Study On The Hypoglycemic Activity And Mechanism Of Two Chinese Medicine Extracts

Diabetes（DM）is a characteristic multifactorial disease with abnormal response and reduced sensitivity to insulin.Usually this chronic metabolic disorder triggers many high-risk events in the body,including hypertension,central obesity,nephropathy,myocardial infarction,cerebrovascular injury and retinopathy.The number of people with DM in the world is rising,especially in China.Type 2 diabetes mellitus（T2DM）,as the most common form of DM,accounts for a large proportion of the the patient population.Insulin resistance（IR）is often defined as a typical pathophysiological feature of T2DM.It is a manifestation of the decrease in the response of the target tissue to insulin metabolic behavior,and is the result of a disorder of the glucose metabolism and gluconeogenesis.Glucose transporter type 4（GLUT4）is the only carrier in fat and muscle that responds to insulin transporting blood glucose into cells.In this study,we aim to use GLUT4 as a reference for exploring potential hypoglycemic drugs.Many of the hypoglycemic drugs currently used to regulate insulin or glucose have serious side effects and a heavy economic burden on patients with poor tolerance.Natural medicinal plants are increasingly used in modern clinical medicine for their superior curative effect and few side effects,as an alternative to T2DM.Therefore,we obtained dandelion chloroform extract（DCE）and ethanolic extract of Folium Sennae（FSE）from two natural plants and studied their hypoglycemic activity and its mechanism of action.Firstly,we studied the DCE.GLU-OX oxidase kit was used to determine the glucose consumption of L6 cells.The dynamic changes of GLUT4 translocation and Ca²⁺level in the stably expressing IRAP-mOrange L6 cells were observed by laser scanning confocal microscopy.Subsequently,a stable myc-GLUT4-mOrange L6 cell line was constructed and the plasma membrane（PM）fusion and insertion event of GLUT4 were monitored by FITC immunofluorescence label.Finally,real time-PCR and western blotting were used to investigate the expression of GLUT4 mRNA/protein and the phosphorylation levels of AMPK,PKB/Akt and PKC activated protein kinases,respectively.The results showed that DCE up-regulated the expression of GLUT4,promoted the translocation of GLUT4 and PM fusion,and ultimately led to glucose uptake.However,unlike PI3K inhibitor wortmannin and PKC inhibitor G?6983,the expression and trafficking of GLUT4 protein mediated by the extract were significantly weakened under AMPK inhibitor compound C.AMPK is the only protein with enhanced expression of phosphorylation in the assay,GLUT4 translocation induced by this extract is not related to Ca²⁺.These results indicate that DCE completes the intracellular activity of GLUT4 through AMPK signaling pathway,suggesting that DCE may be a new hypoglycemic agent for the treatment of T2DM.Secondly,we also explored the relationship between intracellular GLUT4 activity and Ca²⁺in L6 cells under the influence of another natural plant FSE and its specific hypoglycemic mechanism.The FSE not only stimulated glucose uptake,GLUT4expression and translocation,PM fusion,but also triggered intracellular Ca²⁺elevation,AMPK,Akt and PKC phosphorylation.Compound C,wortmannin,G?6983,G protein inhibitor PTX/Gallein and PLC inhibitor U73122 all attenuated FSE-mediated GLUT4translocation.Similarly,in addition to PTX/Gallein and U73122,the IP3R inhibitor 2-APB and 0 mM Ca²⁺-EGTA solutions also partially inhibited the elevation of intracellular Ca²⁺levels.0 mM Ca²⁺+10μM BAPTA-AM has a significant inhibitory effect on GLUT4 activity mediated by FSE.In summary,FSE regulates GLUT4expression and translocation by activating the AMPK,PI3K/Akt,and G protein–PLC–PKC pathways.FSE causes increasing Ca²⁺concentration to promote the fusion of GLUT4 vesicles with PM,allowing glucose uptake.Therefore,FSE may be a potential drug for improving T2DM.The above studies suggest that the discovery of the drug activity and molecular mechanism of natural medicinal plants provides a powerful help for finding a series of drugs that act on GLUT4 hypoglycemic activity,and provides a theoretical basis for the clinical study of insulin resistance or T2DM.