Molecular Control of Fish Lipid Metabolism:

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Presentation transcript:

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 Actual Projected ?!

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 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 HEART ADIPOSE BLOOD OTHER TISSUES LIVER GUT PPARg PPARa FA FA FA LDL BLOOD OTHER TISSUES PPARa FA HDL LIVER FA PPARb FXR FA Bile acids GUT

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 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

+ 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

Plaice PPAR Gene Structures Human PPAR genes are >80kb

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.

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

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

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)

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????