The Effect Of High Fat And High Carbohydrate Diets On Growth Performance,Histology And Transcriptome In Blunt Snout Bream(Megalobrama Amblycephala)

Blunt snout bream(BSB)(Megalobrama amblycephala)is one of the main herbivorous fish species in Chinese freshwater polyculture systems.In recent years,diets formulated for BSB are increasingly maximizing the use of non-protein energy sources(carbohydrates and lipids).This often results in severe body lipid deposition coupled with an increasing outbreak of fatty liver diseases.However,few reports are available concerning the fatty liver disease in this species until now.Normally,fish feeding is influenced by many different factors,including energy and nutrients,which can affect the health of fish and correlate with changes in gene expression,as reported in several fish species,such as rainbow trout and golden pompano.Previous studies of BSB observed changes in gene expression after bacterial challenges.Many studies focused on growth performance and immunity responses of fish fed carbohydrate/lipid diets.However,reports remain scarce regarding the topic of the effects of high fat and high carbohydrate diets on growth performance,histology and transcriptome in BSB,as well as the metabolic mechanisms underlying the obesity associated with such diets.Therefore,we designed four diets,high-fat(HFD;lipid 12.22%),high-carbohydrate(HCBD;carbohydrate 34.13%),high-fat-high-carbohydrate(HFHCD;lipid 10.43%,carbohydrate 30.80%)and control diet,and studied their effects on the physiology of this fish.The growth performance,liver histology and liver transcriptome were evaluated after 8-week test.Based on these results,we further studied gene expression by qRT-PCR,blood biochemistry and metabolite profiles.Comparison of these data among the four dietary groups revealed negative effects of high fat and high carbohydrate diets on liver health and mitochondrial biogenesis.The main results are as follows:1.Effects of high fat and high carbohydrate diets on growth and liver histologyThe results showed that the survival rates were all higher than 94% and not statistically different(p>0.05)among groups.The final weight and weight gain were highest in the HFD group,with a significant difference(p<0.05)between HFD and Control groups,but no significant difference in comparison to the HCBD and HFHCD groups(p>0.05).Feed conversion ratio(FCR)and specific growth rate(SGR)of Control group were significantly different(p<0.05)from HFD and HFHCD,but not significantly different from HCBD(p>0.05)group.These two parameters,FCR and SGR,were the best(lowest and highest,respectively)in the HFD group.Hepatosomatic index(HSI)was significantly different(p<0.05)among Control,HFD and HCBD groups,but in the HFHCD group it was similar(p>0.05)to Control and HFD groups.Increased dietary lipid levels resulted in lower HSI.Condition factor(CF)of the HFHCD group was significantly different(p<0.05)from the HCBD group,but not significantly different(p>0.05)from the other two groups(Control and HFD).Liver histology analyses produced results consistent with the HSI data: Control group exhibited regular hepatocytes with large and spherical nuclei centrally located in moderate cytoplasm and a small number of lipid droplets;whereas HFD,HCBD and HFHCD groups exhibited swollen hepatocytes with large diffused lipid vacuoles,abnormal endothelial cells in the central vein,inflammatory infiltration,and some hypertrophy of hepatocytes.Thus,fatty liver symptoms were apparent in the all three imbalanced diet groups(HFD,HCBD and HFHCD).All these results indicate that,although high fat and high carbohydrate diets can improve the growth performance,they also cause lipid accumulation in hepatocytes,which in turn affects the health of the liver.2.Comparison of transcriptome characteristics among different dietary treatmentsTo understand the transcriptomic response to high fat and high carbohydrate diets,twelve non-normalized c DNA libraries were synthesized from tissues collected from four groups(Control,HFD,HCBD,and HFHCD),and their transcriptomic samples were sequenced by Solexa/Illumina technology.After the transcriptome assembly(average total length per sample = 5.10 Gb),96,429 unigenes were identified.All unigenes were annotated using BLASTX against several public databases: NCBI(Nr),Swiss Prot,COG,KEGG,and GO.The highest sequence similarity(86%)of the assembled unigenes was to zebrafish based on the Nr database.Unigenes(n = 42,563)were assigned to three GO categories: biological processes(14,583 unigenes),cellular components(11,271 unigenes),and molecular functions(14,901 unigenes).KEGG pathways database was used to compare Control with HFD,HCBD and HFHCD groups,whereby ‘2,146',‘1,895' and ‘2,210' unigenes were assigned to a total of 323,319 and 324 pathways,respectively.Among the differentially expressed genes(DEGs)in HFD,HCBD and HFHCD groups,there were 1339,1318,and 1907 up-regulated genes and 4568,4238,and 4799 downregulated genes,respectively.This study extends our understanding of the mechanisms of fatty liver disease in the BSB fed with high fat and high carbohydrate diets,and provides important data for further studies of the lipid and carbohydrate metabolism of this species.3.Negative effects of high-carbohydrates on the health of BSBHigh intake of carbohydrates,associated with obesity,is one of the major causes of fatty liver disease in humans.This part of the study explored how a high-carbohydrate intake affects the liver health.Transcriptome analysis and quantitative real-time PCR(qRT-PCR)indicated that the expression of a number of factors in the insulin signaling pathway of the HCBD fish were significantly(p<0.05)upregulated: INSR,IRS,PI3 K,PDK,AKT,ACC,IL6,AP1,Ch REBP-MLX,PEPCK and FBP;whereas SOCS3,GSK3?,and AMPK were significantly downregulated(p<0.05).Therefore,by regulating the expression of a number of genes related to fatty liver disease,high-carbohydrate diet led to the activation of insulin resistance in hepatocytes and increased lipogenesis in liver.Metabolomic results showed that the high-carbohydrate diet induced significant increases in plasma ?/?-glucose,succinate,and tyrosine;from other studies it was known that this can result in increased hepatic glycogen and triglycerides.Low levels of betaine were found in the livers of the HCBD group.Blood biochemistry analyses revealed that highdensity lipoprotein(HDL),low-density lipoprotein(LDL),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were increased significantly(p<0.05).These results are consistent with the HSI and liver histological results in the first part of the study,suggesting an abnormal liver with excessive lipid accumulation and potential liver damage.This study extends our understanding of how high-carbohydrate diets cause increased fat deposition in the liver,enhanced glycolysis(?/?-glucose)in the plasma,and reduced betaine in the liver.However,it is still needed to further research whether the initial signs of NAFLD will eventually lead to the development of NAFLD after a longer period of time.4.High fat and high carbohydrate diet caused mitochondrial dysfunctionThe above study of the transcriptomic results for HFHCD group also revealed a large number of differentially expressed genes(DEGs)associated with mitochondrial metabolism in all three main categories in the GO database: molecular function,cellular components and biological process.KEGG analysis(2210 DEGs mapped to 324 metabolic pathways)showed that the largest proportion DEGs(831)were associated with human diseases,predominantly(239 DEGs)with neurodegenerative diseases(Alzheimer's,Huntington's and Parkinson's).Further KEGG pathway enrichment analysis found that most significantly enriched metabolic pathways were ‘Parkinson's disease',‘oxidative phosphorylation',‘Alzheimer's disease' and ‘Huntington's disease'.In addition,similar to the results of chapter four,many DEGs were associated with NAFLD pathways,which might be a reflection of mitochondrial function disorder caused by high-fat-high-carbohydrate diet.Because mitochondria play a very important role in the physiology of eukaryotes,mitochondrial dysfunction is the key factor causing metabolic diseases and neurodegenerative disorders.In order to further explore the relationships of high-fat-high-carbohydrate diet with mitochondrial dysfunction and neurodegenerative diseases,we studied the expression of 17 genes associated with mitochondrial metabolism by qRT-PCR.Results showed that FATP,FABP and Ch REBP were significantly increased(p<0.05)in the HFHCD group,which indicated that the intake,transportation and synthesis of fatty acids were all increased.However,expression of genes in the mitochondrial ?-oxidation pathway,namely downregulation of PPAR? and PPAR?(p<0.05)and upregulation of PPAR?,CPTI and CPTII(p<0.05),suggests that lipid catabolism was also increased.These results are different from observation in mammals and our expectations,which is likely to be a reflection of idiosyncrasies of fish metabolism,particularly the tendency of fish to store excess energy as protein,instead of fat.Functions of PPARs in fish are also different from mammals.A number of key genes in the oxidative phosphorylation pathway,CXI,CXII,CXIII,CXV and cytc,and SOD2 were significantly downregulated(p<0.05).This is likely to be a reflection of a metabolic shift in response to a high nutritional load,wherein ATP and NADH levels increase in a number of different cell types,the metabolic balance shifts toward lipid and glycogen storage,and mitochondrial biogenesis is downregulated.Therefore,the excess energy in diets could result not only in mitochondrial dysfunction,but also in downregulated mitochondrial biogenesis.