Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.

Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Free, long-chain, polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes.

Because previous studies showed that polyunsaturated fatty acids can reduce the contraction rate of spontaneously beating heart cells and have antiarrhythmic effects, we examined the effects of the fatty acids on the electrophysiology of the cardiac cycle in isolated neonatal rat cardiac myocytes. Exposure of cardiomyocytes to 10 microM eicosapentaenoic acid for 2-5 min markedly increased the strength of the depolarizing current required to elicit an action potential (from 18.0 +/- 2.4 pA to 26.8 +/- 2.7 pA, P < 0.01) and the cycle length of excitability (from 525 ms to 1225 ms, delta = 700 +/- 212, P < 0.05). These changes were due to an increase in the threshold for action potential (from -52 mV to -43 mV, delta = 9 +/- 3, P < 0.05) and a more negative resting membrane potential (from -52 mV to -57 mV, delta = 5 +/- 1, P < 0.05). There was a progressive prolongation of intervals between spontaneous action potentials and a slowed rate of phase 4 depolarization. Other polyunsaturated fatty acids--including docosahexaenoic acid, linolenic acid, linoleic acid, arachidonic acid, and its nonmetabolizable analog eicosatetraynoic acid, but neither the monounsaturated oleic acid nor the saturated stearic acid--had similar effects. The effects of the fatty acids could be reversed by washing with fatty acid-free bovine serum albumin. These results show that free polyunsaturated fatty acids can reduce membrane electrical excitability of heart cells and provide an electrophysiological basis for the antiarrhythmic effects of these fatty acids.

Products of Cytochrome P450BioI (CYP107H1)-Catalyzed Oxidation of Fatty Acids

[GRAPHICS] Oxidation of tetradecanoic and hexadecanoic acids by cytochrome P450(Biol) (CYP107H1) produces mainly the 11-, 12-, and 13-hydroxy C-14 fatty acids and the 11- to 15-hydroxy C-16 fatty acids, respectively. In contrast to previous reports, terminal hydroxylation is not observed. The enantiospecificity of fatty acid hydroxylation by P450(Biol) was also determined, and the enzyme was shown to be moderately selective for production of the (R)-alcohols.

Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds (radical clocks), which can produce both unrearranged and ring opened products upon oxidation, have been key tools in these investigations. In this study, we introduce several cyclopropyl containing fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation. The probes are shown to be capable of distinguishing radical from cationic intermediates due to the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these probes by P450(BM3) and P450(Biol) gives results consistent with a radical but not a cationic intermediate in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened products gives remarkably homogeneous rates for oxygen rebound of (2-3) x 10(10) s(-1). The effects of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation are discussed.

Cholesterol and fatty acids regulate dynamic caveolin trafficking through the golgi complex and between the cell surface and lipid bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Essential fatty acids for dry eye:a review

Purpose Dry eye is a common complaint often encountered in optometric practice. However, it is a difficult condition to treat as clinical signs do not always correlate with patient symptoms. Essential fatty acids (EFA), particularly omega-3 EFA, may be effective in dealing with the underlying causes. Methods A literature review was carried out on the PubMed, ScienceDirect and Ovid databases. Searches included keywords such as ‘dry eye’, ‘essential fatty acids’ and ‘nutrition’ to find articles relating to the treatment of dry eye syndrome (DES) with omega-3 EFAs. Results Omega-3 and -6 EFAs need to be consumed together within a reasonable ratio to be effective. Currently, typical diets in developed countries lack omega-3 EFA and this results in an overexposure to omega-6. Omega-3 supplementation has an anti-inflammatory effect, inhibiting creation of omega-6 prostaglandin precursors. Omega-3 EFAs also demonstrate anti-inflammatory action in the lacrimal gland preventing apoptosis of the secretory epithelial cells. Supplementation clears meibomitis, allowing a thinner, more elastic lipid layer to protect the tear film and cornea. Conclusion Dietary supplementation of omega-3 EFA has already proven to be effective in coronary heart disease and arthritis. Safety is not a concern as it works synergistically with omega-6 in the body. Evidence suggests that supplementation with omega-3 EFA may be beneficial in the treatment and prevention of DES.

Chemometric modelling to relate antioxidants, neutral lipid fatty acids and flavour components in chicken breast

Relationships among quality factors in retailed free-range, corn-fed, organic, and conventional chicken breasts (9) were modeled using chemometric approaches. Use of principal component analysis (PCA) to neutral lipid composition data explained the majority (93%) of variability (variance) in fatty acid contents in 2 significant multivariate factors. PCA explained 88 and 75% variance in 3 factors for, respectively, flame ionization detection (FID) and nitrogen phosphorus (NPD) components in chromatographic flavor data from cooked chicken after simultaneous distillation extraction. Relationships to tissue antioxidant contents were modeled. Partial least square regression (PLS2), interrelating total data matrices, provided no useful models. By using single antioxidants as Y variables in PLS (1), good models (r2 values > 0.9) were obtained for alpha-tocopherol, glutathione, catalase, glutathione peroxidase, and reductase and FID flavor components and among the variables total mono and polyunsaturated fatty acids and subsets of FID, and saturated fatty acid and NPD components. Alpha-tocopherol had a modest (r2 = 0.63) relationship with neutral lipid n-3 fatty acid content. Such factors thus relate to flavor development and quality in chicken breast meat.

Chlorinated lipids and fatty acids:an emerging role in pathology

Although the existence of halogenated lipids in lower organisms has been known for many years, it is only since the 1990s that interest in their occurrence in mammalian systems has developed. Chlorinated (and other halogenated) lipids can arise from oxidation by hypohalous acids, such as HOCl, which are products of the phagocytic enzyme myeloperoxidase and are generated during inflammation. The major species of chlorinated lipids investigated to date are chlorinated sterols, fatty acid and phospholipid chlorohydrins, and a-chloro fatty aldehydes. While all of these chlorinated lipids have been shown to be produced in model systems from lipoproteins to cells subjected to oxidative stress, as yet only a-chloro fatty aldehydes, such as 2-chlorohexadecanal, have been detected in clinical samples or animal models of disease. a-Chloro fatty aldehydes and chlorohydrins have been found to have a number of potentially pro-inflammatory effects ranging from toxicity to inhibition of nitric oxide synthesis and upregulation of vascular adhesion molecules. Thus evidence is building for a role of chlorinated lipids in inflammatory disease, although much more research is required to establish the contributions of specific compounds in different disease pathologies. Preventing chlorinated lipid formation and indeed other HOCl-induced damage, via the inhibition of myeloperoxidase, is an area of growing interest and may lead in the future to antimyeloperoxidase-based antiinflammatory therapy. However, other chlorinated lipids, such as punaglandins, have beneficial effects that could offer novel therapies for cancer.

Fatty acids and stable isotopes as indicators of early-life feeding and potential maternal resource dependency in the bull shark Carcharhinus leucas

The degree of reliance of newborn sharks on energy reserves from maternal resource allocation and the timescales over which these animals develop foraging skills are critical factors towards understanding the ecological role of top predators in marine ecosystems. We used muscle tissue stable carbon isotopic composition and fatty acid analysis of bull sharks Carcharhinus leucas to investigate early-life feeding ecology in conjunction with maternal resource dependency. Values of δ13C of some young-of-the-year sharks were highly enriched, reflecting inputs from the marine-based diet and foraging locations of their mothers. This group of sharks also contained high levels of the 20:3ω9 fatty acid, which accumulates during periods of essential fatty acid deficiency, suggesting inadequate or undeveloped foraging skills and possible reliance on maternal provisioning. A loss of maternal signal in δ13C values occurred at a length of approximately 100 cm, with muscle tissue δ13C values reflecting a transition from more freshwater/estuarine-based diets to marine-based diets with increasing length. Similarly, fatty acids from sharks >100 cm indicated no signs of essential fatty acid deficiency, implying adequate foraging. By combining stable carbon isotopes and fatty acids, our results provided important constraints on the timing of the loss of maternal isotopic signal and the development of foraging skills in relation to shark size and imply that molecular markers such as fatty acids are useful for the determination of maternal resource dependency.

Fatty acids composition of Chylomicron remnants like particles influences their uptake and induction of lipid accumulation in macrophages.

The influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on their uptake and induction of lipid accumulation in macrophages was studied. CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n−6 or n−3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages derived from the human monocyte cell line THP-1 were used. Lipid accumulation (triacylglycerol and cholesterol) in the cells was measured after incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was determined using CRLPs radiolabelled with [3H]triolein. Total lipid accumulation in the macrophages was significantly greater with palm CRLPs than with the other three types of particle. This was mainly due to increased triacylglycerol concentrations, whereas changes in cholesterol concentrations did not reach significance. There were no significant differences in lipid accumulation after incubation with olive, corn or fish CRLPs. Palm and olive CRLPs were taken up by the cells at a similar rate, which was considerably faster than that observed with corn and fish CRLPs. These findings demonstrate that CRLPs enriched in saturated or monounsaturated fatty acids are taken up more rapidly by macrophages than those enriched in n−6 or n−3 polunsaturated fatty acids, and that the faster uptake rate results in greater lipid accumulation in the case of saturated fatty acid-rich particles, but not monounsaturated fatty acid-rich particles. Thus, dietary saturated fatty acids carried in chylomicron remnants may enhance their propensity to induce macrophage foam cell formation.

Deleterious Effects of Lard-enriched Diet on Tissues Fatty Acids Composition and Hypothalamic Insulin Actions

Altered tissue fatty acid (FA) composition may affect mechanisms involved in the control of energy homeostasis, including central insulin actions. In rats fed either standard chow or a lard-enriched chow (high in saturated/low in polyunsaturated FA, HS-LP) for eight weeks, we examined the FA composition of blood, hypothalamus, liver, and retroperitoneal, epididymal and mesenteric adipose tissues. Insulin-induced hypophagia and hypothalamic signaling were evaluated after intracerebroventricular insulin injection. HS-LP feeding increased saturated FA content in adipose tissues and serum while it decreased polyunsaturated FA content of adipose tissues, serum, and liver. Hypothalamic C20:5n-3 and C20:3n-6 contents increased while monounsaturated FA content decreased. HS-LP rats showed hyperglycemia, impaired insulin-induced hypophagia and hypothalamic insulin signaling. The results showed that, upon HS-LP feeding, peripheral tissues underwent potentially deleterious alterations in their FA composition, whist the hypothalamus was relatively preserved. However, hypothalamic insulin signaling and hypophagia were drastically impaired. These findings suggest that impairment of hypothalamic insulin actions by HS-LP feeding was not related to tissue FA composition.

Effects of Different Fatty Acids and Dietary Lipids on Adiponectin Gene Expression in 3T3-L1 Cells and C57BL/6J Mice Adipose Tissue

Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis.