Liraglutide Ameliorates Non-alcoholic Fatty Liver Disease Through ADCY3 Regulating PI3K/Akt Signaling Pathway

Objective:The high prevalence of nonalcoholic fatty liver disease(NAFLD)among patients with type 2 diabetes has implicated the role of hepatic insulin resistance in the diseases.Glucagon-like peptide 1(GLP-1)was primarily effective in alleviating the insulin resistance of the liver,which was proven to be effective in alleviating NAFLD.Our previous research found that glucagon-like peptide 1 GLP-1 receptor analogue liraglutide can increase the serum adenylate cyclase 3(ADCY 3)levels in newly diagnosed diabetic patients.Also,the serum ADCY 3 level and homeostasis model assessment of insulin resistance(HOMA-IR)was negatively correlated.Animal studies have also found that liraglutide can reduce non-alcoholic fatty lesions in the liver of obese mice and increase the expression of ADCY 3 in the liver of the mice.Liraglutide may cause weight loss at the same time improve insulin resistance in NAFLD,which may be related to the activation of ADCY 3,but its downstream mechanism is not yet clear.Abnormalities in the InsR/IRS/PI3K/Akt insulin signaling pathway play a key role in the occurrence and development of insulin resistance.Liraglutide regulates the insulin function and alleviates insulin resistance in NAFLD may through the InsR/IRS/PI3K/Akt cascade.Liraglutide may restore the liver lipid metabolism through ADCY 3 regulated PI3K/Akt signaling pathway.To better understand the potential mechanism,we have evaluated the pathophysiological effects of Liraglutide on NAFLD via the insulin signaling pathway.Methods:(1)Animal experiment:A 2×2 factorial analysis experiment was designed.High-fat diet(HFD)-induced NAFLD mice with diabetes were treated with Liraglutide 0.2mg/kg for 10 weeks,while the control mice were saline-treated.Blood biochemical analysis is used to detect changes in ALT,AST,TG,TC,HDL-C,LDL-C content.Insulin resistance index(HOMA-IR)was used to evaluate insulin resistance.Liver magnetic resonance imaging–estimated proton density fat fraction(MRI-PDFF)examination to determine liver tissue fat content.Hematoxylin and eosin staining,Oil Red O staining and electron microscopy were used to observe the accumulation of triglycerides in the liver.Masson staining was used to observe liver cell fibrosis.Real-time quantitative PCR(real time PCR)and Western blotting were used to analyze the expression of ?-SMA,InsR,IGF-1R,IRS2,PI3 K,Akt,PKA and ADCY3 in liver tissue at the gene and protein levels,respectively.(2)Cell experiment:HepG2 cells were chosen for this study.Liraglutide at two concentrations were used to culture for oleic acid induced HepG2 cells.Oil Red O staining was used to observe the accumulation of triglycerides in the cells.Real time PCR and Western blotting were used to analyze the expression of PI3 K,Akt,PKA and ADCY3 in HepG2 cells at the gene and protein levels,respectively.Silencing the ADCY 3 gene by small interfering RNA(siRNA)technology.And then,Western blotting was used to analyze the expression of PI3 K,Akt,PKA and ADCY3 after gene silencing.Results: 1.Animal experiment:(1)Liraglutide improves metabolism in the HFD-induced NAFLD mouse model.Compared with the NC group,the BW? FBG and MRI-PIDD levels were significantly increased in the HFD group after 10 weeks of consumption of the high-fat diet(p <0.001).Liver histopathology and HE staining showed hepatocyte swelling,elevated steatosis,rarefaction of the hepatocyte cytoplasm and clumped strands of intermediate filaments in the HFD-fed mice compared to the normal liver structure of the control group.In addition,Oil Red O staining and transmission electron microscopy revealed lipid droplets in hepatocytes in the liver of HFD-fed mice.The data indicates the successful establishment of the mouse model of NAFLD with T2 DM.(2)After subcutaneous injection of liraglutide daily for 10 weeks,further factorial analyses revealed that there were significant differences in BWs,FBG levels,HOMA-IR scores,ALT and AST between NC and HFD mice and also between Liraglutide-treated and saline-treated groups.Compared with the NC group,the BW? FBG? HOMA-IR?ALT?AST and TG were significantly increased in the HFD group(p <0.05).Liraglutide significantly reduced BW,FBG,HOMA-IR,ALT,AST and TG both in the HFD group and NC group(p<0.05).(3)Liraglutide treatment reduced TG deposition in liver tissue: Compared with the NC group,TG levels were significantly increased in the HFD group(p<0.05).After Liraglutide treatment,a decrease in TG expression was observed in both the NC group and the HFD group(p <0.05).Liraglutide treatment reduced Intrahepatocellular Lipid Accumulation in the Mouse Model of HFD-Induced NAFLD.The N+S and N+L groups exhibited a normal liver structure,and lipid droplets and fibrosis were undetectable.The structure of the hepatic lobules of the O+S group was characterized by a large amount of steatosis,and lipid droplets were visible.In contrast,a significant decrease in the severity of fat accumulation was observed in the liver tissue of mice in the O+L group.(4)Liraglutide treatment reduced liver fibrosis in the mouse model of NAFLD.The N+S and N+L groups exhibited a normal liver structure and fibrosis were undetectable.On the contrary,blue collagen Fiber were visible in the O + S group,suggesting the performance of liver fibrosis,while the O + L group could alleviated this condition without obvious fibrosis.The gene and protein expression of ?-SMA in liver tissue were both significantly increased(p <0.05).After liraglutide treatment,the gene and protein expression of ?-SMA significantly decreased(p < 0.05).(5)The factorial analyses revealed insignificant differences in the expression of InsR,IRS2,PI3 K,Akt,PKA and ADCY 3 mRNA associated with the liraglutide treatment and saline treatment between the NC and HFD groups of mouse model.Compared to NC group,relative levels of the InsR,IRS2,PI3 K,Akt and ADCY 3 mRNAs were significantly downregulated in the HFD group(P<0.05).However,after treatment with liraglutide,levels of the InsR,IRS2,PI3 K,Akt,PKA and ADCY 3 mRNAs were increased(P<0.05).(6)The factorial analyses revealed insignificant differences in the expression of InsR+IGF-1R,IRS2,PI3 K,Akt,PKA and ADCY 3 protein associated with the liraglutide treatment and saline treatment between the NC and HFD groups of mouse model.Compared to the NC group,InsR+IGF-1R,IRS2,PI3 K,Akt,PKA and ADCY 3 protein expression were significantly decreased(p <0.05)in the HFD group.However,after treatment with liraglutide,the levels of the InsR+IGF-1R,IRS2,PI3 K,Akt,PKA and ADCY 3 proteins were significantly increased(p <0.05).2?Cell experiment:(1)Liraglutide treatment reduced TG deposition in HepG2 cells: Compared with the NC group,the content of TG in the model group increased significantly(p <0.05)in HepG2 cells induced by oleic acid,and a large number of red lipid droplets appeared in the cytoplasm.Compared with the model group,the TG content of liraglutide 100 nmol/L concentration treatment group and liraglutide200 nmol/L concentration treatment group both decreased(p <0.05).(2)Liraglutide treatment on PI3 K,Akt,PKA,ADCY 3 mRNA expression levels in HepG2 cells induced by oleic acid steatosis.Compared with the model group,the PI3 K mRNA expressions were significantly increased(p <0.05)in the liraglutide 100 nmol/L concentration treatment group.Compared with the normal groups,the Akt mRNA expressions were significantly decreased(p<0.05)in the model group.Compared with the normal group,the ADCY 3mRNA expressions in the model group and liraglutide 200 nmol/L concentration treatment group were both significantly decreased(p <0.05).Compared with model group,the ADCY 3 mRNA expressions significantly increased(p <0.05)in liraglutide 100 nmol/L concentration treatment group.Compared with normal group,the PKA mRNA expressions significantly decreased(p <0.05)in model group.Compared with the model group,the Akt mRNA expressions of liraglutide 100 nmol/L concentration treatment group was significantly increased(p <0.05).(3)Liraglutide treatment on PI3 K,Akt,PKA,ADCY 3 protein expression in HepG2 cells induced by oleic acid steatosis.Compared with the normal group,the pPI3 K p85/PI3 K p85 protein expression were significantly decreased(p<0.05)in both model groups and liraglutide 200 nmol/L concentration treatment group.Compared with the model group,the pPI3 K p85/PI3 K p85protein expression were significantly increased(p <0.05)in liraglutide 100 nmol/ L concentration treatment group.Compared with the normal group,the pAkt /Akt protein expression was significantly decreased(p <0.05)in both model group and liraglutide 200 nmol/L concentration treatment group.Compared with the model group,the pAkt/Akt protein expression was significantly increased(p <0.05)in the liraglutide 100 nmol/L concentration treatment group.Compared with the normal group,the ADCY 3 protein expression was significantly reduced(p <0.05)in both the model group and liraglutide 200 nmol/ L concentration in the treatment group.Compared with the model group,the ADCY 3 protein expression was significantly increased(p <0.05)in liraglutide100 nmol/L concentration treatment group.Compared with the normal group,the PKA protein expression was significantly decreased(p <0.05)in both the model group and liraglutide 200 nmol/L concentration treatment group.Compared with the model group,the PKA protein expression was significantly increased(p <0.05)in liraglutide 100 nmol/L concentration treatment group.(4)The ADCY 3 interference-mediated gene regulation in HepG2 cells offers great potential in PI3K/Akt signal pathways.SiRNA(ADCY 3-siRNA)group ADCY 3,PKA,PI3 K,Akt protein expression significantly down-regulated(p <0.05).Conclusions:(1)Liraglutide can significantly improve the body weight,blood sugar,liver function,insulin resistance index and triglyceride levels of T2 DM combined with the NAFLD model,can reduce liver fatty degeneration and fibrosis.(2)Liraglutide improves liver fibrosis and steatosis in NAFLD may be related to the regulation of PI3 K/Akt insulin signaling pathway.(3)Liraglutide ameliorates NAFLD and improves hepatic steatosis.This mainly medium by ADCY 3 and upregulation of the PI3K/Akt signaling mediators.