Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus)

Abstract

The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

Figure 1. Contents of n-3 LC-PUFA in diets and tissues of experimental fish.

Values (means ± S.E.M.) in bars that do not have the same letter are significantly different (P<0.05) among treatments. The fatty acid contents were expressed as percentage of total fatty acids.

Figure 2. Comparison of the deduced amino acid sequences of FADS2 from Japanese seabass and other fish.

The amino acid sequences were aligned using ClustalX, and identity/similarity shading was based on the BLOSUM62 matrix and the cut off was 75%. Identical residues are shaded black and similar residues are shaded grey. The cytochrome b5-like domain is dot-underlined, the two transmembrane regions are dash underlined, the three histidine-rich domains are solid underlined and asterisks on the top mark the haem-binding motif, H–P–G–G.

Figure 3. Phylogenetic tree of FADS2.

The amino acid sequences of FADS2 used in the phylogenetic tree included those from European sea bass, gilthead sea bream, salmon, zebrafish, other fish species (Atlantic cod, turbot, rabbitfish, barramundi, carp, rainbow trout, and tilapia), mammals (mouse and human), fungus (M. alpina) and nematode (C. elegans). The horizontal branch length is proportional amino acid substitution rate per site. The numbers represent the frequencies with which the tree topology presented here was replicated after 1000 bootstrap iterations.

Figure 4. Tissue distribution of FADS2 in Japanese seabass.

Relative FADS2 mRNA expression was determined by quantitative real-time PCR (qRT-PCR) as described in the Materials and Methods section. Results are expressed as means ± S.E.M. (n = 3). Different letters above the bars denote significant differences among tissues at the P<0.05 level (P = 0.000) as determined by one-way ANOVA followed by Tukey's test (SPSS).

Figure 5. Relative FADS2 mRNA levels in liver of experimental fish.

Relative FADS2 mRNA levels were evaluated by quantitative real-time PCR (qRT-PCR) and expressed relative to β-actin levels. Results are expressed as means ± S.E.M. (n = 3). Different letters above the bars denote significant differences among dietary groups at the P<0.05 level (P = 0.000) as determined by one-way ANOVA followed by Tukey's test (SPSS).

Figure 6. Alignment of FADS2 promoter fragments among Japanese seabass and other fish.

The numbers indicate the sequence position relative to the possible transcriptional start site. Binding sites of SP1 and NF-Y, and sterol regulatory element (SRE) were indicated based on previous work from Zheng et al. and Geay et al. .

Figure 7. Methylation rate of 55 CpG in FADS2 gene promoter of Japanese seabass.

The methylation rate was determined using BSP cloning-based sequencing analysis. Results are expressed as means ± S.E.M. (n = 3). Different letters above the bars denote significant differences among dietary groups at the P<0.05 level (P = 0.000) as determined by one-way ANOVA followed by Tukey's test (SPSS).