Study On The Antidiabetic Activity And Mechanism Of Andrographolide And Caragana Jubata

Glucose is the main energy source for mammalian cells,maintaining normal glucose metabolism is essential for physiological functions.Glucose metabolism depends on the uptake of glucose by cells.Glucose transporter（GLUT）is a family of transmembrane proteins that regulate the transport of extracellular glucose,GLUT4 is one of its members.It mainly exists in insulin sensitive tissues such as fat and skeletal muscle,and plays an important role in the physiopathology of type II diabetes mellitus（T2DM）.When regulating glucose metabolism in patients with T2DM,drugs,appropriate diet or exercise can be used to target the increased expression and transport of GLUT4,so as to reduce the excessive glucose load in the blood of T2DM patients.At present,all kinds of mature hypoglycemic drugs on the market have different ways of action,but they will cause different degrees of side effects.Therefore,it is very necessary to find new drugs for the treatment of T2DM.The aim of this study is to screen natural drugs with antidiabetic activity targeting GLUT4,and to promote the development of new hypoglycemic agents.After preliminary screening,we found that andrographolide（AND）and ethanol extract of Caragana jubata（CJEE）,two natural Chinese herbs with ethnic characteristics,had antidiabetic activity.Meanwhile,we studied the molecular mechanism underlying the hypoglycemic effects of these two drugs.First,we conducted experiments with AND as the research object.After L6skeletal muscle cells were cultured and differentiated into myoduct cells,glucose uptake of L6 cells was detected by glucose oxidase kit.The level of Ca²⁺and the fusion of GLUT4 with plasma membrane in myc-GLUT4-m Orange L6 cells stimulated by AND were detected by confocal laser scanning microscope.The expression and phosphorylation levels of GLUT4,Akt,AMPK and PKC were detected by western blot.The results showed that AND activated the phosphorylation of AMPK and PKC pathways,but did not affect the phosphorylation of Akt pathway.At the same time,G（?）6983 significantly inhibited the expression of AND induced GLUT4 under the addition of three pathway inhibitors Compound C（AMPK pathway inhibitor）,G（?）6983（PKC pathway inhibitor）and Wortmannin（PI3K/Akt pathway inhibitor）,respectively,while Compound C and Wortmannin did not.In addition,AND promoted the increase of Ca²⁺concentration in L6 cells and induced the translocation of GLUT4 and the uptake of glucose in a Ca²⁺dependent manner.Therefore,we suggest that AND promoted the expression of GLUT4 and its fusion with L6 plasma membrane in a Ca²⁺dependent manner through the PKC pathway,so as to enhance the ability of glucose uptake.Secondly,we also studied the hypoglycemic activity and molecular mechanism of CJEE targeting GLUT4 in vivo and in vitro.In vitro,CJEE stimulated glucose uptake and the expression and transport of GLUT4.CJEE also increased the phosphorylation of AMPK and PKC pathways,and promoted intracellular Ca²⁺release in L6 cells.When three pathway inhibitors,Compound C,G（?）6983 and Wortmannin,were incubated with CJEE,respectively,only G（?）6983 significantly inhibited the expression and transport of GLUT4 stimulated by CJEE.In addition,intracellular Ca²⁺significantly affected CJEE-induced GLUT4 translocation and glucose uptake,and both CJEE-induced the glucose uptake and the fusion of GLUT4with the plasma membrane were significantly inhibited when intracellular Ca²⁺was inhibited.In vivo experiments,we established a group of T2DM model mice by high-fat feeding combined with STZ injection.After four weeks of intragastric administration at CJEE,we found that CJEE could reduce the fasting blood glucose level and improve the oral glucose tolerance and insulin tolerance in diabetic mice.It could also improve the fat deposition and fat cell hypertrophy in liver of diabetic mice,remodel the morphology and function of pancreatic islets,and significantly improve the lipid metabolism disorder and insulin resistance induced by diabetes.Therefore,this study screened AND and CJEE,two natural ethnic drugs targeting GLUT4,and studied their antidiabetic activity and molecular mechanism.All the above experiments proved that AND and CJEE have certain antidiabetic activity,which is expected to be a potential drug for treating T2DM.