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 α-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 (α-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 appear 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-triphosphate-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 n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

Polyunsaturated free fatty acids stimulate an increase in cytosolic Ca2+ by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in T cells through a mechanism independent of phosphoinositide turnover

Polyunsaturated free fatty acids (PUFAs) of both w-3 and w-6 series, induce a rapid increase of cytosolic free Ca2+ concentration ([Ca2+]i) in a leukemic T-cell line (JURKAT), measured by the fluorescent indicator fura-2. The early increase in [Ca2+]i was transient, falling to a sustained level which returned to base line after 10-15 min. In Ca2+-free medium, PUFAs still caused an early increase in [Ca2+]i but rapidly returned to basal. Depletion of endoplasmic reticular Ca2+ pool by addition of OKT3 (antibodies to CD3 of the T3-antigen receptor complex) to JURKAT cells (in Ca2+-free medium) abolished the PUFAs-mediated [Ca2+]i increase and vice versa. By using saponin-permeabilized JURKAT cells, the intracellular free Ca2+ released by PUFAs was found to be the non-mitochondrial, ATP-dependent sequestered Ca2+ pool which is sensitive to inositol 1,4,5-trisphosphate. However, PUFAs do not induce any apparent increase in inositol phosphates in JURKAT cells. No Ca2+ influx was detected in JURKAT cells when stimulated with PUFAs. A correlation was observed between both the carbon chain length and the number of double bonds with the ability to mobilize cytosolic free [Ca2+]i in the w-3 PUFAs. These results demonstrate that PUFAs stimulate the release of Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in the endoplasmic reticulum of JURKAT cells via a mechanism independent of inositol lipid hydrolysis.     

Inhibition of receptor-mediated calcium influx in T cells by unsaturated non-esterified fatty acids.

The effect of omega-3, omega-6 and omega-9 unsaturated fatty acids (UFAs) on receptor-mediated Ca2+ entry was investigated in a T-cell line (JURKAT) by using anti-CD3 antibodies (OKT3) to induce intracellular Ca2+ [( Ca2+]i) increase and Ca2+ influx. All the UFAs, as well as Ni2+ ions and 12-O-tetradecanoylphorbol 13-acetate, decreased the OKT3-induced sustained [Ca2+]i increase to basal levels. Although non-esterified fatty acids activate protein kinase C (PKC) [McPhail, Clayton & Snyderman (1984) Science 224, 622-624; Murakami, Chan & Routtenberg (1986) J. Biol. Chem. 261, 15424-15429], studies using H-7 and analysis of the PKC-dependent phosphorylation of 19 and 80 kDa marker substrates ruled out the involvement of PKC in UFA-induced inhibition of Ca2+ entry. Flow-cytometry analysis showed that UFAs do not interfere with antibody-receptor binding. BSA (0.2%, w/v) reversed the effect of UFAs after these fatty acids have decreased the OKT3-induced [Ca2+]i increase to basal levels. The relevance of these findings and possible mechanisms for inhibition by UFAs of receptor-mediated Ca2+ influx were discussed.     

Fatty acid composition of platelet phospholipids and plasma lipids in obese Zucker rats

The purpose of this work was to see whether hyperlipaemia observed in genetically obese Zucker rats (fa/fa) was associated with differences in fatty-acid composition of plasma triacylglycerols, plasma phospholipids and of platelet phospholipids, in comparison with the control lean rats (Fa/-). Results showed that plasma triacylglycerols and phospholipids were increased in obese rats. In triacylglycerols, the amount of saturated and monounsaturated fatty acids was highly increased whereas the amount of the n-6 and n-3 polyunsaturated fatty acids was little modified. In plasma phospholipids, saturated and monounsaturated fatty acids were also increased, as were the n-3 fatty acids (except C 18:3 n-3); the n-6 fatty acids were little increased except C 20:3 n-6 which was markedly increased. These results concerning the amounts of fatty acids have their counterpart in their relative proportions of fatty acids. Data thus obtained suggest that conversion of linoleic acid (C 18:2 n-6) into arachidonic acid (C 20:4 n-6) was decreased in obese rats, particularly the delta 5 desaturation step. On the contrary, conversion of linolenic acid (C 18:3 n-3) into higher polyenes seemed increased. Thrombocytosis was not modified in the obese rat, but the volume of the platelets was increased. Platelet phospholipids exhibited the same modifications as plasma phospholipids but with different magnitude. Saturated and monounsaturated fatty acids were little augmented, n-3 fatty acids were more augmented (except C 18:3 n-3 acid which was unchanged); n-6 fatty acids were not modified except C 20:3 n-6 acid which was highly increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific phospholipid fatty acid composition of brain regions in mice. Effects of n-3 polyunsaturated fatty acid deficiency and phospholipid supplementation

This study examined the effects of dietary alpha-linolenic acid deficiency followed or not by supplementation with phospholipids rich in n;-3 polyunsaturated fatty acid (PUFA) on the fatty acid composition of total phospholipids in 11 brain regions. Three weeks before mating, mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group were supplemented with n;-3 polyunsaturated fatty acids (PUFA) from either egg yolk or pig brain phospholipids for 2 months. Saturated and monounsaturated fatty acid levels varied among brain regions and were not significantly affected by the diet. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all regions. alpha-Linolenic acid deficiency decreased the level of 22:6 n-3 and was compensated by an increase in 22:5 n-6 in all regions. However, the brain regions were affected differently. After the pituitary gland, the frontal cortex, and the striatum were the most markedly affected with 40% reduction of 22:6 n-3. Supplementation with egg yolk or cerebral phospholipids in deficient mice restored a normal fatty acid composition in brain regions except for the frontal cortex. There was a regional distribution of the fatty acids in the brain and the impact of deficiency in alpha-linolenic acid was region-specific. Dietary egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for the recovery of altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.     

Regulation of intracellular calcium levels by polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, in astrocytes: possible involvement of phospholipase A2

Pathological conditions in the brain, such as ischemia, trauma and seizure are accompanied by increased levels of free n-6 and n-3 polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). A neuroprotective role has been suggested for PUFA. For investigation of the potential molecular mechanisms involved in neuroprotection by PUFA, we studied the regulation of the concentration of intracellular Ca2+ ([Ca2+]i) in rat brain astrocytes. We evaluated the presence of extracellular PUFA and the release of intracellular PUFA. Interestingly, only the constitutive brain PUFA AA and DHA, but not eicosapentaenoic acid (EPA) had prominent effects on intracellular Ca2+. AA and DHA suppressed [Ca2+]i oscillation, inhibited store-operated Ca2+ entry, and reduced the amplitudes of Ca2+ responses evoked by agonists of G protein-coupled receptors. Moreover, prolonged exposure of astrocytes to AA and DHA brought the cells to a new steady state of a moderately elevated [Ca2+]i level, where the cells became virtually insensitive to external stimuli. This new steady state can be considered as a mechanism of self-protection. It isolates disturbed parts of the brain, because AA and DHA reduce pathological overstimulation in the tissue surrounding the damaged area. In inflammation-related events, frequently AA and DHA exhibit opposite effects. However, in astrocytes AA and DHA exerted comparable effects on [Ca2+]i. Extracellularly added AA and DHA, but not EPA, were also able to induce the release of [3H]AA from prelabeled astrocytes. Therefore, we also suggest the involvement of phospholipase A2 activation and lysophospholipid generation in the regulation of intracellular Ca2+ in astrocytes.     

Membrane lipid profile alterations are associated with the metabolic adaptation of the Caco-2 cells to aglycemic nutritional condition

Cancer cells can adapt their metabolic activity under nutritional hostile conditions in order to ensure both bioenergetics and biosynthetic requirements to survive. In this study, the effect of glucose deprivation on Caco-2 cells bioenergetics activity and putative relationship with membrane lipid changes were investigated. Glucose deprivation induces a metabolic remodeling characterized at mitochondrial level by an increase of oxygen consumption, arising from an improvement of complex II and complex IV activities and an inhibition of complex I activity. This effect is accompanied by changes in cellular membrane phospholipid profile. Caco-2 cells grown under glucose deprivation show higher phosphatidylethanolamine content and decreased phosphatidic acid content. Considering fatty acid profile of all cell phospholipids, glucose deprivation induces a decrease of monounsaturated fatty acid (MUFA) and n-3 polyunsaturated fatty acids (PUFA) simultaneously with an increase of n-6 PUFA, with consequent drop of n-3/n-6 ratio. Additionally, glucose deprivation affects significantly the fatty acid profile of all individual phospholipid classes, reflected by an increase of peroxidability index in zwitterionic phospholipids and a decrease in all anionic phospholipids, including mitochondrial cardiolipin. These data indicate that Caco-2 cells metabolic remodeling induced by glucose deprivation actively involves membrane lipid changes associated with a specific bioenergetics profile which ensure cell survival.     

Dynamics of fatty acid composition of total lipids during embryonic development of atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L.

Dynamics of fatty acid composition of total lipids was studied for freshwater salmon Salmo salar L. during its embryonic development from blastula (3 hours) up to hatching (108 days) as well as in unfertilized eggs. Stable amount of total and some saturated, monounsaturated and polyunsaturated fatty acids (PUFA) of total lipids was observed during embryonic development. Considerable changes in fatty acid composition were observed at the stage of prelarvae hatching, i.e., significant decrease of (n-6) PUFA (18:2(n-6) and 20:4(n-6)) and (n-3) PUFA and increase of total and some saturated and monounsaturated fatty acids was registered. Change in saturation ratio of membrane lipids justifies the presence of the biochemical mechanism forwarded on regulation of cell membrane enzymes in accordance with the changes of internal physiological processes taking place in the organism and fluctuations of external environmental conditions or the preparation period (as reproduction). Data on peculiarities of transformation and utilization of fatty acids during salmon embryonic development may be used for understanding of their functional role in the developing organism as well as for assessing the quality of the caviar.     

Dynamics of fatty acid composition of total lipids during embryonic development of Atlantic salmon Salmo salar L

Dynamics of fatty acid composition of total lipids was studied for freshwater salmon Salmo salar L. during its embryonic development from blastula (3 hours) up to hatching (108 days) as well as in unfertilized eggs. Stable amount of total and some saturated, monounsaturated and polyunsaturated fatty acids (PUFA) of total lipids was observed during embryonic development. Considerable changes in fatty acid composition were observed at the stage of prelarvae hatching, i.e., significant decrease of (n-6) PUFA (18:2(n-6) and 20:4(n-6)) and (n-3) PUFA and increase of total and some saturated and monounsaturated fatty acids was registered. Change in saturation ratio of membrane lipids justifies the presence of the biochemical mechanism forwarded on regulation of cell membrane enzymes in accordance with the changes of internal physiological processes taking place in the organism and fluctuations of external environmental conditions or the preparation period (as reproduction). Data on peculiarities of transformation and utilization of fatty acids during salmon embryonic development may be used for understanding of their functional role in the developing organism as well as for assessing the quality of the caviar.     

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.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Fatty acid metabolism of isolated mammalian cells

It is now clear that a wide variety of differentiated cells in culture exhibit essentially the full spectrum of mammalian fatty acid metabolism. These cells readily incorporate free fatty acids into membrane phosphoglycerides, modify exogenous fatty acids by desaturation and elongation, and store excess fatty acyl groups, primarily as triacylglycerols. Similarly, many different types of cells synthesize cyclooxygenase and lipoxygenase derivatives of long chain polyunsaturated fatty acids. Furthermore, although the fatty acid composition of cellular phospholipids can be modified by medium supplementation, cells in culture exhibit definite fatty acyl specificities for the various steps of fatty acid activation, transesterification and release. As the extensive repertoire of fatty acid metabolism in mammalian cells has been elucidated, and as the ability to grow differentiated cells in culture has increased, new questions have arisen. There is still much to be learned about the enzymes involved in synthesizing and maintaining the unique fatty acid composition of the different cellular phospholipids and the processes which regulate the desaturation, elongation and retroconversion of polyunsaturated fatty acids. Other areas of great current interest are the mechanisms by which certain long chain polyunsaturated fatty acids are made available for conversion to oxygenated, biologically-active derivatives, the metabolic interactions between different polyunsaturated fatty acids, particularly n-3 and n-6 fatty acids, the cellular roles of the C22 polyunsaturated fatty acids, and the functions of particular molecular species of phospholipids in membrane-mediated events. Further research in these areas will contribute to unravelling the role of fatty acids and fatty acid derivatives in the physiological processes of mammalian cells.     

Influence of the fatty acid composition of lipids in chylomicron remnants derived from fish or corn oil on the lipid profile of cultured rat hepatocytes

The aim of this work was to characterise the lipid and fatty acid composition of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) and to investigate their influence on the fatty acid profiles of the lipids of rat hepatocytes cultured in monolayers. Chylomicrons were prepared from the lymph collected from the thoracic duct of rats given an oral dose of fish or corn oil (high in n-3 and n-6 PUFA, respectively), and remnants were prepared in vitro from such chylomicrons using rat plasma containing lipoprotein lipase. The fatty acids predominating in the oils abounded also in their respective chylomicrons and remnants, especially in triacylglycerols. Chylomicrons as well as remnants contained small amounts of phospholipids and long-chain PUFA that were minor in, or absent from, the dietary oils, evidently provided by the intestinal epithelium. The incubation of hepatocytes for 6 h, with either n-3 or n-6 PUFA-rich remnants (0.25-0.75 mM triacylglycerol) resulted in a dose-dependent increase in the amount of triacylglycerols and phospholipids in the cells, which was not affected further by increasing the incubation time to 19 h. Whereas hepatocyte triacylglycerols mostly incorporated the PUFA predominating in each remnant type, the fatty acid profile of cell phospholipids was virtually unchanged. In addition, irrespective of whether they were enriched in n-3 or n-6 PUFA, remnants promoted a relative decrease in the amount of cholesteryl esters, a minor hepatocyte lipid class poor in PUFA. The results demonstrate that the hepatocyte fatty acid profile is modulated in a lipid-class specific way by the amount and type of dietary PUFA delivered to cells in chylomicron remnants.     

Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary alpha-linolenic acid deficiency

Age and diet-induced variations of phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and alpha-tocopherol concentration in the liver microsomal membrane were studied in male Wistar rats fed a semipurified diet either balanced in n-6 and n-3 polyunsaturated fatty acids (PUFA) (Control) or deprived of alpha-linolenic acid, i.e. n-3 PUFA (Deficient) over two generations. The animals were studied at the age of 6 months (adult) or 24 months (old). Both PHGPx activity and vitamin E level were significantly higher in 24-month old rats as compared to 6-month old rats. By contrast, the thiobarbituric acid reactive substances (TBARS) following stimulated in vitro peroxidation of membrane lipids were markedly lower (P < 0.01) with aging. The fatty acid composition of microsomal membrane phospholipids (PL) was also considerably modified by age. In particular, the levels of arachidonic acid and total n-6 PUFA were lower (P < 0.001) whereas n-3 PUFA levels were higher (P < 0.001) in most PL main classes. The alpha-linolenic acid deficiency markedly influenced these age-related changes. The higher PHGPx activity in the old rats as compared to the adult rats was only significant in those fed the control diet. In the 6-month old rats (but not in the 24-month old rats), the deficient diet led to a higher membrane vitamin E level and to lower TBARS production than the control diet. The results suggest that the nature of dietary PUFA may influence the age-related variations in this pair of membrane antioxidants and also in the fatty acid composition of microsomes.     

Fatty acid composition of membrane bilayers: Importance of diet polyunsaturated fat balance

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.     

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.