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Uptake of apolipoprotein E-rich and apolipoprotein E-poor subfractions of high-density lipoprotein by liver membranes and HepG2 cells.

ABSTRACT: Apolipoprotein (apo) E plays an important role in mediating high-density lipoprotein (HDL) cholesterol transport and uptake by the liver. Evidence for and against the existence of conventional liver receptors for HDL containing apoE have been reported, although the selective uptake of the cholesterol moiety of HDL has been demonstrated. The present study investigated the hepatic uptake of subfractions of HDL separated on the basis of their apoE content. Rabbit HDL and its apoE-rich and apoE-poor subfractions, separated by heparin-Sepharose affinity chromatography, were labelled in their apoprotein moieties with [14C]sucrose and in their cholesteryl ester moiety with 3H. No binding of either subfraction to rabbit liver membranes could be detected. With cultured HepG2 cells, however, there was a high uptake of 3H but a very low uptake of 14C from both HDL subfractions, demonstrating that selective uptake was operating. Addition of unlabelled apoE-poor HDL inhibited the uptake of both labels from the two subfractions to the same extent. These studies, which differed from previously reported investigations by employing native homologous HDL subfractions of known apolipoprotein composition, demonstrated that apoE is not directly involved in the selective uptake of HDL cholesterol by the liver. In the absence of specific binding sites on liver membranes, it is suggested that an alternative mechanism might exist for the clearance of HDL cholesterol from the plasma.

SUBMITTER: Fragoso YD

PROVIDER: S-EPMC1136043 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Obesity favors apolipoprotein E- and C-III-containing high density lipoprotein subfractions associated with risk of heart disease.

Project description:Human HDLs have highly heterogeneous composition. Plasma concentrations of HDL with apoC-III and of apoE in HDL predict higher incidence of coronary heart disease (CHD). The concentrations of HDL-apoA-I containing apoE, apoC-III, or both and their distribution across HDL sizes are unknown. We studied 20 normal weight and 20 obese subjects matched by age, gender, and race. Plasma HDL was separated by sequential immunoaffinity chromatography (anti-apoA-I, anti-apoC-III, anti-apoE), followed by nondenaturing-gel electrophoresis. Mean HDL-cholesterol concentrations in normal weight and obese subjects were 65 and 50 mg/dl (P = 0.009), and total apoA-I concentrations were 119 and 118 mg/dl, respectively. HDL without apoE or apoC-III was the most prevalent HDL type representing 89% of apoA-I concentration in normal weight and 77% in obese (P = 0.01) individuals; HDL with apoE-only was 5% versus 8% (P = 0.1); HDL with apoC-III-only was 4% versus 10% (P = 0.009); and HDL with apoE and apoC-III was 1.5% versus 4.6% (P = 0.004). Concentrations of apoE and apoC-III in HDL were 1.5-2× higher in obese subjects (P ≤ 0.004). HDL with apoE or apoC-III occurred in all sizes among groups. Obese subjects had higher prevalence of HDL containing apoE or apoC-III, subfractions associated with CHD, whereas normal weight subjects had higher prevalence of HDL without apoE or apoC-III, subfractions with protective association against CHD.

| S-EPMC4174008 | biostudies-literature

The Impact of Lipoprotein Apheresis on Oxidative Stress Biomarkers and High-Density Lipoprotein Subfractions.

Project description:Lipoprotein apheresis (LA) treatment results in a substantial reduction of low-density lipoprotein- (LDL-) cholesterol and lipoprotein(a) concentrations, which consequently decreases the rate of cardiovascular events. The additional benefit of LA may be associated with its impact on the composition and quality of high-density lipoprotein (HDL) particles, inflammation, and oxidative stress condition. To verify the effects of LA procedure, the current study is aimed at analyzing the effect of a single apheresis procedure with direct hemadsorption (DALI) and cascade filtration (MONET) on oxidative stress markers and HDL-related parameters. The study included eleven patients with familial hypercholesterolemia and hyperlipoproteinemia(a) treated with regular LA (DALI or MONET). We investigated the pre- and postapheresis concentration of the lipid-related oxidative stress markers 8-isoPGF2, oxLDL, TBARS, and PON-1. We also tracked potential changes in the main HDL apolipoproteins (ApoA-I, ApoA-II) and cholesterol contained in HDL subfractions. A single session of LA with DALI or MONET techniques resulted in a similar reduction of lipid-related oxidative stress markers. Concentrations of 8-isoPGF2 and TBARS were reduced by ~60% and ~30%, respectively. LA resulted in a 67% decrease in oxLDL levels along with a ~19% reduction in the oxLDL/ApoB ratio. Concentrations of HDL cholesterol, ApoA-I, ApoA-II, and PON-1 activity were also reduced by LA sessions, with more noticeable effects seen in the MONET technique. The quantitative proportions between HDL2 and HDL3 cholesterol did not change significantly by both methods. In conclusion, LA treatment with MONET or DALI system has a small nonselective effect on lowering HDL particles without any changes in the protein composition of these particles. Significant reduction in the level of oxidative stress parameters and less oxidation of LDL particles may provide an additional benefit of LA therapy.

| S-EPMC7428943 | biostudies-literature

The isolation and characterization of high-density-lipoprotein subfractions containing apolipoprotein E from human plasma.

Project description:1. Plasma high-density lipoprotein (HDL) was separated by heparin-Sepharose affinity chromatography into a non-bound, apolipoprotein E-poor, and a bound, apolipoprotein E-rich, fraction through the binding effect of Mn2+ in the column buffer. 2. The application of a series of elution buffers in which the concentration of Mn2+ was progressively replaced by Mg2+ resulted in the separation of the bound HDL into five subfractions. 3. Each subfraction migrated a different distance on gradient-gel electrophoresis. Three of the subfractions had RF (relative migration compared with BSA) values within the range of HDL2b. One subfraction contained largely HDL2a, with some material in the regions of HDL2b and HDL3a, and one subfraction spanned the RF regions of HDL2a, HDL3a and HDL3b. 4. The number of molecules, per HDL particle, of cholesteryl ester, non-esterified cholesterol and phospholipid increased with particle size, whereas triacylglycerol passed through a maximum and the number of amino acid residues remained approximately the same. 5. Apolipoprotein (apo) A-I was the major apoprotein in all five subfractions, but the latter differed appreciably in their contents of apo A-II and apo E. 6. The major fatty acid component of each subfraction was linoleic acid, with moderate amounts of C16:0 and C18:1 fatty acids and a smaller content of C18:0, C20:4,n-6 and C22:6,n-3, with no significant difference in composition between the subfractions. 7. This paper provides the first description of a method for the isolation of three subfractions of HDL2b together with other subfractions in quantities that are sufficient for further analytical or metabolic studies.

| S-EPMC1132663 | biostudies-other

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis.

Project description:HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.

| S-EPMC3857142 | biostudies-literature

Lipid exchange of apolipoprotein A-I amyloidogenic variants in reconstituted high-density lipoprotein with artificial membranes.

Project description:High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

| S-EPMC11010956 | biostudies-literature

Acute coronary syndrome remodels the protein cargo and functions of high-density lipoprotein subfractions.

Project description:ObjectivesThis study examined alterations in the functions and proteome of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) isolated from patients with acute coronary syndrome (ACS) compared with control subjects.MethodsWe measured HDL subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and lipid hydroperoxide (LOOH) levels in both male age-matched controls and the ACS group (n = 40/group). Additionally, proteomic analysis was used to monitor changes in the HDL subfraction proteome between controls and ACS subjects.ResultsBoth HDL2 and HDL3 from ACS patients had greater HII and LOOH levels compared with controls (P<0.001); PON1 activity and cholesterol efflux capacity in both HDL2 and HDL3 from the ACS group were significantly less than those of controls (P<0.001). Using proteomic analysis, we demonstrated that, compared with the control group, nine proteins were selectively enriched in HDL3 from subjects with ACS, and ras-related protein Rab-7b was decreased in HDL3. Additionally, in the ACS subjects, 12 proteins were decreased in HDL2 and 4 proteins were increased in HDL2.ConclusionsFunctional HDL subfractions shifted to dysfunctional HDL subfractions during ACS, and the functional impairment was linked to remodeled protein cargo in HDL subfractions from ACS patients.

| S-EPMC3988065 | biostudies-literature

High-density lipoprotein (HDL3)-associated alpha-tocopherol is taken up by HepG2 cells via the selective uptake pathway and resecreted with endogenously synthesized apo-lipoprotein B-rich lipoprotein particles.

Project description:alpha-Tocopherol (alphaTocH) is transported in association with lipoproteins in the aqueous milieu of the plasma. Although up to 50% of circulating alphaTocH is transported by high-density lipoproteins (HDLs), little is known about the mechanisms of uptake of HDL-associated alphaTocH. During the current study, human apolipoprotein (apo)E-free HDL subclass 3 (HDL3) labelled with [14C]alphaTocH was used to investigate uptake mechanisms of HDL3-associated alphaTocH by a permanent hepatoblastoma cell line (HepG2). HDL3-associated alphaTocH was taken up independently of HDL3 holoparticles in excess of apoA-I comparable with the non-endocytotic delivery of cholesteryl esters to cells termed the 'selective' cholesteryl ester uptake pathway. Experiments with unlabelled HDL3 demonstrated net mass transfer of alphaTocH to HepG2 cells. Time-dependent studies with [14C]alphaTocH-labelled HDL3 revealed tracer uptake in 80-fold excess of apoA-I and in 4-fold excess of cholesteryl linoleate. In addition to HLDs, low-density lipoprotein (LDL)-associated alphaTocH was also taken up in excess of holoparticles, although to a lesser extent. These findings were confirmed with unlabelled lipoprotein preparations, in which HDL3 displayed a 2- to 3-fold higher alphaTocH donor efficiency than LDLs (lipoproteins adjusted for equal amounts of alphaTocH). An important factor affecting particle-independent uptake of alphaTocH was the cellular cholesterol content (a 2-fold increase in cellular cholesterol levels resulted in a 2.3-fold decrease in uptake). Pulse-chase studies demonstrated that some of the HDL3-associated alphaTocH taken up independently of holoparticle uptake was resecreted along with a newly synthesized apoB-containing lipoprotein fraction.

| S-EPMC1219451 | biostudies-other

Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

Project description:High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that apolipoprotein (apo) AI and apoAII were present in all 5 subfractions; apoCI was present only in H1, and apoCIII and apoE were most abundantly present in H4 and H5. HDL-associated antioxidant enzymes such as lecithin-cholesterol acyltransferase, lipoprotein-associated phospholipase A2, and paraoxonase 1 were most abundant in H4 and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time-of-flight mass spectrometry. To quantify other proteins in the HDL subfractions, we used the isobaric tags for the relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity, with the exception of H5, which promoted the least efflux activity. In conclusion, anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions, it may be possible to uncover HDL-specific proteins that play a role in disease.

| S-EPMC3919464 | biostudies-literature

Apolipoprotein E3-mediated cellular uptake of reconstituted high-density lipoprotein bearing core 3, 10, or 17 nm hydrophobic gold nanoparticles.

Project description:We have developed a high-density lipoprotein (HDL)-based platform for transport and delivery of hydrophobic gold nanoparticles (AuNPs). The ability of apolipoprotein E3 (apoE3) to act as a high-affinity ligand for the low-density lipoprotein receptor (LDLr) was exploited to gain entry of HDL with AuNPs into glioblastoma cells. AuNPs of 3, 10, and 17 nm diameter, the latter two synthesized by phase transfer process, were solubilized by integration with phospholipids and apoE3, yielding reconstituted HDL (rHDL) bearing AuNPs. Ultraviolet-visible spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm. Transmission electron microscopy (TEM) of rHDL-AuNP revealed roughly spherical particles with AuNPs embedded in the core. The rHDL-AuNP particles displayed robust binding to the LDLr and were internalized by receptor-mediated endocytosis in glioblastoma cells. Confocal microscopy confirmed cellular uptake of AuNPs in the endosomal-lysosomal compartments, while TEM revealed intracellular aggregated AuNPs. Cell viability assay demonstrated that >85% of cells were viable with rHDL-AuNP treatment of 0.1-100 μg/mL for 24 hours. These findings are significant since they offer an effective means of delivering AuNPs across the cell membrane, which is particularly relevant in tumor cells that overexpress LDLr.

| S-EPMC5708192 | biostudies-literature

High density lipoprotein and its apolipoprotein-defined subspecies and risk of dementia.

Project description:Whether HDL is associated with dementia risk is unclear. In addition to apoA1, other apolipoproteins are found in HDL, creating subspecies of HDL that may have distinct metabolic properties. We measured apoA1, apoC3, and apoJ levels in plasma and apoA1 levels in HDL that contains or lacks apoE, apoJ, or apoC3 using a modified sandwich ELISA in a case-cohort study nested within the Ginkgo Evaluation of Memory Study. We included 995 randomly selected participants and 521 participants who developed dementia during a mean of 5.1 years of follow-up. The level of total apoA1 was not significantly related to dementia risk, regardless of the coexistence of apoC3, apoJ, or apoE. Higher levels of total plasma apoC3 were associated with better cognitive function at baseline (difference in Modified Mini-Mental State Examination scores tertile 3 vs. tertile 1: 0.60; 95% CI: 0.23, 0.98) and a lower dementia risk (adjusted hazard ratio tertile 3 vs. tertile 1: 0.73; 95% CI: 0.55, 0.96). Plasma concentrations of apoA1 in HDL and its apolipoprotein-defined subspecies were not associated with cognitive function at baseline or with the risk of dementia during follow-up. Similar studies in other populations are required to better understand the association between apoC3 and Alzheimer's disease pathology.

| S-EPMC7053836 | biostudies-literature

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