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Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK(+) cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology.
Vegan and vegetarian diets supply higher amounts of linoleic acid than those of omnivores. Intakes of alpha-linolenic acid (18:3n-3) are variable, depending on the oils used, but are generally high in vegans. Docosahexaenoic acid (22:6n-3) (DHA) is absent from vegan and many vegetarian diets. Cord plasma and cord artery phospholipid levels of Hindu vegetarians contained less DHA and more docosapentaenoic acid (22:5n-6) compared with those of omnivore control subjects. These differences in fatty acid composition were not statistically related to differences in birth weight, head circumference, or length. In human milk from vegans, vegetarians, and omnivores, the mean percentage in total fatty acids of 18:2n-6 was 23.8%, 19.7%, and 10.9%, respectively; that of 18:3n-3 was 1.36%, 1.25%, and 0.49%, respectively; and that of 22:6n-3 was 0.14%, 0.30%, and 0.37%, respectively. The proportion of DHA in erythrocyte total lipids of infants breast-fed by vegans was 1.9% compared with 3.7% in infants fed a milk formula containing butterfat as the sole source of fat and 6.2% in infants breast-fed by omnivores at 14 weeks postpartum. The ratio of linoleic/alpha-linolenic acid in the diet was predictive of the proportion of eicosapentaenoic acid but not that of DHA in infant red blood cell lipids. It is concluded that the intakes of linoleic acid and DHA are the major determinants of the proportion of DHA in plasma and red blood cell lipids.
This study evaluates the influence of sex on platelet fatty acid (FA) composition, and whether sex differences are conditioned by age. Since plasma FA have a specific relationship with platelet FA their variations with age and sex are also considered. Forty-nine male-female human couples (16-75 years), where within each couple the partners were on qualitatively similar diets and of similar age, were studied. Few differences were found between the whole groups of men and women in platelet FA. A comparison of data on FA in platelet phospholipids (PL) from 3 age groups (16-40, 40-60 and over 60) showed an increase in saturated FA of middle-aged subjects, an age-dependent decrease in 20: 5 in both sexes and of 18: 2 mainly in women. The percentage of plasma phosphatidylserine plus phosphatidylinositol decreased in middle-aged subjects. With regard to the influence of FA of plasma PL on FA of platelet PL, we found a higher correlation coefficient (r) for 16:0 and 18:0 and 20:4 and a lower one for 20:5 in middle-aged men and post-menopausal women. Considering that an increase in saturated FA and 20:4 and a decrease in 20:5 in platelet PL may increase platelet function, the plasma FA influence on platelets may help to explain the higher incidence of CHD in those groups of subjects.
The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.
Bromo-addition products of unsaturated long-chain fatty acid esters have been prepared and chromatographed on thin layers of unmodified silica gel. The polarity of these derivatives was found to be directly related to the number of double bonds of the parent fatty acid from which they were derived. This has been made the basis of a simple method for assessing the relative proportions of the main fatty acid classes in a mixture.
BACKGROUND: The increased consumption of n-6 polyunsaturated fatty acids (PUFA) has been shown to coincide with the increased prevalence of atopic diseases. We aimed to investigate whether maternal diet and atopic status influence the PUFA composition of breast milk and the serum lipid fatty acids of infants. METHODS: Maternal diet was assessed by a food questionnaire. The PUFA composition of breast milk obtained at 3 months from 20 allergic and 20 healthy mothers and of their infants' (10 atopic and 10 nonatopic/group of mothers) serum lipids was analyzed. RESULTS: Although no differences in maternal PUFA intake were observed, the breast milk of allergic mothers contained less gamma-linolenic acid (18:3 n-6) than that of healthy mothers. Similarly, atopic infants had less gamma-linolenic acid in phospholipids than healthy infants, although n-6 PUFA were elevated in other serum lipid fractions in atopic infants. The serum lipid fatty acids in atopic infants did not correlate with those in maternal breast milk. CONCLUSION: Our results suggest that dietary n-6 PUFA are not as readily transferred into breast milk or incorporated into serum phospholipids, but may be utilized for other purposes, such as eicosanoid precursors, in allergic/atopic individuals. Subsequently, high dietary proportions of n-6 PUFA, or reduced proportions of regulatory PUFA, such as gamma-linolenic acid and n-3 PUFA, may be a risk factor for the development of atopic disease.
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BACKGROUND: Dietary intervention trials rely on self-reported measures of intake for assessment of energy and macronutrient composition. Dietary fat intake is of particular interest due to strong associations with pathophysiology. In epidemiological trials phospholipid fatty acid composition may reflect composition of habitual diet, although strong correlations have been identified only for essential polyunsaturated fatty acids (PUFAs). Preliminary evidence shows that saturated fatty acids (SFA) C15:0 and C17:0 may be acceptable biomarkers. This study measured changes in erythrocyte membrane fatty acids during a period of strictly controlled fat feeding to investigate their use as a short-term marker of compliance, particularly for intake of SFAs. RESULTS: This was a randomised cross-over trial in which diet was provided and strictly controlled. 20 healthy, male subjects were given a 40 energy % (en%) fat diet, high in saturated (high-SFA, 20 en%) or unsaturated (high-USFA, 24 en%) fatty acids for 2 periods of 3 weeks. Subjects were residential during intervention with all food and beverages provided. Dietary composition was verified by direct chemical analysis. Blood samples were collected on days 1,7,14, 21 and analysed for red blood cell (RBC) membrane fatty acid composition. Pearson correlation showed RBC fatty acid composition to mimic dietary composition by 3 weeks, but the relationships were weak. Of the SFAs only RBC C16:0 decreased in response to decreased dietary content on high-USFA treatment (ANOVA, diet, P < 0.05). Of the USFAs, higher levels of C18:1 MUFA, C20:4 and C22:6 long chain PUFA on high-USFA diet lead to higher C18:1, C20:4 and C22:6 within RBCs (ANOVA, time*diet, P < 0.05). Pearson's correlation was significant between dietary and RBC fatty acids during the 21d dietary manipulation for C18:1, and C20:5, C22:6 only (P < 0.05). CONCLUSION: RBC membrane fatty acids cannot reliably be used as an independent measure of compliance for dietary SFA intake in short-term studies. The MUFA oleic acid and PUFAs EPA and DHA may be more useful as markers of compliance during short term intervention trials.
The goals of this investigation were, first, to develop a chemical strategy to identify and quantitate the mass of fatty acid which is covalently bound to proteins by thioester linkage in unactivated platelets, and, second, to determine whether exogeneously added fatty acids can alter the fatty acid composition of thioester bound fatty acids. Studies with radiolabeled fatty acids cannot identify and quantitate the actual fatty acids bound to proteins because they permit analysis of only the radiolabeled fatty acids added and their metabolites. Therefore, in the absence of metabolic labeling by radiolabeled fatty acids, we isolated the thioester-linked fatty acids from platelet proteins using hydroxylamine at neutral pH to form fatty acid hydroxamates. The hydroxamates were subsequently converted to fatty acid methyl esters by acid methanolysis for quantitation by gas chromatography-mass spectrometry. Using platelet specimens from 14 subjects, 74% of the fatty acid recovered from the unactivated platelet proteins as thioester linked was palmitate. Importantly, however, 22% was stearic acid, and oleate was 4% of the total thioester bound fatty acid. There was minimal variability (2.6-fold at maximum) between the subjects in the amount of the thioester-linked palmitate and thioester-linked stearate. However, there was substantial variability (>100-fold at maximum) between subjects in the amount of thioester-linked oleate. We also demonstrated that incubation of platelets with exogenous fatty acids can alter the profile of fatty acids bound to platelet proteins by thioester linkages. Incubation of platelets with 100 microM palmitate for 3 h increased the amount of thioester-linked palmitate by up to 26%, and incubation of platelets with 100 microM stearate increased the amount of thioester-linked stearate up to 30%. In support of the observation that radiolabeled fatty acids other than palmitate were shown to be capable of binding to platelet proteins by thioester linkage, our results indicate that the fatty acids actually bound to unactivated platelet proteins include a significant amount of stearate, and variable amounts of oleate, as well as palmitate. In addition, the data show that palmitate and stearate can be increased, as a percentage of total protein-bound fatty acid, by incubation with exogenous palmitate and stearate, respectively.
Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid beta-oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like very-long-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial beta-oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.
Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.
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Polyunsaturated fatty acids (PUFA) have a selective cytotoxic/cytostatic effect on a number of tumor cell lines in culture. Although this process may be enhanced by the addition of iron there is a minimum level of PUFA necessary for potentiation of cell death. Vitamin E blocks PUFA cytotoxicity when added up to 5 days after fatty acid administration. Levels of thio-barbiturate reactive material (TBARM) in the medium rise in parallel with cell death. However, they are not affected by small alterations in temperature or oxygen tension. Incubating cells with PUFA causes marked alterations in the fatty acid patterns of both neutral and phospholipid fractions. Membrane fluidity is increased and the activity of membrane-bound receptors may be influenced directly or through the actions of eicosanoids derived from the exogenous fatty acid. PUFA may be an effective way of influencing tumor growth and a safe approach for the management of human cancer.
The interaction between valproic acid (VPA) and free fatty acids (FFA) was investigated at a systemic level in eight rhesus monkeys. Intralipid emulsion (I.L.) was infused to increase plasma FFA levels. During I.L. treatment, plasma FFA levels were elevated in all cases (12 cases) with increases ranging from 38 to 525% (median, 102%). Valproate free fraction (fp) was elevated in 11 of 12 cases, with increases ranging between 7 and 100% (median, 18%). A positive correlation between the relative increase in FFA and the corresponding increase in fp of VPA was obtained. Intralipid infusion was associated with changes in the systemic clearance of VPA (an increase in five cases and a decrease in seven cases). The intrinsic clearance (Clint) of VPA was decreased in 9 of 12 cases with decreases ranging from 13 to 78% (median, 22%). Urinary excretion of valproate glucuronide was decreased in three of four monkeys during I.L. treatment. A significant positive correlation (p less than 0.05) between the relative increase in FFA levels and the decrease in Clint of VPA were also observed. The mechanism of interaction between VPA and FFA in rhesus monkey is more complex than a simple protein binding displacement and involves at least one metabolic inhibition component such as glucuronidation.
Butter, which is naturally enriched in cis-9, trans-11 conjugated linoleic acid (rumenic acid; RA) and vaccenic acid (VA), has been shown to be an effective anticarcinogen in studies with animal models; however, there has been no examination of the effects of a naturally derived source of VA and RA on atherosclerosis-related biomarkers. The current study was designed to determine the effect of a diet containing VA/RA-enriched butter on plasma lipoproteins and tissue fatty acid profiles in cholesterol-fed hamsters. Male Golden Syrian hamsters were fed diets containing 0.2% cholesterol and 20% added fat as: 1) Control, 20% standard butter (CT); 2) 5% standard butter + 15% VA/RA-enriched butter (EB); 3) 15% standard butter + 5% partially-hydrogenated vegetable oil (VO). After 4 wk, plasma lipoproteins were isolated, cholesterol quantified, and tissue fatty acid profiles determined. Tissue concentrations of VA and RA were increased by consumption of the EB diet compared with both the CT and VO diets, whereas the VO diet increased their concentration compared with the CT diet only. Total and LDL cholesterol concentrations were significantly reduced in hamsters fed EB and VO compared with CT, whereas VLDL cholesterol concentrations were reduced in hamsters fed EB compared with those fed CT and VO. HDL cholesterol concentrations did not differ among treatments. The ratio of potentially atherogenic lipoproteins [VLDL + intermediate density lipoproteins (IDL) + LDL] to antiatherogenic HDL was significantly lower in hamsters fed VA/RA-enriched butter (0.60) than in those fed either control diet (1.70) or the diet containing partially hydrogenated vegetable oil (1.04). Thus, increasing the VA/RA concentration of butter results in a plasma lipoprotein cholesterol profile that is associated with a reduced risk of atherosclerosis.