Molecular mechanisms

In addition to the more applied feed development research, the Nutrition group also investigates the basic mechanisms underpinning this work through biochemical and molecular studies aimed at identifying and characterising the factors controlling and regulating lipid and PUFA metabolism in fish. One major aim of this fundamental research is to provide the basic information, methodologies and tools necessary to develop novel approaches to the design of new feeds and to the selection of strains of fish with improved performance when fed novel feeds. This approach is based on the identification and quantification of key traits at a genotypic level that are important in determining important phenotypic responses to nutritional change.

The initial aim of the long-established LC-PUFA (LC-PUFA) biosynthesis programme was to clone, characterize and determine the genes for enzymes of paramount importance in the conversion of 18:3n-3, abundant in some vegetable oils, to the critical essential omega-3 LC-PUFA, EPA and DHA. A range of cDNAs encoding both fatty acyl desaturase (Fad) and elongase (Elovl) enzymes have been cloned from a variety of freshwater, marine and anadromous fish species, and the gene products functionally characterized by heterologous expression in the yeast Saccharomyces cerevisiae. Gene expression studies included tissue distributions, developmental and nutritional regulation studied through genomic technologies (transcriptomic/microarray and proteomics), and quantitative real-time PCR (qPCR). This work expanded to include a BBSRC-funded study of the genomic structure and organization of the Fad and Elovl genes and, in particular, their up-stream regulatory sequences and the identification of functional promoters, cis elements and their associated transcription factors (see below). Studies on LC-PUFA biosynthesis in a variety of fish species has identified novel and unique Fads capable of Delta-4 desaturation activity, new, alternative (Delta-8) pathways for the biosynthesis of EPA and DHA from C18 precursor fatty acids, and provided further insights to the complex inter-relationships between environment and trophic level in determining essential fatty acid requirements in fish species. This has prompted further studies focused on elucidating the evolutionary history of the Fad and Elovl genes and the LC-PUFA biosynthesis pathway in fish species and vertebrates in general.

The LC-PUFA Synthesis Programme also contributed greatly to work that resulted in the production of a trait-targeted microarray for monitoring gene expression to assess health and performance in Atlantic salmon. The multi-partner BBSRC Exploiting Genomics Project developed the 17K TRAITS/SGP array targeted at traits important in terms of quality and nutritional value of salmon products for the consumer, with the trait of interest investigated in Stirling being LC-PUFA biosynthesis. All the gene features identified and collected for the TRAITS array are now included on custom Atlantic salmon 44K oligoarrays (Agilent) that are being used routinely as major analytical tools to determine genomic effects of a variety of challenges including nutritional and environmental stresses, as well as pathogen challenges.

A further major research programme in the Nutrition Group has aimed to determine the transcriptional control of lipid and fatty acid metabolism, including LC-PUFA biosynthesis, through studies involving the cloning and characterisation of key transcription factors known to be intimately involved in the regulation of lipid metabolism in mammals. Transcription factors and nuclear receptors are proteins that affect gene expression by interacting with DNA and altering the transcription of specific sets of genes. Understanding the mechanisms whereby fish regulate lipid homeostasis, that is the regulation of various lipid biosynthetic and catabolic pathways in specific tissues, is particularly relevant to the optimal formulation of aquaculture feeds. Initially, studies focused on peroxisome proliferator-activated receptors (PPARs) that are critical to the regulation of lipid and energy homeostasis. The fish PPAR programme, funded by the BBSRC and EU FP5 (FPPARS) resulted in the cloning and characterisation of PPARs in plaice and salmon, and functional studies using cell lines, primary cell cultures and in vivo dietary trials sought to determine the roles of PPARs in controlling lipid and fatty acid metabolism in fish. These studies expanded to include studies of other transcription factors including Liver X receptor in salmonids and the nuclear receptors sterol regulatory element-binding proteins, SREBP-1 and SREBP-2, in salmon. All these regulatory factors have been cloned and characterized and studies are in progress to determine their roles in integrated control and regulation of lipid and fatty acid metabolism, particularly focusing on LC-PUFA biosynthesis, using a combination of cell culture studies supported by in vivo dietary trials.