| Blunt snout bream(BSB),Megalobrama amblycephala,is an herbivorous cyprinid fish native to China,with a fast-growing yearly production that currently places it among the top seven species in Chinese aquaculture.Because of high consumption of this fish species,the fish feed has been affected by global trend to use non-protein energy source to replace the protein source in fish feed.The carbohydrate and fat are the source of protein replacement in aquatic animal feed production.The high level of carbohydrate and fat inclusion in fish feed in order to reduce the amount of costly protein by sparing protein from dietary carbohydrate and fat have caused the disease and affected the metabolic disorder in fish.High fat and high carbohydrate diets are known to be risk factors in many diseases.Both can have adverse impacts on different metabolic pathways,enzyme regulation and organ functions.However,little is known about the differences in metabolic changes caused by these imbalanced diets in fish.In this study,we have assessed the impacts of high fat diet(HFD),high carbohydrate diet(HCD)and high-fathigh-carbohydrate diet(HFHCD)on serum and liver metabolites of high fat high carbohydrate diets-fed Megalobrama amblycephala for eight weeks using the 1H nuclear magnetic resonance(NMR)technique.The metabolomics profiles were conducted by comparative analysis and classified it into metabolic pathway.The biochemical analyses were also conducted on the serum of fish fed high fat high carbohydrate diets to confirm the impact on energy metabolism of Megalobrama amblycephala.The main results are as follows:1.The biochemical analysis of HFD group in comparison to control group showed that except of the significantly elevated high-density lipoprotein(HDL)value(p<0.05),the triglyceride(TG),total cholesterol(TCHOL),low-density lipoprotein(LDL),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in serum were not significant difference(p>0.05)in comparison to control group.The metabolomics results of serum and liver metabolites by variance analysis of the cross-validated residuals(CVANOVA)indicate no significant differences(p>0.05)for HFD group in comparison to control group.However,as the p value for the control vs.HFD pair in serum(p=0.059)was barely above the chosen significance threshold(0.05).Hence,the HF diet with 12% fat level did not cause the significant changes of serum metabolites and biochemistry in Megalobrama amblycephala.The results of biochemical analysis were agreed with the metabolomics results in this study.2.The biochemical analysis of HCD group in comparison to control group resulted in the elevated HDL,ALT and AST levels in serum(p<0.05).However,other parameters were not significantly different(p>0.05)in comparison to control group.The metabolomics results showed the elevated ?-/?-glucose and creatine metabolites in serum sample.In addition,the adenosine monophosphate(AMP)and betaine were decreased in liver sample.The elevated creatine is in response to the energy stress.The elevated ?-/?-glucose in glycolysis pathway impacted the metabolites in tricarboxylic acid(TCA)cycle led to the elevated creatine in creatine pathway via AST,aspartate,arginine and guanidinoacetate(GAA).Interestingly,the elevated AST value was relevant with the creatine pathway.Thus,the biochemical results were consistent with metabolomics results of HCD group.The HC diet with 34% of carbohydrate can lead to the metabolites changes in energy pathway where impacted on the glycolysis,TCA cycle and creatine pathways.3.Compared with control group,the biochemical analysis of HFHCD group resulted in the significantly elevated serum LDL and ALT values(p<0.05),whereas,other parameters were not significantly different(p>0.05).The metabolomics results showed the significant changes of serum tyrosine,trimethylamine(TMA),and histidine.Interestingly,the correlation coefficient of inosine(0.59)and hypoxanthine(0.56)were very close with cutoff value(|r|=0.602).However,there were not significant changes in the liver sample(p>0.05).The elevated tyrosine and histidine lead to the metabolites changes in TCA cycle where can further affect to the metabolism of energy unit,ATP,in purine metabolism,resulting in changes of inosine and hypoxanthine-related energy metabolism.The HFHC diet can increase the activity of choline-degraded gut microbiota,resulting in the products of microbiota metabolism(TMA)related with the amount of high fat and high carbohydrates in diet.The HFHC diet with 31% carbohydrate and 10% fat levels resulted in the changes of energy metabolism through tyrosine,histidine,inosine and hypoxanthine.In addition,the diet could affect the metabolism of intestinal microbiota.4.The comparative analysis in other three group pairs of HFD vs.HCD,HFD vs.HFHCD and HCD vs.HFHCD were carried out to understand the affected of each diet.In comparison to HFD group,HCD group showed the significantly(p<0.05)elevated serum ?-/?-glucose,creatine,tyrosine,succinate,hypoxanthine,inosine and TMA metabolites.Moreover,the AMP was decreased in liver sample.The HCD affected on the elevated metabolites in glycolysis(?-/?-glucose).In addition,the glycolysis metabolites can affect to the TCA cycle intermediate(tyrosine and succinate)and creatine pathway(creatine)via AST.The excess of energy affected by HCD resulted in the elevated purine metabolism to meet energy balance,thus resulting in the decreased AMP and elevated inosine and hypoxanthine levels.The elevated TMA in serum of HCD group can be possibly that the microbiota could not well convert the TMA to trimethylamine-N-oxide(TMAO)by flavin monooxygenase(FMO)in the liver,resulted in the excess TMA in serum.In comparison to HFD group,HFHCD group showed the significantly elevated(p<0.05)serum isoleucine,leucine,valine,glutamine,methionine,tyrosine,histidine and TMA metabolites.These metabolites were mainly related in metabolic pathways: TCA cycle,one carbon metabolism and gut microbiota metabolism.The HFHCD group resulted in the excess of the products of carbohydrate and fat metabolism in the TCA cycle,resulting in a consequence the “congestion” of the TCA cycle.The excess of their products in TCA cycle led to a reduced numbers of amino acids entering the cycle,leading to the increase of the seven amino acids concentrations in the serum.However,there were not significant changes in liver sample(p>0.05).In comparison to HCD group,the HFHCD group showed the significantly decreased(p<0.05)serum ?-/?-glucose,TMA,succinate,inosine and hypoxanthine metabolites.However,the serum histidine metabolite was increased.Interestingly,the metabolites changes were similar to the HFD vs.HCD group pair.There were clearly significant changes in glycolysis and TCA cycle(?-/?-glucose and succinate),gut microbiota metabolism(TMA),histidine(histidine)and purine(inosine and hypoxanthine)metabolism.In addition,the histidine was only significantly higher in HFHCD group(p<0.05).The elevated histidine metabolite was possibly of high fatty acid,resulting in the decreased lipolysis and histidine supply,leading to the elevated histidine in the serum.However,there were not significant changes in liver sample(p>0.05).Overall,the high carbohydrate(34%)diet and high-fat-high-carbohydrate diet(10% fat and 31% carbohydrate)resulted in the changes of significant energy metabolism when they were fed to Megalobrama amblycephala for 8 weeks.However,the high fat(12%)diet was not significantly changes in fish metabolites.These results provide an insight into the diet-induced metabolic disorders that often lead to substantial financial losses in the Megalobrama amblycephala aquaculture. |
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