Mechanism On Feed Intake Regulation Of Lateolabrax Japonicus When Fishmeal Was Replaced By Plant Protein

Voluntary anorexia is one of the important factors for limiting ultilization of plant protein in carvirous fish speices.Similar to the mammals,the regulation of appetite and body weight is a complex phenomenon involving elaborate interactions between peripheral and central nervous system?CNS?.However,the key factor and mechanism of feeding preference and feed intake control are not clear.Japanese seabass?Lateolabrax japonicus?,a typical carnivorous fish,is a fishmeal-reliant species and less likely to accept plant proteins.A long-term growth trial were conducted to study the growth performance,feeding intake regulation mechanism of Japanese seabass.Three isonitrogenous and isoenergetic diets were formulated.A basal diet containing 54%fishmeal was used as the control,whereas 50%and 100%of the fishmeal were replaced with a plant protein blend?soybean protein concentrate?SPC?and cottonseed protein concentrate?CPC??.These three experimental diets were designated FM,PPB50 and PPB100,respectively.In the PPB diet,Lys,Met and Thr were balanced using the crystallized amino acids?CAAs?L-Lys-HCl?98%?,DL-Met?98%?and L-Thr?98%?,respectively.Fish with an initial body mass of 12.97±0.03 g were randomly selected for transfer to 60conical fiberglass tanks in a recirculation system,with 25 fish per tank.Four replicates were used for each dietary treatment?12 tanks?for the long-term growth trial,and the other tanks were arranged for sampling?short-term feeding trial?.Fish were fed to apparent satiation twice daily at 8:00 am and 8:00pm for 12 w,which was followed by 3 w of fasting and 3 w resume feeding.For long-term trial tanks,at the end of the 4^th,8^th and 12^th weeks,after a morning meal,the fish were starved for 24 h and then batch weighed.The plasma,hypothalamus and stomach were sampled at 3 d,2 w and 4 w at 3 h,6 h and 24 h postprandially.Samples with the same number as short-term feeding trial were collected at 3h and 24h postprandially after 12 w long-term feeding trial.After the 3 w of fasting and 3 w resume feeding,samples at s3w,r1d and r3w at 3 h and 24 h postprandially were also collected.The results showed that no significant differences were observed in PPB50 group comparing to the FM group.However,Japanese seabass fed PPB100 diet showed extreme anorexia during the first 2 w,then followed by feeding adaptation during 5-8w,but weight gain of PPB100 group was still lower than that of fish fed FM diet.During the 9-12 w,the FR and WG in PPB100 were significantly higher than FM?P<0.05?,indicated a compensation behavior occurred in PPB100 group.The appetite-related gene expressions at mRNA and protein phosphorylation levels of ghrelin/leptin-mTOR/S6K1-NPY/AgRP/POMC pathways in stomach and hypothalamus were detected at different feeding stages?anorexia,adaption,compensation,fasting and resume feeding?in fishmeal group?FM?and group of fishmeal were totally replaced with plant protein blend?PPB?.The considerable negative energy balance induced by food deprivation seemed to stimulate an up-regulation of ghrelin and down-regulation of leptin transcription in the stomach,strongly indicating the effective sensing of hunger in peripheral feed intake organs.However,activation of mTOR in the central nervous system?CNS?decreased the phosphorylation of downstream S6K1,accompanied by further up-regulation of feeding inhibition factors POMC and down-regulation of appetitive peptide AgRP expression in anorectic stage,indicating that the CNS failed to respond effectively to peripheral starvation.Further,the similar results were confirmed in plasma glucose.Plasma ghrelin,peripheral promoting feeding hormone,was higher in PPB than FM in anorexia period and the difference was eliminated after 4 weeks.Surprisingly,plasma NPY,CNS appetite related neuropeptide,shown no difference between two groups in anorexia period.In 4 w-3 h,the reversal expression of AgRP was occured,indicated that Japanese seabass have adapted the plant protein and the central feeding regulatory system was consistent with the peripheral energy state.In 12 w-24 h,the significantly lower ghrelin and higher leptin in plasma indicated that Japanese seabass in PPB group were in a relatively lower appitite status compared to the FM group,however,the significant lower POMC mRNA levvle in hypothalamus could be an important reason for inducing compensatory feeding during this stage.Fasting and refeeding trial were conducted to clarify the mechanism of voluntary anorexia induced by plant protein and passive feeding inhibition caused by starvation in fish.The results shown that as soon as the fish adapted to the plant protein diet,the central feeding regulatory system was consistent with the peripheral energy state.Voluntary anorexia induced by plant protein and passive feeding inhibition caused by starvation were regulated by different way.Higher mRNA expression levels of the anorectic genes mTOR,POMC and leptin and lower expression of the orexigenic gene AgRP were shown at 3 d-3 h than in the s3w treatment.Meanwhile,S6K1 and P-S6K1 protein expression showed that fish produced more appetite signals during feeding restriction than during the anorectic period,with higher expression of the activated form of S6K1,which is phosphorylated at Thr389.These results were consistent with the phenotype of anorexia in Japanese seabass fed a PPB diet during the short-term feeding period?3 d-3 h?and the actively foraging status at s3w.In conclusion,Japanese seabass has ability of adaption to plant protein.Ghrelin/leptin-mTOR-S6K1-AgRP/POMC pathway can efficiently regulate the feeding behavior of Japanese seabass from anorexia to adaptation and even compensatory.The phenotypes of adaptation and even compensatory behavior in carnivorous fish to a diet high in plant protein may be reasonable factors to promote the future breeding of a genotype with high tolerance for plant protein.