The Regulation Of Carnitinne In Fish Nutrional Metabolsim

High fat diets(HFDs)are currently used in aquaculture for their “protein-sparing effect”.However,the HFDs often lead to severe lipid accumulation in liver and abdominal adipose tissues in farmed fish,which induce adverse health effects.Thus,researches on effective additives with lipid-lowering effects have attracted the attention of fish nutritionists.Carnitine increases the mitochondria ?-oxidation capability and lowers lipid content in tissues in mammals.Therefore,carnitine has been used extensively in fish nutrition research and aquaculture.Carnitine has shown to increase growth performance,decrease the body lipid content and provide protein sparing effect in some fish studies.Indeed,the use of carnitine has been shown to reduce lipid metabolism in other fishes.However,such effects have not been observed in some fishes.The contradictory and complicated results of carnitine supplementation in fish have not been clearly explained in fish nutrition research.Moreover,the mechanism of lipid lowering effect is still elusive.The above facts have restricted further utilization of carnitine in fish feed industry.Therefore,regulatory effect of carnitine in fish nutrition should be systemically studied to reveal the reasons for contradicting results.To study the regulatory mechanism of L-carnitine in fish nutritional metabolism,zebrafish were feed with diets supplemented with L-carnitine which established the high-carnitine zebrafish.We further established the low-carnitine zebrafish by feeding them with an inhibitor of carnitine synthesis(mildronate).The low-carnitine Nile tilapia model was also established to evaluate the different functions of L-carnitine and D-carnitine in regulating fish nutritional metabolism.1.The regulation of L-carnitine in the nutritional metabolism of high-carnitine zebrafishExcess fat accumulation has been observed widely in farmed fish;therefore,efficient lipid-lowering factors have obtained high attention in the current fish nutrition studies.Dietary L-carnitine can increase fatty acid ?-oxidation in mammals,but has produced contradictory results in different fish species.To date,the mechanisms of metabolic regulation of L-carnitine in fish have not been fully determined.The present study used zebrafish to investigate the systemic regulation of nutrient metabolism by dietary L-carnitine supplementation.L-carnitine significantly decreased the lipid content in liver and muscle,accompanied by increased concentrations of total and free carnitine in tissues.Meanwhile,L-carnitine enhanced significantly mitochondrial ?-oxidation activities and the expression of carnitine palmitoyltransferase 1 m RNA,whereas it depressed the m RNA expression of adipogenesis-related genes.In addition,L-carnitine caused higher glycogen deposition in the fasting state and increased and decreased the m RNA expressions of gluconeogenesis-related and glycolysis-related genes,respectively.L-carnitine also increased the hepatic expression of m TOR in the feeding state.Taken together,dietary L-carnitine supplementation decreased lipid deposition by increasing mitochondrial fatty acid ?-oxidation and is likely to promote protein synthesis.However,the L-carnitine-enhanced lipid catabolism caused a decrease in glucose utilization.Therefore,L-carnitine has comprehensive effects on nutrient metabolism in fish.2.The regulation of L-carnitine in the nutritional metabolism of low carnitine zebrafishThe contradiction and complication of the L-carnitine effect has restricted the recognition of its role in fish nutritional metabolism.We used carnitine deficient fish model to study the carnitine function in order to enhance our knowledge on carnitine regulation in nutritional metabolism.To establish a low-carnitine zebrafish model and illustrate its characteristics on nutrient metabolism,male zebrafish were fed with an inhibitor of carnitine synthesis(mildronate at 0.05% body weight/day)for six weeks,then the fish were fasted for one week.Carnitine and triglyceride(TG)concentrations,fatty acid(FA)?-oxidation capability and other molecular and biochemical assays of lipid,glucose and protein metabolism were measured in the fed and fasted states.The results indicated that,mildronate markedly decreased hepatic carnitine concentrations in the fed and fasted states,but in muscles it only decreased carnitine concentrations in the fasted state.Liver TG concentrations increased by more than 50% in both nutritional states in mildronate-treated fish.Mildronate decreased the efficiency of liver mitochondrial ?-oxidation,increased the hepatic m RNA expression of genes related to FA ?-oxidation and lipolysis,and decreased the expression of lipogenesis genes.Mildronate decreased whole body glycogen content,increased glucose metabolism rate and upregulated the expression of glucose uptake and glycolysis genes.Mildronate also increased whole body protein content and hepatic m RNA expression of mechanistic target of rapamycin(m TOR)and decreased the expression of a protein catabolism-related gene in the fed state.Liver,rather than muscle,was the primary organ targeted by mildronate.In short,mildronate-induced low-carnitine zebrafish showed decreased mitochondrial FA ?-oxidation efficiency,greater lipid accumulation and altered glucose and protein metabolism.This model could be used as a novel fish model for future nutrient metabolism studies.3.The different function of L-carnitine and D-carnitine in in the nutritional metabolism of Nile tilapiaIn practice,carnitine is presented as L-carnitine and D-carnitine.However,the different function of the two carnitines in fish nutritional metabolism is currently not clear.Consequently,it is difficult for fish nutritionists to choose the appropriate type between the two carnitines,which has limited the further application of carnitine in the feed industry.To establish the low carnitine Nile tilapia,male fish were fed with an inhibitor of carnitine synthesis(mildronate at 1000 mg/kg and 25 g/kg diet body weight per day)for four weeks.The results showed that,the total carnitine significantly decreased in liver,muscle and plasma.The low carnitine Nile tilapia model,were then separately fed with 0.4 g/kg of L-carnitine and D-carnitine diets for six weeks.The results of showed that,the L-carnitine increased the acyl-carnitine content in liver and decreased the total lipid in liver,muscle and adipose tissue and decreased the plasm TG content.Moreover,it downregulated the expression of gene-related to detoxification(cyp1a).L-carnitine improved the dyslipidemia of low carnitine Nile tilapia.Compared with the L-carnitine,the D-carnitine decreased the acyl-carnitine content in liver,increased the plasm TG content and the total lipid in liver and adipose tissue,upregulated the expression of genes related to mitochondrial and peroxisomal ?-oxidation(cpt1 and aco)and downregulated the expression of cyp1 a.In addition,D-carnitine did not affect the plasm glucose and liver glycogen,but separately upregulated and downregulated the expression of genes related to glycolysis and gluconeogenesis in liver,respectively.D-carnitine also upregulated the expression of the gene related to amino acid catabolism(gcn2)in the liver.Meanwhile,D–carnitine upregulated the expression of inflammation(il-1?,tgf?1)in the liver,increased the plasma MDA and enhanced the m RNA expression of oxidative stress(gst)and apoptosis(casp8)genes.The metabolomics analysis showed that the D–carnitine decreased the acyl-carnitine and increased phospholipid.D–carnitine also changed the pyruvate pathway to produce more lactic acid and D-citramalic acid.Moreover,D–carnitine decreased five essential amino acids.Furthermore,D–carnitine raised the product of tricarboxylic acid(TCA)cycle(fumaric acid,phosphoric acid)and oxidative phosphorylation(AMP,phosphoric acid).In general,L-carnitine decreased the lipid content by increasing the acyl-carnitine content while D–carnitine increased the lipid deposition by decreasing the acyl-carnitine content and caused liver lipotoxicity.D-carnitine enhanced the amino acid catabolism,but not glycolysis,to produce more energy to maintain the higher activity of TCA cycle and oxidative phosphorylation.Therefore,compared with the L-carnitine,which was identified to play biological functions,the D-carnitine did not show biological activities,suggesting it is not the effective form of carnitine.