Effect And Mechanism Of Liraglutide On Liver Fat Content In Obese Type 2 Diabetes Patients With Nnonalcoholic Fatty Liver Disease

With the improvement of economic level and the change of people’s lifestyle,the global prevalence of nonalcoholic fatty liver disease(NAFLD)has increased significantly.Epidemiological survey results show that the prevalence of NALFD in China is as high as 29.8%,and the prevalence of NAFLD in type 2 diabetes and obese patients has further increased,with a higher risk of liver fibrosis.Although the progress of NAFLD is slow,if it progresses to liver fibrosis,the all-cause mortality rate will increase and seriously threaten human health.At present,the pathogenesis of NAFLD is not completely clear,and lack of effective treatment.Therefore,it is of great clinical significance to explore the pathogenesis and therapeutic targets of NAFLD.Ferroptosis which characterized by oxidative stress and cell lipid peroxidation caused by intracellular iron overload is a new type of cell death.Studies found that ferroptosis may lead to hepatic fibrosis from simple hepatocyte steatosis.Patients with NAFLD have abnormal iron metabolism and liver iron accumulation and iron chelator can improve the liver pathology.Inhibition of ferroptosis may become a new strategy for the treatment of NAFLD.Liraglutide is a glucagon-like peptide-1(GLP-1)receptor agonist,which can stimulate proliferation and differentiation of β cell and inhibit secretion of glucagon,thereby inhibiting appetite and delaying gastric emptying.Recent studies have shown that in addition to the above effects,liraglutide also has the effects of antioxidant stress,improving vascular endothelial function and reducing intrahepatic fat accumulation.The latest research shows that liraglutide can inhibit ferroptosis in the liver of diabetes mice and alleviate oxidative stress injury in the liver.In this study,obese type2 diabetes patients with NAFLD were treated with liraglutide.The effects of liraglutide on body weight,body composition,glucose and lipid metabolism,iron metabolism,oxidative stress and liver fat content were observed.Through the induction of HepG2 cells by palmitic acid/oleic acid,to explore whether ferroptosis is involved in the pathogenesis of NAFLD and whether liraglutide can play a beneficial role by inhibiting ferroptosis.Part 1 Effect of liraglutide on liver fat content in obese type 2 diabetes patients with nonalcoholic fatty liver diseaseObjective: To observe the effect of liraglutide on body composition,glucose and lipid metabolism,iron metabolism,oxidative stress and liver fat content in obese type 2 diabetes patients with NAFLD.Methods: Forty five obese type 2 diabetes patients with NAFLD aged18-65 years old were enrolled,totally 24 weeks.They were treated with liraglutide and given routine diet and exercise guidance.Blood pressure,height,weight,waist circumference of the subjects before and after treatment were measured,and body mass index(BMI)was calculated;Biochemical indexes before and after treatment were measured: fasting blood glucose,blood lipids,glycosylated hemoglobin(Hb A1c),transaminase,serum albumin;Iron metabolism index: serum iron,hepcidin,serum ferritin;Oxidative stress index: superoxide dismutase(SOD)and malondialdehyde(MDA).Body composition and controlled attenuation parameters(CAP)were also measured.Compare the changes of above indicators before and after the treatment.Analyze the correlation between the change of CAP and the difference of clinical indicators and the influencing factors of the change of CAP.Observe the safety of liraglutide at the same time.Results:1.After 24 weeks of treatment by liraglutide,the body weight,body mass index,waist circumference,skeletal muscle content,body fat content,body fat rate and visceral fat area of the subjects were significantly lower than those before treatment(P<0.05).2.24 weeks treatment of liraglutide decreased the levels of fasting blood glucose,low density lipoprotein cholesterol,triglyceride,glycosylated hemoglobin,alanine aminotransferase and aspartate aminotransferase,insulin resistance index,ferritin and hepcidin of the subjects(P<0.05);High density lipoprotein cholesterol significantly increased(P<0.05).Systolic blood pressure,diastolic blood pressure,total cholesterol and serum albumin decreased compared with those before treatment but there were no statistical difference(P>0.05).The serum iron level was slightly higher than that before treatment but there was no statistical difference(P>0.05).3.Compared with baseline before treatment,after 24 weeks of treatment by liragutide,the level of MDA in the subjects decreased and the activity of SOD increased(P<0.05).4.After 24 weeks of treatment by liraglutide,the CAP of the subjects was significantly lower than that before treatment(P<0.05).5.Correlation analysis shows that the reduction of CAP was positively correlated with the changes in body weight,body mass index,waist circumference,visceral fat area,ferritin,hepcidin and MDA(r=0.630,0.661,0.355,0.360,0.324,0.638,0.496,P<0.05)and negatively correlated with the changes in high-density lipoprotein cholesterol and SOD(r=-0.372,-0.495,P<0.05).Multiple linear regression analysis shows that the changes of body mass index,ferritin,hepcidin and MDA were independent influencing factors of the reduction of CAP(P<0.05).6.After treatment with liraglutide,3 patients(6.7%)suffered from intolerable diarrhea,nausea and anorexia but the symptoms gradually relieved after stopping treatment.During the study,8 patients(17.8%)suffered from nausea and anorexia,2 patients(4.4%)suffered from constipation,2 patients(4.4%)suffered from weakness,1 patient(2.2%)suffered from headache and1 patient(2.2%)suffered from palpitation.With the adjustment of diet structure and the extension of treatment,the symptoms relieved.No hypoglycemia and serious adverse events occurred during the treatment.Summary:1.Treatment with liraglutide for 24 weeks can significantly reduce weight,waist circumference,body fat content and visceral fat area and improve body composition in obese type 2 diabetes patients with NAFLD,which have no malnutrition.2.Treatment with liraglutide for 24 weeks can significantly improve the disorder of glucose and lipid metabolism,reduce insulin resistance,reduce the level of alanine aminotransferase and aspartate aminotransferase and improve liver function in obese type 2 diabetes patients with NAFLD.3.Treatment with liraglutide for 24 weeks can significantly reduce liver fat content,reduce liver iron accumulation,improve the ability to resist oxidative stress and reduce lipid peroxidation in obese type 2 diabetes patients with NAFLD.4.After 24 weeks of treatment with liraglutide,the decline of liver fat content in obese type 2 diabetes patients with NAFLD is closely related to the decrease of body weight,the improvement of iron metabolism and the decrease of lipid peroxidation.Liraglutide may improve the accumulation of fat in the liver of NAFLD patients by inhibiting ferroptosis.5.Treatment with liraglutide alone for obese type 2 diabetes patients with NAFLD is well tolerated,low incidence of hypoglycemia and with high safety.Part 2 Liraglutide inhibits ferroptosis in HepG2 cells induced by palm-itic acid/oleic acid and reduces the content of intracellular lipidsObjective: To explore whether ferroptosis is involved in the pathogenesis of NAFLD and the intervention mechanism of liraglutide in HepG2 cells induced by palmitic acid/oleic acid.Methods: HepG2 cells were divided into control group(Con group):cells were incubated in MEM medium containing 10% FBS for 24 hours;free fatty acids group(FFA group): cells were incubated in MEM medium containing 200μM/100μM palmitic acid/oleic acid for 24h;liraglutide intervention group(FFA+LI group): cells were incubated in MEM medium containing 100 n M liraglutide and 200μM/100μM palmitic acid/oleic acid for24 hours.The content of triglyceride in cells of each group was detected and the lipid deposition was observed by oil red O staining.The content of glutathione(GSH),malondialdehyde(MDA),iron and the activity of superoxide dismutase(SOD)in each group were measured.Real time PCR and western bloting were used to detect the content of gene and protein expression of recombinant solute carrier family 7 member11(SLC7A11),glutathione peroxidase 4(GPX4),transcription factor E2 related factor 2(Nrf2)and transferrin receptor 1(TFR1)in each group.Results:1.Compared with Con group,the content of orange lipid droplets and triglycerides in HepG2 cells increased significantly in FFA group;After intervention with liraglutide,the contents of orange lipid droplets and triglycerides in HepG2 cells were significantly decreased in FFA+LI group,but still higher than those in Con group(P<0.05).2.Compared with Con group,the SOD activity and the level of GSH in HepG2 cells were significantly decreased in FFA group;After intervention with liraglutide,the SOD activity and the level of GSH in HepG2 cells were significantly higher in FFA+LI group,but still lower than those in Con group(P<0.05).3.Compared with Con group,the level of MDA and iron content in HepG2 cells were significantly higher in FFA group;After intervention with liraglutide,the level of MDA and iron content in HepG2 cells in FFA+LI group were significantly lower than those of FFA group,but still higher than those in Con group(P<0.05).4.Compared with Con group,the protein expression of SLC7A11 in HepG2 cells in FFA group were significantly lower(P<0.05).After intervention with liraglutide,the protein expression of SLC7A11 increased in FFA+LI group,but there was no significant differences compared with FFA group(P>0.05).Compared with Con group,the protein expression of Nrf2 and GPX4 in HepG2 cells were significantly decreased in FFA group.After intervention with liraglutide,compared with FFA group,the protein expression of Nrf2 and GPX4 in HepG2 cells were significantly higher in FFA+LI group,but still lower than those in Con group(P<0.05).The protein expression of TFR1 in HepG2 cells was significantly increased in FFA group compared with Con group.After intervention with liraglutide,compared with FFA group,the protein expression of TFR1 in HepG2 cells decreased significantly in FFA+LI group,but was still higher than those in Con group(P<0.05).5.Compared with Con group,the m RNA expression of SLC7A11 in HepG2 cells in FFA group were significantly lower(P<0.05).After intervention with liraglutide,the m RNA expression of SLC7A11 increased in FFA+LI group,but there was no significant differences compared with FFA group(P>0.05).Compared with Con group,the m RNA expression of Nrf2 and GPX4 in HepG2 cells were significantly decreased in FFA group.After intervention with liraglutide,compared with FFA group,the m RNA expression of Nrf2 and GPX4 in HepG2 cells were significantly higher in FFA+LI group,but still lower than those in Con group(P<0.05).The m RNA expression of TFR1 in HepG2 cells was significantly increased in FFA group compared with Con group.After intervention with liraglutide,compared with FFA group,the m RNA expression of TFR1 in HepG2 cells decreased significantly in FFA+LI group,but was still higher than those in Con group(P<0.05).Summary:1.The lipid content increased in HepG2 cells induced by palmitic acid/oleic acid and liraglutide can reduce the content of intracellular lipid.2.HepG2 cells induced by palmitic acid/oleic acid showe the characeristics of ferroptosis,such as decreased antioxidant stress activity,iron overload and increased cell lipid peroxidation;At the same time,the expression of ferroptosis related proteins SLC7A11,Nrf2 and GPX4 decreases and the expression of TFR1 increases,suggesting that ferroptosis is involved in the pathogenesis of NAFLD.3.Liraglutide can improve the antioxidative stress ability of HepG2 cells induced by palmitic acid/oleic acid,reduce intracellular iron overload,and reduce cell lipid peroxidation through up-regulate ferroptosis related protein expression of Nrf2 and GPX4 and down-regulate TFR1,thereby inhibiting ferroptosis.Conclusion:1.Liraglutide may improve liver lipid accumulation in obese type 2diabetes patients with NAFLD by inhibiting ferroptosis.2.HepG2 cells induced by palmitic acid/oleic acid exhibit characteristics of ferroptosis such as reduced antioxidant stress capacity,iron overload,and increased cell lipid peroxidation.3.Liraglutide inhibits ferroptosis through upregulating the expression of ferroptosis related proteins such us Nrf2 and GPX4 and downregulating the expression of TFR1 and reduces lipid deposition in HepG2 cells induced by palmitic acid/oleic acid.