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 Actual Projected Annual production stable at 1.1 to 1.4 million tons ?!

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

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

7. 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, ,  and  PPARs Coactivator proteins

8. BLOOD FA Bile acids GUT LIVER ADIPOSE HEART INNATE IMMUNE OTHER TISSUES LDL HDL PPAR  PPAR  PPAR  FXR

9. 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-CoA dehydrogenase, 3-hydroxy, 3- methylglutaryl-CoA synthase; Uncoupling protein I

10. 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

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

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

13. Plaice PPAR Gene Structures Human PPAR genes are >80kb

14. 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.

15. 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.

16. A/BCDE/F Ligand-independent transactivation (phosphorylation?) DNA-binding, Dimerisation, Co-activator- binding Ligand-binding, Co-activator-binding 20%90%70% PPAR structure and function PPARRXR E/F CC A/B DNA PROMOTER

17. PPSASSDL PP SA SSDL ACO-AGSTA.2 CC Species PPAR Probe  PPAR RXR PPRE DNA-binding domains PPRE EMSA

18. 1 2 3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13 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 CARLA: Sea Bream PPAR  LBD A Ligands Association values (no ligand=1) 35 S PPAR-LBD Co-activator protein SRC1 Ligand Complex Formation PAGE Autorad

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

21. 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

22. 1 2 3 4 5 6 7 8 9 10 PPAR  PPAR  PPAR  1. Liver, 2. Kidney, 3. Intestine, 4. Gill, 5. Heart, 6. Spleen, 7. White muscle, 8. Red muscle, 9. Brain, 10. Adipose Sea Bream PPAR Tissue Expression Profile RNase protection

23. Immunochemical analysis Anti PPAR  Anti PPAR  Tissue expression profile of PPARs in sea bream

24. 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

25. Dietary Trial PPAR  - Liver and Heart- Fatty acid oxidation- – Conjugated linolenic acid (CLA), 16:1, 18:1 ??? PPAR  - All tissues- Function? – 16:1 PPAR  - Adipose - Fat Sorage – ??? Diet- 16:1 (+ 18:3+18-2) + CLA