Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids

Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.     

Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation

Rates of conversion of alpha-linolenic acid (alpha-LNA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3) by the mammalian brain and the brain's ability to upregulate these rates during dietary deprivation of n-3 polyunsaturated fatty acids (PUFAs) are unknown. To answer these questions, we measured conversion coefficients and rates in post-weaning rats fed an n-3 PUFA deficient (0.2% alpha-LNA of total fatty acids, no DHA) or adequate (4.6% alpha-LNA, no DHA) diet for 15 weeks. Unanesthetized rats in each group were infused intravenously with [1-(14)C]alpha-LNA, and their arterial plasma and microwaved brains collected at 5 minutes were analyzed. The deficient compared with adequate diet reduced brain DHA by 37% and increased brain arachidonic (20:4n-6) and docosapentaenoic (22:5n-6) acids. Only 1% of plasma [1-(14)C]alpha-LNA entering brain was converted to DHA with the adequate diet, and conversion coefficients of alpha-LNA to DHA were unchanged by the deficient diet. In summary, the brain's ability to synthesize DHA from alpha-LNA is very low and is not altered by n-3 PUFA deprivation. Because the liver's reported ability is much higher, and can be upregulated by the deficient diet, DHA converted by the liver from circulating alphaLNA is the source of the brain's DHA when DHA is not in the diet.     

Associations of maternal prenatal dietary intake of n-3 and n-6 fatty acids with maternal and umbilical cord blood levels

Maternal n-3 and n-6 polyunsaturated fatty acid (PUFA) status may influence birth outcomes and child health. We assessed second trimester maternal diet with food frequency questionnaires (FFQs) (n=1666), mid-pregnancy maternal erythrocyte PUFA concentrations (n=1550), and umbilical cord plasma PUFA concentrations (n=449). Mean (SD) maternal intake of total n-3 PUFA was 1.17 g/d (0.43), docosahexaenoic and eicosapentaenoic acids (DHA+EPA) 0.16 g/d (0.17), and total n-6 PUFA 12.25 g/d (3.25). Mean maternal erythrocyte and cord plasma PUFA concentrations were 7.0% and 5.2% (total n-3), 5.0% and 4.6% (DHA+EPA), and 27.9% and 31.4% (total n-6). Mid-pregnancy diet–blood and blood–blood correlations were strongest for DHA+EPA (r=0.38 for diet with maternal blood, r=0.34 for diet with cord blood, r=0.36 for maternal blood with cord blood), and less strong for n-6 PUFA. The FFQ is a reliable measure of elongated PUFA intake, although inter-individual variation is present     

Chronic dietary n-6 PUFA deprivation leads to conservation of arachidonic acid and more rapid loss of DHA in rat brain phospholipids

To determine how the level of dietary n-6 PUFA affects the rate of loss of arachidonic acid (ARA) and DHA in brain phospholipids, male rats were fed either a deprived or adequate n-6 PUFA diet for 15 weeks postweaning, and then subjected to an intracerebroventricular infusion of ³H-ARA or ³H-DHA. Brains were collected at fixed times over 128 days to determine half-lives and the rates of loss from brain phospholipids (J_out). Compared with the adequate n-6 PUFA rats, the deprived n-6-PUFA rats had a 15% lower concentration of ARA and an 18% higher concentration of DHA in their brain total phospholipids. Loss half-lives of ARA in brain total phospholipids and fractions (except phosphatidylserine) were longer in the deprived n-6 PUFA rats, whereas the J_out was decreased. In the deprived versus adequate n-6 PUFA rats, the J_out of DHA was higher. In conclusion, chronic n-6 PUFA deprivation decreases the rate of loss of ARA and increases the rate of loss of DHA in brain phospholipids. Thus, a low n-6 PUFA diet can be used to target brain ARA and DHA metabolism.     

Increased arachidonic acid concentration in the brain of Flinders Sensitive Line rats, an animal model of depression

Depression may be associated with impaired membrane PUFA composition, especially decreased n-3 PUFA. This assumption has not been tested at the level of brain tissue. Moreover, most studies were confounded by dietary variability. We examined the FA composition of selected brain areas in an animal model of depression, the Flinders Sensitive Line (FSL) rat, and compared the findings with those in controls fed identical diets. In all brain regions studied, the concentration of arachidonic acid (AA) was significantly higher in the FSL rats: in the hypothalamus by 21%, in the nucleus accumbens by 24%, in the prefrontal cortex by 31%, and in the striatum by 23%. No significant differences were observed for n-3 PUFA or for the saturated and monounsaturated FAs. Our results confirm the existence of altered brain PUFA composition in an animal model of depression. The finding of increased AA, an n-6 PUFA, rather than decreased n-3 PUFA, emphasizes the importance of both PUFA families in the pathophysiological processes underlying depression. The FSL rat is a useful tool for further elucidation of the FA disturbances in depression.     

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 M isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 M, compared with no fatty acid addition (from 99.0 ±: 1.0% to 30.7 ± 5.2% (p < 0.05) for DHA and 23.8 ± 2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7 ± 1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r_ss) of atrial myocytes compared with no addition of fatty acid (control) (from r_ss = 0.203 ±0.004 to 0.159 ± 0.004 (p < 0.01) for DHA, 0.166 ± 0.001 (p < 0.01) for EPA and 0.186 ±0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.     

A low omega-6 polyunsaturated fatty acid (n-6 PUFA) diet increases omega-3 (n-3) long chain PUFA status in plasma phospholipids in humans

This study aimed to determine the effect of reducing the dietary linoleic acid (LA) intake from ~5% to <2.5% energy (%E) on n-3 long chain PUFA (LCPUFA) status in humans. Thirty-six participants followed a <2.5%E LA diet for 4 weeks. Nutrient intakes were estimated from diet diaries and blood samples were collected for assessment of fatty acid composition in plasma and erythrocyte phospholipids. LA intakes were reduced from 4.6%E to 2%E during the low LA intervention (P<0.001) while n-3 LCPUFA intakes were unchanged. LA and total n-6 PUFA content of plasma and erythrocyte phospholipids were significantly reduced after the low LA diet phase (P<0.001). The n-3 LCPUFA content of plasma phospholipids was significantly increased after the low LA diet compared to baseline (6.22% vs. 5.53%, P<0.001). These data demonstrate that reducing LA intake for 4 weeks increases n-3 LCPUFA status in humans in the absence of increased n-3 LCPUFA intake.     

The Benefits and Risks of n-3 Polyunsaturated Fatty Acids

There is a growing number of animal models and clinical trials of n-3 polyunsaturated fatty acid (PUFAs) supplementation in disease. Epidemiologic and biochemical studies have suggested beneficial effects of n-3 PUFAs. But also, the use of n-3 PUFAs has some potential toxicological risks that can be circumvented by careless processing, storing, and preserving the PUFAs. The use of n-3 PUFAs is safe if appropriate preparations and dosages are selected. Much research is needed to clarify their use under different disease conditions. The newly established clinical and nutritional facts on n-3 PUFAs will induce industry to develop food products based on this knowledge.     

Chronic nutritional deprivation of n-3 α-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n-3) were markedly reduced in brains of third-generation (F3) rats nutritionally deprived of alpha-linolenic acid (18 : 3 n-3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n-6), an arachidonic acid (AA, 20 : 4 n-6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3-generation n-3-deficient rats, compared with control concentrations. Brain DPA-CoA could be measured in the deprived but not control rats, and AA-CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA- and DHA-selective enzymes, respectively. Competition among n-3 and n-6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n-3 deprivation), or conversion to bioactive eicosanoids and other metabolites.     

Antiarrhythmic Mechanisms of n-3 PUFA and the Results of the GISSI-Prevenzione Trial

The purpose of this paper is twofold: on the one hand, to confirm the positive results on n-3 PUFA from the overall results Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GlSSI)-Prevenzione trial; on the other, to summarize and describe how the results of an important trial can help generate hypotheses either on mechanisms of action or on differential results in particular subgroups of patients, as well as test the pathophysiological hypotheses that have accompanied in the years the story of the hypothesized mechanisms of action of a drug. GISSI-Prevenzione was conceived as a pragmatic population trial on patients with recent myocardial infarction and it was conducted in the framework of the Italian public health system. In GISSI-Prevenzione, 11,323 patients were enrolled in a clinical trial aimed at testing the effectiveness of n-3 polyunsaturated fatty acids (PUFA) and vitamin E. Patients were invited to follow Mediterranean dietary habits, and were treated with up-to-date preventive pharmacological interventions. Long-term n-3 PUFA at 1 g daily, but not vitamin E at 300 mg daily, was beneficial for death and for combined death, non-fatal myocardial infarction, and stroke. All the benefit, however, was attributable to the decrease in risk for overall (−20%), cardiovascular (−30%), and sudden death (−45%). At variance from the orientation of a scientific scenario largely dominated by the “cholesterol-heart hypothesis”, GISSI-Prevenzione results indicate n-3 PUFA (virtually devoid of any cholesterol-lowering effect) as a relevant pharmacological treatment for secondary prevention after myocardial infarction. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico (GISSI) Associazione Nazionale Medici Cardiologi Ospedalieri (ANMCO), Italy. Istituto di Ricerche Farmacologiche Mario Negri - Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy *The names of GISSI-Prevenzione Investigators are reported in Lancet 1999; 354:447–55. (40)     

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.     

Effect of n-3 and n-6 fatty acids on hepatic microsomal lipid metabolism: a time course study

The present study examines the time dependent effects of n-6 and n-3 polyunsaturated fatty acids on liver microsomal lipid metabolism in FVB mice fed a diet supplemented with a mixture of free fatty acids (mainly 18:3n-6 and 20:5n-3) at 25 mg/g diet. Significant changes in the fatty acid composition of total liver and microsomal lipids were observed after 7 days on the diets. Thereafter, some animals remained on the same diet while others were fed a diet supplemented with hydrogenated coconut oil (HCO). With the exception of 20:5n-3 which showed a slower recovery, establishment of the HCO pattern was rapid indicating that the diet-induced changes could be easily reversed. The unsaturation index, the cholesterol/phospholipid ratio and the microviscosity of the microsomal membranes were not affected by these dietary manipulations. Unsaturated fatty acid supplementation reduced the activity of ⁹ desaturase by 50%. Feeding the HCO diet to mice previously fed the EPA/GLA diet led to a progressive increase in ⁹ desaturase activity, reaching 80% of the day zero values after 14 days. The monoene content of hepatic total lipids reflected, in most cases, the changes in enzyme activity. This study shows that a low dose of a n-3 and n-6 free fatty acid mixture increases the quantities of members of the n-3 family, without loss of n-6 fatty acids in microsomal membranes and modifies the activity of ⁹ desaturase without altering the microsome physicochemical parameters.     

Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids

Male rat pups (21 days old) were placed on a diet deficient in n-3 polyunsaturated fatty acids (PUFAs) or on an n-3 PUFA adequate diet containing alpha-linolenic acid (alpha-LNA; 18 : 3n-3). After 15 weeks on a diet, [4,5-3H]docosahexaenoic acid (DHA; 22 : 6n-3) was injected into the right lateral cerebral ventricle, and the rats were killed at fixed times over a period of 60 days. Compared with the adequate diet, 15 weeks of n-3 PUFA deprivation reduced plasma DHA by 89% and brain DHA by 37%; these DHA concentrations did not change thereafter. In the n-3 PUFA adequate rats, DHA loss half-lives, calculated by plotting log10 (DHA radioactivity) against time after tracer injection, equaled 33 days in total brain phospholipid, 23 days in phosphatidylcholine, 32 days in phosphatidylethanolamine, 24 days in phosphatidylinositol and 58 days in phosphatidylserine; all had a decay slope significantly greater than 0 (p < 0.05). In the n-3 PUFA deprived rats, these half-lives were prolonged twofold or greater, and calculated rates of DHA loss from brain, Jout, were reduced. Mechanisms must exist in the adult rat brain to minimize DHA metabolic loss, and to do so even more effectively in the face of reduced n-3 PUFA availability for only 15 weeks.     

Peroxidation of n-3 and n-6 polyunsaturated fatty acids in the acidic tumor environment leads to ferroptosis-mediated anticancer effects

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Dietary ratio of n-6 to n-3 polyunsaturated fatty acids and periodontal disease in community-based older Japanese: a 3-year follow-up study

The longitudinal relationship between dietary n-6 to n-3 PUFAs ratio and periodontal disease in 235 Japanese subjects for whom data were available for the years 2003-2006 was investigated. PUFAs intake was assessed at baseline with a brief-type self-administered diet history questionnaire. Full-mouth periodontal status, measured as the clinical attachment level (CAL), was recorded at baseline and once a year for 3 years. The number of teeth with a change in the loss of CAL ≥3 mm at any site over a year was calculated as ‘periodontal disease events’. Poisson regression analysis was conducted, with dietary n-6 to n-3 PUFAs ratio as the main predictor, to estimate its influence on periodontal disease events.A high dietary n-6 to n-3 PUFAs ratio was significantly associated with greater number of periodontal disease events. The findings suggest the dietary n-6 to n-3 PUFAs ratio is associated with periodontal disease among older Japanese.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.     

Comparison of the bioavailability of docosapentaenoic acid (DPA, 22:5n-3) and eicosapentaenoic acid (EPA, 20:5n-3) in the rat

Based on the results from a human study which showed significantly reduced incorporation of DPA compared with EPA into chylomicrons, this study was designed to test if dietary DPA was significantly less absorbed than EPA. Male Sprague Dawley rats were randomly assigned to three groups of six, and were fed a semi-synthetic high fat diet (23.5% fat) for 9 days. The test omega 3 fatty acids (EPA and DPA, 250 mg/animal/day, free fatty acid form) or olive oil (250 mg/animal/day) were added to the high fat diet on days 5, 6 and 7. Dietary EPA and DPA appeared in the faeces on days 6, 7 and 8, with the total amount of DPA excreted being 4.6-fold greater than that of EPA. The total amount of faecal fat did not differ significantly between the groups. At the conclusion of the study (day 9), it was found that liver DPA, EPA and total n-3 LC-PUFA levels were significantly increased by both DPA and EPA feeding compared with the olive oil fed control group. In the heart, DPA feeding increased the DPA content and both DPA and EPA feeding increased the total n-3 LC-PUFA levels. This study showed that DPA and EPA, both provided in free form, are metabolised differently, despite being chemically similar.