Changes in the morphology of cell-size liposomes in the presence of cholesterol: Formation of neuron-like tubes and liposome networks

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.     

Changes in the morphology of cell-size liposomes in the presence of cholesterol: formation of neuron-like tubes and liposome networks

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.     

Melittin-lipid bilayer interactions and the role of cholesterol

The membrane-destabilizing effect of the peptide melittin on phosphatidylcholine membranes is modulated by the presence of cholesterol. This investigation shows that inclusion of 40 mol % cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes reduces melittin's affinity for the membrane. It is significant that the presence of cholesterol does not increase the amount of membrane-associated melittin needed to cause maximum leakage from, or major structural rearrangements of, the liposomes. Furthermore, comparison of microscopy and leakage data suggests that melittin-induced leakage occurs via different mechanisms in the cholesterol-free and cholesterol-supplemented systems. In the absence of cholesterol, leakage of carboxyfluorescein takes place from intact liposomes in a manner compatible with the presence of small melittin-induced pores. In the presence of cholesterol, on the other hand, adsorption of the peptide causes complete membrane disruption and the formation of long-lived open-bilayer structures. Moreover, in the case of cholesterol-supplemented systems, melittin induces pronounced liposome aggregation. Cryotransmission electron microscopy was used, together with ellipsometry, circular dichroism, turbidity, and leakage measurements, to investigate the effects of melittin on phosphatidylcholine membranes in the absence and presence of cholesterol. The melittin partitioning behavior in the membrane systems was estimated by means of steady-state fluorescence spectroscopy measurements.     

The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA: Cholesterol acyltransferase substrate pool in liver microsomal fraction

The incubation at 37°C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA: cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these values was also the activation energy for the increase in acyl-CoA: cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.     

Bile salt micelle can sustain more cholesterol in the intermicellar aqueous phase than the maximal aqueous solubility

The intermicellar aqueous phase in equilibrium with micelle plays an important role in the uptake of sterol. To test the hypothesis whether cholesterol concentration in the intermicellar aqueous phase of a micellar solution is similar to its maximal aqueous solubility, cholesterol concentration in the intermicellar aqueous phase of a bile salt-cholesterol solution and maximal aqueous cholesterol solubility were quantitatively determined by capillary gas-liquid chromatography after filtration. Cholesterol concentration in the intermicellar aqueous phase increased linearly with cholesterol concentration in the micellar solution and reached 1.3 microM at its micellar solubility limit, while the maximal aqueous solubility of cholesterol was (1.2-1.4) x 10(-8) M. The intermicellar monomer concentration of taurocholate was 5.8 mM in which 26 x 10(-8) M cholesterol was solubilized. The results indicate the presence of a cholesterol concentration in the intermicellar aqueous phase that is significantly higher than its maximal aqueous solubility, which can be ascribed primarily to the presence of an intermicellar concentration of bile salt.     

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.     

Phase separation of cholesterol and the interaction of ethanol with phosphatidylserine-cholesterol bilayer membranes

Thermotropic and structural effects of ethanol on phosphatidylserine (PS) membranes containing up to 0.4 mol fraction cholesterol were investigated by differential scanning calorimetry, X-ray diffraction and fluorescence spectroscopy. It was found that in the presence of cholesterol, 10% (v/v) added ethanol depresses the melting temperature of the phospholipid by approximately 2 degrees C, similar to what was observed in the absence of cholesterol. Below the melting temperature the progressive disordering effect of added cholesterol is weakly enhanced by the presence of ethanol. In the liquid crystalline state, the marked decrease in the thickness of the bilayer which ethanol causes in the absence of cholesterol (Chem. Phys. Lipids 92 (1998) 127), is also observed in its presence. We conclude that, in contrast to what has been observed for zwitterionic phospholipids, high concentrations of cholesterol do not diminish the interaction of ethanol with PS membranes. With addition of 10% (v/v) ethanol, crystalline cholesterol diffraction, an indication of phase separation of the sterol, appears at mol fraction cholesterol 0.34, as compared to 0.3 in the absence of ethanol (Chem. Phys. Lipids 92 (1998) 71).     

Pulmonary Surfactant Protein SP-C Counteracts the Deleterious Effects of Cholesterol on the Activity of Surfactant Films under Physiologically Relevant Compression-Expansion Dynamics

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes. However, an excess of cholesterol has been associated with impaired surface activity of surfactant. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In this study, we analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol, and the ability of lipid/protein complexes containing cholesterol to form and respread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. SP-C modulates the effect of cholesterol to reduce the enthalpy associated with the gel-to-liquid-crystalline melting transition in dipalmitoylphosphatidylcholine (DPPC) bilayers, as analyzed by differential scanning calorimetry. The presence of SP-C affects more subtly the effects of cholesterol on the thermotropic properties of ternary membranes, mimicking more closely the lipid composition of native surfactant, where SP-C facilitates the miscibility of the sterol. Incorporation of 1% or 2% SP-C (protein/phospholipid by weight) promotes almost instantaneous adsorption of suspensions of DPPC/palmitoyloleoylphospatidylcholine (POPC)/palmitoyloleoyl-phosphatidylglycerol (POPG) (50:25:15, w/w/w) into the air-liquid interface of a captive bubble, in both the absence and presence of cholesterol. However, cholesterol impairs the ability of SP-C-containing films to achieve very low surface tensions in bubbles subjected to compression-expansion cycling. Cholesterol also substantially impairs the ability of DPPC/POPC/POPG films containing 1% surfactant protein SP-B to mimic the interfacial behavior of native surfactant films, which are characterized by very low minimum surface tensions with only limited area change during compression and practically no compression-expansion hysteresis. However, the simultaneous presence of 2% SP-C practically restores the compression-expansion dynamics of cholesterol- and SP-B-containing films to the efficient behavior shown in the absence of cholesterol. This suggests that cooperation between the two proteins is required for lipid-protein films containing cholesterol to achieve optimal performance under physiologically relevant compression-expansion dynamics.     

Effect of albumin on the solubility of cholesterol in bile

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.     

Understanding the mechanism of LCAT reaction may help to explain the high predictive value of LDL/HDL cholesterol ratio.

Traditionally, lecithin:cholesterol acyltransferase (LCAT) role in the reverse cholesterol transport (RCT) has been considered "antiatherogenic" as the cholesterol esterification is the prerequisite for the formation of mature high density lipoprotein (HDL) particles and may create a gradient necessary for the flow of unesterified cholesterol (UC) from tissues to plasma. However, newer data suggest that a higher esterification rate is not necessarily protective. Here we review the available data on the role of LCAT in RCT and propose that the LCAT-mediated esterification of plasma cholesterol promotes RCT only in the presence of sufficient concentrations of HDL2 while this reaction may be atherogenic in the presence of high concentration of plasma low density lipoprotein (LDL) cholesterol Thus, the "protective" or potentially "atherogenic" role of LCAT depends on the quality of HDL and concentration of LDL. This hypothesis is consistent with the known high predictive value of LDL/HDL cholesterol ratio.     

On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone.

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.     

Studies on the interaction of cholesterol with soluble and insoluble elastins

The exchange of ³H-cholesterol in an aqueous solution of taurodeoxycholate with insoluble elastin and kappa-elastin peptides has been quantitatively studied. At higher cholesterol concentrations, and in similar experimental conditions, 0.3 μg and 0.2 μg of cholesterol could be adsorbed, respectively, on to 1 mg insoluble elastin and 1 mg kappa-elastin coacervate. Therefore, the above amounts of cholesterol fixed per mg elastin represent minimal estimates. The replacement of Na⁺ ions by Ca²⁺ ions increased the cholesterol binding both of fibrous and of soluble elastins. The binding curves tend towards a limiting value in the presence of Na⁺, but no saturation effect was observed in the presence of Ca²⁺. The replacement of sodium ions by calcium ions in the taurodeoxycholate solutions also lowered the coacervation temperature of the kappa-elastin peptides. Fibrous elastin retained more cholesterol at 65°C than at 37°C, suggesting the importance of hydrophobic interactions in cholesterol elastin association. These results suggest that conformational changes could be induced in both soluble and insoluble forms of elastin by calcium ions increasing their affinity for cholesterol. This enhancement of cholesterol fixation by elastin in the presence of Ca²⁺ ions may well have a physiopathological importance. So does also the fact that elastin-peptides exhibit a higher affinity in the presence of Ca²⁺ for cholesterol than insoluble elastin, suggesting the possibility of increased cholsterol (and calcium) fixation as elastin is degraded in situ by elastases.     

Thermotropic properties of mixtures of negatively charged phospholipids with cholesterol in the presence and absence of Li+ or Ca2+ ions

Mixtures of cholesterol with dipalmitoylphosphatidylserine or phosphatidic acid were investigated by differential scanning calorimetry. As in mixtures of natural phosphatidylserine with cholesterol (Bach, D. (1984) Chem. Phys. Lipids 35, 385-392), also here phase separation of cholesterol at molar ratios of 2:1 (phospholipid:cholesterol) and below is observed. The limited solubility of cholesterol in negatively charged phospholipids is found to be independent of the nature of the acyl chain residues, and independent of whether the negative charge resides on both COO- and PO- groups (as in phosphatidylserine) or on PO- only (as in phosphatidic acid). The separate cholesterol phase is also seen by DSC in mixtures of natural phosphatidylserine or phosphatidic acid with cholesterol in the presence of Ca2+; and in phosphatidylserine/cholesterol mixtures in the presence of Li+, by DSC and X-ray diffraction.     

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii: A 2H-NMR study

We have investigated by ²H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of Acholeplasma laidlawii B. This is the only ²-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristics condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the ²H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.     

Fatty acids and cholesterol: effect on the interaction of theophylline with bovine serum albumin

The binding of theophylline (Th, 11-840 microM) to bovine serum albumin (BSA, 10 microM) using microdialysis technique in the presence of fatty acids (2.5-50.0 microM) and cholesterol (20-500 nM) indicates that fatty acids and cholesterol inhibit the binding of Th to BSA. The maximum inhibition (90.5%) occurs in presence of acetic acid (AA) followed by lauric acid (LA, 83.3%), palmitic acid (PA, 72.2%), oleic acid (OA, 44.4%) and cholesterol (22.2%). Fatty acids and cholesterol also decrease the number of binding sites and the affinity for the binding of Th to BSA. Such a decrease is maximum in the presence of AA followed by LA, PA, OA and cholesterol. Complete abolition of the low affinity binding site in the presence of AA indicates that the low affinity binding is predominantly ionic in nature while the high affinity binding involves ionic and other type(s) of unidentified force(s). This makes high affinity binding stronger than low affinity binding.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

In vitro regulation of bovine adrenal cortical acyl-CoA: cholesterol acyltransferase and comparison with the rat liver enzyme

Acyl-CoA:cholesterol acyltransferase activity in bovine adrenal cortical microsomes was increased by preincubation of microsomes in vitro in the presence of MgCl2. The acyltransferase activity in the microsomes could be inhibited by further incubation in the presence of ATP/MgCl2. These effects appear to complement the known ATP-dependent activation of adrenal cytosolic cholesterol ester hydrolase, which is consistent with the role of the hydrolase in supplying cholesterol for steroidogenesis. These effects are, however, opposite to those recently demonstrated for the rat liver and intestine. Acyl-CoA:cholesterol acyltransferase activity in rat liver can be increased by the addition of cholesterol, as substrate, or by 25-hydroxycholesterol. Such activation was not observed in adrenal microsomal preparations, further suggesting that the mechanisms of regulation of cholesterol esterification differs between these tissues.     

Defining raft domains in the plasma membrane

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol‐based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid‐ordered (Lo)‐phase domains in giant unilamellar vesicles, Lo‐phase‐like domains formed at lower temperatures in giant PM vesicles, and detergent‐resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid‐like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non‐raft domains, as defined here, in the PM.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996