Study On Lipotoxicity Response In Zebrafish Liver Induced By Palmitic Acid

Palm oil(PO)is the second most abundant vegetable oil in the world and has been used in fish diets widely.PO contains high level of palmitic acid.However,accumulation of excess PA leads to cell dysfunction or cell death,namely lipotoxicity.Studies have identified that high concentration(250-500 μM)of PA induced ER stress and apoptosis in mammal cells.However,the PA lipotoxicity has never been investigated in warm water finfish and their hepatocytes.In the first part of this study,we established a warmwater finfish model using zebrafish and fed 1-month old zebrafish either low fat(LF)diet,high fat(HF)diet or 3 PA incorporated high fat diets(PA4%,PA8%,PA12%).The PA accumulation in liver was detected after 2 weeks.The serum alanine transaminase(ALT),serum aspartate aminotransferase(AST),liver ALT,caspase3,triacylglycerol(TAG)and expression of gene markers related to liver damage and ER stress were detected after 4 weeks.Liver PA level increased with dietary PA supplementation.The serum ALT level was increased by 40.7% and 59.5% in PA 8% and 12% groups compared with the HF group(P< 0.05).The serum AST level was 28.8% and 28.0% higher in PA8% and 12% groups versus the HF group(P=0.009 and P=0.029,respectively).The expression of UPR markers(Grp78,Chop)was up-regulated in PA12% group(2.2 and 2.7-fold,respectively,P< 0.05)compared with HF group.The results revealed that dietary PA induced liver damage and ER stress in zebrafish liver.In the second part of this study,we used ZFL cell model to research the mechanism involved in PA lipotoxicity to zebrafish liver.The responses of ZFL to PA were analyzed by Real-Time Cell Analyzer(RTCA),Alarmablue assay and flow cytometry at 24 h.The potential pathways related to PA toxicity in ZFL were investigated by real time qPCR,gene silencing and pathway blocking with specific inhibitors.In ZFL,PA induced apoptosis in a concentration-and time-dependent manners.The expression of UPR sensors,Grp78 and Grp94,was up-regulated by 50 μM PA(3.3 and 12.1-fold,respectively,P<0.01)and 100 μM PA(3.0 and 6.2-fold,respectively,P<0.01)compared with the control.Blocking experiments indicated that PERK-CHOP and IRE1α-JNK pathways were involved in PA-induced apoptosis in ZFL.The results revealed PERK-CHOP and IRE1α-JNK pathways were involved in PA lipotoxicity to zebrafish liver.In the third part of this study,we used larvae model to verify whether the defined ER stress pathways in ZFL are involved in PA lipotoxicity in vivo.We fed 5-dpf zebrafish larvae 8% PA diet for 1 week and tested the expression of gene markers involved in liver damage,ER stress and autophagy.In zebrafish larvae model,PA diet induced ER stress and the expression of Atg4 b,Atg9a and Saa2 were decreased after blocking PERK-CHOP pathway(P<0.01).The results revealed PERK-CHOP was involved in PA lipotoxicity to zebrafish larvae.PA damaged liver of warm water finfish via activation of ER stress,which suggested the limitation of high PO inclusion in the aquafeeds for warm water finfish culture.