Study On The Mechanisms Of Cordycepin Activates AMPK And Regulates Lipid Metabolism

Cordycepin, also known as a nucleoside analog3’-deoxyadenosine, is a bioactive compound present in traditional Chinese medicine Cordyceps. Recently, many pharmaco-logical properties of cordycepin have been reported, including antiviral, antitumor and antifungal activity. Preliminary studies on hamsters fed a high-fat diet (HFD) found that cordycepin significantly decreased lipid in plasma, especially on TG levels. Western blot and luciferase gene assay indicate that effects of cordycepin to suppression TG synthesis have poor correlation with PPAR-y. Therefore, we believe that cordycepin lowering blood lipids mechanism is different from general antihyperlipidemics.Recently, cordycepin was shown to function as an activator of AMP-activated protein kinase (AMPK) through in vivo and in vitro study. Molecular docking results indicate that cordycepin binds to AMP-activated protein kinase with high affinity. The hydrogen bonds provide attractive forces between molecules. Therefore, we hypothesize that the effects of cordycepin regulate lipid metabolism may be related with AMPK, a sensor of cellular energy status. Effects of cordycepin on Oleic acid-induced lipid accumulation model in HepG2cells were observed by molecular biology, proteomics and RNA interference means were uesd to make sure whether AMPK is the main targets of cordycepin on regulation of lipids synthesis.MTT assay were used to investigate the action of growth inhibiting of cordycepin on human hepatocarcinoma cell line HepG2. Regulations of cordycepin on expression of phospho-AMPK were observed in HepG2treated with Oleic acid. The lipid accumulation in cells was observed by Oil-Red O stain and cells were collected for the estimation of cholesterol(TC), triglyceride(TG). HPLC, western blot and RNA interference were used to observe regulation effects of cordycepin on AMPK signaling pathway. The regulation of CCS on protein expression of AMPKγ and phospho-AMPK in AMPKγ silence cells were detected by western blot analysis.The data showed that cordycepin in different concentrations did not inhibition the activity of cells. Treatment with CCS can significantly increase the levels of phospho-AMPK in lipid accumulated cells, and the level of TC, TG were reduced. Firstly, we determined the intracellular AMP level and AMP:ATP ratio in HepG2cells treated with cordycepin using UPLC. We did not observe changes in the intracellular AMP concentration or the AMP:ATP ratio, which suggests that cordycepin actives AMPK through an AMP-independent pathway. Second, AMPK phosphorylation by the upstream AMPK kinases CaMKK and LKB1were tested by Western blot and RNA interference. Our result indicated that cordycepin-mediated AMPK activation is independent of CaMKK and LKB1activities. Finally the lentiviral which can ellectively inhibited protein expression levels of AMPKγ1were used to infect HepG2cells. Our data clearly show that cordycepin-mediated AMPK activation and regulation of intracellular TC, TG level was significantly impaired by the knockdown of the AMPKγ1subunit.Collectively, cordycepin-mediated AMPK activation is independent of intracellular AMP: ATP ratio or CaMKK and LKB1activities. These data indicate that cordycepin activates AMPK by directly binding and regulating AMPKy subunit, AMPK is the main target of cordycepin regulates lipid metabolism.