Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells

The effects of linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA) were compared to that of palmitic acid (PA), on inflammatory responses in human monocytic THP-1 cells. When cells were pre-incubated with fatty acids for 2-h and then stimulated with lipopolysaccharide for 24-h in the presence of fatty acids, secretion of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNFalpha) was significantly decreased after treatment with LA, ALA, and DHA versus PA (P < 0.01 for all); ALA and DHA elicited more favorable effects. These effects were comparable to those for 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and were dose-dependent. In addition, LA, ALA, and DHA decreased IL-6, IL-1beta, and TNFalpha gene expression (P < 0.05 for all) and nuclear factor (NF)-kappaB DNA-binding activity, whereas peroxisome proliferator-activated receptor-gamma (PPARgamma) DNA-binding activity was increased. The results indicate that the anti-inflammatory effects of polyunsaturated fatty acids may be, in part, due to the inhibition of NF-kappaB activation via activation of PPARgamma.     

Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids

Human subjects consuming fish oil showed a significant suppression of cyclooxygenase-2 (COX-2) expression in blood monocytes when stimulated in vitro with lipopolysaccharide (LPS), an agonist for Toll-like receptor 4 (TLR4). Results with a murine monocytic cell line (RAW 264.7) stably transfected with COX-2 promoter reporter gene also demonstrated that LPS-induced COX-2 expression was preferentially inhibited by docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), the major n-3 polyunsaturated fatty acids (PUFAs) present in fish oil. Additionally, DHA and EPA significantly suppressed COX-2 expression induced by a synthetic lipopeptide, a TLR2 agonist. These results correlated with the preferential suppression of LPS- or lipopeptide-induced NF kappa B activation by DHA and EPA. The target of inhibition by DHA is TLR itself or its associated molecules, but not downstream signaling components. In contrast, COX-2 expression by TLR2 or TRL4 agonist was potentiated by lauric acid, a saturated fatty acid. These results demonstrate that inhibition of COX-2 expression by n-3 PUFAs is mediated through the modulation of TLR-mediated signaling pathways. Thus, the beneficial or detrimental effects of different types of dietary fatty acids on the risk of the development of many chronic inflammatory diseases may be in part mediated through the modulation of TLRs.     

Hydrogenation of polyunsaturated fatty acids by human colonic bacteria

Emulsions of the fatty acids linoleic (C18:2 n-6), alpha-linolenic (C18:3 n-3) and arachidonic acid (C20:4 n-6) were incubated for 4 h under anaerobic conditions with human faecal suspensions. Linoleic acid was significantly decreased (P < 0.001) and there was a significant rise (P < 0.05) in its hydrogenation product, stearic acid. Linolenic acid was also significantly decreased (P < 0.01), and significant increases in C18:3 cis-trans isomers (P < 0.01) and linoleic acid (P < 0.05) were seen. With each acid, there were non-significant increases in acids considered to be intermediates in biohydrogenation. The study provides evidence that bacteria from the human colon can hydrogenate C18 essential polyunsaturated fatty acids. However, with arachidonic acid there was no evidence of hydrogenation.     

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.     

Comparative utilization of n-3 polyunsaturated fatty acids by cultured human Y-79 retinoblastoma cells

The Y-79 retinoblastoma cell, a cultured human line derived from the retina, was utilized as a model for investigating the metabolism of n-3 polyunsaturated fatty acids in neural tissue. When cultures were incubated with 5 microM linolenic (18:3), eicosapentaenoic (20:5) or docosahexaenoic (22:6) acids, a low concentration probably representative of physiologic levels, the amount incorporated was 20:5 congruent to 18.3 greater than 22:6. Regardless of which fatty acid was provided, 65-75% of the total uptake accumulated in phosphatidylethanolamine and ethanolamine plasmalogen, suggesting that these phospholipids play an important role in n-3 polyunsaturated fatty acid metabolism. A small amount of 22:6 was converted to 20:5, which was recovered in phosphatidylinositol and phosphatidylserine. Therefore, one metabolic function of 22:6 may be to serve as an intracellular storage pool for the formation of 20:5 through retroconversion. When any of the n-3 polyunsaturates was available, the main fatty acid that accumulated in the cell phospholipids was 22:6. The extent to which 22:6 accumulated, however, depended on the particular n-3 polyunsaturated fatty acid that was available. This suggests that the 22:6 content of a neural cell, and any cellular function dependent on 22:6 content, may be regulated by changes in the type of n-3 polyunsaturate available to the nervous system.     

Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts

Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form.     

Conjugated EPA activates mutant p53 via lipid peroxidation and induces p53-dependent apoptosis in DLD-1 colorectal adenocarcinoma human cells

Both conjugated linoleic acid (CLA), which contains conjugated double bonds, and eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, have antitumor effects. Hence, we hypothesized that a combination of conjugated double bonds and an n-3 highly unsaturated fatty acid may produce a stronger antitumor effect, and we have previously shown that conjugated EPA (CEPA), prepared by alkaline treatment of EPA, induces strong and selective apoptosis in vitro and in vivo, with the mechanism proceeding via lipid peroxidation. In this study, we examined CEPA-induced gene expression in DLD-1 colorectal adenocarcinoma human cells carrying a mutant p53, in order to understand the details of CEPA-induced apoptosis via lipid peroxidation. DNA microarray analysis of 9970 genes was performed by comparison of CEPA-treated DLD-1 cells with untreated DLD-1 cells, thereby allowing determination of the differential gene expression profile induced by CEPA in these cells. CEPA treatment caused up-regulation of expression of genes induced by p53 and activation of the mitochondrial apoptosis pathway via Bax and the death pathway via TRAIL, leading to apoptosis of DLD-1 cells. In addition, activation of the mutant p53 was also induced by CEPA, and these effects showed lipid-peroxidation dependency. This is the first such gene expression analysis of the effects of CEPA, and our results confirm that CEPA induces lipid peroxidation, activates mutant p53, and causes p53-dependent apoptosis in DLD-1 cells.     

A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as β-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser³⁰⁷ phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.     

The biosynthesis of polyunsaturated fatty acids by rat sertoli cells.

1. The biosynthesis of polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series was investigated in cultured Sertoli cells. 18:2n-6, 18:3n-6, 20:2n-6, 18:3n-3 and 20:3n-3 were added individually at a concentration of 20 mumol to culture media. 2. Maximum incorporation of 20- and 22-carbon PUFA into membrane lipids was observed after 72 hr of incubation with all the exogenous substrates used. 3. As reported in other cell systems, the delta 6 desaturation was the first rate-limiting step; the major factor regulating this activity was the concentration of linoleic acid or alpha-linolenic acid in the medium. 4. Our data show that the delta 5-desaturation represents a second regulatory step in PUFA biosynthesis. 5. The sum of n-6 and n-3 PUFA of the 22 carbon chain length constantly represented between 11 and 12% of total fatty acids, regardless of the exogenous substrate used. 6. Our kinetic studies of the incorporation of PUFA of the n-6 and n-3 series did not permit detection of a delta 8 desaturase activity.     

Decrease in adhesion of cells cultured in polyunsaturated fatty acids

The addition of long chain unsaturated fatty acids (linoleic, linolenic and arachidonic acids) to BHK cells reduces the cell to substrate adhesion, causes morphological changes and alters the cellular growth properties. The new characteristics are similar to those of transformed cells. The data indicate that the effects are probably due to actual changes in the surface membrane lipids and not due to prostaglandin synthesis.     

Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.     

Membrane polyunsaturated fatty acids and lithium-sodium countertransport in human erythrocytes

Two groups of individuals, 26 normotensive normolipemic and 37 normotensive hyperlipemic, all without family history of hypertension have been selected in attempt to demonstrate whether Li-Na countertransport of erythrocytes is influenced by plasma and membrane lipid composition. The maximal rate of Li-Na countertransport was elevated in hyperlipemics (0.344 +/- 0.168 vs 0.220 +/- 0.074 mmol/l erythrocytes/h). This difference is highly significant. Hyperlipemics had different composition of membrane lipids than normals. The most important variations were: increase of palmitic, palmitoleic and total saturated fatty acids (SFA) as well as increase of cholesterol/phospholipids ratio (C/PL); in contrast, hyperlipemics had a reduced amount of linoleic acid and total unsaturated fatty acids (UFA) as well as total polyunsaturated fatty acids (PUFA). Consequently, UFA/SFA and PUFA/SFA ratios were lower than in normals. Li-Na countertransport was negatively correlated with the amount of PUFA (P less than 0.02), whereas it was positively correlated with the following parameters: oleic/linoleic ratio (p less than 0.02), monounsaturated fatty acids/polyunsaturated fatty acids ratio (p less than 0.03) as well as with the SFA + monounsaturated fatty acid/PUFA ratio (p less than 0.03). These findings suggest that the V max of Li-Na countertransport in erythrocytes is influenced by the lipid composition of the membrane.     

Adaptive thermogenesis by dietary n-3 polyunsaturated fatty acids: Emerging evidence and mechanisms

Brown/beige fat plays a crucial role in maintaining energy homeostasis through non-shivering thermogenesis in response to cold temperature and excess nutrition (adaptive thermogenesis). Although numerous molecular and genetic regulators have been identified, relatively little information is available regarding thermogenic dietary molecules. Recently, a growing body of evidence suggests that high consumption of n-3 polyunsaturated fatty acids (PUFA) or activation of GPR120, a membrane receptor of n-3 PUFA, stimulate adaptive thermogenesis. In this review, we summarize the emerging evidence that n-3 PUFA promote brown/beige fat formation and highlight the potential mechanisms whereby n-3 PUFA require GPR120 as a signaling platform or act independently. Human clinical trials are revisited in the context of energy expenditure. Additionally, we explore some future perspective that n-3 PUFA intake might be a useful strategy to boost or sustain metabolic activities of brown/beige fat at different lifecycle stages of pregnancy and senescence. Given that a high ratio of n-6/n-3 PUFA intake is associated with the development of obesity and type 2 diabetes, understanding the impact of n-6/n-3 ratio on energy expenditure and adaptive thermogenesis will inform the implementation of a novel nutritional strategy for preventing obesity.     

Odd-chain polyunsaturated fatty acids in thraustochytrids

A series of unusual odd-chain fatty acids (OC-FA) were identified in two thraustochytrid strains, TC 01 and TC 04, isolated from waters off the south east coast of Tasmania, Australia. FA compositions were determined by capillary GC and GC–MS, with confirmation of polyunsaturated fatty acids (PUFA) structure performed by analysis of 4,4-dimethyloxazoline derivatives. PUFA constituted 68–74% of the total FA, with the essential PUFA; eicosapentaenoic acid (20:5ω3, EPA), arachidonic acid (20:4ω6, AA) and docosahexaenoic acid (22:6ω3, DHA), accounting for 42–44% of the total FA. High proportions of the saturated OC-FA 15:0 (7.1% in TC 01) and 17:0 (6.2% in TC 04) were detected. The OC-FA 17:1ω8 was present at 2.8% in TC 01. Of particular interest, the C₂₁ PUFA 21:5ω5 and 21:4ω7 were detected at 3.5% and 4.1%, respectively, in TC 04. A proposed biosynthesis pathway for these OC-PUFA is presented. It is possible that the unsaturated OC-PUFA found previously in a number of marine animals were derived from dietary thraustochytrids and they could be useful biomarkers in environmental and food web studies.