The Effect Of Endoplasmicreticulum Stress On Lipogenesis Andthe Molecular Mechanism In Grasscarp(Ctenopharyngodon Idellus)

For energy mobilization,adipocytes can accumulate triglycerides.Similar to mammals,excessive accumulation of lipids can also trigger a series of metabolic diseases in fish adipocytes.The endoplasmic reticulum is a sensitive receptor for nutrients and is involved in the synthesis and transport of intracellular lipids.Endoplasmic reticulum stress(ERS)is induced by intracellular and extracellular stimuli,and subsequent unfolded protein reaction(UPR)can alleviate ERS.Moreover,both the classical UPR pathway and the glycogen synthetase kinase 3β(GSK-3β),which is independent of the UPR pathway,can regulate lipid metabolism in mammalian adipose tissue and liver.However,the effect of ERS on lipid accumulation in grass carp adipocytes is unclear.In this study,the methods of primary adipocyte culture,gene cloning and other cell and molecular biological were used to analyze the effect of ERS on lipid accumulation in adipocytes of grass carp and its mechanism.The results obtained are as follows:1.Analysis of lipid metabolism of grass carp adipocytes induced by oleic acid(OA)To build a lipid accumulation model for adipocytes,grass carp preadipocytes were obtained by enzymatic digestion in vitro.After the adipocytes grew to confluent(approximately 7 days),400 μM OA was added to the medium for 48 hours.CCK-8was used to determine cell viability,nile red staining and DAPI staining were used to observe lipid accumulation in adipocytes,kits were used to detect intracellular triglyceride content and glycerol release in culture medium,RT-PCR was used to detect the m RNA expression levels of lipid metabolism-related genes.The results showed that(1)400 μM OA significantly increased cell viability(P <0.01),the number of red lipid droplets increased and the triglyceride content in adipocytes increased significantly(P <0.01);(2)The m RNA expression levels of lipolysis related genes ATGL,HSLa,HSLb and fatty acid β-oxidation related genes PPARα,CPT1αwere significantly up-regulated(P <0.05),the glycerol content in the medium also increased significantly(P <0.01);(3)The m RNA expression levels of adipogenesis-related genes PPARγ and lipogenesis-related genes SREBP1 c,FAS,ACCα,DGAT2 were significantly increased(P <0.05).These results indicated that exogenous OA promotes the processes of lipid synthesis and lipolysis.The role of lipid synthesis may be greater than that of lipolysis,or the lipolysis-related genes were regulated by post-translational regulation,which ultimately resulted in phenotype oflipid accumulation.2.ERS mediates lipid accumulation in grass carp adipocytes mediated by UPR pathwayIn order to investigate the effect of ERS on lipid accumulation,adipocytes were pre-incubated with ERS inhibitor Sodium 4-phenylbutyrate(4-PBA)(0,5,10,15,20μM)for 4 hours,and then co-incubated with 400 μM OA for 48 h.Nile red staining and DAPI staining were used to observe the accumulation of lipids in adipocytes.The content of cellular triglycerides was quantified.RT-PCR was used to detect the m RNA expression levels of ERS and lipid metabolism related genes.The results showed that(1)The m RNA expression levels of classic UPR-related genes GRP78,ATF6,PERK,e IF2α and CHOP were significantly up-regulated by treatment with 400 μM OA(P<0.05),indicating that ERS was activated during lipids accumulation in adipocytes;(2)The m RNA expression levels of GRP78,PERK,e IF2α,CHOP,IRE1α and ATF6 and the protein expression levels of GRP78 were significantly reduced(P <0.05),indicating that ERS was relieved by treatment with 10 μM 4-PBA;(3)After treated with 10 μM 4-PBA,the number of red lipid droplets were decreased,and the cellular triglycerides were significantly decreased(P <0.05);(4)The m RNA expression levels of adipogenesis-related genes PPARγ,C/EBPα and lipogenesis-related genes FAS,ACCα,SCD1,DGAT1 a,DGAT1b and DGAT2 both were significantly reduced(P<0.05).These results showed that ERS is activated during OA-induced lipid accumulation in grass carp adipocytes,and the classical UPR pathway involved in lipid accumulation in adipocytes.3.Glycogen synthase kinase-3β(GSK-3β)of grass carp(Ctenopharyngodon idella):synteny,structure,tissue distribution and expression in oleic acid(OA)-induced adipocytesStudies on mammals have found that ERS not only activates UPR to participate in lipid metabolism,but also regulates lipid accumulation by activating GSK-3β.In order to investigate the role of GSK-3β in grass carp lipid accumulation,its two paralogs,GSK-3β1 and GSK-3β2 were isolated and characterized,encoding peptides of 421 and 457 amino acids,respectively.We used bioinformatics analysis,RT-PCR to detect tissue expression pattern of GSK-3β1 and GSK-3β2 and their m RNA expression levels during lipid accumulation in adipocytes.The results showed that(1)Alignment of grass carp GSK-3β deduced amino acid sequences with select human,mouse,pig,chicken,zebrafish and common carp showed that the protein wasconserved.However,two paralogs of the GSK-3β had great variation in gene structure:the GSK-3β1 contained eleven exons while the GSK-3β2 contained nine exons;(2)Grass carp GSK-3β1 and GSK-3β2 may have originated from the teleost-specific genome duplication(TSGD)event;(3)The two paralogs were expressed in a wide range of tissues,GSK-3β1 was most expressed in adipose tissue,GSK-3β2 was most expressed in liver;(4)During lipid accumulation in adipocytes,GSK-3β1,GSK-3β2,and β-catenin m RNA expression levels were significantly up-regulated(P <0.05).These results have shown that GSK-3β may be involved in the lipid accumulation of grass carp adipocytes through the GSK-3β/β-catenin pathway.In summary,ERS was activated during the lipid accumulation of grass carp adipocytes.The classical UPR pathway which was triggered by ERS involved in lipid accumulation in adipocytes.Furthermore,GSK-3β,which was independent of the classical UPR,may participate in the process of lipid accumulation.