Effects Of Dietary Carbohydrates On The Activities And Gene Expression Of Carbohydrate Metabolic Enzymes In Turbot (Scophthalmus Maximus)

Feeding trials were conducted to investigate the effects of dietary carbohydrateon the activities and gene expression of carbohydrate metabolic enzymes in turbot(Scophthalmus maximus). The results of the present study are as follows:1. A3×4two-factorial experiment was conducted to investigate the effects ofdietary carbohydrate sources (glucose, sucrose and dextrin) and levels (0,5%,15%and28%) on the activities of glycolytic and gluconeogenic enzymes in turbot. Eachdiet was fed to triplicate groups of turbot in a flow-through water system for9weeks.Activities of hexokinase (HK), glucokinase (GK),6-phosphofructokinase-1(PFK),pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK) andfructose-1,6-bisphosphatase (FBPase) were examined. The results showed that asignificant increase in hepatic GK and PK activity were noticed with the increase ofdietary carbohydrate level from0to15%. However, there was a lack of furtherincrease of them with28%of dietary glucose or dextrin. HK and PFK activities werenot significantly affected by dietary carbohydrate levels (P>0.05). Activities ofFBPase, not PEPCK, were significantly increased by5%of dietary glucose. Dietssupplemented with dextrin depressed hepatic FBPase and PEPCK activity incomparison to that without carbohydrate supplements. Activities of these twoenzymes were not significantly affected by dietary sucrose levels (P>0.05). The datasuggested that diets supplemented with15%of glucose or dextrin was effective inenhancing liver glycolytic enzyme activity. Dietary dextrin was more effective thanglucose in depressing liver gluconeogenic enzyme activities. Only GK activity wassignificantly promoted by28%of dietary sucrose, and no significant effect of dietarysucrose levels on the other enzymes was found.2. In the present study, the cDNA of glycolytic and gluconeogenic enzymes were cloned from the liver of turbot by rapid amplification of cDNA ends (RACE)technique. The results are presented as follows:(1) The full length of glucokinase(GK) was2226bp, consisting an open reading frame (ORF) of1434bp. The deducedprotein has478amino acid residues and shares high identity with GK of Giltheadseabream. Some conserved functional sites (ATP binding site, glucose binding site,N-linked glycosylation site) were also found in turbot GK amino acid. Tissuedistribution of GK mRNA in liver, spleen, intestine, heart, gill, muscle and brain ofturbot was analyzed by SYBR real-time PCR, the expression level of GK mRNA inthe liver was significantly higher than others.(2) A full-length cDNA coding hepaticfructose-1,6-bisphosphatase(FBPase) was cloned from turbot. The cDNA was1314bpwith a1014bp open frame encoding337amino acids. The amino acid sequence has ahigh similarity to hepatic FBPase of other species, the percent identity compared withJapanese sea bass is96%. Turbot FBPase mRNA were found to be higher expressedin liver, kidney than in the heart and gill. It would be necessary to define muscleFBPase exist in turbot or not in the future.(3) In this study we had obtained twoglucose-6-phosphatase (G6Pase) isoforms from the liver of turbot. The full lengthsequence of G6Pase1cDNA obtained was1641bp which the open reading frameencodes360amino acids, while G6Pase2cDNA obtained was1357bp which theopen reading frame encodes352amino acids. The results showed that the expressionof the G6Pase1mRNA in the liver was2.6times more than the expression of theG6Pase2mRNA. Some conserved functional sites were found in turbot G6Pase1through multiple alignment and phylogenetic analysis. G6Pase1could be the basictype, and G6Pase2was the other type.(4) Full length cDNA sequence of the cytosolic(cPEPCK) and mitochondrial (mPEPCK) form of phosphoenolpyruvatecarboxykinase were obtained from the liver of turbot. The full length of cPEPCKcDNA was2833bp with a1875bp open reading frame (ORF) encoding624aminoacids, and the full length of mPEPCK cDNA was3124bp with a1908bp open readingframe (ORF) encoding635amino acids. Both the two PEPCK forms had theconserved oxaloacetate binding domain, GTP binding domain and Mg2+binding domain. The proportion of mPEPCK in the liver of turbot was about70%, whilecPEPCK was about30%. The results indicated that those carbohydrate metabolicenzyme gene had more similarity to terrestrial vertebrates, and they could befunctional in turbot. Our result provided some important information for the researchof nutrition regulation about turbot.3. Studies were conducted to investigate the effects of dietary carbohydratesources (glucose, sucrose and dextrin) and levels (0,15%and28%) on transcriptionalexpression of carbohydrate enzymes of turbot. The results were that:(1) Dietarycarbohydrate could significantly up-regulate the expression levels of GK in liver ofturbot compared with those fed with control diet. The expression levels of GK had nosignificant difference in fish fed with diets supplemented with15%or28%glucose,while GK expression was significantly lower in fish fed28%sucrose or dextrin dietthan those fed15%sucrose or dextrin diet, respectively.(2) Expression of hepaticmPEPCK of turbot was independent of diet. Expression of cPEPCK was independentof the diet supplemented with glucose or sucrose, but was down-regulated by the dietsupplemented with dextrin.(3) Experimental diet added with glucose or sucrose didnot affect the transcription of FBPase, while expression of FBPase was significantlylower in fish fed with dextrin compared with the control group.(4) The geneexpression of G6Pase1was independent of dietary carbohydrate, and the geneexpression of G6Pase2was independent of dietary dextrin. However, lowerexpression was found in fish fed28%glucose diet compared with those fed15%glucose, higher expression was observed at15%sucrose group than in the controlgroup. In conclusion, Dietary glucose was more effective than dextrin to up-regulatethe expression levels of GK. Dietary dextrin could down-regulate the expressionlevels of FBPase, cPEPCK, while cannot regulate the expression level of G6Pase1.while expressions of those genes were affected by glucose or sucrose diets. HepaticmPEPCK of turbot is not under nutritional regulation.