Influenza virus replication in lung epithelial cells depends on redox‐sensitive pathways activated by NOX4‐derived ROS

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus‐induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4‐derived ROS in activating redox‐regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4‐mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.     

Polyunsaturated fatty acids and choline in dairy goats nutrition: Production and health benefits

The potential to modify milk fatty acid composition and milk production by dietary administration of marine oils rich in n-3 PUFAs in goats diets is reviewed. Moreover animal and human health implications are considered. Role of nutrition in dairy goats for enhancing content of CLA in milk fat is also discussed. At last, rumen protected choline supplementation is evaluated to improve productive performance and metabolic health. While the effects of n-3 PUFAs administration on goat productive performance seem to depend on many factors, fish oil administration has been extensively shown to lower average concentration of C18:0 and saturated fatty acids, with a relative increase of C16:1, C18:3 n-3 and very long-chain n-3 PUFAs. Positive results have been evidenced in animals health following administration of EPA and DHA from fish oil, leading to increased phagocytic activity with no effects on extracellular ROS production in incubated goats cells. The nutritional and health properties of goat's milk could be further improved by increasing the content of CLA in milk fat. Provision of PUFAs from fresh pasture and plant lipids, mainly C18:2 n-6 and C18-3 n3 which serve as precursor for trans C18:1 formation in the rumen, have proved to enhance content of CLA in goat milk fat. Marine oils rich in n-3 PUFAs have been shown to be very effective at increasing CLA content in bovine milk, but very scarce data are available on dairy goats.Rumen protected choline has been show to increase productive performance, particularly milk production, fat percentage, and fat and protein yield without detrimental effects on methyl groups, thus reducing BHBA plasma content and hepatocellular lipid accumulation around transition.However the magnitude of the production response seems to be affected by the composition of the diet, and other factors as already reported for n-3 PUFAs administration.     

Transgenic conversion of omega-6 into omega-3 fatty acids in a mouse model of Parkinson's disease

We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.     

Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs

Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.     

Comparison of stearidonic acid and alpha-linolenic acid on PGE2 production and COX-2 protein levels in MDA-MB-231 breast cancer cell cultures

The bioactivity of stearidonic acid (SDA, 18:4n-3) and alpha-linolenic acid (LNA, 18:3n-3) on cyclooxygenase-2 (COX-2) enzyme expression and prostaglandin E2 (PGE2) production has not been evaluated. This investigation examined the effects of SDA and LNA on PGE2 biosynthesis and COX-2 protein and mRNA levels in MDA-MB-231 human breast cancer cells. Cells were supplemented with SDA, LNA, linoleic acid and arachidonic acid (AA) at concentrations ranging from 10 to 200 microM. At 50 and 200 microM, both SDA and LNA treatments and their combinations reduced PGE2 production as compared with AA. At 50 microM, SDA treatment also lowered the COX-2 protein level as compared with the vehicle, but this reduction was not observed with the LNA treatment. Gas chromatographic analysis of fatty acids in cellular lipids of breast cancer cells revealed that SDA led to significantly greater concentrations of 20:5n-3 and other long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) (20:4n-3, 22:4n-3 and 22:5n-3) as compared with the LNA treatment. Both SDA and LNA reduced the level of 20:4n-6; however, SDA was more effective than LNA in decreasing the ratio of n-6/n-3 PUFAs in cells. In addition, SDA was more potent than LNA in suppressing the expression of the COX-2 gene, which was associated with the reduction in the levels of nuclear factor kappa B and peroxisome proliferator-activated receptor gamma mRNA. This study showed that although PGE2 production in MDA-MB-231 breast cancer cells was not significantly different between the SDA and LNA treatments, SDA was more effective than LNA in converting into LC n-3 PUFAs and in reducing COX-2 protein and mRNA levels.     

A modified <Emphasis Type="Italic">n</Emphasis>–3 fatty acid desaturase gene from <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis> produced high proportion of DHA and DPA in transgenic mice

The functions of polyunsaturated fatty acids (PUFAs) have been widely investigated. In mammals, levels of n-3 PUFAs are relatively low compared to those of n-6 PUFAs. Either a lack of n-3 PUFAs or an excess of n-6 PUFAs could potentially cause health problems in humans. Hence, methods to increase the amount of n-3 PUFAs in diet have been intensely sought. In this study, we demonstrated that the n-3 fatty acid desaturase gene (sFat-1) synthesized from revised and optimized codons based on roundworm Caenorhabditis briggsae genomic gene for enhanced expression in mammals was successfully expressed in Chinese hamster ovary (CHO) cells and significantly elevated cellular n-3 PUFA contents. We generated sFat-1 transgenic mice by introducing mammal expression vector DNAs containing the sFat-1 gene into regular mice through the method of microinjection. Fatty acid compositions were then altered and the levels of docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acid (DPA, 22:5n-3) were greatly increased in these transgenic mice. Various types of tissues in the transgenic mice produced many types of n-3 PUFAs, such as alpha-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), DPA, and DHA, for example, muscle tissues of the transgenic mice contained 12.2% DHA, 2.0% DPA, and 23.1% total n-3 PUFAs. These research results demonstrated that the synthesized sFat-1 gene with modified and optimized codons from C. briggsae possess functional activity and greater capability of producing n-3 PUFAs, especially DHA and DPA, in transgenic mice.     

Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition

The hypolipidemic fibrates have been identified as agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a critical role in the regulation of cardiac fatty acid metabolism. Despite the widespread clinical use of fibrates, their role in myocardial oxidative stress and fatty acid composition is less known. In this study, male Sprague-Dawley rats were treated with either vehicle (olive oil, 1 ml/kg) or clofibrate (300 mg/kgday i.p.) for 1-14 days. Lipid peroxidation in heart homogenate was determined by thiobarbituric acid reactive substance (TBARS) assay. Results show that hearts from clofibrate-treated rats are more susceptible to FeSO(4)-induced TBARS production. The antioxidants including catalase and glutathione-related enzymes were marginally affected. We demonstrated that myocardial fatty acid composition was dramatically altered by clofibrate treatment. In hearts from clofibrate-treated rats, the principal n-6 polyunsaturated fatty acids (PUFAs), linoleic acid (C18:2 n-6) and arachidonic acid (C20:4 n-6), was significantly reduced, while the content of the principal n-3 PUFA, docosahexaenoic acid (C22:6 n-3), was markedly increased. The overall effect was to reduce n-6/n-3 ratio and increase the unsaturation extent of myocardial fatty acids. Functional study showed that hearts from clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The data shows that clofibrate has a profound impact on cardiac fatty acid composition, which may contribute to its cardioprotective effect.     

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.     

Contrasting effects of membrane enrichment with polyunsaturated fatty acids on phospholipid composition and cholesterol efflux from cholesterol-loaded J774 mouse or primary human macrophages

A high consumption of polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, is atheroprotective. PUFAs incorporation into membrane phospholipids alters the functionality of membrane proteins. We studied the consequences of the in vitro supplementation of several PUFAs on the FA profiles and on ABCA1-dependent cholesterol efflux capacities from cholesterol-loaded macrophages. Arachidonic acid (AA, C20:4 n-6) and, to a lesser extent, eicosapentaenoic acid (EPA, C20:5 n-3), dose-dependently impaired cholesterol efflux from cholesterol-loaded J774 mouse macrophages without alterations in ABCA1 expression, whereas docosahexaenoic acid (DHA, C22:6 n-3) had no impact. AA cells exhibited higher proportions of arachidonic acid and adrenic acid (C22:4 n-6), its elongation product. EPA cells exhibited slightly higher proportions of EPA associated with much higher proportions of docosapentaenoic acid (C22:5 n-3), its elongation product and with lower proportions of AA. Conversely, both EPA and DHA and, to a lesser extent, AA decreased cholesterol efflux from cholesterol-loaded primary human macrophages (HMDM). The differences observed in FA profiles after PUFA supplementations were different from those observed for the J774 cells. In conclusion, we are the first to report that AA and EPA, but not DHA, have deleterious effects on the cardioprotective ABCA1 cholesterol efflux pathway from J774 foam cells. Moreover, the membrane incorporation of PUFAs does not have the same impact on cholesterol efflux from murine (J774) or human (HMDM) cholesterol-loaded macrophages. This finding emphasizes the key role of the cellular model in cholesterol efflux studies and may partly explain the heterogeneous literature data on the impact of PUFAs on cholesterol efflux.     

NADPH oxidase 4 is required for interleukin-1β-mediated activation of protein kinase Cδ and downstream activation of c-jun N-terminal kinase signaling in smooth muscle

Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by proinflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Members of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle (VSM) cells as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1β-dependent activation of PKCδ and increases IL-1β-stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1β increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1β-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1β-dependent activation of JNK. Further studies showed that the IL-1β-dependent upregulation of inducible NO synthase expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1β-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox-sensitive mediator of IL-1β-dependent signaling and downstream activation of inflammatory mediators in VSM cells.     

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (− 28% for 70 μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages.     

Fatty acid composition of individual polar lipid classes from marine macrophytes

Major glycolipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)) and phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)] as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from Anfeltia tobuchiensis (Rhodophyta), Laminaria japonica, Sargassum pallidum (Phaeophyta), Ulva fenestrata (Chlorophyta) and Zostera marina (Embriophyta), harvested in the Sea of Japan. GC analysis of their fatty acid (FA) composition revealed that the n-6 polyunsaturated FAs (PUFAs) shared the most part of the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG. In algae, it was related to the prevalence of 20:4n-6 over 20:5n-3 in non-photosynthetic lipids. Percentage of n-6 PUFAs as well as the sum of n-3 and n-6 PUFAs decreased in the following sequence: PC-->PE-->PG. The saturation increased in the lines of MGDG-->DGDG-->SQDG and PC-->PE-->PG. PG was close to SQDG by the level of saturation. Distribution of C(18) and C(20) PUFAs in polar lipids depended on taxonomic position of macrophytes. Balance between C(18) and C(20) PUFAs was preferably shifted to the side of C(20) PUFAs in PC and PE that was observed in contrast to glycolipids and PG from L. japonica containing both series of FAs. The set of major FAs of polar lipid classes can essentially differ from each other and from total lipids of macrophytes. For example, MGDG was found to accumulate characteristic fatty acids 16:4n-3, 16:3n-3, 18:3n-6 and 18:4n-3, 20:3n-6 in U. fenestrata, Z. marina, L. japonica and S. pallidum, respectively.     

C19 odd-chain polyunsaturated fatty acids (PUfas) are metabolized to C21-PUfas in a rat liver cell line, and curcumin, gallic acid, and their related compounds inhibit their desaturation

It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the A6 desaturation of C24-PUFAs in rat liver cells.     

The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice

Abstract

In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1–5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1–3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.     

(Pro)renin Receptor Mediates Both Angiotensin II-Dependent and -Independent Oxidative Stress in Neuronal Cells

The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.     

Inhibitory effect of n-3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells

Abnormal activity of the cardiac Na+/Ca2+ exchanger (NCX1) can affect intracellular Ca2+ homeostasis and cause arrhythmias. The n-3 polyunsaturated fatty acids (PUFAs), however, may prevent arrhythmias. To test the effect of PUFAs on the cardiac NCX1 current (I(NCX1)), the canine NCX1 cDNA was expressed in human embryonic kidney (HEK293t) cells. The average density of I(NCX1) was 10.9+/-2.6 pA/pF (n=44) in NCX1-transfected cells and eicosapentaenoic acid (EPA, C20:5n-3) significantly inhibited I(NCX1) The suppression of I(NCX1) by EPA was concentration-dependent with an IC50 of 0.82+/-0.27 microM. EPA had a similar effect on outward or inward I(NCX1). Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) also significantly inhibited I(NCX1), whereas the saturated fatty acid, stearic acid (SA, C18:0), did not. Our data demonstrate that the n-3 PUFAs significantly suppress cardiac I(NCX1), which is probably one of their protective effects against lethal arrhythmias.     

Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the CAREMA Cohort Study

Background

Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20∶5n-3) and DHA (C22∶6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20∶4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20∶4n-6 to C20∶3n-6 and C18∶3n-6 to C18∶2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence.

Methods

We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors.

Results

The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20∶5n-3 and C22∶6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk.

Conclusion

In this prospective cohort study, we observed a reduced CHD risk with an increased C20∶4n-6 to C20∶3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies.     

Partitioning of polyunsaturated fatty acid oxidation between mitochondria and peroxisomes in isolated rat hepatocytes studied by HPLC separation of oxidation products

The extent of mitochondrial and peroxisomal contribution to beta-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO(2), while peroxisomal beta-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO(2) were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly beta-oxidized only one cycle in peroxisomes.     

The immunomodulatory effects of novel beta-oxa, beta-thia, and gamma-thia polyunsaturated fatty acids on human T lymphocyte proliferation, cytokine production, and activation of protein kinase C and MAPKs

We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (beta-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of beta-oxa, beta-thia, and gamma-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated beta-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-beta production. However, beta-oxa 25:6n-3 and beta-oxa 21:4n-3 displayed lower inhibitory action on IFN-gamma production. Surprisingly, beta-oxa 23:4n-6 and beta-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The beta-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two gamma-thia PUFA tested, gamma-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but gamma-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, beta-thia 23:4n-6 and beta-thia 21:3n-6, were studied in more detail and shown to have an IC(50) of 1-2 muM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.