CORDIS provides links to public deliverables and publications of HORIZON projects.
Links to deliverables and publications from FP7 projects, as well as links to some specific result types such as dataset and software, are dynamically retrieved from OpenAIRE .
Deliverables
Genes and corresponding cDNAs encoding three distinct PPAR isotypes have been identified in plaice (Pleuronectes platessa). In Atlantic salmon (Salmo salar) an second PPAR beta isoform has been identified in addition to the three other isotypes. Of the three fish isotypes the deduced protein of PPAR beta exhibits the highest homology with its mammalian counterpart in both the DNA and ligand binding domains (DBD and LBD, respectively). Plaice PPAR alpha and gamma are also highly homologous to their mammalian and salmon counterparts in the DBD. However, both plaice isotypes have an extended LBD, as compared either to mammalian or salmon PPARs.
Furthermore, in both palice and salmon PPAR gamma two important amino acid substitutions have been observed in relation to their mammalian homologue, both involving residues implicate in ligand binding in the human receptor. The above indicate the ligand binding properties of the fish PPAR gamma are potentially different from those of its mammalian homologue.
The genomic organization of the fish PPARs is remarkably different from that observed in mammalian genes in that the LBD in the plaice alpha and beta and salmon beta receptors is encoded by three exons; the LBD of plaice PPAR gamma is encoded by four exons; while those of salmon PPAR alpha and gamma are encoded by only two exons as is also the case for all mammalian PPARs.
This result represents the first demonstration of the existence of three PPAR isotypes in fish and suggest that the structure and function of these receptors has evolved before the divergence of the osteichtyan and amphibian/mammalian lineages. Therefore, the study of these receptors in fish can result in significant knowledge concerning fatty acid and lipid metabolism in lower vertebrates.
An antibody specific for the marine fish PPAR gamma has been developed through the use of synthetic peptides corresponding to a highly conserved region within the A/B domain of the marine fish receptors This antibody has been tested with various success on bacterially expressed receptor, on in vitro translated receptor, and on fish tissue protein extracts.
Improving the efficiency of this antibody could result in an important tool for the study of marine fish PPARs, through immunocytochemical, imunohistological, and ELISA applications. Improved efficiency could result in the commercial exploitation of this antibody. Furthermore, the development process (peptide sequence etc.) has the potential to be patented.
Genes and corresponding cDNAs encoding three distinct PPAR isotypes have been identified in sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). Of the three fish isotypes the deduced protein of PPAR beta exhibits the highest homology with its mammalian counterpart in both the DNA and ligand binding domains (DBD and LBD, respectively). Fish PPAR alpha and gamma are also highly homologous to their mammalian counterparts in the DBD. However, both isotypes have an extended LBD, as compared to mammalian PPARs.
Furthermore, in the fish PPAR gamma two important amino acid substitutions have been observed in relation to their mammalian homologue, both involving residues implicate in ligand binding in the human receptor. The above indicate the ligand binding properties of the fish PPAR gamma are potentially different from those of its mammalian homologue.
The genomic organization of the fish PPARs is remarkably different from that observed in mammalian genes in that the LBD in the fish receptors alpha and beta is encoded by three exons and that of gamma by four exons, as compared to only two exons encoding the LBD of either isotype in mammals.
This result represents the first demonstration of the existence of three PPAR isotypes in fish and suggest that the structure and function of these receptors has evolved before the divergence of the osteichtyan and amphibian/mammalian lineages. Therefore, the study of these receptors in fish can result in significant knowledge concerning fatty acid and lipid metabolism in lower vertebrates.
Protocols have been developed for the primary cultures of hepatocytes, adipocytes and enterocytes from Atlantic salmon and sea bream. These cells have been tested for the induction of PPAR expression and the expression of PPAR-target genes, as well as for global changes in fatty acid and lipd metabolism in response to treatment with PPAR ligands.
The above developed systems have the potential to be used for the study of a variety of other processes in addition to the PPAR-regulated lipid metabolism.
Quantitative real-time PCR (Q-PCR) protocols have been developed and have been successfully used to determine PPAR isotype expression in tissues of sea bream as well as in primary hepatocytes and adipocytes from the same species. The method is based on the amplification of a short region of the cDNA of each of the three sea bream PPAR isotypes using specific primers and specific Taq-man fluorescent probes. As internal standard (reference gene) the sea brean alpha-tubulin has been used.
The protocol results in great sensitivity and accuracy in determining PPAR expression and presents the potential of numerous applications in various aspects of PPAR biology in sea bream. With appropriate adaptations (species-specific primers), this protocol can be used in the determination of PPAR expression in virtually any fish species.
Five experimental feeds for sea bream and Atlantic salmon have been developed and tested on fish growing in an aquaculture set-up. Three of the diets were based on vegetable oils (rapeseed, linseed, soybean) chosen because the major fatty acid constituents in these oils were found to be potent fish PPAR activators. In the remaining two diets fish oil was supplemented with either 2 or 4% CLA.
This result demonstrates the effects of the dietary lipid composition on the growth of fish and on the lipid content and lipid tissue composition of the fish. Furthermore, it establishes CLA as a fat lowering nutrient in fish tissues. Thus, CLA can be used as a lead compound for the further development of commercially viable nutritional supplements with effects similar to those of CLA.
The DNA binding properties of the PPAR isotypes from plaice, sea bream, Atlantic salmon, and sea bass have been studied by the electrophoretic mobility shift assay (EMSA) using in vitro translated PPAR proteins and a variety of PPAR response elements of both mammalian and piscine origin. This result demonstrates that fish PPARs, like their mammalian homologues heterodimerize with RXR and recognize response elements of the DR-1 type.
The ligand binding properties of the fish PPARs were studied through the coactivator-dependent receptor ligand assay (CARLA) and it was demonstrated that fatty acids, eicosanoids, and synthetic hypolipidemic drugs can induce the interactions of the ligand binding domain of the receptors with the mammalian steroid receptor coactivator-1(SRC-1).
The above, besides establishing the DNA and ligand binding properties of the fish PPARs also demonstrate that the mechanisms involved in PPAR transactivation have evolved before the divergence of the osteichthyan and mammalian lieages. Therefore, further optimization of the CARLA assay could result in the identification of novel ligands for the fish PPARs with potential use in commercial fish feeds.
An important finding is the identification of conjugated linoleic acid (CLA) as fish PPAR ligand that has been applied in experimental fish diets with promising results.
The transactivation properties of the plaice, sea bream, and Atlantic salmon PPARs have been studied in two fish-derived, established cell lines, i.e. the sea bass larval (SBL) and the Atlantic salmon (AS) cell lines.
This result demonstrates that the above cell lines can be successfully used for transfection purposes with PPARs and PPAR-regulated reporter constructs in order to identify specific fish PPAR activators.
A comprehensive analysis of the effects of fish diets based on vegetable oils and or supplemented with CLA on the expression of the PPAR isotypes and potential PPAR-target genes. This result demonstrates that the lipid composition of the fish diets can have significant effects on the expression of the PPAR genes and therefore to the global lipid and fatty acid metabolism in fish tissues.
By establishing measurable parameters at both the molecular and biochemical levels, it provides the necessary background for the future evaluation of novel dietary formulations in fish nutrition and "nutreomics".
Complete or partial cDNAs encoding fatty acid metabolising enzymes have been identified in both sea bream and Atlantic salmon. Specifically, from sea bream a complete cDNA for carnitine palmitoyltransferase 1 (CPT1) and a partial cDNA for glutathione S-transferase A (GSTA) have been isolated. From Atlantic salmon, a partial cDNA for CPT1 has been identified. These cDNAs along with those encoding a fatty acyl elongase and a fatty acyl desaturase (delta 5/6) from sea bream and Atlantic salmon and the GSTA from plaice, that have been previously described, constitute a significant number of potential PPAR-target genes and thus can be used as markers of PPAR-regulated fatty acid metabolism. Important is to note the identification of a partial cDNA encoding the acyl-CoA oxidase from sea bass.
Additional cDNAs that have been identified in the frame of this project, although unrelated to fatty acid metabolism, include the comple coding sequence of alpha-amylase from sea bream and sea bass, a partial cDNA for alpha-tubulin from sea bream, sea bass, and Atlantic salmon, a partial cDNA for beta-actin from sea bream and sea bass, and a partial cDNA for the Atlantic salmon glyceraldehyde phosphate dehydrogenase (GAPDH).
As most of these cDNAs have not been previously reported in the above species, they provide the opportunity for a more thorough examination of their regulation in response to nutritional stimuli or other factors that could affect their expression.
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