Transcript No Slide Title

Molecular Control of Fish
Lipid Metabolism:
Isolation and Characterisation of
Peroxisome Proliferator-Activated
Receptor (PPAR) Genes from Fish
Species
PHARM
0%
INDUS
8%
FISH OIL USE
2000
1990
Annual production stable at
1.1 to 1.4 million tons
PHARM
INDUS
2%
10%
AQUA
16%
Actual
AQUA
54%
AGRI
34%
AGRI
76%
2005
PHARM
2%
2010
INDUS
0%
INDUS
12%
PHARM
2%
AGRI
1%
AGRI
9%
Projected
?!
AQUA
77%
AQUA
97%
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
PPARs
9-cisRA
PUFA
PPAR
RXR
LIGAND
BINDING
LIGAND
BINDING
hinge
A/B
A/B
DNA BINDING
AAGTCAnAAGTCA
GENE TRANSCRIPTON
CHROMOSOM E
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, a, b/d and g
INNATE
IMMUNE
PPARg
HEART
ADIPOSE
PPARa
PPARg
FA
FA
FA
LDL
BLOOD
OTHER
TISSUES
PPARb
PPARa
FA
HDL
LIVER
FA
FXR
FA
GUT
Bile acids
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, 3methylglutaryl-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
Stategy for PPAR Gene and cDNA Isolation
Partial digest
Genomic DNA
ligate
+
l bacteriophage arms
RT-PCR
Isolate and sequence
cDNA
Isolate and
sequence gene
Package, plate on
lawn of E. coli and
screen with
hybridisation probe
Plaice PPAR Gene
Structures
1 kb
7kb
pPPARa
*
*
4.5kb
pPPARb
pPPARg
10kb
**
Human PPAR genes are >80kb
xlPPAR a
100
hsPPAR a
100
ggPPAR a
97
ssPPARa
ppPPAR a
99
dlPPPAR a
100
saPPAR a
99
ssPPARb1
95
99
ppPPAR b
dlPPAR b
100
99
saPPAR b
hsPPAR b
100
99
ggPPAR b
100
xlPPAR b
xlPPAR g
89
hsPPAR g
ggPPAR g
100
Phylogenetic plot of
PPAR sequences.
ssPPARg
ppPPAR g
100
dlPPAR g
100
81
saPPAR g
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.
PPAR structure and function
Ligand-independent
transactivation
(phosphorylation?)
DNA-binding,
Dimerisation,
Co-activatorbinding
A/B
C
20%
90%
Ligand-binding,
Co-activator-binding
D
E/F
70%
PPAR
RXR
E/F
E/F
A/B C
C A/B
DNA PROMOTER
EMSA
Performed with in vitro
translated fish PPARs
and plaice RXR and
mouse ACO gene
promoter oligo
PPAR Transactivation Assays
Co-transfect to cells in culture
(Multiwell plates)
Ligate constitutive gene
promoter to PPARgene
CMV
PPRE
PPAR cDNA
CMV
CAT gene
PPAR
Ligate a PPAR response
element (PPRE) to CAT
reporter gene
PPAR
PPAR cDNA
PPRE
RXR
CAT gene
PPRE
CAT
Measure CAT
(Muliwell ELISA)
CAT gene
Treat cells with potential PPAR
activators
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
• 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????