Lipid composition of Limulus photoreceptor membranes.

The lipid composition has been determined for rhabdomeric photoreceptor membranes of Limulus, and these data are compared with those from photoreceptor membranes of albino rats. The comparison is of interest because the membranes of these two photoreceptor cells regulate ionic transport differently during the response to illumination. 1. Phospholipid class composition of Limulus is similar, but not identical, to that of rats. The major differences are a greater percentage of sphingomyelin in Limulus and a greater percentage of phosphatidylethanolamine in the rat. 2. Ethanolamine plasmalogens, not observed in rat photoreceptor membranes, are present in Limulus photoreceptor fractions. 3. The level of cholesterol in Limulus is higher than that usually reported for vertebrate rod outer segments. 4. The predominant polyunsaturated fatty acids of Limulus photoreceptor membrane phospholipids are 20: 4(n-6) and 20: 5(n-3) with only traces of 22: 6(n-3). This is in sharp contrast with the large percentages of 22: 6(n-3) found in rat photoreceptors. 5. The fatty acid distributions of both membrane systems are highly unsaturated, but the ratio of (n-3) to (n-6) polyunsaturates is only 1.7 for Limulus as compared to 4.6 for rat.     

Dietary triacylglycerol modulates sodium-dependent D-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent D-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as (n - 3) unsaturated fatty acids and lower percentages of monounsaturated and (n - 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0-20:5, 16:0-22:6 and 18:0-20:5 and lower levels of 18:0-18:2 and 16:0-18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na+-dependent D-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity (Vmax) than butter fat membrane vesicles but a similar Km for glucose.     

Lipid peroxidation modifies the picture of membranes from the "Fluid Mosaic Model" to the "Lipid Whisker Model"

The “Fluid Mosaic Model”, described by Singer and Nicolson, explain both how a cell membrane preserves a critical barrier function while it concomitantly facilitates rapid lateral diffusion of proteins and lipids within the planar membrane surface. However, the lipid components of biological plasma membranes are not regularly distributed. They are thought to contain “rafts” - nano-domains enriched in sphingolipids and cholesterol that are distinct from surrounding membranes of unsaturated phospholipids. Cholesterol and fatty acids adjust the transport and diffusion of molecular oxygen in membranes. The presence of cholesterol and saturated phospholipids decreases oxygen permeability across the membrane. Alpha-tocopherol, the main antioxidant in biological membranes, partition into domains that are enriched in polyunsaturated phospholipids increasing the concentration of the vitamin in the place where it is most required. On the basis of these observations, it is possible to assume that non-raft domains enriched in phospholipids containing PUFAs and vitamin E will be more accessible by molecular oxygen than lipid raft domains enriched in sphingolipids and cholesterol. This situation will render some nano-domains more sensitive to lipid peroxidation than others. Phospholipid oxidation products are very likely to alter the properties of biological membranes, because their polarity and shape may differ considerably from the structures of their parent molecules. Addition of a polar oxygen atom to several peroxidized fatty acids reorients the acyl chain whereby it no longer remains buried within the membrane interior, but rather projects into the aqueous environment “Lipid Whisker Model”. This exceptional conformational change facilitates direct physical access of the oxidized fatty acid moiety to cell surface scavenger receptors.     

Comparative aspects of sperm membrane fatty acid composition in silver (Vulpes vulpes) and blue (Alopex lagopus) foxes, and their relationship to cell cryopreservation

Cryogenic protocols have been developed for the storage of farmed silver fox (Vulpes vulpes) spermatozoa. However, these same protocols and modifications of these protocols have failed to satisfactorily preserve spermatozoa collected from farmed blue foxes (Alopex lagopus). Because cryogenic success has been linked to membrane composition, the plasma membrane lipid composition of farmed blue fox and silver fox spermatozoa was studied. Silver fox spermatozoal membranes have significantly higher levels of docosapentaenoic acid (DPA; 22:5, n-6) compared to blue fox spermatozoa, and blue fox spermatozoal membranes have significantly higher levels of stearic acid (18:0). Silver fox spermatozoal membranes not only have a higher ratio of unsaturated/saturated membrane fatty acids, but also higher levels of membrane desmosterol and cholesterol.     

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.     

Membrane lipid metabolism in Acholeplasma laidlawii A EF 22. Influence of cholesterol and temperature shift-down on incorporation of fatty acids and synthesis of membrane lipid species

1. Membrane lipid metabolism in Acholeplasma laidlowii A EF 22 has been studied under different conditions by applying three different techniques for changing membrane viscosity: fatty acid and cholesterol supplementation and temperature changes. 2. The molar relationship between the two dominating membrane lipids, monoglucosyldiglyceride and diglucosyldiglyceride, is to a large extent determined by membrane viscosity properties. This is shown by the varying metabolic responses occurring during incorporation of different fatty acids with and without cholesterol and by temperature shift-down experiments. Higher viscosity in membranes stimulates synthesis of monoglucosyldiglyceride at the expense of diglucosyldiglyceride. Synthesis of phospho and phosphoglucolipids is affected as well. 3. Temperature shift-down from 37 degrees C to 17 degrees C results in an immediate synthesis of monoglucosyldiglyceride accompanied by an increased incorporation of unsaturated fatty acids into this lipid. Synthesis of the other membrane lipid species (containing more unsaturated fatty acids) lags behind temporarily. 4. Incorporation from an equimolar mixture of palmitic and oleic acids together with cholesterol yields greater amounts of oleic acid in membrane lipids than incorporation in the absence of cholesterol, indicating that incorporation is viscosity dependent. 5. Studies of precursor relationships reveal that all main lipids have an active turnover which differs depending on membrane composition and conditions. Furthermore, this turnover proceeds with different intra-lipid pools. 6. Isolated membranes contain no detectable lipolytic enzymes capable of hydrolyzing membrane phospho or glycolipids. It is suggested that lipid turnover is partly mediated by enzymatic interlipid conversions, thus not allowing intermediates to accumulate.     

Fast Diffusion of Very Long Chain Saturated Fatty Acids across a Bilayer Membrane and Their Rapid Extraction by Cyclodextrins: IMPLICATIONS FOR ADRENOLEUKODYSTROPHY*

Abnormalities in the transport of saturated very long chain fatty acids (VLCFA; >C18:0) contribute to their toxic levels in peroxisomal disorders of fatty acid metabolism, such as adrenoleukodystrophy and adrenomyeloneuropathy. We previously showed that VLCFA desorb much slower than normal dietary fatty acids from both albumin and protein-free lipid bilayers. The important step of transbilayer movement (flip-flop) was not measured directly as a consequence of this very slow desorption from donors, and the extremely low aqueous solubility of VLCFA precludes addition of unbound VLCFA to lipid membranes. We have overcome these limitations using methyl-β-cyclodextrin to solubilize VLCFA for rapid delivery to “acceptor” phosphatidylcholine vesicles (small and large unilamellar) and to cells. VLCFA binding was monitored in real time with the fluorescent probe fluorescein-labeled phosphatidylethanolamine in the outer membrane leaflet, and entrapped pyranine was used to detect flip-flop across the membrane. The upper limit of the rate of flip-flop across the membrane was independent of temperature and media viscosity and was similar for model raft and non-raft membranes as well as living cells. We further showed that cyclodextrins can extract VLCFA rapidly (within seconds) from vesicles and cells, which have implications for the mechanism and potential alternative approaches to treat adrenoleukodystrophy. Because VLCFA diffuse through the lipid bilayer, proteins may not be required for their transport across the peroxisomal membrane.     

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Biosynthetic labelling of membrane lipids of eukaryotic cells in tissue culture by a novel type of fluorescent fatty acids.

W-Anthryl labelled fatty acids with hydrocarbon chains of different lengths (C8, C11, C15) and different degrees of unsaturation have been incorporated into the membrane lipids of three different cell lines in tissue culture by addition of these 3H-labelled precursor fatty acids to the growth medium. The cell lines were baby hamster kidney cells (BHK 21), Chang liver cells and the RN6 cell line derived from a chemically induced Schwannoma tumor cell clone. Cell growth was normal. The quantitative analysis on the basis of radioactivity determinations demonstrated that the fluorescent-labelled fatty acids were introduced into the neutral lipid fraction (triglycerides, diglycerides, and cholesterol esters, all present in small amounts), but mainly into the phospholipid classes phosphatidylcholine, -ethanolamine and -serine, and to a lesser extent, as N-acyl component of sphingolipids (sphingomyelins, ceramides, mono- and diglycosylceramides). Cell fractionation studies indicated that the membranes of all subcellular particles were labelled with the fluorescent probes in their lipid moieties. These w-anthryl fatty acids are the first type of fluorescent lipid precursors which can be incorporated biosynthetically in vivo into membrane lipids of eukaryotic cells. The effective incorporation of the bulky fluorescent anthryl group in the terminal position of fatty acids of different chain lengths into the complex membrane lipids of the cell gives proff of 1) their uninhibited membrane transport, 2) their activation by the acyl-CoA synthetase and 3) their substrate properties for the O- acyl and N-acyl transferases in phospho- and sphingolipid biosynthesis.     

Fatty acids are rapidly delivered to and extracted from membranes by methyl-��-cyclodextrin

We performed detailed biophysical studies of transfer of long-chain fatty acids (FAs) from methyl-β-CD (MBCD) to model membranes (egg-PC vesicles) and cells and the extraction of FA from membranes by MBCD. We used i) fluorescein phosphatidylethanolamine to detect transfer of FA anions arriving in the outer membrane leaflet; ii) entrapped pH dyes to measure pH changes after FA diffusion (flip-flop) across the lipid bilayer; and iii) soluble fluorescent-labeled FA binding protein to measure the concentration of unbound FA in water. FA dissociated from MBCD, bound to the membrane, and underwent flip-flop within milliseconds. In the presence of vesicles, MBCD maintained the aqueous concentration of unbound FA at low levels comparable to those measured with albumin. In studies with cells, addition of oleic acid (OA) complexed with MBCD yielded rapid (seconds) dose-dependent OA transport into 3T3-L1 preadipocytes and HepG2 cells. MBCD extracted OA from cells and model membranes rapidly at concentrations exceeding those required for OA delivery but much lower than concentrations commonly used for extracting cholesterol. Compared with albumin, MBCD can transfer its entire FA load and is less likely to extract cell nutrients and to introduce impurities.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

Lipid traffic: the ABC of transbilayer movement

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.     

Cytosolic fatty acid binding proteins catalyze two distinct steps in intracellular transport of their ligands

Cytosolic long-chain fatty acid binding proteins (FABPs) are found in tissues that metabolize fatty acids. Like most lipid binding proteins, their specific functions remain unclear. Two classes have been described. Membrane-active FABPs interact directly with membranes during exchange of fatty acids between the protein binding site and the membrane, while membrane-inactive FABPs bind only to fatty acids that are already in aqueous solution. Despite these binding proteins, most fatty acids in cell cytoplasm appear to be bound to membranes. This paper reviews data suggesting that FABPs catalyze transfer of fatty acids between intracellular membranes, often across considerable intracellular distances. This process occurs in three distinct steps: dissociation of the fatty acid from a donor membrane, diffusion of the fatty acid across the intervening water layer, and binding to an acceptor membrane. Membrane-active FABPs catalyze dissociation of the fatty acid from the donor membrane and binding to the acceptor membrane, while membrane-inactive FABPs catalyze diffusion of fatty acids across the aqueous cytosol. Thus, FABPs catalyze all three steps in intracellular transport. A simple quantitative model has been developed that predicts the rate of intracellular transport as a function of the concentration, affinity and diffusional mobility of the binding protein. Different FABPs may have evolved to match the specific transport requirements of the cell type within which they are found.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.     

Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics

Although the most exogenous lipids enter the cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics, and response to lipid transfer proteins. SCP-2 expression decreased the mass of cholesterol and lyso-bis-phosphatidic acid [LBPA], as well as the ratios of cholesterol/phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5- and 3.8-fold, respectively, from lysosomal membranes isolated from SCP-2 expressing cells as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t(1/2)>4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells. These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore, the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.