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.

Ubiquitous Promoters Direct the Expression of Fatty Acid Delta-6 Desaturase from Nile Tilapia (Oreochromis niloticus) in Saccharomyces cerevisiae.

In general, promoters have significant influence on recombinant protein production. Herein, we compared the performance of actin (pACT), phosphoglycerate kinase (pPGK), and translational elongation factor (pTEF) promoters for driving the expression of fatty acid delta-6 (Δ6) desaturase from Nile tilapia (Oreochromis niloticus; Oni-fads2) in Saccharomyces cerevisiae. Our results showed that by applying real-time RT-PCR, the highest level of Oni-fads2 mRNA was observed in S. cerevisiae carrying the expression vector driven by pTEF promoters. Exogenous substrate C18:2n-6 was used to determine Δ6 activity by quantitatively determining the C18:3n-6 product. The results showed that highest Δ6 desaturation was observed when using pTEF as a promoter. Recombinant S. cerevisiae cells expressing Oni-fads2 driven by pTEF were tested with the substrate C18:3n-3, and Δ6 desaturation efficiently converted C18:3n-3 to C18:4n-3. Furthermore, crude extract of recombinant yeast also exhibited Δ6 activity. Thus, recombinant S. cerevisiae cells expressing Oni-fads2 driven by the pTEF promoter have potential as a yeast factory for the sustainable production of long-chain polyunsaturated fatty acids.

Δ6-Desaturase from Mortierella alpina: cDNA cloning, expression, and phylogenetic analysis.

The open reading frame of the Δ(6)-desaturase gene was isolated from Mortierella alpina W15 and the gene was cloned into a pPIC3.5K vector. The vector was transformed into Pichia pastoris GS115 and expression was induced with methanol. The Δ(6)-desaturase expressed in P. pastoris GS115 catalyzed the conversion of linoleic acid to γ-linolenic acid but not the conversion of α-linolenic acid to octadecatetraenoic acid. The results indicate that the Δ(6)-desaturase gene from M. alpina W15 has substrate specificity in different organisms. Phylogenetic analysis revealed that Δ(6)-desaturase genes can be divided into four monophyletic groups. This work paves the way for further study of the functions of Δ(6)-desaturase in fatty acid metabolism and its three-dimensional structure.

Hepatic lipid composition and stearoyl-coenzyme A desaturase 1 mRNA expression can be estimated from plasma VLDL fatty acid ratios.

BACKGROUND: Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the limiting step of monounsaturated fatty acid synthesis in humans and is an important player in triglyceride generation. SCD1 has been repeatedly implicated in the pathogenesis of metabolic and inflammatory diseases. Therefore it is of great importance to determine SCD1 activity in human samples. In this study we aimed to evaluate a hepatic SCD1 activity index derived from plasma VLDL triglyceride composition as a tool to estimate hepatic SCD1 expression in humans. Additionally, we further evaluated commonly used fatty acid ratios [elongase, de novo lipogenesis, and Delta5 and Delta6 desaturase] in plasma VLDL and hepatic lipid fractions. DESIGN AND METHODS: Liver biopsies and plasma samples were simultaneously collected from 15 individuals. Plasma VLDL was obtained by ultracentrifugation. Hepatic and plasma VLDL lipids were fractionated by thin-layer chromatography, and the fatty acid composition of each fraction was analyzed by gas chromatography. Hepatic SCD1 expression was determined by real-time PCR. RESULTS: Hepatic SCD1 mRNA expression was associated with the product/precursor ratios (16:1/16:0 and 18:1/18:0) of hepatic lipid fractions. The 16:1/16:0 ratio in hepatic and VLDL triglycerides as well as the 18:1/18:0 ratio in plasma VLDL were closely associated with hepatic SCD1 expression. The hepatic de novo lipogenesis index from triglycerides was associated with expression of lipogenic genes [fatty acid synthase (FASN), acetyl-Coenzyme A carboxylase alpha (ACACA), and sterol regulatory element binding transcription factor 1 (SREBP-1)] and is closely reflected by the de novo lipogenesis index in VLDL triglycerides. CONCLUSION: We demonstrated for the first time that hepatic SCD1 expression can be estimated noninvasively from routine blood samples by measuring the SCD1 activity index in fasting plasma VLDL.

A minimal model for hepatic fatty acid balance during fasting: application to PPAR alpha-deficient mice.

The purpose of this study is to identify the hierarchy of importance amongst pathways involved in fatty acid (FA) metabolism and their regulators in the control of hepatic FA composition. A modeling approach was applied to experimental data obtained during fasting in PPARalpha knockout (KO) mice and wild-type mice. A step-by-step procedure was used in which a very simple model was completed by additional pathways until the model fitted correctly the measured quantities of FA in the liver. The resulting model included FA uptake by the liver, FA oxidation, elongation and desaturation of FA, which were found active in both genotypes during fasting. From the model analysis we concluded that PPARalpha had a strong effect on FA oxidation. There were no indications that this effect changes during the fasting period, and it was thus considered to be constant. In PPARalpha KO mice, FA uptake was identified as the main pathway responsible for FA variation in the liver. The models showed that FA were oxidized at a constant and small rate, whereas desaturation of FA also occurred during fasting. The latter observation was rather unexpected, but was confirmed experimentally by the measurement of delta-6-desaturase mRNA using real-time quantitative PCR (QPCR). These results confirm that mathematical models can be a useful tool in identifying new biological hypotheses and nutritional routes in metabolism.

Hypermethylation of Fads2 and altered hepatic fatty acid and phospholipid metabolism in mice with hyperhomocysteinemia.

Alterations in lipid metabolism may play a role in the vascular pathology associated with hyperhomocysteinemia (HHcy). Homocysteine is linked to lipid metabolism through the methionine cycle and the synthesis of phosphatidylcholine (PC) by phosphatidylethanolamine (PE) methyltransferase, which is responsible for the synthesis of 20-40% of liver PC. The goal of the present study was to determine if the reduced methylation capacity in HHcy is associated with alterations in liver phospholipid and fatty acid metabolism. Mice heterozygous for disruption of cystathionine beta-synthase (Cbs+/-) fed a diet to induce HHcy (HH diet) had higher (p<0.001) plasma total homocysteine (30.8+/-4.4 microM, mean+/-S.E.) than C57BL/6 mice (Cbs+/+) fed the HH diet (7.0+/-1.1 microM) or Cbs+/+ mice fed a control diet (2.3+/-0.3 microM). Mild and moderate HHcy was accompanied by lower adenosylmethionine/adenosylhomocysteine ratios (p<0.05), higher PE (p<0.05) and PE/PC ratios (p<0.01), lower PE methyltransferase activity (p<0.001), and higher linoleic acid (p<0.05) and lower arachidonic acid (p<0.05) in PE. Mice with moderate HHcy also had higher linoleic acid and alpha-linolenic acid (p<0.05) and lower arachidonic acid and docosahexaenoic acid (p<0.05) in liver PC. The first step in the desaturation and elongation of linoleic acid and linolenic acid to arachidonic acid and docosahexaenoic acid, respectively, is catalyzed by Delta6-desaturase (encoded by Fads2). We found hypermethylation of the Fads2 promoter (p<0.01), lower Fads2 mRNA (p<0.05), and lower Delta6-desaturase activity (p<0.001) in liver from mice with HHcy. These findings suggest that methylation silencing of liver Fads2 expression and changes in liver fatty acids may contribute to the pathology of HHcy.

Diet and gene expression: delta-5 and delta-6 desaturases in healthy Chinese and European subjects.

AIM: To compare the composition of fatty acids (FAs) in diet, and the expression of delta-6 desaturase (D6D) and delta-5 desaturase (D5D) genes in peripheral blood mononuclear cells (PBMCs) between Chinese and Europeans. METHODS: Three-day dietary records from 20 subjects from Beijing, China (n = 10) and Kent, UK (n = 10) were analysed. Expression of PBMC D6D and D5D genes of the subjects was determined using RT-PCR. RESULTS: The dietary intake of Chinese subjects contained less saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), but more essential fatty acids (EFAs) than that of Europeans. Levels of expression of PBMC D6D and D5D genes of Chinese subjects were significantly lower than those of Europeans. A significant positive correlation was found between dietary intake of total SFAs and total MUFAs and expression of PBMC D6D and D5D genes, but a significant negative correlation between dietary intake of linoleic acid (LA) and alpha-linolenic acid (LNA) and the expression of PBMC D6D and D5D genes. CONCLUSION: Intake of high SFAs and MUFAs appears to increase expression of PBMC D6D and D5D genes, whilst high EFAs intake appears to decrease expression of PBMC D6D and D5D genes. A follow-up study of the expression of D6D and D5D genes in Chinese who live in European countries with high SFA and MUFA diets would be of interest.

Distribution and metabolism of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) by oral supplementation in rats.

We compared the dietary effects of dihomo-gamma-linolenic acid (DGLA) contained in the DGLA oil produced by a fungus with gamma-linolenic acid (GLA) on the fatty acid composition. Wistar rats were fed with three kinds of oil for two weeks as follows: (i) control group: corn oil; (ii) GLA group: borage oil; (iii) DGLA group: DGLA oil/safflower oil = 55:45. The DGLA concentrations in the liver, serum, and brain of the DGLA group were higher than those of the GLA oil group. We also examined the dose effect of DGLA. The DGLA levels in the liver, serum, and brain significantly increased with increasing dosage of DGLA in the diet. DGLA administration significantly increased the ratio of PGE1/PGE2 in the rat plasma. The mechanism for GLA administration to improve atopic eczema is thought to involve an increase in the concentration of DGLA metabolized from GLA, so these results suggest that the dietary effect of DGLA would be more dominant than GLA.

Effect of dietary conjugated linoleic acid (CLA) on lipid composition, metabolism and gene expression in Atlantic salmon (Salmo salar) tissues.

Dietary conjugated linoleic acid (CLA) affects fat deposition and lipid metabolism in mammals, including livestock. To determine CLA effects in Atlantic salmon (Salmo salar), a major farmed fish species, fish were fed for 12 weeks on diets containing fish oil or fish oil with 2% and 4% CLA supplementation. Fatty acid composition of the tissues showed deposition of CLA with accumulation being 2 to 3 fold higher in muscle than in liver. CLA had no effect on feed conversion efficiency or growth of the fish but there was a decreased lipid content and increased protein content after 4% CLA feeding. Thus, the protein:lipid ratio in whole fish was increased in fish fed 4% CLA and triacylglycerol in liver was decreased. Liver beta-oxidation was increased whilst both red muscle beta-oxidation capacity and CPT1 activity was decreased by dietary CLA. Liver highly unsaturated fatty acid (HUFA) biosynthetic capacity was increased and the relative proportion of liver HUFA was marginally increased in salmon fed CLA. CLA had no effect on fatty acid Delta6 desaturase mRNA expression, but fatty acid elongase mRNA was increased in liver and intestine. In addition, the relative compositions of unsaturated and monounsaturated fatty acids changed after CLA feeding. CLA had no effect on PPARalpha or PPARgamma expression in liver or intestine, although PPARbeta2A expression was reduced in liver at 4% CLA feeding. CLA did not affect hepatic malic enzyme activity. Thus, overall, the effect of dietary CLA was to increase beta-oxidation in liver, to reduce levels of total body lipid and liver triacylglycerol, and to affect liver fatty acid composition, with increased elongase expression and HUFA biosynthetic capacity.

Highly unsaturated fatty acid synthesis in marine fish: cloning, functional characterization, and nutritional regulation of fatty acyl delta 6 desaturase of Atlantic cod (Gadus morhua L.).

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a delta6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5'-UTR, a 375-bp 3'-UTR, and an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed delta6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the delta6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the delta6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the delta6 desaturase appears to be barely functional in cod under the conditions tested.

[Heteroexpression of Rhizopus arrhizus delta6-fatty acid desaturase gene in Pichia pastoris].

Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.