| Objective:The purpose of this study is to investigate whether liraglutide,a glucagon-like peptide 1(GLP-1)analogue,can improve insulin resistance(IR)of rat L6 skeletal muscle cells(L6 cells)incubated with palmitic acid(PA)through Sestrin2(Sesn2)-mediated autophagy.Method:1.The IR model of L6 cells was established by PA induction,and the specific experimental groups were as follows:(1)L6 cells were treated with liraglutide at concentrations of 10,100 and 1000nmol/L to observe the effect of liraglutide on cell activity;(2)L6 cells were treated with 0.2,0.4,0.6 and0.8mmol/L PA,and the optimal intervention concentration of PA was selected.(3)Control group(Con),PA 0.6 mmol/L treatment group(PA),PA 0.6mmol/L combined with liraglutide 10 nmol/L treatment group(PA+Lir10group),PA 0.6 mmol/L combined with liraglutide 100 nmol/L(PA+Lir100group)and PA 0.6 mmol/L combined with liraglutide 1000 nmol/L treatment group(PA+Lir1000 group).Cell activity was detected by CCK-8 method.Glucose concentration in cell medium was determined by glucose oxidase-catalase method.The expression of Sesn2 protein,IR-associated GLUT4,Akt,p-Akt and autophagy associated proteins LC3 I,LC3II and P62 were detected by Western Blot.The mrna expressions of Sesn2,GLUT4,LC3 II and P62 were detected by real-time quantitative polymerase chain reaction(RT-PCR).2.Inhibition of intracellular autophagy level with autophagy inhibitor3-Methyladenine(3-MA),specific group: Control group(Con group),autophagy inhibitor 3-MA 5 mmol/L treatment group(3-MA group),3-MA 5mmol/L combined with 0.6 mmol/L PA treatment group(3-MA+PA group),3-MA5 mmol/L,PA0.6mmol/L and liraglutide 100nmol/L combined treatment group(3-MA+PA+Lir100 group).The protein expressions of Sesn2,GLUT4,Akt,p-Akt,LC3 I,LC3II and P62 were detected by Western Blot.3.L6 cells were transfected with a small interfering RNA(si RNA)technique.According to different treatments,the cells were divided into control group(Con group),transfection negative group(si-Con group),transfection si-Sesn2 group(si-Sesn2 group),transfection si-Sesn2 after PA0.6mmol/L group(PA+si-Sesn2 group),transfection si-Sesn2 after PA0.6Five groups were treated with mmol/L and liraglutide 100nmol/L(Lir100+PA+si-Sesn2 group).Glucose concentration in L6 cell medium was detected by glucose oxidase-catalase method.The protein levels of Sesn2,GLUT4,Akt,p-Akt,LC3 I,LC3II and P62 and m RNA levels of Sesn2,GLUT4,LC3 II and P62 were detected by Western Blot and RT-PCR.Results:1.IR model of L6 cells induced by PA and the intervention results of different concentrations of liraglutide.1.1 Comparison of cell activity among different groups.Compared with Con group,there was no significant change in the activity of L6 cells treated with liraglutide at concentrations of 10,100 and1000nmol/L(P>0.05).Compared with Con group,the activity of L6 cells in PA group decreased significantly at 0.6 mmol/L concentration(P<0.001).Compared with PA group,the cell activity in PA+Lir10,PA+Lir100 and PA+Lir1000 groups was increased(P<0.001).Compared with PA+Lir10group,the cell activity in PA+Lir100 group and PA+Lir1000 group was increased(P<0.001).1.2 Comparison of glucose levels in cell culture media of different groups.Compared with Con group,glucose concentration in L6 skeletal muscle cell medium in PA group was significantly higher(P<0.001).Compared with PA group,after liraglutide intervention,glucose concentration in L6 skeletal muscle cell medium in PA+Lir10,PA+Lir100 and PA+Lir1000 groups was significantly lower than that in PA group,with statistical significance(P<0.05 or P<0.001).Compared with PA+Lir10 group,glucose concentration in L6 cell medium in PA+Lir100 and PA+Lir1000 groups was significantly decreased,and the difference was statistically significant(P<0.001).1.3 Expression of IR-related factors in different groups of cells.Compared with Con group,the GLUT4 protein and m RNA expression and p-Akt/Akt protein ratio in L6 cells of PA group were significantly down-regulated,with statistical significance(P<0.05 or P<0.01).Compared with PA group,the GLUT4 protein and m RNA expression and p-Akt/Akt protein ratio in PA+Lir10 group were not significantly changed after liraglutide treatment(P>0.05);the GLUT4 protein and m RNA expression and p-Akt/Akt protein ratio in PA+Lir100 and PA+Lir1000 groups were significantly increased(P<0.05 or P<0.01).1.4 Expression of Sesn2 and changes of autophagy in cells of each group.Compared with Con group,the expression of Sesn2 protein and m RNA in L6 cells in PA group decreased,with statistical significance(P< 0.05 or P<0.01).Compared with PA group,the expression of Sesn2 protein and m RNA in L6 cells in PA+Lir10 group did not change after liraglutide intervention,and the difference was not statistically significant(P>0.05);the expression of Sesn2 protein and m RNA in L6 cells of PA+Lir100 group and PA+Lir1000group were increased,and the difference was statistically significant(P<0.05).Compared with Con group,LC3 II/LC3 I protein ratio and LC3 II m RNA expression in L6 cells of PA group were decreased,while P62 protein and m RNA expression levels were increased,with statistical significance(P< 0.05 or P<0.01).Compared with PA group,the m RNA expression of P62 in L6 cells of PA+Lir10 group decreased after liraglutide intervention,and the difference was statistically significant(P<0.05);LC3 II/LC3 I protein ratio and LC3 II m RNA expression in L6 cells of PA+Lir100 group and PA+Lir1000 group were increased,while P62 protein and m RNA expression levels were decreased,with statistical significance(P<0.05 or P<0.01).2.Changes of autophagy level and IR-related factors in each group after administration of autophagy inhibitorCompared with Con group,after the intervention of autophagy inhibitor3-MA,LC3II/LC3 I protein ratio in 3-MA group was decreased,and P62 protein expression was increased,with statistical significance(P<0.05).Compared with 3-MA+PA group,LC3II/LC3 I protein ratio and P62 protein expression in 3-MA+PA+Lir100 group had no significant changes after liraglutide intervention(P>0.05).Compared with Con group,the p-Akt/Akt protein ratio and GLUT4 protein expression in L6 cells of 3-MA group were down-regulated,and the differences were statistically significant(P<0.05 or P<0.01).Compared with the 3-MA intervention group,the P-Akt /Akt protein ratio and GLUT4 protein expression in L6 cells of the 3-MA+PA group were down-regulated after incubation with PA,and the differences were statistically significant(P<0.05).Compared with 3-MA+PA group,the p-Akt/Akt protein ratio and GLUT4 protein expression in L6 cells of 3-MA+PA+Lir100 group had no significant changes after liraglutide intervention(P>0.05).3.Changes in Sesn2 expression after transfection with si-RNA knockdown3.1 Changes of Sesn2 expression level in each group after si-RNA transfection.Compared with Con group,there were no significant differences in the expression of Sesn2 protein and m RNA in L6 cells of si-Con group(P>0.05).Compared with si-Con group,the expression levels of Sesn2 protein and m RNA in L6 cells transfected with si-Sesn2 group were significantly decreased,with statistical significance(P<0.05).Compared with PA+si-Sesn2 group,there was no significant increase of Sesn2 protein in L6 cells of Lir100+PA+si-Sesn2 group after liraglutide intervention(P>0.05),indicating that Sesn2 expression was successfully suppressed.3.2 Changes of autophagy level in each group after si-RNA transfection.Compared with Con group,LC3 II/LC3 I protein ratio,LC3 II m RNA expression,P62 protein and m RNA expression in L6 cells of si-Con group had no significant changes(P>0.05).Compared with si-Con group,LC3 II/LC3 I protein ratio and LC3 II m RNA expression in si-Sesn2 group and PA+si-Sesn2 group were decreased,while P62 protein and m RNA expression were increased,with statistical significance(P<0.05).Compared with PA+si-Sesn2 group,LC3 II/LC3 I protein ratio,LC3 II m RNA and P62 protein and m RNA expression levels in L6 cells of Lir100+PA+si-Sesn2 group were not significantly changed after liraglutide intervention(P>0.05).3.3 Changes of IR-related glucose concentration,GLUT4 expression and p-Akt/Akt protein ratio in each group after si-RNA transfectionCompared with Con group,glucose concentration,GLUT4 protein and m RNA and p-Akt/Akt protein ratio in cell medium of si-Con group had no significant changes(P>0.05).Compared with si-Con group,glucose concentration in cell culture-medium of si-Sesn2 group increased,GLUT4 protein and m RNA expression decreased(P<0.05 or P<0.01).GLUT4 protein and m RNA and p-Akt/Akt protein ratio decreased in L6 cell culture-medium of PA+Sesn2 group with increased glucose concentration,and the differences were statistically significant(P<0.05 or P<0.01).Compared with PA+si-Sesn2 group,there were no significant changes in glucose concentration,GLUT4 protein and m RNA and p-Akt/Akt protein ratio in Lir100+PA+si-Sesn2 group after liraglutide intervention(P>0.05).Conclusions:1 High lipid culture resulted in decreased Sesn2 expression,autophagy inhibition and IR in L6 cells.Liraglutide restored Sesn2 expression and autophagy levels and improved IR in high lipid culture L6 cells in a concentration-dependent manner.2 Liraglutide improves the IR of hyperlipid-induced L6 cells by enhancing Sesn2-mediated autophagy levels. |
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