| Part1:Effect of recombinant human glucagon-like peptide-1(7-36)on the expression of Akt?p70S6K in human hepatocytes line HL7702ObjectiveThe objective of this study was to investigate the effect of recombinant human glucagon-like peptide-1(7-36)[ rh GLP-1(7-36)]on the expression of protein kinase B(Akt)and ribosomal protein S6 kinase(p70S6K)in HL7702 cells,and to find the best time.The mechanisms of rh GLP-1(7-36)were also discussed.MethodsHuman liver HL7702 cell line were cultured to exponential phase,HL7702 cell line was cultured in the medium containing 100 n M rh GLP-1(7-36).The expression of Akt,p-Akt(Ser473),p-Akt(Thr308)and p-70S6 K was determined at 0min,10 min,60 min and 90 min.The above protein expression was determined by Western Blot.ResultsThe expression of p-Akt(Thr308)protein increased with time and increased significantly at 90 min(P=0.001).The expression of p-p70S6K(Thr389)protein increased with time and increased significantly at 60 min and 90 min(P=0.045,P=0.004).There was no change in the expression level of Akt,p-Akt(Ser473)and p70S6 K protein in all groups.Conclusionrh GLP-1(7-36)promotes phosphorylation of Akt protein and p70S6 K protein,and the proteins increase with time and has the strongest effect at 90 min.This result provides a basis for studying the direct effect of GLP-1 on hepatocytes.Part2:The effect in Akt/m TOR/p70S6 K and Akt/GSK3 pathways by rh GLP-1(7-36)?tacrolimus?rh GLP-1(7-36) plus tacrolimus in human hepatocytes line HL7702ObjectiveThe HL7702 cells were treated by rh GLP-1(7-36)plus tacrolimus,the objective of which is to investigate the effect in Akt/m TOR/p70S6 K and Akt/GSK3 pathways by the combination in liver.MethodsHL7702 cells were cultured at the logarithmic growth phase,then they were divided into control group and experimental group.The experimental group were divided into Group Tac?Group GLP-1?Group GLP-1+Tac,and they were treated with 5mg/L concentrations of tacrolimus ? 100 n M concentrations of rh GLP-1(7-36)?5mg/L concentrations of tacrolimus combined with 100 n M concentrations of rh GLP-1(7-36)with fresh media for 90 min,respectively,and the control group was incubated with fresh media without tacrolimus and rh GLP-1(7-36)for 90 min.Western blot analysis was used to detect the total protein and phosphorylation levels of protein kinase B(Akt)?glycogen synthase kinase(GSK3)?glycogen synthase(GS)?ribosomal protein S6 kinase(p70S6K).Results1?Compared with the control group,the expression of Akt protein and p-Akt(Ser473)protein showed no changes in experimental groups,but the expression level of p-Akt(Thr308)protein was significantly increased(Tac group P=0.03,GLP-1 group P=0.01,GLP-1+Tac goup P=0.004).Compared with Tac group,there were no significant changes in the total protein and phosphorylation level of Akt in GLP-1+Tac goup.Compared with GLP-1,there were no significant changes in the total protein and phosphorylation level of Akt in GLP-1+Tac goup.2?Compared with the control group,the expression of p70S6 K protein showed no change in experimental groups,but the expression level of p-p70S6K(Thr389)protein was significantly increased(Tac group P=0.03,GLP-1 group P=0.02,GLP-1+Tac goup P=0.0047).Compared with Tac group,there were no significant changes in the total protein and phosphorylation level of p70S6 K protein in GLP-1+Tac goup.Compared with GLP-1,there were no significant changes in the total protein and phosphorylation level of p70S6 K in GLP-1+Tac goup.3?Compared with the control group,the expression of GSK3? protein and GSK3? protein showed no change in experimental groups;compared with the control group,the expression level of p-GSK3?(Ser21)protein wassignificantly decreased in the Tac group(P=0.02),but increased in the GLP-1 group and GLP-1+Tac group(P=0.003,P=0.01);compared with the control group,the expression level of p-GSK3?(Ser9)protein was significantly decreased in the Tac group(P=0.02),but increased in the GLP-1 group and GLP-1+Tac group(P=0.002,P=0.001).Compared with Tac group,there were no significant changes in the expression of GSK3? protein and GSK3? protein,but the expressions of p-GSK3?(Ser21)protein and p-GSK3?(Ser9)protein in GLP-1+Tac group were significant increased(all P<0.001).Compared with GLP-1,there were no significant changes in the total protein and phosphorylation level of GSK3 in GLP-1+Tac goup.4?Compared with the control group,the expression of GS protein showed no change in experimental groups,and the expression level of p-GS(Ser641)protein was significantly increased in Tac group(P=0.048),but decreased in GLP-1 group and GLP-1+Tac goup(all P=0.03).Compared with Tac group,there was no significant change in the expression of GS,but decreased in the expression of p-GS(Ser641)(P=0.001).Compared with GLP-1,there were no significant changes in the total protein and phosphorylation level of GS in GLP-1+Tac goup.Conclusion Under the conditions of this experiment:1?Akt/m TOR/p70S6 K signaling pathway was activated by Tac,which may promote fat synthesis by this way,and GSK3/GS signaling pathway was activated by Tac,which may inhibit GS activation to inhibit the production of glucose synthesis.2?GSK3/GS signaling pathway was inhibited by GLP-1,which may activate GS to induce the glucose synthesis.3?The activation of Tac by inhibiting GSK3/GS signaling pathway can be inhibited by GLP-1,which means GLP-1 may activate GS to induce the glucose synthesis.This study provides a certain experimental basis for the treatment of PTDM caused by tacrolimus by GLP-1 hypoglycemic agents. |
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