Alisol A 24-acetate Promotes Glucose Uptake And Alleviates Insulin Resistance In C2C12 Myotubes

Alisma orientalis(Sam.)Juzep is an traditional Chinese medicine with long cultivation history.Pharmacological researches have revealed its multiple bioactivities including diuresis,anti-inflammatory,anti-tumor,anti-hepatic steatosis,hypolipidemic and hypoglycemic.Studies have shown that crude extract of Alisma orientalis(Sam.)Juzep reduces blood sugar and protects pancreatic islets in diabetic rats.However,its effective substance and underlying mechanism still remain unclear.Alisol A-24-acetate(AA-24-a)is one of the main active triterpenes isolated from Alisma orientalis(Sam.)Juzep.Determination of AA-24-a is the quality control of Alisma orientalis(Sam.)Juzep.Through preliminary screening of bioactive substances in Alisma orientalis(Sam.)Juzep,we found that AA-24-a had significant effect on promoting glucose uptake in C2C12 myotubes.Herein,we investigated effects of AA-24-a on glucose metabolism and insulin sensitivity as well as its underlying mechanism in C2C12 myotubes and insulin resistant C2C12 myotubes induced by palmitate.Firstly,C2C12 myotubes were used to investigate the effect of AA-24-a on glucose uptake and its underlying mechanism.Cell glucose uptake was determined by cell 2-NBDG uptake and cell membrane protein was isolated to examine GLUT4 translocation.We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes.We further tested relative protein and gene expression levels by Western Blotting and q-PCR.While AA-24-a had no effect on phosphorylation levels of IRS1 at site of Tyr632 or AKT at site of Ser473 and Thr308,it do significantly upregulated phosphorylation levels of AMPK,ACC,AS160 and p38.This finding suggests that effects of AA-24-a on glucose metabolism may be associated with the AMPK pathway.Pre-treated with AMPK inhibitor Compound C reversed upregulation of glucose uptake,GLUT4 tanslocation and phosphorylation levels of AMPK,ACC,AS160 and p38 by AA-24-a.Pre-treated with Ca MKKβ inhibitor STO-609 also had the same effects,which proves AA-24-a promotes glucose uptake through Ca MKKβ-AMPK-p38/AS160 pathway.Furthermore we discovered that AA-24-a increased expression levels of mitochondrial biogenesis related gene ppargc1 a,tfam,nrf1,nrf2,tfb1 m and tfb2 m,which implies its potential mitochondrial function improving activity.After that,we prepared palmitate-BSA medium by which insulin resistant C2C12 myotubes were induced.We investigated effects of AA-24-a on insulin sensitivity in insulin resistant C2C12 myotubes and its underlying mechanism.Palmitate impaired insulin sensitivity,inhibiting insulin-mediated glucose uptake as well as insulin signalling transduction in C2C12 myotubes.AA-24-a restored phosphorylation levels of IRS1 at site of Tyr632 and AKT at site of Ser473 and Thr308 as well as glucose uptake under insulin stimulation compared with the model group,which manifests ameliorating of insulin resistance.At the meantime,AA-24-a upregulated phosphorylation levels of AMPK,AS160 and downregulated expression level of ER stress monitor Bi P.AMPK inhibitor Compound C blocked restoring effect of AA-24-a on insulin pathway and glucose uptake,and also weakened upregulation of phosphorylation levels of AMPK,AS160 and downregulation of expression level of Bi P.This proves that the insulin resistance ameliorating activity of AA-24-a is AMPKdependent.In summary,this study unveiled that AA-24-a upregulated phosphorylation levels of AMPK via Ca MKKβ in C2C12 myotubes and promoted glucose uptake via Ca MKKβ-AMPK-p38/AS160 pathway.In terms of insulin resistant C2C12 myotubes,AA-24-a alleviated insulin resistance by activation of AMPK which not only inhibited ER stress and its adverse effect on insulin pathway but also activated phosphorylation level of AS160,and consequently promoted insulin-mediated glucose uptake.Our findings clarify the effective substance and target of Alisma orientalis(Sam.)Juzep and provide scientific evidence for utilization and modernization of traditional Chinese medicine Alisma orientalis(Sam.)Juzep.Hopefully,this study may shed light on a new natural phytochemicals in possession of hypoglycemic and insulin resistance alleviating activity.