Liraglutide Ameliorates Hepatic Steatosis In Diabetes By Regulating ADCY3-LXR/RXR Signal Pathway

Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease worldwide.NAFLD patients often have components of the metabolic syndrome,such as obesity,type 2 diabetes mellitus(T2DM),hyperlipidemia,and hypertension.Among these comorbidities,T2 DM seems to be the most important risk factor for NAFLD and the most important clinical predictor of adverse clinical outcomes such as advanced liver fibrosis and mortality.Epidemiological studies have found that in T2 DM patients,the prevalence of NAFLD can be as high as 70%.Diabetes can not only be accompanied by NAFLD,but also make NAFLD worse.Therefore,a deeper understanding of the relationship between liver metabolic abnormalities and T2 DM will help us re-understand the pathogenesis of T2 DM and provide a basis for the formulation of new T2 DM preventive measures.GLP-1 is a peptide secreted from intestinal L cells.Nevertheless,circulating GLP-1 is rapidly identified and cleaved by dipeptidyl peptidase-4(DPP-4),so its half-life is just 2minutes in vivo.Synthetic GLP-1 receptor agonists are resistant to DPP-4 and have an extended half-life,which make it possible for clinical treatment.Liraglutide is an acylated analog of human GLP-1 with 97% homology with native GLP-1 and its half-life is about 11 to 15 hours.Previous studies have shown that GLP-1 improves liver steatosis without relying on weight loss.Recent studies have shown that liraglutide can also reduce fat accumulation in the liver of people with diabetes and NAFLD.However,the mechanism by which liraglutide improves NAFLD is not clear.Adenosine cyclase 3(ADCY3)is a member of ADCY family of ten related cyclases that catalyze the conversion of ATP to c AMP.About 70% of GLP-1biological function is mediated by c AMP signaling pathway.Based on our clinical research,we found that liraglutide can increase the expression of ADCY3 in serum of newly diagnosed obese and non-obese diabetic patients.The level of serum ADCY 3 is negatively correlated with the insulin resistance index(HOMA-IR).Based on our animal experiments,Liraglutide can alleviate NAFLD in obese mice by up-regulating the expression of ADCY3 in the liver.The expression of ADCY3 is negatively correlated with body weight,Homeostasis model of assessment-IR(HOMA-IR)and liver fat area.Liraglutide can up-regulate the ADCY3 / c AMP / PKA metabolic pathway in the liver.Therefore,liraglutide can improve liver lipid metabolism through the ADCY3 / c AMP / PKA metabolic pathway,but the downstream factors that regulate it have not been identified.The main pathological feature of NAFLD is hepatic lipid accumulation,accompanied by liver steatosis.Liver X receptor(LXR)plays an important role in the regulation of cholesterol homeostasis and adipogenesis.LXR-? forms a heterodimer with retinoid X receptor(RXR)and directory binds cis-element on the SREBP-1c promoter,leading to transcriptional activation,and has been established as a dominant activator of SREBP-1c expression.In the current studies it was found that protein kinase A(PKA),a mediator of glucagon/c AMP,a fasting signaling,suppresses SREBP-1c by modulating the activity of LXR-?,a dominant activator of SREBP-1c expression.Therefore,we speculate that GLP-1 can improve liver steatosis by increasing ADCY3 expression and then up-regulating PKA activity,which changes the LXR / RXR-SREBP-1c of the fat metabolism pathway.Part One Effects of liraglutide on lipid metabolism in HFD-induced diabetic and NAFLD miceObjectives: To observe the effect of GLP-1 analog liraglutide on lipid metabolism in HFD-induced diabetic and NAFLD mice.Methods: C57BL/6J mice(5 weeks old,male)were purchased from Animal Experiment Center of Guangxi Medical University.The experimental mice were maintained in a specific pathogen-free room with a 12-hr light/dark cycle.In the first week,all mice were fed with a normal rodent chow diet(5% fat wt/wt).After then,the mice were randomly divided into two groups,ie,NC group: the mice fed with a normal chow diet(5% fat wt/wt)and HFD group: the mice fed with high-sucrose-high-fat diet(HFD)(D12492,60% fat wt/wt).Body weight(BW)and fasting blood glucose(FBG)levels were monitored weekly.After 10 weeks of chow diet or HFD feeding,MRI of liver was performed to confirm the success of modeling.In HFD group,the mice with FBG levels >13.9 mmol/L(250 mg/d L)for 3 consecutive days were considered to be diabetic,and the mice were considered obese when their BW exceeded normal weight by at least20%.The diabesity mice and obese were divided into two groups: a normal saline-treated group(O+S)and a Liraglutide-treated group(O+L).The control mice were also divided into two groups: a normal saline-treated group(N+S)and a Liraglutide-treated group(N+L).The mice were treated with a daily subcutaneous injection of Liraglutide(0.2 mg/kg)or normal saline for 10 weeks.At the end of treatment,all mice were fasted for 8 hrs,anesthetized and sacrificed for blood and tissue collection.Collected Fresh liver tissues for HE staining,Masson staining,oil red O staining,and electron microscopy.Hepatic?-SMA mRNA expression levels were measured by real-time quantitative polymerase chain reaction(RT-q PCR).Hepatic ?-SMA protein expression levels were measured by Western bloting.Serum levels of insulin,ALT,AST,lipid and TG level in liver tissue were determined via enzymatic methods using commercial kits.The HOMA-IR was used to assess IR.Results:1.Before liraglutide treatment,the BW,FBG and MRI-PDFF of mice in HFD group exhibited significantly higher than the mice in the NC group(p <0.05).Histopathological examination in liver of HFD mice with HE staining exhibited hepatocyte ballooning,elevated steatosis,rarefaction of hepatocytic cytoplasm and clumped strands of intermediate filaments and NC group displayed normal liver architectures.2.BW,FBG,TG,ALT and AST were significantly higher in HFD group than in NC group(p <0.05).Liraglutide treatment can significantly decreased BW,FBG,TG,ALT and AST(p <0.05).HOMA-IR scores was significantly higher in HFD group than in NC group(p<0.05).Liraglutide treatment can significantly decreased HOMA-IR scores(p <0.05).No significant differences in TC,LDL-C and HDL-C were observed between in HFD group than in NC group or between liraglutide-treated mice and saline-treated mice.3.We used HE staining to investigate the histopathological changes in the liver and used Oil Red O staining and transmission electron microscopy to visualize lipid droplets.The N+S and N+L groups displayed normal liver architectures.The normal structure of hepatic lobules in mice in the O+S group was characterized by massive fatty degeneration.In contrast,a significant decrease in the severity of fat accumulation was observed in the liver tissues of mice in the O+L group.Lipid droplets were undetectable in the livers of mice in the N+S and N+L groups but could be seen in the livers of mice in the O+S group.Conversely,the livers of mice in the O+L group exhibited a significant decrease in lipid droplet accumulation.TG levels in the hepatic tissue was significantly higher in HFD group than in NC group(p <0.05).Liraglutide treatment can significantly decreased TG levels in the hepatic tissue(p <0.05)4.Liraglutide reduced liver fibrosis.The hepatic levels of ?-SMA mRNA and protein were significantly higher in HFD group than in NC group(p <0.05).Liraglutide treatment can significantly decreased the hepatic levels of ?-SMA mRNA and protein(p<0.05).The fibrosis were undetectable in the livers of mice in the N+S and N+L groups but could be seen in the livers of mice in the O+S group.Conversely,the fibrosis disappeared in the O+L group.Conclusions:1.High-sucrose-high-fat feeding C57 BL / 6J is an effective method to establish T2 DM combined with NAFLD model.2.Liraglutide can significantly reduce the BW,HOMA-IR,liver function and TG level of T2 DM combined with NAFLD model,and ameliorates hepatic steatosis and fibrosis.Part two Liraglutide ameliorates hepatic steatosis in diabetes by regulating ADCY3-PKA/LXR/RXR signal pathwayObjectives: To observe the changes of signal molecules in ADCY3 / PKA /LXR / RXR signal pathway regulated by liraglutide,and to explore the effect of liraglutide on the steatosis of liver and Hep G2 cells in HFD induced T2 DM combined with NAFLD mice.Methods:1.Animal studies Mice feeding and intervention were same as part one.After 10 weeks,the mice were fasted overnight and anaesthetized.The livers were immediately dissected,frozen in liquid nitrogen and stored at-80°C until analysed.Hepatic ADCY3,PKA,LXR,RXR and SREBP-1c mRNA expression levels were measured by real-time quantitative polymerase chain reaction(RT-q PCR).Hepatic ADCY3,PKA,LXR-?,RXR and SREBP-1c protein expression levels were measured by Western bloting.2.Cells studies Hep G2 Cells(treated with oleic acid(0.3 mmol / L))were cultured with liraglutide in different concentrations(0,100 and 200 nmol/l),and after 48 hours,the cells were harvested for western blotting and RT-q PCR.ADCY3,PKA,LXR,RXR and SREBP-1c mRNA expression levels were measured by RT-q PCR.ADCY3,PKA,LXR-?,RXR and SREBP-1c protein expression levels were measured by western bloting.TG levels in cells were determined via enzymatic methods using commercial kits.Reduce the expression level of ADCY3 in hep G2 cells by siRNA technology,and then measured the expression levels of ADCY3,PKA,LXR-?,RXR and SREBP-1c protein by Western bloting.Results:1.The hepatic levels of ADCY3 mRNA and protein were significantly lower in HFD group than in NC group(p <0.01).Liraglutide treatment can significantly increased the hepatic levels of ADCY3 mRNA and protein(p <0.05).The hepatic levels of LXR,RXR,SREBP-1c mRNA and protein were significantly higher in HFD group than in NC group(p <0.01).Liraglutide treatment can significantly decreased the hepatic levels of LXR,RXR,SREBP-1c mRNA and protein(p <0.05). 2.After intervention with different concentrations of liraglutide(0,100,200nmol/l)on steatotic Hep G2 cells: compared with the normal control group,ADCY3 mRNA levels were decreased in the model group and 200nmol/L liraglutide treatment group.Compared with the model group,the expression of ADCY3 mRNA in the 100 nmol/L liraglutide group was increased,and the 200nmol/L liraglutide group was not significant compared with the model group;Compared with the normal control group,PKA mRNA levels was decreased in the model group.Compared with the model group,the expression of PKA mRNA in the 100 nmol/L liraglutide group and 200 nmol/L liraglutide group were increased,but it was significantly higher in the 100 nmol/L liraglutide group than in 200 nmol/L liraglutide group.Compared with the normal control group,LXR mRNA levels were increased in the model group and 200nmol/L liraglutide treatment group.Compared with the model group,the expression of LXR mRNA in the 100 nmol/L liraglutide group was decreased.No significant differences in the expression level of RXR,SREBP-1c mRNA after different concentrations of liraglutide.Spearman correlation analysis showed that ADCY3,PKA,LXR,RXR and SREBP-1c mRNA levels had no correlated with the concentrations of liraglutide.3.After intervention with different concentrations of liraglutide(0,100,200nmol/l)on steatotic Hep G2 cells: compared with the normal control group,ADCY3,PKA protein levels were decreased in the model group and 200nmol/L liraglutide treatment group.Compared with the model group,ADCY3,PKA protein levels in the 100 nmol/L liraglutide group was increased,and the 200nmol/L liraglutide group was not significant compared with the model group;Compared with the normal control group,LXR-?,SREBP-1c protein levels were increased in the model group and 200 nmol/L liraglutide group.Compared with the model group,the expression of LXR-?,SREBP-1c protein levels in the100 nmol/L liraglutide group were decreased,but it was no significant differences in the 200 nmol/L liraglutide group.Compared with the normal control group,RXR protein levels was increased in the model group.Compared with the model group,the expression of RXR protein levels in the 100 nmol/L liraglutide group was decreased,but it was no significant differences in the 200nmol/L liraglutide group.Spearman correlation analysis showed that ADCY3,PKA,LXR-?,RXR and SREBP-1c protein levels had no correlated with the concentrations of liraglutide.4.Effect of different concentrations of liraglutide on TG levels in steatotic Hepa G2 cells: Compared with the normal control group,TG levels was increased in the model group,100 nmol/L liraglutide group and 200 nmol/L liraglutide group.Compared with the model group,the TG levels in the 100 nmol/L liraglutide group and 200 nmol/L liraglutide group were decreased,but it was significantly lower in the 100 nmol/L liraglutide group than in 200 nmol/L liraglutide group.Spearman correlation analysis showed that TG levels had no correlated with the concentrations of liraglutide.5.Reduce the expression level of ADCY3 in hep G2 cells by siRNA technology,and then measured the expression levels of ADCY3,PKA,LXR-?,RXR and SREBP-1c protein by Western bloting.The results showed that ADCY3 and PKA proteins were significantly down-regulated,and LXR-? and RXR proteins were significantly up-regulated in the siRNA group.Conclusions:Liraglutide can up-regulate hepatic ADCY3 / PKA expression levels and down-regulate hepatic LXR-?,RXR and SREBP-1c expression levels.The effect of liraglutide on hepatic ADCY3,PKA,LXR-?,RXR and SREBP-1c expression levels in T2 DM combined with NAFLD model were significantly higher than those in normal contral mice.Liraglutide can up-regulate hepatic ADCY3 / PKA expression levels and down-regulate hepatic LXR-?,RXR and SREBP-1c expression levels in steatotic Hep G2 cells.After SiRNA Silencing ADCY3 Gene,it was found that the expression of PKA decreased and the expression of LXR and RXR increased,suggesting that ADCY3 has a negative regulation effect on LXR-?/RXR.Lilaglutide is likely to ameliorate NAFLD by regulating the expression of LXR-?,RXR and SREBP-1c through ADCY3 / PKA.