Study On Energy Metabolism And Compensatory Growth Molecular Regulation Mechanism Of Silver Pomfret(Pampus Argenteus) During The Process Of Starvation And Re-feeding

After starvation or lack of nutrition,fish would show compensatory growth when they were re-fed,and this phenomenon had an important relationship with metabolic level of fish,but the specific mechanism of compensatory growth was still unclear.Pampus argenteus had great sport ability but the hunger tolerance was very poor,and unavailable model of compensatory growth had negitave effects on the growth and development of the fish.In view of this,to further investigate the specific mechanism of compensatory growth and available model of compensatory growth in Pampus argenteus,in present study,we first selected the compensatory growth mode which the fish fast recovered the body weight and other indicators under different starvation and re-feeding mode;in this optimal mode,we sequenced the transcriptome of liver,muscle and intestine of Pampus argenteus during the process of re-feeding after starvation,and analyzed the metabolism and metabolic regulation related KEGG pathway;we sequenced the metabonomics of muscle of Pampus argenteus during the process of re-feeding after starvation,and screen the differential energy metabolites and KEGG pathway highly enriched by differential energy metabolites;Based on the transcriptome and metabolome,we verified the key genes in metabolism and metabolic regulation related KEGG pathway by RT-qPCR,and verified of IGF1 protein by Western blot（WB）;we established Pampus argenteus muscle cell line（PaM）and a starvation and recovery model in this cell line（cell activity compensation recovery model）,and we verified the key genes in metabolism and metabolic regulation related KEGG pathway in RT-qPCR and WB,and the similarity between this model and the starvation and re-feeding model of Pampus argenteus（compensatory growth model）was confirmed.In this cell model,the key gene IGF1 and its upstream and downstream genes in compensatory growth related regulation pathway were interfered by si RNA,and then RT-qPCR was used to further prove that the correlation regulation pathway played an important role in compensatory growth.The specific contents and research results were as follows:（1）The relationship between body weight and body length was that W=0.0382L^2.9052（R²=0.9473,n=141）under normal feeding;the average body length of 5-month-old silver pomfret was 10 cm and the average fatness was 2.9.We set one control group and three stavation and re-feed groups（S7R21,S14R14 and S21R7）.The results showed that the compensatory growth of silver pomfret was not obvious in the S7R21 group and S21R7group;the compensatory growth of silver pomfret was obvious in the S14R14group,and the body weight,fatness and height of silver pomfret were almost the same as those of the control group,and the relationship among body weight,fatness and body length reached the normal standard.After the end of starvation treatment,the food intake of the three experimental groups was basically higher than that of the normal group within1-2 days,but then was similar to that of the control group,and the average food intake after re-feeding was consistent with that of the control group.We set the S14R14 group as test group in sebsequent experiments.（2）After starvation,a large number of energy catabolism related items were enriched in GO term and KEGG pathway in liver and muscle of Pampus argenteus,and KEGG pathways such as protein digestion and absorption（ko04974）and fat metabolism and absorption（ko04975）were significantly enriched in intestine.In the transcriptome of silver pomfret,many go term and KEGG pathways of anabolism were significantly enriched in muscle and liver,but fat metabolism and protein metabolism were still highly enriched in intestine;In those tissues,PPAR signaling pathway（ko03320）and AMPK signaling pathway（ko04152）were also enriched.（3）After starvation,lipid substances such as LPE 20:3,LPC 18:0 and amino acid substances such as L-threonine,L-tyrosine and glycine were significantly down regulated,short chain lipid substances such as LPC 18:0 were exhausted,and long chain lipid substances such as LPE 20:3,LPG 20:0 and protein substances were consumed for energy supplement.In the process of re-feed,carbohydrate metabolites such as D-glucose6-phosphate and dihydroxyacetone phosphate,lipid metabolites such as LPS 16:1,LPC20:5,LPA 22:6 and amino acid metabolites such as lysine and citrulline were recovered rapidly,and there were only amino acid metabolites such as L-Isoleucine and Ornithine being recovered rapidly in later period of re-feed.The results of rapid recovery of this energy storage substance were similar to those of transcriptome.（4）The key differential genes of energy metabolism and related regulatory pathways in the process of re feeding after starvation were screened and verified by RT-qPCR.In muscle tissue,the expression of pgm,CALR,TER,FABP2 b and ACLY were down-regulated firstly and then up-regulated,while the expression of ACSL,TSC2,MLYCD were up-regulated firstly and then down-regulated;In liver,the expression of SCP2,FBF,UGT,IDH1 and ERG25 were down-regulated firstly and then up-regulated,while the expression of PECI were up-regulated firstly and then down-regulated;in intestinal tissue,the expression of FDFT1,PPAR,SSR2,SDH1 and FABP2 a were down-regulated firstly and then up-regulated,while the expression of PRSS were up-regulated firstly and then down-regulated;The expression trend of IGF1 and IGFBP3in the three tissues was firstly up-regulated and then down-regulated;The expression of GH did was raised but not significantly in the brain,but it was increased during starvation;according to WB results,the expression trend of IGF1 protein in the three tissues was first increased then dropped.（5）Pampus argenteus muscle cell line（PaM）was cultured in L-15 medium containing 20%fetal bovine serum（FBS）and was passaged at the ratio of 1:1 to 1:2.At present,it has survived for more than 200 days in vitro and was passaged to 40 generations.The 50μg/L concentration of HGF,IGF1 and GH growth factor was selected for PaM culture.A starvation and recovery model of PaM cultured in serum-free medium for 9 h/3h normal serum medium was established.In this model,the cell activity decreased significantly under starvation,but increased significantly after recovery,which was higher than that of the control group,resulting in an activity compensation recovery effect.In this model,GHR,IGF1,IGF1R,IGFBP3,mTOR and AMPK were verified by RT-qPCR,and IGF1 was also verified by WB,which showed that the expression trend of these genes in vitro was similar to that in Pampus argenteus.Through RNAi of GHR,IGF1 and IGF1R genes,we found that the cell activity did not increase significantly after recovery and was much lower than that of the control group,and we analyzed the expression of was used to analyze GHR,IGF1,IGF1R,IGFBP3,mTOR and AMPK by RT qPCR,which verified the important regulatory role of IGF1 accumulation in GH-GHR-IGF1-IGF1R pathway on compensatory growth of Pampus argenteus.Combined with the above results,silver pomfret showed a complete compensatory growth effect after 14 days of starvation and 14 days of re-feed.In this starvation process,carbohydrate and lipid were consumed firstly,and then protein was consumed as energy source.Fat accumulated rapidly after 14 days of starvation,resulting in rapid growth,and protein was mainly recovered in the later stage of starvation.For food intake,silver pomfret did not rely on the long-term increase in food intake,leading to the rapid growth.A large amount of IGF1/IGFBP3 complexes were accumulated during starvation.After recovery,there were a great quantity of free IGF1 dissociated from IGFBP3,which promoted the anabolism and feed conversion ratio and resulted in the compensatory growth of silver pomfret.