Metabolic Regulation Mechanism Of PPARα In Glucose And Lipid Utilization And Heath Of Nile Tilapia(Oreochromis Niloticus)

Carbohydrates are important non-protein enegy sources of fish,and they are the most economical component in aquatic feeds due to their high availability and relatively low price,therefore the inclusion of a high level of carbohydrate has been widely used in aquatic feed.Although high carbohydrate diets have been currently used in aquaculture to partly replace expensive dietary protein,so-called the“protein sparing effect”,however,the problem of excessive fat accumulation caused by high carbohydrate diets should not be ignored.Recently,the potential regulatory mechanism to reduce lipid deposition in fish has attracted high attention.However,it is not clear whether the increase of lipid catabolism can alleviate the negative effect of high carbohydrate in fish.The peroxisome proliferator-activated receptor-α（PPARα）belongs to a nuclear receptor superfamily and plays a critical role in lipid metabolism in mammals and fish.However,the regulatory mechanismof PPARαon energy metabolism in fish,especially in glucose and lipid metabolic homeostasis is also unclear.Therefore,it is particularly important to clarify the function and regulation mechanism of PPARαin fish energy metabolism.In the present study,Nile tilapia（Oreochromis niloticus）was used as a model fish to systematically investigate the role of PPARαin nutrients utilization and antibacterial immunity and its biochemical and molecular regulation mechanism of glucose and lipid metabolism homeostasis.The methods of ¹⁴C-labeled single nutrient metabolic tracking test,biochemical and molecular biological,western blot and metabolomic analysis were used to in our present stdy.First,Nile tilapia was treated with the PPARαactivators（gemfibrozil and fenofibrate）,to confirm the activation of PPARαand explore the effects of PPARαactivation on improving carbohydrate and lipid metabolism and antibacterial immunity.Secondly,in order to further explore the regulatory effects of PPARαon the energy metabolism of Nile tilapia;in this study,we investigated the regulatory mechanism of PPARαactivation on the oxygen consumption rate,intracellular oxygen and hypoxia-inducible factors,as well as insulin signaling pathway and glucose oxidative phosphorylation.The main results of our present study are presented as follows:1.Effects of PPARαactivation on lipid catabolism and tolerance to high-carbohydrate diet of Nile tilpaiaPPARαplays an important regulatory role in the maintenance of glucose and lipid homeostasis,and has been extensively studied in mammals.However,studies on the effects of PPARαon glucose and lipid metabolism in fish are limited.In order to investigate the regulatory role of PPARαin glucose and lipid metabolism.Nile tilaia（3.03±0.11 g）were fed with three diets:control（30%carbohydrate）,HCD（45%carbohydrate）and HCG（HCD supplemented with 200 mg/kg gemfibrozil,an agonist of PPARα）for eight weeks.Our results showed that the mRNA and protein expression of PPARαwere largely upregulated in gemfibrozil treatment group.PPARαactivation significantly decreased the HIS,MFI,total lipid of whole fish,as well as triglyceride and glycogen contents of liver also significantly downregulated in the fish fed with high-carbohydrate diet.In contrast,the content of lactate in liver was significantly increased.PPARαactivation also increased the levels of glucose and lactate in serum,but decreased serum concentrations of triglyceride,free fatty acids and MDA in the fish fed with high-carbohydrate diet.Furthermore,the PPARα-activated fish showed lower activity of AST and higher activity of SOD and CAT.In addition,PPARαactivation also significantly increased the mRNA expression of glycolysis and lipolysis related genes.Overall,the present study indicated that 1)PPARαactivation could help reduce the negative effects in fish fed with high carbohydrate diet by improving lipid catabolism;2)PPARαactivation may change the energy pattern of Nile tilapia by promoting lipid catabolism.2.Effects of PPARαactivation on lipid catabolism and the antibacterial immunity of Nile tilapiaPPARαplays critical physiological roles in energy metabolism,antioxidation and immunity of mammals,however,these functions have not been fully understood in fish.In order to prove that PPARαmay be also improve the health of fish by regulating energy metabolism.In the present study,Nile tilapia were fed with fenofibrate,for six weeks,and subsequently challenged with Aeromonas hydrophila.The results showed that PPARαwas efficiently activated by fenofibrate through increasing mRNA and protein expressions and protein dephosphorylation.PPARαactivation increased significantly mitochondrial fatty acidβ-oxidation efficiency,the copy number of mitochondrial DNA and expression of monoamine oxidase（MAO）,a marker gene of mitochondria.Meanwhile,PPARαactivation also increased significantly the mRNA expression of mitochondrial respiratory chain complex enzymes.The fenofibrate-fed fish had higher survival rate when exposed to A.hydrophila.Moreover,the fenofibrate-fed fish also had higher activities of immune and antioxidative enzymes,and gene expressions of anti-inflammatory cytokines,while had lower expressions of pro-inflammatory cytokine genes.Taken together,PPARαactivation improved the ability of Nile tilapia to resist A.hydrophila,mainly through enhancing mitochondrial fatty acidsβ-oxidation,immune and antioxidant capacities,as well as inhibiting inflammation.The results of the above two studies indicated that PPARαcould be activated in Nile tilapia by exogenous ligands,and it also could improve the utilization of dietary glucose and lipid and antibacterial immunity of Nile tilapia by enhancing the lipolytic efficiency and regulating energy metabolism.Therefore,the regulatory role of PPARαin energy metabolism deserves further study.3.Regulation of PPARαactivation on glucose anaerobic glycolysis of Nile tilapiaIn the previous study in this thesis found that PPARαactivation can increase the activity of anaerobic glycolysis and improve the utilization of high-carbohydrate diet of Nile tilapia.In order to explore the regulatory mechanism of PPARαactivation in anaerobic glycolysis.Nile tilapia（initial weight,12.42±1.80 g）were performing with control diets（Ctrl）and fenofibrate diets（FB）for 4-week to investigate the regulation of PPARαon anaerobic glycolysis.The results of these section showed that fenofibrate significantly increased the expression of PPARαmRNA and protein expression in liver.PPARαactivated mainly increased the levels of pyruvate and lactate in serum,and also increased the content of lactate in liver.In addition,the gene expression of key enzymes in glycolysis and the pentose phosphate pathway were largely upregulated in liver,while the gene expression of key enzymes in the gluconeogenesis pathway did not change significantly.Our results also found that PPARαactivated significantly increased the activity of lactate dehydrogenase（LDH）,and the mRNA and protein expressions of lactate dehydrogenase A（LDHA）were significantly upregulated in liver.Furthermore,we conducted metabolic tracking tests by intraperitoneally injecting individual fish with ¹⁴C-labeled lactate.Fenofibrate-fed fish increased significantly ¹⁴CO₂ release from[1-¹⁴C]-lactate oxidation,while PPARαactivated reduced significantly the contents of resered ¹⁴C-lipid and ¹⁴C-glycogen in whole fish.Furthermore,metabonomics analysis result of liver showed that PPARαactivation significantly increased the intermediate metabolites of glycolysis and pentose phosphate pathway,whereas the opposite occured in the lipid metabolites.Taken together,the results of this part indicated that the activated PPARαincreased the anaerobic glycolysis pathway and also improved the ability to scavenge lactate of Nile tilapia.The present results confirmed that PPARαactivation can effectively activate the biochemical processes of anaerobic glycilysis.4.Effects of PPARαactivation on intracellular oxygen levels and expression of hypoxia-inducible factors in Nile tilapiaIn the previous study,we have learned that the PPARαactivation increases glucose anaerobic glycolysis and the production of lactate.In order to explore the regulatory mechanism of PPARαactivation on anaerobic glycolysis,we focused on the effects of PPARαon intracellular oxygen and hypoxia-inducible factors in the present study.A 4-week trial was conducted,and 120 Nile tilapia（initial weight,12.42±1.80 g）were randomly divided into control group（Ctrl）and fenofibrate treatment group（FB）.Our results showed that PPARαactivation decreased the content of triglyceride in serum and liver,as well as decreased HIS,MFI and total lipid of whole fish.In addition,PPARαactivation increased the hepatic mitochondrialβ-oxidation efficiency.Of note,PPARαactivation also significantly increased the oxygen consumption rate of Nile tilapia.The result of intracellular oxygen monitoring showed that PPARαactivation significantly reduced the levels of oxygen in intracellular.Furthermore,PPARαactivation also significantly increased the mRNA and protein expression of HIF1αand HIF3αin liver.Overall,our results indicated that PPARαactivated regulates anaerobic glycolysis by reducing intracellular oxygen and activating hypoxia-inducible factors expression in Nile tilapia.5.Regulation of PPARαactivation on glucose oxidative phosphorylation and its signaling pathway of Nile tilapiaAlthough the effect of PPARαon glucose utilization has been extensively studied in mammals,studies on the regulation of glucose oxidative utilization in fish PPARαare limited and the mechanism involved is unclear.After confirming the regulation mechanism of PPARαon the process of anaerobic glycolysis,this study further explored the regulation mechanism of PPARαon the oxidative phosphorylation of glucose in Nile tilapia.In the present study,a four weeks fenofibrate feeding trial was performed.Our study showed that PPARαactivation significantly increase the levels of glucose and insulin in serum,while reduced significantly the glycogen content of liver.Glucose tolerance test found that PPARαactivation reduced significantly the glucose clearance in Nile tilapia.We conducted metabolic tracking tests by intraperitoneally injecting individual fish with ¹⁴C-labeled glucose.PPARαactivation significantly reduced the oxidation rate of ¹⁴C-glucose.Furthermore,PPARαactivation significantly reduced the contents of reserved ¹⁴C-lipid,while there were no significant differences in the contents of reserved ¹⁴C-glycogen and ¹⁴C-protein in the body.Our study on insulin signaling pathway and oxidative phosphorylation of glucose found that PPARαactivation significantly reduced the phosphorylation level of Akt.Besides,the mRNA and protein expression of TRIB2 were significantly up-regulated in PPARα-activated fish.PPARαactivation significantly increased the mRNA and protein expression of PDK2 and PDK4 in liver,while the mRNA and protein expression of PDHE1αwere significantly down-regulated.However,the level of phosphorylation of PDHE1αwas significantly increased in PPARα-activated fish.Of note,the effect of PPARαactivation on muscle is weak.Overall,our results in this section indicated that in Nile tilapia 1)PPARαinhibits the insulin signaling pathway by activating TRIB2;2)PPARαactivation reduces oxidative phosphorylation of glucose in Nile tilapia by regulating PDK2/PDK4-PDHE1αaxis.In conclusion,PPARαcan be effectively activated by exogenous ligamds in fish,and it improves the utilization of glucose and lipid in diets and antibacterial immunity by enhancing lipolysis efficiency and regulating energy metabolism.In addition,PPARαchanges the glucose metabolism pattern by enhancing lipolysis efficiency and regulating the TRIB2/Akt-PDK/PDHE1αsignaling pathway,thereby maintaining the homeostasis of glucose and lipid metabolism.