Effect Of Liraglutide On Expression Of Key Enzymes PEPCK And G6pase In Liver Gluconeogenesis And Its Mechanism

Objective:To investigate the effects of liraglutide on the expression of glycogenic key enzymes phosphoenolpyruvate carboxylase(PEPCK)and glucose-6-pho sphatase(G6Pase)in liver of diabetic mice and Hep G2-IR(Hep G2 insulin resistance)cells and their interaction mechanism,and to further explore the mechanism.Methods:1.In vivo study: The mice were randomly divided into three groups:liraglutide intervention group(male KK-AY diabetic mice,n=5),which was intraperitoneally injected with liraglutide(250ug/kg);Normal control group(male C57 mice,n=5)was intraperitoneally injected with the same volume of normal saline.Model control group(male KK-AY diabetic mice,n=5)was intraperitoneally injected with the same volume of normal saline.Mouse in the three groups were fed in the same environment and injected with drugs once a day from 16:00 to 17:00 every day for 8 weeks.After the drug intervention,the random blood glucose of mouse was detected.After the mice were killed,the liver tissue protein of mouse was extracted completely.The protein expressions of G6 Pase,PEPCK,Fox O1(forkhead transcription factor 1),Cry1(cryptochrome 1)and E3 ubiquitin-based protease DDB1(DNA damage-binding protein 1)were further detected by Western blot.2.The in vitro experiment was divided into two parts: the first part was the control group,i.e.the normal control group of Hep G2 cells;IR(insulin resistance)group,i.e.,model control group,which was treated with insulin intervention,and Hep G2-IR model was established;In liraglutide inter vention group,on the basis of Hep G2-IR,liraglutide(1000nmol/L)was used to intervene Hep G2-IR cells for 24h;Liraglutide +Exendin-(9-39)group:liraglutide(1000nmol/L)and Exendin-(9-39)(500nmol/L)for 24 h based on Hep G2-IR.The second part was the control group,namely the Hep G2 normal control group.Control +DMSO group: the same amount of DMSO was used to intervene Hep G2 cells to a certain extent,and the treated group was used as the solvent control group,in order to eliminate the interference of DMSO on the experimental results;IR+DMSO group: Insulin intervention in Hep G2 cells to establish Hep G2-IR model(same method as above);MG132 interven tion group: Hep G2-IR cells were treated with MG132(500NMO /L)for 12 h on the basis of Hep G2-IR.After the intervention,cell proteins were extracted from each group.Results Western Blot was used to detect the protein expression trends of PEPCK,G6 Pase,Fox O1,Cry1 and DDB1,and p< 0.05 was considered statistically significant.Results:1.In vivo experiment: The blood glucose measured in the liraglutide intervention group was significantly decreased compared with diabetic mice.WB results showed that:Compared with the normal control group,the protein expression levels of G6 Pase,Pepck,Fox O1 and DDB1 in the liver of diabetic mice were significantly increased,while the protein expression level of Cry1 was decreased.After the intervention of liraglutide,the protein expression levels of G6 Pase,Pepck,Fox O1 and DDB1 in the liver of diabetic mice were decreased.Meanwhile,The related expression of CRY1 showed a gradually increasing trend,and the difference was statistically significant(P < 0.05).2.In vitro experiment: the glucose content in supernatant of Hep G2 cells did not decrease after insulin intervention for 36 hours,while the protein expression levels of PEPCK and G6 Pase were increased by WB detection,which proved the existence of insulin resistance(IR);In the presence of IR,the protein expression level of DDB1 and Fox O1 was increased,while the protein expression level of CRY1 was decreased.The opposite trend appeared after the intervention of liraglutide,that is,the protein expression levels of G6 Pase and PEPCK,Fox O1 and DDB1 were decreased,but the protein expression level of CRY1 was significantly increased.The difference was statistically significant(p<0.05).Exendin-(9-39)was co-incubated with liraglutide to counteract the effect of liraglutide on protein expression.After the intervention of MG132 in Hep G2-IR cells,the expression of DDB1 was not significantly different,but the expression of CRY1 was significantly increased compared with the IR group,while the expression levels of G6 Pase,PEPCK and Fox O1 itself were decreased,with good statistical significance(p< 0.05).Conclusion:Liraglutide can down-regulate the expression of key glycogenic enzyme s G6 Pase and PEPCK in the liver of diabetic mice and Hep G2-IR cells,and its effect may be through down-regulation of E3 ubiquitin protease DDB1,thereby inhibiting the ubiquitination degradation of CRY1 and affecting the expression of Fox O1-mediated glycogenic key enzymes.