Jiang Baoping, Le Liang, Zhai Wei, Wan Wenting, Hu Keping, Yong Peng, He Chunnian, Xu Lijia, Xiao Peigen
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 151 Malianwa North, Haidian District, Beijing 100193, China; Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 151 Malianwa North, Haidian District, Beijing 100193, China.
Phytomedicine. 2016 Aug 15;23(9):891-900. doi: 10.1016/j.phymed.2016.05.004. Epub 2016 May 19.
Our previous study has shown that Coreopsis tinctoria increases insulin sensitivity and regulates hepatic metabolism in high-fat diet (HFD)-induced insulin resistance rats. However, it is unclear whether or not marein, a major compound of C. tinctoria, could improve insulin resistance. Here we investigate the effect and mechanism of action of marein on improving insulin resistance in HepG2 cells.
We investigated the protective effects of marein in high glucose-induced human liver carcinoma cell HepG2. In kinase inhibitor studies, genistein, LY294002, STO-609 and compound C were added to HepG2 cells 1h before the addition of marein. Transfection with siRNA was used to knock down LKB1, and 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG), an effective tracer, was used to detect glucose uptake.
The results showed for the first time that marein significantly stimulates the phosphorylation of AMP-activated protein kinase (AMPK) and the Akt substrate of 160kDa (AS160) and enhanced the translocation of glucose transporter 1 (GLUT1) to the plasma membrane. Further study indicated that genistein (an insulin receptor tyrosine kinase inhibitor) altered the effect of marein on glucose uptake, and both LY294002 (a phosphatidylinositol 3-kinase inhibitor) and compound C (an AMP-activated protein kinase inhibitor) significantly decreased marein-stimulated 2-NBDG uptake. Additionally, marein-stimulated glucose uptake was blocked in the presence of STO-609, a CaMKK inhibitor; however, marein-stimulated AMPK phosphorylation was not blocked by LKB1 siRNA in HepG2 cells. Marein also inhibited the phosphorylation of insulin receptor substrate (IRS-1) at Ser 612, but inhibited GSK-3β phosphorylation and increased glycogen synthesis. Moreover, marein significantly decreased the expression levels of FoxO1, G6Pase and PEPCK.
Consequently, marein improved insulin resistance induced by high glucose in HepG2 cells through CaMKK/AMPK/GLUT1 to promote glucose uptake, through IRS/Akt/GSK-3β to increase glycogen synthesis, and through Akt/FoxO1 to decrease gluconeogenesis. Marein could be a promising leading compound for the development of hypoglycemic agent or developed as an adjuvant drug for diabetes mellitus.
我们之前的研究表明，金鸡菊可提高高脂饮食（HFD）诱导的胰岛素抵抗大鼠的胰岛素敏感性并调节肝脏代谢。然而，金鸡菊的主要成分紫铆因是否能改善胰岛素抵抗尚不清楚。在此，我们研究紫铆因改善HepG2细胞胰岛素抵抗的作用及作用机制。
我们研究了紫铆因对高糖诱导的人肝癌细胞HepG2的保护作用。在激酶抑制剂研究中，在添加紫铆因前1小时，将染料木黄酮、LY294002、STO-609和化合物C添加到HepG2细胞中。使用小干扰RNA（siRNA）转染敲低肝激酶B1（LKB1），并使用一种有效的示踪剂2-（N-（7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基）氨基）-2-脱氧葡萄糖（2-NBDG）检测葡萄糖摄取。
结果首次表明，紫铆因可显著刺激AMP活化蛋白激酶（AMPK）和160kDa的Akt底物（AS160）的磷酸化，并增强葡萄糖转运蛋白1（GLUT1）向质膜的转位。进一步研究表明，染料木黄酮（一种胰岛素受体酪氨酸激酶抑制剂）改变了紫铆因对葡萄糖摄取的影响，并且LY294002（一种磷脂酰肌醇3激酶抑制剂）和化合物C（一种AMP活化蛋白激酶抑制剂）均显著降低了紫铆因刺激的2-NBDG摄取。此外，在存在CaMKK抑制剂STO-609的情况下，紫铆因刺激的葡萄糖摄取受到阻断；然而，在HepG2细胞中，紫铆因刺激的AMPK磷酸化未被LKB1 siRNA阻断。紫铆因还抑制胰岛素受体底物（IRS-1）在丝氨酸612处的磷酸化，但抑制糖原合成酶激酶-3β（GSK-3β）的磷酸化并增加糖原合成作用。此外，紫铆因显著降低叉头框蛋白O1（FoxO1）、葡萄糖-6-磷酸酶（G6Pase）和磷酸烯醇式丙酮酸羧激酶（PEPCK）的表达水平。
因此，紫铆因通过CaMKK/AMPK/GLUT1促进葡萄糖摄取、通过IRS/Akt/GSK-3β增加糖原合成以及通过Akt/FoxO1减少糖异生，改善了HepG2细胞中高糖诱导的胰岛素抵抗。紫铆因可能是一种有前途的先导化合物，可用于开发降血糖药物或作为糖尿病辅助药物。
