Liraglutide Improves Hepatic Lipid Accumulation By Inducing Autophagy Via AMPK/mTOR Pathway

BackgroundNonalcoholic fatty liver disease(NAFLD)is a common chronic liver disease caused by abnormal lipid deposition in the liver.It has been reported that type 2 diabetes(type 2 Diabetes Mellitus,T2DM),obesity,hyperlipidemia,etc.alone or together constitute NAFLD susceptibility factors.The occurrence of cardiovascular disease and kidney disease also increased risk of higher mortality.The Glucagon-Like Peptide-1(GLP-1)is a kind of physiological peptide hormone secreted mainly by small intestinal L cells,acting on pancreatic β cells,and has glucose-dependent insulin secretion.Recent studies have shown that GLP-1 receptors are expressed on human hepatocytes and GLP-1 plays an important role in reducing liver fat content.Liraglutide(LRG)is a long-acting analogue of GLP-1,which overcomes the characteristics of natural GLP-1 degradation,and exerts the synergistic effect of GLP-1,including hypoglycemic,weight and lipid loss.Autophagy is a relatively conservative self-decomposition process in the organism.Recent studies have found that autophagic abnormalities are closely related to insulin resistance,abnormal liver lipid metabolism,and apoptosis.Animal experiments confirmed that in the early stages of fat accumulation,autophagy plays a protective role in liver function by the effect on lipid metabolism,but long-term chronic lipid load to the liver cell autophagy levels.MTOR plays an important role in the development of autophagy.Studies have shown that mTOR upstream pathway protein AMPK phosphorylation increases can inhibit mTOR,thereby enhancing autophagy.But the current role of liraglutide in hepatocellular steatosis and the mechanism of action is not clear.In the present study,we first set up an obesity mouse model with HFD and oleate-palmitate-induced human L-O2 cells.Then we explored the effects of LRG on liver fat and intracellular lipids,and the role of autophagy was determined.Finally,AMPK/mTOR signaling pathway was detectedin order to reveal the potential molecularmechanisms underlying theautophagy-promoting role of LRG.Part Ⅰ:Liraglutide improves simple hepatic steatosis by enhancing autophagyObjectiveIn this part,an in vivo model of simple hepatic steatosis was established.First,we measured the effect of liraglutide(LRG)on hepatocyte steatosis,and then observed whether this improvement of liraglutide was related to autophagy.And it may provide the theoretical and experimental basis for clinical application.Method60 male C57BL/6 mice were randomly selected as normal control group,and the others were treated with high fat diet(HFD)to construct hepatic steatosis model.The body weight,food intake and blood glucose were monitored every week.After two weeks of feeding,two mice from each group were sacrificed.Liver tissue was measured by HE staining and blood lipid analysis to evaluate the effect of modeling.After the success of the model,the rats were divided into four groups:high fat group,liraglutide group,chloroquine(CQ,autophagy inhibitor)group,liraglutide +chloroquine group.(IPGTT)and peritoneal insulin tolerance test(IPITT)were used to detect the effects of high fat and liraglutide on islet function in mice.The levels of triglyceride TG,cholesterol TC,low density lipoprotein LDL,high density lipoprotein HDL,liver fat content,transmission electron microscopy,immunohistochemistry,and the levels of autophagy were detected by Western blot.All values were expressed as mean ± standard misuse,with GraphPad Prism 5 software for paired t test,when P<0.05,the difference was statistically significant.Result1.Effects of Liraglutide on Body Weight and Feed Intake of High Fat Feeding MiceAfter 12 weeks of high fat diet,the body weight of mice was significantly higher than that of normal control group.The food intake was slightly higher,but the difference was not statistically significant.Compared with the high fat group,theliraglutide group began to decrease significantly from the 4th week,and the decrease was more obvious at the 8th week.There was no significant difference in the high fat level of liraglutide group.2.Effects of Liraglutide on Islet Function of High Fat-fed MiceIPGTT experiments showed that glucose tolerance was decreased in high-fat group mice,and glucose tolerance was increased after liraglutide.IPITT experiments showed that:high-fat group of mice with insulin resistance,decreased insulin sensitivity,and insulin sensitivity was increased after liraglutide intervention.3.Effects of Liraglutide on Blood Lipid in Mice Fed with High FatCompared with the control group,the levels of TC,TG and HDL in the high-fat group were significantly higher than those in the control group,and the LDL had no significant change in LDL group.4.Effects of Liraglutide on Liver in High Fat-fed Mice4.1Liver weight,liver lipid changesThe liver weight of the mice in the high fat group was significantly higher than that in the control group,and the liver weight of the liraglutide group was significantly decreased.The levels of TC and TG in the liver of the high fat group were significantly higher than those in the control group.After treatment with liraglutide,TC and TG in the liver were significantly lower than those in the high fat group.4.2 Histopathological changes in liver tissueThe mice in the control group had sharp edges in the liver and were bright red.The liver of the high fat group was diffuse and the edge became blunt.The volume of the liver in the liraglutide group was smaller and the edge was sharper.HE staining showed high fat in the liver found a large number of fat vacuoles,liver cell steatosis area reached 2/3,liver cells arranged disorder;liraglutide group of liver tissue fat vacuoles reduced,the liver cells arranged neatly,normal.4.3 Changes of autophagy levelsin liver tissueTransmission electron microscopy showed a small amount of autophagy in the liver tissue of the control group.High fat group showed a lot of lipid vacuoles,but no autophagosomes.Liraglutide group showed less lipid vacuoles,and a large number of autophagosomes.Immunohistochemistry showed a small amount of LC3B expression in the liver tissue of the control group.The expression of LC3B in the high fat group was rare,and the expression of LC3B in the liraglutide group was significantly increased.Western blot analysis showed that the expression of P62 in the high fat group was higher than that in the control group,and the expression level of P62 was significantly decreased after the treatment with liraglutide.The expression level of LC3B in the high fat group was lower than that in the control group and the expression level of LC3B was significantly increased after liraglutide treatment.The expression of P62 and LC3B in the higher lipid group was significantly increased in the chloroquine group.The expression of LC3B was the highest in the liraglutide +chloroquine group,and the expression level of P62 was between the two groups.After the action of chloroquine,the liver size and liver fat content were higher than the high fat group.Combined with liraglutide can reduce the protective effect of liraglutide on the liver.Hepatocellular carcinoma HE staining showed that the fatty acidity of the cells in the chloroquine group was significantly decreased,and the fat fraction of liraglutide + chloroquine group was significantly decreased.Conclusion1.Liraglutide can improve lipid deposition in simple hepatocyte steatosis models;2.Autophagy may be one of the mechanisms by which liraglutide relieves the simple hepatic cell steatosis.Part Ⅱ:AMPK/mTOR pathway is involved in liraglutide induced autophagy in hepatocyte steatosis of L-02 cellsObjectiveIn this part,an in vitro model of simple hepatic steatosis was established.First,we examined the effect of liraglutide on the improvement of hepatic steatosis,and then observed whether this improvement of liraglutide was related to autophagy.Finally,the effect of liraglutide on the improvement of liver cell steatosis is through which pathway to play a role.Providing a certain scientific basis for its clinical application andproviding new targetsfor NAFLD treatment.MethodHuman normal liver cell line L-O2 cells were cultured in 10%fetal bovine serum DMEM medium.The control group was replaced with fresh common medium.The model group was replaced with 1 mM free fatty acid mixture(Free Fatty Acid,FFA,oleic acid:palmitic acid = 2:1)for 24 hours to establish an in vitro model of hepatic steatosis.The results were evaluated by oil red O staining and determination of liver cell fat content.After modeling success,the patients were divided into four groups:model group,liraglutide 25nM group,liraglutide 50nM group,liraglutide 100nM group.(100nM),the rats were divided into 6 groups:model group,liraglutide group,bafilomycin A1(Bafilomycin Al,Baf,30nM,(Comp C,20 μM,AMPK inhibitor)group,liraglutide + Comp C group.First;the effects of each group on the lipid content of hepatocytes and the changes of autophagy intensity were evaluated.Finally,AMPK/mTOR pathway was detected to explore the possible pathways of autophagy.All values were expressed as mean ± standard misuse,with GraphPad Prism 5 software for paired t test,when P<0.05,the difference was statistically significant.Result1.Effect of liraglutide on lipid content in FFA-induced L-02 cellsOil red O staining showed a large number of red lipid droplets in the model group.The contents of TC and TG in the cells were significantly higher than those in the control group.The content of TC and TG in L-02 cells decreased significantly in 100nM group compared with the model group,and the dose was dose-dependent.2.Changes in autophagy levels of L-02 cells induced by FFATransmission electron microscopy showed a small amount of autophagy in the control group.The model group showed a lot of lipid vacuoles,no autophagosomes.Liraglutide-treated group had less lipid vacuoles,showing a large number of autophagosomes.Immunofluorescence showed that LC3B expression was observed in the model group,and the expression of LC3B was significantly increased in the liraglutide group and most in the 100 nM group.Western blot analysis showed that the expression level of P62 was the highest in the model group and the expression level of P62 was significantly decreased in the 100nM group after treatment with liraglutide.The expression level of LC3B was the lowest in the model group and the expression level of LC3B in the 100nM group.The expression of P62 and LC3B in the Baf group was higher than that in the model group.The expression of LC3B was the highest in the liraglutide and Baf group,and the expression of P62 was between the two single use.The lipid droplets and lipid content of FFA-induced L-02 cells were not significantly different from those in the model group after the action of Baf.After combination with liraglutide,the lipid droplets and liquefaction of liraglutide+Baf group were between the two groups.Immunofluorescence showed that the expression of LC3B was consistent with that Western blot.3.AMPK/mTOR path detectionThe expression of AMPK phosphorylation and Beclin 1 in the liraglutide-treated group was higher than that in the model group and the highest in the 100 nM group.MTOR phosphorylation was highest in the model group and decreased after treatment with liraglutide.After the application of AMPK inhibitor Comp C,the expression of LC3B was decreased and the expression of P62 was increased.At the same time,the content of FFA induced L-02 cells increased.Liraglutide can partially reverse the effect of AMPK inhibitors.Oil red O staining revealed that intracellular lipid accumulation was increased slightly in Comp C treated group,and then decreased significantly when co-used with LRG.Intracellular lipid profiles showed that TC and TG levels were higher in Comp C treated group compare with FFA treated group,and dramatically reduced after treated with LRG together.Conclusion1.Liraglutide can improve lipid deposition in simple hepatic steatosis models;2.Autophagy may be one of the mechanisms by which liraglutide relieves simple hepatic steatosis;3.AMPK/mTOR signaling may be involved in the liraglutide-induced autophagy in hepatocytes.Full text conclusion1.Liraglutide has a certain effect on simple hepatic steatosis in a dose-dependent manner.2.Autophagy may be one of the mechanisms by which liraglutide relieves simple hepatic cell steatosis;3.AMPK/mTOR signaling pathways may be involved in the liraglutide-induced autophagy in hepatocytes.