Studies On PI3KR1 And AKT1 Genes Of Largemouth Bass (Micropterus Salmoides) And The Regulation Of Glucose Metabolism And Nonspecific Immunity By Dietary Resveratrol

Study 1: The full-length sequences of phosphatidylinositol 3-kinase p85(PI3KR1)and protein kinase B1(AKT1)genes of largemmouth bass were cloned by homologous cloning and rapid amplification of c DNA end(RACE)techniques.On this basis,multiple sequence alignment and phylogenetic tree analysis were performed for the above genes.The expression difference of PI3KR1 and AKT1 gene in tissues was detected by real-time quantitative PCR.The results are as follows:1.The full length of PI3KR1 of largemouth bass was 4170 bp,including 245 bp in the 5 ’non-coding region,1389 bp in the open reading frame,and 2536 bp in the 3’ non-coding region,encoding 462 amino acids in total.The cloned PI3KR1 gene of largemouth bass was similar to that of large yellow croaker.From the phylogenetic tree results,the amino acid sequence of PI3KR1 gene of largemouth bass is closely related to the deep fissure blackfish and large yellow croakers,but is far related to mammals.2.The full length of Akt1 gene is 3672 bp,including 346 bp in the 5 ’non-coding region,1422 bp in the open reading frame,and 1904 bp in the 3’ non-coding region,encoding 473 amino acids in total.The cloned AKT1 gene of largemouth bass was similar to that of large yellow croaker.From the evolutionary tree results,the amino acid sequence of AKT1 gene of largemouth bass is close to that of European bass and large yellow croaker,but far from that of mammals.Study 2: Effects of dietary Resveratrol(RSV)supplemental levels on growth performance,liver glycogen accumulation,glucose metabolism,and nonspecific immunity of largemouth bass(33.76 ± 0.12 g BW).Two isonitrogenous and isolipid diets were formulated with 5% starch and 10% starch,respectively,as positive control(LC)and negative control(HC).On the basis of negative control group,0.05%,0.10%,0.25% and 0.50% resveratrol were added in gradient.After fasting for 24 hours,fish of similar size and vigorous initial body weight of 33.76 ± 0.12 g were randomly assigned to 18 fish tanks(800L water volume),with 30 fish in each tank.The experiment was carried out for 9 weeks by feeding the above feed twice at 8 am and 4 pm.The breeding experiment was carried out for 9 weeks.During the experiment,the circulating water in the breeding system was physically filtered and then disinfected by UV.Water temperature was kept at 27 ± 1℃,p H 7.2 ± 0.2,and natural light was used.The results are as follows:1.Effects of resveratrol supplementation on growth,body composition and feed utilization of experimental fish.There was no significant effect on the survival ratio of fish in all groups(P > 0.05).Compared with low carbohydrate diet group(LC),high carbohydrate diet(HC)decreased the specific growth rate of experimental fish,but the effect was not significant(P > 0.05).The intake of high carbohydrate diet significantly increased the hepatosmatic index of largemouth bass,but the supplementation of resveratrol significantly decreased the hepatosmatic index increase induced by high carbohydrate diet(P < 0.05).The increase of dietary carbohydrate level had no significant effect on the viscerosomatic index of largemouth bass(P > 0.05).Compared with HC treatment group,resveratrol supplementation decreased the viscerosomatic index of largemouth bass in high carbohydrate diet(P > 0.05),and the viscerosomatic index of 0.25% resveratrol treatment group was significantly lower than that of 0.50% resveratrol treatment group(P < 0.05).There was no significant difference incondition factor of largemouth bass among all treatment groups(P > 0.05).The intake of high carbohydrate diet significantly decreased the feeding rate(P < 0.05),while the addition of resveratrol had no significant effect on the feeding rate(P > 0.05).The feed efficiency ratio and protein efficiency rate of largemouth bass in high carbohydrate diet group were significantly higher than those in low carbohydrate group(P < 0.05),and the supplementation of resveratrol had no significant effects on the above two indexes(P > 0.05).There were no significant differences in whole body composition,muscle composition and liver moisture of largemouth bass among treatment groups(P > 0.05).0.10% resveratrol treatment group significantly increased the liver protein content of largemouth bass in high carbohydrate diet group(P < 0.05);the liver fat content of largemouth bass in 0.25% and 0.50% resveratrol groups was significantly higher than that in 0.10% resveratrol group(P < 0.05).The glycogen contents in liver and muscle of largemouth bass in HC group were significantly higher than those in LC group(P < 0.05),and the addition of resveratrol significantly decreased the accumulation of glycogen in liver and muscle of largemouth bass induced by high glucose diet(P < 0.05).2.Effects of resveratrol supplementation on postprandial serum glucose,insulin signaling pathway and gene expression related to glucose metabolism in experimental fishAt 24 h postprandiall,there was no significant difference in the blood glucose content of largemouth bass among all treatment groups(P > 0.05).At 0,1,3,6 and 12 postprandial time points,the supplementation of resveratrol significantly reduced the increase in blood glucose content of largemouth bass induced by high glucose diet(P < 0.05).The increase of dietary sugar level significantly decreased the expression of SIRT1,while the supplementation of 0.10% and 0.25% resveratrol significantly increased the expression of SIRT1(P < 0.05).There was no significant difference in the expression of insulin receptor(IR)in largemouth bass among all treatment groups(P > 0.05).Phosphatidylinositol-3-kinase p85(PI3KR1)expression of largemouth bass in HC group was significantly lower than that in LC group(P < 0.05),and resveratrol supplementation had no significant effect on the expression of PI3KR1(P > 0.05).The increase of dietary sugar level had no significant effect on the expression of protein kinase B1(AKT1)of largemouth bass(P > 0.05),but the expression of protein kinase B1(AKT1)of largemouth bass in 0.10% and 0.25% resveratrol groups was significantly increased compared with HC group(P < 0.05).High carbohydrate diet significantly increased the expression of forkhead transcription factor O1(FOXO1)of largemouth bass(P < 0.05),while resveratrol supplementation significantly decreased the expression of FOXO1(P < 0.05).Compared with HC group,resveratrol supplementation significantly increased the expressions of glucokinase(GK)and 6-phosphofructokinase(PFKL)in largemouth bass(P < 0.05).The pyruvate kinase(PK)expression of largemouth bass was not significantly different among treatment groups(P < 0.05).The high glucose diet did not significantly affect the expression of the gluconeogenesis related gene catalytic subunit of glucose-6-phosphate(G6PC),fructo-1,6-bisphosphatase-1(FBP1)and phosphoenolpyruvate hydroxylase(PEPCK)(P > 0.05).The 0.25% and 0.50% resveratrol groups significantly decreased the increase in the expression of Glucose-6-phosphate catalytic subunit(G6PC)of largemouth bass induced by high glucose diet(P < 0.05).Compared with HC group,resveratrol supplementation significantly increased the expressions of fructose-1,6-bisphosphatase-1(FBP1)and phosphoenolpyruvate hydroxylase(PEPCK)(P < 0.05).3.Effect of resveratrol on nonspecific immunity of experimental fishThe increase of dietary sugar level significantly decreased the lysozyme content in serum and liver of largemouth bass(P < 0.05),and the addition of resveratrol significantly increased the decrease of lysozyme content(P < 0.05).There were no significant differences in the contents of C3,C4 in serum and C3 in liver of largemouth bass among treatment groups(P > 0.05).The intake of high carbohydrate diet significantly increased the liver C4 content(P < 0.05),and the 0.25% and 0.50% resveratrol groups significantly decreased the liver C4 content increase caused by high carbohydrate diet(P < 0.05).There was no significant difference in Rela expression among all treatment groups(P > 0.05).The expression of interleukin 1β(IL-1β)in largemouth bass was not affected by dietary sugar level.Compared with HC group,the supplementation of resveratrol significantly decreased the expression of interleukin-1β(IL-1β)(P < 0.05).The high carbohydrate intake significantly increased the expression of tumor necrosis factor α(TNF-α)in largemouth bass(P < 0.05).The increase of dietary sugar level had no significant effects on the expression of interleukin-10(IL-10)and transforming growth factor β(TGF-β)of largemouth bass(P > 0.05),the addition of resveratrol significantly promoted the expression of interleukin-10(IL-10)and transforming growth factor β(TGF-β)of largemouth bass(P < 0.05).