Molecular Control of Fish Lipid Metabolism:

Isolation and Characterisation of Peroxisome Proliferator-Activated Receptor (PPAR) Genes from Fish Species

FISH OIL USE

Actual

Projected

Annual production stable at 1.1 to 1.4 million tons

?!

Fish Oil Replacement

• Fat Deposition?• Nutritional Quality?• Disease Resistance?

• Need a better understanding of underlying physiology

Peroxisome proliferator-activated receptors

• PPARs– Transcription factors– Control genes involved in lipid homeostasis– Activated by PUFA and their eicosanoid

derivatives

PPAR RXR

PUFA 9-cisRA

GENE TRANSCRIPTON

LIGAND BINDING

DNA BINDINGCHROMOSOME

A/BA/Bhinge

LIGAND BINDING

AAGTCAnAAGTCA

PPRE

•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

BLOOD

FA

FA

FA

FA

FA

FA

Bile acids

GUT

LIVER

ADIPOSEHEART

INNATEIMMUNE

OTHERTISSUES

LDL

HDL

PPAR

PPAR PPAR

PPAR

PPAR FXR

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

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

Plaice as a model

• Marine species– Highly dependent on fish oil

• Small genome- small genes – Facilitates gene isolation from lambda phage

libraries• Also salmon, sea bream and sea bass

Genomic DNA

Partial digest

bacteriophage arms

ligatePackage, plate on lawn of E. coli and

screen with hybridisation probe

Isolate and sequence geneRT-PCR

Isolate and sequence cDNA

Stategy for PPAR Gene and cDNA Isolation

+

Plaice PPAR Gene Structures

Human PPAR genes are >80kb

* *

* *

1kb

pPPARa

pPPARb

pPPARg

7kb

4.5kb

10kb

saPPAR

PPAR

ppPPAR

81

ssPPAR

100

PPAR

hsPPAR

xPPAR

100

100

89

xPPAR

PPAR

hsPPAR

sPPAR

PPAR

ppPPAR

99

ssPPAR1

100

100

95

99

sPPAR

PPPAR

ppPPAR

99

ssPPAR

100

PPAR

hsPPAR

xPPAR

97

99

100

99

100

100

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.

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.

A/B C D E/F

Ligand-independent transactivation (phosphorylation?)

DNA-binding,Dimerisation,Co-activator-binding

Ligand-binding,Co-activator-binding

20% 90% 70%

PPAR structure and function

PPAR RXR

E/FE/F

CCA/B A/B

DNA PROMOTER

EMSA

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

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 genePPRE

Measure CAT(Muliwell ELISA)

PPAR Transactivation Assays

CAT

PPAR

PPAR RXR

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

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

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– No good natural Fatty Acid Ligands

• Diet- 16:1 + 18:3 + CLA????