Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of substrate composition on the esterification and hydrolysis activity of lysosomal acid sterol ester hydrolase

Lipid microemulsions with various core and surface lipid compositions were prepared by co-sonication of cholesteryl esters, triolein (TO), egg phosphatidylcholine (egg PC), and cholesterol. The heterogeneous emulsion particle mixture was purified by gel filtration and particles with the size and general organization of low density lipoproteins were obtained. These lipid microemulsion particles were used for studies of the cellular metabolism of lipoprotein-derived cholesterol and cholesteryl esters as catalyzed by the enzyme acid sterol ester hydrolase (EC 3.1.1.13). The hydrolysis of cholesteryl oleate (CO) was more than twice and that of cholesteryl linoleate (CL) more than three times faster than the hydrolysis of cholesteryl stearate (CS) over the temperature range 25-39.6 degrees C. Both the synthesis and hydrolysis of cholesteryl esters were insensitive to the physical state of the microemulsion cores. The synthesis of cholesteryl esters by this enzyme was also insensitive to the ratios of cholesterol and egg PC in the microemulsion surface layers. Incorporation of triolein into the microemulsion cholesteryl ester core slightly increased the rate of cholesteryl ester synthesis. A decreasing fatty acyl chain length (C18:0 to C14:0) and an increasing degree of unsaturation (C18:0 to C18:2) enhanced the synthesis rate. It is suggested that the hydrolysis and synthesis of cholesteryl esters in microemulsions (and lipoproteins) take place only in the particle surface layer and that the rate of catalysis is directly dependent on the amount of substrate in this surface layer.     

Effects of sphingomyelin on apolipoprotein E- and lipoprotein lipase-mediated cell uptake of lipid particles

It has been reported that human plasma sphingomyelin (SM) levels are positively and independently related to coronary artery disease. The lipoprotein surface is mainly formed by phosphatidylcholine (PC) and SM together with cholesterol and apolipoproteins. However, the influence of SM on the cell uptake of triglyceride-rich lipoproteins and remnants is poorly understood. To clarify the role of SM in lipoprotein uptake, we prepared lipid emulsions containing triolein, PC and SM as model particles of lipoproteins. Apolipoprotein E (ApoE) binding studies revealed that incorporation of SM into the emulsion surface reduced the binding capacity of apoE without changing the affinity. Surface SM reduced apoE-mediated uptake of emulsions by HepG2 cells because of the decreased amount of binding apoE. Apolipoproteins C-II and C-III inhibited the apoE-mediated uptake of SM containing emulsions more effectively. The stimulatory effect of lipoprotein lipase (LPL) on emulsion uptake was decreased by replacing surface PC with SM. These results suggest that SM-induced changes in the binding properties of apolipoproteins and LPL correlate with decreased hepatic uptake of lipid particles.     

Role of apolipoproteins in cholesterol efflux from macrophages to lipid microemulsion: proposal of a putative model for the pre-beta high-density lipoprotein pathway.

Lipid microemulsion of phospholipid and triglyceride with the size of low-density lipoprotein was capable of removing cholesterol from cholesterol-loaded mouse peritoneal macrophages, resulting in reduction of intracellularly accumulated cholesteryl ester. Apolipoproteins (apo) A-I, A-II, C-III, and E bound to the surface of the microemulsion did not modulate the interaction of the microemulsion with the cells in terms of the cholesterol efflux. The cholesterol removal by the microemulsion was enhanced by some 30% only when apoA-I, -A-II, and -E were present in excess to provide their free forms in the medium, but apoC-III did not show such an effect even by its excess amount. The kinetics including the results with apoC-III were consistent with a model that the apparent enhancement was due to generation of pre-beta high-density lipoprotein (HDL)-like particles upon the interaction of free apolipoproteins with macrophages [Hara, H., & Yokoyama, S. (1991) J. Biol. Chem. 266, 3080-3086]. However, pre beta-HDL-like particle was not detected after 6- and 24-h incubation in the medium where cholesterol efflux to the emulsion was maximally enhanced by the apolipoproteins, and cholesterol and phospholipids removed from the cells were all found with the microemulsions. It was also shown separately that the lipids in pre beta-HDL-like particles generated by apoA-I and macrophages were rapidly, within the order of minutes, transferred to the apo-lipoprotein-covered microemulsions when they were incubated together. Thus, the data were consistent with a model that the free form of certain apolipoproteins, such as apoA-I, -A-II, and -E but not apoC-III, generates pre beta-HDL-like particles with cellular lipids in situ and these particles act as mediators for cholesterol transfer from the cells to other lipoproteins.     

13C MAS NMR studies of crystalline cholesterol and lipid mixtures modeling atherosclerotic plaques.

Cholesterol and cholesteryl esters are the predominant lipids of atherosclerotic plaques. To provide fundamental data for the quantitative study of plaque lipids in situ, crystalline cholesterol (CHOL) and CHOL/cholesteryl ester (CE) mixtures with other lipids were studied by solid-state nuclear magnetic resonance with magic-angle-sample spinning. Highly distinctive spectra for three different crystalline structures of CHOL were obtained. When CHOL crystals were mixed with isotropic CE oil, solubilized CHOL (approximately 13 mol % CHOL) was detected by characteristic resonances such as C5, C6, and C3; the excess crystalline CHOL (either anhydrous or monohydrate) remained in its original crystalline structure, without being affected by the coexisting CE. By use of 13C-enriched CHOL, the solubility of CHOL in the CE liquid-crystalline phase (approximately 8 mol %) was measured. When phosphatidylcholine was hydrated in presence of CHOL and CE, magic-angle-sampling nuclear magnetic resonance revealed liquid-crystalline CHOL/phosphatidylcholine multilayers with approximately an equal molar ratio of CHOL/phosphatidylcholine. Excess CHOL existed in the monohydrate crystalline form, and CE in separate oil or crystalline phases, depending on the temperature. The magic-angle-sampling nuclear magnetic resonance protocol for identifying different lipid phases was applied to intact (ex vivo) atherosclerotic plaques of cholesterol-fed rabbits. Liquid, liquid-crystalline, and solid phases of CE were characterized.     

Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.     

Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.     

Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.     

Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans. Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency

The extent to which lipid and apolipoprotein (apo) concentrations in tissue fluids are determined by those in plasma in normal humans is not known, as all studies to date have been performed on small numbers of subjects, often with dyslipidemia or lymphedema. Therefore, we quantified lipids, apolipoproteins, high density lipoprotein (HDL) lipids, and non-HDL lipids in prenodal leg lymph from 37 fasted ambulant healthy men. Lymph contained almost no triglycerides, but had higher concentrations of free glycerol than plasma. Unesterified cholesterol (UC), cholesteryl ester (CE), phosphatidylcholine (PC), and sphingomyelin (SPM) concentrations in whole lymph were not significantly correlated with those in plasma. HDL lipids, but not non-HDL lipids, were directly related to those in plasma. Lymph HDLs were enriched in UC. However, as the HDL cholesterol/non-HDL cholesterol ratio in lymph exceeded that in plasma, whole lymph nevertheless had a lower UC/CE ratio than plasma. Lymph also had a significantly higher SPM/PC ratio. The lymph/plasma (L/P) ratios of apolipoproteins were as follows: A-IV > A-I and A-II > C-III and E > B. Comparison with the L/P ratios of seven nonlipoprotein proteins suggested that apoA-IV was predominantly lipid free. Concentrations of apolipoproteins A-II, A-IV, C-III, and E in lymph, but not of apolipoproteins A-I or B, were positively correlated with those in plasma. The L/P ratios of apolipoproteins B, C-III, and E in two subjects with lipoprotein lipase (LPL) deficiency, and of apolipoproteins A-I and A-IV in a subject with lecithin:cholesterol acyltransferase (LCAT) deficiency, were low relative to those in normal subjects. Thus, the concentrations of lipids, apolipoproteins, and lipoproteins in human tissue fluid are determined only in part by their concentrations in plasma. Other factors, including the actions of LPL and LCAT, are at least as important.     

Enhancement of the human plasma lipid transfer protein reaction by apolipoproteins

Transfer of cholesteryl ester between triacylglycerol/phospholipid microemulsions catalyzed by human plasma lipid transfer protein was investigated with a pyrene-containing analogue of which fluorescent properties depend on its concentration in the core of the microemulsions. The transfer of pyrene-cholesteryl ester between the emulsions was increased by the transfer protein linearly with its concentration, but maximally only to the extent of twice as much as spontaneous transfer in the given experimental conditions. When human apolipoproteins A-I or A-II are present in the reaction mixture enough to saturate the surface of the emulsion, the enhancement of the pyrene-cholesteryl ester transfer reaction by the transfer protein was 7.5-times more than in the absence of the apolipoproteins while the rate of spontaneous transfer was not affected significantly by the apolipoproteins. Bovine serum albumin did not have such an effect. Furthermore, the enhancement of the lipid transfer protein reaction by apolipoprotein A-I was linearly proportional to the percent saturation of the surface of the microemulsion with the apolipoprotein.     

Properties of the products formed by the activity of serum opacity factor against human plasma high-density lipoproteins

Serum opacity factor from Streptococcus pyogenes transfers the cholesteryl esters (CE) of approximately 100,000 plasma high-density lipoprotein particles (HDL) to a CE-rich microemulsion (CERM) while forming neo HDL, a cholesterol-poor HDL-like particle. HDL, neo HDL, and CERM are distinct. Neo HDL is lower in free cholesterol and has lower surface and total microviscosities than HDL; the surface polarity of neo HDL and HDL are similar. CERM is much larger than HDL and richer in cholesterol and CE. Although the surface microviscosity of HDL is higher than that of CERM, they have similar total microviscosities because cholesterol partitions into the neutral lipid core. Because of its unique surface properties apo E preferentially associates with the CERM. In contrast, the composition and properties of neo HDL make it a potential acceptor of cellular cholesterol and its esterification. Thus, neo HDL and CERM are possible vehicles for improving cholesterol transport to the liver.     

Simultaneous determination of hydroperoxides of phosphatidylcholine, cholesterol esters and triacylglycerols by column-switching high-performance liquid chromatography with a post-column detection system

A method for the simultaneous determination of hydroperoxides of phosphatidylcholines (PC), triacylglycerols (TG) and cholesterol esters (CE) has been developed. A sample was separated into a combined TG and CE hydroperoxides fraction and a PC hydroperoxides fraction on a short silica column. The fractions were introduced into an ODS column and another silica column by a valve-switching device. The PC hydroperoxides were monitored by a post-column detection system with diphenyl-1-pyrenylphosphine, and the TG and CE hydroperoxides were monitored by another switching device. With this system, the hydroperoxides were determined at the picomole level within 32 min. Their detection limits were 2–4 pmol at a signal-to-noise ratio of 3, and the relative standard deviations of the peak areas were 1.6–3.1%. This method was successfully applied to determine lipid hydroperoxides in human plasma.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids

Bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (CHOL) are commonly used as systems to model the raft-lipid domain structure believed to compartmentalize particular cell membrane proteins. In this work, micropipette aspiration of giant unilamellar vesicles was used to test the elasticities, water permeabilities, and rupture tensions of single-component PC, binary 1:1 PC/CHOL, and 1:1 SM/CHOL, and ternary 1:1:1 PC/SM/CHOL bilayers, one set of measurements with dioleoyl PC (DOPC; C18:1/C18:1 PC) and the other with stearoyloleoyl PC (SOPC; C18:0/C18:1 PC). Defining the elastic moduli (K_A), the initial slopes of the increase in tension (σ) versus stretch in lipid surface area (α_e) were determined for all systems at low (15°C) and high (32-33°C) temperatures. The moduli for the single-component PC and binary phospholipid/CHOL bilayers followed a descending hierarchy of stretch resistance with SM/CHOL > SOPC/CHOL > DOPC/CHOL > PC. Although much more resistant to stretch than the single-component PC bilayers, the elastic response of vesicle bilayers made from the ternary phospholipid/CHOL mixtures showed an abrupt softening (discontinuity in slope), when immediately subjected to a steady ramp of tension at the low temperature (15°C). However, the discontinuities in elastic stretch resistance at low temperature vanished when the bilayers were held at ∼1 mN/m prestress for long times before a tension ramp and when tested at the higher temperature 32-33°C. The elastic moduli of single-component PC and DOPC/CHOL bilayers changed very little with temperature, whereas the moduli of the binary SOPC/CHOL and SM/CHOL bilayers diminished markedly with increase in temperature, as did the ternary SOPC/SM/CHOL system. For all systems, increasing temperature increased the water permeability but decreased rupture tension. Concomitantly, the measurements of permeability exhibited a prominent correlation with the rupture tension across all the systems. Together, these micromechanical tests of binary and ternary phospholipid/CHOL bilayers demonstrate that PC hydrocarbon chain unsaturation and temperature are major determinants of the mechanical and permeation properties of membranes composed of raft microdomain-forming lipids.     

Changes in mammary uptake of free fatty acids,triglyceride, cholesterol and phospholipid in relation to milk synthesis during lactation in goats

Uptake of free fatty acids (FFA), triglycerides (TG), cholesterol (CHOL) and phospholipids (PL) was measured in both mammary glands of dairy goats during lactation. Arterial concentrations of TG, CHOL and PL as well as arterio-venous difference (AV) and extraction rate (E) for TG were highest in goats with the highest dietary feed intake. AV were linearly related to arterial concentrations for the four lipid classes, and arterial concentrations of CHOL were linearly related to output of lactose, protein and fat in milk. Arterial supply, and not mammary synthetic activity, is the main determinant of mammary FFA, TG and CHOL uptake.     

Apolipoprotein A-I is necessary for the in vivo formation of high density lipoprotein competent for scavenger receptor BI-mediated cholesteryl ester-selective uptake

The severe depletion of cholesteryl ester (CE) in steroidogenic cells of apoA-I(-/-) mice suggests that apolipoprotein (apo) A-I plays a specific role in the high density lipoprotein (HDL) CE-selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. The nature of this role, however, is unclear because a variety of apolipoproteins bind to SR-BI expressed in transfected cells. In this study the role of apoA-I in SR-BI-mediated HDL CE-selective uptake was tested via analyses of the biochemical properties of apoA-I(-/-) HDL and its interaction with SR-BI on adrenocortical cells, hepatoma cells, and cells expressing a transfected SR-BI. apoA-I(-/-) HDL are large heterogeneous particles with a core consisting predominantly of CE and a surface enriched in phospholipid, free cholesterol, apoA-II, and apoE. Functional analysis showed apoA-I(-/-) HDL to bind to SR-BI with the same or higher affinity as compared with apoA-I(+/+) HDL, but apoA-I(-/-) HDL showed a 2-3-fold decrease in the V(max) for CE transfer from the HDL particle to adrenal cells. These results indicate that the absence of apoA-I results in HDL particles with a reduced capacity for SR-BI-mediated CE-selective uptake. The reduced V(max) illustrates that HDL properties necessary for binding to SR-BI are distinct from those properties necessary for the transfer of HDL CE from the core of the HDL particle to the plasma membrane. The reduced V(max) for HDL CE-selective uptake likely contributes to the severe reduction in CE accumulation in steroidogenic cells of apoA-I(-/-) mice.     

Domain formation in model membranes studied by pulsed-field gradient-NMR: the role of lipid polyunsaturation

The effects of increased unsaturation in the sn-2 fatty acyl chain of phosphatidylcholines (PCs) on the lipid lateral diffusion have been investigated by pulsed-field gradient NMR. Macroscopically oriented bilayers containing a monosaturated PC, egg sphingomyelin, and cholesterol (CHOL) have been studied at temperatures between 0 degrees C and 60 degrees C, and the number of double bonds in the PC was one, two, four, or six. For PC bilayers, with and without the incorporation of egg sphingomyelin and CHOL, the lateral diffusion increased with increasing number of double bonds, as a consequence of the increased headgroup area caused by the unsaturation. Addition of CHOL caused a decrease in lipid diffusion due to the condensing effect of CHOL on the headgroup area. Phase separation into large domains of liquid-disordered and liquid-ordered phases were observed in the ternary systems with PCs containing four and six double bonds, as evidenced by the occurrence of two lipid diffusion coefficients. PC bilayers with one or two double bonds appear homogeneous on the length scales probed by the experiment, but the temperature dependence of the diffusion suggests that small domains may be present also in these ternary systems.     

Pathways in the formation of human plasma high density lipoprotein subpopulations containing apolipoprotein A-I without apolipoprotein A-II

The lecithin:cholesterol acyltransferase (LCAT)-induced transformation of two discrete species of model complexes that differ in number of apolipoprotein A-I (apoA-I) molecules per particle was investigated. One complex species (designated 3A-I(UC)-complexes) contained 3 apoA-I per particle, was discoidal (13.5 X 4.4 nm), and had a molar composition of 22:78:1 (unesterified cholesterol (UC):egg yolk phosphatidylcholine (egg yolk PC):apoA-I). The other complex species (designated 2A-I(UC)complexes) containing 2 apoA-I per particle was also discoidal (8.4 X 4.1 nm) and had a molar composition of 6:40:1. Transformation of 3A-I(UC)complexes by partially purified LCAT yielded a product (24 hr, 37 degrees C) with a cholesteryl ester (CE) core, 3 apoA-I, and a mean diameter of 9.2 nm. The 2A-I(UC)complexes were only partially transformed to a core-containing product (24 hr, 37 degrees C) which also had 3 apoA-I; this product, however, was smaller (diameter of 8.5 nm) than the product from 3A-I(UC)complexes. Transformation of 3A-I(UC)complexes appeared to result from build-up of core CE directly within the precursor complex. Transformation of 2A-I(UC)complexes, however, followed a stepwise pathway to the product with 3 apoA-I, apparently involving fusion of transforming precursors and release of one apoA-I from the fusion product. In the presence of low density lipoprotein (LDL), used as a source of additional cholesterol, conversion of 2A-I(UC)complexes to the product with 3 apoA-I was more extensive. The transformation product of 3A-I(UC)complexes in the presence of LDL also had 3 apoA-I but was considerably smaller in size (8.6 vs. 9.2 nm, diameter) and had a twofold lower molar content of PC compared with the product formed without LDL. LDL appeared to act both as a donor of UC and an acceptor of PC. Transformation products with 3 apoA-I obtained under the various experimental conditions in the present studies appear to be constrained in core CE content (between 13 to 22 CE per apoA-I; range of 9 CE molecules) but relatively flexible in content of surface PC molecules they can accommodate (between 24 to 49 PC per apoA-I; range of 25 PC molecules). The properties of the core-containing products with 3 apoA-I compare closely with those of the major subpopulation of human plasma HDL in the size range of 8.2-8.8 nm that contains the molecular weight equivalent of 3 apoA-I molecules.     

A Langmuir film approach to elucidating interactions in lipid membranes: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/cholesterol/metal cation systems

The interactions between two membrane lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol (CHOL), were studied in Langmuir films using surface pressure isotherms and Brewster angle microscopy. The DPPE/CHOL interactions were probed for chosen monolayer and subphase (Na(+), Ca(2+)) composition at 20, 25, and 30 degrees C. The results obtained show that DPPE and CHOL are miscible for the cholesterol mol fractions x(CHOL)=0.3-0.5. Cholesterol induces condensation of the DPPE monolayers. The most significant condensation of the DPPE/CHOL monolayers was observed in the presence of Ca(2+) ions in the subphase at x(CHOL)=0.4. The negative deviation of the molecular surface area (MMA) additivity from the ideal behavior together with negative values of excess free enthalpy of mixing in the monolayers were interpreted in terms of attractive interactions between lipid molecules.     

Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes

The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P相似文献