The opposing effects of n-3 and n-6 fatty acids

Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids, and in westernized diet the predominant dietary PUFAs are n-6 fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acid is arachidonic acid, which is converted to prostaglandins, leukotrienes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. Typical n-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are competitive substrates for the enzymes and products of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived eicosanoids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids are ligands/modulators for the nuclear receptors NFkappaB, PPAR and SREBP-1c, which control various genes of inflammatory signaling and lipid metabolism. n-3 Fatty acids down-regulate inflammatory genes and lipid synthesis, and stimulate fatty acid degradation. In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival.     

Plasma fatty acid levels in autistic children

Phospholipid fatty acids are major structural components of neuronal cell membranes, which modulate membrane fluidity and hence function. Evidence from clinical and biochemical sources have indicated changes in the metabolism of fatty acids in several psychiatric disorders. We examined the phospholipid fatty acids in the plasma of a population of autistic subjects compared to mentally retarded controls. Our results showed a marked reduction in the levels of 22: 6n-3 (23%) in the autistic subjects, resulting in significantly lower levels of total (n-3) polyunsaturated fatty acids (PUFA) (20%), without significant reduction in the (n-6) PUFA series, and consequently a significant increase in the (n-6)/(n-3) ratio (25%). These variations are discussed in terms of potential differences in PUFA dietary intake, metabolism, or incorporation into cellular membranes between the two groups of subjects. These results open up interesting perspectives for the investigation of new biological indices in autism. Moreover, this might have new therapeutic implications in terms of child nutrition.     

Docosahexaenoic acid affects insulin deficiency- and insulin resistance-induced alterations in cardiac mitochondria

The effect of docosahexaenoic acid (DHA) intake on cardiac mitochondrial function was evaluated in permeabilized fibers in insulin deficiency and insulin resistance in rats. The insulin-deficient state was obtained by streptozotocin injection 2 mo before investigations. Insulin resistance was obtained by feeding a 62% fructose diet for 3 mo. DHA was incorporated in the diet to modify the fatty acid composition of cardiac membranes, including mitochondria. Insulin deficiency decreased mitochondrial creatine kinase (mi-CK) activity and mitochondrial sensitivity to ADP. DHA intake prevented these alterations. Moreover, the insulin-deficient state significantly decreased n-3 polyunsaturated fatty acids (PUFA) and slightly increased n-6 PUFA in both cardiac and mitochondrial membranes, inducing a significant increase in the n-6-to-n-3 ratio. DHA intake maintained high myocardial and mitochondrial DHA content. Insulin deficiency also decreased glutamate- and palmitoylcarnitine-supported mitochondrial respiration, but DHA intake did not prevent these effects. In contrast, insulin resistance did not affect mi-CK activity or sensitivity to ADP. However, insulin resistance influenced the myocardial fatty acid composition with decreased n-6 and n-3 PUFA contents and increased monounsaturated fatty acid content. Only slight alterations were observed in mitochondrial fatty acid composition, and they were corrected by DHA intake. Moreover, insulin resistance decreased the glutamate-supported respiration, and DHA intake did not influence this effect. In conclusion, the impairment of cardiac mitochondrial function was more pronounced in the insulin-deficient state than in insulin resistance. The modification of fatty acid composition of cardiac and mitochondrial membranes by DHA partially prevented the mitochondrial alterations induced in the two models.     

Polyunsaturated fatty acids,neuroinflammation and well being

The innate immune system of the brain is principally composed of microglial cells and astrocytes, which, once activated, protect neurons against insults (infectious agents, lesions, etc.). Activated glial cells produce inflammatory cytokines that act specifically through receptors expressed by the brain. The functional consequences of brain cytokine action (also called neuroinflammation) are alterations in cognition, mood and behaviour, a hallmark of altered well-being. In addition, proinflammatory cytokines play a key role in depression and neurodegenerative diseases linked to aging. Polyunsaturated fatty acids (PUFA) are essential nutrients and essential components of neuronal and glial cell membranes. PUFA from the diet regulate both prostaglandin and proinflammatory cytokine production. n-3 fatty acids are anti-inflammatory while n-6 fatty acids are precursors of prostaglandins. Inappropriate amounts of dietary n-6 and n-3 fatty acids could lead to neuroinflammation because of their abundance in the brain and reduced well-being. Depending on which PUFA are present in the diet, neuroinflammation will, therefore, be kept at a minimum or exacerbated. This could explain the protective role of n-3 fatty acids in neurodegenerative diseases linked to aging.     

Low dietary fish-oil threshold for myocardial membrane n-3 PUFA enrichment independent of n-6 PUFA intake in rats

Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil.     

Modulation of enzymatic activities by n-3 polyunsaturated fatty acids to support cardiovascular health

Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.     

Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Abstract

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for β-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and α-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.     

Docosahexaenoic acid (DHA) and the developing central nervous system (CNS) - Implications for dietary recommendations

The accretion of docosahexaenoic acid (DHA) in membranes of the central nervous system is required for the optimum development of retina and brain functions. DHA status is determined by the dietary intake of n-3 polyunsaturated fatty acids (PUFA), both the metabolic precursor α-linolenic acid (α-LNA) and DHA. Clinical studies have shown that feeding term or premature infants with formula low in total n-3 PUFA may alter the maturation of visual acuity. Moreover, feeding infants over the first 6 mon of life with formula containing adequate α-LNA, but no DHA, did not sustain the same cerebral accretion of DHA as that of breast-fed infants. Whether lower DHA accretion in brain of formula-fed term infants impairs neurophysiological performances is not clearly established. Contradictory data have been published, possibly owing to confounding factors such as maternal intakes and/or genetic variations in PUFA metabolism. Nevertheless, a large corpus of data is in favor of the recommendation of regular dietary intakes of DHA (during at least the first 6 mon of life) and suggest that DHA should be added in formulas at the level generally found in human milk (0.2-0.3 wt% of total fatty acids). The maternal intake of n-3 PUFA during pregnancy and lactation is also crucial, since the n-3 PUFA are provided during perinatal development through placental transfer and maternal milk, which determines the DHA status of the newborn and consequently impacts on post-natal development of brain and visual functions. Whether more clinical studies are needed to control and improve the impact of DHA maternal intakes on the progeny’s neurodevelopment, several commissions recommended by precaution that DHA average intake for pregnant and lactating women should be of 200-300 mg/day.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

n-3 Polyunsaturated fatty acids—Physiological relevance of dose

n-3 Polyunsaturated fatty acids (PUFA) are widely used for chemotheraphy/chemoprevention of chronic diseases. However, the molecular mechanism(s) by which the bioactive n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) modulate effector pathways are not fully elucidated. Multiple experimental approaches, including use of animal models, cell lines, and human clinical trials, have been utilized to dissect the complex effectors. It is imperative to link these different experimental approaches together in order to interpret outcomes in the context of human physiology and pathophysiology. Unfortunately, the adoption of a broad array of model systems and a wide range of fatty acid exposures (i.e. doses) has made it difficult to interpret biological outcomes. Therefore, in this mini-review we discuss the impact of (a) molecular structure of bioactive fatty acids, (b) dose relevance relative to human consumption, (c) enrichment of fatty acids in sera and tissues following dietary intake, and (d) limitations of cell/tissue culture studies.     

Dietary fats and membrane function: implications for metabolism and disease

Lipids play varied and critical roles in metabolism, with function dramatically modulated by the individual fatty acid moities in complex lipid entities. In particular, the fatty acid composition of membrane lipids greatly influences membrane function. Here we consider the role of dietary fatty acid profile on membrane composition and, in turn, its impact on prevalent disease clusters of the metabolic syndrome and mental illness. Applying the classical physiological conformer-regulator paradigm to quantify the influence of dietary fats on membrane lipid composition (i.e. where the membrane variable is plotted against the same variable in the environment--in this case dietary fats), membrane lipid composition appears as a predominantly regulated parameter. Membranes remain relatively constant in their saturated (SFA) and monounsaturated (MUFA) fatty acid levels over a wide range of dietary variation for these fatty acids. Membrane composition was found to be more responsive to n-6 and n-3 polyunsaturated fatty acid (PUFA) levels in the diet and most sensitive to n-3 PUFA and to the n-3/n-6 ratio. These differential responses are probably due to the fact that both n-6 and n-3 PUFA classes cannot be synthesised de novo by higher animals. Diet-induced modifications in membrane lipid composition are associated with changes in the rates of membrane-linked cellular processes that are major contributors to energy metabolism. For example, in the intrinsic activity of fundamental processes such as the Na+/K+ pump and proton pump-leak cycle. Equally, dietary lipid profile impacts substantially on diseases of the metabolic syndrome with evidence accruing for changes in metabolic rate and neuropeptide regulation (thus influencing both sides of the energy balance equation), in second messenger generation and in gene expression influencing a range of glucose and lipid handling pathways. Finally, there is a growing literature relating changes in dietary fatty acid profile to many aspects of mental health. The understanding of dietary lipid profile and its influence on membrane function in relation to metabolic dysregulation has exciting potential for the prevention and treatment of a range of prevalent disease states.     

Synergistic effects of stress and omega-3 fatty acid deprivation on emotional response and brain lipid composition in adult rats

The aim was to determine the consequences of multi-generational n-3 polyunsaturated fatty acids (PUFA) deficiency on emotional response in rats subjected to maternal separation (MS) as chronic early life stress. Pups fed a control or an n-3 PUFA deficient diet were daily separated for 2 weeks before weaning. In adult rats, reward response was assessed by sucrose consumption and reactivity to novelty using openfield test. Both n-3 PUFA deficiency and MS increased reward response and impulsivity. Moreover, nutritional deficiency and stress acted in synergy to elevate sucrose intake by 80%, compared to control conditions. n-3 PUFA deprivation induced a depletion of docosahexanoeic acid of brain membranes by 70% compensated by increase in 22:5 n-6 and arachidonic acid (AA) levels. The diet-induced AA increase was, however, significantly higher in MS rats. This suggests that n-3 PUFA deficit could be an environmental risk increasing vulnerability to depressive-like response induced by chronic stress.     

Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases.     

Myogenic differentiation and lipid-raft composition of L6 skeletal muscle cells are modulated by PUFAs

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells.     

Interactive effects of dietary (n-3) polyunsaturated fatty acids and chronic ethanol intoxication on synaptic membrane lipid composition and fluidity in rats.

The influence of dietary polyunsaturated fatty acids on fatty acid composition, cholesterol and phospholipid content as well as 'fluidity' (assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probes) of brain synaptic plasma membranes (SPM) and their interactions with chronic ethanol effects were studied in rats fed for two generations with diets either devoid of (n-3) fatty acids (sunflower oil diet), rich in alpha-linolenic acid (soya oil diet) or in long chain (n-3) fatty acids (sunflower + cod liver oil diet). Results were compared with rats fed standard lab chow. Sunflower oil led to an increase in the (n-6)/(n-3) ratio in the membranes with an increase of the 'fluidity' at membrane apolar level; sunflower + cod liver oil decreased the (n-6)/(n-3) ratio without affecting membrane 'fluidity' while no difference was seen between the SPM of rats fed soya oil and standard diet. After 3 weeks alcohol intoxication in rat fed the standard diet: oleic alpha-linoleic acids and cholesterol levels were increased, arachidonic acid and the double bond index/saturated fatty acids were decreased and there was a decrease of 'fluidity' in the lipid core of the SPM. Soya oil almost totally abolished these usually observed changes in the SPM fatty acids composition but increased oleic acid and cholesterol without any change in fluidity. Sunflower oil led to the same general alterations of fatty acid as seen with standard diet but to a greater extent, with decrease of the 'fluidity" at the apolar level and in the region probed by TMA-DPH. When sunflower oil was supplemented with cod liver oil, oleic and alpha-linoleic acids were increased while the 'fluidity' of the apolar core of SPM was decreased. So, the small changes in fatty acid pattern seem able to modulate neural properties i.e. the responses to a neurotoxic like ethanol. A structurally specific role of PUFA is demonstrated by the pernicious effects of the alpha-linolenic acid deficient diet which are not totally prevented by the supply of long chain (n-3) PUFA.