Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C₁₈ PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C₁₈ PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

Rainbow trout (Oncorhynchus mykiss) Elovl5 and Elovl2 differ in selectivity for elongation of omega-3 docosapentaenoic acid

The synthesis of the omega-3 long-chain polyunsaturated fatty acids (LCPUFA)  eicosapentaenoic acid (EPA; 20:5n− 3) and docosahexaenoic acid (DHA; 22:6n − 3) from dietary α-linolenic acid (ALA; 18:3n − 3) requires three desaturation and three elongation steps in vertebrates. The elongation of EPA to docosapentaenoic acid (DPA; 22:5n − 3) can be catalysed by the elongase enzymes Elovl5 or Elovl2, but further elongation of DPA to 24:5n − 3, the penultimate precursor of DHA, is limited to Elovl2, at least in mammals. Elovl5 enzymes have been characterised from seventeen fish species but Elovl2 enzymes have only been characterised in two of these fish. The essentiality of Elovl2 for DHA synthesis is unknown in fish. This study is the first to identify an Elovl2 in rainbow trout (Oncorhynchus mykiss) and functionally characterise the Elovl5 and Elovl2 using a yeast expression system. Elovl5 was active with C_18–20 PUFA substrates and not C₂₂ PUFA. In contrast, Elovl2 was active with C_20–22 PUFA substrates and not C₁₈ PUFA. Thus, rainbow trout is dependent on Elovl2 for DPA to 24:5n − 3 synthesis and ultimately DHA synthesis. The expression of elovl5 was significantly higher than elovl2 in liver. Elucidating this dependence on Elovl2 to elongate DPA and the low elovl2 gene expression compared with elovl5 are critical findings in understanding the potential for rainbow trout to synthesize DHA.     

Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of four fatty acyl elongases and a novel desaturase capacity

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the ‘Sprecher pathway’. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs.     

The fatty acid chain elongase,Elovl1, is required for kidney and swim bladder development during zebrafish embryogenesis

Very long chain fatty acids are required for sphingolipid synthesis, lipid homeostasis, myelin formation, epidermal permeability, and retinal function. Seven different enzymes are known to be involved in the elongation cycle of fatty acids, with different chain-length specificities. Elovl1 is one of those enzymes whose function has been linked mainly to the synthesis of sphingolipids and the epidermal barrier. However, the role of Elovl1 in organogenesis is not clear. In zebrafish, 2 Elovl1 genes, elovl1a and elovl1b, are highly expressed in the swim bladder, and elovl1b is also expressed in the kidney. We found that both elovl1 knockdown embryos contain increased levels of long chain fatty acids from carbon number 14 to 20 as compared to control embryos. Oil-Red-O staining shows that yolk lipid consumption is greatly reduced, whereas lipid droplets accumulate within the swim bladder. Notably, knockdown of either elovl1a or elovl1b affects the expression of genes involved in swim bladder development and impairs inflation of the swim bladder. Consistent with its expression in the pronephros, knockdown of elovl1b alone affects the expression of genes required for kidney development and reduces renal clearance. Our findings strongly suggest that both elovl1 genes are a key determinant of swim bladder and kidney development in zebrafish, which may be comparatively applicable to lung and kidney development in humans.     

Dietary LC-PUFA and environmental salinity modulate the fatty acid biosynthesis capacity of the euryhaline teleost thicklip grey mullet (Chelon labrosus)

The capacity to biosynthesise long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) depends upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. The presence of a Δ5/Δ6 desaturase enabling the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) through the “Sprecher pathway” has been reported in Chelon labrosus. Research in other teleosts have demonstrated that LC-PUFA biosynthesis can be modulated by diet and ambient salinity. The present study aimed to assess the combined effects of partial dietary replacement of fish oil (FO) by vegetable oil (VO) and reduced ambient salinity (35 ppt vs 20 ppt) on the fatty acid composition of muscle, enterocytes and hepatocytes of C. labrosus juveniles. Moreover, the enzymatic activity over radiolabelled [1-¹⁴C] 18:3n-3 (α-linolenic acid, ALA) and [1-¹⁴C] 20:5n-3 (eicosapentaenoic acid, EPA) to biosynthesise n-3 LC-PUFA in hepatocytes and enterocytes, and the gene regulation of the C. labrosus fatty acid desaturase-2 (fads2) and elongation of very long chain fatty acids protein 5 (elovl5) in liver and intestine was also investigated. Recovery of radiolabelled products including stearidonic acid (18:4n-3, SDA), 20:5n-3, tetracosahexaenoic acid (24:6n-3, THA) and 22:6n-3 in all treatments except FO35-fish, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA and DHA from ALA is present and active in C. labrosus. Low salinity conditions upregulated fads2 in hepatocytes and elovl5 in both cell types, regardless of dietary composition. Interestingly, FO20-fish showed the highest amount of n-3 LC-PUFA in muscle, while no differences in VO-fish reared at both salinities were found. These results demonstrate a compensatory capacity of C. labrosus to biosynthesise n-3 LC-PUFA under reduced dietary supply, and emphasise the potential of low salinity conditions to stimulate this pathway in euryhaline fish.     

Neph3 associates with regulation of glomerular and neural development in zebrafish

Neph3 (filtrin) is a membrane protein expressed in the glomerular epithelial cells (podocytes), but its role in the glomerulus is still largely unknown. To characterize the function of Neph3 in the glomerulus, we employed the zebrafish as a model system. Here we show that the expression of neph3 in pronephros starts before the onset of nephrin and podocin expression, peaks when the nephron primordium differentiates into glomerulus and tubulus, and is then downregulated upon glomerular maturation. By histology, we found that neph3 is specifically expressed in pronephric podocytes at 36 hpf. Furthermore, disruption of neph3 expression by antisense morpholino oligonucleotides results in distorted body curvature and transient pericardial edema, the latter likely reflecting perturbation of glomerular osmoregulatory function. Histological analysis of neph3 morphants reveals altered glomerular morphology and dilated pronephric tubules. The phenotype of neph3 morphants, curved body and pericardial edema, is rescued by wild-type zebrafish neph3 mRNA. In addition to glomerulus, neph3 is highly expressed in the developing brain and specific regions of mature midbrain and hindbrain. In line with this, neph3 morphants show aberrant brain morphology. Collectively, the expression of neph3 in glomerulus and brain together with the morphant phenotype imply that neph3 is a pleiotropic gene active during distinct stages of tissue differentiation and associates directly in the regulation of both glomerular and neural development.     

Upregulated mRNA expression of desaturase and elongase,two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

Background  

Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.     

Transcriptional control mechanisms of genes of lipid and fatty acid metabolism in the Atlantic salmon (Salmo salar L.) established cell line, SHK-1

The regulatory control mechanisms of lipid and fatty acid metabolism were investigated in Atlantic salmon. We identified sterol regulatory element binding protein (SREBP) genes in salmon and characterised their response, and the response of potential target and other regulatory genes including liver X receptor (LXR), to cholesterol and long-chain polyunsaturated fatty acids (LC-PUFA) in the salmon established cell line, SHK-1. Two cDNAs for SREBPs homologous to mammalian SREBP-1 and SREBP-2 were characterised. We identified three groups of genes whose expression responded differently to the treatments. One group of genes, including cholesterol biosynthetic genes, showed increased expression in response to lipid depletion but supplementary cholesterol or LC-PUFA had no further effect. The expression of a second group of genes belonging to fatty acid biosynthetic pathways, included fatty acid synthase, Δ6 and Δ5 fatty acyl desaturases, also increased after lipid depletion but this was negated by cholesterol or by LC-PUFA supplementation. The expression of a third group of genes including acyl-CoA oxidase, HMG-CoA reductase and Elovl5 elongase was increased by cholesterol treatment but was not affected by lipid depletion or by LC-PUFA. This same pattern of expression was also shown by liver X receptor (LXR), indicating that acyl-CoA oxidase, HMG-CoA reductase and Elovl5 are possible direct targets of LXR. This suggests that salmon Elovl5 may be regulated differently from mammalian Elovl5, which is an indirect target of LXR, responding to LXR-dependent increases in SREBP-1.