The Regulatory Mechanisms Of Mitochondrial Fatty Acid β- Oxidation In Energy Homeostasis Maintenance Of Fish

The metabolism of amino acids,glucose and fatty acids is always in a dynamic balance in cells,which constitutes to the cellular energy homeostasis.The imbalance of energy homeostasis is an important factor of metabolic disorders of organisms,including human beings,land animals,fish and so on.In the current aquaculture,there are many prevalent metabolic disorders characterized by severe lipid accumulation,which would induce a series of adverse effects,such as slow growth,low immunity,and decreased nutritional quality.These strongly influence the quality and safety of aquatic products,which have seriously hindered the sustainable development of aquaculture.Therefore,it is particularly important to explore the regulatory mechanism of energy homeostasis in fish.The mitochondrial fatty acidβ-oxidation（FAO）system,namely carnitine palmitoyltransferase（CPT）mitochondrial membrane shuttle-through system,is an important energy metabolic process in cells and plays a crucial role in maintaining cellular energy homeostasis.In mammals,the mitochondrial FAO system is regarded as an important target to regulate lipid metabolism,and is conducive to study the regulation mechanism of energy homeostasis.L-carnitine and CPT1 are two key regulatory factors in the mitochondrial FAO process and have become the pivotal regulatory targets in the study of mitochondrial FAO function.In the present study,to explore the regulatory role of mitochondrial FAO in energy homeostasis of fish and the related molecular mechanisms,we used the low-carnitine tilapia established by inhibiting endogenous L-carnitine synthesis with inhibitor,and the cpt1b knockout zebrafish generated with gene-editing technology.In this study,we used the biochemical methods,transcriptomic and metabolomics analysis,isotope-labeled nutrients tracer technique,RT-PCR and western blot analysis,histological analysis,cell biology methods,and many nutritional measurement methods,and investigated systematically and thoroughly the effects of mitochondrial FAO inhibition on fish energy metabolism and the important signaling pathways involved,as well as the regulation effects of mitochondria FAO system on fish with intake of high-energy diets.We found that both low-carnitine tilapia and cpt1b deficiency zebrafish significantly reduced the mitochondrial FAO activity and suggested that inhibited mitochondrial FAO system triggered a metabolic reprogramming,accompanied by the increased carbohydrate utilization and protein deposition,and the AMPK/AKT-mTOR signaling cascades are involved in this process.In addition,this study also demonstrated that the mitochondrial FAO system is an important regulatory target for fish to adapt to high-energy diets intake.1.Exploring preliminarily the metabolic characteristics of the low-carnitine tilapiaL-carnitine,as one of the important regulatory factors of mitochondrial FAO,plays an important role in lipid catabolism and energy homeostasis.In this study,the low-carnitine tilapia was established by feeding Nile tilapia（Oreochromis niloticus）with diets of 1000 mg/kg.d mildronate（MD,L-carnitine synthesis inhibitor）supplementation for 6 weeks.We investigated preliminarily the effects of inhibited mitochondrial FAO on nutrient metabolism of tilapia through the physiological,biochemical and molecular methods.After 6-week feeding of MD diets,we found the free carnitine content and mitochondrial FAO capacity were significantly decreased in liver and muscle,and the peroxidosomal FAO activity and mitochondrial proliferation were increased.The inhibited mitochondrial FAO tilapia increased total lipid content of whole body,serum triglyceride（TG）and non-esterified fatty acid（NEFA）concentrations,and the hepatic TG content.Meanwhile,RT-PCR results showed that the mRNA expression of genes related to FAO process genes（cpt1a,cpt1b and aox1）and lipogenesis（dgat2 and srebp1）were significantly increased in liver and muscle.However,mitochondrial FAO inhibition did not affect the activity of AST and ALT and malondialdehyde（MDA）in serum,nor the expression of genes related to cellular damage and inflammation in liver and muscle.Moreover,the results of glucose metabolism showed that inhibited mitochondrial FAO significantly reduced the concentrations of serum glucose and insulin,and improved insulin sensitivity,inducing the increase of glucose uptake and glycolysis to accelerate the blood glucose clearance.The inhibited mitochondrial FAO tilapia increased activity of glycolytic enzyme（PK and HK）and the content of glycolytic products（pyruvate and acetyl-CoA）in liver and muscle,and reduced the gluconeogenesis and glycogen synthesis,resulting in the decrease of the glycogen content in tissues.In addition,inhibited mitochondrial FAO increased the total protein content of tilapia and muscle and the levels of amino acids in muscle,decreased the concentration of serum total amino acid（TAA）through reducing the expression of genes related to amino acid catabolism（asns,glud1,atf4and gcn2）.Taken together,we suggested that inhibited mitochondrial FAO increased carbohydrate metabolism and protein deposition in tilapia.Therefore,this work found that the inhibition of mitochondrial FAO could change the nutrient metabolic balance of tilapia.2.Exploring the biochemical mechanism of altered enegy mebaolism balance induced by inhibited mitochondrial FAO in tilapiaTo more thoroughly and systematically identify the the biochemical mechanism of altered enegy mebaolism balance induced by inhibited mitochondrial FAO in tilapia,we used the isotope-labeled nutrients tracer technique,and transcriptomic and metabolomics analysis,and detailedly analyzed the changes of nutrient metabolism processes and metabolites in the low-carnitine tilapia.The intraperitoneal（i.p.）injection of ¹⁴C-labeled nutrients tracer test results showed that the inhibited mitochondrial FAO tilapia significantly reduced oxidation of ¹⁴C-labeled palmitic acid,which was mainly deposited as ¹⁴C-labeled lipid in body.However,after ¹⁴C-labeled glucose injection,the content of ¹⁴CO₂ released and the deposition of ¹⁴C in body were respectively increased and decreased,and the deposition of ¹⁴C-labeled protein was increased in body.In addition,inhibited mitochondrial FAO tilapia increased ¹⁴C-labeled protein deposition in body after ¹⁴C-labeled amino acid injection.These results further suggest that inhibited mitochondrial FAO alter the utilization of nutrient in tilapia that is more inclined to break down glucose and deposit lipid and protein.The transcriptomic analysis results showed that inhibited mitochondrial FAO changed the hepatic metabolic pattern of tilapia,and the differentially expressed genes（DEGs）mainly in the metabolic processes（75.68%）and metabolic signaling pathways（10.22%）through the enrichment analysis of the KEGG pathway.The inhibited mitochondrial FAO tilapia up-regulated the expression of mitochondrial and peroxisomal FAO genes（acs,cpt1a,cpt2,acox,acot and vlacs）and down-regulated the expression of lipolysis genes（pnpla3,lipea,pld4 and lipin3）.In addition,the genes expression of glycolysis（gckr,gck and pfk）and pyruvate metabolism gene（phha）were up-regulated,and the genes expression of gluconeogenesis（pck1 and g6p）and glycogen synthesis（pgd and gys1）were down-regulated.Moreover,the genes expression of amino acid catabolism（glud1b,atf3 and asns）,proteolytic process（xpnpep2,cstba and ctsc）,and a series of genes related to proteolysis in proteasomes were significantly down-regulated,and many genes related amino acids conversation were up-regulated.These results indicate that inhibited mitochondrial FAO increased compensatorily genes expression in FAO process,reduced lipid catabolism,and changed the metabolic processes contributed to carbohydrate utilization and protein deposition.The metabolomics analysis results displayed that inhibited mitochondrial FAO tilapia were detected 39 different metabolites in liver tissue,most of which were increased lipids.There were some reduced intermediate metabolites of carbohydrate,and several increased essential amino acids.In short,inhibited mitochondrial FAO influenced the expression of genes in nutrient metabolism pathway,and changed the levels of intracellular metabolites,and thus remodeled cellular energy homeostasis.3.Investigation on the molecular mechanism of remodeled energy homeostasis induced by inhibited mitochondrial FAOTo elucidate the regulatory mechanism of inhibited mitochondrial FAO induced metabolic homeostasis reprogramming in tilapia,we analyzed the activity of proteins in regulatory signaling pathway in vivo and in vitro,by using western blot analysis and inhibitor of protein.We found that that inhibited mitochondrial FAO tilapia increased the hepatic expression of AMPK protein,and the proteins in insulin signaling pathway（p-AKT and IR）in liver and muscle,indicating that the elevated expression of AMPK triggered the AKT signaling pathway activation to improved insulin sensitivity.In addition,the expression of proteins in the mTOR signaling pathway（p-mTOR and p-S6）were increased in liver and muscle,indicating the activation of the protein synthesis.Then,we treated primary hepatocytes of tilapia with 1 mM MD for 24 and 36 h,and found that the mitochondrial FAO activity was inhibited in 36 h treatment hepatocytes,which increased ¹⁴C-labeled glucose oxidation and decreased ¹⁴C-labeled amino acid breakdown,through the activation of AKT and mTOR signaling pathways.To further explore the relationship between AKT and mTOR proteins,we used respectively AKT inhibitor（MK-2206 2HCl,MK）and mTOR inhibitor（rapamycin,Rap）in the primary hepatocytes with inhibited mitochondrial FAO,and discovered that the expression of p-mTOR was significantly decreased with the inhibition of AKT activity,while the inhibition of mTOR activity had no effect on the expression of AKT,indicating that the activation of mTOR would depend on the activity of AKT.These results indicated that inhibited mitochondrial FAO would activate the AMPK/AKT-mTOR signaling cascades to maintain cellular metabolic homeostasis.4.Investigation on the metabolic characteristics and the regulation mechanism of energy homeostasis in cpt1b knockout zebrafishTo avoid side-effects of inhibitor of L-carnitine synthesis,we generated cpt1b knockout systemically zebrafish by using gene-editing techniques CRISPR/Cas9,and identified the characteristics of energy metabolism and the regulatory mechanism involved in maintaining metabolic homeostasis.We found that the larvae of cpt1b mutant showed lower behavior activity and oxygen consumption rate（OCR）,and increased lipid accumulation in whole body.The adult of cpt1b mutant showed higher weigh gain rate（WG）,decreased mitochondrial FAO activity,increased lipid content in tissues and whole body through up-regulating hepatic expression of lipid anabolism genes.Moreover,the adult of cpt1b mutant reduced the levels plasma glucose and insulin,and increase glucose clearance capacity resulting from the improvement of insulin sensitivity.They increased the hepatic expression of glut2 and glycolysis genes,and reduced reducing the genes expression of gluconeogenesis and glycogen synthesis,resulting in the decrease of hepatic glycogen and muscle and the increase of the concentration of pyruvate and acetyl-CoA in muscle,through the activation of AKT.In addition,the cpt1b mutant increased the protein content of whole body and the amino acid level of muscle,reduced the expression of genes related to amino acid catabolism,accompanied by the activation of the mTOR.The isotopic tracer results showed that the cpt1b mutant increased glucose oxidation,reduced the breakdown of fatty acid and amino acid,and promoted the protein deposition from glucose.These data indicate that cpt1b deficiency inhibits mitochondrial FAO activity,increases glucose utilization,and promotes protein deposition.The AKT-mTOR signaling pathway is involved in the reprogramming of energy homeostasis,which further verifies the important role of mitochondrial FAO in the regulation of energy homeostasis in fish.5.Inhibited mitochondrial FAO impairs adaptation to high-fat diet in Nile tilapia The intake of high-fat diet（HFD）often cause imbalanced of energy homeostasis and has some adverse effects in fish.This study was conducted to examine the regulatory roles of carnitine and its related mitochondrial FAO in the adaptation to HFD intake of Nile tilapia.Nile tilapia were fed with HFD（13%lipid）,HFD+MD（1000 mg/kg body per day）or a normal fat diet（NFD,7%lipid）for eight weeks.After the feeding trail,the fish fed with HFD showed higher hepatic free carnitine content and FAO activities,and similar levels of serum TG and whole body lipid.However,the HFD+MD-fed fish remarkably decreased carnitine content and mitochondrial FAO efficiency in tissues than those in the HFD-fed fish,and increased contents of serum FFA and TG,whole body lipid and hepatic TG.Moreover,the HFD-fed fish up-regulated the expression of the genes associated to FAO,lipid transport and lipolysis.Nevertheless,the fish fed with HFD+MD showed lower transcriptional levels of the genes related to lipolysis and lipid transport,and higher lipogenesis genes.These results indicate that the adaptive changes in the fish fed with HFD were eliminated by inhibited mitochondrial FAO due to the lower carnitine level,and show that carnitine and its related mitochondrial FAO activity play important roles in the adaptation to HFD intake in fish.This study illustrates that in the practical usage of HFD in aquaculture,the endogenous carnitine concentration and mitochondrial FAO activities should be important checkpoints.6.Inhibited mitochondrial FAO improves glucose utilization in Nile tilapia fed a high-carbohydrate dietFish are regarded as poor users of dietary carbohydrate and often display a prolonged hyperglycemia and lipid deposition after high-carbohydrate diet（HCD）feeding.In this context,we have identified,for the first time in tilapia and zebrafish,inhibited mitochondrial FAO with MD would ameliorate glucose utilization.This study was performed to elucidate the effects of mitochondrial FAO inhibition with MD on nutrient metabolism in Nile tilapia fed HCD.Nile tilapia（6.13±0.11 g）were fed HCD（45%cornstarch）,HCD+MD（1000 mg/kg body weight per day）or a normal diet（NCD,30%cornstarch）for eight weeks.After the feeding trail,we observed that fish fed HCD showed higher hepatic free carnitine content and FAO activities,whereas MD supplementation remarkably decreased these indexes,indicating the attenuated FAO reaction.In addition,dietary MD significantly reduced HCD-induced high serum glucose and insulin,and hepatic glycogen,lactate and pyruvate,accompanied by improved glucose clearance,suggesting that FAO inhibition benefits insulin sensitivity and glucose utilization.Moreover,HCD increased the values of hepatosomatic index（HSI）and mesenteric fat index（MFI）,the lipid content of whole body and tissues,the activity of serum ALT and AST,and hepatic MDA,indicating that HCD induced lipid deposition and hepatic damage.However,these adverse effects were all reversed with MD treatment.Additionally,MD supplementation increased whole body protein,suggesting inhibited mitochondrial FAO promoted protein deposition in fish with HCD intake.Furthermore,HCD-fed fish down-regulated the expression of genes associated to glucose uptake,glycolysis,FAO,and lipolysis,yet up-regulated genes related to lipogenesis and proteolysis.Nevertheless,MD addittion showed positive effects on transcriptional levels of these genes.These results strongly indicate that inhibited mitochondrial FAO improves glucose utilization and eliminates the adverse effects induced by HCD intake in juvenile Nile tilapia.This work further reinforce the conclusion that mitochondrial FAO plays an important roles in regulating energy homeostasis to adapt to high-energy diets intake in fish.