Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells

In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis.     

Arachidonic and docosahexaenoic acids differentially affect the expression of fatty acyl-CoA oxidase, protein kinase C and lipid peroxidation in HepG2 cells.

Arachidonic (AA) and docosahexaenoic (DHA) acids (5-20 microM), when supplemented to human hepatoma HepG2 cells, which are depleted in these long-chain polyunsaturated fatty acids in conventional culture conditions, enhance the expression of acyl-CoA oxidase (ACOX), the first enzyme in the peroxisomal beta-oxidation cycle. DHA is effective at lower concentrations (at 5 microM) and to a greater extent (about 60% increment) than AA (about 40%) at 20 microM. Protein kinase C (PKC) appears to be involved in the activity of AA on ACOX, but not in that of DHA, since only the effect of AA is prevented by the PKC inhibitor Staurosporine, and since a remarkable elevation of the PKC activator diacylglycerol occurs only after AA supplementation. AA also induces elevation of lipoperoxides, favoured by the relative vitamin E deficiency occurring in cultured cells, and this effect, which is prevented by supplementation of the vitamin, may contribute to PKC activation.     

Polyunsaturated fatty acids stimulate phosphatidylcholine synthesis in PC12 cells

The synthesis of phospholipids in mammalian cells is regulated by the availability of three critical precursor pools: those of choline, cytidine triphosphate and diacylglycerol. Diacylglycerols containing polyunsaturated fatty acids (PUFAs) apparently are preferentially utilized for phosphatide synthesis. PUFAs are known to play an important role in the development and function of mammalian brains. We therefore studied the effects of unsaturated, monounsaturated and polyunsaturated fatty acids on the overall rates of phospholipid biosynthesis in PC12 rat pheochromocytoma cells. Docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) all significantly stimulated the incorporation of (14)C-choline into total cellular phospholipids. In contrast, monounsaturated oleic acid (OA) and the saturated palmitic (PA) and stearic (SA) acids did not have this effect. The action of DHA was concentration-dependent between 5 and 50 microM; it became statistically significant by 3 h after DHA treatment and then increased over the ensuing 3 h. DHA was preferentially incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS), while AA predominated in phosphatidylcholine (PC).     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Docosahexaenoic Acid Is a Major n-3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µ M ) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.     

Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism

Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that overexpression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6, formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 overexpression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any fatty acids into phospholipids (PLs) or triacylglycerides (TAGs) was unknown. Here we show that Acsl6 overexpression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, oleic acid (OA) and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 overexpression. Additionally, the level of total cellular PLs was increased in Acsl6 overexpressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 overexpression increased the incorporation of [(14)C]-labeled OA, AA, or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of fatty acids into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its overexpression increases DHA and AA internalization primarily during the first 24 h of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth.     

Interaction of brain fatty acid-binding protein with the polyunsaturated fatty acid environment as a potential determinant of poor prognosis in malignant glioma

Malignant gliomas are the most common adult brain cancers. In spite of aggressive treatment, recurrence occurs in the great majority of patients and is invariably fatal. Polyunsaturated fatty acids are abundant in brain, particularly ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Although the levels of ω-6 and ω-3 polyunsaturated fatty acids are tightly regulated in brain, the ω-6:ω-3 ratio is dramatically increased in malignant glioma, suggesting deregulation of fundamental lipid homeostasis in brain tumor tissue. The migratory properties of malignant glioma cells can be modified by altering the ratio of AA:DHA in growth medium, with increased migration observed in AA-rich medium. This fatty acid-dependent effect on cell migration is dependent on expression of the brain fatty acid binding protein (FABP7) previously shown to bind DHA and AA. Increased levels of enzymes involved in eicosanoid production in FABP7-positive malignant glioma cells suggest that FABP7 is an important modulator of AA metabolism. We provide evidence that increased production of eicosanoids in FABP7-positive malignant glioma growing in an AA-rich environment contributes to tumor infiltration in the brain. We discuss pathways and molecules that may underlie FABP7/AA-mediated promotion of cell migration and FABP7/DHA-mediated inhibition of cell migration in malignant glioma.     

Suppression of platelet-activating factor generation and modulation of arachidonate metabolism by dietary enrichment with (n-9) eicosatrienoic acid or docosahexaenoic acid in mouse peritoneal cells

Several studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) suppress platelet-activating factor (PAF) generation in leukocytes of humans and rodents, which is associated with the antagonism of arachidonic acid metabolism. Dietary eicosatrienoic acid (20:3n-9, ETrA) is also suggested to antagonize arachidonic acid (AA) metabolism, but its effect on PAF generation in leukocytes has not been defined. In the present study, we investigated the effects of an ETrA-rich diet on PAF generation and AA metabolism in mouse peritoneal cells, which were compared with those of a docosahexaenoic acid (DHA)-rich diet. Mice were fed a diet supplemented with a lipid preparation rich in ETrA, a DHA-rich fish oil (FO) or palm oil (PO) for 3 weeks, and peritoneal cells containing more than 80% of monocytes/macrophages were obtained. The peritoneal cells in the DHA and ETrA diet groups generated upon zymosan stimulation a smaller amount of PAF than cells in the PO diet group. In the peritoneal cells of the DHA diet group, AA contents in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly lower than those in cells of the PO diet group, but those in phosphatidylinositol (PI) were not significantly different between the two dietary groups. A considerable amount of ETrA was incorporated into the peritoneal cells of the ETrA diet group, and AA was reduced as compared with the PO diet group. These changes occurred preferentially in PI but to a less extent in PC and PE. The amount of free AA released by the peritoneal cells upon zymosan stimulation was significantly reduced in the DHA diet group as compared with that in the PO diet group, whereas AA release was similar between the PO and ETrA diet groups. In conclusion, the effects of dietary ETrA on AA content in the phospholipid subclasses and AA release were quite different from those of dietary DHA, although both diets suppressed PAF generation in mouse peritoneal cells to a similar extent.     

Modification of phospholipids fatty acid composition in reuber H35 hepatoma cells: effect on HMG-CoA reductase activity

There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.     

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Alteration of fatty acid composition of LM cells by lipid supplementation and temperature.

Alteration of the fatty acid composition of monolayer cultures of LM cells grown in chemically defined medium was achieved by supplementation with fatty acids complexed to bovine serum albumin. Phospholipids containing up to 40% linoleate were found in cells grown in medium containing 20 mu g of linoleate/ml. Incorporation of linoleate into phospholipids reached a plateau after 12-24 hr, and cells remained viable for at least 3-4 days. Although linoleic, linolenic, and arachidonic acids were incorporated into LM cells equally well, only the latter was elongated by these cells under these experimental conditions. Nonadecanoic acid was incorporated to a lesser extent than the polyunsaturated fatty acids. Phosphatidylcholine and phosphatidylethanolamine of LM cells had different fatty acid compositions; phosphatidylethanolamine contained more longer chain and unsaturated fatty acids. Cells were also grown in the absence of choline and presence of choline analogs such as N,N-dimethylethanolamine, N-methylethanolamine, 3-amino-1-propanol, and 1-2-amino-1-butanol. The analog phospholipids in these cells had fatty acid compositions which were intermediate between those of phosphatidylethanolamine and phosphatidylcholine of control cells grown in the presence of choline. Linoleate was found in both phosphatidylcholine and phosphatidylethanolamine of cells supplemented with linoleate. The sphingolipid fraction of these cells, however, did not contain significant amounts of linoleate. When linoleate was present in the phospholipids, compensatory decreases in the oleate and palmitoleate content of phospholipids were observed. Lowering of the growth temperature to 28 degrees produced an increase in unsaturate fatty acid content of the phospholipids. When linoleate was supplied to cells grown at 28 degrees, there was no further increase in the unsaturated fatty acid composition of the phospholipids. Using both fatty acid supplementation and lowered growth temperature, LM cell membranes can be produced which have phospholipids with vastly different fatty acid compositions.     

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.     

Dietary effects on brain fatty acid composition: the reversibility of n-3 fatty acid deficiency and turnover of docosahexaenoic acid in the brain, erythrocytes, and plasma of rhesus monkeys

Rhesus monkeys given pre- and postnatal diets deficient in n-3 essential fatty acids develop low levels of docosahexaenoic acid (22:6 n-3, DHA) in the cerebral cortex and retina and impaired visual function. This highly polyunsaturated fatty acid is an important component of retinal photoreceptors and brain synaptic membranes. To study the turnover of polyunsaturated fatty acids in the brain and the reversibility of n-3 fatty acid deficiency, we fed five deficient juvenile rhesus monkeys a fish oil diet rich in DHA and other n-3 fatty acids for up to 129 weeks. The results of serial biopsy samples of the cerebral cortex indicated that the changes of brain fatty acid composition began as early as 1 week after fish oil feeding and stabilized at 12 weeks. The DHA content of the phosphatidylethanolamine of the frontal cortex increased progressively from 3.9 +/- 1.2 to 28.4 +/- 1.7 percent of total fatty acids. The n-6 fatty acid, 22:5, abnormally high in the cerebral cortex of n-3 deficient monkeys, decreased reciprocally from 16.2 +/- 3.1 to 1.6 +/- 0.4%. The half-life (t 1/2) of DHA in brain phosphatidylethanolamine was estimated to be 21 days. The fatty acids of other phospholipids in the brain (phosphatidylcholine, -serine, and -inositol) showed similar changes. The DHA content of plasma and erythrocyte phospholipids also increased greatly, with estimated half-lives of 29 and 21 days, respectively. We conclude that monkey cerebral cortex with an abnormal fatty acid composition produced by dietary n-3 fatty acid deficiency has a remarkable capacity to change its fatty acid content after dietary fish oil, both to increase 22:6 n-3 and to decrease 22:5 n-6 fatty acids. The biochemical evidence of n-3 fatty acid deficiency was completely corrected. These data imply a greater lability of the fatty acids of the phospholipids of the cerebral cortex than has been hitherto appreciated.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Effects of long-term supplementation of eicosapentanoic and docosahexanoic acid on the 2-, 3-series prostacyclin production by endothelial cells.

We studied the effects of polyunsaturated fatty, acids such as arachidonic acid [20:4 (n-6)], eicosapentanoic acid [EPA, 20:5 (n-3)], and docosahexanoic acid [DHA, 22:6 (n-3)] on the changes of lipid profiles and prostacyclin production by cultured bovine aortic endothelial cells. The amounts of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha) and delta17-6-keto-PGF1alpha, non-enzymatic metabolites of prostacyclin (PGI2 and PGI3) in culture medium were measured by gas chromatography/selected ion monitoring. Endothelial cells were supplemented for five passages with arachidonic acid, EPA, or DHA, and the fatty acids of cell lipids and prostacyclin production in cultured medium were quantified. From the fatty acid analysis, the amounts of docosapentaenoic acid [22:5 (n-3)] were significantly increased in EPA-grown cells. In DHA-grown cells, the amounts of EPA were slightly increased compared to control cells. These cells produced similar amounts of PGI2 as the controls, but larger amounts of PGI3 under basal conditions. These findings suggest that EPA, docosapentaenoic acid, and DHA are interconverted to each other, and anti-aggregatory effects of EPA or DHA may be partially due to the stimulation of prostacyclin formation in endothelial cells.     

Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca(2+) load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca(2+)-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca(2+) load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.