NR4A1 Affects Glucose Metabolism Dysfunction In C2C12 Myotube Cells Induced By Thapsigargin Through PI3K/AKT Signaling Pathway

Objective:Diabetes mellitus(DM)is a major chronic disease in China,which is characterized by impaired glucose metabolism due to Insulin resistance(IR)or secretion defects and elevated blood glucose levels.Persistent hyperglycemia is a key risk factor for many metabolic diseases.Skeletal muscle is the main site of blood glucose homeostasis.Under insulin stimulation,skeletal muscle promotes glucose uptake mainly through Phosphatidylinositol-3-kinase/Akt kinases(PI3K/ Akt)pathway.Orphan nuclear receptor4A1(NR4A1)is a kind of nuclear receptor.Many studies have proved that NR4A1 can participate in glucose metabolism regulation through various signaling pathways.NR4A1 participates in the glucose metabolism regulation,while PI3K/AKT pathway is the main regulatory pathway of glucose metabolism under insulin activation,both of them play an important role in glucose metabolism.Therefore,in this study,C2C12 myotube cells were used as experimental subjects,and Thapsigargin was used to construct a glucose metabolism damage model to explore whether there is a correlation between NR4A1 and PI3K/AKT signaling pathway,thus affecting glucose metabolism of C2C12 myotube cells.Methods:Experiment 1:(1)C2C12 cells cultured in vitro were induced for 0-6 days.Cells were collected at 0,2,4 and 6 days respectively.NR4A1 and markers of myotube differentiation Myogenic differentiation Antigen(Myo D)and Myogenin(Myo G),were detected by q PCR at different time points.(2)The differentiated C2C12 myotube cells were divided into NC group and insulin group.Serum-free medium was changed to starve for 6h.Insulin group was stimulated with 20 n M insulin for 10 min and NC group was treated with solvent PBS for 10 min.The phosphorylation levels of PI3 K and AKT were detected by Western blot.(3)The differentiated C2C12 myotube cells were treated with different Thapsigargin concentration gradients for 24 h,and CCK-8 method detected the cell viability.The concentration with no significant effect on cell viability was selected for subsequent experiments.(4)The differentiated C2C12 myotube cells were divided into NC group,Anhydrous ethanol solvent group and Thapsigargin group.NC group did not receive special treatment.Anhydrous ethanol solvent group added the solvent anhydrous ethanol as control.Thapsigargin group added 1μM Thapsigargin.All three groups were treated for 24 h.After 18 h,the serum-free medium was changed to starve for6 h,and 20 n M insulin was added for 10 min.Glucose consumption was detected by glucose kit,and Western blot detected NR4A1,p-PI3 K and p-AKT protein expressions.Experiment 2:(1)C2C12 cells were infected with lentivirus overexpressing NR4A1 and empty lentivirus.Purinomycin was added for screening.The overexpressing NR4A1C2C12 cells were named Ad-NR4A1 and the corresponding Ad-NC.The cells were observed under fluorescence microscope,and the infection efficiency was verified by q PCR and Western blot.(2)After screening,the cells were divided into Ad-NC,AdNC+Thapsigargin,Ad-NR4A1+Thapsigargin,and induced until day 6.After liquid exchange,the Ad-NC group was added with the solvent anhydrous ethanol,and the AdNC+Thapsigargin group and Ad-NR4A1+Thapsigargin group were added with 1μM Thapsigargin for 24 h.After 18 h,the serum-free medium was changed to starve for 6h,and 20 n M insulin was added for 10 min.Glucose consumption was detected by glucose kit,and Western blot detected NR4A1,p-PI3 K and p-AKT protein expressions.Results:Result 1:(1)Most of C2C12 cells differentiated into myotube cells 4-6 days after induction.QPCR results showed that the expression of Myo D and Myo G increased gradually with the extension of times(all P < 0.01),and the expression of NR4A1 in the differentiated myotube cells increased significantly(P < 0.01).(2)After insulin stimulation of C2C12 myotube cells,the PI3K/AKT pathway was activated(P < 0.01).(3)CCK-8 showed that the cell viability had no significant change when Thapsigargin concentration was 0.2-1μM(P > 0.05).When Thapsigargin concentration was higher than1μM(2μM、6μM),cell viability began to decrease(all P < 0.01).(4)Compared with NC group,anhydrous ethanol had no significant effects on glucose consumption,NR4A1 and PI3K/AKT pathway expression(all P > 0.05).Compared with anhydrous ethanol group,glucose consumption,NR4A1,p-PI3 K and p-AKT expression level were decreased after Thapsigargin treatment(all P <0.01).Result 2:(1)C2C12 cells overexpressing NR4A1 were successfully constructed.Fluorescence indicated successful virus infection,and Western blot and q PCR showed that NR4A1 was significantly up-regulated in Ad-NR4A1 group compared with Ad-NC group(all P<0.01).(2)Compared with Ad-NC+Thapsigargin group,glucose consumption was increased(P <0.05),and p-PI3 K,p-AKT signaling pathway were activated in AdNR4A1+Thapsigargin group after insulin stimulation(P <0.05,P <0.01).Conclusions:1.Thapsigargin inhibits glucose metabolism in C2C12 myotube cells.2.NR4A1 improves glucose metabolism dysfunction induced by Thapsigargin in C2C12 myotube cells,which may play a role through PI3K/AKT signaling pathway.