Fatty acid control of nitric oxide production by macrophages

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FEBS Letters xxx (2006) xxx–xxx

FEBS 30727 No. of Pages 9

6 May 2006; Disk UsedARTICLE IN PRESS

Fatty acid control of nitric oxide production by macrophages

Thais Martins de Limaa,*, Larissa de Sa Limab, Cristoforo Scavoneb, Rui Curia

a Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof Lineu Prestes, 1524,05508-900, Sao Paulo, Brazil

b Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof Lineu Prestes, 1524, 05508-900, Sao Paulo, Brazil

Received 17 April 2006; accepted 26 April 2006

Available online

Edited by Sandro Sonnino

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Abstract Modulation of macrophage functions by fatty acids(FA) has been studied by several groups, but the effect of FAon nitric oxide production by macrophages has been poorlyexamined. In the present study the effect of palmitic, stearic,oleic, linoleic, arachidonic, docosahexaenoic and eicosapentae-noic acids on NF-jB activity and NO production in J774 cells(a murine macrophage cell line) was investigated. All FA testedstimulated NO production at low doses (1–10 lM) and inhibitedit at high doses (50–200 lM). An increase of iNOS expressionand activity in J774 cells treated with a low concentration ofFA (5 lM) was observed. The activity of NF-jB was time-depen-dently enhanced by the FA treatment. The inhibitory effect of FAon NO production may be due to their cytotoxicity, as observedby loss of membrane integrity and/or increase of DNA fragmen-tation in cells treated for 48 h with high concentrations. The re-sults indicate that, at low concentrations FA increase NOproduction by J774 cells, whereas at high concentrations theycause cell death.� 2006 Published by Elsevier B.V. on behalf of the Federation ofEuropean Biochemical Societies.

Keywords: Fatty acid; Nitric oxide; NF-jB; iNOS expression;iNOS activity; Cell death

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RR1. Introduction

NO produced by activated macrophages has been shown to

regulate antimicrobial and antitumor activities. However, NO

production in excess causes tissue damage that is associated

with acute and chronic inflammation [1]. NO is synthesized

from LL-arginine by NO synthase (NOS) using NADPH and

oxygen as cosubstrates [2]. Macrophages stimulated with lipo-

polysaccharide (LPS) and pro-inflammatory cytokines such as

interferon-c (IFN-c) and tumor necrosis factor a (TNF-a) [3,4]

produce large amounts of NO through inducible NOS (iNOS)

activity. The expression of iNOS is regulated by the nuclear

factor kappa B (NF-jB) in several cell types, including macro-

phages [5,6]. NF-jB plays an important role in controlling

inflammatory gene activation [7]. This transcription factor is

usually found in the cytosol as a heterodimer complex with

its inhibitory protein, IjB. When cells are stimulated with

LPS, phorbol esther or inflammatory cytokines, IjB is phos-

phorylated by IjB kinase and degraded. IjB phosphorylation

*Corresponding author. Fax: +55 11 30917285.E-mail address: [email protected] (T.M. de Lima).

0014-5793/$32.00 � 2006 Published by Elsevier B.V. on behalf of the Feder

doi:10.1016/j.febslet.2006.04.091

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OOdissociates the dimmer and allows NF-jB to translocate to the

nucleus, where it activates target genes, including iNOS [8].

Recent studies have shown that fatty acids (FA) can modu-

late NF-jB activation. In human monocytic THP-1 cells, lino-

leic (LA), a-linolenic (ALA) and docosahexaenoic (DHA) acids

decreased NF-jB DNA-binding activity [9]. Palmitic acid (PA)

increased NF-jB activity in 3T3-L1 adipocytes [10] and peri-

cytes [11]. On the other hand, palmitic (PA), oleic (OL) and lin-

oleic (LA) acids induced iKKb activation and decreased NF-jB

activity in endothelial cells [12]. Eicosapentaenoic (EPA) and

docosahexaenoic (DHA) acids decreased LPS induced NF-jB

activation in human kidney-2 (HK-2) cells [13].

Several authors have shown modulation of macrophage

functions by FA [14–17]. The x � 6 polyunsaturated FA usu-

ally stimulate the inflammatory response, whereas x � 3 FA

have been considered as anti-inflammatory agents. The effect

of FA has been examined on cytokine, eicosanoids and reac-

tive oxygen species production [18–20], and adhesion molecule

expression [17,21]. However, the effect of FA on NO produc-

tion and iNOS expression in macrophages has been poorly

examined [22–25]. The studies have shown that x � 3 FA,

especially DHA, markedly suppress NO production and iNOS

expression in murine macrophages. An increase in NO produc-

tion by macrophages from animals fed x � 3 FA rich diets has

been also reported [26,27]. Up to now, however, there is no re-

port comparing the effect of the more abundant FA in plasma,

palmitic (saturated) and oleic acids (monounsaturated, x � 9),

with x � 3 (DHA and EPA) and x � 6 (linoleic and arachi-

donic) FA.

As described above, NO production and iNOS expression are

regulated by NF-jB activation, and the activity of this tran-

scription factor can be modulated by FA. This information

led us to investigate the effect of various FA on NF-jB activity

and NO production in J774 cells (a murine macrophage cell

line). The following FA were studied: palmitic (PA), stearic

(SA), oleic (OA), linoleic (LA), arachidonic (AA), docosahexa-

enoic (DHA) and eicosapentaenoic (EPA) acids.

9394959697

2. Materials and methods

2.1. ReagentsRPMI-1640 medium, HEPES, penicillin and streptomycin were

purchased from Invitrogen (Carlsbad, CA, USA). Fatty acids, LPS,sulfanilamide, naphthylene diamine dihydrochloride, sodium pyro-phosphate and sodium orthovanadate were obtained from Sigma (St.Louis, MO, USA). Ethanol and phosphoric acid were purchased fromMerck (Frankfurter, Germany). Sodium bicarbonate and sodium ni-trite were purchased from Labsynth products (Diadema, SP, Brazil).DAF-DA was obtained from Molecular Probes (Eugene, OR, USA).

ation of European Biochemical Societies.

mailto:[email protected]

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Reagents for SDS–PAGE and immunoblotting were from Bio-RadLaboratories (Richmond, CA, USA). Antibodies were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA, USA).

2.2. Culture conditions and fatty acid treatmentJ774 cells were grown in RPMI-1640 medium containing 10% fetal

calf serum (FCS). This medium was supplemented with glutamine(2 mM), HEPES (20 mM), streptomycin (10000 lg/mL), penicillin(10000 UI/mL) and sodium bicarbonate (24 mM). Cells were grownin 75 cm2 flasks containing 0.5–1 · 106 cells per mL. The cells werekept in a humidified atmosphere containing 5% CO2 at 37 �C.

Cells were treated with various concentrations (1–200 lM) of pal-mitic (PA), stearic (SA), oleic (OA), linoleic (LA), arachidonic (AA),eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids for differ-ent periods (from 3 up to 48 h). The fatty acids were dissolved in eth-anol. The final concentration of ethanol in the culture medium did notexceed 0.5%. This concentration of ethanol is not toxic to the cells asreported by Siddiqui et al. [28].

2.3. Determination of nitric oxideThe content of nitrite was measured in the supernatant of cultured

cells based on the method described by Ding et al. [29]. Cells (5 · 105

per well) were seeded in 96 well plates and treated with 2.5 lg per mLLPS and different concentrations of FA for 48 h. At the end of the cul-ture period, 50 lL of the supernatant were removed and incubated withan equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, 2.5% H3PO4) at room temperature for10 min. The absorbance was determined at 550 nm. Nitrite concentra-tion was determined by using sodium nitrite as standard.

2.4. Determination of iNOS protein expressionJ774 cells were seeded in 25 cm flasks and treated with 2.5 lg per mL

LPS and 5 lM of the FA for 6, 12 and 24 h. At the end of the incuba-tion period, cells were immediately homogenized in 150 lL extractionbuffer (100 mM Trizma, pH 7.5; 10 mM EDTA; 10% sodium dodecylsulfate (SDS); 100 mM NaF; 10 mM sodium pyrophosphate; 10 mMsodium orthovanadate; at 100 �C) for 30 s. Samples were boiled for5 min and centrifuged at 12000 rpm, for 40 min, at 4 �C. Aliquots ofsupernatants were used for the measurement of total protein content,as described by Bradford [30]. Equal amount of protein of each sample(70 lg) was separated using 6% SDS–polyacrylamide gel. Westernblotting was carried out following the method described by Towbinet al. [31]. The proteins of the gel were transferred to a nitrocellulosemembrane at 120 V for 1 h. Non-specific bounds were blocked by incu-bating the membranes with 5% defatted milk in basal solution (10 mMTrizma, pH 7.5; 150 mM NaCl; 0.05% Tween 20) at room tempera-ture, for 2 h. Membranes were washed in basal solution three timesfor 10 min each and then incubated with anti-iNOS antibody in basalsolution containing 3% defatted milk, at room temperature, for 3 h.Membranes were washed three times for 10 min each and incubatedwith anti-IgG antibody linked to horseradish peroxidase in basal solu-tion containing 1% defatted milk, at room temperature, for 1 h. Fol-lowing another washing, membranes were incubated with substratefor peroxidase and chemiluminescence enhancer (Amersham Biosci-ences, Upsalla, SW) for 1 min and immediately exposed to X-ray filmfor 30 min. Films were then revealed in the conventional manner. Bandintensities were analysed using the ScionImage software (Scion Corpo-ration, MD, USA).

2.5. Measurement of iNOS activityiNOS activity was measured in J774 cells treated for 12, 24 and 48 h

with 2.5 lg per mL LPS and 5 lM FA. This assay is based on the bio-chemical conversion of LL-arginine to LL-citrulline by iNOS. Cells werehomogenized in ice-cold Tris–HCl buffer (20 mM Tris–HCl, 10 mMEDTA, and 10 mM EGTA, pH 7.4) using a Teflon homogenizer.The homogenates were centrifuged at 12000 · g for 5 min at 4 �C.Supernatants were removed and NOS assay was performed by incubat-ing (37 �C for 20 min) 150 lg (20 lL) of protein in a final volume of60 lL of assay mixture containing 50 mM Tris–HCl, 6 lM tetrahydro-biopterin, 2 lM FAD, 2 lM FMN, 10 mM NADPH, 100 mM LL-argi-nine/LL-[H3]-arginine (5 lCi/mL), 1 mM EDTA/EGTA. The reactionwas stopped with 1 mL of ice-cold stop buffer (50 mM HEPES and5 mM EDTA, pH 5.5) and 100 lL of cation-exchange resin (Dowex,

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Na+ form, equilibrated with 50 mM HEPES, pH 5.5) was added toeach reaction mixture to remove the excess of LL-[H3]-arginine. Aliquotswere collected into vials, scintillation liquid (6 mL) was added andradioactivity was quantified in a scintillation counter (Packard TRICARB 2100 TR Counters, Downers Grove, IL, USA). Protein concen-trations in samples were determined as described by Bradford [30] withbovine serum albumin as standard.

2.6. Electrophoretic mobility shift assayNFjB activation was evaluated after treatment of the cells for 3, 6,

12, 24 and 48 h with 5 lM fatty acids; concentrations in which a highproduction of NO was observed.

Nuclear extract from J774 cells was obtained as previously described[31]. Double-stranded oligonucleotides containing the NF-jB (5 0-AGTTGAGGGGACTTTCCCAGGC-3 0) consensus binding site [33]were end-labeled using T4 PNK and [c-32P]ATP (Amersham Biosci-ences). Binding reactions of the probes (30000 cpm) were performedwith 10 lg proteins from nuclear extract, at room temperature, for20 min, in 20 lL of the binding buffer consisting of 20 mM HEPES,pH 7.6, 50 mM KCl, 10% glycerol, 0.2 mM EDTA, 1 mM DTT and2 lg polydeoxyinosinic–deoxycytidylic acid (poly[dI–dC]). Competi-tive binding assays were conducted under the same conditions withthe addition of 2 pmol (100-fold molar excess) of unlabeled competitoroligonucleotides. The DNA–protein complexes were electrophoresedon 4% non-denaturing polyacrylamide gels, at 4 �C, in 45 mM Tris,45 mM borate and 1 mM EDTA buffer. The gels were dried and sub-jected to autoradiography. The blots were analysed by scanner densi-tometry (Image Master 1D�, Amersham Biosciences) and the resultsof the binding activity were expressed as arbitrary units.

2.7. Determination of fatty acid cytotoxicityFatty acid cytotoxicity was assessed by flow cytometry after treat-

ment of the cells for 48 h with high concentrations of fatty acids. Atthe end of the culture period, 0.5 mL of medium containing cells wereused to evaluate the membrane integrity. In this assay, 50 lL of a pro-pidium iodide (PI) solution (100 lg per mL in saline buffer) were addedto the cells. Propidium iodide is a highly water-soluble fluorescentcompound that cannot pass through intact membranes and is generallyexcluded from viable cells. It binds to DNA by intercalating betweenthe bases with little or no sequence preference. After 5 min incubationat room temperature, the cells were evaluated in a FACScalibur flowcytometry equipment (Becton Dickinson, CA, USA) by using the CellQuest software. Fluorescence was measured using the FL2 channel(Orange-red fluorescence – 585/42 nm). Ten thousand events were ana-lysed per experiment.

We also determined the percentage of cells with fragmented DNAafter the treatments using propidium iodide. In this assay, cells wereresuspended in a solution containing detergents that permeabilize thecells, which promptly incorporate the dye into DNA. Briefly, 0.5 mLof medium containing cells were centrifuged at 1000 · g, for 10 min,at 4 �C. The pellet was gently resuspended in 300 lL hypotonic solutioncontaining 50 lg/mL propidium iodide, 0.1% sodium citrate, and 0.1%Triton X-100. The cells were then incubated for 2 h at 4 �C. Fluores-cence was measured and analysed by flow cytometry as described above.

Both the lost of membrane integrity and/or DNA fragmentationwere considered signs of toxicity, regardless which one was first ob-served.

2.8. Statistical analysisResults are presented as means ± S.E.M. of 6–9 determinations from

2–3 experiments. Comparisons with control were performed by analy-sis of variance (2-way-ANOVA). Significant differences were analysedby the Bonferroni post-tests (Graph Pad Prism 4 – Graph Pad Soft-ware Inc., San Diego, CA, USA). The level of significance was set atP < 0.05.

3. Results

3.1. Effect of fatty acids on NO production

All FA tested stimulated NO production at low doses but

showed inhibitory effect at high concentrations (Fig. 1). The

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stimulatory effect of the FA was more pronounced at concen-

trations between 2.5 and 5 lM (P < 0.001). At 5 lM, palmitic

acid was the less effective, inducing an increase of 32% in NO

production. Arachidonic acid, on the other hand, was the most

potent, increasing in 119% the production of NO. The crescent

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Fig. 1. Effects of fatty acids on NO production of J774 cells. The concentratreatment for 48 h with palmitic, stearic, oleic, linoleic, arachidonic, docosahLPS are shown. The supernatant was incubated with an equal volume of Grare presented as means ± S.E.M. of 16 determinations from four experiment

order of stimulatory effect at 5 lM was: PA (32%), SA (60%),

DHA (79%), LA (83%), EPA (89%), OA (94%), and AA

(117%). PA and EPA inhibited NO production at 50 lM. SA

suppressed NO production at 100 lM, AA and DHA at

150 lM, and OA and LA at 200 lM.

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tion (lM) of nitrite measured in the supernatant of cultured cells afterexaenoic and eicosapentaenoic acids in the presence of 2.5 lg per mL

iess reagent and the absorbance was determined at 550 nm. The valuess. * P < 0.001 for comparison with control.

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3.2. Effect of fatty acids on iNOS expression

In order to investigate the mechanisms involved in the regu-

lation of NO production by FA, we determined iNOS protein

expression in J774 cells treated for 6, 12 and 24 h with 5 lM

FA in the presence of LPS (2.5 lg/mL). Cells treated with

PA did not present changes in iNOS expression when com-

pared with those treated with LPS and ethanol at any period

of treatment. SA and OA treated cells presented higher iNOS

expression after 12 h (P < 0.001). Cells treated with LA, AA,

DHA and EPA showed increased iNOS expression after 6 h

of treatment (P < 0.001) and this effect remained up to 24 h

(Fig. 2).

3.3. Involvement of NFjB activation on modulation of iNOS

56 expression by FA

Treatment of J774 cells with 5 lM FA altered NFjB activa-

tion as observed in Fig. 3. However, the period of incubation

for the effect of FA to be observed varied considerably. LPS

caused NFjB activation and presented the more potent effect

after 3 h treatment (115%) but the addition of ethanol (vehicle)

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Fig. 2. Effects of fatty acids on LPS induced iNOS expression. Cells were trWhole cell lysates were dissolved in a sample buffer and submitted to 8% Spolyclonal antibody. Band intensities were analysed using the ScionImagecompared to the respective control (LPS and ethanol). Controls received an aexperiments. * P < 0.001, # P < 0.01 for comparison with control.

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diminished NFjB activation by 16.6 ± 3.2% (means ± S.E.M.

of five experiments). PA, DHA and EPA increased NFjB acti-

vation after 3 h treatment, when compared to cells treated with

LPS and ethanol, by 51%, 52% and 62%, respectively. AA ex-

erted its stimulatory effect after 6 h of treatment (36%) and

reached its peak after 24 h (106%). OA increased NFjB activa-

tion after 12 h (39%). SA and LA did not affect NFjB activa-

tion at any period of treatment tested.

3.4. Effect of fatty acids on iNOS activity

Cells treated with AA for 12 h showed higher iNOS activity

when compared with the correspondent control (ethanol 12 h).

Exposure for 24 h to PA also led to an increased of iNOS

activity. Cells treated with OA, LA, DHA and EPA presented

higher iNOS activity after 48 h treatment. SA did not present

significant effect on iNOS activity (Fig. 4).

3.5. Effect of fatty acids on cell viability and DNA fragmentation

In order to evaluate if the inhibitory effect of the FA on NO

production at high concentrations was due to their cytotoxic-

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eated for 6, 12 and 24 h with 2.5 lg per mL LPS and 5 lM of the FA.DS–PAGE. Western blotting was performed using mouse anti-iNOSsoftware (Scion Corporation) and are expressed as relative values

rbitrary value of 1. The values are presented as means ± S.E.M. of three

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Fig. 3. Results of the electrophoretic mobility shift assay. (Right) Nuclear extracts prepared from J774 cells that had been treated with 2.5 lg per mLLPS and 5 lM of the FA for 3, 6, 12, 24 and 48 h were used for protein–DNA binding reactions in the presence of the radio-labeled probe(�30000 cpm), as described in Section 2. A: control, B: ethanol, C: LPS 2.5 lg per mL, D: LPS 2.5 lg per mL + ethanol, E: PA 5 lM, F: SA 5 lM,G: OA 5 lM, H: LA 5 lM, I: AA 5 lM, J: DHA 5 lM, K: EPA 5 lM. (Left) Band intensities were analysed using the ScionImage software (ScionCorporation) and are expressed as relative values compared to the respective control. Controls received an arbitrary value of 1. The values arepresented as means ± S.E.M. of three experiments. * P < 0.001, P < 0.05 for comparison with cells treated with LPS + ethanol.




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Fig. 4. Effects of fatty acids on iNOS activity. Cells were treated for 12, 24 and 48 h with 2.5 lg per mL LPS and 5 lM of the FA. iNOS activity wasdetermined after quantification of radioactivity of the LL-citrulline produced after conversion of LL-arginine to LL-citrulline by iNOS. The values arepresented as means ± S.E.M. of three experiments. * P < 0.001, # P < 0.01, P < 0.05 for comparison with the respective control (ethanol and LPS).




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ity, the percentage of cells with intact cell membrane and with

fragmented DNA after the treatment for 48 h was determined

(Fig. 5). All FA induced loss of membrane integrity, except

AA. Cells treated with PA (50 lM), SA (100 lM), OA

(200 lM), LA (200 lM), DHA (150 lM) and EPA (100 lM)

presented a significant decrease in cell membrane integrity by

34%, 26%, 12%, 24%, 27% and 18%, respectively. The treat-

ments also induced DNA fragmentation, except for LA. An in-

crease in the percentage of cells with fragmented DNA was

observed after treatment with PA (50 lM), SA (100 lM), OA

(200 lM), AA (150 lM), DHA (150 lM) and EPA (100 lM)

by 2-, 1.9-, 1.7-, 2.1-, 1.7- and 1.6-fold, respectively. Cells trea-

ted with ethanol, the vehicle used for FA preparation, did not

present loss of membrane integrity or induction of DNA frag-

mentation, indicating that the concentration of ethanol used

(0.5%) is not cytotoxic.

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The effect of different concentrations of various FA on nitric

oxide production by J774 cells and the possible mechanisms in-

volved were investigated in this study.

J774 cells cultivated for 48 h with FA and LPS showed high

production of NO as compared to control cells, especially at

low concentrations (1–10 lM). Cells treated with LA, AA,

DHA and EPA showed a stimulatory effect up to 25 lM. On

the other hand, higher concentration (50–200 lM) inhibited

NO production. These results seem controversial but they

may help to explain the discrepancy in the literature. Many

studies observed a decrease in NO production by mice macro-

phages and cell lineages after exposure to FA [22–25], whereas

others found an increase [27,34]. In addition to cell type and

period of stimulation, these controversial results may be also

due to the different concentrations of the FA used. The inhib-

itory effect of FA on NO production may be due to their cyto-

toxicity, as observed by loss of membrane integrity and/or

increase of DNA fragmentation in cells treated for 48 h with

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Phigh concentrations. FA toxicity has been reported in several

cell types, as a concentration and time-dependent effect of

these metabolites [35,36].

The stimulatory effect of FA was also observed by fluores-

cence microscopy using DAF-DA. Cells treated for 12 and

24 h presented high intracellular NO content, which dimin-

ished after 48 h of exposure to FA. These results corroborate

with the low level of NO in the medium of cells treated with

FA for 24 h (data not shown).

High production of NO is achieved when the expression of

inducible NOS is stimulated [3]. J774 cells treated with LPS

and FA (5 lM) presented higher expression of iNOS protein

than cells treated with LPS and ethanol (vehicle). The period

of incubations where they exerted their effects varied, and

may be related to the activity of the nuclear factor kappa B

(NF-jB). Activation of NF-jB can trigger inflammatory re-

sponses by transcriptional induction of several pro-inflamma-

tory proteins and enzymes that generate mediators of

inflammation (e.g. iNOS) [37,38]. J774 cells presented signifi-

cant NF-jB activity in basal conditions (absence of LPS and

FA) that was increased by two fold after LPS addition. The

FA tested, with exception of SA and LA, stimulated NF-jB

activation, and this effect occurred prior to or at the same incu-

bation period where an increase in iNOS protein level was ob-

served.

Several studies have shown a decrease in NF-jB activation

by DHA and EPA [39–44]. However, Maziere et al. [45] ob-

served an increase of NFjB activation in fibroblasts treated

with DHA and EPA and Camandola et al. [46] stated that

EPA (45 lM) exerts no effect on the nuclear translocation of

NF-jB in the human promonocytic cell line U937. In the pres-

ent study, DHA and EPA stimulated NO production in J774

cells at low concentrations (1–25 lM). Concomitantly, an in-

crease of NF-jB activation was observed at 5 lM, indicating

that this effect on NF-jB may raise iNOS protein level and,

consequently, NO production.

Stimulatory effect of PA, AA and OA on the nuclear trans-

location of NF-jB has also been observed in different cells

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B

* *

Fig. 5. Effects of fatty acids on cell membrane integrity and DNA fragmentation. The percentage of cells with intact membrane (A) and fragmentedDNA (B) after treatment for 48 h with palmitic (PA), stearic (SA), oleic (OA), linoleic (LA), arachidonic (AA), docosahexaenoic (DHA) andeicosapentaenoic (EPA) acids are shown. Cells were stained with a saline buffer containing propidium iodide to assess membrane integrity. A buffercontaining citrate, triton X-100 and propidium iodide was used to assess DNA fragmentation. The values are presented as means ± S.E.M. of 9determinations from three experiments. * P < 0.001 for comparison with control (ethanol – OH).




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types, such as L6 myotubes, mouse C2C12 myoblasts, skeletal

muscle cells, human promonocytic cell line U937, human

endothelial cells and mouse macrophages [46–51]. On the other

hand, no effect on NF-jB activation and iNOS expression was

observed in insulinoma (INS)-1E cells, human fibroblasts and

human endothelial cells treated with OA [52,53].

In the periods of incubation studied, LA and SA did not af-

fect NF-jB activation but increased iNOS expression after 6

and 12 h, respectively. Other transcriptional factors are in-

volved in iNOS expression, as the octamer factor (Oct)

[54,55], signal transducer and activator of transcription-1a

(STAT-1a) [56–58], cAMP-induced transcription factors such

as cAMP-responsive element binding protein (CREB) and

CCAAT-enhancer box binding protein (C/EBP) [59,60], and

activating protein-1 (AP-1) [61]. SA and LA may have acti-

vated one of these transcriptional factors and increased iNOS

expression.

Three proteins that interact with iNOS and regulate its

activity have been recently identified. In the central nervous

system, the protein kalirin appears to inhibit iNOS by pre-

venting enzyme dimerization [62]. In murine macrophages,

a 110-kDa protein (named NAP110) has been identified that

directly interacts with the amino-terminus of iNOS, thereby

preventing dimer formation and inhibiting NOS activity [63].

Stable overexpression of Rac2 in RAW 264.7 cells increased

LPS-induced nitrite generation and iNOS activity without

measurably affecting iNOS protein abundance [64]. Our re-

sults suggest that FA are also modulators of iNOS activity

in J774 cells.

The results presented herein demonstrate that FA stimulate

NO production by J774 cells at low concentrations (1–10 lM)

and inhibit it at high doses (50–200 lM). The stimulatory effect

of most FA on NO production is time dependent, involves

NF-jB activation, and increases iNOS expression and activity.

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390

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The inhibitory effect of FA on NO production by J774 cells is

mainly due to their cytotoxicity.
455456457458459460461462463464465
5. Uncited reference

[32].

Acknowledgements: The authors are grateful to the technical assistanceof G. de Souza, J.R. Mendonca and E.P. Portioli. This research is sup-ported by FAPESP, CNPq, and CAPES.

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Fatty acid control of nitric oxide production by macrophages

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