Abstract
The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5Mt with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50–100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5Mt showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bcMt showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5Mt and Δ6bcMt demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5Mt demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.
Highlights
The health benefits of fish oil eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) have been demonstrated by many studies
Two groups of Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mutated salmon, Δ6abc/5Mt and Δ6bcMt were generated as previously described in Atlantic salmon[33]
The current study sought to understand in vivo functions of Atlantic salmon desaturases and to evaluate their levels of in vivo functional redundancy in long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis using two groups of CRISPR-mutated salmon, Δ6abc/5Mt with mutated Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads[2] genes and Δ6bcMt where only Δ6fads2-b and Δ6fads2-c were mutated
Summary
The health benefits of fish oil eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) have been demonstrated by many studies. The capacity for LC-PUFA synthesis in any species depends on complementary activities of fatty acyl desaturases (Fads2) and elongases of very long chain fatty acid (Elovls). In assessing LC-PUFA biosynthetic capacity of Atlantic salmon and other salmonids, such as rainbow trout, Artic charr and brown trout, numerous in vitro studies involving heterologous expression[13,16,17,20,21] and fatty acid desaturation/elongation assays in both hepatocytes and enterocytes[22,25,26] have been performed. To broaden and provide detailed insight into the in vivo functions of genes encoding salmon LC-PUFA biosynthetic enzymes, as well as understand nutritional and transcriptional regulation of LC-PUFA biosynthesis, in vivo functional studies are required. Our data further suggest Srebp-1 as a major transcription regulator of salmon LC-PUFA biosynthesis and show that the status of endogenous LC-PUFA synthesis as well as dietary LC-PUFA composition control expression of srebp-1
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