence the cellular ATP level lowered by CAPE and 36-25A, indicating that CAPE and 36-25A may decrease the cellular ATP level as an upstream to activate AMPK and also change Akt signaling pathway to trigger the translocation of GLUT4 to the plasma membrane and increase cellular glucose uptake and glycogen content. Conclusion: From the results of this study, we discover that CAPE and 36-25A can increase glucose uptake in HepG2 cells which is related to the activation of AMPK and Akt. We also confirm that CAPE and 36-25A activate AMPK by lowering cellular ATP level, but what is the detailed mechanism of CAPE and 36-25A in decreasing the cellular ATP level and how they activate Akt still need some deeper research. The activation of AMPK and Akt and the regulation between these two signaling pathways are the next issue of our interest.