The Role Of SCD1-AMPK Pathway Mediated-lipophagy In The Pathogenesis Of NAFLD

Background and Objective: Non-alcoholic fatty liver disease(NAFLD)is a pathological syndrome characterized by the hepatic steatosis and in the absence of significant alcohol intake or any other primary cause.NAFLD is a common chronic liver disease worldwide,and the aberrant lipid deposition in hepatocytes is the basis for its development.Therefore,exploring how to prevent the abnormal intrahepatic lipids is of great significance for seeking new therapeutic approaches for NAFLD.Stearoyl-Co A desaturase 1(SCD1)involved in high-fat induced hepatic steatosis,however other current researches about SCD1 focused on the regulation of triglyceride synthesis as well as fatty acid beta-oxidation.Increasing evidence indicates that lipophagy,as a selective way of autophagy,is thought to be a new mechanism in regulating lipid metabolism and shows beneficial effects in NAFLD.AMP-activated protein kinase(AMPK),as a cellular energy sensor,plays an important role in the regulation of autophagy,and its activity is influenced by SCD1.Our own previous study reavled that the inhibition of SCD1 could activate the AMPK signaling pathway,thereby enhancing autophagy and promoting apoptosis in human hepatocellular carcinoma.However,whether SCD1 regulates lilophagy in NAFLD via AMPK signaling pathway to affect its development has not been reported.In this study,we established a model of mouse primary hepatocyte steatosis by PA stimulation and observed the relationship between the expression of SCD1 and AMPK,lipophagy.Next we observed the effects of altered SCD1 expression on lipid deposition,AMPK and lipophagy in primary hepatocytes.Then we enhanced or inhibited SCD1 expression and AMPK activity simultaneously to explore whether SCD1 regulates lilophagy via AMPK to affect lipid deposition in primary hepatocytes.On this basis,we observed the effects of inhibition of SCD1 expression in vivo on hepatic steatosis in mice.The results of this study suggested that SCD1-AMPK-lipophagy pathway may be a potential therapeutic target for NAFLD.Methods: I.Relationship between the expression of SCD1 and AMPK,lipophagy in the hepatocyte model of steatosis.1.The CCK8 method was used to screen the optimal stimulated concentration of PA for primary hepatocyte.2.After primary hepatocytes were stimulated by PA for 24 h,TG assay kit and oil red O staining were applied to determine lipid deposition.3.After primary hepatocytes were stimulated by PA for 24 h,the protein expression levels of SCD1,AMPK,LC3 and p62 were detected by Western-blot.II.Effects of altered SCD1 expression on lipid deposition,AMPK and lipophagy in primary hepatocytes.1.After primary hepatocytes were transfected with si RNA-SCD1 for 48 h,Western-blot was used to detect the protein expression level of SCD1 to screen and validate the silencing effect of si RNA-SCD1.2.Primary hepatocytes were stimulated by PA for 24 h after transfected with si RNA-SCD1 for 48 h.Primary hepatocytes were divided into 4 groups: control group,si RNA-SCD1 group,PA group,PA+ si RNA-SCD1 group.TG assay kit and oil red O staining were applied to determine lipid deposition.3.The protein expression levels of SCD1,AMPK,LC3 and p62 in primary hepatocytes of the above groups were determined by Western-blot.4.After primary hepatocytes were transfected with SCD1 overexpression adenoviruse(SCD1-OE)for 48 h,Western-blot was used to detect the protein expression level of SCD1 to verify the overexpression effect of SCD1-OE.5.Primary hepatocytes were stimulated by PA for 24 h after transfected with SCD1-OE for 48 h.Primary hepatocytes were divided into 4 groups: control group,SCD1-OE group,PA group,PA+ SCD1-OE group.TG assay kit and oil red O staining were applied to determine lipid deposition.6.The protein expression levels of SCD1,AMPK,LC3 and p62 in primary hepatocytes of the above groups were determined by Western-blot.III.The role of SCD1-AMPK pathway mediated-lipophagy on lipid deposition in primary hepatocytes.1.Primary hepatocytes were stimulated by PA for 24 h and Dorsomophin for 2h after transfected with si RNA-SCD1 for 48 h.Primary hepatocytes were divided into 8 groups: control group,si RNA-SCD1 group,Dorsomophin group,si RNA-SCD1+Dorsomophin group,PA group,PA+si RNA-SCD1 group,PA+Dorsomophin group,PA+si RNA-SCD1+ Dorsomophin group.TG assay kit and oil red O staining were applied to determine lipid deposition.2.The protein expression levels of SCD1,AMPK,LC3 and p62 in primary hepatocytes of the above groups were determined by Western-blot.3.Transmission electron microscope was used to observe the distribution of autophagosomes and autolysosomes in primary hepatocytes of the above groups.4.Primary hepatocytes were stimulated by PA for 24 h and A-769662 for 2h after transfected with SCD1-OE for 48 h.Primary hepatocytes were divided into 8 groups: control group,SCD1-OE group,A-769662 group,SCD1-OE+A-769662 group,PA group,PA+SCD1-OE group,PA+ A-769662 group,PA+SCD1-OE+A-769662 group.TG assay kit and oil red O staining were applied to determine lipid deposition.5.The protein expression levels of SCD1,AMPK,LC3 and p62 in primary hepatocytes of the above groups were detected by Western-blot.6.Transmission electron microscope was used to observe the distribution of autophagosomes and autolysosomes in primary hepatocytes of the above groups.IV.Effects of inhibition of SCD1 expression in vivo on hepatic steatosis,AMPK and lipophagy in mice.1.Mice were divided into 3 groups:NCD group(normal chow diet),HFD group(high fat diet),CAY10566 group(high fat diet combined with CAY10566 intraperitoneal injection).Mice were fed for 14 weeks.The liver function and blood lipid levels of each group of mice were detected by automatic biochemical analyzer.2.Hepatic steatosis was detected by HE staining and oil red O staining.3.The protein expression level of SCD1,AMPK,LC3 and p62 in the liver of each group of mice were detecd by Western-blot.4.The distribution of autophagosomes and autolysosomes in liver tissues was observed by transmission electron microscope.Results: I.Relationship between the expression of SCD1 and AMPK,lipophagy in the hepatocyte model of steatosis.1.CCK8 assay data showed that 324?M was the best PA stimulated concentration for primary hepatocyte.2.After PA stimulation for 24 h,TG contents and lipid droplets were increased in primary hepatocytes.3.Western-blot analysis revealed that SCD1 and p62 protein expression were increased in PA-stimulated primary hepatocytes,while p-AMPK/t-AMPK and LC3-II/LC3-I protein expression were decreased.II.Effects of altered SCD1 expression on lipid deposition,AMPK and lipophagy in primary hepatocytes.1.We used si RNA to knock down SCD1 in primary hepatocytes,Western-blot confirmed decreased SCD1 levels in si RNA-SCD1-414-transfected cells compared with the control groups.2.After inhibiting the expression of SCD1,the TG contents and lipid droplets in primary hepatocytes was significantly reduced.3.Western-blot analysis revealed that inhibition the expression of SCD1 led to the stimulation of AMPK signaling and induced lipophagy.4.We used SCD1 overexpression adenoviruse to enhance the expression of SCD1 in primary hepatocytes,Western-blot confirmed increased SCD1 levels in these cells compared with the control groups.5.After overexpression of SCD1,the TG contents and lipid droplets in primary hepatocytes was significantly increased.6.Western-blot analysis revealed that overexpression of SCD1 led to the inactivation of AMPK signaling and suppresses lipophagy.III.The role of SCD1-AMPK pathway mediated-lipophagy on lipid deposition in primary hepatocytes.1.The TG contents and lipid droplets were decreased in PA+si RNA-SCD1+Dorsomorphin group compared with PA+ Dorsomorphin group,but increased compared to PA+si RNA-SCD1 group.2.Western-blot analysis revealed that the expression of SCD1 in PA+Dorsomophin group did not change significantly compared with PA group.And treatment with Dorsomorphin markedly decreased the p-AMPK/t-AMPK.Suppresses lipophagy was observed in PA+si RNA-SCD1+Dorsomophin group compared with the PA+si RNA-SCD1 group,but induced compared to PA+ Dorsomophin group.3.There was fewer autophagosomes and autolysosomes in hepatocytes treated with PA compared with that in hepatocytes without PA treatment.si RNA-SCD1 leaded to increase of autophagosomes and autolysosomes in hepatocytes.Inhibition of AMPK obviously decreased the amount of autophagosomes and autolysosomes in the hepatocytes treated with si RNA-SCD1.4.The TG contents and lipid droplets were increased in PA+SCD1-OE+ A-769662 group compared with PA+ A-769662 group,but decreased compared to PA+ SCD1-OE group.5.Western-blot analysis revealed that the expression of SCD1 in PA+ A-769662 group did not change significantly compared with PA group.And treatment with A-769662 markedly increased the p-AMPK/t-AMPK.Induced lipophagy was observed in PA+SCD1-OE+ A-769662 group compared with PA+ SCD1-OE group,but suppressed compared to PA+ A-769662 group.6.SCD1-OE leaded to decrease of autophagosomes and autolysosomes in hepatocytes.Activation of AMPK obviously increased the amount of autophagosomes and autolysosomes in the hepatocytes treated with SCD1-OE.IV.Effects of inhibition of SCD1 expression in vivo on hepatic steatosis,AMPK and lipophagy in mice.1.The liver function and blood lipid level in HFD group were significantly higher than that in NCD group,while that in CAY10566 group were significantly lower compared with HFD group.2.The hepatic tissue steatosis in HFD group was significantly higher than that in NCD group,while that in CAY10566 group were significantly lower compared with HFD group.3.Western-blot analysis revealed that the expression of SCD1 in HFD group was significantly higher compared with NCD group,whileCAY10566 treatment markedly decreased SCD1 expression.Inactivation of AMPK signaling and suppresses lipophagy were observed in HFD group compared with NCD group,while stimulation of AMPK signaling and lipophagy were observed in CAY10566 group compared to HFD group.4.Among the three groups,the number of autophagosomes and autolysosomes in NCD group was the highest.There was no distribution of autophagosomes and autolysosomes in HFD group.Comparing with NCD group,autophagosomes and autolysosomes were decreased in CAY10566 group,but increased significantly compared with HFD group.Conclusion: PA treatment could induce lipid deposition in primary hepatocytes,accompanied by increased expression of SCD1,inactivation of AMPK signaling and suppresses lipophagy.Inhibition of SCD1 expression enhanced the activity of AMPK and lipophagy and decreased lipid deposition in primary hepatocytes,whereas overexpression of SCD1 decreased the activity of AMPK and lipophagy and increased lipid deposition in primary hepatocytes.SCD1 negatively regulated lipophagy through inactivation of the AMPK to affect lipid deposition.Inhibition of SCD1 expression in vivo could alleviate hepatic steatosis induced by high-fat diet,enhanced the activity of AMPK and lipophagy.In summary,SCD1-AMPK-lipophagy pathway can be used as a potential therapeutic target for NAFLD.