Stearidonic and γ-linolenic acids in echium oil improves glucose disposal in insulin resistant monkeys.

Echium oil (EO) contains stearidonic acid (18:4), a n-3 polyunsaturated fatty acids (PUFAs), and gamma-linolenic acids (18:3), a n-6 PUFA that can be converted to long chain (LC)-PUFAs. We aimed to compare a safflower oil (SO)-enriched diet to EO- and fish oil (FO)-enriched diets on circulating and tissue PUFAs levels and glycemic, inflammatory, and cardiovascular health biomarkers in insulin resistant African green monkeys. In a Latin-square cross-over study, eight monkeys consumed matched diets for 6 weeks with 3-week washout periods. Monkeys consuming FO had significantly higher levels of n-3 LC-PUFAs and EO supplementation resulted in higher levels of circulating n-3 LC-PUFAs and a significant increase in dihomo-gamma linolenic acid (DGLA) in red blood cells and muscle. Glucose disposal was improved after EO consumption. These data suggest that PUFAs in EO supplementation have the capacity to alter circulating, RBC and muscle LC-PUFA levels and improve glucose tolerance in insulin-resistant monkeys.

Advanced age, but not anergy, is associated with altered serum polyunsaturated fatty acid levels.

Unknown factors present in the serum of older adults impair lymphocyte function and may be responsible for anergy (absence of delayed-type hypersensitivity (DTH)) present in many older adults. Polyunsaturated fatty acids (PUFAs) and their metabolites are immunomodulatory and may play a role in clinical conditions of advanced age, including immune dysfunction. We hypothesized that PUFAs could be the factor(s) present in serum that contribute to impaired immune responses in older adults. Prior studies of serum PUFAs in older adults neither adequately control dietary PUFA intake, nor investigated the relationship of PUFAs and DTH responses. We determined serum PUFA concentrations in young adults with normal immune responses, and older adults with impaired (anergic elderly) or normal immunity (nonanergic elderly) before and after administering a standardized diet. After controlling for dietary intake, advancing age was associated with markedly higher serum concentrations of arachidonic acid (AA), dihomo-gamma-linoleic acid (DGLA), and eicosapentaenoic acid (EPA) and a lower AA:EPA ratio. Other serum PUFAs and the AA:DGLA ratio were unaffected by age. However, there was no difference between older adults with or without anergy. These data suggest advanced age is associated with marked alterations of serum PUFAs that are only apparent after strictly controlling dietary intake. However, there was no association of serum PUFA concentrations with DTH status among older adults.

Enhancement of mast cell survival: a novel function of some secretory phospholipase A(2) isotypes.

This study tested the hypothesis that certain secretory phospholipase A(2) (sPLA(2)) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA(2) activity and sPLA(2) receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA(2), prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA(2) hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA(2) did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA(2). Additionally, both wild-type and catalytically inactive group IB PLA(2) induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin V(FITC) binding and only partially inhibited thymidine incorporation in sPLA(2)-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin V(FITC) binding and thymidine incorporation in mast cells maintained with IL-3. sPLA(2) enhanced phosphoinositide 3'-kinase activity, and a specific inhibitor of phosphoinositide 3'-kinase reversed the antiapoptotic effects of sPLA(2). Likewise, sPLA(2) increased the degradation of I-kappaBalpha, and specific inhibitors of nuclear factor kappa activation (NF-kappaB) reversed the antiapoptotic effects of sPLA(2). Together, these experiments reveal that certain isotypes of sPLA(2) enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA(2) receptors rather than sPLA(2) catalytic products.

Early de novo gene expression is required for 15-deoxy-Delta 12,14-prostaglandin J2-induced apoptosis in breast cancer cells.

Cyclopentenone prostaglandin derivatives of arachidonic acid are potent inducers of apoptosis in a variety of cancer cell types. Several investigators have shown that the terminal derivative of prostaglandin J(2) (PGJ(2)) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), but 15dPGJ(2) effects can be mediated by PPARgamma-dependent and PPARgamma-independent mechanisms. Here we report that 15dPGJ(2) regulates early gene expression critical to apoptosis. Specifically, 15dPGJ(2) induces potent and irreversible S phase arrest that is correlated with expression of genes critical to cell cycle arrest and apoptosis, including the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Inhibition of RNA or protein synthesis abrogates apoptosis induced by 15dPGJ(2) in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor-alpha or CD95/Fas ligand. Additionally, 15dPGJ(2) induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ(2)-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

Addition of eicosapentaenoic acid to gamma-linolenic acid-supplemented diets prevents serum arachidonic acid accumulation in humans.

Previous studies reveal that supplementation of human diets with gamma-linolenic acid (GLA) reduces the generation of lipid mediators of inflammation and attenuates clinical symptoms of chronic inflammatory disorders such as rheumatoid arthritis. However, we have shown that supplementation with this same fatty acid also causes a marked increase in serum arachidonate (AA) levels, a potentially harmful side effect. The objective of this study was to design a supplementation strategy that maintained the capacity of GLA to reduce lipid mediators without causing elevations in serum AA levels. Initial in vitro studies utilizing HEP-G2 liver cells revealed that addition of eicosapentaenoic acid (EPA) blocked Delta-5-desaturase activity, the terminal enzymatic step in AA synthesis. To test the in vivo effects of a GLA and EPA combination in humans, adult volunteers consuming controlled diets supplemented these diets with 3.0 g/d of GLA and EPA. This supplementation strategy significantly increased serum levels of EPA, but did not increase AA levels. EPA and the elongation product of GLA, dihomo-gamma-linolenic acid (DGLA) levels in neutrophil glycerolipids increased significantly during the 3-wk supplementation period. Neutrophils isolated from volunteers fed diets supplemented with GLA and EPA released similar quantities of AA, but synthesized significantly lower quantities of leukotrienes compared with their neutrophils before supplementation. This study revealed that a GLA and EPA supplement combination may be utilized to reduce the synthesis of proinflammatory AA metabolites, and importantly, not induce potentially harmful increases in serum AA levels.

Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans.

To understand the in vivo metabolism of dietary gamma-linolenic acid (GLA), we supplemented the diets of 29 volunteers with GLA in doses of 1.5-6.0 g/d. Twenty-four subjects ate controlled eucaloric diets consisting of 25% fat; the remaining subjects maintained their typical Western diets. GLA and dihomo-gamma-linolenic acid (DGLA) increased in serum lipids of subjects supplemented with 3.0 and 6.0 g/d; serum arachidonic acid increased in all subjects. GLA supplementation with 3.0 and 6.0 g/d also resulted in an enrichment of DGLA in neutrophil phospholipids but no change in GLA or AA levels. Before supplementation, DGLA was associated primarily with phosphatidylethanolamine (PE) of neutrophil glycerolipids, and DGLA increased significantly in PE and neutral lipids after GLA supplementation. Extending the supplementation to 12 wk did not consistently change the magnitude of increase in either serum or neutrophil lipids in subjects receiving 3.0 g/d. After GLA supplementation, A23187-stimulated neutrophils released significantly more DGLA, but AA release did not change. Neutrophils obtained from subjects after 3 wk of supplementation with 3.0 g/d GLA synthesized less leukotriene B4 (P < 0.05) and platelet-activating factor. Together, these data reveal that DGLA, the elongase product of GLA, but not AA accumulates in neutrophil glycerolipids after GLA supplementation. The increase in DGLA relative to AA within inflammatory cells such as the neutrophil may attenuate the biosynthesis of AA metabolites and may represent a mechanism by which dietary GLA exerts an anti-inflammatory effect.

Metabolism of gammalinolenic acid in human neutrophils.

Gammalinolenic acid (GLA), when provided as a dietary supplement, has been reported to improve clinical symptoms of several inflammatory disorders. The goal of the current study was to examine the metabolism of GLA and its relationship to arachidonic acid (AA) in the human neutrophil. Initial studies indicated that neutrophils provided GLA in vitro rapidly elongate it (by two carbons) to dihomogammalinolenic acid (DGLA). The bulk of this newly formed DGLA is incorporated into neutral lipids and specifically triacylglycerides. Neutrophils from volunteers supplemented with GLA as borage oil also had elevated quantities of DGLA but not GLA, when compared with neutrophils from volunteers not consuming the GLA supplement. To determine whether DGLA could be mobilized from cellular glycerolipids, neutrophils were stimulated with ionophore A23187 and fatty acid levels were determined. DGLA and AA were both released during stimulation, and the quantities of DGLA mobilized increased threefold after in vitro GLA supplementation. Exogenously provided DGLA was converted to one major metabolite during cell stimulation; this product migrated on reverse-phase HPLC with the 15-lipoxygenase product, 15-hydroxy-eicosa-trienoic acid (15-HETre). Both 15-HETre and DGLA (provided exogenously) inhibited the formation of leukotriene B4, (LTB4) and 20-hydroxy-leukotriene B4 (20-OH-LTB4). The IC50 for 15-HETre inhibition of both LTR, and 20-OH-LTB4 in A23187-stimulated neutrophils was 5 microM. This inhibition could be reversed by removing the compounds from the cells. Taken together, these data reveal that there are enzymes within the human neutrophil that metabolize GLA or its elongation product DGLA, and that the metabolism of GLA and AA may interact at a number of critical junctures.

Migration of human inflammatory cells into the lung results in the remodeling of arachidonic acid into a triglyceride pool.

Increasing evidence suggests that the metabolism of arachidonic acid (AA) may be different in inflammatory cells isolated from blood or migrating into tissues. To explore the possibility that changes in AA metabolism between blood and tissue inflammatory cells could be due in part to a different content or distribution of AA in glycerolipid classes, we studied these parameters in six human inflammatory cells isolated from blood (eosinophils, monocytes, neutrophils, and platelets) or from the lung tissue (mast cells and macrophages). Lung cells generally had a higher total cellular content of AA than that found in the blood cells. In addition, both mast cells and macrophages had a large endogenous pool of AA associated with triglycerides (TG), containing 45 and 22% of their total cellular AA, respectively. To address the hypothesis that cells migrating into the lung had a higher cellular level of AA and a larger AA pool in TG, we studied neutrophils isolated from the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome. BAL neutrophils had a fourfold increase in cellular AA as compared with blood neutrophils and contained 25% of their AA in TG versus 3% in blood neutrophils. BAL neutrophils also had a higher number of cytoplasmic lipid bodies (8 +/- 3/cell) relative to blood neutrophils (2 +/- 1/cell). High concentrations of free AA were also found in the cell-free BAL fluid of adult respiratory distress syndrome patients. To explore whether changes in BAL neutrophils may be due to the exposure of the cells to high concentrations of exogenous AA found in BAL, we incubated blood neutrophils in culture with AA (10-100 microM) for 24 h. Neutrophils supplemented with AA had a 10-fold increase in the amount of AA associated with TG and a sixfold increase in the number of lipid bodies. In addition, supplementation with AA induced a dose-dependent formation of hypodense cells. Taken together, these data indicate that human inflammatory cells undergo a fundamental and consistent remodeling of AA pools as they mature or enter the lung from the blood. These biochemical and morphological changes can be mimicked in vitro by exposing the cells to high levels of AA. This mechanism may be responsible for the changes in AA mobilization and eicosanoid metabolism observed in tissue inflammatory cells.

Dietary n-3 fatty acid effects on neutrophil lipid composition and mediator production. Influence of duration and dosage.

Healthy volunteers supplemented their usual Western diets with Promega fish oil supplement (eicosapentaenoic acid [EPA], 0.28 g; docosahexaenoic acid [DCHA], 0.12 g; other n-3 fatty acids 0.10 g per capsule) using three protocols. Initial experiments (protocol 1 and 2) investigated the kinetics of incorporation of n-3 fatty acids into serum and neutrophil lipids after 10 capsules/d of Promega. EPA was rapidly detected in both serum and neutrophil lipids; the arachidonic acid (AA) to EPA ratio in neutrophil phospholipids showed a maximal reduction of 49:1 to 8:1 within 1 wk of beginning supplementation. EPA was preferentially incorporated into phosphatidyl-ethanolamine and phosphatidylcholine but not phosphatidylinositol. Long-term supplementation for up to 7 wk did not influence the AA/EPA ratio or the distribution of EPA among neutrophil phospholipids in a manner that was not observed after the first week. Neutrophils produced similar quantities of platelet-activating factor and slightly lower quantities of leukotriene B4 during long-term supplementation when compared with presupplementation values. Experiments examining the influence of Promega dosage indicated that the AA/EPA ratio in neutrophil lipids decreased in a dose-dependent manner. Only when the dose was increased to 15 capsules/d was there a reduction in the AA/DCHA ratio in neutrophil lipids. The quantity of AA in neutrophil lipids remained relatively constant at all supplement doses. Taken together, the current study demonstrates the capacity of n-3 fatty acids provided with a Western diet to be rapidly incorporated into neutrophil lipids. However, dietary n-3 fatty acids appear not to significantly reduce arachidonate content within neutrophil phospholipids. Constant arachidonate levels may account for the lack of large reductions in the biosynthesis of lipid mediators by neutrophils after fish-oil supplementation.

Factors that influence the proportions of platelet-activating factor and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine synthesized by the mast cell.

Recent studies have demonstrated that inflammatory cells can be divided into two groups depending on the type of 2-acetylated phospholipids [1-radyl-2-acetyl-sn-glycero-3-phosphocholine (GPC)] they produce: those that produce predominantly platelet-activating factor (PAF), and those that produce predominantly its 1-acyl analogue (1-acyl-2-acetyl-GPC; AAGPC) [Triggiani, Schleimer, Warner & Chilton (1991) J. Immunol. 147, 660-666]. The present study has examined the factors that regulate the production of these two molecules in mouse bone marrow-derived mast cells (BMMC). Initial experiments indicated that PAF and AAGPC were catabolized by BMMC in a differential manner via two pathways: the first, exclusive for AAGPC, involved a 1-acyl hydrolase that removed the long chain at the sn-1 position of the molecule, and the second, common to AAGPC and PAF, involved acetylhydrolase that removed the acetate at the sn-2 position of the two molecules. Experiments were next designed to identify conditions where the differential catabolism of AAGPC and PAF could be eliminated in order to uncover other factors that regulate the proportions of AAGPC and PAF produced. Phenylmethanesulphonyl fluoride (PMSF) completely blocked the 1-acylhydrolase activity while having little or no effect on the acetyl hydrolase activity, thereby eliminating the influence of the catabolic pathway unique to AAGPC. Moreover, PMSF did not alter the release of arachidonic acid from phospholipid subclasses. PMSF-treated BMMC produced larger quantities of AAGPC than of PAF. The AAGPC/PAF ratio detected in PMSF-treated BMMC was very similar to the ratio of arachidonate contained in and released from 1-acyl-/1-alkyl-linked phosphatidylcholine (PC). BMMC supplemented with arachidonic acid in culture for 3 days increased their total arachidonic acid content in PC as well as the ratio of 1-acyl-2-arachidonoyl-GPC to 1-alkyl-2-arachidonoyl-GPC. These changes resulted in parallel and significant increases in both the total amount of 1-radyl-2-acetyl-GPC and the AAGPC/PAF ration in BMMC. These data indicate that the AAGPC/PAF ratio produced by inflammatory cells is regulated by at least two factors: (1) differential catabolism of these two molecules, and (2) the distribution of arachidonate in 1-acyl- and 1-alkyl-2-arachidonyl-GPC. These observations support the concept of a common pathway for AAGPC and PAF biosynthesis in which the two precursor molecules are 1-acyl-2-arachidonoyl-GPC and 1-alkyl-2-arachidonoyl-GPC, respectively.

Evidence that increasing the cellular content of eicosapentaenoic acid does not reduce the biosynthesis of platelet-activating factor.

Our study has examined platelet-activating factor (PAF) biosynthesis in neutrophils from individuals on a fish oil-enriched diet and in mast cells enriched with eicosapentaenoic acid (EPA) in vitro. Neutrophils isolated from males who were fed fish oil supplement (EPA; 2.8 g/day) for 5 wk contained large quantities of eicosapentaenoate in phosphatidylcholine (PC) and phosphatidylethanolamine and less in phosphatidylinositol. The ratio arachidonate/eicosapentaenoate in PC and phosphatidylethanolamine decreased from greater than 10 before the enriched diet to approximately 3 after the diet. The putative precursor of PAF, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC) contained the bulk of eicosapentaenoate in PC subclasses with smaller quantities found in 1-acyl and 1-alk-1'-enyl linked species. Ionophore A23187-stimulated neutrophils produced similar quantities of PAF before and after enriched diet. Neutrophils during normal diet acylated 1-O-alkyl-2-lyso-GPC only with arachidonate whereas neutrophils from individuals on enriched diet transferred both arachidonate and eicosapentaenoate into exogenously-provided 1-O-alkyl-2-lyso-GPC. This allowed for the labeling of neutrophils with 1-O-[3H]-alkyl-2-arachidonoyl-GPC (before diet) as well as neutrophils with 1-O-[3H]-alkyl-2-eicosapentaenoyl-GPC and 1-O-[3H]-alkyl-2-arachidonoyl-GPC (after diet). Neutrophils after diet converted similar quantities of these labeled precursors to labeled PAF upon stimulation as those before the diet. Analysis of the nature of the long chain acyl residue remaining in the sn-2 position of 1-alkyl-2-acyl-GPC after cell stimulation indicated that arachidonate and eicosapentaenoate were both released from 1-O-alkyl-2-acyl-GPC at comparable rates. Finally, in vitro supplementation of murine mast cells (PT-18) with arachidonic acid or EPA caused a marked increase in the amount of PAF produced by the cell without having any effect on histamine release. Data from these experiments suggest that EPA is incorporated into a PAF precursor pool. However, this appears not to inhibit PAF production because phospholipase A2 can use eicosapentaenoate- as well as arachidonate-containing phospholipids in the initial step of PAF biosynthesis.

Influence of immunologic activation and cellular fatty acid levels on the catabolism of platelet-activating factor within the murine mast cell (PT-18).

The present study has examined the catabolism of 1-O-[3H]hexadecyl-2-acetyl-GPC (C16-PAF) and of 1-O-octadecyl-2-acetyl-GPC (C18-PAF) in spleen-derived PT-18 murine mast cells (mast cells). Mast cells catabolized exogenous PAF into two inactive metabolites, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC). The rate of conversion of C16-PAF to metabolites was more rapid than that of C18-PAF. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC formed during the metabolism of PAF revealed that arachidonic acid (20:4) was the major fatty acyl chain incorporated at the sn-2 position. However, 25% of newly formed 1-O-alkyl-2-acyl-GPC was reacylated with docosahexaenoic acid (22:6). The influence of cellular fatty acid content on PAF catabolism was further explored in mast cells in which the ratio of fatty acids within cellular phosphoglycerides had been altered by supplementing the cells with various fatty acids in culture. Mast cells supplemented with 20:4 or 22:6 converted PAF to 1-O-alkyl-2-acyl-GPC at a significantly higher rate than non-supplemented cells. In contrast, cells supplemented with linoleic acid (18:2) metabolized PAF at rates similar to non-supplemented cells. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC derived from the metabolism of PAF in 20:4-supplemented cells indicated that 20:4 was incorporated exclusively into the sn-2 position. Conversely, 22:6-supplemented cells incorporated predominantly 22:6 at the sn-2 position of 1-alkyl-2-lyso-GPC. Supplementation with 18:2 had no effect on the acylation pattern seen in newly formed 1-O-alkyl-2-acyl-GPC. Activation of passively sensitized mast cells with antigen or with ionophore A23187 significantly enhanced the rate of catabolism of exogenously-provided PAF but had no effect on the acylation pattern of 1-O-alkyl-2-acyl-GPC. Experiments performed with the soluble fraction of the cells showed that acetyl hydrolase activity was increased in mast cells stimulated with antigen. In addition, supernatant fluids from antigen or ionophore-treated mast cells converted PAF to lysoPAF, suggesting that acetyl hydrolase activity was released during cell activation. These data indicate that the ability of mast cells to catabolize PAF to inactive metabolites is influenced by cell activation and by the cellular levels of certain fatty acids.