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The A549 cell line in which is derived from adenocarcinoma human alveolar basal epithelial cells has been used as a model for cancer research. This cell line has shown good suitability as a model for respiratory studies due to presence of type II alveolar functionality. Additionally, this cell type secretes surfactant that contains phospholipids in which plays a major role in reducing alveolar surface tension. The objective of this study was to evaluate the morphological features of the A549 cells upon the exposure to low and high doses of omega fatty acids. Sterile aseptic techniques were followed throughout the experimentations by following standard lab protocols. Morphological evaluation was conducted by using two different standard staining protocols namely: hematoxylin & eosin (H-E) stain was used (to evaluate general qualitative assessment of cell characteristics); and the alcian blue stain (to identify mucin production and assess the ability of the cells to produce phosolipid surfactant). A549 cells were treated with a low and high concentration of flaxseed oil, providing omega 3-6-9 fatty acids and allowed to incubate for 24, 48 and 72 hours. The results of this study revealed: (i) at the end 24 hours of incubation, the cells showed slight indication of growth qualitatively compared to the control group; (ii) at the end 48 and 72 hours of incubation, an increase in growth was observed with islands of confluent cells forming when compared to the control group; (iii) maintenance of cell functionality was observed by evidence of alcian blue mucopolysaccharide staining in and around the peripheral of the cell; (iv) no visible signs of debris or cytotoxicity were noted at both low and high doses and at all three incubation intervals. The results of this pilot study suggest that the exposure of omega fatty acids to A549 cells can provide an adequate matrix for defining and identifying the components of an asthma inflammatory condition. Further investigations are highly recommended to assess the quantitative measurements of cell viability, integrity and actual cell count as well as functional capacity. Keywords: A549 cell line, omega fatty acids, mucopolysaccharide, surfactant, hematoxylin and eosin, alcian blue, INTRODUCTION Omega-3 fatty acid in dietary oils, such as flaxseed or fish oil, is described as an anti-inflammatory agent that reduces inflammation throughout the body. Omega-3 is part of a [...]

Merkel MJ, Liu L, Cao Z, Packwood W, Young J, AIkayed N J, Van Winkle DM. Inhibition of soluble epoxide hydrolase preserves cardiomyocytes: role of STAT3 signaling. Am J Physiol Heart Circ Physiol 298: H679-H687, 2010. First published December 11, 2009; doi: 10.1152/ajpheart.00533.2009.--Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs), primarily 14,15-EET. EETs are derived from arachidonic acid via P-450 epoxygenases and are cardioprotective. We tested the hypothesis that sEH deficiency and pharmacological inhibition elicit tolerance to ischemia via EET-mediated STAT3 signaling in vitro and in vivo. In addition, the relevance of single nucleotide polymorphisms (SNPs) of EPHX2 (the gene encoding sEH) on tolerance to oxygen and glucose deprivation and reoxygenation and glucose repletion (OGD/RGR) was assessed in male C57BL\6J (WT) or sEH knockout (sEHKO) cardiomyocytes by using transactivator of transcription (TAT)-mediated transduction with sEH mutant proteins. Cell death and hydrolase activity was lower in Arg287Gln EPHX2 mutants vs. nontransduced controls. Excess 14,15-EET and SEH inhibition did not improve cell survival in Arg287Gln mutants. In WT cells, the putative EET receptor antagonist, 14,15-EEZE, abolished the effect of 14,15-EET and sEH inhibition. Cotreatment with 14,15-EET and SEH inhibition did not provide increased protection. In vitro, STAT3 inhibition blocked 14,15-EET cytoprotection, but not the effect of SEH inhibition. However, STAT3 small interfering RNA (siRNA) abolished cytoprotection by 14,15EET and sEH inhibition, but cells pretreated with JAK2 siRNA remained protected. In vivo, STAT3 inhibition abolished 14,15-EETmediated infarct size reduction. In summary, the Arg287Gln mutation is associated with improved tolerance against ischemia in vitro, and inhibition of sEH preserves cardiomyocyte viability following OGD/ RGR via an EET-dependent mechanism. In vivo and in vitro, 14,15EET-mediated protection is mediated in part by STAT3. eicosanoid; oxygen and glucose deprivation; reoxygenation and glucose repletion; 14,15-EET; N-adamantanyl-N'-dodecanoic acid urea doi: 10.1152/ajpheart.00533.2009

Essential fatty acid (EFA) deficiency in mice has been associated with increased bile production, which is mainly determined by the enterohepatic circulation (EHC) of bile salts. To establish the mechanism underlying the increased bile production, we characterized in detail the EHC of bile salts in EFA-deficient mice using stable isotope technique, without interrupting the normal EHC. Farnesoid X receptor (FXR) has been proposed as an important regulator of bile salt synthesis and homeostasis. In [Fxr.sup.-/-] mice we additionally investigated to what extent alterations in bile production during EFA deficiency were FXR dependent. Furthermore, we tested in differentiating Caco-2 cells the effects of EFA deficiency on expression of FXR-target genes relevant for feedback regulation of bile salt synthesis. EFA deficiency-enhanced bile flow and biliary bile salt secretion were associated with elevated bile salt pool size and synthesis rate (+146 and +42%, respectively, P < 0.05), despite increased ileal bile salt reabsorption (+228%, P < 0.05). Cyp7a1 mRNA expression was unaffected in EFA-deficient mice. However, ileal mRNA expression of Fgf15 (inhibitor of bile salt synthesis) was significantly reduced, in agreement with absent inhibition of the hepatic bile salt synthesis. Bile flow and biliary secretion were enhanced to the same extent in EFA-deficient wild-type and [Fxr.sup.-/-] mice, indicating contribution of other factors besides FXR in regulation of EHC during EFA deficiency. In vitro experiments show reduced induction of mRNA expression of relevant genes upon chenodeoxycholic acid and a selective FXR agonist GW4064 stimulation in EFA-deficient Caco-2 cells. In conclusion, our data indicate that EFA deficiency is associated with interrupted negative feedback of bile salt synthesis, possibly because of reduced ileal Fgf15 expression. fgf15; FGF19; small intestine; stable isotope dilution

Iron deficiency (ID) is the most prevalent worldwide nutritional deficiency. Groups at risk of developing ID anemia are infants and pregnant women, even in industrialized countries. Our goal in this study was to evaluate the long-term consequences of maternal ID on the offspring's fatty acid and eicosanoid metabolism, behavior, and spatial memory. Female guinea pigs consumed iron-sufficient (IS) and--deficient (ID) diets for 14 d before mating and throughout pregnancy and lactation. Dietary iron restriction resulted in ID in pregnant females. On postnatal d 9, all offspring (ID and IS) were weaned to the IS diet and at 42 d, all offspring were iron replete. Locomotion was tested in pups on postnatal d 24 and 40 and spatial memory from d 25 to 40. Pups from the ID group were significantly more active in the open field at both times of testing, whereas spatial memory, tested in a Morris water maze, was comparable in both groups. On postnatal d 42, liver, RBC, and brain fatty acid composition were measured. Dihomogammalinolenic [20:3(n-6)], docosapentaenoic [22:5(n-3)], and docosahexaenoic [22:6(n-3)] acid contents were significantly higher in brain phospholipids of offspring born to ID dams. Prostaglandin [E.sub.2] and [F.sub.2[alpha]] concentrations were also significantly higher in brains of offspring born to ID dams. This demonstrates that moderate ID during gestation and lactation results in alterations of brain fatty acid and eicosanoid metabolism and perturbation in behavior in adult offspring.

Essential fatty acid (EFA) deficiency in mice decreases plasma triglyceride (TG) concentrations and increases hepatic TG content. We evaluated in vivo and in vitro whether decreased hepatic secretion of TG-rich very low-density lipoprotein (VLDL) contributes to this consequence of EFA deficiency. EFA deficiency was induced in mice by feeding an EFA-deficient (EFAD) diet for 8 wk. Hepatic VLDL secretion was quantified in fasted EFAD and EFA-sufficient (EFAS) mice using the Triton WR-1339 method. In cultured hepatocytes from EFAD and EFAS mice, VLDL secretion into medium was measured by quantifying [[sup.3]H]-labeled glycerol incorporation into TG and phospholipids. Hepatic expression of genes involved in VLDL synthesis and clearance was measured, as were plasma activities of lipolytic enzymes. TG secretion rates were quantitatively similar in EFAD and EFAS mice in vivo and in primary hepatocytes from EFAD and EFAS mice in vitro. However, EFA deficiency increased the size of secreted VLDL particles, as determined by calculation of particle diameter, particle sizing by light scattering, and evaluation of the TG-to-apoB ratio. EFA deficiency did not inhibit hepatic lipase and lipoprotein lipase activities in plasma, but increased hepatic mRNA levels of apoAV and apoCII, both involved in control of lipolytic degradation of TG-rich lipoproteins. EFA deficiency does not affect hepatic TG secretion rate in mice, but increases the size of secreted VLDL particles. Present data suggest that hypotriglyceridemia during EFA deficiency is related to enhanced clearance of altered VLDL particles. hepatic lipoprotein secretion: lipoprotein clearance; hypotriglyceridemia

This study examined the effects of diets deficient (D) in linoleic [18:2(n-6)] and linolenic acid [18:3(n-3)] at 0.8 and 0.05% energy, respectively, or adequate (C)in 18:2(n-6) and 18:3(n-3) at 8.3 and 0.8% energy, respectively, without (-) or with (+) 0.2% energy arachidonic [20:4(n-6)] and 0.16% energy docosahexaenoic [22:6(n-3)] acid in piglets fed from birth to 18 d. Frontal cortex dopaminergic and serotoninergic neurotransmitters and phospholipid fatty acids were measured. Piglets fed the D- diet had significantly lower frontal cortex dopamine, 3,4-dihydroxyphenylacetic (DOPAC), homovanillic acid (HVA), serotonin and 5-hydroxyindoleacetic acid (5-HIAA) concentrations than did piglets fed the C- diets. Frontal cortex dopamine, norepinephrine, DOPAC, HVA, serotonin and 5-HIAA were higher in piglets fed the D+ compared to those fed the D- diet (P< 0.05) and not different between piglets fed the D+ and those fed the C- diets or the C- and C+ diets. Piglets fed the Ddiet had lower frontal cortex phosphatidylcholine (PC) and phosphatidylinositol (PI) 20:4(n-6) and PC and phosphatidylethanolamine (PE) 22:6(n-3) than did piglets fed the C- diet (P KEY WORDS: essential fatty acids; docosahexaenoic acid; arachidonicacid; dopaminergic; serotoninergic, piglets

Semipurified diets incorporating either perilla oil [high in [Alpha]-linolenate, 18:3(n-3)] or safflower oil [high in linoleate, 18:2(n-6)] were fed to senescence-resistant SAMR1 mouse dams and their pups. Male offspring at 15 mo were examined using behavioral tests. In the open field+ test, locomotor activity during a 5-min period was significantly higher in the safflower oil group than in the perilla oil group. Observations of the circadian rhythm (48 h) of spontaneous motor activity indicated that the safflower oil group was more active than the perilla oil group during the first and second dark periods. The total number of responses to positive and negative stimuli was higher in the safflower oil group than in the perilla oil group in the light and dark discrimination learning test, but the correct response ratio was lower in the safflower oil group. The difference in the (n-6)/(n-3) ratios of the diets reflected the proportions of (n-6) polyunsaturated fatty acids, rather than those of (n-3) polyunsaturated fatty acids in the brain total fatty acids, and in the proportions of (n-6) and (n-3) polyunsaturated fatty acids in the total polyunsaturated fatty acids of the brain phospholipids. These results suggest that in SAMR1 mice, the dietary [Alpha]-linolenate/linoleate balance affects the (n-6)/(n-3) ratio of brain phospholipids, and this may modify emotional reactivity and learning ability. KEY WORDS: senescence-accelerated mouse, [Alpha]-linolenic acid, linoleic acid, open field activity, circadian rhythm, discrimination learning

Effects of dietary essential fatty acids on visual attention were explored longitudinally in infant rhesus monkeys with a visual paired-comparison paradigm. Sets of primate faces and of patterns were presented at Weeks 2, 5, 9, and 13 to 9 infants deficient in [Omega]-3 fatty acids and 8 fed a standard nursery diet. Familiarization to 1 member of each pair preceded simultaneous presentation of both stimuli. Infants fed the deficient diet showed longer individual looks in both immediate and 24-hr tests. Duration of looks decreased with age to familiar but not to novel stimuli. The proportion of time looking at the novel stimulus (% novel) increased with age but was not affected by diet. Look duration and % novel were differentially affected and may reflect different underlying processes.

Quantitative variations of polyunsaturated fatty acids (PUFA) were studied in various tissues: red blood cells (RBC), hepatic microsomes, kidney, skeletal muscle and heart of young rats fed either a control diet (n = 7) or an essential fatty acid (EFA)-deficient diet (n = 7). After 4 wk, the EFA-[deficient rats had significantly lower proportions of (n-6) and (n-3) fatty acids in RBC, hepatic microsomes and kidney than the control group. Paradoxically, normal proportions of arachidonic acid [20:4(n-6)] and 5,8,11,14,17-eicosapentaenoic acid [20:5(n-3)] were retained in heart and skeletal muscle despite generally lower proportions of the precursors, 18:2(n-6) and 18:3(n-3). Morever, absolute levels of 20:4(n-6) and 20:5(n-3) in skeletal muscle of the EFA deficient group were significantly higher than in controls and 22:5(n-3) and 22:6(n-3) levels were comparable. This suggests that fatty acid proportions alone, without any consideration of long-chain polyunsaturated fatty acid quantities, may not reflect the (n-6) and (n-3) PUFA status of individual tissues. This study indicates that diet-[induced changes in the PUFA composition of RBC, which are often used in clinical investigations, do not fully reflect the changes in the fatty acid composition of organs, and that individual tissues respond differently to EFA deficiency. The conservation of proportional and absolute levels of 20:5(n-3) and 20:4(n-6), and the decrease in the more unsaturated homologues in the heart, suggest that this organ may avidly retain 20:5(n-3) and 20:4(n-6) in order to maintain eicosanoid production. J. Nutr. 126: 3040-3045, 1996. INDEXING KEY WORDS: * fatty acids * essential fatty acid deficiency * tissue membranes * rats

A study of the influence of prostaglandins in renal sensory receptor stimulation by examining rats on essential fatty acid-deficient (EFAD) diet and control rats on standard diet reveals that EFAD died-fed rats exhibit depletion of tissue arachidonate and its metabolites, caused by the suppression of ipsilateral afferent renal nerve activity enhancement due to increased ureteral pressure. The ipsilateral afferent nerve activity response to ureteral pressure increase is reestablished on addition of the arachidonate metabolite prostaglandin E2 (PGE2). EFAD diet-fed rats also exhibit reduced response of afferent renal nerve activity to bradykinin renal pelvic perfusion, indicating that arachidonic acid metabolites such as PGE2 are involved in renal pelvic sensory receptor stimulation.

The effect of a diet deficient in essential fatty acid (EFA) on hibernation patterns were studied in yellow-bellied marmots. Significantly shorter hibernation periods were noted among EFA-deficient subjects. Results show that EFAs regulate body temperatures during hibernation. However, the observation that animals hibernate despite diets deficient in EFA suggest that EFAs are not requisites to hibernation.

The large proportions of arachidonic acid (20:4 omega-6) and docosahexaenoic acid (22:6 omega-3) in brain and retina structural lipids are important for normal central nervous system function. Study of dietary requirements for omega-6 and omega-3 fatty acids for brain growth is difficult in the human infant because of variabilities in human milk fatty acids, lack of correspondence between blood lipids and central nervous system (CNS) lipids and ethical limits of tissue analyses. Comparison of nutrient requirements, lipid digestion, absorption and transport and milk and brain lipids and the timing of intestinal maturation and the brain growth spurt during fetal-infant development show the piglet to be an excellent choice for studies relevant to lipid nutrition of term gestation infants. Of practical relevance, piglets are easily hand-fed and, with a relatively large body size and rapid growth, provide ample tissue for analyses of the effects of diet fat on specific CNS cell membranes. Use of the piglet to define omega-6 and omega-3 fatty acid requirements and the effects of addition of marine oils to formulas and the lactating mother's diet on the developing CNS are described.

The objective of this study was to investigate whether dietary trans fatty acids (TFA) during the pre- and postnatal periods would exacerbate the effects of marginal essential fatty acid (EFA) status on growth, brain long-chain polyunsaturated fatty acids (LC-PUFA) and behavioral development in [B6D2F.sub.2] mice. Pregnant [B6D2F.sub.1] females were randomly assigned to one of the following three diets: marginal EFA plus 22% trans 18:1 (mEFA + TFA); marginal EFA (mEFA); and control (CON). The total 18:1 content in all diets was similar. The offspring were weaned and maintained on the same diets. Both the mEFA and mEFA + TFA groups had reduced growth and brain weight compared with CON, but did not differ from one another. As expected, the mEFA and mEFA + TFA groups had reduced docosahexaenoic acid [DHA; 22:6(n-3)]) and increased 22:5(n-6) concentrations in brain phosphatidylcholine (PC) and phosphatidylethanolamine (PE) compared with the CON group, but again did not differ from one another. Reversal learning in the T-water maze was significantly slower in the mEFA + TFA groups compared with the mEFA group and both were slower than the CON group. These findings illustrate that TFA combined with a marginal EFA status do not exacerbate the effects of marginal EFA status on growth or brain LC-PUFA. However, long-term effects of dietary TFA during the pre- and postnatal period on behavioral development and neural function should be investigated in future studies. J. Nutr. 131: 1568-1573, 2001. KEY WORDS: * dietary trans fatty acids * essential fatty acid status * arachidonic acid * docosahexanoic acid * behavioral development

The effects of dietary restriction of a single essential amino acid (EAA) on insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP)-1 were investigated in rats. Rats were fed experimental diets containing amino acid (AA) mixtures in which the concentrations of all EAA were at levels recommended by the National Research Council (control), in which a single EAA was restricted to 20% of that of the control diets ([Leu.sup.-], [Lys.sup.-], [Met.sup.-] or [Thr.sup.-]), or in which the diet was devoid of amino acids ([AA.sup.-]). To eliminate the effect of differences in energy intake, rats were fed the mean amount of food as consumed by the [AA.sup.-] group on the previous day. Growth was significantly retarded in rats fed diets restricted in just one EAA compared with that of rats fed the control diet, and further growth retardation was observed in rats fed the [AA.sup.-] diet. On the other hand, the plasma IGF-I concentrations in the groups with a single EAA restriction or in the [AA.sup.-] group were 66% (P [is less than] 0.05) and 50% (P [is less than] 0.05) of that of the control group, respectively. The effect of any single EAA restriction was not significantly different from that of total AA deprivation. The plasma IGFBP-1 concentration in the control group did not differ from that of rats fed diets with the single EAA restrictions except for methionine restriction, but it was ~6-fold greater in the [AA.sup.-] group. Differences in plasma IGFBP-1 concentration under these conditions could be explained by differences in hepatic IGFBP-1 mRNA contents. Based on these results, we conclude that restriction of single EAA does not affect IGFBP-1 synthesis in vivo, although the deprivation of a single EAA has been reported to increase IGFBP-1 production in hepatocyte cultures. Our results also indicated that a single EAA restriction decreased IGF-I production but did not affect IGFBP-1 production. The present study suggests that not only plasma IGF-I, but also IGFBP-1, affects the magnitude of growth retardation in vivo. J. Nutr. 130: 2910-2914, 2000. KEY WORDS: * essential amino acid * insulin-like growth factor-binding protein-1 * mRNA * rats

Essential fatty acid (EFA) incorporation into phospholipid is influenced by chloride channels, suggesting that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) may regulate aspects of EFA metabolism. The objective of this study was to determine whether the [Delta]F508 mutation in the CFTR lowers 18:2(n-6) levels in phospholipid. Control cells, CF cells and CF cells transfected with the 'normal' CFTR gene or the [Delta]F508 CFTR gene were cultured for 3-5 d and used to determine [1-[sup.14]C]18:2(n-6) incorporation into cell lipids. CF cells exhibited low 18:2(n-6) levels in phospholipid, reduced [1-[sup.14]C]18:2(n-6) incorporation into phospholipid (50% of control) and greater [1-[sup.14]C]18:2(n-6) incorporation into the triacylglycerol fraction (400% of control; P [is less than] 0.05). Kinetic modeling of time course data for [1-[sup.14]C] 18:2(n-6) incorporation revealed a loss of metabolic control over the intracellular partitioning of 18:2(n-6) between phospholipid and triacylglycerol pools in CF cells. Expression of the normal CFTR gene in transfected CF cells increased chloride efflux and the incorporation of [1-[sup.14]C]18:2(n-6) into phospholipid and triacylglycerol fractions. The increased incorporation of [1-[sup.14]C]18:2(n-6) into phospholipid was attributed to significantly increased incorporation of [1-[sup.14]C]18:2(n-6)into phosphatidylcholine and phosphatidylinositol. In CF cells expressing the defective [Delta]F508 CFTR gene, conversion of [1-[sup.14]C]18:2(n-6) to 20:4(n-6) by desaturation-chain elongation was 1.8-fold greater (P [is less than] 0.05) than observed for CF cells transfected with the normal gene. The observations suggest that CF results in a defect in the utilization of 18:2(n-6), which is attributed in part to the defective CFTR. J. Nutr. 130: 2870-2875, 2000. KEY WORDS: * cystic fibrosis * essential fatty acids * humans * epithelial cell * membrane

Increasing efforts have been made to determine the distribution and concentration of trans fatty acids in milk, due to the importance of lipids in infant growth and development. In general, trans fatty acid concentration of milk reflects trans fatty acid intake, but insufficient data are available to assess the effects of dietary trans fatty acids on maternal milk. Thus, controlled studies are needed to establish whether there is a dose-response relationship and whether trans fatty acids could affect the concentration of essential fatty acids (EFA), long-chain polyunsaturated fatty acids (PUFA) and the (n-6)/(n-3) ratio in milk. Three groups of six rats each were fed for 10 wk one of three diets differing in trans fatty acid concentration (Control, 0 mol/100 mol; high trans concentration (H), 14.5 mol/100 mol; very high trans concentration (VH), 30 mol/100 mol), but containing the same proportions of linoleic and [Alpha]-Iinolenic acids and a ratio of 18:2(n-6)/18:3(n-3) of about 7:1. Trans fatty acids were incorporated into maternal milk in a dose-dependent manner. In addition, rats fed trans isomers had greater linoleic acid levels than controls. The proportion of [Alpha]-linolenic acid in milk was lower in the VH group, and the (n-6)/(n-3) cis PUFA ratio in milk of the VH group was greater than that in controls. Total long-chain PUFA levels did not differ among groups. These results suggest that high intakes of trans fatty acids affect the EFA concentration but not that of long-chain PUFA of rat milk, provided that EFA are supplied in sufficient amounts. J. Nutr. 130: 847-851, 2000. KEY WORDS: * trans fatty acids * rats * milk * EFA * PUFA