Dietary black soldier fly oil enhances growth performance, flesh quality, and health status of largemouth bass (Micropterus salmoides).

The study aimed to assess the effects of dietary black soldier fly oil (BSFO) on the growth performance, flesh quality, and health status of largemouth bass (Micropterus salmoides). Six iso-nitrogenous and isolipid diets were formulated by substituting fish oil and soybean oil (1/2, wt/wt) with BSFO in percentages of 0%, 20%, 40%, 60%, 80%, and 100%, respectively. The diets were fed to 960 fish (initial body weight = 16.5 g) in four replicates for 8 weeks. Indicators related to growth performance, body composition, hematology, flesh quality, expression of genes related to inflammatory cytokines and apoptosis, and the response of fish to Aeromonas veronii challenge were analyzed. The results showed that the weight gain rate was numerically improved in all BSFO substitution groups, ranging from 9.3% to 44.0% compared to the control group. The highest survival rate and the lowest hepatosomatic index and condition factor were observed in the BFSO20 group. In terms of flesh quality, the water-holding capacity of the dorsal muscle was elevated with higher levels of dietary BSFO. However, significant changes in texture properties (cohesiveness, gluing, and chewiness) were observed in the BSFO20 group (P < 0.05). Six hematological parameters related to glycolipid and liver function were optimized in most of the BFSO substitution groups. Furthermore, the expressions of six inflammation- and apoptosis-related genes (IL-1ß, Bcl-xl, BAX, caspase8, TNF-α, and IL-10) were significantly affected by dietary BSFO (P < 0.05). Following bacterial challenge, the seven-day cumulative survival rates of fish were considerably increased from 10.0% in the control group to 60.0% and 66.7% in the BSFO80 and BSFO100 groups, respectively. One-variable linear regression analysis revealed that various parameters related to fish growth, flesh quality, and health status were significantly influenced by dietary BSFO substitution levels in a dose-dependent manner (P < 0.05). In conclusion, substituting around 20% of dietary fish oil and soybean oil with BSFO is promising in improving the growth performance and flesh quality of M. salmoides. However, to enhance immunity and disease resistance, it is recommended to further increase the inclusion of BSFO in the diet.

GPR120-ERK1-Srebp1c signaling pathway regulates long-chain polyunsaturated fatty acids biosynthesis in marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost reported to possess long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic ability; its regulatory mechanisms have been investigated at the transcriptional and posttranscriptional levels, but little is known about its regulation at the cellular signaling level. The present study investigated the regulatory role of the G-protein-coupled receptor 120 (GPR120) signaling pathway in LC-PUFA biosynthesis in rabbitfish. S. canaliculatus hepatocyte line (SCHL) cells treated with GRP120 agonists (TUG891 and GW9508) showed significantly lower docosahexaenoic acid (DHA) content and mRNA levels of the key genes involved in LC-PUFA biosynthesis, encoding Δ6/Δ5 Fads2, Elovl5, and transcriptional factor Srebp1c. Transcriptome analysis of the treated SCHL cells showed significantly lower mRNA levels of genes encoding extracellular signal-regulated kinase 1 (ERK1), AMP-activated protein kinase (AMPKα2), target of rapamycin (TORC2) and Srebp1c, suggesting that these proteins are potentially involved in the GRP120 signaling pathway. Moreover, treatment of SCHL cells with signaling chemicals of ERK1, AMPKα2, TORC2, and Srebp1c confirmed the involvement of the ERK1-Srebp1c signaling pathway in the regulation of LC-PUFA biosynthesis. The mRNA levels of Srebp1c, Δ6/Δ5 fads2 and elovl5 were significantly lower in cells treated with PUFAs (linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, DHA) but higher in those treated with ERK1 inhibitors (U0126 and CI-1040). CI-1040-treated cells showed significantly higher DHA content, but the other treatment groups (except PD98059) showed significantly lower DHA content. These results indicate that the GPR120-ERK1-Srebp1c signaling pathway regulates rabbitfish LC-PUFA biosynthesis, representing a novel regulatory mechanism in vertebrates.

Insulin can up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and stimulatory protein 1 (Sp1) in marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors catalyzed by fatty acyl desaturases (Δ6/Δ5 Fads, Δ4 Fads) and elongases of very long chain fatty acids (Elovls). Previously, we predicted the existence of insulin (INS) response elements (IREs) including nuclear factor Y (NF-Y) and sterol regulatory element (SRE) in the core promoter region of rabbitfish Δ6/Δ5 fads and Δ4 fads. To clarify the potential regulatory effect and mechanism of INS in LC-PUFA biosynthesis, INS responding region was identified at -456 bp to + 51 bp of Δ6/Δ5 fads core promoter, but not in Δ4 fads promoter. Moreover, a unique stimulatory protein 1 (Sp1) element was predicted in the INS responding region of Δ6/Δ5 fads. Subsequently, SRE, NF-Y and Sp1 elements were proved as IREs in Δ6/Δ5 fads promoter. The up-regulation of INS on gene expression of Srebp-1c, Sp1, Δ6/Δ5 fads and elovl5 as well as the LC-PUFA biosynthesis was further demonstrated in S. canaliculatus hepatocyte line (SCHL) cells, but no influence was detected on Δ4 fads. Besides, inhibitors of transcription factors Srebp-1c (Fatostatin, PF-429242) and Sp1 (Mithramycin) could inhibit the gene expression of Srebp-1c, Δ6/Δ5 fads and elovl5, and abolish the up-regulation of INS on these genes' expression and LC-PUFA biosynthesis. These results indicated that INS could up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and Sp1 in rabbitfish S. canaliculatus, which is the first report in teleost.

Dietary calcium pyruvate could improve growth performance and reduce excessive lipid deposition in juvenile golden pompano (Trachinotus ovatus) fed a high fat diet.

Excessive lipid deposition in farmed fish is a challenge in the aquaculture industry. To study the effect of dietary calcium pyruvate (CaP) on lipid accumulation in fish, we used a high fat diet (HFD) to establish a lipid accumulation model in juvenile golden pompano (Trachinotus ovatus) and supplemented with 0%, 0.25%, 0.50%, 0.75% and 1.0% CaP (diets D0-D4, respectively). After 8-week feeding in floating cages, dietary CaP significantly improved growth performance, which peaked in fish fed diet D3. Supplementation of CaP significantly decreased whole body lipid content in fish fed D2-D4 and hepatosomatic index and liver lipid content in fish fed D3 and D4. Serum and hepatic antioxidant indices, including glutathione, catalase and superoxide dismutase, showed generally increasing trends in fish fed diets with CaP. In addition, increasing dietary CaP increasingly reduced hepatic activities of hexokinase, phosphofructokinase and pyruvate kinase involved in glycolysis, and increased glycogen contents of the liver and muscle. Dietary CaP up-regulated the liver mRNA expression of pparα, cpt1, hsl and fabp1, but down-regulated expression of srebp-1, fas and acc. In conclusion, 0.75% CaP improved growth performance and reduced excessive lipid deposition by affecting fatty acid synthesis and lipolysis in juvenile T. ovatus fed HFD.

Insulin activates LC-PUFA biosynthesis of hepatocytes by regulating the PI3K/Akt/mTOR/Srebp1 pathway in teleost Siganus canaliculatus.

Insulin is well known an important metabolic regulator in glucose and lipid metabolism. It has been proved to activate long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in mammals, but little is known about such a role in fish. To explore the effects and molecular mechanisms of insulin in fish LC-PUFA biosynthesis, we treated the rabbitfish S. canaliculatus hepatocyte line (SCHL) cells with 65 nM insulin for 12 h, and the results showed that the mRNA levels of genes encoding the key enzymes and transcription factor involved in rabbitfish LC-PUFA biosynthesis such as Δ6Δ5 fads2, elovl5 and srebp1, as well as those of PI3K pathway genes including pdk1, akt2 and mtor increased significantly. Moreover, SCHL cells treated with different PI3K/Akt pathway inhibitors (LY294002, Wortmannin, AKTi-1/2) alone or combined with insulin decreased the mRNA levels of PI3K/Akt/mTOR downstream signaling genes, including Δ6Δ5 fads2, Δ4 fads2, elovl5, elovl4 and srebp1. While PI3K/Akt agonists (740 Y-P, IGF-1, SC-79) had the opposite results. The results of fatty acid composition analysis of hepatocytes showed that insulin stimulation increased the Δ6Δ5 Fads2-dependent PUFA desaturation indexes, while Elovl5-dependent PUFA elongation indexes had upward trends, and consequently LC-PUFA contents increased. Taken together, these results indicated that insulin activated LC-PUFA biosynthesis probably through PI3K/Akt/mTOR/Srebp1 pathway in S. canaliculatus hepatocytes.

Growth and Fatty Acid Composition of Black Soldier Fly Hermetia illucens (Diptera: Stratiomyidae) Larvae Are Influenced by Dietary Fat Sources and Levels.

A 16-day rearing trial was performed to investigate the influence of two supplemental levels (5% and 10%) of six dietary fat sources (linseed oil, peanut oil, coconut oil, soybean oil, lard oil and fish oil) on the growth, development and nutrient composition of black solider fly larvae. Our results demonstrated that the pre-pupa rate of larvae was linearly influenced by dietary C18:0, C18:3n-3 and C18:2n-6 content (pre-pupa rate = 0.927 × C18:0 content + 0.301 × C18:3n-3 content-0.258 × C18:2n-6 content p < 0.001)), while final body weight was linearly influenced by that of C16:0 (final body weight = 0.758 × C16:0 content, p = 0.004). Larval nutrient composition was significantly affected by dietary fat sources and levels, with crude protein, fat and ash content of larvae varying between 52.0 and 57.5, 15.0 and 23.8, and 5.6 and 7.2% dry matter. A higher level of C12:0 (17.4-28.5%), C14:0 (3.9-8.0%) and C16:1n-9 (1.3-4.3%) was determined in larvae fed the diets containing little of them. In comparison, C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 proportions in larvae were linearly related with those in diets, with the slope of the linear equations varying from 0.39 to 0.60. It can be concluded that sufficient C16:0, C18:0 and C18:3n-3 supply is beneficial for larvae growth. Larvae could produce and retain C12:0, C14:0, and C16:1n-9 in vivo, but C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 could only be partly incorporated from diets and the process may be enhanced by a higher amount of dietary fat. Based on the above observation, an accurately calculated amount of black soldier fly larvae could be formulated into aquafeed as the main source of saturated fatty acids and partial source of mono-unsaturated and poly-unsaturated fatty acids to save fish oil.

Regulation of long-chain polyunsaturated fatty acid biosynthesis in teleost fish.

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA, C20-24), including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are involved in numerous biological processes and have a range of health benefits. Fish have long been considered as the main source of n-3 LC-PUFA in human diets. However, the capacity for endogenous biosynthesis of LC-PUFA from C18 PUFA varies in fish species based on the presence, expression and activity of key enzymes including fatty acyl desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) proteins. In this article, we review progress on the identified Fads and Elovl, as well as the regulatory mechanisms of LC-PUFA biosynthesis both at transcriptional and post-transcriptional levels in teleosts. The most comprehensive advances have been obtained in rabbitfish Siganus canaliculatus, a marine teleost demonstrated to have the entire pathway for LC-PUFA biosynthesis, including the roles of transcription factors hepatocyte nuclear factor 4α (Hnf4α), liver X receptor alpha (Lxrα), sterol regulatory element-binding protein 1 (Srebp-1), peroxisome proliferator-activated receptor gamma (Pparγ) and stimulatory protein 1 (Sp1), as well as post-transcriptional regulation by individual microRNA (miRNA) or clusters. This research has, for the first time, demonstrated the involvement of Hnf4α, Pparγ and miRNA in the regulation of LC-PUFA biosynthesis in vertebrates. The present review provides readers with a relatively comprehensive overview of the progress made into understanding LC-PUFA biosynthetic systems in teleosts, and some insights into improving endogenous LC-PUFA biosynthesis capacity aimed at reducing the dependence of aquafeeds on fish oil while maintaining or increasing flesh LC-PUFA content and the nutritional quality of farmed fish.

Identification of miR-145 as a Key Regulator Involved in LC-PUFA Biosynthesis by Targeting hnf4α in the Marine Teleost Siganus canaliculatus.

Fish, particularly marine species, are considered as the major source of long-chain polyunsaturated fatty acids (LC-PUFA) in the human diet. The extent to which fish can synthesize LC-PUFA varies with species and is regulated by dietary fatty acids and ambient salinity. Therefore, to enable fish to produce more LC-PUFA, comprehending the mechanisms underlying the regulation of LC-PUFA biosynthesis is necessary. Here, the regulatory roles of miR-145 were investigated in the marine teleost rabbitfish Siganus canaliculatus. The hepatic abundance of miR-145 was lower in rabbitfish reared in low salinity (10 ppt) in comparison with that of those cultured in seawater (32 ppt), while the opposite pattern was observed for the transcripts of the transcription factor hepatocyte nuclear factor 4 alpha (Hnf4α), known to affect rabbitfish LC-PUFA biosynthesis. Rabbitfish hnf4α was identified as a target of miR-145 by luciferase reporter assays, and overexpression of miR-145 in the S. canaliculatus hepatocyte line (SCHL) markedly reduced the expression of Hnf4α and its target genes involved in LC-PUFA biosynthesis, namely, Δ4 fads2, Δ6Δ5 fads2, and elovl5. The opposite pattern was observed when miR-145 was knocked down in SCHL cells, with these effects being attenuated by subsequent hnf4α knockdown. Moreover, increasing endogenous Hnf4α by the knockdown of miR-145 increased the expression of LC-PUFA biosynthesis genes and enhanced the synthesis of LC-PUFA in both SCHL cells and rabbitfish in vivo. This is the first report to identify miR-145 as a key effector of LC-PUFA biosynthesis by targeting hnf4α, providing a novel insight into the mechanisms of the regulation of LC-PUFA biosynthesis in vertebrates.

miR-26a mediates LC-PUFA biosynthesis by targeting the Lxrα-Srebp1 pathway in the marine teleost Siganus canaliculatus.

MicroRNAs have been recently shown to be important regulators of lipid metabolism. However, the mechanisms of microRNA-mediated regulation of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis in vertebrates remain largely unknown. Herein, we for the first time addressed the role of miR-26a in LC-PUFA biosynthesis in the marine rabbitfish Siganus canaliculatus The results showed that miR-26a was significantly down-regulated in liver of rabbitfish reared in brackish water and in S. canaliculatus hepatocyte line (SCHL) incubated with the LC-PUFA precursor α-linolenic acid, suggesting that miR-26a may be involved in LC-PUFA biosynthesis because of its abundance being regulated by factors affecting LC-PUFA biosynthesis. Opposite patterns were observed in the expression of liver X receptor α (lxrα) and sterol regulatory element-binding protein-1 (srebp1), as well as the LC-PUFA biosynthesis-related genes (Δ4 fads2, Δ6Δ5 fads2, and elovl5) in SCHL cells incubated with α-linolenic acid. Luciferase reporter assays revealed rabbitfish lxrα as a target of miR-26a, and overexpression of miR-26a in SCHL cells markedly reduced protein levels of Lxrα, Srebp1, and Δ6Δ5 Fads2 induced by the agonist T0901317. Moreover, increasing endogenous Lxrα by knockdown of miR-26a facilitated Srebp1 activation and concomitant increased expression of genes involved in LC-PUFA biosynthesis and consequently promoted LC-PUFA biosynthesis both in vitro and in vivo These results indicate a critical role of miR-26a in regulating LC-PUFA biosynthesis through targeting the Lxrα-Srebp1 pathway and provide new insights into the regulatory network controlling LC-PUFA biosynthesis and accumulation in vertebrates.

Effects of dietary n-3 highly unsaturated fatty acids levels on growth, lipid metabolism and innate immunity in juvenile golden pompano (Trachinotus ovatus).

To investigate the effects of dietary n-3 highly unsaturated fatty acids (HUFA) levels on growth, lipid metabolism and innate immunity in juvenile golden pompano Trachinotus ovatus, a marine carnivorous teleost, a total of 450 fish (average body weight: 14.84 g) were randomly distributed into 18 cages at sea, each dietary group with three cages and respectively fed six diets (D1-D6) with 2.30% (D1), 0.64% (D2), 1.00% (D3), 1.24% (D4), 1.73% (D5), or 2.10% (D6) n-3 HUFA. Here, D1 with fish oil as lipid source was set as control, while D2-D6 used a mixed vegetable oil as lipid source and supplemented with docosahexaenoic acid- (DHA) and eicosapentaenoic acid- (EPA) enriched oils to adjust the n-3 HUFA levels. After 8 weeks feeding, the daily growth coefficient (DGC), specific growth rate (SGR) and feed efficiency ratio (FER) showed no significant difference among the six dietary groups (P > 0.05). The levels of EPA and DHA in serum and liver increased with the dietary n-3 HUFA levels. The activity of total superoxide disumutase (T-SOD) in serum of fish fed D4 and D5 were significantly higher than that of the other groups, whereas the opposite was true for serum IL-1ß and IL-6 levels as well as liver malondialdehyde (MDA) content. The mRNA levels of genes related to hepatic lipid metabolism including sterol regulatory element-binding protein-1 (srebp-1), fatty acid binding protein 1 (fabp1), peroxisome proliferators-activated receptor alpha (pparα), elongase of very long-chain fatty acids 5 (elovl5) and fatty acyl desaturase 2 (fads2) were down-regulated in fish fed the diets with high n-3 HUFA levels, while those of apolipoprotein b 100 (aprob 100) and carnitine palmitoyl transferase 1 (cpt1) increased significantly as increasing n-3 HUFA levels up to 1.73% (D2-D5), but decreased in the 2.10% n-3 HUFA group (D6). In addition, the expression levels of genes related to innate immunity including interleukin-10 (il-10) and transforming growth factor ß1 (tgf-ß1) increased significantly when dietary n-3 HUFA increased from 0.64% to 1.73%, whereas the opposite was true for the expression levels of nuclear factor kappa-B (nf-κb), interleukin-1ß (il-1ß), interleukin-6 (il-6) and interleukin-8 (il-8). Overall, the results indicated that dietary n-3 HUFA at 1.24-1.73% (D4-D5) can effectively improve fatty acid profiles, lipid metabolism, antioxidant capacity and immune response of golden pompano.

The miR-15/16 Cluster Is Involved in the Regulation of Vertebrate LC-PUFA Biosynthesis by Targeting pparγ as Demonostrated in Rabbitfish Siganus canaliculatus.

Post-transcriptional regulatory mechanisms play important roles in the regulation of long-chain (≥ C20) polyunsaturated fatty acid (LC-PUFA) biosynthesis. Here, we address a potentially important role of the miR-15/16 cluster in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. In rabbitfish, miR-15 and miR-16 were both highly responsive to fatty acids affecting LC-PUFA biosynthesis and displayed a similar expression pattern in a range of rabbitfish tissues. A common potential binding site for miR-15 and miR-16 was predicted in the 3'UTR of peroxisome proliferator-activated receptor gamma (pparγ), an inhibitor of LC-PUFA biosynthesis in rabbitfish, and luciferase reporter assays revealed that pparγ was a potential target of miR-15/16 cluster. In vitro individual or co-overexpression of miR-15 and miR-16 in rabbitfish hepatocyte line (SCHL) inhibited both mRNA and protein levels of Pparγ, and increased the mRNA levels of Δ6Δ5 fads2, Δ4 fads2, and elovl5, key enzymes of LC-PUFA biosynthesis. Inhibition of pparγ was more pronounced with co-overexpression of miR-15 and miR-16 than with individual overexpression in SCHL. Knockdown of miR-15/16 cluster gave opposite results, and increased mRNA levels of LC-PUFA biosynthesis enzymes were observed after knockdown of pparγ. Furthermore, miR-15/16 cluster overexpression significantly increased the contents of 22:6n-3, 20:4n-6 and total LC-PUFA in SCHL with higher 18:4n-3/18:3n-3 and 22:6n-3/22:5n-3 ratio. These suggested that miR-15 and miR-16 as a miRNA cluster together enhanced LC-PUFA biosynthesis by targeting pparγ in rabbitfish. This is the first report of the participation of miR-15/16 cluster in LC-PUFA biosynthesis in vertebrates.

Hepatocyte nuclear factor 4α (Hnf4α) is involved in transcriptional regulation of Δ6/Δ5 fatty acyl desaturase (Fad) gene expression in marine teleost Siganus canaliculatus.

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.

The catadromous teleost Anguilla japonica has a complete enzymatic repertoire for the biosynthesis of docosahexaenoic acid from α-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.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n-6 to 18:3n-6, 18:2n-6 to 20:2n-6, and 18:3n-3 to 20:3n-3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Effects of dietary lipid sources on the intestinal microbiome and health of golden pompano (Trachinotus ovatus).

Replacement of fish oil (FO) with vegetable oils (VO) in diets is economically desirable for the sustainable development of the aquaculture industry. However, inflammation provoked by FO replacement limited its widely application in fish industry. In order to understand the mechanism of VO-induced inflammation, this study investigated the impact of different dietary vegetable oils on the intestinal health and microbiome in carnivorous marine fish golden pompano (Trachinotus ovatus). Three diets supplemented with fish oil (FO, rich in long-chain polyunsaturated fatty acids), soybean oil (SO, rich in 18:2n-6) and linseed oil (LO, rich in 18:3n-3), respectively, were fed on juvenile golden pompano for 8 weeks, and the intestinal histology, digestive enzymes activities, immunity and antioxidant indices as well as intestinal microbiome were determined. The results showed that dietary SO significantly impaired intestinal health, and decreased the number and height of intestinal folds, and muscle thickness, as well as the zonula occludens-1 (zo-1) mRNA expression in intestine. Moreover, the two dietary VO significantly decreased the amylase and lipase activities in intestine, and reduced the trypsin activity in the dietary SO group. Furthermore, the two VO diets increased intestinal acid phosphatase (ACP) activity, while intestinal lysozyme (LZM) activity and serum diamine oxidase (DAO) activity in the SO group were also significantly increased (P < 0.05). Analysis of the intestinal microbiota showed that the two VO diets significantly increased the abundance of intestinal potentially pathogenic bacteria (Mycoplasma and Vibrio) and decreased proportions of intestinal probiotics (Bacillus and Lactococcus), especially in the dietary SO group. These results indicate that complete replacement of FO with VO in diets would induce intestinal inflammation and impair intestinal function, which might be due to changes in intestinal microbiota profiles, and that dietary SO would have a more negative effect compared to dietary LO on intestinal health in T. ovatus.

Comparison of the growth performance and long-chain PUFA biosynthetic ability of the genetically improved farmed tilapia (Oreochromis niloticus) reared in different salinities.

To compare the growth and biosynthetic ability of long-chain PUFA (LC-PUFA) of the genetically improved farmed tilapia (GIFT) (Oreochromis niloticus) in different water salinities, an 8-week feeding trial was conducted on the GIFT juveniles at 0, 12 and 24 ‰ (parts per thousand; ppt), respectively, with three isonitrogenous (32 %) and isolipidic (8 %) diets (D1-D3). Diet D1 with fish oils (rich in LC-PUFA) as lipid source was used as the control, while D2 and D3 with vegetable oil (free LC-PUFA) blends as lipid source contained different ratios of linoleic acid (LA, 18 : 2n-6) and α-linolenic acid (ALA, 18 : 3n-3) at 4·04 (D2) and 0·54 (D3), respectively. At the end of feeding trial, the growth performance of D2 and D3 groups under all salinity treatments was as good as that of D1 group, which indicates that the GIFT juveniles may convert dietary LA and ALA into LC-PUFA to meet the requirement of essential fatty acids for normal growth and physiology. When fed the same diets, GIFT at 12 ppt had a better growth performance coupled with a higher liver and muscle arachidonic acid content than those in freshwater. Furthermore, brackish water (24 ppt) significantly promoted the mRNA levels of elongase 5 of very long-chain fatty acids (elovl5) and peroxisome proliferator-activated receptor α (pparα) in liver, when compared with freshwater. These results suggest that the GIFT may display better growth performance together with a relatively higher endogenous LC-PUFA biosynthetic ability under brackish water (12 and 24 ppt), probably through improving the expression of elovl5 and pparα in liver.

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.

Pparγ Is Involved in the Transcriptional Regulation of Liver LC-PUFA Biosynthesis by Targeting the Δ6Δ5 Fatty Acyl Desaturase Gene in the Marine Teleost Siganus canaliculatus.

As the first marine teleost demonstrated to have the ability of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis from C18 PUFA precursors, the rabbitfish Siganus canaliculatus provides us a unique model for clarifying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts aiming at the replacement of dietary fish oil (rich in LC-PUFA) with vegetable oils (rich in C18 PUFA precursors but devoid of LC-PUFA). In the study of transcription regulation of gene encoding the Δ6Δ5 fatty acyl desaturase (Δ6Δ5 Fads), a rate-limiting enzyme catalyzing the first step of LC-PUFA biosynthesis in rabbitfish, a binding site for the transcription factor (TF), peroxisome proliferator-activated receptor γ (Pparγ), was predicted in Δ6Δ5 fads2 promoter by bioinformatics analysis, and thus the present study focused on the regulatory roles of Pparγ on Δ6Δ5 fads2. First, the activity of the Δ6Δ5 fads2 promoter was proved to be downregulated by pparγ overexpression and upregulated by treatment of Pparγ antagonist (GW9662), respectively, in HEK 293T cells with the dual luciferase reporter assay. Pparγ was further confirmed to interact with the promoter by electrophoretic mobility shift assay. Moreover, in S. canaliculatus hepatocyte line (SCHL) cells, GW9662 decreased the expression of pparγ together with increase of Δ6Δ5 fads2 mRNA. Besides, Δ6Δ5 fads2 expression was increased by pparγ RNAi knockdown and reduced by its mRNA overexpression. Furthermore, knockdown of pparγ induced a high conversion of 18:3n-3 to 18:4n-3 and 18:2n-6 to 18:3n-6, while pparγ mRNA overexpression led to a lower conversion of that, and finally a significant decrease of 20:4n-6(ARA), 20:5n-3(EPA), and 22:6n-3(DHA) production. The results indicate that Pparγ is involved in the transcriptional regulation of liver LC-PUFA biosynthesis by targeting Δ6Δ5 fads2 in rabbitfish, which is the first report of Pparγ involvement in the regulation of LC-PUFA biosynthesis in teleosts.

Hnf4α Is Involved in LC-PUFA Biosynthesis by Up-Regulating Gene Transcription of Elongase in Marine Teleost Siganus canaliculatus.

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.

miR-146a is involved in the regulation of vertebrate LC-PUFA biosynthesis by targeting elovl5 as demonstrated in rabbitfish Siganus canaliculatus.

Rabbitfish Siganus canaliculatus is the first marine teleost demonstrated to have the ability to synthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and thus provides us a unique model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. MicroRNAs (miRNAs) were shown to play important roles in the regulation of LC-PUFA biosynthesis in rabbitfish at posttranscriptional level in our previous studies. Here, we focused the roles of miR-146a in such regulation. The expression of miR-146a displayed an inverse pattern with that of elongase 5 (Elovl5), a key enzyme catalyzing the elongation of C18 (18:4n3 and 18:3n6) and C20 (20:5n3 and 20:4n6) PUFA in the LC-PUFA biosynthesis, in vivo in liver of rabbitfish reared under different salinities, as well as in vitro in S. canaliculatus hepatocyte line (SCHL) cells incubated with different fatty acids. Bioinformatics analysis predicted that miR-146a may target the 3'UTR of elovl5 directly, which was confirmed by the dual luciferase reporter assays in HEK 293T cells. Overexpression of miR-146a significantly downregulated the expression of elovl5 in SCHL cells, while knockdown of miR-146a showed an opposite effect. Moreover, up-regulation of miR-146a in SCHL cells significantly suppressed the elongation indexes 20:3n6/18:3n6, 20:4n3/18:4n3 and 22:5n3/20:5n3 associated with Elovl5 catalyzing activity, and consequently reduced the contents of LC-PUFA. These results indicate that miR-146a is involved in the regulation of LC-PUFA biosynthesis through inhibiting the mRNA expression and activity of Elovl5 in rabbitfish, which was for the first time to focus on the role of miR-146a in LC-PUFA biosynthesis in vertebrates and will provide a new insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleosts.