Abstract
The rabbitfish Siganus canaliculatus is the first marine teleost shown to be able to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors catalyzed by two fatty acyl desaturases (fad) including Δ4 Fad and Δ6/Δ5 Fad as well as two elongases (Elovl4 and Elovl5). Previously, hepatocyte nuclear factor 4α (Hnf4α) was demonstrated to be predominant in the transcriptional regulation of two fads. To clarify the regulatory mechanisms involved in rabbitfish lipogenesis, the present study focused on the regulatory role of Hnf4α to elovl5 expression and LC-PUFA biosynthesis. Bioinformatics analysis predicted two potential Hnf4α elements in elovl5 promoter, one binding site was confirmed to interact with Hnf4α by gel shift assays. Moreover, overexpression of hnf4α caused a remarkable increase both in elovl5 promoter activity and mRNA contents, while knock-down of hnf4α in S. canaliculatus hepatocyte line (SCHL) resulted in a significant decrease of elovl5 gene expression. Meanwhile, hnf4α overexpression enhanced LC-PUFA biosynthesis in SCHL cell, and intraperitoneal injection to rabbitfish juveniles with Hnf4α agonists (Alverine and Benfluorex) increased the expression of hnf4α, elvol5 and Δ4 fad, coupled with an increased proportion of total LC-PUFA in liver. The results demonstrated that Hnf4α is involved in LC-PUFA biosynthesis by up-regulating the transcription of the elovl5 gene in rabbitfish, which is the first report of Hnf4α as a transcription factor of the elovl5 gene in vertebrates.
Highlights
Long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic acid (EPA; 20:5n-3), arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) are cell membranes components, precursors of lipogenesis [1]
Up to the present day, freshwater fish and salmonid species can convert C18 PUFAs into LC-PUFAs through a series of desaturation and elongation steps catalyzed by fatty acid desaturases (Fad) and elongases of very long-chain fatty acids (Elovl), while most marine teleost are inefficient in LC-PUFA biosynthesis in vivo or lack such a capability [7]
To clarify the overall regulatory mechanisms of LC-PUFA biosynthesis in rabbitfish, the present study focused on the promoter analysis of elovl5 gene and regulatory role of hepatocyte nuclear factor 4α (HNF4α) to that gene
Summary
Long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic acid (EPA; 20:5n-3), arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) are cell membranes components, precursors of lipogenesis [1]. As for the teleost elovl gene, it has been cloned and functionally characterized in about twenty fish species [9], and its regulatory mechanism in marine teleost lipogenesis has been reported mainly at the transcriptional level [10]. Laying Hens and his group demonstrated that estrogen could promote hepatic LC-PUFA biosynthesis by regulating Elovl at post-transcriptional level, suggesting that there were different regulatory mechanisms between mammals and teleost [24]. All the key enzymes for LC-PUFA biosynthesis including the ∆4 fatty acyl desaturase (Fad) (the first report in vertebrates), ∆6/∆5 bifunctional Fad (the first report in marine fish) and two elongases of very long-chain fatty acids (Elovl and Elovl5) were characterized in this species, rabbitfish is a good model for us to study the regulatory mechanisms involved in LC-PUFA biosynthesis of teleosts [26,27]. The results could increase our understanding of the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates, which would contribute to the optimization and/or enhancement of the LC-PUFA pathway in teleosts
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