DHA-treated Cells Research Articles

The omega-3 fatty acid, DHA, decreases neuronal cell death in association with altered zinc transport

Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid in neuronal cell membranes. We hypothesize that DHA induces a decrease in neuronal cell death through reduced ZnT3 expression and zinc uptake. Exposure of M17 cells to DHA-deficient medium increased the levels of active caspase-3, relative to levels in DHA-replete cells, confirming the adverse effects of DHA deficiency in promoting neuronal cell death. In DHA-treated M17 cells, zinc uptake was 65% less and ZnT3 mRNA and protein levels were reduced in comparison with DHA-depleted cells. We propose that the neuroprotective function of DHA is exerted through a reduction in cellular zinc levels that in turn inhibits apoptosis.

Growth inhibitory effects of dihydroartemisinin on pancreatic cancer cells: involvement of cell cycle arrest and inactivation of nuclear factor-κB

In a recent publication, we have shown that dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin, inhibits growth of pancreatic cancer cells in vitro and in vivo mediated by its anti-proliferative and pro-apoptotic effects. As it has been shown that the apoptosis might be induced due to cell cycle arrest, and that transcriptional factor nuclear factor-kappa B (NF-kappaB) plays vital roles in the apoptosis of pancreatic cancer cells, we extend our study to investigate the effects of DHA on cell cycle progression and NF-kappaB activity in pancreatic cancer cells to further reveal the anticancer effects of DHA on pancreatic cancer. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Changes in the expression of cell cycle-associated proteins were detected using Western blot analysis. Measurement of NF-kappaB activity was performed with immunoblot analyzing the nuclear protein expression of NF-kappaB/p65 and ELISA detecting the NF-kappaB DNA-binding activity. The treatment with DHA resulted in a dose-dependent G(0)/G(1) cell cycle arrest and regulated the expression of some cyclins, cdks and cdk inhibitors that involved in the G(0)/G(1) cell cycle progression such as cyclin E, cdk2, cdk4 and p27(Kip1) in pancreatic cancer BxPC-3 and AsPC-1 cells. The translocation and DNA-binding activity of NF-kappaB were inhibited in DHA-treated cells in a dose-dependent manner, indicated the inactivation effects of DHA in pancreatic cancer cells. Together with our previous observations, our data show that DHA induces cell cycle arrest and apoptosis in pancreatic cancer cells, and this effect might be due to inhibition of NF-kappaB signaling. We suggest that DHA could be developed as a novel agent against pancreatic cancer.

Docosahexaenoic Acid Activates Some SREBP‐2 Targets Independent of Cholesterol and ER Stress in SW620 Colon Cancer Cells

The SREBP-2 transcription factor is mainly activated by low cellular cholesterol levels. However, other factors may also cause SREBP-2 activation. We have previously demonstrated activation of SREBP-2 by the polyunsaturated fatty acid docosahexaenoic acid (DHA) in SW620 colon cancer cells. Despite activation of SREBP-2, only a few target genes were induced and cholesterol biosynthesis was reduced. In the present study, gene expression analysis at early time points verified the previously observed SREBP-2 target gene expression pattern. Activation of SREBP-2 using siRNAs targeting Niemann Pick C1 protein (NPC1) led to increased expression of all SREBP target genes examined, indicating that activation of some SREBP-2 target genes is inhibited during DHA-treatment. Cholesterol supplementation during DHA treatment did not abolish SREBP-2 activation. We also demonstrate that activation of SREBP-2 is independent of ER stress and eIF2alpha phosphorylation, which we have previously observed in DHA-treated cells. Thapsigargin-induced ER stress repressed expression of SREBP-2 target genes, but with a different pattern than observed in DHA-treated cells. Moreover, oleic acid (OA) treatment, which does not induce ER stress in SW620 cells, led to activation of SREBP-2 and induced a target gene expression pattern similar to that of DHA-treated cells. These results indicate that DHA and OA may activate SREBP-2 and inhibit activation of SREBP-2 target genes through a mechanism independent of cholesterol level and ER stress.

Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages

The long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of fish oil, eicosapentanoic (EPA) and docosahexanoic (DHA) acids are considered cardioprotective. Inflammation elicited by macrophages is increasingly recognised in the aetiology of metabolic syndrome. This study investigated the differential anti-inflammatory potential of EPA and DHA through cytokine production and nuclear factor (NF)-κB signalling in a human macrophage model. We investigated the dependency of LC n-3 PUFA immune-modulation on concentration and duration of lipopolysaccharide (LPS) activation. Interleukin (IL)-1β, IL-6 and tumor necrosis factor-α secretion from EPA, DHA and control cells were differentially limited by LPS concentration. In all cases, there was no benefit in activating cells with >0.1 μg/ml LPS. LC n-3 PUFA decreased proinflammatory cytokines production, an effect modulated by LPS concentration. Expression of the transcription factor NF-κB p65 was significantly reduced in the nucleus and retained in the cytoplasm of EPA- and/or DHA-treated macrophages during 5-h activation with 0.1 μg/ml LPS. Nuclear binding of p65 was significantly reduced in EPA- and DHA-treated cells at 2-h LPS activation. Over the time course, expression of nuclear IκBα was significantly reduced, cytoplasmic NF-κB p50 significantly increased and cytoplasmic cleaving enzyme IκB inhibitor complex significantly reduced in LC n-3 PUFA-treated cells. EPA and DHA down-regulated the production of proinflammatory cytokines associated with the aetiology of metabolic syndrome, NF-κB transcriptional activity and upstream cytoplasmic signalling events. Immune responses are dynamic, and the present study suggests a nutrient sensitive window of LPS activation at which EPA and DHA are strongly anti-inflammatory.

In vitro fatty acid enrichment of macrophages alters inflammatory response and net cholesterol accumulation

Dietary long-chain PUFA, both n-3 and n-6, have unique benefits with respect to CVD risk. The aim of the present study was to determine the mechanisms by which n-3 PUFA (EPA, DHA) and n-6 PUFA (linoleic acid (LA), arachidonic acid (AA)) relative to SFA (myristic acid (MA), palmitic acid (PA)) alter markers of inflammation and cholesterol accumulation in macrophages (MPhi). Cells treated with AA and EPA elicited significantly less inflammatory response than control cells or those treated with MA, PA and LA, with intermediate effects for DHA, as indicated by lower levels of mRNA and secretion of TNFalpha, IL-6 and monocyte chemoattractant protein-1. Differences in cholesterol accumulation after exposure to minimally modified LDL were modest. AA and EPA resulted in significantly lower MPhi scavenger receptor 1 mRNA levels relative to control or MA-, PA-, LA- and DHA-treated cells, and ATP-binding cassette A1 mRNA levels relative to control or MA-, PA- and LA-treated cells. These data suggest changes in the rate of bidirectional cellular cholesterol flux. In summary, individual long-chain PUFA have differential effects on inflammatory response and markers of cholesterol flux in MPhi which are not related to the n position of the first double bond, chain length or degree of saturation.

Dietary Fatty Acids Differentially Regulate Production of TNF‐α and IL‐10 by Murine 3T3‐L1 Adipocytes

Obesity correlates with increased production of adipocyte-derived cytokines, which may contribute to a chronic subclinical inflammation seen in obese individuals. This study evaluated the ability of specific fatty acids to modulate production of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory cytokine, interleukin-10 (IL-10), in murine 3T3-L1 adipocytes. Effects on nuclear factor-kappaB (NF-kappaB), a key transcriptional activator of the inflammatory cascade, and suppressor of cytokine signaling 3 (SOCS-3), a negative regulator of cytokine signaling, were also determined. Adipocytes were incubated for 24 and 48 h with and without 50 or 500 micromol/l of palmitic acid, oleic acid, or docosahexaenoic acid, (DHA). Effects on gene expression and protein secretion of TNF-alpha and IL-10 were determined using real-time PCR and a murine multipex RIA kit. SOCS-3 expression was determined by northern blotting and NF-kappaB binding activity was assessed using a commercially available assay. Adipocytes treated for 24 h with palmitic acid exhibited a 70% increase in TNF-alpha production and up to a 75% decrease in IL-10 production, relative to untreated cells. In contrast, DHA treatment had no effect on TNF-alpha, but increased IL-10 production twofold. No effect of oleic acid was seen on either TNF-alpha or IL-10 production. Similar results were obtained during a 48-h incubation. Furthermore, NF-kappaB DNA-binding activity increased fourfold in response to palmitic acid and decreased 60% in response to DHA. Expression of SOCS-3 increased twofold in DHA-treated cells. In aggregate, these results suggest that dietary fatty acids act directly on adipocytes to modulate cytokine production. As circulating fatty acids levels are chronically elevated in obese individuals, this effect may account in part for obesity-associated inflammation.

Dihydroxyacetone‐induced death is accompanied by advanced glycation endproduct formation in selected proteins of Saccharomyces cerevisiae and Caenorhabditis elegans

Advanced glycation endproduct (AGE) formation is an important mechanism for protein deterioration during diabetic complications and ageing. The effects on AGE formation following dihydroxyacetone (DHA) stress were studied in two model organisms, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Total protein AGEs, detected using an anti-N(epsilon)-carboxyalkyllysine-specific monoclonal antibody, displayed a strong correlation to DHA-induced yeast cell mortality in the wild-type and hypersensitive as well as resistant mutant strains. During DHA-induced cell death we also detected AGEs as the formation of acidic protein modifications by 2-D PAGE. Furthermore, we confirmed AGE targets immunologically on 2-D gel-separated protein extracted from DHA-treated cells. AGE modification of several metabolic enzymes (Eno2p, Adh1p, Met6 and Pgk1p) and actin (Act1p) displayed a strong correlation to DHA-induced cell death. DHA was toxic to C. elegans even at low concentration and also in this organism AGE formation accompanied death. We propose the use of DHA as a model AGE-generating substance for its apparent lack of a clear oxidative stress connection, and yeast and worm as model organisms to identify genetic determinants of protein AGE formation.

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Docosahexaenoic acid facilitates cell maturation and β-adrenergic transmission in astrocytes

The effects of docosahexaenoic acid (DHA; 22:6 n-3), a major omega-3 PUFA in the mammalian brain, on the structure and function of astrocytes were studied using primary cultures from rat cerebra. Gas-liquid chromatography of methyl esters of FAs isolated from cultures exposed to individual FAs, namely, stearic acid, linoleic acid, arachidonic acid, and DHA, showed alterations in the lipid profiles of the membranes, with a preferential incorporation of the FA to which the cells were exposed. Immunofluorescence studies demonstrated that unlike treatment with other FAs, after which the astrocytes remained as immature radial forms, DHA-treated astrocytes showed distinct differentiation, having morphology comparable to those grown in normal serum-containing medium. Receptor binding studies to determine the concentration of various neurotransmitter receptors showed that DHA selectively increased the number of beta-adrenergic receptors (beta-ARs) compared with FA-untreated controls, suggesting a greater role of DHA on beta-AR expression in membranes. This was also reflected by an increase in downstream events of the beta-AR pathways, such as the induction of protein kinase A and glycogen turnover by isoproterenol (ISP), a beta-AR agonist in DHA-treated cells. Moreover, ISP completely transformed DHA-treated cells into mature astrocytes bearing long processes, as in cells grown under normal conditions. Together, our observations suggest that DHA plays a unique role in facilitating some of the vital functions of astrocytes in the developing brain.

Docosahexaenoic acid induces ciap1 mRNA and protects human endothelial cells from stress-induced apoptosis

Induction of apoptosis represents a potential reaction of endothelial cells (ECs) after injury of the vascular endothelium. Beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in vascular diseases are widely recognized although the responsible mechanisms are not fully understood. Because it is not known whether PUFAs modulate EC apoptosis, we investigated the effects of n-3 and n-6 PUFAs on 4-hydroxynonenal (HNE)-induced EC apoptosis by annexin V staining and caspase-3 activation assays. Pretreatment with the n-3 fatty acid docosahexaenoic acid (DHA) reduced HNE-induced EC apoptosis. DHA-treated cells did not show the pronounced drop in intracellular GSH after HNE exposure seen in vehicle- or n-6 arachidonic acid-treated cells. This is most likely due to increased GSH levels in DHA-treated cells. Furthermore, DHA pretreatment increased ciap1 mRNA levels and transfection of cIAP1 small interfering RNA abolished the protective effect of DHA in HNE-induced apoptosis in HUVECs. Thus pretreatment of HUVECs with DHA reduces HNE-induced oxidative stress and apoptosis, and the protective effects of DHA seem to be dependent on cIAP1. The results provide a possible new mechanism for the atheroprotective effects of n-3 fatty acids in vascular disease.

Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA, 22:6n-3), modulate immune responses and exert beneficial immunosuppressive effects, but the molecular mechanisms inhibiting T-cell activation are not yet elucidated. Lipid rafts have been shown to play an important role in the compartmentalization and modulation of cell signaling. We investigated the role of DHA in modulating the lipid composition in lipid rafts and membrane subdomain distribution of interleukin-2 (IL-2) receptor signaling molecules. We found that DHA altered lipid components of rafts and modified the IL-2-induced Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway by partially displacing IL-2 receptors from lipid rafts. We fractionated plasma membrane subcellular compartments and discovered that certain amounts of STAT5a and STAT5b existed in detergent-resistant plasma membrane fractions of T-cells. After DHA treatment, STAT5a and STAT5b were not detected in lipid raft fractions and were located in detergent-soluble fractions. These data demonstrate for the first time that DHA alters the lipid composition of membrane microdomains and suppresses IL-2 receptor signaling in T-cells. Thus, our data provide evidence for a functional modification in lipid rafts by DHA treatment and explain PUFA-mediated immunosuppressive effects.

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells

n−3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 μM of either C20:4n−6 (ARA), or C20:5n−3 (EPA), or C22:6n−3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n−6 and C22:5n−3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n−3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.

Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide.

Recent studies from our laboratory have demonstrated that ascorbate potentiated enzymatic synthesis of nitric oxide (NO) from polymorphonuclear leukocytes (PMNs). NO is known to modulate various function of PMNs such as chemotaxis, adherence, aggregation, and generation of reactive oxygen species (ROS). The role of ascorbate in the PMN phagocytosis, ROS generation, and apoptosis was thus evaluated in the present study. Ascorbate and its oxidized and cell-permeable analog, dehydroascorbate (DHA), did not affect the phagocytosis but enhanced ROS generation and apoptosis following treatment with Escherichia coli or arachidonic acid. A detailed investigation on the DHA-mediated response indicated that inhibitors of DHA uptake, reduced nicotinamide adenine dinucleotide phosphate oxidase, NO synthase, or ROS scavengers attenuated ROS generation. In DHA-treated cells, enhanced generation of peroxynitrite was also observed; thus, ascorbate-mediated ROS and reactive nitrogen species generation might mediate cytotoxicity toward the ingested microbes and subsequently, augmented PMN apoptosis. Results of the present study have helped in delineating the role of ascorbate in the modulation of NO-mediated ROS generation from PMNs.

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose–response effect demonstrated that 5 or 10 μM DHA has no effect on Jurkat cell survival; however, 15 μM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 μM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.

Docosahexaenoic Acid and Vitamin E Can Reduce Human Monocytic U937 Cell Apoptosis Induced by Tumor Necrosis Factor

The effects of polyunsaturated fatty acids and vitamin E on tumor necrosis factor (TNF)-induced apoptosis of human monocytic U937 cells was explored to assess to what extent these nutrients could attenuate apoptosis. Preincubation of U937 cells with arachidonic acid for 24 h did not affect TNF-induced apoptosis. Eicosapentaenoic acid slightly but significantly reduced the proportion of apoptotic cells only when apoptosis was induced by TNF without cycloheximide (CHI). In contrast, preincubation with docosahexaenoic acid (DHA) greatly (40 approximately 70%) attenuated apoptosis induced by stimulation with either TNF or TNF + CHI for 3 h. The inhibition of apoptosis was accompanied by enrichment of DHA in membrane phospholipids, indicating that DHA probably exerted its inhibitory activity after being incorporated into the phospholipids. Vitamin E also played a role as a partial inhibitor of apoptosis 3 h after TNF addition. This vitamin could further reduce the apoptosis of DHA-treated cells, and such an additive effect was obvious when apoptosis was induced at a low frequency. Longer-range stimulation of U937 cells with TNF showed that inhibition of apoptosis by preincubating cells with either DHA or vitamin E was not significant 9 h after TNF addition, but that preincubation with both DHA and vitamin E could reduce the proportion of apoptotic cells even at this time point. Our findings suggested that ingestion of nutrients such as DHA and vitamin E might exert beneficial effects on organ dysfunction associated with various TNF-related diseases.

Effects of eicosapentaenoic acid and docosahexaenoic acid on plasma membrane fluidity of aortic endothelial cells.

We investigated the relative effects of n-3 eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on the plasma membrane fluidity of endothelial cells (EC) cultured from the thoracic aorta by determining fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its cationic derivative trimethylamino-DPH (TMA-DPH). Fluidity assessed by TMA-DPH demonstrated no significant differences in plasma membranes of vehicle (dimethyl sulfoxide; DMSO)-, EPA-, and DHA-treated EC. Plasma membrane fluidity assessed by DPH polarization, however, was significantly higher in the order of DHA > EPA > DMSO. Total cholesterol content decreased significantly by 28.4 and 15.9% in the plasma membranes of DHA- and EPA-treated cells, respectively. Total phospholipid content remained unaltered in the plasma membranes of the three groups of cells; however, the molar ratio of total cholesterol to phospholipid decreased significantly only in the membranes of DHA-treated EC. The unsaturation index in the plasma membranes of EPA- and DHA-treated cells increased by 35.7 and 64.3%, respectively, compared with that in the plasma membranes of control cells. The activities of catalase and glutathione peroxidase in the whole-cell homogenates, and levels of lipid peroxides in either the whole-cell homogenates or in plasma membrane fractions were not altered in EPA- or DHA-treated EC. These results indicate that the influence of DHA is greater than that of EPA in increasing plasma membrane fluidity of vascular EC. We speculate that the greater effect of DHA compared to EPA is due to its greater ability to decrease membrane cholesterol content or the cholesterol/phospholipid molar ratio, or both, and also to its greater ability in elevating the unsaturation index in the plasma membranes of EC.

Docosahexaenoic acid lowers phosphatidate level in human activated lymphocytes despite phospholipase D activation

N–3 polyunsaturated fatty acids from marine oil have been shown to decrease T cell-mediated immune function both in animals and humans, and to inhibit the mitogen-induced lymphoproliferative response when added to lymphocyte culture medium. As phosphatidic acid (PA) is a key mediator of the mitogenic process, the present study aims to investigate whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, the main n–3 fatty acids from fish oil, are able to alter the mitogen-induced synthesis of PA, when added to the culture medium of human peripheral blood mononuclear cells (PBMC). Incubation of PBMC in a medium containing 5 μM DHA bound to 5 μM human delipidated serum albumin induced a 2–fold increase in the basal PA mass whereas incubation with EPA, in the same conditions, had no effect. In contrast, both fatty acids markedly reduced the concanavalin A (ConA)-induced production of PA as compared with untreated cells. Paradoxically, phospholipase D (PLD) activity, evidenced by the synthesis of phosphatidylbutanol, was only detected in DHA-treated cells further stimulated by ConA, indicating that both DHA and ConA are required for PLD activation. Similarly, an increased diacylglycerol (DAG) mass was only observed in DHA-treated cells stimulated by ConA, whereas no modification occurred in control or EPA-treated cells stimulated or not by ConA. Furthermore, 1-butanol suppressed the ConA-induced increase of DAG mass observed in DHA-treated cells, indicating that phosphatidate was the source of the newly synthesized diacylglycerol. Altogether, these results show that, in concanavalin A-activated human peripheral blood mononuclear cells, docosahexaenoate stimulates both phospholipase D and phosphatidate phosphohydrolase activities, which ultimately results in an increased diacylglycerol production at the expense of phosphatidate.—Bechoua, S., M. Dubois, G. Némoz, M. Lagarde, and A-F. Prigent. Docasahexaenoic acid lowers phosphatidate level in human activated lymphocytes despite phospholipase D activation. J. Lipid Res. 1998. 39: 873–883.

Effect of Docosahexaenoic Acid on Lipoprotein Synthesis and Secretion by Cultured Eel Hepatocytes.

We investigated effect of docosahexaenoic acid on lipoprotein synthesis and secretion by cultured eel hepatocytes. When eel hepatocytes were incubated with 1 mM docosahexaenoic acid (DHA) at 28 degrees C for 24 h, triacylglycerol (TG), free cholesterol (FC), and cholesteryl ester (CE) syntheses from 14C-acetate increased 9.7-, 1.6-, and 8.5-fold, respectively, and the specific activity of TG was twofold that of control. These results indicate the stimulative effect of DHA on lipid synthesis. However, the radioactivity of the lipoprotein secreted by DHA-treated cells incubated with 14C-acetate and 3H-leucine reduced to 55 and 60%, respectively, compared to that by control cells, but DHA did not affect the incorporation of 3H-leucine into other secreted protein. Furthermore, the amount of the secreted lipoprotein per 24 h by DHA-treated cells also reduced to 17%. These results show that DHA inhibits the secretion of lipoprotein. Intracellular lipids such as TG, PL, and TC increased by 4.8, 1.6, and 1.8 times, respectively, in DHA-treated cells. The remarkable increase in TG in DHA-treated cells seems to be due to the stimulative effect of DHA on TG synthesis and an inhibitory effect of DHA on lipoprotein secretion, since about 70% of the secreted lipoprotein consists of TG. The chemical composition and apoprotein profile of the lipoprotein secreted by DHA-treated and control cells were almost the same. These results suggest that DHA inhibits the assembly or secretion of the lipoprotein in eel hepatocytes.