Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence.

3-Methyl-branched fatty acids, as phytanic acid, undergo peroxisomal alpha-oxidation in which they are shortened by 1 carbon atom. This process includes four steps: activation, 2-hydroxylation, thiamine pyrophosphate dependent cleavage and aldehyde dehydrogenation. The thiamine pyrophosphate dependence of the third step is unique in peroxisomal mammalian enzymology. Human pathology due to a deficient alpha-oxidation is mostly linked to mutations in the gene coding for the second enzyme of the sequence, phytanoyl-CoA hydroxylase.     

Phytanic acid alpha-oxidation: identification of 2-hydroxyphytanoyl-CoA lyase in rat liver and its localisation in peroxisomes.

Phytanic acid is broken down by alpha-oxidation in three steps finally yielding pristanic acid. The first step occurs in peroxisomes and is catalysed by phytanoyl-CoA hydroxylase. We have studied the second step in the alpha-oxidation pathway, which involves conversion of 2-hydroxyphytanoyl-CoA to pristanal catalysed by 2-hydroxyphytanoyl-CoA lyase. To this end, we have developed a stable isotope dilution gas chromatography-mass spectrometry assay allowing activity measurements in rat liver homogenates. Cell fractionation studies demonstrate that in rat liver 2-hydroxyphytanoyl-CoA lyase is localised in peroxisomes. This finding may have important implications for inherited diseases in man characterised by impaired phytanic acid alpha-oxidation.     

Peroxisomal lipid degradation via β-and α-oxidation in Mammals

Peroxisomal β-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol). In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are β-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA, dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of β-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened, via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized. 3-Methyl-branched fatty acids such as phytanic acid are not directly β-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal β-oxidation. In the human and the rat, α-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO₂. The activation, hydroxylation and cleavage reactions and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO₂ occurs mainly in the cytosol.     

Identification of pristanal dehydrogenase activity in peroxisomes: conclusive evidence that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which, due to the methyl-group at the 3-position, can not undergo beta-oxidation unless the terminal carboxyl-group is removed by alpha-oxidation. The structure of the phytanic acid alpha-oxidation machinery in terms of the reactions involved, has been resolved in recent years and includes a series of four reactions: (1) activation of phytanic acid to phytanoyl-CoA, (2) hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, (3) cleavage of 2-hydroxyphytanoyl-CoA to pristanal and formyl-CoA, and (4) oxidation of pristanal to pristanic acid. The subcellular localization of the enzymes involved has remained enigmatic, with the exception of phytanoyl-CoA hydroxylase and 2-hydroxyphytanoyl-CoA lyase which are both localized in peroxisomes. The oxidation of pristanal to pristanic acid has been claimed to be catalysed by the microsomal aldehyde dehydrogenase FALDH encoded by the ALDH10-gene. Making use of mutant fibroblasts deficient in FALDH activity, we show that phytanic acid alpha-oxidation is completely normal in these cells. Furthermore, we show that pristanal dehydrogenase activity is not fully deficient in FALDH-deficient cells, implying the existence of one or more additional aldehyde dehydrogenases reacting with pristanal. Using subcellular localization studies, we now show that peroxisomes contain pristanal dehydrogenase activity which leads us to conclude that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes.     

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.     

Alpha-oxidation of 3-methyl-substituted fatty acids in rat liver.

3-Methyl-substituted fatty acids are first oxidatively decarboxylated (alpha-oxidation) before they are degraded further via beta-oxidation. We synthesized [1-14C]phytanic and 3-[1-14C]methylmargaric acids in order to study their alpha-oxidation in isolated rat hepatocytes, rat liver homogenates and subcellular fractions. alpha-Oxidation was measured as the production of radioactive CO2. In isolated hepatocytes, maximal rates of alpha-oxidation amounted to 7 and 10 nmol/min x 10(8) cells with phytanic acid and 3-methylmargaric acid, respectively. At equimolar substrate concentrations, alpha-oxidation of branched fatty acids was approximately 10- to 15-fold slower than the beta-oxidation of the straight chain palmitate. In whole liver homogenates, rates of alpha-oxidation that equaled 60 to 70% of those observed in the hepatocytes were obtained. Optimum rates required O2, NADPH, Fe3+, and ATP. Fe3+ could be replaced by Fe2+ and ATP could be replaced by a number of other phosphorylated nucleosides and even inorganic phosphate without loss of activity. NADH could substitute for NADPH but not always with full restoration of activity. A variety of other cofactors and metal ions was either inhibitory or without effect. Scavengers of reactive oxygen species, known to be formed during the NADPH-dependent microsomal reduction of ferric-phosphate complexes, were without effect on alpha-oxidation. No evidence was found for the accumulation of NADPH-dependent or Fe(3+)-dependent reaction intermediates. Subcellular fractionation of liver homogenates demonstrated that alpha-oxidation was located predominantly, if not exclusively, in the endoplasmic reticulum. alpha-Oxidation, measured in microsomal fractions, was not inhibited by CO, cytochrome c, or ferricyanide, indicating that NADPH cytochrome P450 reductase and cytochrome P450 are not involved in alpha-oxidation. Our results indicate that, contrary to current belief, alpha-oxidation is catalyzed by the endoplasmic reticulum. The cofactor requirements suggest that alpha-oxidation involves the reduction of Fe3+ by electrons from NADPH and that it is stimulated by phosphate ions and nucleotides.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.     

Preparation of N-acylated proteins modified with fatty acids having a specific chain length using an insect cell-free protein synthesis system

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.     

Chemical diagnosis of inherited defects of fatty acid metabolism and ketogenesis

Urinary organic acid profiles in patients with inherited defects of fatty acid metabolism and ketogenesis are described. Medium-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, multiple acyl-CoA dehydrogenase, and 3-hydroxy-3-methyl-glutaryl-CoA lyase deficiencies can be recognized at the metabolite level. Data on long-chain acyl-CoA dehydrogenase and systemic carnitine deficiencies are scarce. In the latter disorders, dicarboxylic aciduria is rather nonspecific and points to a modest omega-oxidation of long chain fatty acids.     

The enzymic formation of long chain aldehydes and alcohols by alpha-oxidation of fatty acids in extracts of cucumber fruit (Cucumis sativus).

1. An enzyme system that catalyses the alpha-oxidation of fatty acids to shorter chain products is present in acetone powders of cucumber fruits. 2. In the absence of NAD+, the predominant product from palmitic acid is pentadecanal. Addition of NAD+ gives rise to a homologous series of n-alkanals, the concentrations of which are in the same order as that reported in the volatile products formed on homogenization of cucumbers, i.e. C15 greater than C14 greater than C13 greater than C12. 3. Pentadecan-1-ol is also formed from palmitic acid in the absence of added NAD+; C15, C14 and C13 n-alkanols are produced in the presence of NAD+. 4. The substrate specificity for saturated fatty acids is in the order C12 less than C14 greater than C16 greater than C18. Unsaturated C18 acids are oxidized more readily than stearic acid. 5. The alpha-oxidation system is inhibited by dithiothreitol, cysteine, imidazole and certain metal ligands (CN-, N3-, diphenylthiocarbazone) but not by EDTA. 6. Differences between the alpha-oxidation system in cucumber and those previously reported in other plants are discussed.     

alpha-oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an alpha-dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid.

A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523-1537). Linolenic acid incubated at 23 degrees C with preparations containing the recombinant enzymes underwent alpha-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z), 14(Z)-heptadecatrienal (83%), an alpha-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 degrees C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z), 12(Z),15(Z)-octadecatrienoic acid in alpha-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same alpha-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z), 15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 degrees C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 degrees C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for alpha-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic alpha-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into alpha-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both alpha-dioxygenases, and that members of this new class of enzymes catalyze the first step of alpha-oxidation in plant tissue.     

Maternal dietary n-3 fatty acids alter cardiac ventricle fatty acid composition, prostaglandin and thromboxane production in growing chicks

The effects of feeding n-6 and n-3 fatty acids to broiler hens on cardiac ventricle fatty acid composition, and prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) production of hatched chicks were investigated. Fertile eggs obtained from hens fed diets supplemented with 3.5% sunflower oil (Low n-3), 1.75% sunflower+1.75% fish oil (Medium n-3), or 3.5% fish oil (High n-3) were incubated. The hatched chicks were fed a diet containing 18:3 n-3, but devoid of longer chain n-6 and n-3 fatty acids for 42 days. Arachidonic acid content was lower in the cardiac ventricle of High n-3 and Medium n-3 compared to Low n-3 birds for up to 2 weeks (P<0.002). Long chain n-3 fatty acids were higher in the cardiac ventricle of chicks from hens fed High and Medium n-3 diets when compared to chicks from hens fed the Low n-3 diet. Differences in long chain n-3 fatty acids persisted up to four weeks of age (P<0.001). Peripheral blood mononuclear cells (PBMNC) of 7-day-old High n-3 broilers produced significantly lower PGE2 and TXA2 than PBMNC from Low n-3 and Medium n-3 birds. These results indicate that maternal dietary n-3 fatty acids increases cardiac ventricle n-3 fatty acids while reducing arachidonic acid and ex vivo PGE2 and TXA2 production during growth in broiler chickens.     

Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of greater than 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.     

Fatty acid desaturase 2 (FADS2) but not FADS1 desaturates branched chain and odd chain saturated fatty acids

Branched chain fatty acids (BCFA) and linear chain/normal odd chain fatty acids (n-OCFA) are major fatty acids in human skin lipids, especially sebaceous gland (SG) wax esters. Skin lipids contain variable amounts of monounsaturated BCFA and n-OCFA, in some reports exceeding over 20% of total fatty acids. Fatty acid desaturase 2 (FADS2) codes for a multifunctional enzyme that catalyzes Δ4-, Δ6- and Δ8-desaturation towards ten unsaturated fatty acids but only one saturate, palmitic acid, converting it to 16:1n-10; FADS2 is not active towards 14:0 or 18:0. Here we test the hypothesis that FADS2 also operates on BCFA and n-OCFA. MCF-7 cancer cells stably expressing FADS1 or FADS2 along with empty vector control cells were incubated with anteiso-15:0, iso-16:0, iso-17:0, anteiso-17:0, iso-18:0, or n-17:0. BCFA were Δ6-desaturated by FADS2 as follows: iso-16:0 → iso-6Z-16:1, iso-17:0 → iso-6Z-17:1, anteiso-17:0 → anteiso-6Z-17:1 and iso-18:0 → iso-6Z-18:1. anteiso-15:0 was not desaturated in either FADS1 or FADS2 cells. n-17:0 was converted to both n-6Z-17:1 by FADS2 Δ6-desaturation and n-9Z-17:1 by SCD Δ9-desaturation. We thus establish novel FADS2-coded enzymatic activity towards BCFA and n-OCFA, expanding the number of known FADS2 saturated fatty acid substrates from one to six. Because of the importance of FADS2 in human skin, our results imply that dysfunction in activity of sebaceous FADS2 may play a role in skin abnormalities associated with skin lipids.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).