Characterization Of Autophagy And Its Regulatory Mechanism In Zinc Influencing Lipid Metabolism Of Yellow Catfish Pelteobagrus Fulvidraco

Autophagy is a conserved catabolic process in which intracellular long-lived proteins,superfluous or damaged organelles are delivered to lysosomes for degradation and recycling.Autophagy has been acknowledged to provide a survival strategy that supplies nutrients,regenerates organelle,cleans the metabolic waste and maintains cellular homeostasis for cell survival under adverse environment.As a catabolic machinery,autophagy could degrade hepatocellular lipid droplets through a process termed lipophagy,which is essential for maintaining the homeostasis on lipid metabolism.Our previous study indicated that Zinc?Zn?,an essential micronutrient,significantly influenced lipid metabolism in a fish species of significance for aquaculture,yellow catfish Pelteobagrus fulvidraco,but further mechanism remained unknown.Whether the Zn-induced changes in lipid metabolism is accompanied by activated autophagy?Whether autophagy acts as a mediator in the Zn-induced effects on lipid metabolism?What is the potential mechanism?The purpose of this study is to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating autophagy.Using yellow catfish as experimental animal model,we first isolated and identified the full-length cDNA sequences of 20 autophagy-related genes involved in autophagosome formation.Then,we characterised the transcriptional responses of those genes to dietborne and waterborne Zn.Last,we demonstrated the relative contribution of Zn²⁺/Ca²⁺/CaMKK?/AMPK pathways to activate autophagy and lipid turnover in response to Zn.Results are as follows:1 Molecular characterization and function analysis for the autophagy-related genes from yellow catfishThe full-length cDNA sequences of 20 autophagy-related genes involved in autophagosome membrane initiation,nucleation,expansion,closure and maturation?ULK1A,ULK1B,ATG3,ATG4A,ATG4B,ATG4C,ATG4Da,ATG4Db-1,ATG4Db-2,ATG5,Beclin1,ATG7,MAP1LC3A,MAP1LC3B,ATG9A,ATG9B,ATG13-1,ATG13-2,ATG101,SQSTM1?were successfully obtained by RT-PCR and RACE methods from P.fulvidraco.More ATG4 and ULK1 members were firstly identified in fish that might have arisen by teleost-specific whole genome duplication events.In this work,as a preliminary step to unravel the physiological role of the autophagy-related genes,we determined the tissue distribution patterns of these genes.Their mRNAs were widely expressed in various tissues,but at variable levels,indicating expression of the autophagy-related genes had tissue specificity and species specificity.All of these members shared similar domain structure to their orthologous genes of vertebrates.Phylogenetic analysis further identified these genes,and confirmed their classification.These results provide evidence that the autophagic process can also be fully reconstituted in fish,which opens the possibility to further explore the physiological function of autophagy and the mechanism of induction,regulation and execution of autophagy in fish.2 The effects of dietborne and waterborne Zn addition?different exposure pathways?on the hepatic autophagy in yellow catfishThe effects of different exposure pathways?dietborne and waterborne Zn?on the autophagy of liver were investigated.The experiment was divided into two parts.In part 1,P.fulvidraco were fed 8.83?low Zn?,19.20?middle Zn?or 146.65?high Zn?g Zn kg^-1diet for 56 days.In part 2,P.fulvidraco were exposed to three Zn concentrations?0.007±0.001,0.253±0.004 and 0.514±0.070 mg Zn/L,respectively?for 56 days.Electron microscopic?EM?observations demonstrated that dietborne and waterborne Zn increased the amount of hepatic autophagic vacuoles,indicating autophagy activation.Extra Zn addition in water or diets induced mRNA expression of ULK1A,ULK1B,LC3B,ATG4Da,ATG4Db,ATG3,ATG7,ATG9B and SQSTM1.These genes involved in autophagosome membrane initiation,expansion and maturation may mediate the changes of Zn-induced hepatic lipid metabolism of yellow catfish under different Zn exposure pathways.Transcriptional activation of the key autophagy genes plays an important role in the activation of autophagy induced by Zn.The present study found that Zn could activate autophagy in vivo,which would contribute to our understanding of the molecular basis for physiological functions of Zn,and also provide new insights into roles and mechanisms of autophagy in the physiological response of Zn.3 Roles and mechanisms of autophagy in the regulation of Zn on hepatic fat deposition and lipid metabolism in yellow catfishAccording to previous studies,Zn deficiency is associated with higher risk for fatty liver and impaired lipid profile.Clinical trials in humans,animal and in vitro studies demonstrate that Zn plays an essential protective role in regulation of lipid accumulation and metabolism.Autophagy has been shown to regulate lipid metabolism.Whether Zn alleviates fat deposition via the activation of autophagy.The underlying mechanism of Zn action still needs further study.Utilizing flow cytometry,laser confocal microscopy and transmission electron microscope combine with BODIPY 493/503 staining,determination of TG concentrations and Oil Red O Staining,we determined the effects of Zn on lipid deposition in vivo and in vitro.Utilizing western blot,flow cytometry,laser confocal microscopy and transmission electron microscope combine with MDC staining,OA staining,determination of LC3B protein level and Lyso-Tracker Red Staining,we determined whether Zn administration activates autophagy.Results show that Zn increases hepatocytes lipid clearance and autophagosome formation in a dose-dependent manner in hepatocytes.We used pharmacological inhibitors?CQ and 3-MA?of autophagic and lysosomal function to demonstrate that whether the autophagy was directly involved in the Zn-induced reduction of lipid accumulation.3-MA or CQ not only reduced Zn induced autophagosome formation,but also significantly decreased Zn-mediated reduction of intracellular lipid,strongly suggesting involvement of autophagy in Zn-induced lipid depletion.The co-localization analysis of the autolysosomes and the lipid droplets in hepatocytes co-stained with Lyso Tracker?red?and BODIPY 493/503?green?,which indicated the induction of lipophagy.EM images corroborated these findings by showing lipid droplets integrated with the autolysosomes in Zn-treated cells.More NEFA release,activated lipolysis and inhibited lipogenesis further confirmed the coupling of autophagic induction and lipid turnover following Zn treatment.We corroborated that Zn reduced lipid accumulation and stimulates lipolysis in hepatocytes by an autophagy-mediated lipophagy dependent pathway.To understand the possible mechanism by which Zn activates autophagy and lipid depletion in hepatocytes,the effects of Zn supplementation on AMPK activity were determined.Zn upregulated AMPK activity,indicating Zn activated signaling pathways involving AMPK.The Zn-induced increased autophagy,lipolysis and hepatocellular NEFA content,and the decreased of TG content and lipogenesis was partly reversed by the presence of CaMKK?/AMPK pathway inhibitor?Compound C and STO-609?.Newport green staining(Zn²⁺fluorescent probe)demonstrated a dose-dependent increase in intracellular free Zn²⁺of hepatocytes treated by Zn.TPEN?chelating agent of Zn?blocked lipid clearance and autophagosome formation by Zn supplementation.Results show that Zn induced upregulation of autophagy and lipid depletion is cytosolic free Zn²⁺-dependent.The hepatocytes exhibited a time-dependent increased in Ca²⁺fluxes as measured with a Ca²⁺fluorescent probe Fluo-4,AM.Moreover,TPEN pretretment followed by Zn incubation blocked the increase of intracellular Ca²⁺induced by Zn administration.These data indicated that the Zn²⁺/Ca²⁺/CaMKK?/AMPK signaling pathway indeed mediated the Zn-induced autophagy and lipid turnover.We elucidated the roles and mechanisms of autophagy in the regulation of Zn on hepatic fat deposition and lipid metabolism.These results extends our understanding into Zn nutrition and physiological function of Zn,and provides novel insights on the mechanism and prevention of fatty liver disease in fish.