The Role Of Liraglutide On Autophagy In Lipotoxic Liver Injury

Objective:At present,the incidence of non-alcoholic fatty liver disease（NAFLD）is increasing.In developed countries,about 80%of adults with obesity or diabetes suffer from NAFLD.It is generally believed that NAFLD is closely related to insulin resistance,obesity,diabetes,dyslipidemia and other metabolic syndrome components,and is considered as the clinical manifestations of metabolic syndrome on liver^[2].NAFLD includes simple fatty liver,fatty hepatitis and fatty liver-related cirrhosis,which can develop into liver cancer^[3].It has been confirmed that even simple fatty liver is related with cardiovascular and cerebrovascular diseases,and thus destroy the quality of life.So the prevention and the control of NAFLD has become the focus of today’s medicine.The pathogenesis of NAFLD has not been fully understood,and the"second strike"theory is currently widely accepted.The first is insulin resistance that result in excessive intake of free fatty acids（FFA）and the steatosis;the second is the complex effects of oxidative stress,lipid peroxidation and abnormal cytokines on the local inflammatory changes of hepatic lobules and the steatohepatitis.So the essence of NAFLD is a series of changes caused by excessive lipid deposition in liver.Based on previous studies,autophagy is currently considered closely related to intrahepatic lipid deposition because of its function on degrading fat in hepatocytes.In living bodies,autophagy is a relatively fixed process of auto-catabolism,which"self-purge"some of the accumulated protein aggregates and defective organelles in cells and maintains the stability of the intracellular environment.Lipid drops（LDs）are one of the biodegradation substrates^[4].Autophagy annexes and decomposes the LDs by the lysosomal pathway,and thus maintains the balance of intracellular lipid metabolism.In the NAFLD model,previous study found the decreased activity of intrahepatic autophagy,dysfunction of mitochondrial,and the presence of insulin resistance and endoplasmic reticulum stress^[5].In liver cells of rats fed with long-term high fat diet,the decreased autophagic activity was thought to be the decompensation of autophagy to metabolize because of excessive lipid deposition under long-term high-fat stimulation.^[6]In cases of starvation,high temperature,hypoxia,protein aggregation and pathogen infection,intracellular autophagy is activated to irradiate the above pressures and then recovers to normal level,and the regulation process is mediated by different signaling pathways^[7,8].The AMP-activated protein kinase（AMPK）pathway is thought to play an important role in the regulation of autophagy^[9].AMPK is a protein kinase that regulates energy metabolism in cells.Its main feature is the ability to bind to AMP and regulate the activity of the enzyme by AMP-sensed cell energy levels and metabolic balance levels.In normal cells,AMPK pathway can inhibit the synthesis of metabolic pathways,the activation of catabolic pathway,and promote cell survival by the use of energy to improve the adaptation of cells to metabolic stress^[10].As the AMPK pathway can act as an intracellular energy receptor conduction signal,and autophagy is also affected by changes of intracellular energy,so the role of AMPK pathway on autophagy has been widespread concern of many researchers.Many studies have shown that the AMPK/mTOR pathway may be an important pathway for autophagy^[11].AMPK can induce autophagy by activating the TSC1 protein to inhibit the activity of mTOR.Nowadays,the pathogenesis of NAFLD is not clear,and there is no specific medicine for NAFLD.Among the many interventions,lifestyle changes and weight loss remain the first choice for NAFLD interventions.But there are still some limitations,that is,poor compliance,and the intervention of drugs are always required^[12].Liraglutide is a new type of drug for the treatment of diabetes,which belongs to the glucagon-like peptide 1（GLP-1）analogue.GLP-1 is a peptide hormone in the human body,and can regulate glucose metabolism,thus result in the control of blood glucose levels,weight loss,avoid hypoglycemia and other effects.Studies have confirmed the presence of GLP-1 receptors（GLP-1R）in hepatocytes,and liraglutide can act on the liver by direct acting on GLP-1R.Armstron MJ et al^[13]pointed out that the application of liraglutide in the treatment of diabetic patients for 26 weeks,liver transaminase and other biochemical indicators were improved significantly.Shvetank^[14]suggested that GLP-1 protected the liver from cell apoptosis caused by fatty acids by promoting autophagy and suppressing endoplasmic reticulum stress dysfunction.This provides a new direction for the prevention of GLP-1 on further deterioration of hepatic steatosis in NAFLD patients.In the study,NAFLD models were generated by high-fat diet on rat in vivo,and by palmitate fatty acid exposure on HepG2 cells in vitro.Then we treated rats and cells with liraglutide and examined its effects on autophagy in the rat liver and the cultured cells.The possible molecular mechanism of liraglutide interfering autophagy was illustrated by using AMPK inhibitors.This work is divided into two parts,and the main contents are as follows:1.Study on the effect of liraglutide on autophagy in both NAFLD rats and palmitic acid induced lipid-toxicity-HepG2 cells2.Study on the possible mechanism of liraglutide on autophagy in HepG2 cells.Methods:1.NALFD rat models were induced by high-fat diet and evaluated by Hematoxylin-eosin（HE）staining.2.The control group rats were given normal saline.The high-fat diet group rats were divided into four groups treated with different doses（0,50,100,or 200μg/kg）of liraglutide.All reagents were injected subcutaneously twice daily for 4 weeks.3.Serum lipid profiles（TC,TG,HDL-C,LDL-C,FFA,ALT and AST）were determined using an auto-analyzer.4.HepG2 cells were cultured.Lipotoxicity was induced by 400μmol/L palmitic acid in HepG2 cells,and treated with different doses of liraglutide（10 nmol/L,50nmol/L,100 nmol/L,500 nmol/L）.Cell viabilities were determined by the Methyl thiazolyldiphenyl-tetrazolium bromide（MTT）assay.5.Autophagosomes of rat liver were observed by transmission electron microscopy.6.Real-time quantitative reverse transcription polymerase chain reaction（real-time RT-PCR）was used to detect the expression of autophagy-related protein mRNA,such as Light microarray-associated protein 1 chain 3（MAP1-LC3,LC3）,Beclin1 protein and autophagy related gene 7（Atg7）,in the NAFLD liver and HepG2cells.7.Western blot was used to detect the protein expression of autophagy-related proteins LC3,Beclin1 and Atg7 in NAFLD rats hepatocytes s and HepG2 cells.8.Western blot was used to detect the protein expression of AMP-activated protein kinase（AMPK）,phosphorylated adenylate activated protein kinase（p-AMPK）,TSC1protein（tuberous sclerosis-1,TSC1）,the mammalian target of rapamycin（mTOR）and phosphorylated mTOR protein in NAFLD rats hepatocytes and HepG2 cells.Results:Ⅰ.NAFLD rat model was successfully generated by 12-week of high-fat diet.At the end of the 12th week,hepatocytes in the NC group were in normal shape,and the hepatic cords were neat without any obvious lipid droplets vacuoles and inflammation.However,the hepatocytes in the HF group have steatosis,lobular inflammation,and ballooning.The NAFLD activity scores for steatosis,lobular inflammation,and ballooning in the HF group were much higher than those in the NC group（P<0.01）.It indicates that the NAFLD rat models have been successfully established by high-fat diet.Ⅱ.Effects of liraglutide on liver histomorphology in NAFLD ratsAt the 16th week,liver cells of high fat（HF）group were filled with the various sizes of fat vacuoles.The signs of inflammation can be observed in liver leaflets,and the liver cells were balloon-like.Liraglutide can decrease the bubbles,inflammatory infiltration and balloon-like changes,especially in the moderate-and high-dose group.Ⅲ.Effect of liraglutide on the whole-body metabolism in NAFLD ratsThe average body weight of all groups is similar at the beginning of this study.At the end of 16th week,there was a significant increase in body weight,TG,TC,LDL,FFA,AST,ALT,liver tissue weight（as a percentage of body weight）and reduction in HDL in the HF group compared with the NC group（P<0.01）.After liraglutide treatment for 4 weeks（at the end of the 16th week）,body weight and biochemical parameters have been significantly improved,especially in the high dose treatment group（200L）（P<0.01）.Ⅳ.The effects of liraglutide on the survival and TG content of the HepG2 cells induced by lipotoxicityUnder lipotoxic environment,liraglutide promoted cell survival,with the best potentiation at l00nmol/L concentration（P<0.01）.In addition,liraglutide could improve TG content,and treatment at l00nmol/L produced the best effect（P<0.01）.Ⅴ.Effects of liraglutide on autophagy of liver in NAFLD rats.The number of autophagosomes in the HF group was significantly lower than that in the NC group.And the number of autophagosomes in the 50L,100L and 200L groups was significantly higher than that in the HF group.The mRNA and protein levels of LC3-II,Beclin1,Atg7 in liver tissue of HF group was significantly lower than that of NC group（P<0.05）.The mRNA and protein levels of LC3-II Beclin1,Atg7were increased after lilaglutide intervention.The differences were statistically significant.（P<0.05）Ⅵ.Effects of liraglutide on autophagy in HepG2 cellsIn palmitic acid（PA）group,the mRNA and protein levels of LC3,Beclin1 and Atg7 were significantly lower than that in BSA group（P<0.01）.The mRNA and protein levels of LC3,Beclin1 and Atg7 in cells cotreated with PA and10nmol/L-500nmol/L liraglutide were higher than that in PA group（P<0.01）.Ⅶ.The effect of liraglutide on autophagy of HepG2 cell in the presence of AMPK pathway inhibitor.The levels of LC3,Beclin1 and Atg7 in HepG2 cell were decreased in PA group（400mmol/L palmitic acid）compared with the control group（BSA group）（P<0.01）.The levels of LC3,Beclin1 and Atg7 were significantly higher in the liraglutide intervention group（100G group）than those of PA group（P<0.01）.The levels of autophagy-related proteins LC3,Beclin1 and Atg7 were significantly lower in the inhibition group（100G+C）containing AMPK pathway inhibitor,palmitic acid（400mmol/L）and liraglutide（100nmol/L）,than those of 100G group（P<0.01）.Ⅷ.Effects of liraglutide on AMPK pathway-associated proteins in the presence of AMPK pathway inhibitionThe levels of p-AMPK/AMPK,TSC1,p-mTOR/mTOR were decreased（P<0.01）in PA group（400mmol/L palmitic acid）compared with the control group（BSA group）.And the levels of p-AMPK/AMPK,TSC1 and p-mTOR/mTOR were increased in the liraglutide intervention group（100G group）compared with the PA group（p<0.01）.The levels of p-AMPK/AMPK,TSC1,p-mTOR/mTOR were significantly lower than those of 100G group in the inhibition group（100G+C）containing AMPK pathway inhibitor,palmitic acid（400mmol/L palmitic acid）and liraglutide（100nmol/L）（P<0.01）.Conclusion:1.Liraglutide can reverse the NAFLD.In NAFLD rats,the level of autophagy was significantly decreased,and the level of autophagy was increased after liraglutide intervention.The palmitic acid can produce lipid toxicity and decrease the level of autophagy in HepG2 cells.The level of autophagy recovered after liraglutide intervention.Therefore,the protection of liraglutide on hepatocytes is related with its up-regulation on autophagy.2.AMPK pathway was inhibited in the HepG2 cells with palmitic acid treatment.Liraglutide can activate the AMPK pathway,which was further suppressed by the AMPK pathway inhibitors.Our data suggest that liraglutide may regulate the autophagic effect through the AMPK signaling pathway.