1. Molecular Control of Fish Lipid Metabolism:
Isolation and Characterisation of Peroxisome Proliferator-Activated Receptor (PPAR) Genes from Fish Species

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

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

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

6. PPARs 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 HEART ADIPOSE BLOOD OTHER TISSUES LIVER GUT PPARg PPARaFA 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-CoA dehydrogenase, 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. Plaice as a model Marine species Small genome- small genes
Highly dependent on fish oil
Small genome- small genes
Facilitates gene isolation from lambda phage libraries
Also salmon, sea bream and sea bass

11. + Stategy 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
Isolate and sequence gene
RT-PCR
Isolate and sequence cDNA

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 C C
A/B
DNA PROMOTER

16. EMSA
Performed with in vitro translated fish PPARs and plaice RXR and mouse ACO gene promoter oligo

17. PPAR Transactivation Assays
Ligate constitutive gene promoter to PPARgene
Co-transfect to cells in culture
(Multiwell plates)
CMV
PPAR cDNA
CMV
PPAR cDNA
PPRE
CAT gene
PPAR
Ligate a PPAR response element (PPRE) to CAT reporter gene
PPRE
CAT gene
PPAR
RXR
PPRE
CAT gene
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. 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

21. 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
No good natural Fatty Acid Ligands
Diet- 16:1 + 18:3 + CLA????