| In recent years,high-fat diet has been widely used by aquaculturists because of its cost-effective,wide source and obvious growth promotion.Although high-fat diet can save protein,reduce cost and accelerate growth,it also brings potential side effects,such as lipid metabolism disorder and endoplasmic reticulum stress,which threatens fish farming industry.Blunt snout bream（Megalobrama amblycephala）as a famous freshwater species in China,whose world production has reached 826,178 tons in 2016 and increases annually under rapidly aquacultural developing.Compared with other teleost fish,blunt snout bream is more susceptible to high-fat diet-induced lipid metabolic disorder in the liver,due to its relatively low hepatosomatic index（the ratio of liver weight to body weight）.Therefore,our research used the species to study the relationship between lipid metabolism and endoplasmic reticulum stress,so as to provide feasible suggestions for avoiding hepatic steatosis in aquaculture.The experiments included three parts:Experiment 1 Effects of different fat levels on growth performance,mitochondrial damage and endoplasmic reticulum stress in blunt snout bream Megalobrama amblycephalaThis study aimed to explore the effect of dietary lipid levels on growth performance,mitochondrial damage,and endoplasmic reticulum stress fat deposition and endoplasmic reticulum stress of blunt snout bream through changing.Four experiment diets were low fat diet（3%fat,LFD）,normal fat diet（6%fat,NFD）,high fat diet（11%fat,HFD）,and high fat diet added with 100 mg/kg simvastatin（HFD+S）,respectively.240 Megalobrama amblycephala juveniles（41.83±0.05 g）were randomly assigned to four groups,and per group had four duplications at a rate of 15 fish/cage.The experiment period was 9 weeks.The results showed that high-fat diet（HFD）remarkably increased VSI,IPF,whole-body lipid,liver and muscle lipid contents,plasma levels of LDL-C,TC and TG contents,cytosolic cytochrome c activity and Ca2+ content and the transcriptions of GRP78,IRE1,XBP1s and SREBP1,whereas the opposite was true for plasma HDL-C content,SDH and Na+,K+-ATPase activities in the mitochondria,Ca2+-ATPase activity in the ER and the transcriptions of XBPlu,PPARa and CPT I.In addition,simvastatin supplementation remarkably decreased VSI,liver and muscle lipid contents,plasma LDL-C and TC levels,cytosolic cytochrome c activity and Ca2+ content and the transcriptions of GRP78,IRE1,XBP1s and SREBP1,while the opposite was true for plasma HDL-C level,Ca2+-ATPase activity and the transcriptions of XBP1u,PPARa and CPT I.Furthermore,the histological results showed that large lipid droplets and ultrastructural impairments of the mitochondria,nucleus,and ER were present in hepatocytes of fish fed the HFD,while simvastatin supplementation had the potential to alleviate these damages.Taken together,these results indicated that there was an interaction between lipid deposition and endoplasmic reticulum stress induced by HFD.Experiment 2 Molecular characterization and tissue distribution of inositol-requiring enzyme 1（IRE1）in Megalobrama amblycephalaThis study aimed to characterize the full-length cDNA of IRE1 from fish Megalobrama amblycephala,and investigate its tissue distribution.The full-length cDNA covered 3665 bp,with an open reading frame（ORF）of 3096 bp,which encodes 1031 amino acids,35 bp of 5’-untranslated region（UTR）and 534 bp of 3’UTR.The IRE1 protein has a calculated molecular weight（Mw）of 116536.18 Da and isoelectric point（PI）of 6.17.This AA sequence contains several conserved functional sites,including one signal peptide（Metl-Ser32）,one luminal domain（Glu39-Ser307）,one domain of the Serine/Threonine kinase（Asn570-Trp835）,one RNase domain（Lys839-Tyr963）,one activation loop（Ser712-Gly735）,two N-linked glycosylation sites（Asn185 and Asn533）and a lot of phosphorylation sites.The amino acid sequence alignment of IRE1 showed the high identity with carp（the homology is 92%）,and less identity with both human（the homology is 74%）.The highest expression was observed in trunk kidney followed by brain and spleen.The expression levels in gill,muscle,and head kidney were all moderate,while significantly lower expression levels were detected in liver,intestine,adipose,skin,and heart.Experiment 3 High-fat diet induces aberrant hepatic lipid secretion in blunt snout bream by activating endoplasmic reticulum stress-associated IRE1/XBP1 pathwayThe aim of this study was to investigate the effect of high-fat diet on hepatic lipid secretion and endoplasmic reticulum stress in blunt snout bream.The experimental fish（average weight:41.84±0.07 g）were randomly divided into control group（6%fat）and high-fat diet group（11%fat）,and the experimental period was 9 weeks.The feed utilization efficiency was evaluated at the end of the trial.The liver samples of both groups were harvested for molecular analysis and histological evaluation.Compared to the Control group,the high-fat diet group showed no effects on energy intake in blunt snout bream.However,high-fat diet resulted in a massive accumulation of lipid and pathological structural alternations,and disrupted expression of lipid transport-related genes and endoplasmic reticulum stress in the liver of the fish.In vitro,after exposure of the isolated primary hepatocytes from blunt snout bream to oleic acid,the cells showed increased intracellular TG accumulation,decreased VLDL secretion,which was attributed to altered expression levels of lipid transport-related genes through the activated IRE1/XBP1 signaling.The oleic acid-induced detrimental effects were alleviated by co-incubating the cells with an IER1 inhibitor,4μ8c.In conclusion,high-fat diet could lead to aberrant lipid secretion by activating the ER stress-associated IRE1/XBP1 pathway.Inhibiting the activity of IRE 1 represents a promising target to rescue the side-effects of high-fat diet on the liver function of blunt snout bream. |
PDF Full Text Request