fPPARs

Cloning and functional analysis of fish Peroxisome Proliferator-Activated Receptors

The transcriptional control of lipid metabolism in farmed fish species

fPPAR groupGrigorios Krey, Efthimia Antonopoulou, Evridiki BoukouvalaNational Agricultural Research Foundation, Fisheries Research Institute, Kavala, Greece

Michael J. Leaver, Douglas R. Tocher, Tariq EzazInstitute of Aquaculture, University of Stirling, Stirling, UK

Jose M. Bautista, Amalia Diez MartinMol Biol and Biochem IV, Veterinary Faculty, Universidad Complutense de Madrid, Spain

Alex ObachNutreco Aquaculture Research Centre, Stavanger, Norway.

Guillermo BoresCripesa, Port Pesquer, Tarragona, Spain

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 BINDING

CHROMOSOME

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

Coactivator proteins

BLOOD

FA

FA

FA

FA

FA

FA

Bile acids

GUT

LIVER

ADIPOSEHEART

INNATEIMMUNE

OTHERTISSUES

LDL

HDL

PPAR

PPAR PPAR

PPAR

PPARFXR

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

Species

Plaice (Pleuronectes platessa) Atlantic salmon (Salmo salar)

Sea bream (Sparus aurata) Sea bass (Dicentrarchus labrax)

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 cDNAs

Stategies for PPAR Gene and cDNA Isolation

+

Plaice and SalmonSea bass and Sea bream

PCR of conserved regions

Plaice PPAR Gene Structures

Human PPAR genes are >80kb

* *

* *

1kb

pPPARa

pPPARb

pPPARg

7kb

4.5kb

10kb

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.

xlPPAR

ggPPAR

hsPPAR100

ssPPAR

saPPAR

dlPPAR

ppPPAR

85

99

89

100

xlPPAR

ggPPAR

hsPPAR

ssPPAR1

ssPPAR2

saPPAR

dlPPAR

ppPPAR

99

87

99

99

97

98

xlPPAR

ggPPAR

hsPPAR96

ssPPAR

saPPAR

dlPPPAR

ppPPAR

99

100

100

96

99

100

100

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

PP SA SS DL PP SA SS DL

ACO-A GSTA.2

C C

Species

PPAR

Probe

PPAR RXR

PPRE

DNA-binding domains

PPRE

EMSA

0,00

0,50

1,00

1,50

2,00

2,50

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 LBDA

Ligands

Ass

ocia

tion

val

ues

(no

liga

nd=

1)

35S

35S

PPAR-LBDCo-activator protein

SRC1

SRC1

Ligand

Complex Formation

PAGE

Autorad

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

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 ProfileRNase protection

Immunochemical analysis

Anti PPAR Anti PPAR

Tissue expression profile of PPARs in sea bream

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– ???

• Diet- 16:1 (+ 18:3+18-2) + CLA