The Mechanism Of Berberine Enhanced Glucose Metabolism Through P38MAPK In Skeletal Muscle

Objective:Insulin resistance（IR）,which means dysfunctional of glucose uptake into the target peripheral tissue,like skeletal muscle and adipose tissue,is one oimportant role in the pathogenesis of type 2 diabetes mellitus（T2DM）.Skeletal muscle is an important tissue that utilizes glucose and maintains blood glucose balance,glucose transporter 4（GLUT4）is the main glucose transporter of skeletal muscle,and its protein expression and activity are related to IR.Mostly,the drugs for ameliorating IR are focused on promoting GLUT4 translocation,the p38MAPK signaling pathway playsanimportantroleinIRbyregulatingGLUT4activityand translocation.Berberine（BBR）,which belongs to the alkaloids of isoquinoline,has been demonstrated to have many pharmacological activities,such as anti intestinal bacterial infection,antiarrhythmic,promoting insulin secretion and ameliorating IR.The improvement of IR is related to the regulation of GLUT4 protein expression and activity,but the mechanism is not very clear.Therefore,we used T2DM mouse model and C2C12 skeletal muscle cells model in this study,and based on molecular docking simulation to study whether BBR improves IR through p38MAPK signaling pathways to regulate GLUT4 protein expression and activity,this study will further clarify the mechanism of BBR in improving IR,and provide a better experimental basis for the prevention and treatment of T2DM.Methods:1.BBR improved glucose metabolism in C2C12 myotubesC2C12 Myotubes model was established and treated with different dose berberine(0.25,0.5,1μmol·L^-1).Glucose consumption was determined by the glucose oxidase method,glucose uptake was measured by 2-NBDG method.Molecular docking simulation studies of the interaction between BBR and insulin receptor（InsR）,GLUT1,GLUT4 were performed on computer.InsR-β,GLUT1,GLUT4,p-p38MAPK,p38MAPK protein levels were measured by Western blot.The translocation of GLUT4 to plasma membrane was detected by immunofluorescence assay.2.BBR improved glucose metabolism in T2DM mouse modelTo develop T2DM model,C57BL/6J mice were treated with high fat diet（45%）combine with 120mg/kg STZ injection intraperitoneally.The diabetic rat models were randomly divided into 6 groups,model group,positive control group,BBR low,medium,high dose Group and medium dose BBR combined with cinnamon aldehyde（CA）（10:1）.After 6 weeks,fasting blood glucose（FBG）,fast serum insulin level（FINS）and homeostasis model assessment for insulin resistance（HOMA-IR）were measured.Results:1.The expreimental results in C2C12 myotubes were as follow:（1）C2C12 myotubes differentiated successfully:C2C12 were cultured for 4-5days,in morphology,cell gradually became longer,and more than 80%of the myoblasts could differentiate into multinuclear myotubes;Compared with undifferentiated C2C12,the expression of myogenin in myotubes significantly increased within 4 days,which indicated that the induced differentiation was successful and could be used in subsequent experiments.（2）BBR had no toxicity below the concentration of 2.5μmol·L^-1:Treated before and after differention C2C12 with 0、2.5、10、40μmol·L^-1concentration of BBR for24h,found that 40μmol·L^-1had no significant effects on cell activity before differentiation;2.5μmol·L^-1had no significant effects on cell activity after differentiation,differentiated cell morphology changed obviously at dose of 10,40μmol·L^-1,so BBR concentration at 2.5μmol·L^-1and below had no toxicity to C2C12 myotubes.（3）The glucose metabolism effects of BBR in C2C12 myotubes:we used GOD-POD and 2-NBDG methods and found that BBR significantly increased glucose consumption and glucose uptake in C2C12 myotubes,which meant that BBR can increase glucose metabolism in C2C12 myotubes.（4）Molecular docking simulation studies between BBR andGLUT4:The results showed that interactions between berberine and GLUT4 involvedπ-π,meanwhile,interactions with InsR、GLUT1 involved hydrogen bonds,which means that BBR may had better activity.The docking score of berberine with insulin receptor（InsR）,GLUT1 and GLUT4 were-5.370,-8.117,-7.274,which meant combination between BBR and InsR、GLUT1、GLUT4.（5）The effect of BBR in GLUT4 and phosphorylation level of p38MAPK:We found that although GLUT1 and GLUT4 total protein expression had no differences,but BBR inhibited the expression of InsR-β,and BBR can stimulate GLUT4 moved to cell membrane and meanwhile increase phosphorylation level of p38MAPK.2.The expreimental results in vivo study were as follow:（1）C57BL/6J mice fed a high fat（45%calories）diet for 8 weeks and then120mg/kg STZ injected intraperitoneally,FBG≧11.1mmol/L was choosed as T2DM model,then randomly divided into 6 groups for the next study;（2）BBR decreased FBG、FINS、HOMA-IR,and BBR combined with CA works better than BBR alone.Conclusion:1.BBR can increase glucose metabolism in skeletal muscle cells,stimulate the translocation of GLUT4,which may through inhibiting InsR-β、increasing phosphorylation levels of p38MAPK.2.BBR can ameliorate T2DM mouse model insulin resistance,and the combination with CA has an better effect.