DHA-containing Phospholipids Research Articles

Docosahexaenoic acid disrupts lipid raft clustering and enhances proliferation and survival of EL4 lymphomas

An emerging molecular mechanism by which docosahexaenoic acid (DHA) acyl chains modify cellular function is through changes in lipid raft organization. Biophysical studies in liposomes show that DHA does not directly modify rafts; instead, it incorporates into non‐rafts to modify the lateral organization of proteins such as the major histocompatibility complex (MHC) class I. Here we tested predictions of the model at a cellular level by incorporating DHA and eicosapentaenoic acid (EPA) into the membranes of EL4 lymphomas. Quantification of fluorescence microscopy images showed that DHA, but not EPA, diminished lipid raft clustering by incorporating into rafts and changed MHC I lateral organization by increasing the fraction of the non‐raft protein into rafts. We next addressed the functional consequences of treating EL4 cells given changes in raft organization are associated with suppression of cellular proliferation. Both EPA and DHA enhanced the proliferation and survival of EL4 cells relative to controls. Taken together, our findings are not in agreement with biophysical studies; therefore, we propose a new model, which reconciles contradictory viewpoints from biophysical and cellular studies, to explain how DHA‐containing phospholipids modify rafts. Furthermore, our data highlight the notion that n‐3 PUFAs exert differential functional effects depending on the cell type.

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.

Regulation of Membrane Proteins by Dietary Lipids: Effects of Cholesterol and Docosahexaenoic Acid Acyl Chain-Containing Phospholipids on Rhodopsin Stability and Function

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature ( T m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process ( E a) was determined from DSC thermograms by four separate methods. Both T m and E a varied with bilayer composition. Cholesterol increased the T m both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E a in the absence of DHA, but raised E a in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T m for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability ( E a) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Docosahexaenoic acid domains: the ultimate non-raft membrane domain

What distinguishes polyunsaturated fatty acids (PUFAs) from less unsaturated fatty acids is the presence of a repeating CH–CH 2–CH unit that produces an extremely flexible structure rapidly isomerizing through conformational states. Docosahexaenoic acid (DHA) with 6 double bonds is the most extreme example. The focus of this review is the profound impact that the high disorder of DHA has on its interaction with cholesterol when the PUFA is incorporated into membrane phospholipids. Results from a battery of biophysical techniques are described. They demonstrate an aversion of DHA for the sterol that drives the lateral segregation of DHA-containing phospholipids into liquid disordered ( l d) domains that are depleted in cholesterol. These domains are compositionally and organizationally the antithesis of lipid rafts, the much-studied liquid ordered ( l o) domain that is enriched in predominantly saturated sphingolipids and cholesterol. We hypothesize that the introduction of DHA-rich domains into the plasma membrane where they coexist with lipid rafts is the origin, in part, of the astonishing diversity of health benefits that accrue from dietary consumption of DHA. According to our model, changes in the conformation of signaling proteins when they move between these disparate domains have the potential to modulate cell function.

Membrane phospholipid composition may contribute to exceptional longevity of the naked mole-rat ( Heterocephalus glaber): A comparative study using shotgun lipidomics

Phospholipids containing highly polyunsaturated fatty acids are particularly prone to peroxidation and membrane composition may therefore influence longevity. Phospholipid molecules, in particular those containing docosahexaenoic acid (DHA), from the skeletal muscle, heart, liver and liver mitochondria were identified and quantified using mass-spectrometry shotgun lipidomics in two similar-sized rodents that show a ∼9-fold difference in maximum lifespan. The naked mole rat is the longest-living rodent known with a maximum lifespan of >28 years. Total phospholipid distribution is similar in tissues of both species; DHA is only found in phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS), and DHA is relatively more concentrated in PE than PC. Naked mole-rats have fewer molecular species of both PC and PE than do mice. DHA-containing phospholipids represent 27–57% of all phospholipids in mice but only 2–6% in naked mole-rats. Furthermore, while mice have small amounts of di-polyunsaturated PC and PE, these are lacking in naked mole-rats. Vinyl ether-linked phospholipids (plasmalogens) are higher in naked mole-rat tissues than in mice. The lower level of DHA-containing phospholipids suggests a lower susceptibility to peroxidative damage in membranes of naked mole-rats compared to mice. Whereas the high level of plasmalogens might enhance membrane antioxidant protection in naked mole-rats compared to mice. Both characteristics possibly contribute to the exceptional longevity of naked mole-rats and may indicate a special role for peroxisomes in this extended longevity.

The role of polyunsaturated lipids in membrane raft function

Docosahexaenoic acid (DHA), with 22 carbons and six double bonds, is the longest, most unsaturated fatty acid commonly found in humans. It represents the extreme example of an omega-3 (n-3) polyunsaturated fatty acid. Since early epidemiology studies, DHA has been linked to alleviation of an enormous number of human afflictions, including heart disease, cancer and neurological disorders. How one simple molecule can affect so many seemingly unrelated abnormalities has been a contentious question for many years. One research direction has investigated events that follow the uptake of DHA into animal cell plasma membrane phospholipids. Summarized here is a variety of membrane properties impacted by the incorporation of DHA. DHA’s dynamic shape, consisting of multiple configurations, is very different from what its static, stick structure would indicate. DHA-containing phospholipids have a wide hydrophobic base compared with their hydrophilic head and so induce nega tive curvature strain that severely impacts the activity of a variety of important membrane proteins. The unusual structure means that DHA-rich membranes are also surprisingly thin and support high permeability, compression, fusion and flip-flop rates. DHA does not exist in an environment that is independent from other membrane lipids. Of particular interest is the interaction of DHA-containing phospholipids with the major lipid raft components cholesterol and sphingomyelin. From a wide variety of biophysical studies, primarily done on model lipid monolayers and bilayers, a new hypothesis is proposed suggesting that DHA may alter plasma membrane lipid raft structure and hence essential cell signaling events. A fundamental role for DHA affecting a feature common to all cells, membrane structure and function, may explain its wide variety of reputed health benefits. Keywords: docosahexaenoic acid; lipid rafts; membrane structure; polyunsaturated fatty acids; n-3

A Novel Phosphatidylcholine Which Contains Pentadecanoic Acid at sn-1 and Docosahexaenoic Acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."

The role of polyunsaturated lipids in membrane raft function

Docosahexaenoic acid (DHA), with 22 carbons and six double bonds, is the longest, most unsaturated fatty acid commonly found in humans. It represents the extreme example of an omega-3 (n-3) polyunsaturated fatty acid. Since early epidemiology studies, DHA has been linked to alleviation of an enormous number of human afflictions, including heart disease, cancer and neurological disorders. How one simple molecule can affect so many seemingly unrelated abnormalities has been a contentious question for many years. One research direction has investigated events that follow the uptake of DHA into animal cell plasma membrane phospholipids. Summarized here is a variety of membrane properties impacted by the incorporation of DHA. DHA’s dynamic shape, consisting of multiple configurations, is very different from what its static, stick structure would indicate. DHA-containing phospholipids have a wide hydrophobic base compared with their hydrophilic head and so induce nega tive curvature strain that severely impacts the activity of a variety of important membrane proteins. The unusual structure means that DHA-rich membranes are also surprisingly thin and support high permeability, compression, fusion and flip-flop rates. DHA does not exist in an environment that is independent from other membrane lipids. Of particular interest is the interaction of DHA-containing phospholipids with the major lipid raft components cholesterol and sphingomyelin. From a wide variety of biophysical studies, primarily done on model lipid monolayers and bilayers, a new hypothesis is proposed suggesting that DHA may alter plasma membrane lipid raft structure and hence essential cell signaling events. A fundamental role for DHA affecting a feature common to all cells, membrane structure and function, may explain its wide variety of reputed health benefits. Keywords: docosahexaenoic acid; lipid rafts; membrane structure; polyunsaturated fatty acids; n-3

Oleic and Docosahexaenoic Acid Differentially Phase Separate from Lipid Raft Molecules: A Comparative NMR, DSC, AFM, and Detergent Extraction Study

We have previously suggested that the ω-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) may in part function by enhancing membrane lipid phase separation into lipid rafts. Here we further tested for differences in the molecular interactions of an oleic (OA) versus DHA-containing phospholipid with sphingomyelin (SM) and cholesterol (CHOL) utilizing 2H NMR spectroscopy, differential scanning calorimetry, atomic force microscopy, and detergent extractions in model bilayer membranes. 2H NMR and DSC (differential scanning calorimetry) established the phase behavior of the OA-containing 1-[ 2H 31]palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d 31)/SM (1:1) and the DHA-containing 1-[ 2H 31]palmitoyl-2-docosahexaenoyl- sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d 31)/SM (1:1) in the absence and presence of equimolar CHOL. CHOL was observed to affect the OA-containing phosphatidylethanolamine (PE) more than the DHA-containing PE, as exemplified by >2× greater increase in order measured for the perdeuterated palmitic chain in 16:0-18:1PE-d 31/SM (1:1) compared to 16:0-22:6PE-d 31/SM (1:1) bilayers in the liquid crystalline phase. Atomic force microscopy (AFM) experiments showed less lateral phase separation between 16:0-18:1PE-rich and SM/CHOL-rich raft domains in 16:0-18:1PE/SM/CHOL (1:1:1) bilayers than was observed when 16:0-22:6PE replaced 16:0-18:1PE. Differences in the molecular interaction of 16:0-18:1PE and 16:0-22:6PE with SM/CHOL were also found using biochemical detergent extractions. In the presence of equimolar SM/CHOL, 16:0-18:1PE showed decreased solubilization in comparison to 16:0-22:6PE, indicating greater phase separation with the DHA-PE. Detergent experiments were also conducted with cardiomyocytes fed radiolabeled OA or DHA. Although both OA and DHA were found to be largely detergent solubilized, the amount of OA that was found to be associated with raft-rich detergent-resistant membranes exceeded DHA by almost a factor of 2. We conclude that the OA-PE phase separates from rafts far less than DHA-PE, which may have implications for cellular signaling.

Concentration of Docosahexaenoic Acid-containing Phospholipid through Lipozyme IM-mediated Hydrolysis

A selective partial hydrolyses of egg-yolk lecithin from fish-oil fed hens and squid-skin lecithin were carried out with immobilized Rhizomucor miehei lipase (Lipozyme IM) in n-hexane media in order to concentrate docosahexaenoic acid (DHA) -containing phospholipids. DHA were abundant in phospholipid fractions that had recovered from the partial hydrolysates than the corresponding substrates i.e. egg-yolk lecithin and squid lecithin. Thus, it was clear that DHA ester bonds were less susceptible against hydrolysis than other fatty acid ester bonds. Optimum water activity (aw) for Lipozyme IM to concentrate DHA-containing phospholipids was aw=0.44. Under this aw, DHA showed to three fold increase compared to that of the original egg-yolk lecithin from fish-oil fed hens, after 8 h reaction at 40°C. Water activity-controlled partial hydrolysis with Lipozyme IM was borned out to be a useful method for concentrating functional phospholipids.

Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an ( n − 3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As ( n − 3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of ( n − 3) fatty acids from the diet, most likely due to retention of membrane-bound ( n − 3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Inhibitory effect of docosahexaenoic acid-containing phospholipids on 5-lipoxygenase in rat basophilic leukemia cells

5-Lipoxygenase has been recognized to be an important enzyme that catalyzes the first step in leukotriene production. In this study we examine whether or not phospholipids containing docosahexaenoic acid (DHA) affect 5-lipoxygenase activity of a rat basophilic leukemia cell line (RBL-1). Among the synthesized phospholipids examined, 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (1-oleoyl-2-DHA-PC) was found to be the most potent inhibitor of 5-lipoxygenase. The inhibition was dose-dependent and the ID 50 value was 4.0 μM. When the fatty acid at the sn-2-position was replaced by other unsaturated fatty acids, the inhibitory activity decreased with decreasing numbers of both carbon atoms and double bonds in the fatty acids. Substitution at the 1-position of the DHA-containing PC also affected the inhibitory potency. If oleic acid was substituted with palmitic acid, the inhibition activity was completely abolished. Lineweaver-Burk plot analysis showed that the inhibition of 5-lipoxygenase by 1-oleoyl-2-DHA-PC was non-competitive. The inhibition by this synthesized phospholipid was very specific to 5-lipoxygenase; that is, it did not extend to fatty acid cyclooxygenase, 12-lipoxygenase or 15-lipoxygenase. These results suggest that endogenously existing DHA-containing phospholipids may affect 5-lipoxygenase activity and thus control leukotriene biosynthesis in vivo.

Omega‐3 fatty acid modification of membrane structure and function. II. Alteration by docosahexaenoic acid of tumor cell sensitivity to immune cytolysis

Docosahexaenoic acid (DHA, 22:6) is a long-chain omega-3 fatty acid abundant in cold water fish; it is the most unsaturated fatty acid found in biologic systems and is reported to alter membrane structure. To explore DHA's effect on membrane function, we have fused tumor cells with synthetic phosphatidylcholine (PC) containing stearic acid in the sn-1 position and DHA in the sn-2 position (18:0, 22:6 PC) and have found the lipid-modified tumor cells to be more sensitive to cytolysis by alloreactive cytotoxic T lymphocytes. Cold target competition experiments suggested that fusion of tumor plasma membranes with 18:0, 22:6 PC produced a qualitative change in expression of surface antigens recognized by cytotoxic T lymphocytes. We monitored the expression of various epitopes on tumor cells by complement-mediated lysis and radioimmunoassay with monoclonal antibodies against H-2 class I antigens. Our results suggest that membrane-bound DHA increases the expression of some epitopes while decreasing the expression of others and that different tumor lines vary in the magnitude of DHA's effect. Our findings are consistent with a model in which DHA-containing phospholipids segregate into membrane domains, in turn altering the expression of membrane proteins.