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.     

Selective effects of isomeric cis and trans fatty acids on fatty acyl delta 9 and delta 6 desaturation by human skin fibroblasts

Human skin fibroblasts incorporate and actively desaturate long-chain fatty acids. Growth of these cells in lipid-free medium can be used to enhance delta 9 and delta 6 desaturation of [14C]stearate and [14C]linoleate, respectively. Medium supplementation with cis fatty acids inhibits delta 9 desaturation; effectiveness as inhibitors is linoleate (9c,12c-18:2) greater than oleate (9c-18:1) greater than vaccenate (11c-18:1). Linoelaidate (9t,12t-18:2), trans-vaccenate (11t-18:1) and saturated fatty acids are without effect; elaidate (9t-18:1) appears stimulatory. By contrast, the trans fatty acids elaidate and linoelaidate are potent inhibitors of delta 6 desaturation; inhibition by trans-vaccenate is 50% of that of elaidate. Desaturation of [14C]linoleate is only slightly inhibited by oleate, cis-vaccenate, or (6c,9c,12c)-linolenate. The relative effectiveness of isomeric cis- and trans-octadecenoic acids as inhibitors of delta 9 and delta 6 desaturation in intact human cells is different from that found in microsomal studies. The cell culture system can thus be important in evaluating physiological effects of isomeric fatty acids on cellular metabolic processes.     

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.     

Metabolism of saturated and polyunsaturated fatty acids by normal and Zellweger syndrome skin fibroblasts.

The metabolism of 1-11C-labelled derivatives of palmitic (C16:0), arachidonic (C20:4,n-6) lignoceric (C21:0) and tetracosatetraenoic (C24:4,n-6) acids was studied in normal skin fibroblast cultures and in cultures of fibroblasts from peroxisome-deficient (Zellweger's syndrome) patients. Radiolabelled products of the fatty acids included carbon dioxide. C14-24 saturated and mono-unsaturated fatty acids formed from released acetate either by synthesis de novo or by elongation of endogenous fatty acids, fatty acids formed by 2-6-carbon elongation of added substrates, and a number of water-soluble compounds, some of which were tentatively identified as the amino acids glutamine, glutamic acid and asparagine. The labelled amino acids were found predominantly in the culture medium. Zellweger's syndrome fibroblasts showed a marked decrease in radiolabelled carbon dioxide and water-soluble-product formation from (I-14C)-labelled arachidonic, tetracosatetraenoic and lignoceric acids but not from [I-14C]palmitic acid, and the production of radiolabelled C14-18 fatty acids was also diminished. However, the elongation of individual fatty acids was either normal or above normal. Our data support the view that the oxidation of 20:4, 24:4 and 24:0 fatty acids in cultured skin fibroblasts takes place largely in peroxisomes, and further that the acetyl-CoA released by the beta-oxidation process is available for the synthesis of fatty acids and amino acids. We speculate that the generation of C2 units used for synthesis is a major peroxisomal function and that this function is absent or greatly impaired in Zellweger's syndrome cells.     

{Omega}-3 and {omega}-6 polyunsaturated fatty acids block HERG channels

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na⁺, Ca²⁺, and several K⁺ channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, -6) and docosahexaenoic acid (DHA, -3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 µM, AA and DHA blocked HERG channels, at the end of 5-s pulses to –10 mV, to a similar extent (37.7 ± 2.4% vs. 50.2 ± 8.1%, n = 7–10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by –5.1 ± 1.8 mV (n = 10, P < 0.05) and –11.2 ± 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 ± 3.9 mV (n = 4; P < 0.05) and +15.8 ± 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to –120 mV (IO). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA. K⁺ channel; membrane currents; ion channels; arrhythmia; antiarrhythmics     

Dietary polyunsaturated fatty acids (C18:2 omega6 and C18:3 omega3) do not suppress hepatic lipogenesis

Omega 3 polyunsaturated fatty acids are promoted as beneficial in the prevention of metabolic and cardiovascular diseases. In general, dietary omega 3 fatty acids are derived from plant sources as linolenic acid (LNA, C18:3 omega3) the precursor to eicosapentaenoic acid (EPA, C20:5 omega3) and docosahexaenoic acid (DHA, C22:6 omega3). However, it remains unclear if the polyunsaturated fatty acid (PUFA) LNA can provide the same health benefits as the very long chain highly unsaturated fatty acids (HUFA) EPA and DHA generally derived from oily fish. In this study, mice were fed synthetic diets containing lard (low in PUFA and HUFA), canola oil (to supply PUFA), or a mixture of menhaden and arasco (fish and fungal) oils (to supply HUFA) for 8 weeks. The diets were neither high in calories nor fat, which was supplied at 6%. The lard and canola oil diets resulted in high levels of hepatic triglycerides and cholesterol and elevation of lipogenic gene expression. By comparison livers from mice fed the fish/fungal oil diet had low levels of lipid accumulation and more closely resembled livers from mice fed standard laboratory chow. SREBP1c and PPARgamma gene and protein expression were high in livers of animals fed diets containing lard or canola oil compared with fish/fungal oil. Hepatic fatty acid analyses indicated that dietary PUFA were efficiently converted to HUFA regardless of source. Therefore, differences in hepatic lipid levels and gene expression between dietary groups were due to exogenous fatty acid supplied rather than endogenous pools. These results have important implications for understanding the regulation of hepatic lipogenesis by dietary fatty acids.     

Beta-oxidation of very-long-chain fatty acids and their coenzyme A derivatives by human skin fibroblasts

The beta-oxidation of lignoceric acid (C24:0), hexacosanoic acid (C26:0), and their coenzyme A derivatives was investigated in human skin fibroblast homogenates. The cofactor requirements for oxidation of lignoceric acid and hexacosanoic acid were identical but were different from their coenzyme A derivatives. For example, lignoceric acid and hexacosanoic acid oxidation was strictly ATP dependent whereas the oxidation of the corresponding coenzyme A derivatives was ATP independent. Also the rate of oxidation of coenzyme A derivatives of lignoceric acid or hexacosanoic acid was much higher compared to the free fatty acids. In patients with Zellweger's syndrome, X-linked adrenoleukodystrophy and infantile Refsum's disease, the beta-oxidation of lignoceric and hexacosanoic acids was defective whereas the oxidation of their corresponding coenzyme A derivatives was nearly normal. The results presented in this communication suggest strongly that the beta-oxidation of very-long-chain fatty acids occurs exclusively in peroxisomes. However, the coenzyme A derivatives of very-long-chain fatty acids can be oxidized in mitochondria as well as in peroxisomes. The inability of the mitochondrial system to oxidize free fatty acids may be due to its inability to convert them to their corresponding coenzyme A derivatives. Our results suggest that a specific very-long-chain fatty acyl CoA synthetase may be required for the activation of the free fatty acids and that this synthetase may be deficient in patients with Zellweger's syndrome and possibly X-linked adrenoleukodystrophy, as well. The results presented suggest that substrate specificity and the subcellular localization of the synthetase may regulate the beta-oxidation of very-long-chain fatty acids in the cell.     

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.     

Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids

We assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with fish oil. We also evaluated their effect on lipogenesis and its related enzymes. Rats were administered 100 mg/kg/d fish oil, by oral gavage, for 30 days. The fatty acid profile of total liver lipids was determined by gas–liquid chromatography coupled to mass spectrometry. Individual phospholipid classes and their molecular species were quantified by ESI-MS/MS. Omega 3 fatty acids readily incorporated into hepatic phospholipids, decreased stearoyl-CoA desaturase 16, stearoyl-CoA desaturase, delta 6 desaturase, and delta 5 desaturase activities (calculated as product/substrate ratio) and decreased the “lipogenesis index”, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. Our results show that long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases' activity in the non-steatotic liver. In addition to corroborating advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis.     

Protein synthesis and degradation in isolated muscle. Effect of omega 3 and omega 6 fatty acids.

The ability of derivatives of the essential fatty acids linoleic acid (C18:2, omega 6) and alpha-linolenic acid (C18:3, omega 3) to stimulate rates of protein synthesis and degradation was investigated in isolated intact muscles from fasted rabbits. Both omega 6 derivatives examined, arachidonic acid (C20:4, omega 6) and dihomo-gamma-linolenic acid (C20:3, omega 6), when added at concentrations up to 1 microM, stimulated the rate of protein synthesis and the release of prostaglandin F2 alpha (PGF2 alpha). Metabolites of the omega 6 series, namely eicosapentaenoic acid (C20:5, omega 3) and docosahexaenoic acid (C22:6, omega 3), were without effect on the rate of protein synthesis and resulted in a decrease in the release of PGF2 alpha. None of the fatty acids had a significant effect on the rate of protein degradation. Although insulin (100 mu units/ml) also stimulated rates of protein synthesis when added alone, none of the omega 3 or omega 6 fatty acids, when added with insulin at concentrations of 0.2 microM, potentiated the effect of the hormone.     

Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts

Epoxyeicosatrienoic acids (EETs), the eicosanoid biomediators synthesized from arachidonic acid by cytochrome P450 epoxygenases, are inactivated in many tissues by conversion to dihydroxyeicosatrienoic acids (DHETs). However, we find that human skin fibroblasts convert EETs mostly to chain-shortened epoxy-fatty acids and produce only small amounts of DHETs. Comparative studies with [5,6,8,9,11,12,14,15-(3)H]11,12-EET ([(3)H]11,12-EET) and [1-(14)C]11,12-EET demonstrated that chain-shortened metabolites are formed by removal of carbons from the carboxyl end of the EET. These metabolites accumulated primarily in the medium, but small amounts also were incorporated into the cell lipids. The most abundant 11, 12-EET product was 7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2), and two of the others that were identified are 9, 10-epoxyoctadecadienoic acid (9,10-epoxy-18:2) and 5, 6-epoxytetradecaenoic acid (5,6-epoxy-14:1). The main epoxy-fatty acid produced from 14,15-EET was 10,11-epoxyhexadecadienoic acid (10, 11-epoxy-16:2). [(3)H]8,9-EET was converted to a single metabolite with the chromatographic properties of a 16-carbon epoxy-fatty acid, but we were not able to identify this compound. Large amounts of the chain-shortened 11,12-EET metabolites were produced by long-chain acyl CoA dehydrogenase-deficient fibroblasts but not by Zellweger syndrome and acyl CoA oxidase-deficient fibroblasts. We conclude that the chain-shortened epoxy-fatty acids are produced primarily by peroxisomal beta-oxidation. This may serve as an alternate mechanism for EET inactivation and removal from the tissues. However, it is possible that the epoxy-fatty acid products may have metabolic or functional effects and that the purpose of the beta-oxidation pathway is to generate these products.     

L-glutamine and palmitate catabolism in pancreatic islets from rats depleted in long-chain polyunsaturated omega 3 fatty acids

The catabolism of D-glucose was recently found to be impaired in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids. The specificity of this alteration was now investigated by characterizing the oxidative fate of endogenous nutrients in islets preincubated with either L-[U-14C]glutamine or [U-14C]palmitate and then incubated variously in the absence of D-glucose, presence of the hexose or presence of metabolic poisons. Relative to their radioactive content after preincubation, the production of 14CO2 by islets prelabelled with [U-14C]glutamine was higher in omega3-depleted rats than control animals. The enhancing action of D-glucose upon such production was impaired, however, in the omega3-depleted rats. The net uptake of 14C-palmitate and absolute value for 14CO2 output were both increased in omega3-depleted rats, whilst the ratio between 14CO2 output and islet radioactive content was decreased in the same animals. The inhibition of 14CO2 production by metabolic poisons was comparable in all cases. These results are consistent with recent findings on such items as the availability of endogenous amino acids and uptake of unesterified fatty acids in extrapancreatic sites of the omega3-depleted rats. They also support the view that the alteration of D-glucose metabolism in the islets of the latter animals may be attributable, in part at least, to alteration of glucokinase kinetics by high intracellular acyl-CoA levels.     

Differential induction of electrophile-responsive element-regulated genes by n-3 and n-6 polyunsaturated fatty acids

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.     

Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology

Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions.     

Peroxisome proliferators enhance linoleic acid metabolism in rat liver. Increased biosynthesis of omega 6 polyunsaturated fatty acids

The alterations by peroxisome proliferators of metabolism of linoleic acid in rat liver were studied. Administration of P-chlorophenoxyisobutyric acid (clofibric acid) enhanced in vivo conversion of linoleic acid to its desaturated and/or elongated metabolites, 6,9,12-octadecatrienoic acid, 8,11,14-eicosatrienoic acid, and arachidonic acid, whereas the formation of 11,14-eicosadienoic acid was decreased. These changes observed in vivo were confirmed in vitro to be due to the increases in activities of delta 6 desaturation of linoleic acid to 6,9,12-octadecatrienoic acid (18.4 times), delta 8 desaturation of 11,14-eicosadienoic acid to 8,11,14-eicosatrienoic acid (3.4 times), and delta 5 desaturation of 8,11,14-eicosatrienoic acid to arachidonic acid (4.1 times). No considerable changes in activities of chain elongation of either linoleic acid or 6,9,12-octadecatrienoic acid were observed. The increases in the activities of three desaturations by clofibric acid were prevented by the treatment of rats with cycloheximide. The inductions of delta 6 and delta 5 desaturations were brought about by the treatment of rats with 2,2'-(decamethylenedithio)diethanol or di-(2-ethylhexyl)-phthalate, peroxisome proliferators structurally unrelated to clofibric acid, as well. These changes in metabolism of linoleic acid by clofibric acid were consistent with the changes in mass proportion of omega 6 fatty acids in hepatic lipid. Physiological significance of the marked changes in linoleic acid metabolism by peroxisome proliferators was discussed.     

Long-chain fatty acyl-coenzyme A-induced inhibition of glucokinase in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids

The metabolism of D-glucose was recently reported to be impaired in pancreatic islets from second generation rats depleted in long-chain polyunsaturated omega3 fatty acids. Considering the increased clearance of circulating non-esterified fatty acids prevailing in these rats, a possible inhibition of glucokinase in insulin-producing cells by endogenous long-chain fatty acyl-CoA was considered. The present study was mainly aimed at assessing the validity of the latter proposal. The activity of glucokinase in islet homogenates, as judged from the increase in D-glucose phosphorylation rate in response to a rise in the concentration of the hexose represented, in the omega3-depleted rats, was only 81.8 +/- 4.8% (n = 11; p < 0.005) of the paired value recorded in control animals. This coincided with the fact that the inclusion of D-glucose 6-phosphate (3.0 mM) and D-fructose 1-phosphate (1.0 mM) in the assay medium resulted in a lesser fractional decrease of D-glucose phosphorylation in omega3-depleted rats than in control animals. Moreover, whereas palmitoyl-CoA (50 microM) decreased the activity of glucokinase by 38.0 +/- 6.0% (n = 4; p < 0.01) in islet homogenates from normal rats, the CoA ester failed to affect significantly the activity of glucokinase in islet homogenates from omega3-depleted rats. These findings afford direct support for the view that glucokinase is indeed inhibited by endogenous long-chain fatty acyl-CoA in islets from omega3-depleted rats, such an inhibition probably participating to the alteration of D-glucose catabolism prevailing in these islets.     

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 different types of polyunsaturated fatty acids on cholesterol esterification in human fibroblasts.

We have enriched human fibroblasts with oleic acid, with linoleic acid and with eicosapentaenoic acid. The accumulation of cholesteryl esters in the cells and the rate of esterification of cholesterol by microsomal acyl-CoA:cholesterol acyltransferase (ACAT) were measured in these cells. Cholesteryl ester levels were lower in cells enriched with eicosapentaenoic acid compared with cells enriched with oleate or linoleate. We also observed significantly lower ACAT activities in the microsomes from fibroblasts enriched with the n-3 polyunsaturated fatty acids relative to cells enriched with oleic acid or linoleic acid. We suggest that the presence of n-3 polyunsaturated fatty acids might suppress cholesteryl ester accumulation and inhibit atherogenesis.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.