Elongation of long-chain fatty acids in rabbitfish Siganus canaliculatus: Cloning, functional characterisation and tissue distribution of Elovl5- and Elovl4-like elongases

Elongases of very long-chain fatty acids (Elovl) catalyse the rate-limiting step of the elongation pathway that results in net 2 C elongation of pre-existing fatty acyl chains. As the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) is particularly relevant in fish, Elovl involved in the pathway have been investigated in various studies. Here we report the molecular cloning, functional characterisation and tissue distribution of two distinct elovl-like cDNAs isolated from the herbivorous marine teleost Siganus canaliculatus. Unlike the carnivorous marine fish previously investigated, we hypothesise that the rabbitfish has an enhanced LC-PUFA biosynthetic capability as previously anticipated in a former study on fatty acyl desaturases (Fad). The results of the present study showed that rabbitfish expresses at least two elovl cDNAs, which have high homology in sequence and function to Elovl5 and Elovl4 elongases that have been investigated previously in other fish species. Furthermore, the results confirm that the activities of the Elovl5 and Elovl4 enzymes enable rabbitfish to perform all the elongation reactions required for the biosynthesis of the physiologically essential C20-22 LC-PUFA including eicosapentaenoic (20:5n-3), arachidonic (20:4n-6) and docosahexaenoic (22:6n-3, DHA) acids, as well as the less common very long-chain fatty acids (greater than C24). Rabbitfish is thus the first marine teleost in which genes encoding Fad and Elovl enzymes, with all the activities required for the production of DHA from C18 PUFA, have been characterised. Highlights Rabbitfish possess at least two fatty acid elongases. The elongases were functionally characterised as Elovl5 and Elovl4 types.  The elongases possess all the activities necessary for the biosynthesis of DHA.  Elovl4 is capable of synthesising very long-chain fatty acids up to C36 in length.  Rabbitfish represent a marine fish not dependent upon dietary EPA and DH

The miR-33 gene is identified in a marine teleost: a potential role in regulation of LC-PUFA biosynthesis in Siganus canaliculatus

As the first marine teleost demonstrated to have the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18PUFA precursors, rabbitfishSiganus canaliculatusprovides a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. Here the potential roles of miR-33 in such regulation were investigated. The miR-33 gene was identified within intron 16 of the gene encoding sterol regulatory element-binding protein 1 (Srebp1), an activator of LC-PUFA biosynthesis. Expression of miR-33 in rabbitfish tissues correlated with that ofsrebp1, while its expression in liver was highly responsive to ambient salinities and PUFA components, factors affecting LC-PUFA biosynthesis. Srebp1 activation promoted the expression of Δ4 and Δ6 Δ5 fatty acyl desaturases (Fad), key enzymes for LC-PUFA biosynthesis, accompanied by elevated miR-33 abundance in rabbitfish hepatocytes. miR-33 overexpression induced the expression of the twofad, but suppressed that of insulin-induced gene 1 (insig1), which encodes a repressor blocking Srebp proteolytic activation and has targeting sites of miR-33. These results indicated that miR-33, cooperating with Srebp1, may be involved in regulation of LC-PUFA biosynthesis by facilitatingfadexpression, probably through targetinginsig1. To our knowledge, this is the first report of the participation of miR-33 in LC-PUFA biosynthesis in vertebrates

Investigating long-chain polyunsaturated fatty acid biosynthesis in teleost fish: Functional characterization of fatty acyl desaturase (Fads2) and Elovl5 elongase in the catadromous species, Japanese eel Anguilla japonica

The capacity for endogenous production of LC-PUFA from PUFA in euryhaline or diadromous fish is largely unknown other than for Atlantic salmon (Salmo salar), an anadromous species, which displays a freshwater pattern. The aim of the present study was to characterize the enzymes of the LC-PUFA pathway in Japanese eel (Anguilla japonica), the most important catadromous species currently being farmed. cDNAs of two key genes were cloned and functional assays showed they encoded a desaturase (Fads2) with D6 and D8 activity and an elongase (Elovl5) with activity towards C18 and C20 PUFA, with activities similar to marine fish and an D6/D8 activity ratio similar to Atlantic salmon. Furthermore, tissue distribution of the mRNA showed a clear marine pattern with highest expression in brain and eye. Phylogenetic analysis placed the eel cDNAs in line with classical taxonomy. The data suggest that diadromous species display a pattern of LC-PUFA biosynthesis capacity that likely reflects the environmental and nutritional influence of their early life stages rather than those of adult fish. Future studies aim to establish the full range of PUFA desaturases and elongases in Japanese eel and to provide further insight to the importance and relevance of LC-PUFA biosynthesis in fish species and the influence of diadromy

Hnf4α is involved in the regulation of vertebrate LC-PUFA biosynthesis: insights into the regulatory role of Hnf4α on expression of liver fatty acyl desaturases in the marine teleost Siganus canaliculatus

Long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is an important metabolic pathway in vertebrates, especially fish, considering they are the major source of n-3 LC-PUFA in the human diet. However, most fish have only limited capability for biosynthesis of LC-PUFA. The rabbitfish (Siganus canaliculatus) is able to synthesize LC-PUFA as it has all the key enzyme activities required including Δ6Δ5 Fads2, Δ4 Fads2, Elovl5 and Elovl4. We previously reported a direct interaction between the transcription factor Hnf4α and the promoter regions of Δ4 and Δ6Δ5 Fads2, which suggested that Hnf4α was involved in the transcriptional regulation of fads2 in rabbitfish. For further functionally investigating it, a full-length cDNA of 1736 bp encoding rabbitfish Hnf4α with 454 amino acids was cloned, which was highly expressed in intestine, followed by liver and eyes. Similar to the expression characteristics of its target genes Δ4 and Δ6Δ5 fads2, levels of hnf4α mRNA in liver and eyes were higher in fish reared at low salinity than those reared in high salinity. After the rabbitfish primary hepatocytes were respectively incubated with Alverine, Benfluorex or BI6015, which were anticipated agonists or antagonist for Hnf4α, the mRNA level of Δ6Δ5 and Δ4 fads2 displayed a similar change tendency with that of hnf4α mRNA. Furthermore, when the mRNA level of hhf4α was knocked down using siRNA, the expression of Δ6Δ5 and Δ4 fads2 also decreased. Together, these data suggest that Hnf4α is involved in the transcriptional regulation of LC-PUFA biosynthesis, specifically, by targeting Δ4 and Δ6Δ5 fads2 in rabbitfish

Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases

Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n−3/18:2n−6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish

Cloning and characterization of ∆6/∆5 fatty acyl desaturase (Fad) gene promoter in the marine teleost Siganus canaliculatus

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding ∆6/∆5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C18 PUFA to LC-PUFA. First, 2044 bp promoter sequence was cloned by genome walking, and the sequence from -456 bp to + 51bp was determined as core promoter by progressive deletion mutation. Moreover, binding sites of transcription factors (TF) such as CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), nuclear factor Y (NF-Y), activated protein 1 (AP1), sterol regulatory element (SRE), hepatocyte nuclear factor 4α (HNF4α) and peroxisome proliferator activated receptor γ (PPARγ) were identified in the core promoter by site-directed mutation and functional assays. Moreover, NF-1 and HNF4α were confirmed to interact with the core promoter region by gel shift assay and mass spectrometry. This is the first report of the promoter structure of a ∆6/∆5 Fad in a marine teleost, and a novel discovery of NF-1 and HNF4α binding to the ∆6/∆5 Fad promoter

miR-24 is involved in vertebrate LC-PUFA biosynthesis as demonstrated in marine teleost Siganus canaliculatus

Recently, microRNAs (miRNAs) have emerged as crucial regulators of lipid metabolism. However, the miRNA-mediated regulatory mechanism on long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remains largely unknown. Here, we address a potentially important role of miRNA-24 (miR-24) in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. miR-24 showed significantly higher abundance in liver of rabbitfish reared in brackish water than in seawater for fish fed vegetable oil diets and in S. canaliculatus hepatocyte line (SCHL) cells incubated with alpha-linolenic acid (ALA) than the control group. Similar expression patterns were also observed on the expression of sterol regulatory element-binding protein-1 (srebp1) and LC-PUFA biosynthesis related genes. While opposite results were observed on the expression of insulin-induced gene 1 (insig1), an endoplasmic reticulum membrane protein blocking Srebp1 proteolytic activation. Luciferase reporter assays revealed rabbitfish insig1 as a target of miR-24. Knockdown of miR-24 in SCHL cells resulted in increased Insig1 protein, and subsequently reduced mature Srebp1 protein and expression of genes required for LC-PUFA biosynthesis, and these effects could be attenuated after additional insig1 knockdown. Opposite results were observed with overexpression of miR-24. Moreover, increasing endogenous insig1 by knockdown of miR-24 inhibited Srebp1 processing and consequently suppressed LC-PUFA biosynthesis in rabbitfish hepatocytes. These results indicate a potentially critical role for miR-24 in regulating LC-PUFA biosynthesis through the Insig1/Srebp1 pathway by targeting insig1. This is the first report of miR-24 involved in LC-PUFA biosynthesis and thus may provide knowledge on the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates

The catadromous teleost Anguilla japonica has a complete enzymatic repertoire for the biosynthesis of docosahexaenoic acid from alpha-linolenic acid: Cloning and functional characterization of an Elovl2 elongase

The Japanese eel Anguilla japonica is a catadromous fish species with considerable farming scale. Previous studies showed that dietary α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) satisfied essential fatty acid requirements in eel, which suggested that Japanese eel should have a complete pathway for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA). However, existing knowledge was insufficient to explain the molecular basis of LC-PUFA biosynthetic capacity in eel. In order to further characterize this pathway in eel, a full-length cDNA of a putative fatty acyl elongase was isolated, with the ORF encoding a protein with 294 amino acids. The putative elongase displayed high homology to Elovl2 of other teleosts. Functional characterization by heterologous expression in yeast showed the protein product of the cDNA had high activity towards C20 and C22 PUFA substrates and low activity towards C18 PUFA substrates, characteristic of Elovl2 elongases. Tissue distribution of the elovl2 mRNA showed highest expression in brain and eyes, which was different from freshwater and anadromous species. This may reflect an important role for this enzyme in the in situ endogenous biosynthesis of docosahexaenoic acid (DHA) in neural tissues in eel. This is the first report of an Elovl2 in a catadromous teleost and demonstrates that Japanese eel has a complete enzyme repertoire required for the endogenous biosynthesis of DHA via the Sprecher pathway. These data have increased our knowledge of the diversity of LC-PUFA biosynthesis in vertebrates, and provided further insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleost fish

Genome wide identification and functional characterization of two LC-PUFA biosynthesis elongase (elovl8) genes in rabbitfish (Siganus canaliculatus)

Elongases of very long-chain fatty acids (Elovls) catalyze the rate-limiting step of the elongation pathway that results in net 2‑carbon elongation of pre-existing fatty acyl chains. As a set of crucial enzymes involved in the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, Elovls of fish have been investigated extensively in recent years. In the present study, we first identified two novel fish-specific elovl genes (named as elovl8a and elovl8b) from the herbivorous marine teleost rabbitfish (Siganus canaliculatus) by genomic survey and molecular cloning methods. Subsequently, their functional characteristics, tissue distribution patterns and transcriptional changes in response to different nutritional states were investigated. Full-length coding sequences of the elovl8a and elovl8b genes were 804 and 792 bp, encoding 267 and 263 amino acids, respectively. Multiple alignment, genomic synteny and phylogenetic analyses further suggested that elovl8 genes were unique to teleosts. Functional characterization by heterologous expression in yeast showed that Elovl8b could elongate C18 (18:2n-6, 18:3n-3 and 18:4n-3) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids (PUFA) to longer-chain polyunsaturated fatty acids (LC-PUFA) whereas Elovl8a lacked this ability. In vitro, the expression of elovl8b but not elovl8a in rabbitfish hepatocytes was significantly up-regulated by incubation with 18:2n-6, 18:3n-3, 20:4n-6 and 20:5n-3, respectively. In vivo, compared with fish oil, dietary vegetable oil enriched in C18 PUFA enhanced the expression of elovl8b in rabbitfish brain, liver, intestine and gill. These findings suggest that elovl8b but not elovl8a is a novel active member of the Elovl protein family involved in the LC-PUFA biosynthesis pathway in rabbitfish, and provide novel insight into the mechanisms of LC-PUFA biosynthesis in teleost

Causal link between hypothyroidism and gastric cancer risk: insights gained through multivariable Mendelian randomization and mediation analysis

BackgroundGastric cancer (GC) is the third leading cause of cancer death worldwide, and hypothyroidism has been identified as a potential influencing factor. Despite known associations between hypothyroidism and various cancers, the causal link between hypothyroidism and GC and potential mediators of this relationship remains unclear. This study aimed to clarify these relationships using Mendelian randomization (MR).MethodsUtilizing genetic variant information from the FinnGen and MRC Integrative Epidemiology Unit open genome-wide association studies (GWAS) databases, we conducted univariable and multivariable MR analyses to explore the causal relationship between hypothyroidism and GC risk. The analysis was adjusted for confounders such as BMI, smoking status, and alcohol intake, and included mediator MR analysis to examine the role of high cholesterol.ResultsWe identified a significant inverse association between hypothyroidism and GC risk (OR = 0.93, 95% CI= 0.89–0.98, P = 0.003), with no evidence of reverse causation or pleiotropy. Adjustments for Helicobacter pylori infection weakened this association. Mediator analysis highlighted high cholesterol levels, chronic hepatitis B infection, and diabetes/endocrine disease status as significant mediators of the protective effect of hypothyroidism on GC risk.ConclusionOur findings suggest that hypothyroidism may confer a protective effect against GC, mediated in part by high cholesterol and other factors. These results underscore the importance of thyroid function and metabolic health in GC risk, offering new insights for preventive strategies and highlighting the need for further research into these complex associations