The Activation And The Related Regulatory Mechanism Of PPARα In Lipid Metabolism Of Nile Tilapia, Oreochromis Niloticus

A series experiments were conducted symstemically and comprehensively to investigate the mechanism, action taget and the lipid metabolic regulation of PPARa activation in Nile tilapia, the methods of nutrition, biochemical, molecular biological and transcriptomic as well as the means of cell culture and isotope labelling, were studied by the examination the PPARa structure, tissue-specific expression and its metabolic regulation. In the present study, NtPPARa was firstly cloned, identified, and the tissue mRNA expression was studied. Secondly, the activation mechanism of NtPPARα was verified in both primary hepatocytes and living fish primarily by the decreased phosphorylation of NtPPARα, or the supplemental increased expression of NtPPARα mRNA and protein. A hepatic transcriptomic study was followed to draw out the whole regulatory pathways of PPARα activation in the tilapia. Thirdly. 10-week feeding trial with high or low fat diet and 4-week following fenofibrate feeding trial were conducted to investigate if high fat diet and the body lipid background will affect the expression of PPARα mRNA and the hypolipidemia effects of fenofibrate in Nile tilapia. Lastly, an 8-weeks trial was conducted to evaluate whether the hypolipidemic effect of PPARα activation could benefit weight gain.The main results of this study are presented as follows:1. PPARα clone and tissue specific expression in Nile tilapia (Oreochromis niloticus)Although the key regulatory functions of mammalian PPARα in metabolism have been thoroughly studied, the molecular mechanisms and the metabolic regulation of PPARα activation in fish are less known. In the present study of Nile tilapia. NtPPARα was firstly cloned, identified, and examined its expression pattern at 11 tissues. The full-length cDNA of PPARα in Nile tilapia was cloned by RACE (Rapid Amplification of cDNA Ends), and the protein structures, physical, chemical properties and their functional domain structures which encoded by PPARα gene were analyzed by bioinformatics method. The results showed that cDNA sequences of PPAR a in T. ovatus were 1686 bp, and the open reading frame was 1428 bp, a 5’-untranslated region (UTR) of 167bp, and a 3’-UTR of 91 bp, encoding 475 amino acids (GenBank accession No. AHI50297). The protein was a nuclear protein without signal peptide and trans-membrane. The 3D-structure of PPAR a contains 16 alpha helixes,3 beta turns, and extended strands, random coils. There were some typical structures in PPAR a, including a DNA-binding domain and a ligand binding domain which can integrate ligands and activate PPARa.That the sequences of Nile tilapia PPAR a were moderate homologous with those cloned from human, mouse, zebrafish and African Clawed Frog (63.7%-67.6%). It should be noted that the LBD of NtPPARa had 18 excess AA residues (from 252 to 269) compared to the homologs in human and mouse, and this phenomenon was also observed in other fishes (grass carp, rainbow trout and zebra fish), which only had 6,2,2 excess AA residues, respectively. As LBD is the ligand binding domain, which might result in functional divergence in PPARa activation. Phylogenetic tree analysis of PPAR a protein showed that the nearest relationship existed between Nile tilapia and gilthead seabream among all the species mentioned above, and the lowest homology was human. Using quantitative real-time RT-PCR, we observed that PPAR a mRNA was expressed highest in brain followed by heart and liver, and the lowest in spleen. These results were in accordance with the status in mammalians that PPARa is expressed in tissues with high fatty acid catabolism.2. The mechanisms and metabolic regulation of PPARa activation in Nile tilapia (Oreochromis niloticus)In order to clarify the NtPPARa activation and its activated mechanism in vivo and in vitro. Nile tilapia was performing with agonist (fenofibrate) or fasting for four weeks, and the activation mechanism of NtPPARa. was verified in both primary hepatocytes and living fish primarily by the decreased phosphorylation of NtPPARa. or the supplemental increased expression of NtPPARa mRNA and protein. A 4-week fenofibrate feeding or fasting trial was then performed to investigate the metabolic regulation effects of NtPPARa, respectively. A transcriptomic study was also followed. The results indicated that fenofibrate treatment mainly decreased the content of triglyceride and 18C-series fatty acids in liver, and increased the levels of catabolism of 1-14C palmitate in vivo, hepatic mitochondrial (3-oxidation efficiency and cytochrome b DNA quantity, and carnitine palmitoyltransferase-1a mRNA expression. Fenofibrate treatment also increased serum concentrations of glucose, insulin and lactate. Compared to fenofibrate, fasting had stronger hypolipidaemic and gene regulatory effects. Taken together, we concluded that in tilapia 1) the metabolic regulation of specific NtPPARa activation mainly targets to liver and is relatively moderate; 2) dephosphorylation is the basal NtPPARa activation mechanism rather than the enhanced mRNA and protein expression; 3) the activated NtPPARa plays hypolipidaemic effects mainly through increasing activities and number of hepatic mitochondrial; 4) PPARa activation would also affect carbohydrate metabolism through affecting the energy homeostasis balanced among nutrients.3. Diet or lipid deposition background affects hypolipidemia effects of fenofibrate in Nile tilapia (Oreochromis niloticus)Although the effect of high fat diet (HFD) on PPARa activation and lipid deposition on hypolipidemia effect of fenofibrate have been thoroughly studied in mammalians, the effect of which in fish are less known. In the present study, Nile tilapia was firstly assigned with HFD or low fat diet (LFD) for ten-week to examine the effect of HFD group on PPARa activation. Secondly,24 fish from first feeding trial was selected for a 4-week fenofibrate feeding trial to investigate if the lipid intake background will affect hypolipidemia effects of fenofibrate in Nile tilapia. The results indicated that high fat diet, when compared with LFD, couldn’t induce the mRNA expression of PPARa, and FABP4 was largely downregulated in all detected tissues except adipose tissue, while inflammation related gene TGFβ1 presented extremely high expression in liver and genes related lipolysis(ATGL and HSL) and regulation factor of adipogenesis (SREBP1c) were highly expressed in the adipose tissue; Fenofibrate supplementation significantly increased the expression of PPARa mRNA and protein in HFD group and resulted in significantly decreased the hepatic triglyceride and TGF(31 express, but increased the levels of catabolism of 1-14C palmitate and hepatic mitochondrial enzyme activity at each group. Almost no effect on muscle and adipose tissue. Fenofibrate treatment also decreased serum concentrations of triglyceride, total cholesterol but increased the concentrations of high density lipoprotein (HDL). Taken together, we concluded that in tilapia 1) PPARa activation couldn’t be induced by HFD, while HFD intake mainly targets to adipose tissue result in increased its lipid metabolism; 2) HFD may result in inflammation and lipidperoxidation to some extent; 3) the activated NtPPARa plays hypolipidaemic effects mainly depend on diet or body lipid background rather than lipid deposition of tissue per se; 4) Liver is the target tissue response to Fenofibrate supplement; 5) PPARa activation could lower the hepatic lipid effectively and would also alleviate inflammation through affecting the expression of cytokine TGFβ1.4. The effect of different DP/DE ratio affects hypolipidemia and protein-sparing effects of fenofibrate in Nile tilapia (Oreochromis niloticus)An 8-weeks trial was conducted to evaluate whether the hypolipidemic effect of PPARa activation could benefit weight gain. In the present study, four diet made from two protein (fishmeal, soymeal, casein, gelatin) level,28%,38%, and two fat (soy oil, fish oil) level,7%,14%, are named 28P7L,28P14L,38P7L and 38P14L. respectively. And each group of feed was supplemented with Fenofibrate (Fenofibrate groups) or not (control groups).480 Nile tilapia (initial weight,8.03±0.03g) were randomly divided into 24 tanks, and assigned with eight group of different DP/DE ratio diet supplemented with fenofibrate or not. Afterward, the body parameters, blood parameters and QPCR indices were investigate at the end of this trial. The results indicated that the moisture and protein content of each fenofibrate group exhibit no significant difference when compared to that of each control group; the weight gain rate and protein efficiency from 28% protein fenofibrate groups were significantly decreased, while the hepatosomatic index and food coefficient were significantly increased; And fenofibrate significantly decreased the plasma LDL from all groups, HDL from high protein groups, total amino acid and ammonia from 28P7L, while whose TG and MDA were significantly increased. Fenofibrate also upregulated and downregulated the genes related lipid catabolism anabolism (CPT1a, FAS, ALCY, GPAT) and (ACO, DGAT2), downregulated the expression of genes related lipoprotein (LDLR, MTP, ApoB). In short, the weight gain rate from 28% protein fenofibrate groups were evidently decreased when compared to that of control groups; no fenofibrate group has a significantly benefit for growth, except the 38P7L diet group appear to has an increasing trend; Taken together, we concluded that in this trial: 1) hypolipidemic effect of PPARa activation could only occure at high protein level of diet in Nile tilapia; 2) PPARa activation plays a role of hypolipidaemic effect and has a trend to benefit weight gain and protein-sparing effect at 38P7L diet, rather than causing an impair effect on hepatic lipid metabolism and lipid peroxidation at 28P7L. In conclusion, the hypolipidemic effect of PPARa activation in Nile tilapia was closely related to diet protein level.