The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Characteristics of fatty acid composition of lipids in higher plant vacuolar membranes

The fatty acid composition of vacuolar membrane lipids from plant storage tissues and their genesis have been studied. A high content of unsaturated fatty acids (up to 77%) was observed in lipids of these membranes. Linoleic acid prevailed in vacuolar lipids of carrot and red beet (54.2 and 44.2%, respectively). Linolenic acid prevailed in vacuolar lipids of garden radish and turnip (39.7 and 33.9%, respectively). Regarding saturated fatty acids, vacuolar lipids of garden radish, carrot, and red beet contained predominantly palmitic acid (up to 20-24%). Unsaturated fatty acids, petroselinic (C18: 1omega12), cis-vaccenic (C18: 1omega7), hexatrien-7,-10,-13-oic (C16:3omega3) and others, were observed in vacuolar lipids of roots. These acids are usually synthesized in chloroplasts, and their presence in vacuolar lipids can be associated either with the transport of metabolites to the vacuole, or with endocytosis during vacuolar formation in the plant cell. The specific features of fatty acid composition of tonoplast lipids apparently are closely related to the tonoplast unique fluidity and mobility required for running osmotic processes in the cell and for forming transport protein assemblies.     

Cholesterol exchange as a function of cholesterol/phospholipid mole ratios.

The activation energy (Ea) for cholesterol exchange between dioleoyl phosphatidylcholine vesicles and erythrocyte 'ghosts' is measured as a function of molar percentage of cholesterol in both donor and acceptor membranes. A sharp increase in Ea occurs (from 39.9kJ/mol to 84kJ/mol) when the molar percentage of cholesterol decreases from 30 to 20%.     

Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes

Fatty acid binding proteins (FABP) are a family of 14-15 kDa proteins found in high abundance in many mammalian cell types. The physiological functions of the FABP remain unknown. It is also not known whether each FABP has a unique function, or whether all FABP function in a similar manner in their respective tissues. In this report the rate of transfer of anthroyloxy-labeled free fatty acid (ffa) from FABP to phospholipid bilayers is monitored using a fluorescence resonance energy transfer assay. A comparison is made between heart muscle FABP and liver FABP, and the results show that the rate of ffa transfer from the heart protein is an order of magnitude greater than the rate of transfer from the liver protein. Ffa transfer rates from both liver and heart FABP are independent of acceptor concentration and composition, suggesting that, at least in the case of model membrane acceptor vesicles, the mechanism of transfer is via aqueous diffusion rather than via collision of FABP with membranes. Since the rate of ffa transfer is likely to be important to cellular ffa traffic, these studies suggest that heart FABP may function differently within the myocyte than does liver FABP within the hepatocyte.     

Interactions of proteins and cholesterol with lipids in bilayer membranes

Mixtures of lipids and proteins, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$, of the lipid and aggregated below $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$. For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present.In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50–200 nm in length, around smooth patches of lipid.Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperarture of the lipid is discussed.     

The solubility of cholesterol and its exchange between membranes

It has been proposed that exchange between membrane cholesterol pools occurs by desorption of molecules into the aqueous environment rather than by formation of a transitory collision complex between the membranes. The rate of exchange is likely to be determined by the rate of dissociation of cholesterol from the membrane bilayer and by the concentration of cholesterol monomers or aggregates of cholesterol molecules in solution. The aim of this study was to measure the effects of agents known to increase cholesterol exchange rates on cholesterol solubility, critical micellar concentration and on the activation energy of exchange. A comparison was also made with regard to these parameters, of the exchange of cholesterol to that of 4-cholesten-3-one, another steroid which exchanges more rapidly than cholesterol. Acetone and dimethylsulphoxide increased cholesterol exchange between liposomes and erythrocytes, but only modestly increased the apparent solubility of cholesterol in saline and had no effect on the activation energy of the exchange process. However, acetone and dimethylsulphoxide increased the critical micellar concentration of the cholesterol 3-fold, although tetraethylammonium iodide, which had a smaller effect on exchange, did not. 4-Cholesten-3-one had a lower solubility and critical micellar concentration than that of cholesterol, but had the same activation energy for exchange. It is concluded that the apparent solubility of steroid aggregates are unlikely to determine the rate of exchange, but that agents which substantially increase exchange also increase the critical micellar concentration. The low critical micellar concentration of cholestenone suggests that the actual monomer concentration in an exchange system is low and that the rate of dissociation of the molecules from the liposomes must determine the exchange rate. This is not reflected in the activation energy measurements since these are a composite of all the elements of the exchange process.     

Infrared spectroscopic study of stratum corneum model membranes prepared from human ceramides, cholesterol, and fatty acids

The outermost layer of the skin, the stratum corneum, consists of corneocytes surrounded by lipid domains. The main lipid classes in stratum corneum are cholesterol, ceramides (CER), and free fatty acids forming two crystalline lamellar phases. However, only limited information is available on whether the various lipid classes participate in the same crystalline lattices or if separate domains are formed within the lipid lamellae. In this article infrared spectroscopic studies are reported of hydrated mixtures prepared from cholesterol, human CER, and free fatty acids. Evaluation of the methylene stretching vibrations revealed a conformational disordering starting at approximately 60 degrees C for all mixtures. Examination of the rotational ordering (scissoring and rocking vibrations) of mixtures prepared from equimolar cholesterol and CER with a variation in the level of free fatty acids showed that at lower free fatty acid content orthorhombic and hexagonal domains coexist in the lipid lamellae. Increasing the fatty acid level to an equimolar cholesterol/CER/fatty acid mixture reveals the dominant presence of an orthorhombic lattice, confirming x-ray diffraction studies. Replacing the protonated free fatty acid chains by their perdeuterated counterparts demonstrates that free fatty acids and CER participate in the same orthorhombic lattice up to a level of slightly less than 1:1:0.75 cholesterol/CER/free fatty acids molar ratio but that free fatty acids also form separate domains within the lipid lamellae at equimolar ratios at room temperature. However, no evidence for this has been observed at 32 degrees C. Extrapolating these findings to the situation in stratum corneum led us conclude that in stratum corneum, fatty acids and CER participate in the orthorhombic lattice at 32 degrees C, the skin temperature.     

N-3 fatty acids and membrane microdomains: From model membranes to lymphocyte function

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease.     

Exclusion of a cholesterol analog from the cholesterol-rich phase in model membranes

Vesicles of phosphatidylcholine/cholesterol mixtures show a wide composition range with coexistence of two fluid phases, the 'liquid disordered' (cholesterol-poor) and 'liquid ordered' (cholesterol-rich) phases. These systems have been widely used as models of membranes exhibiting lateral heterogeneity (membrane domains). The distributions of two fluorescent probes (a fluorescent cholesterol analog, NBD-cholesterol, and a lipophilic rhodamine probe, octadecylrhodamine B) in dimyristoylphosphatidylcholine/cholesterol vesicles were studied, at 30 degrees C and 40 degrees C. The steady-state fluorescence intensity of both probes decreases markedly with increasing cholesterol concentration, unlike the fluorescence lifetimes. The liquid ordered to liquid disordered phase partition coefficients K(p) were measured, and values much less than unity were obtained for both probes, pointing to preference for the cholesterol-poor phase. Globally analyzed time-resolved energy transfer results confirmed these findings. It is concluded that, in particular, NBD-cholesterol is not a suitable cholesterol analog and its distribution behavior in phosphatidylcholine/cholesterol bilayers is in fact opposite to that of cholesterol.     

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.     

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.     

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.     

Modulators of cellular cholesterol homeostasis as antiproliferative and model membranes perturbing agents

Cholesterol is an important component of mammalian cell membranes affecting their fluidity and permeability. Together with sphingomyelin, cholesterol forms microdomains, called lipid rafts. They play important role in signal transduction forming platforms for interaction of signal proteins. Altered levels of cholesterol are known to be strongly associated with the development of various pathologies (e.g., cancer, atherosclerosis and cardiovascular diseases). In the present work, the group of compounds that share the property of affecting cellular homeostasis of cholesterol was studied. It contained antipsychotic and antidepressant drugs, as well as the inhibitors of cholesterol biosynthesis, simvastatin, betulin, and its derivatives. All compounds were demonstrated to be cytotoxic to colon cancer cells but not to non-cancerous cells. Moreover, the most active compounds decreased the level of free cellular cholesterol. The interaction of drugs with raft-mimicking model membranes was visualized. All compounds reduced the size of lipid domains, however, only some affected their number and shape. Membrane interactions of betulin and its novel derivatives were characterized in detail. Molecular modeling indicated that high dipole moment and significant lipophilicity were characteristic for the most potent antiproliferative agents. The importance of membrane interactions of cholesterol homeostasis-affecting compounds, especially betulin derivatives, for their anticancer potency was suggested.     

Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of D. melanogaster on media supplemented with [1-¹³C]-palmitate lead to a family of ¹³C-palmitate-labeled fatty acid amides in the fly heads. The [1-¹³C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.     

Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

Polar lipids and fatty acid composition ofThermus strains from New Zealand

The polar lipids and fatty acid composition ofThermus aquaticus YT-1 and YS 041,T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed toT. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding toT. filiformis Wai33 A1,T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported inThermus spp. for the first time.     

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.     

Hypertriglyceridemia and hypercholesterolemia: effects of drug treatment on fatty acid composition of plasma lipids and membranes

The effect of atorvastatin, simvastatin and gemfibrozil on fatty acid composition of plasma phospholipids (PL), cholesterol esters (CE), triglycerides (TG) and red cell membrane ghosts (G) has been determined in appropriate sample populations of individuals with hypertriglyceridemia (HTG) or hypercholesterolemia (HCHL). Treatments were appropriate for the condition, gemfibrozil for HTG and a statin for HCHL. Modifications depend on the drug and lipid fraction examined. Both classes of drugs modify fatty acid composition but gemfibrozil modifications are more numerous and dramatic than are the modifications by statins. Gemfibrozil produces major modifications in fatty acid composition, which are both fatty acid and lipid class specific but generally decreases SFA and increases PUFA (mainly n6) and increases the proportion of fatty acids with chain length of 18C or more. Statins tend to increase chain length but have less effect on saturation. Notably, all three drugs increased arachidonic acid (AA) in PL and CE. Statins decreased gamma-linoleic acid (GLA) in PL and CE but gemfibrozil only increased GLA in TG.