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