| Topmouth culter (Erythroculter ilishaeformis bleeker) belongs to Erythroculter Berg,Cultrinae, Cyprinidae, Cypriniformes. It is a kind of freshwater economic fish. It has a bigsize, and grows fast with dean white flesh tasting fine, tender and delicious. As a kind ofsuperior fish, it has become quite popular among general public and enjoyed a quite highprice in the markets of Jiangsu and Zhejiang provinces and Shanghai Municipality. Thewild topmouth culter is also a kind of fierce carnivorous fish which mainly feeds on livefishes. However, during the whole culture period, we can apply artificial feed to the fish. Inorder to study the capability of utilizing carbohydrate in the diet by topmouth culter, thispaper described the isolation and cloning of cDNA sequence of carbohydrate metabolizingenzymes, i.e., Glucokinase (GK, EC 2.7.1.2), Phosphoenolpyruvate carboxykinase (PEPCK,E.C.4.1.1.32), and glucose-6-phosphatase (G6Pase, EC 3.1.3.9), and the measurement ofexpression levels of GK,PEPCK and G6Pase mRNA as well as effects on their enzymesactivities of fast, feed taking and daily taking of various carbohydrate and lipid contents.On the one hand, the paper accumulated data describing molecular control mechanism ofCarbohydrate metabolizing enzymes by fish. On the other hand, the paper also providedreference and guidance to the development of aquaculture diet formula of topmouth culter.When the total energy of daily intake maintaining identical, 720 individuals of topmouthculter(40.73±0.44g) were divided into carbohydrate-free group (including no carbohydrateand crude protein 63.38%, and Ether Extract 9.13%), medium-carbohydrate group(including digestive carbohydrate 14.45%, crude protein 50.14%, Ether Extract 9.92%),high carbohydrate group (including digestive carbohydrate 23.98%, crude protein 40.11%,Ether Extract 9.94%) and high lipid group(including digestive carbohydrate 14.98%, crudeprotein 40.88%, Ether Extract 19.93%). Each group had three redundancies and was cultured for eight weeks. After that, indicators of gene expression level and activities ofcarbohydrate metabolic enzymes, i.e., GK,PEPCK and G6Pase as well as fish growth,hepatosomatic indices, plasma biochemical indices and muscle nutritional compositionwere measured.1 Cloning of GK, PEPCK and G6Pase genes of topmouth culterThis paper utilized RT-PCR and RACE (Rapid Amplification of cDNA Ends) methodsand isolated and cloned for the first time the whole sequence of GK, PEPCK and G6Pase oftopmouth culter. The result indicated the whole cDNA length of GK of topmouth culter was2054 bp, including 63 bp5' untranslated regions, 1428 bp open reading frames and 563bp3' untranslated regions containing Poly(A) signal AATAAA [excluding Poly(A)]. Theopen reading frame was totally coded 476 amino acids and calculated molecular weight53.74 kDa. This sequence contained the conservative binding sites of ATP and glucose inthe GK of vertebrates. It's identities were as high as 86%-96%with the GK of other fishes,and 79%-80% with the GK of Africa clawed toad (Xenopus laevis) and human (Homosapiens). The whole cDNA length of PEPEK of topmouth culter was 2 564 bp, including111 bp5' untranslated regions, 1911 bp open reading frames and 542 bp3' untranslatedregions containing Poly(A) signal AATAAA [excluding Poly(A)]. The reading frame wastotally coded 636 amino acids and calculated molecular weight 69.65 kDa. The topmouthculter PEPCK appears to possess the PEPCK-specific domain to bind oxaloacetate as wellas kinase-1 and kinase-2 motifs to bind the GTP triphosphate chain and Mg~2, respectively.The results revealed the topmouth culter PEPCK shared 83%-96% sequence identity withother fishes, and 50%-69% identity with other animals including clawed frog, and amoeba(Dictyostelium discoideum). The whole cDNA length of G6Pase of topmouth culter was 1900 bp[excluding Poly(A)], including 49 bp5' untranslated regions, 1068 bp open readingframe and 778 bp3' untranslated regions containing signal AATAAA [excluding Poly(A)].The open reading frame was totally coded 355 amino acids and calculated molecular weight39.89 kDa. It's identities were as high as 95%with the G6Pase of zebra fish (Danio rerio),and 63% with the G6Pase of mouse (Mus musculus), dog (Canis familiaris) and human. Itsidentities with G6Pase of clawed frog, gilthead seabream (Sparus aurata L.) and spottedgreen pufferfish (Tetraodon nigroviridis) were 69%,55% and 76% respectively. Thisindicated that the sequences of GK, PEPCK and G6Pase isolated from topmouth culter hadvery high identities with those of other animals, and they were activities. 2 Effect on the performance of topmouth culter by carbohydrate and lipid atvarious levels in the dietUnder the conditions of identical-quantity daily intake level, there were not manyeffects (P＞0.05) on mortality, weight increase rate, specific growth rate, feed co-efficient,fullness. Coefficient, fish body moisture, lipid and liver moisture of topmouth culter by thediets from carbohydrate-free group (high-protein group), medium-carbohydrate group,high-carbohydrate group,and high-lipid group. However, under comparison, weightincrease rate, and specific growth rate were the highest and feed coefficient was the lowestin the high-carbohydrate group; Weight increase rate, and specific growth rate were thelowest and feed coefficient was the highest lowest in the carbohydrate-free group (highprotein group). Hepatosomatic indices of the fish from high-carbohydrate group was veryobviously(P＜0.05) higher than those for medium-carbohydrate group and high-lipid group,and increased by 28.81% and 25.42%. In addition, Hepatosomatic indices from high-lipidgroup distinguishably increased by 56.73% than that from medium-carbohydrate group.Compared with carbohydrate-free group, crude protein and ash for high-carbohydrategroup and high-lipid group significantly decreased (P＜0.05) by 8.25% and 12.88% as wellas 8.99% and 9.07%; Crude protein and ether extract noticeably increased (P＜0.05) by12.76% and 44.88% as well as 16.50% and 9.07%. Compared with medium-carbohydrategroup, crude protein and ether extract in the high-carbohydrate group and high-lipid groupnoticeably (P＜0.05) increased by 4.48% and 55.27% as well 7.93% and 58.23%. Crudeprotein and ash from fish muscle of carbohydrate-free group(high protein group) differedmarkedly with those of high-carbohydrate group and high-lipid group (P＜0.05), increasingrespectively by 8.25% and 12.88% as well as 8.99% and 9.07%; Except for non-effect (P＞0.05) of moisture on diet for the hepatic-pancreas, crude protein and ether extract contentsof hepatic-pancreas of carbohydrate-free group (high protein group) and medium-carbohydrate group were apparently lower (P＜0.05) that those of high-carbohydrate groupand high-lipid group, and didn't show self-evident effects compared to other groups. Thisindicated that too high content of single nutrient might cause the excess of nutrients ininternal organs and caused the viserosomatic indices and hepatosomatic indices becomingtoo big. Meanwhile, it also indicated that intake of too big amount of carbohydrate and lipidmight turn part of them into protein and lipid of hepatic-pancreas to store.After taking in diet, the general trend of blood glucose appeared increasing beforebecoming smooth, with blood glucose levels after three hours of diet intake and after fasting for 48 hours showing no prominence in difference (P＞0.05), blood glucose levelsbetween 3 and 6 hours showing big increase, blood glucose levels after 6 hours appearingsmooth and blood glucose levels after 24 hours markedly being higher (P＜0.05) than thoseof fasting for 48 hours. All this showed that like most fishes, topmouth culter had thephenomenon of continuous high blood glucose level. Compared with the blood glucoselevels, it demonstrated high-carbohydrate group was markedly higher thanmedium-carbohydrate group, carbohydrate-free group (high protein group), and high-lipidgroup(P＜0.05); However, the differences among medium-carbohydrate group,carbohydrate-free group and high-lipid group did not appear salience. Plasma glucosecontents reached maximum for high-carbohydrate group after 6 hours of diet intake, but forhigh-lipid group after 12 hours of diet intake. Comparison of blood sugar values of variousgroups showed that blood sugar values of carbohydrate-free group and medium-carbohydrate group were lower than those of high-carbohydrate group and high-lipid group.This result demonstrated that the higher carbohydrate level in the diet, the higher plasmaglucose levels in fish body. After diet intake, plasma concentrations of triglyceridesincrease before decreasing, and reached maximum 3 hours after diet application. Fromfasting to 6 hours of diet ingestion, plasma concentration of triglycerides reached maximumin high-carbohydrate group, then carbohydrate-free group and high-lipid group, andreached minimum in medium-carbohydrate group; From 6 hours to 24hours, plasmaconcentration of triglycerides reached maximum in high-lipid group, similar tohigh-carbohydrate group, then carbohydrate-free group, and minimum in medium-carbohydrate group. This determined that carbohydrate in the diet can more easily promotethe creation of plasma concentration of triglycerides than the lipid in the diet. The finalresult, however, indicated that the excess of single nutrient in diet would cause the increaseof plasma concentration of triglycerides. But, there existed no marked difference of plasmaconcentration of triglycerides of various groups after fasting. This indicated that the diet ofthe experiment didn't have a long-term effect on plasma concentration of triglycerides offish body. After diet intake, the general trend of plasma concentration of cholesterolincreased before decreasing, reaching maximum after 3 to 6 hours of diet application,except the change of carbohydrate-free group (high-protein group) was not marked andminimum. The analysis showed that after 48 hours of fasting, the fish plasmaconcentrations of cholesterol of various groups didn't show marked difference (P＞0.05),indicating that carbohydrate and lipid levels in diet did not have effect on plasma concentration of cholesterol of topmouth culter.3 Effect on the gene expression of GK, PEPCK, G6Pase of topmouth culter bythe carbohydrate and lipid at various levels in the dietThe fluorescence quantitative RT-PCR was utilized to takeβ-actin as internal standardfor the measurement of relative expression amounts of GK, PEPCK, G6Pase mRNA oftopmouth culter liver after various times of intake of carbohydrate-free, medium-carbohydrate, high-carbohydrate and high-lipid diets. The result revealed that the geneexpression of carb0hydrate-free group and medium-carbohydrate group GK were markedly(P＜0.05) lower than high-carbohydrate group and high-lipid group, with maximum of GKgene expression of high-carbohydrate group appeared after 6 hours of diet intake, andmaximum of GK gene expression of high-lipid group appeared after 12 hours of diet intake,being identical to the blood sugar value. This indicated that high-carbohydrate and highlipid in the diet could induce the GK gene expression of topmouth culter liver, and GK genecould be controlled at InRNA level.PEPCK gene expression of topmouth culter liver in medium-carbohydrate,high-carbohydrate and high-lipid group was not marked after diet intake. The expression ofthis gene of carbohydrate-free group, however, was different from those of other groups:there were no marked difference among fasting, 3 and 6 hours of diet intake, while the geneexpression appeared minimum after 12 hours of diet intake and maximum after 24 hours ofdiet intake. After 6 to 12 hours of diet ingestion, the expression amount of PEPCK gene invarious groups were quite near to one another and did not have marked difference. Betweenmedium-carbohydrate group and high-lipid group, the expression of PEPCK gene after dietintake did not have marked difference, too. Therefore, the carbohydrate and lipid in dietwould not have effect on the expression of PEPCK gene expression. For mammals, PEPCKappeared in two forms of isomer: PEPCK-C (cytoplast-type) and PEPCK-M (mitochondri-on type), of which, only PEPCK-C was controlled by nutrient and hormone, whilePEPCK-M was nearly constant. This experiment discovered that isolated PEPCK geneexpression was not effected by the intake and nutrients in diet, which was identical to thePEPCK gene expression isolated from rainbow trout (Oncorhynchus mykiss), GiltheadBream (Sparus aurata), and Atlantic salmon (Salmo salar) and therefore they might be allPEPCK-M type.The study on G6Pase gene expression of topmouth culter revealed that medium-carbohydrate group had minimum G6Pase gene expressin amount and its intakedid not have significant effect on expression of the gene(P＞0.05), but G6Pase geneexpression of carbohydrate-free group and high-lipid group appeared a trend of increasing,though the expression amount change did not have noticeable difference (P＞0.05). For thehigh-carbohydrate group, the gene expression amount after fasting was markedly higherthan any other group(P＜0.05): after 3 hours of diet intake, expression amount would bedecreasing, followed by continuous increase to maximum after 12 hours of diet intake, i.e.,somewhat higher than the amount when fasting (P＞0.05), or being 4.86 times than theexpression amount than carbohydrate-free group when fasting, and after 24 hours of dietintake, the gene expression amount dropped below the amount when fasting (P＜0.05). Thisbeing the reason, high carbohydrate might induce the gene expression. At the same time,the gene expression amount of high-lipid group and medium-carbohydrate group (lowlipid), high-lipid group was markedly higher than low-lipid group. Therefore, the high lipidalso had certain inducing effect on the gene expression. Judged from G6Pase geneexpression, amount of carbohydrate-free group,medium-carbohydrate group and high-lipidgroup was not effected by diet intake partially explained the reason of continuous bloodglucose after fish intake diet. In mammals, nutrients in the diet effect the expression ofG6Pase gene, but fish might existed the expression of G6Pase gene due to differentmolecule mechanism and different studies on fishes also had different results.4 Effect on activities of GK, PEPCK, G6Pase of topmouth culter by thecarbohydrate and lipid at various levels in the dietThe results of measurement of Hexokinase (HK, EC 2.7.1.1 ) activity of topmouthculter liver after diet intake discovered that except HK activity change of liver inmedium-carbohydrate group was not marked and minimum, all other groups showed thetrend of increasing, then decreasing and followed by increasing again, and reachedmaximum after 3 to 6 hours of diet application. Only in carbohydrate-free group (highprotein group), 3 hours after diet ingestion markedly upgraded (P＜0.05) HK activity ofliver. This indicated diet intake had inducing effect on HK expression. However, thecarbohydrate and lipid levels in diet did not marked effect on HK, while the protein levelmight have an inducing effect. Glucose Dehydrogenase, (GDH, EC 1.1.1.47) activity afterdiet intake generally increased followed by decreasing. Various groups would decreasecontinuously after reaching maximum after 3 hours of diet intake and reach minimum after 24 hours of diet intake. GDH activity of liver after 3 hours diet intake (P＜0.05).However, GDH activity of various groups exhibited no marked difference. GK activityafter diet intake generally appeared increase while no GK activity was measured out forthe fasting of various group. GK enzyme activity after 3 hours of diet intake forCarbohydrate-free group could be measured out, toO. GK enzyme activity in liver ofhigh-carbohydrate group after 6 to 24 hours was remarkably increased (P＜0.05). Thecomparison among various groups, GK enzyme activity of high-carbohydrate group wasthe highest. This showed that compared with protein and lipid in the diet, carbohydrate inthe feed would more easily induce the increase of GK enzyme activity.After diet intake, liver PEPCK enzyme activity of various groups all appeared sharpdecrease followed by being stable and slow. After 6 to 24 hours of diet intake, the liverPEPCK activity would be markedly reduced (P＜0.05). Comparison among PEPCK enzymeactivity discovered that from fasting to 3 hours of diet application, liver PEPCK enzymeactivity was the highest in carbohydrate-free group, while the lowest in high-lipid group,and did not show much difference for medium-Carbohydrate group and high-carbohydrategroup. Liver PEPCK enzyme activity was also the highest after 3 and 24 hours after dietintake for carbohydrate-free group, and then high-lipid group. The high-carbohydrate groupand medium-carbohydrate group did not show marked difference, though the differencewere all not marked. This indicated that fasting might induce the increase of PEPCKactivity. Ingestion different carbohydrate and lipid levels did not have big effect on PEPCKactivities.The general trend of G6Pase activity reduced followed by increase for various groups.It would reached minimum after 3 hours of diet intake for various groups, sharplyincreased followed being stable and slow after 3 to 6 hours of diet intake. From fasting to3 hours after diet intake, liver G6Pase activity was maximum in carbohydrate-free group,then in medium-carbohydrate group, high-carbohydrate group, and minimum in high-lipidgroup. From 3 hours to 6 hours after diet intake, liver G6Pase activity inhigh-carbohydrate group was comparatively the maximum, while did not differ much forthe other 3 groups. From 6 hours to 24 hours after diet intake, liver G6Pase activity inhigh-lipid group was comparatively the maximum, then medium-Carbohydrate group andCarbohydrate-free group, and was minimum in high-carbohydrate group. Compared withfasting, G6Pase activity was reduced markedly in carbohydrate-free group 3 hours afterdiet intake, while G6Pase activity was markedly (P＜0.05) increased in medium- carbohydrate group 6 to 24 hours after diet ingestion. G6Pase activity was markedly(P＜0.05) increased in high-carbohydrate group 6 hours and 24 hours after diet application.G6Pase activity was also markedly increased in high-lipid group 24 hours after dietapplication (P＜0.05) while the other difference was not so marked. This indicated thatcarbohydrate and lipid did not have an obvious effect on G6Pase, but prolonged timewould have an increasing trend of the gene expression.
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