Lipid Metabolism Regulation And Transcriptional Activity Of PPARγ In Nile Tilapia

With the advantages of good growth performance and high stress resistance, Nile tilapia (Oreochromis niloticw) has become a worldwide fanned fish species, as an important model species whose whole genome information has been released, making it more convenient to be studied in terms of fish nutritional physiology. PPARy (Peroxisome proliferator activated receptor), widely exists in veterbrates, was a master regulator in animal lipid metabolism. However, the physiological role, molecular structure, and transcriptional activity of PPARy in fish were still unknown. In this study, in order to explore the physiological role of PPARy in Nile tilapia, molecular cloning, cell culture, quantitative real time PCR, western blotting, transcriptomic analysis, double luciferase reporter system, promoter trapping, and other approaches have been used in the following four studies:1) The role of PPARy in the regulation of lipid homostasis,2) Molecular characterization and transcriptional activity of PPARy,3) Effects of agonist rosiglitazone and antagonist GW9662 of mammalian PPARy on the lipid metabolism of Nile tilapia, and 4) Molecular characterization and nutritional regulation of ACOX1-the target gene of PPARy.1. The role of PPARy in the regulation of lipid homostasisNatural selection endows animals with the abilities to store lipids when food was abundant and to synthesize lipids when it was limited. However, the relevant adaptive strategy of lipid metabolism has not been clearly elucidated in fish. Therefore, the present study examined the systemic metabolic strategies of Nile tilapia and the role of PPARy in maintaining lipid homeostasis when fed with low or high fat diets. Three diets with different lipid contents (1%,7% and 13%) were formulated and fed to tilapias for 10 weeks continuously. At the end of the feeding trial, the growth rate, hepatic somatic index, and the triglyceride contents of serum, liver, muscle and adipose tissue were comparable among three groups, while the total body lipid contents and the mass of adipose tissue increased with the increased dietary lipid levels. Overall quantitative PCR, western blotting and transcriptomic assays indicated that the liver was the primary responding organ to low-fat diet feeding, and the elevated glycolysis and accelerated biosynthesis of fatty acids in the liver was likely to be the main strategies of tilapia towards low fat intake. By contrast, excess ingested lipids were preferentially stored in adipose tissue through increasing the capability of fatty acid uptake, fatty acid synthesis and triglyceride synthesis. Increasing the numbers of adipocytes mediated by PPARγ, but not enlarging size, may be the main strategy of Nile tilapia in response to continuous high-fat diet feeding. This was the first time that we illuminated the systemic adaptation of lipid metabolism responding to low fat or high fat diet in fish, and our results shed new light on fish physiology.2. Molecular characterization and transcriptional activity of PPARγ in Nile tilapiaPPARγ, which widely exists in vertebrates, was the key regulator in lipid metabolism. However, the molecular structure and transcriptional activity of PPARγ in fish were still unclear. Moreover, the results of the study above already showed that Nile tilapia PPARγ (NtPPARγ) participated in the regulation of adipocytes proliferation. So it’s necessary to further investigate the molecular structure and transcriptional activity of NtPPARγ. In this study, PPARγ was cloned from Nile tilapia. NtPPARγ along with retinoid X-receptor α (NtRXRα) plasm id were transfected into HEK-293 cells to explore the mechanism of NtPPARγ in the transcriptional regulation in fish. Two transcripts of NtPPARy varied at the 5’-untranslated region and the DNA binding domain (DBD) was highlγ conserved. Thirty-nine extra amino acid residues in the ligand binding domain (LBD) in Nile tilapia were different from those in human, and this region made NtPPARγ has three more alpha-helix in LBD. What’s more, the two transcripts both were highly expressed in liver, intestine and kidney, even though they showed different expression profiles in 11 tissues. The transcriptional activity assay showed that NtPPARy collaborates with NtRXRa to regulate the expression of Nile tilapia fatty acid binding protein 4 (FABP4). the compartment of which have been identified as the target gene of PPARy in human. In conclusion, the DBD in PPARy was highly conserved, while the LBD was moderately conserved in vetebrates. In Nile tilapia, the PPARy collaborates with RXRa to perform transcriptional regulation of FABP4 at least in vitro. The plasmid system established in this study along with a cell line from Nile tilapia will be useful tools for the further functional study of PPARy in fish.3. Effects of agonist rosiglitazone and antagonist GW9662 of mammalian PPARy on the lipid metabolism of Nile tilapiaAgonist and antagonist treatment of the specific gene were something like obtaining gain of function and loss of function study, two efficient strategies for the study of gene function, so mammalian PPARy agonist rosiglitazone (Rosi) and antagonist GW9662 (GW9) adminstration were conducted to see whether they function in Nile tilapia as that in mammals. We can further explore the physiological role of NtPPARy based on efficient activation and inactivation. Two lipid level (standard diets 4%, SD and high fat diets 15%, HFD) diets were formulated with three different additives (control, Rosi (15 mg/kg), and GW9 (10 mg/kg), respectively). At the end of 6-week feeding, visceral somatic index (VSI) in HFD was significantly higher than that in SD. Hepatic somatic index (HSI) and hepatic triglyceride (TG) were also higher in HFD, indicative of excess lipid deposition in liver. TG content in serum of HFD was significantly higher than that of SD, suggestive of the disturbance of lipid homostasis. But there was no any difference among control, Rosi and GW9. regardless of SD or HFD. So the more efficient way of drug diliver-intraperitoneal (IP) injection was applied. Four different doses were used for Rosi (0,0.6,18.0 and 54 mg/kg) and GW9 (0,0.4,12.0 and 36 mg/kg). The samples were analysized after IP injection done for three consecutive days. The results showed that there was no difference among control and GW9 in terms of TG, FFA and glucose contents in serum. Rosiglitazone treatment didn’t affect the glucose level in serum, but decreased the TG level and increased FFA content in serum. The results of quantitative PCR indicated that the decrease of TG content were contributed to the lower expression of FA synthesis, TG synthesis and VLDL apoprotein genes after rosiglitazone administration. The activation of lipolysis in muscle was the contributor of FFA increase in serum. However, rosiglitazone IP injection didn’t affect the mRNA expression of PPARy in fat, liver and muscle. In the future, it needs to be studied whether rosiglitazone only affect the PPARy activity via post-transcriptional modification, which further drive some physiological effect. In summary, this study showed that short-term rosiglitazone administration via IP injection can activate lipolysis in muscle of Nile tilapia.4. Molecular characterization and nutritional regulation of ACOX1-one target gene of PPARyPlenty of lipid messengers were produced in peroxisome, the proliferation of which was regulated by PPARy. Peroxisomal beta-oxidation, in which acyl-coenzyme A oxidase 1 (ACOX1) was the first rate-limiting enzyme, provided substrates for the production of these lipid messengers. The explorement of the structure and function of ACOX1 gene was important for the study of PPARy regulation of bioactive lipids production in peroxisome. So two isoforms of acyl-coenzyme A oxidase 1 were firstly identified in Nile tilapia (O. niloticus) in this study. And the results showed that ACOX1 isoforml (ACOXlil) and ACOX1 isoform2 (ACOXli2) were encoded by the single gene with 661 amino acids in length. The coding region of both isoforms consisted of 14 exons. The residues from 89 to 193 in ACOXlil were encoded by exon 3b, while in ACOXli2 they were encoded by exon 3a. Homologous alignment analysis indicated that the varied region (the residues from 89 to 193) of ACOXlil was more conserved than ACOXli2 in vertebrates (Mammalia, Aves, Amphibia and Pisces). The mRNA expression level of ACOXlil and ACOXli2 was detected separately in eleven tissues and the results indicated that ACOXi 1 expression was the highest in liver followed by kidney and brain, while the expression of ACOXi2 was the highest in kidney followed by liver. The normalized levels of both transcript variants were comparable in most tissues, however the level of ACOXI i2 was significantly higher than that of ACOXlil in white muscle and kidney (5.1 fold and 3.1 fold), and ACOXlil was significantly higher than ACOXli2 in gill and brain (4.8 fold and 1.9 fold). In different nutritional states, the expression levels of both isoforms in liver were comparable between fasting and most of post-feeding time points, except that the expression at 3 hours post-feeding was significantly lower than others. The expression of ACOXlil in the kidney also showed the similar pattern, indicating the lowest expression at 8 hours post-feeding, however, no significant change was seen in ACOX2i2 among all nutritional states. These results suggested that ACOXlil and i2 may play different roles in tissues, and their expression levels were differently modulated by nutritional stage.