| Excess lipid accumulation and related metabolism disorders,such as fatty liver and obesity,occur widely in farmed fish.However,the mechanisms of lipid metabolism,particularly lipid catabolism,are not well understood in fish,which greatly hinders the development of artificial regulation of lipid deposition in fish.The mechanism of lipid metabolism is relatively perfect in mammalian.Lipid droplets(LDs)catabolism at present is thought to via lipophagy,a new way to degrade fat except to lipolysis.Lipophagy,which is a form of autophagy in mammals,is a biological process in which LDs are degraded through the lysosomal degradative pathway.During lipophagy,LDs sequestrated in autophagosomes are delivered to lysosomes,which fuse to form autophagosomes and then develop into autophagolysosomes,leading to degradation of LDs.A numbers of studies have demonstraced that lipophagy takes part in lipid metabolism and energy homeostasis in mammals.So we suppose whether lipophay can be used as a way to improve the fat deposition in fish.However there no evidence has been provided for lipophagy in fish,which is the largest population of vertebrates.Therefore,we attempt to answer the following quentions in the study:(1)Whether lipophagy exists in fish.(2)Whether lipophagy plays important roles in lipid metabolism of fish.(3)Whether there has a relationship between lipophagy and lipolysis.In the present study,zebrafish and a zebrafish liver cell line(ZFL)were used in experiments.Electron microscopy and the autophagic marker protein of the microtubule-associated protein 1 light chain 3(LC3)were used to verify the existence of lipophagy in zebrafish and ZFL cells.Chloroquine(CQ)and 3-methyladenine(3-MA),which have been widely used as a biochemical inhibitor of lipophagy,and fasting were applied as pharmacological and nutritional regulation of autophagy,respectively,to clarify lipid metabolism-related biochemical,cellular,and molecular characteristics of fish when lipophagic activity changes.Moreover,a transcriptomic assay was performed to investigate the systemic metabolic changes in zebrafish with dysfunctional lipophagy.Finally,we tested the potential interplay between lipophagy and lipolysis in ZFL cells.In the present study,we observed the structures of LDs sequestered in autophagic vacuoles in the liver of fasting zebrafish and a zebrafish liver cell line(ZFL)using electronic microscopy.Moreover,mRNA and protein expression of the autophagy marker LC3 increased during fasting.Which clarify that fasting can induce lipophagy in zebrafsih.Inhibiting autophagy with chloroquine and 3-methyladenine significantly increased LD content and decreased fatty acid ?-oxidation and esterification activities in zebrafish liver and ZFL cells.This finding indicates that the decreases in TG-sourced FFAs caused by inhibiting lipophagy not only reduced FA breakdown activity,but also suppressed lipogenesis.The transcriptomic date of fish liver showed that inhibiting lipophagy depressed the expression of most genes in lipid catabolism and lipogenesis,but upregulated the genes involved in glycolysis,gluconeogenesis,and proteolysis,to maintain the energy homeostasis in fish.Furthermore,the dysfunction of lipophagy inhibited the activity of lipolysis,and the same consequence in inhibited lipolysis,which suggested that there may be a conspiracy relation between lipophagy and lipolysis.Altogether,this is the first study to demonstrate the existence of lipophagy and its essential regulatory roles in lipid metabolism in fish.Moreover,lipophagy is very likely to be an important evolutionarily conserved cellular process for maintaining energy homeostasis in vertebrates. |
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