A vertebrate fatty acid desaturase with Δ5 and Δ6 activities

Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity. The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.     

Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants

We report the identification of bifunctional Delta12/omega3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea. The bifunctional activity of these desaturases distinguishes them from all known Delta12 or omega3 fatty acid desaturases. The omega3 desaturase activity of these enzymes also shows a broad omega6 fatty acid substrate specificity by their ability to convert linoleic acid (LA), gamma-linolenic acid, di-homo-gamma-linolenic acid, and arachidonic acid to the omega3 fatty acids, alpha-linolenic acid (ALA), stearidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid (EPA), respectively. Phylogenetic analysis suggests that omega3 desaturases arose by independent gene duplication events from a Delta12 desaturase ancestor. Expression of F. moniliforme Delta12/omega3 desaturase resulted in high ALA content in both Yarrowia lipolytica, an oleaginous yeast naturally deficient in omega3 desaturation, and soybean. In soybean, seed-specific expression resulted in 70.9 weight percent of total fatty acid (%TFA) ALA in a transformed seed compared with 10.9%TFA in a null segregant seed and 53.2%TFA in the current best source of ALA, linseed oil. The ALA/LA ratio in transformed seed was 22.3, a 110- and 7-fold improvement over the null segregant seed and linseed oil, respectively. Thus, these desaturases have potential for producing nutritionally desirable omega3 long-chain polyunsaturated fatty acids, such as EPA, with a significantly improved ratio of omega3/omega6 long-chain polyunsaturated fatty acids in both oilseeds and oleaginous microbes.     

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.     

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.     

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ⁶-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ⁹-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ⁶ or Δ⁹ positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ⁶-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ⁹-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ⁹-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ⁹-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.     

Fatty acid composition and estimated desaturase activities are associated with obesity and lifestyle variables in men and women

It is known that the fatty acid (FA) composition in serum cholesteryl esters to a certain extent mirrors not only the FA composition of dietary fat, but also the endogenous FA synthesis, where desaturases play an important part. A surrogate measure of delta9-, delta6- and delta5-desaturase activity can be calculated as a [product:precursor] fatty acid ratio. Delta9-desaturase activity is known to be high in conditions like diabetes, atherosclerosis and obesity. The aim of the present study was to relate the proportions of individual fatty acids in serum cholesteryl esters, as well as estimated desaturase ratios to markers of obesity and lifestyle variables (smoking, physical activity and dietary fat). We also studied gender differences. These relationships were studied in a reference population consisting of men (n=554) and women (n=295) who took part in a health survey concerning coronary heart disease in Sweden. We found positive and significant correlations between markers of obesity and the proportions of 16:0, 16:1 (n-7), 18:0, 18:3 (n-6), 20:3 (n-6), 20:4 (n-6), 20:5 (n-3), delta9 and delta6 activities, and an inverse correlation to delta5 activity and 18:2 (n-6). These relationships were independent of age and physical activity and in some cases of body mass index (BMI). For each standard deviation (SD) increase of delta9 and delta6 activities, the risk of being overweight was increased by about 60%, whereas the risk was reduced to about 30% for every SD increase of delta5 activity. Women were found to have significantly higher levels of delta9 and lower levels of delta6 desaturase activities than men. In conclusion, this study shows that a changed FA profile in serum cholesteryl esters and estimated desaturase activities are associated with obesity and lifestyle factors in men and women.     

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.     

Altered levels of n-6/n-3 fatty acids in rat heart and storage fat following variable dietary intake of linoleic acid

Fat-supplemented dies enriched with linoleic acid by the addition of 12% w/w sunflower seed oil or proportionally reduced in linoleic acid by addition of 12% mutton fat were fed to rats for 18 months before the fatty acid composition of perirenal storage fat and myocardial membranes (phospholipids) was determined. Although the fatty acid composition of perirenal fat generally reflected that of the diet, there was an inverse relationship between the consumption of n-6 and the deposition of n-9 fatty acids. In addition, enhanced deposition of oleic acid (18:1, n-9) appears to be related to the dietary intake of stearic acid (18:0). In contrast, in myocardial membranes the n-3 polyunsaturated fatty acids are found to be increased when the intake of n-6 polyunsaturated fatty acids is reduced. This is particularly evident for docosahexaenoic acid (22:6, n-3) which is significantly increased in phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol fractions of myocardial membranes, when the mutton fat diet was fed. After feeding the sunflower seed oil diet, the increased consumption of linoleic acid produced only small changes in the 18:2, n-6 content of cardiac phosphatidylcholine and phosphatidylethanolamine. These major classes of membrane phospholipids also showed only small increases in 20:4, n-6. In diphosphatidylglycerol, increased 18:2, n-6 also followed increased dietary intake, but this was not accompanied by increased 20:4, n-6. These changes in myocardial phospholipid fatty acid composition are similar to those observed after short-term feeding reported previously and confirm that changes in dietary n-6/n-3 fatty acid intake affect the fatty acid composition of both myocardial membranes and storage fat. These changes persist for the duration of the feeding period.     

Effect of the linolenic acid content of the mother's diet on the polyunsaturated fatty acid composition of subcellular fractions in brain development in the rat

In order to determine precisely the respective roles of linolenic acid and linoleic acid in the maternal diet on rat brain subcellular fractions during development, we used two diets with different percentages of linolenic acid (18:3 n-3). The animals were fed peanut oil (group A) or soybean oil (group B) during pregnancy and throughout lactation. Nature and amount of essential fatty acids had no incidence on saturated and monounsaturated fatty acid distributions in myelin, synaptosomal, mitochondrial and microsomal fractions. In adult rats, all subcellular fractions are marked by an increase of n-3 fatty acid and a decrease of n-6 fatty acid levels in group B compared to group A. In 15-day-old animals, on the contrary, only the synaptosomal fractions are significantly affected by the diet. Independent of diet, brain development is marked by a decrease of n-6 fatty acids in all subcellular fractions; on the other hand, the n-3 fatty acid level is increased in the synaptosomal and mitochondrial fractions, and decrease in the myelin and microsomal fractions. The sum of (n-3 + n-6) fatty acids remains constant in group B and in group A in all subcellular fractions. Finally, under our experimental conditions, we found no marked effect of diet composition upon linoleic acid conversion to arachidonic acid; only the delta 4-7-10-13-16-docosapentaenoic acid (22:5 n-6) level decreased in group B. delta 7-10-13-16-19-Docosapentaenoic acid (22:5 n-3) seemed to be a better substrate for delta 4 desaturase than delta 7-10-13-16-docosatetraenoic acid (22:4 n-6).     

Retinal function in rats and guinea-pigs reared on diets low in essential fatty acids and supplemented with linoleic or linolenic acids

Rats were reared into a third generation on diets deficient in essential fatty acids supplemented with linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) with the object of depleting the retina of n-6 or n-3 fatty acids. In the rats fed 18:2 n-6 the percentage by weight of 22:6 n-3 in retinal fatty acids fell from 22.5 to 8.5% in first-generation animals but then remained unchanged in second and third generations. There was no difference in b-wave amplitudes of the electroretinogram between the rats fed 18:2 n-6 and those fed 18:3 n-3. In guinea-pigs fed purified diets low in 18:3 n-3 the percentage by weight of 22:6 n-3 in retinas fell from 8 to less than 0.5% by the third generation. However, there were no statistical differences in the b-wave amplitudes between these animals and those reared on a commercial diet. It is concluded that if n-3 fatty acids are involved in retinal function their role is too subtle to be detected by standard electroretinographic techniques.     

Effect of ethanol on polyunsaturated fatty acid biosynthesis in hepatocytes from spontaneously hypertensive rats

BACKGROUND: Polyunsaturated fatty acids (PUFA) play a major role in membrane structures that are modified during alcoholism. PUFA are also precursors of second messengers-eicosanoids-involved in the regulation of blood pressure. Alcohol has been related to hypertension and to alterations in liver PUFA metabolism. We investigated the effects of ethanol on PUFA biogenesis in hepatocytes of Wistar Kyoto (WKY) rats and Spontaneously Hypertensive Rats (SHR). The effects of a diet enriched with n-3 PUFA, which is known to modulate hypertension, were also studied. METHODS: Isolated hepatocytes from male normotensive Wistar Kyoto (WKY) rats and SHR were incubated for 60 min in the presence of labeled linoleic acid and DGLA, which are precursors of the limiting desaturation steps of PUFA biosynthesis, into a medium containing different concentrations of ethanol. Hepatocytes from SHR that were fed a diet supplemented with n-3 PUFA were incubated with the same precursors. RESULTS: First, the hepatic biogenesis of PUFA is dependent on the level of ethanol in the incubation medium. Second, Delta5 desaturase was more sensitive than Delta6 desaturase to changes in alcohol concentration. Third, in SHR, a tremendous decrease of arachidonic acid biosynthesis was evidenced in alcohol-intoxicated hepatocytes; the effect was reinforced when ethanol concentration was high, mainly for Delta5 desaturase. Fourth, in the presence of ethanol, the biogenesis of PUFA was altered in isolated hepatocytes from SHR that were fed the diet supplemented with n-3 PUFA, particularly via an inhibition of Delta5 desaturation. CONCLUSIONS: Our study showed that hepatocyte PUFA biogenesis is dependent on ethanol concentration. Ethanol strongly inhibits the synthesis of PUFA in hepatocytes from SHR, which can explain the deficit of prostaglandin precursors observed in cardiovascular diseases linked to ethanol intoxication. n-3 PUFA supplemented diet reinforces the inhibition of arachidonic acid synthesis, likely by a substrate competition toward Delta5 desaturation. This in vitro approach provides a better understanding of the effects of ethanol on fatty acid metabolism in relation to hypertension.     

Fatty acid profile of erythrocyte membranes as possible biomarker of longevity

Offspring of long-lived individuals are a useful model to discover biomarkers of longevity. The lipid composition of erythrocyte membranes from 41 nonagenarian offspring was compared with 30 matched controls. Genetic loci were also tested in 280 centenarians and 280 controls to verify a potential genetic predisposition in determining unique lipid profile. Gas chromatography was employed to determine fatty acid composition, and genotyping was performed using Taqman assays. Outcomes were measured for erythrocyte membrane percentage content of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids (omega-6 and omega-3), geometrical isomers of arachidonic and oleic acids, and total trans-fatty acids. Also, allele and genotyping frequencies at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci were considered. Erythrocyte membranes from nonagenarian offspring had significantly higher content of C16:1 n-7, trans C18:1 n-9, and total trans-fatty acids, and reduced content of C18:2 n-6 and C20:4 n-6. No association was detected at endothelial-nitric oxide synthase and delta-5/delta-6 and delta-9 desaturase loci that could justify genetic predisposition for the increased trans C18:1 n-9, monounsaturated fatty acids and decreased omega-6 synthesis. We concluded that erythrocyte membranes derived from nonagenarian offspring have a different lipid composition (reduced lipid peroxidation and increased membrane integrity) to that of the general population.     

Effect of dietary cholesterol on the release of prostacyclin from the mesenteric vascular bed in diabetic rat

Streptozotocin-induced diabetic female rats and age-matched control rats were fed a regular chow with or without the addition of 1% cholesterol in the diet. The release of 6-keto-PGF1 alpha, a prostacyclin metabolite, from the mesenteric vascular bed was significantly increased in diabetic rats. The production of PGI2 in diabetic rats was significantly reduced whereas that in the control was not affected by cholesterol feeding. The examination of the fatty acid composition of phospholipids from the mesenteric vascular bed indicated that proportions of stearic (18:0), linoleic (18:2n-6) and dihomo-gamma-linolenic (20:3n-6) acids were higher whereas those of oleic (18:1n-9) was lower in diabetic rats than those in the controls. Cholesterol feeding had no significant effect on the levels of arachidonic acid (20:4n-6) in the controls but it significantly decreased those in diabetic rats. It is suggested that cholesterol feeding lowers the release of PGI2 from the mesenteric vascular bed possibly because of a reduced level of arachidonic acid, the major precursor for prostaglandin synthesis. This could be due to an impairment of delta-5 desaturase.     

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.     

Abnormal regulation of serum lipid fatty acid profiles in short gut rats fed parenteral nutrition with lipid

Despite absence of essential fatty acid deficiency (EFAD), increases in arachidonic acid to linoleic acid ratios occur in serum phospholipid of patients treated with chronic total parenteral nutrition (TPN). The parenteral lipid component of TPN contains abundant linoleate; thus low phospholipid linoleate may reflect increased conversion to arachidonate. Arachidonic acid excess has been associated with a proinflammatory milieu through increased eicosanoid production and might contribute to the increases in inflammatory markers seen in home TPN patients. We investigated fatty acid metabolism in a rodent model of malabsorption. We hypothesized that short gut rats would metabolize parenteral lipid differently from intact rats. We performed laparotomy and 80% small bowel resection (or sham surgery) in rats. Sixteen sham and 16 short gut rats were randomly assigned to TPN with lipid or fat-free TPN. After 5 days, weight loss was similar in all groups. Analysis of serum phospholipids demonstrated that 20:3omega9 (eicosatrienoic acid) was relatively increased in fat-free TPN groups, irrespective of surgery type, as were distal very long chain omega3 class fatty acids, as anticipated. Uniquely, both nutrition (TPN/lipid v fat-free TPN) and surgery type (sham v short gut) were significant in determining arachidonic acid levels. Relatively elevated arachidonate occurred in both groups of fat-free rats, suggesting increased Delta6 and/or Delta5 desaturase activity, as expected. In contrast, giving TPN/lipid lowered arachidonate (suggesting appropriately downregulated desaturases) in sham animals, but not in short gut animals. Ratios of arachidonic and di-homo-gamma-linolenic to linoleic acids further suggested increased turnover of precursor omega6 to arachidonic acid in short gut rats given lipid compared with the other groups. These preliminary data show that intravenous (IV) lipid gave rise to serum lipid fatty acid profiles that differed in short gut and sham rats. The short gut rat may have a heightened hepatic desaturase activity, inappropriate for the quantity of linoleic acid provided parenterally. Therefore, the short gut rat is an appropriate model to study further arachidonic acid excess in home TPN patients.     

Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: functional and biochemical consequences in normoxic and hypoxic conditions

The respective roles of exogenous polyunsaturated fatty acids on the lipid composition, physiological properties and enzyme release was investigated on isolated cardiac muscle cells in normoxia and hypoxia. Rat neonatal ventricular myocytes were grown for 5 days in conventional serum-supplemented medium. Cells were then incubated for 24 h in fully chemically-defined media featuring a balanced fatty acid composition containing either linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) as sole polyunsaturated fatty acid source. Transmembrane potentials were monitored with microelectrodes and contractions with a photoelectric device. The radio of n-6 to n-3 phospholipid fatty acids increased from 6.3 in control cells to 20.2 in cells exposed to n-6 fatty acids (SM6) and decreased to 1.4 in those exposed to n-3 fatty acids (SM3). These modifications had no influence on the electrical and mechanical activities and on automaticity in normoxic conditions. The action potential depression under hypoxia was less severe in SM6 cells, whereas there was a better electrophysiological recovery upon reoxygenation in SM3 cells. However, the loss of lactate dehydrogenase during sustained hypoxic treatment was not affected by changes in phospholipid fatty acid pattern. These results suggest that the effect of the polyunsaturated fatty acid balance depends on the cellular function under study and on the environmental conditions.     

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.     

Metabolism of long-chain polyunsaturated fatty acids in rat kidney cells

The metabolism of long-chain polyunsaturated fatty acids is characterized in tissues, such as liver and heart, especially from studies based on isolated cells incubated with radiolabelled fatty acid substrates. Differently, only little is known about the metabolism of fatty acids in the kidney. It is controversial whether the kidney possesses the ability to desaturate long-chain fatty acids or whether kidney cells are dependent on performed polyunsaturated fatty acids transported from the liver. In this study we used isolated rat kidney cells obtained by a perfusion technique. The cells were incubated with [1-(14)C]-labelled 18:3(n-3) or 20:3(n-6) fatty acids which were incorporated into complex lipids or desaturated/elongated. The lipids were separated by thin-layer chromatography and high-performance liquid chromatography. The present study shows that isolated kidney cells take up and esterify labelled long-chain polyunsaturated fatty acids in a time- and concentration-dependent manner. We have also demonstrated that isolated rat kidney cells only to a minor extent Delta6-desaturate labelled 18:3(n-3) to 18:4 (n-3). Conversely, the Delta 5-desaturation of 20:3(n-6) to 20:4(n-6) is far more active. It may thus be concluded that the kidney, at least in part, must obtain its C(20) and C(22) fatty acids from the circulation, while the active Delta5-desaturase suggests that preformed C(20) fatty acids can be converted to more unsaturated homologues in the kidney.     

Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ^6,9,12) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a Δ⁶-fatty acid desaturase using linoleic acid (C18:2 Δ^9,12) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ⁶-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 Δ^6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ⁶-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b₅.