Metabolic Regulation Mechanism Of PPAR? 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-??PPAR??belongs to a nuclear receptor superfamily and plays a critical role in lipid metabolism in mammals and fish.However,the regulatory mechanismof PPAR?on 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 PPAR?in fish energy metabolism.In the present study,Nile tilapia?Oreochromis niloticus?was used as a model fish to systematically investigate the role of PPAR?in 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 PPAR?activators?gemfibrozil and fenofibrate?,to confirm the activation of PPAR?and explore the effects of PPAR?activation on improving carbohydrate and lipid metabolism and antibacterial immunity.Secondly,in order to further explore the regulatory effects of PPAR?on the energy metabolism of Nile tilapia;in this study,we investigated the regulatory mechanism of PPAR?activation 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 PPAR?activation on lipid catabolism and tolerance to high-carbohydrate diet of Nile tilpaiaPPAR?plays 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 PPAR?on glucose and lipid metabolism in fish are limited.In order to investigate the regulatory role of PPAR?in 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 PPAR??for eight weeks.Our results showed that the mRNA and protein expression of PPAR?were largely upregulated in gemfibrozil treatment group.PPAR?activation 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.PPAR?activation 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 PPAR?-activated fish showed lower activity of AST and higher activity of SOD and CAT.In addition,PPAR?activation also significantly increased the mRNA expression of glycolysis and lipolysis related genes.Overall,the present study indicated that 1)PPAR?activation could help reduce the negative effects in fish fed with high carbohydrate diet by improving lipid catabolism;2)PPAR?activation may change the energy pattern of Nile tilapia by promoting lipid catabolism.2.Effects of PPAR?activation on lipid catabolism and the antibacterial immunity of Nile tilapiaPPAR?plays 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 PPAR?may 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 PPAR?was efficiently activated by fenofibrate through increasing mRNA and protein expressions and protein dephosphorylation.PPAR?activation increased significantly mitochondrial fatty acid?-oxidation efficiency,the copy number of mitochondrial DNA and expression of monoamine oxidase?MAO?,a marker gene of mitochondria.Meanwhile,PPAR?activation 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,PPAR?activation improved the ability of Nile tilapia to resist A.hydrophila,mainly through enhancing mitochondrial fatty acids?-oxidation,immune and antioxidant capacities,as well as inhibiting inflammation.The results of the above two studies indicated that PPAR?could 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 PPAR?in energy metabolism deserves further study.3.Regulation of PPAR?activation on glucose anaerobic glycolysis of Nile tilapiaIn the previous study in this thesis found that PPAR?activation 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 PPAR?activation 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 PPAR?on anaerobic glycolysis.The results of these section showed that fenofibrate significantly increased the expression of PPAR?mRNA and protein expression in liver.PPAR?activated 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 PPAR?activated 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 PPAR?activated reduced significantly the contents of resered ¹⁴C-lipid and ¹⁴C-glycogen in whole fish.Furthermore,metabonomics analysis result of liver showed that PPAR?activation 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 PPAR?increased the anaerobic glycolysis pathway and also improved the ability to scavenge lactate of Nile tilapia.The present results confirmed that PPAR?activation can effectively activate the biochemical processes of anaerobic glycilysis.4.Effects of PPAR?activation on intracellular oxygen levels and expression of hypoxia-inducible factors in Nile tilapiaIn the previous study,we have learned that the PPAR?activation increases glucose anaerobic glycolysis and the production of lactate.In order to explore the regulatory mechanism of PPAR?activation on anaerobic glycolysis,we focused on the effects of PPAR?on 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 PPAR?activation decreased the content of triglyceride in serum and liver,as well as decreased HIS,MFI and total lipid of whole fish.In addition,PPAR?activation increased the hepatic mitochondrial?-oxidation efficiency.Of note,PPAR?activation also significantly increased the oxygen consumption rate of Nile tilapia.The result of intracellular oxygen monitoring showed that PPAR?activation significantly reduced the levels of oxygen in intracellular.Furthermore,PPAR?activation also significantly increased the mRNA and protein expression of HIF1?and HIF3?in liver.Overall,our results indicated that PPAR?activated regulates anaerobic glycolysis by reducing intracellular oxygen and activating hypoxia-inducible factors expression in Nile tilapia.5.Regulation of PPAR?activation on glucose oxidative phosphorylation and its signaling pathway of Nile tilapiaAlthough the effect of PPAR?on glucose utilization has been extensively studied in mammals,studies on the regulation of glucose oxidative utilization in fish PPAR?are limited and the mechanism involved is unclear.After confirming the regulation mechanism of PPAR?on the process of anaerobic glycolysis,this study further explored the regulation mechanism of PPAR?on the oxidative phosphorylation of glucose in Nile tilapia.In the present study,a four weeks fenofibrate feeding trial was performed.Our study showed that PPAR?activation significantly increase the levels of glucose and insulin in serum,while reduced significantly the glycogen content of liver.Glucose tolerance test found that PPAR?activation reduced significantly the glucose clearance in Nile tilapia.We conducted metabolic tracking tests by intraperitoneally injecting individual fish with ¹⁴C-labeled glucose.PPAR?activation significantly reduced the oxidation rate of ¹⁴C-glucose.Furthermore,PPAR?activation 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 PPAR?activation significantly reduced the phosphorylation level of Akt.Besides,the mRNA and protein expression of TRIB2 were significantly up-regulated in PPAR?-activated fish.PPAR?activation significantly increased the mRNA and protein expression of PDK2 and PDK4 in liver,while the mRNA and protein expression of PDHE1?were significantly down-regulated.However,the level of phosphorylation of PDHE1?was significantly increased in PPAR?-activated fish.Of note,the effect of PPAR?activation on muscle is weak.Overall,our results in this section indicated that in Nile tilapia 1)PPAR?inhibits the insulin signaling pathway by activating TRIB2;2)PPAR?activation reduces oxidative phosphorylation of glucose in Nile tilapia by regulating PDK2/PDK4-PDHE1?axis.In conclusion,PPAR?can 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,PPAR?changes the glucose metabolism pattern by enhancing lipolysis efficiency and regulating the TRIB2/Akt-PDK/PDHE1?signaling pathway,thereby maintaining the homeostasis of glucose and lipid metabolism.