Study Of The Role And Mechanism Of LASS2 Gene In Hepatic Lipid Metabolism

Objective:To explore changes in expression of LASS2 in a model of obesity and hepatic steatosis and to understand the relationship between LASS2 and hepatic lipid metabolism.Methods:The expression levels of LASS2 m RNA and protein in liver tissues of male C57BL/6J,ob/ob,db/db,KK-Ay,and APOE^-/-mice were detected by real-time fluorescence quantitative PCR and western blot methods,respectively.C57BL/6J mice were randomly divided into two groups:a general diet group and a high-fat diet group?all mice were fed the general or high-fat diet for 16 weeks?.Steatosis was induced in primary hepatocytes and Hepa1-6 hepatoma cells by free fatty acids?FFA?,and the expression levels of LASS2 m RNA and protein in each group were detected by quantitative PCR and western blot,respectively.Results:Compared with those in C57BL/6J mice,the m RNA and protein expression levels of LASS2 in liver tissues of ob/ob,db/db,KK-Ay,and APOE-/-mice were significantly decreased?P<0.01?.The protein expression levels of LASS2 in the liver of C57BL/6J mice fed the high-fat diet or in FFA-treated hepatocytes?mouse primary hepatocytes and Hepa1-6 hepatoma cells?were significantly downregulated compared with mice fed the chow diet or in cells without FFA treatment?P<0.01?,respectively,but there were no significant differences in m RNA expression levels?P>0.05?.Conclusion:LASS2 may be related to lipid metabolism and the occurrence and development of hepatic steatosis.Objective: To investigate the effects of LASS2 on hepatic lipid metabolism and related pathway genes in mice with nonalcoholic fatty liver disease?NAFLD?induced by a high-fat diet.Methods: Healthy male C57BL/6J mice were randomly divided into a chow diet?CHD?group and high-fat diet?HFD?group.Mice in the HFD group were injected with AAV-TBG?liver tissue-specific promoter?recombinant expression vector of LASS2 or controls via tail vein after feeding for 16 weeks,in four subgroups: overexpression control group?HFD-GFP?,LASS2 overexpression group?HFD-m LASS2?,knockdown control group?HFD-sh GFP?,and LASS2 knockdown group?HFD-shm LASS2?.Weekly weight changes were recorded.Four weeks after injection of the AAV recombinant expression vector,an intravenous glucose tolerance test was conducted and levels of serum biochemical indices including blood lipids,alanine aminotransferase?ALT?,and aspartate aminotransferase?AST?were measured.Liver tissue sections were stained with hematoxylin and eosin?HE?for pathological analysis,and the contents of hepatic triglycerides?TG?and total cholesterol?TC?were detected using enzymatic methods.The m RNA and protein levels of LASS2,fatty acid synthesis-related genes?FAS,SREBP1?,lipolysis-related genes?ATGL?,cholesterol synthesis-related genes?SREBP2,HMGCR?,and fatty acid ?-oxidation-related genes?CPT1A?in liver were detected by real-time quantitative PCR and western blotting,respectively.Results: Compared with the HFD-GFP or HFD-sh GFP groups,the m RNA and protein levels in liver tissue of mice in the HFD-m LASS2 or HFD-sh m LASS2 group were significantly upregulated or downregulated,respectively?all P<0.01?,suggesting that the LASS2 overexpression and knockdown in vivo models were established successfully.Compared with those of the CHD group,the body weight and liver weight of mice in the HFD group were significantly increased?P<0.05?.Analysis of HE-stained tissue revealed obvious steatosis in liver tissue in the HFD group.In addition,body weight and liver weight were significantly lower in the HFD-m LASS2 group than in the HFD-GFP group.Fasting blood glucose,impaired glucose tolerance,and serum biochemical indices?ALT,AST,TG,TC,high-density lipoprotein-cholesterol,low-density lipoprotein-cholesterol?were significantly higher in the HFD group than in the CHD group.Compared with the HFD-GFP group,intraperitoneal glucose tolerance was significantly improved in the HFD-m LASS2 group,and levels of biochemical indices in serum and contents of hepatic TG and TC were significantly decreased?P<0.05?.The m RNA and protein levels of FAS,SREBP1,SREBP2,and HMGCR were significantly decreased in the HFD-m LASS2 group?all P<0.01?,and ATGL and CPT1 A m RNA and protein expression levels were significantly increased?P<0.01?.The opposite results were observed in the HFD-shm LASS2 group.Conclusion: LASS2 may improve disorders of lipid metabolism by regulating genes related to fatty acid synthesis,lipid decomposition,cholesterol synthesis,and fatty acid ?-oxidation.Overexpression of LASS2 has a protective effect in the process of NAFLD and vice versa.Objective: To investigate the effects of LASS2 on lipid accumulation and lipid metabolism pathway-related genes in free fatty acid?FFA?-induced hepatocytes.Methods: Hep G2 hepatoma cells were transfected with LASS2 overexpression adenovirus?Adv-h LASS2-GFP?for 48 h,and the LASS2-GFP fusion protein was localized at the subcellular level using confocal laser scanning microscopy.Mouse primary hepatocytes?MPHs?and Hepa1-6 hepatoma cells were isolated and cultured.Steatosis of hepatocytes was induced by a combination of FFAs,and hepatocytes were transfected with Adv-m LASS2-GFP or knockdown adenovirus?Adv-shm LASS2-GFP?.Changes in lipid accumulation were observed following oil red O staining,and the contents of triglycerides?TG?in hepatocytes were analyzed by a microbiochemical method.The m RNA and protein relative expression of LASS2,fatty acid synthesis-related genes?FAS,SREBP1,ACC?,lipolysis-related genes?ATGL,HSL?,cholesterol synthesis-related genes?SREBP2,HMGCR?and fatty acid ?-oxidation-related genes?PPAR?,CPT1A?in MPHs and Hepa1-6 cells or Hep G2 cells were detected by real-time quantitative PCR and western blotting,respectively.Results: The LASS2-GFP fusion protein was localized mainly in the nuclear membrane and cytosol,as observed by confocal laser scanning microscopy.Compared with the NC,LOC?Adv-GFP?,and Lsh C?Adv-sh GFP?groups,relative m RNA and protein levels of LASS2 in the LO?Adv-m LASS2 or Adv-h LASS2?and Lsh?Adv-shm LASS2?groups were significantly upregulated or downregulated?P<0.001?,indicating that the LASS2 overexpression or knockdown in vitro model was established successfully.In MPHs and Hepa1-6 cells,lipid accumulation induced by FFAs was significantly improved in the LO group compared with the NC and LOC groups,as shown by oil red O staining,and intracellular TG content was significantly decreased in the LO group?all P<0.01?.In addition,the m RNA and protein expression levels of FAS,SREBP1,SREBP2,and HMGCR in the LO group were significantly downregulated?all P<0.01?.We detected no significant difference in protein expression levels of t-ACC and HSL?P>0.05?.The p-ACC/t-ACC ratio,p-HSL/HSL ratio,and m RNA and protein levels of ATGL,CPT1 A,and PPAR? were significantly upregulated?P<0.05?.The effects of LASS2 overexpression on these genes in Hep G2 cells were consistent with these results.The opposite results were observed in the Lsh group?MPHs and Hepa1-6 cells?.Conclusion: We verified in vitro that LASS2 improved FFA-induced lipid accumulation in hepatocytes by regulating fatty acid synthesis,lipolysis,cholesterol synthesis,and fatty acid ?-oxidation related genes.Objective: To explore the molecular mechanisms by which LASS2 regulates hepatic lipid metabolism.Methods: Free fatty acids?FFA?-induced or non-FFA-induced Hepa1-6 cells were transfected with Adv-GFP or Adv-m LASS2 for 48 h,and the protein complexes interacting with LASS2 were identified by co-immunoprecipitation/liquid chromatography-mass spectrometry?Co-IP/ LC-MS?.All identified proteins were analyzed using Gene Ontology?GO?,Eukaryotic orthologous groups?KOGs?and the Pathway database,and key enriched proteins were analyzed.The interaction between LASS2 and the key protein NDUFS2 was preliminarily verified by Co-IP western blot.Eukaryotic expression vectors of LASS2 and NDUFS2 were constructed and co-transfected into FFA-induced or non-FFA-induced Hepa1-6 cells.Co-localization of LASS2 and NDUFS2 was confirmed by confocal laser scanning microscopy.FFA-induced Hepa1-6 cells were transfected with p EGFP-m LASS2 or pm Cherry-Flag-m NDUFS2,and the interaction between LASS2 and NDUFS2 was confirmed by western blot.FFA-induced MPHs and Hepa1-6 cells were transfected with Adv-GFP and Adv-m LASS2.To further verify the interaction between LASS2 and NDUFS2,related cascade signal pathways?mt ROS/AMPK/m TORC1? were analyzed.The levels of mitochondrial reactive oxygen species?mt ROS?were assessed by flow cytometry and confocal laser scanning microscopy,autophagy/lipophagy was observed by transmission electron microscopy,and the levels of AMPK,raptor,and autophagy-related proteins?LC3II/I,p62,Beclin-1,Atg3,Atg5,Atg7?were detected by western blot.Results: According to CO-IP/LC-MS and analysis using GO,KOGs,Pathway,STRING,and other databases,NDUFS2/OXPHOS was identified as the key protein interacting with LASS2 to be verified.Co-IP western blot results confirmed the interaction between LASS2 and NDUFS2.The results of confocal laser scanning microscopy showed that LASS2 was co-localized with NDUFS2 in Hepa1-6 cells.The western blot results showed that the levels of NDUFS2 were downregulated by LASS2 overexpression?P<0.01?,but levels of LASS2 did not change in the NDUFS2 overexpression group?P>0.05?.Compared with the control group,the levels of mt ROS in LASS2 overexpression MPHs and Hepa1-6 cells were significantly increased?all P<0.01?.The phosphorylation of AMPK?,the ratio of LC3II/LC3 I,and the levels of Beclin-1,ATG3,ATG5,and ATG7 were increased and the phosphorylation levels of Raptor and p62 protein were significantly decreased in LASS2 overexpression hepatocytes?all P<0.01?.The opposite results were observed in the LASS2 knockdown group.Transmission electron microscopy showed that there were more autophagy lysosomes,autophagosomes,and lipophagosomes in LASS2 overexpressed FFA-treated Hepa1-6 cells compared with the control group.Conclusion: LASS2 plays a protective role in FFA-induced hepatocellular steatosis by regulating the AMPK/raptor/autophagy?lipophagy?cascade signaling pathway.The underlying mechanism may be an increase in mt ROS mediated by binding of NDUFS2/OXPHOS-related proteins,which may also be one of the possible mechanisms by which LASS2 regulates hepatic lipid metabolism.