Trans-intestinal cholesterol efflux is not mediated through high density lipoprotein

Transintestinal cholesterol efflux (TICE) provides an attractive target to increase body cholesterol excretion. At present, the cholesterol donor responsible for direct delivery of plasma cholesterol to the intestine is unknown. In this study, we investigated the role of HDL in TICE. ATP-binding cassette protein A1 deficient (Abca1(-/-)) mice that lack HDL and wild-type (WT) mice were intravenously injected with chylomicron-like emulsion particles that contained radiolabeled cholesterol that is liberated in the liver and partly reenters the circulation. Both groups secreted radiolabeled cholesterol from plasma into intestinal lumen and TICE was unaltered between the two mouse models. To further investigate the role of HDL, we injected HDL with radiolabeled cholesterol in WT mice and Abca1(-/-)×Sr-b1(-/-) mice that lack HDL and are also unable to clear HDL via the liver. The intestines of both mice were unable to take up and secrete radiolabeled cholesterol from HDL via TICE. Although a generally accepted major player in the hepatobiliary route-based cholesterol excretion, HDL plays no significant role in TICE in mice.     

Recycling of apolipoprotein E is not associated with cholesterol efflux in neuronal cells

After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a process which is associated with cholesterol efflux. Recycling and cholesterol efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in maintaining cholesterol homeostasis in the brain. The isoform apoE4 is associated with an increased risk of Alzheimer's disease and it has been postulated that high intracellular cholesterol levels promote the amyloidogenic processing of amyloid precursor protein. Therefore we investigated the cellular processing of different apoE isoforms as well as the associated cholesterol efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing lipoproteins resulted in the expected recycling while, as seen in non-neuronal cells, recycling of apoE4 was significantly reduced. However, despite these differences in apoE recycling, there was no difference in rates of cholesterol efflux. Therefore we conclude that in this neuronal cell model the reduced recycling of apoE4 does not affect cellular cholesterol metabolism.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Interaction of human serum high density lipoprotein apoprotein with phospholipids

The interaction of the apoprotein of human serum high density lipoprotein-3 (apo HDL₃)¹¹ with aqueous dispersion of natural and synthetic phospholipids (PL) was investigated at a temperature above the transitions of the PL hydrocarbon chains and also above their critical micellar concentration. This protein is known to contain two major polypeptides: apo A-I and apo A-II. The protein:PL mixtures (weight ratio, 2, 1 or 0.5) were subjected to sonic irradiation and then fractionated by either CsCl or D₂O-sucrose density gradient ultracentrifugation. Usually three bands were obtained, the relative mass distribution of which depended upon the nature of the PL and the ratio of the interacting components: one band contained the PL-poor protein (d 1.28 g/ml), another, the uncombined PL (d ? 1.08 g/ml), and the third band, both protein and PL. This band, which was considered to represent the actual complex, had a hydrated density intermediate between those of either apo HDL₃ or PL alone, a particle weight of 80,000 to 170,000 (i.e., less than that of intact HDL₃), a morphology by electron microscopy which depended on the materials and experimental conditions employed and circular dichroic spectra     

Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.     

Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux

In atherosclerotic lesions, macrophages store lipid in cytoplasmic inclusions and lysosomes. Regression studies show that lysosomal lipid is not as easily cleared as cytoplasmic inclusion lipid. Macrophages enriched with mildly oxidized low density lipoprotein (oxLDL) accumulate cholesteryl ester (CE) and free cholesterol (FC) in lysosomes. We examined whether lysosomal stores of cholesterol from oxLDL are cleared from THP-1 and mouse macrophages. As in previous studies, oxLDL-enriched THP-1 macrophages accumulated substantial lysosomal cholesterol. Surprisingly, less than 12% of oxLDL-derived lysosomal CE was cleared to efficient FC acceptors (e.g., cyclodextrins, apolipoprotein/phosphatidylcholine vesicles, and fetal bovine serum). Filipin staining showed that lysosomes of oxLDL-treated THP-1 cells contained FC, and despite removal of most of the cell FC (70--80%) by incubation with cyclodextrins, filipin staining of FC in lysosomes did not diminish. Also, when THP-1 macrophages were incubated with [(3)H]CE oxLDL, 73--76% of the [(3)H]CE was retained in a lysosomal hydrolysis resistant pool. In contrast, greater than 90% of acetylated low density lipoprotein (acLDL) [(3)H]CE was hydrolyzed. Furthermore, [(3)H]FC liberated from oxLDL [(3)H]CE was released at a slower rate to cyclodextrins than was [(3)H]FC from acLDL [(3)H]CE. In contrast, only 27% of oxLDL [(3)H]CE was resistant to hydrolysis in mouse macrophages, and the [(3)H]FC generated from oxLDL and acLDL [(3)H]CE was released to cyclodextrins at similar rates. We conclude that lack of hydrolysis and efflux of oxLDL cholesterol is not exclusively inherent in oxLDL, but also requires specific cell factors present in one cell type but not the other.--Yancey, P. G., and W. G. Jerome. Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux. J. Lipid Res. 2001. 42: 317--327.     

Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin: cholesterol acyltransferase

HDL plays an initial role in reverse cholesterol transport by mediating cholesterol removal from cells. During infection and inflammation, several changes in HDL composition occur that may affect the function of HDL; therefore, we determined the ability of acute-phase HDL to promote cholesterol removal from cells. Acute-phase HDL was isolated from plasma of Syrian hamsters injected with lipopolysaccharide. Cholesterol removal from J 774 murine macrophages by acute-phase HDL was less efficient than that by control HDL because of both a decrease in cholesterol efflux and an increase in cholesterol influx. LCAT activity of acute-phase HDL was significantly lower than that of control HDL. When LCAT activity of control HDL was inactivated, cholesterol efflux decreased and cholesterol influx increased to the level observed in acute-phase HDL. Inactivation of LCAT had little effect on acute-phase HDL. In GM 3468A human fibroblasts, the ability of acute-phase HDL to remove cholesterol from cells was also lower than that of normal HDL. The impaired cholesterol removal, however, was primarily a result of an increase in cholesterol influx without changes in cholesterol efflux. When control HDL in which LCAT had been inactivated was incubated with fibroblasts, cholesterol influx increased to a level comparable to that of acute-phase HDL, without any change in cholesterol efflux. These results suggest that the ability of acute-phase HDL to mediate cholesterol removal was impaired compared with that of control HDL and the lower LCAT activity in acute-phase HDL may be responsible for this impairment. The decreased ability of acute-phase HDL to remove cholesterol from cells may be one of the mechanisms that account for the well-known relationship between infection/inflammation and atherosclerosis.     

Lipid monolayers: interactions with the apoprotein of high density plasma lipoprotein

Monolayer techniques were used to study the interactions of various lipids (cholesterol, lysophosphatidyl choline, phosphatidal ethanolamine, phosphatidyl choline, sphingomyelin, stearic acid, and lipids extracted from plasma high density lipoproteins and very low density lipoprotein) with the lipid-free protein subunit of rat plasma high density lipoprotein and with rat plasma albumin. The proteins were injected under the lipid monolayer at fixed area, and the increase in surface pressure (decrease in surface tension) was measured as a function of time. With all lipids, both the rate and magnitude of this increase were greater with the apolipoprotein than with albumin. The degree of film penetration of pure lipid films (at an initial film pressure of 15 dynes/cm) by the two proteins followed the same order: cholesterol > phosphatidal ethanolamine > phosphatidyl choline > stearic acid > sphingomyelin > lysophosphatidyl choline. Other variables studied were protein concentration, initial film pressure, and pH. Two distinctive properties of the apolipoprotein were the penetration of lipid films at pressures above the collapse pressure of the protein, and the formation of a film even at low salt concentration. High surface activity and strong interaction of HDL-protein with lipid monolayers may be associated with the flexibility of the protein molecule due to absence of disulfide bridges. The unusual surface activity of HDL-protein may be intimately related to the mechanism of formation of the lipoprotein.     

SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.     

Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.

The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.     

Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.     

Total cholesterol,low density lipoprotein cholesterol,and high density lipoprotein cholesterol and coronary heart disease in Scotland.

OBJECTIVE--To investigate long term changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations and in measures of other risk factors for coronary heart disease and to assess their importance for the development of coronary heart disease in Scottish men. DESIGN--Longitudinal study entailing follow up in 1988-9 of men investigated during a study in 1976. SETTING--Edinburgh, Scotland. SUBJECTS--107 men from Edinburgh who had taken part in a comparative study of risk factors for heart disease with Swedish men in 1976 when aged 40. INTERVENTION--The men were invited to attend a follow up clinic in 1988-9 for measurement of cholesterol concentrations and other risk factor measurements. Eighty three attended and 24 refused to or could not attend. MAIN OUTCOME MEASURES--Changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations, body weight, weight to height index, prevalence of smoking, and alcohol intake; number of coronary artery disease events. RESULTS--Mean serum total cholesterol concentration increased over the 12 years mainly due to an increase in the low density lipoprotein cholesterol fraction (from 3.53 (SD 0.09) to 4.56 (0.11) mmol/l) despite a reduction in high density lipoprotein cholesterol concentration. Body weight and weight to height index increased. Fewer men smoked more than 15 cigarettes/day in 1988-9 than in 1976. Blood pressure remained stable and fasting triglyceride concentrations did not change. The frequency of corneal arcus doubled. Alcohol consumption decreased significantly. Eleven men developed clinical coronary heart disease. High low density lipoprotein and low high density lipoprotein cholesterol concentrations in 1976, but not total cholesterol concentration, significantly predicted coronary heart disease (p = 0.05). Almost all of the men who developed coronary heart disease were smokers (91% v 53%, p less than 0.05). CONCLUSION--Over 12 years the lipid profile deteriorated significantly in this healthy cohort of young men. Smoking, a low high density lipoprotein concentration and a raised low density lipoprotein concentration were all associated with coronary heart disease in middle aged Scottish men, whereas there was no association for total cholesterol concentration. The findings have implications for screening programmes.     

Interaction of high density lipoprotein particles with membranes containing cholesterol

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78A diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.     

Synthetic high density lipoprotein particles. Application to studies of the apoprotein specificity for selective uptake of cholesterol esters

Particles closely resembling rat high density lipoproteins (HDL) in terms of equilibrium density profile and particle size were prepared by sonication of apoA-I with a microemulsion made with egg lecithin and cholesterol oleate. These particles, like authentic HDL, allowed selective uptake of their cholesterol ester moieties by cultured cells without parallel uptake of the particle itself. That uptake was saturable and competed by HDL. In rats, the plasma decay kinetics and sites of uptake of a cholesteryl ether tracer were similar whether that tracer was incorporated into synthetic or authentic HDL. Synthetic particles containing other apoproteins were made by generally the same method, but using in place of apoA-I either a mixture of rat apoCs or apoE that was either competent or reductively methylated to prevent interaction with the B/E receptor. These particles, of lower density and larger Stokes radius than those made with apoA-I, also allowed selective uptake of cholesterol esters, albeit with a lower degree of selectivity than in the case of apoA-I. Thus a specific apoprotein component in the subject lipoprotein particle is not required for selective uptake. However, selective uptake was shown to be a function of particle density or size, and part of the difference in rates of selective uptake from the particles made with various apoproteins was explained by their differences in density or size.     

Identification and characterization of mixed disulphide complexes of E apoprotein in high density lipoprotein of subjects with acute alcoholic hepatitis.

Two E apoprotein complexes have been isolated from the plasma high density lipoprotein fraction of patients with alcoholic hepatitis. Both were mixed disulphide complexes and could be dissociated into subunits with β-mercaptoethanol but not with 1% sodium dodecyl sulphate or 8 M urea. One of the complexes (molecular weight 46,000) was identified as an E-AII unit and the other, of molecular weight 106,000 was consistent in properties with an E trimer. The latter has not been described before and the E-AII complex has been reported once previously in plasma of patients with Type III hyperlipo-proteinaemia. It is proposed that the presence of E complexes in abnormal disease states may affect the normal recognition of E monomer by cells thereby altering the subsequent fate of their host lipoproteins and their lipid constituents.