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fPPARs Cloning and functional analysis of fish Peroxisome Proliferator-Activated Receptors

fPPARs Cloning and functional analysis of fish Peroxisome Proliferator-Activated Receptors The transcriptional control of lipid metabolism in farmed fish species. fPPAR group Grigorios Krey, Efthimia Antonopoulou, Evridiki Boukouvala

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fPPARs Cloning and functional analysis of fish Peroxisome Proliferator-Activated Receptors

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  1. fPPARs Cloning and functional analysis of fish Peroxisome Proliferator-Activated Receptors The transcriptional control of lipid metabolism in farmed fish species

  2. fPPAR group Grigorios Krey, Efthimia Antonopoulou, Evridiki Boukouvala National Agricultural Research Foundation, Fisheries Research Institute, Kavala, Greece Michael J. Leaver, Douglas R. Tocher, Tariq Ezaz Institute of Aquaculture, University of Stirling, Stirling, UK Jose M. Bautista, Amalia Diez Martin Mol Biol and Biochem IV, Veterinary Faculty, Universidad Complutense de Madrid, Spain Alex Obach Nutreco Aquaculture Research Centre, Stavanger, Norway. Guillermo Bores Cripesa, Port Pesquer, Tarragona, Spain

  3. FISH OIL USE Annual production stable at 1.1 to 1.4 million tons Actual Projected ?!

  4. Fish Oil Replacement • Fat Deposition? • Nutritional Quality? • Disease Resistance? • Need a better understanding of underlying physiology

  5. Peroxisome proliferator-activated receptors • PPARs • Transcription factors • Control genes involved in lipid homeostasis • Activated by PUFA and their eicosanoid derivatives

  6. PPARs Coactivator proteins • PPARs are members of nuclear hormone receptor family • PPARs bind as heterodimer with RXR to PPRE • PPARs are activated by fatty acid (PUFA) ligands • Three forms in mammals, a, b/d and g

  7. INNATE IMMUNE PPARg HEART ADIPOSE PPARa PPARg FA FA FA LDL BLOOD OTHER TISSUES PPARa FA HDL LIVER FA PPARb FXR FA Bile acids GUT

  8. PPARs and Lipid Homeostasis • Transport • Apolipoprotien AI, AII, CIII, Liver fatty acid binding protein; Fatty acid transport protein; CD36 • Biosynthesis • Acetyl-CoA synthase; Malic enzyme; Stearoyl-CoA desaturase I • Storage • Adipocyte lipid binding protein; Phosphoenolpyruvate carboxylase • Metabolism • Acyl-CoA oxidase; Bifunctional enzyme; Carnitine palmitoyltransferase; CYP4A1, 4A6; Lipoprotein lipase; Medium chain Acyl-CoAdehydrogenase, 3-hydroxy, 3-methylglutaryl-CoA synthase; Uncoupling protein I

  9. Strategy • Do fish have PPARs? • Construct and screen genomic libraries • What are their ligand activation profiles? • Express fish PPAR genes in cell culture • Diet formulation • Use results to produce a rational framework for fish oil replacement

  10. Species Atlantic salmon (Salmo salar) Plaice (Pleuronectes platessa) Sea bass (Dicentrarchus labrax) Sea bream (Sparus aurata)

  11. Stategies for PPAR Gene and cDNA Isolation Partial digest ligate + Package, plate on lawn of E. coli and screen with hybridisation probe Genomic DNA l bacteriophage arms PCR of conserved regions Isolate and sequence gene RT-PCR Plaice and Salmon Isolate and sequence cDNAs Sea bass and Sea bream

  12. Plaice PPAR Gene Structures • Human PPAR genes are >80kb

  13. Phylogenetic plot of PPAR sequences. xl. Xenopus laevis; hs, Homo sapiens; gg, Gallus gallus; ss, Salmo salar; pp, Pleuronectes platessa; dl, Dicentrarchus labrax; sa, Sparus aurata.

  14. Southern Blot. SstI restricted plaice DNA was hybridised to the probes generated from the first coding exons of the three plaice PPAR genes, or the DNA-binding region. Sizes of fragments correspond to those predicted from the gene sequences.

  15. PPAR structure and function DNA-binding, Dimerisation, Co-activator-binding Ligand-independent transactivation (phosphorylation?) Ligand-binding, Co-activator-binding A/B C D E/F 20% 90% 70% PPAR RXR E/F E/F A/B A/B C C DNA PROMOTER

  16. PP SA SS DL PP SA SS DL EMSA Species PPAR a b g a b g b g g a b g a b g a b g C C DNA-binding domains PPAR RXR PPRE PPRE Probe ACO-A GSTA.2

  17. CARLA: Sea Bream PPARa LBD 01 2 3 4 5 6 7 8 9 10 11 12 13 A 35S PAGE Autorad SRC1 Complex Formation Association values (no ligand=1) Ligand 1 2 3 4 5 6 7 8 9 10 11 12 13 35S Ligands SRC1 PPAR-LBD • 1. No ligand, 2. Phytanic acid, 3. DHA, 4. Arachidonic acid, 5. EPA 6. ETYA, 7. Linolenic acid, 8. Linoleic acid, 9. CLA (10E, 12Z), 10. CLA (9Z, 11E), 11. CLA (9E, 11E), 12. CLA mix, 13. 8(S)-HETE, A. SRC1, 0. GST-CLA mix Co-activator protein

  18. PPAR Transactivation Assays Co-transfect to cells in culture (Multiwell plates) Ligate constitutive gene promoter to PPARgene PPAR cDNA CMV PPAR cDNA CMV PPRE CAT gene PPAR Ligate a PPAR response element (PPRE) to CAT reporter gene PPRE CAT gene RXR PPAR CAT gene PPRE Treat cells with potential PPAR activators CAT Measure CAT (Muliwell ELISA)

  19. Plaice PPAR Tissue Expression Profile Lane 1, liver; 2, kidney; 3, small intestine; 4, gill; 5, heart; 6, spleen; 7, white muscle; 8, red muscle; 9, brain; 10, visceral adipose

  20. 1 2 3 4 5 6 7 8 9 10 PPARg PPARb PPARa Sea Bream PPAR Tissue Expression Profile RNase protection 1. Liver, 2. Kidney, 3. Intestine, 4. Gill, 5. Heart, 6. Spleen, 7. White muscle, 8. Red muscle, 9. Brain, 10. Adipose

  21. Tissue expression profile of PPARs in sea bream Immunochemical analysis Anti PPAR a Anti PPAR g

  22. Next Steps • PPAR activators in primary hepatocytes and adipocytes • Determine fatty acid profiles and metabolic indices • Gene expression profiling • Dietary trial with salmon and sea bream • Measure growth, gene expression, fatty acid profiles

  23. Dietary Trial • PPARa - Liver and Heart- Fatty acid oxidation- • Conjugated linolenic acid (CLA), 16:1, 18:1 ??? • PPARb - All tissues- Function? • 16:1 • PPARg - Adipose - Fat Sorage • ??? • Diet- 16:1 (+ 18:3+18-2) + CLA

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