Cellular cholesterol efflux.

Efflux of free cholesterol (FC) continues even when cellular FC mass is unchanged. This reflects a recirculation of preformed FC between cells and extracellular fluids which has multiple functions in cell biology including receptor recycling and signaling as well as cellular FC homeostasis. Total FC efflux is heterogeneous. Simple diffusion to mature high density lipoprotein (HDL), mainly via albumin as intermediate, initiates FC net transport driven by plasma lecithin:cholesterol acyltransferase activity. A second major efflux component reflects protein-facilitated transport from cell surface domains (caveolae, rafts) driven by FC binding to lipid-poor, pre-beta-migrating HDL (pre-beta-HDL). Facilitated efflux from caveolae, unlike simple diffusion, is highly regulated. Neither ABC1 (the protein defective in Tangier disease) nor other ATP-dependent transporters now appear likely to contribute directly to FC efflux. Their role is limited to the initial formation of a particle precursor to circulating pre-beta-HDL, which recycles without further lipid input from ATP-dependent transporter proteins. Lipid-free apolipoprotein A-I, previously considered a surrogate for pre-beta-HDL, has a reactivity much lower than that of native lipoprotein FC acceptors.     

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.     

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.     

Caveolin-1 does not affect SR-BI-mediated cholesterol efflux or selective uptake of cholesteryl ester in two cell lines

Free cholesterol (FC) has been reported to efflux from cells through caveolae, which are 50-100 nm plasma membrane pits. The 22 kDa protein caveolin-1 is concentrated in caveolae and is required for their formation. The HDL scavenger receptor BI (SR-BI), which stimulates both FC efflux and selective uptake of HDL-derived cholesteryl ester (CE), has been reported to be concentrated in caveolae, suggesting that this localization facilitates flux of FC and CE across the membrane. However, we found that overexpression of caveolin-1 in Fischer rat thyroid (FRT) cells, which lack caveolin-1 and caveolae, or HEK 293 cells, which normally express very low levels of caveolin-1, did not affect FC efflux to HDL or liposomes. Transient expression of SR-B1 did not affect this result. Similarly, caveolin-1 expression did not affect selective uptake of CE from labeled HDL particles in FRT or HEK 293 cells transfected with SR-BI. We conclude that basal and SR-BI-stimulated FC efflux to HDL and liposomes and SR-BI-mediated selective uptake of HDL CE are not affected by caveolin-1 expression in HEK 293 or FRT cells.     

Induction of cellular cholesterol efflux to lipid-free apolipoprotein A-I by cAMP

In the present study apolipoprotein-mediated free cholesterol (FC) efflux was studied in J774 macrophages having normal cholesterol levels using an experimental design in which efflux occurs in the absence of contributions from cholesteryl ester hydrolysis. The results show that cAMP induces both saturable apolipoprotein (apo) A-I-mediated FC efflux and saturable apo A-I cell-surface binding, suggesting a link between these processes. However, the EC50 for efflux was 5-7-fold lower than the Kd for binding in both control and cAMP-stimulated cells. This dissociation between apo A-I binding and FC efflux was also seen in cells treated for 1 h with probucol which completely blocked FC efflux without affecting apo A-I specific binding. Thus, cAMP-stimulated FC efflux involves probucol-sensitive processes distinct from apo A-I binding to its putative cell surface receptor. FC efflux was also dramatically stimulated in elicited mouse peritoneal macrophages, suggesting that cAMP-regulated apolipoprotein-mediated FC efflux may be important in cholesterol homeostasis in normal macrophages. The presence of a cAMP-inducible cell protein that interacts with lipid-free apo A-I was investigated by chemical cross-linking of 125I-apo A-I with J774 cell surface proteins which revealed a Mr 200 kDa component when the cells were treated with cAMP.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

Insulin stimulates the tyrosine phosphorylation of caveolin

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin- sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin- associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton- insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin- dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.     

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.     

Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption

Human plasma high-density lipoproteins (HDL) are important vehicles in reverse cholesterol transport, the cardioprotective mechanism by which peripheral tissue-cholesterol is transported to the liver for disposal. HDL is the target of serum opacity factor (SOF), a substance produced by Streptococcus pyogenes that turns mammalian serum cloudy. Using a recombinant (r) SOF, we studied opacification and its mechanism. rSOF catalyzes the partial disproportionation of HDL into a cholesteryl ester-rich microemulsion (CERM) and a new HDL-like particle, neo HDL, with the concomitant release of lipid-free (LF)-apo A-I. Opacification is unique; rSOF transfers apo E and nearly all neutral lipids of approximately 100,000 HDL particles into a single large CERM whose size increases with HDL-CE content (r approximately 100-250 nm) leaving a neo HDL that is enriched in PL (41%) and protein (48%), especially apo A-II. rSOF is potent; within 30 min at 37 degrees C, 10 nM rSOF opacifies 4 microM HDL. At respective low and high physiological HDL concentrations, LF-apo A-I is monomeric and tetrameric. CERM formation and apo A-I release have similar kinetics suggesting parallel or rapid sequential steps. According to the reaction products and kinetics, rSOF is a heterodivalent fusogenic protein that uses a docking site to displace apo A-I and bind to exposed CE surfaces on HDL; the resulting rSOF-HDL complex recruits additional HDL with its binding-delipidation site and through multiple fusion steps forms a CERM. rSOF may be a clinically useful and novel modality for improving reverse cholesterol transport. With apo E and a high CE content, CERM could transfer large amounts of cholesterol to the liver for disposal via the LDL receptor; neo HDL is likely a better acceptor of cellular cholesterol than HDL; LF-apo A-I could enhance efflux via the ATP-binding casette transporter ABCA1.     

Apo A-II participates in HDL–liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL–liposome interaction may influence the anti-atherogenic properties of liposomes.     

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [(3)H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [(3)H]cholesterol when apo A-I, HDL(3), or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r(2) = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r(2) = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [(3)H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Exchange of Apolipoprotein A-I between Lipid-associated and Lipid-free States: A POTENTIAL TARGET FOR OXIDATIVE GENERATION OF DYSFUNCTIONAL HIGH DENSITY LIPOPROTEINS*

An important event in cholesterol metabolism is the efflux of cellular cholesterol by apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins (HDL). Lipid-free apoA-I is the preferred substrate for ATP-binding cassette A1, which promotes cholesterol efflux from macrophage foam cells in the arterial wall. However, the vast majority of apoA-I in plasma is associated with HDL, and the mechanisms for the generation of lipid-free apoA-I remain poorly understood. In the current study, we used fluorescently labeled apoA-I that exhibits a distinct fluorescence emission spectrum when in different states of lipid association to establish the kinetics of apoA-I transition between the lipid-associated and lipid-free states. This approach characterized the spontaneous and rapid exchange of apoA-I between the lipid-associated and lipid-free states. In contrast, the kinetics of apoA-I exchange were significantly reduced when apoA-I on HDL was cross-linked with a bi-functional reagent or oxidized by myeloperoxidase. Our observations support the hypothesis that oxidative damage to apoA-I by myeloperoxidase limits the ability of apoA-I to be liberated in a lipid-free form from HDL. This impairment of apoA-I exchange reaction may be a trait of dysfunctional HDL contributing to reduced ATP-binding cassette A1-mediated cholesterol efflux and atherosclerosis.     

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.     

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Characteristics of the cholesterol efflux induced by novel seminal phospholipid-binding proteins

Our recent results indicated that the major proteins of bovine seminal plasma (collectively called BSP proteins) stimulate cholesterol efflux from fibroblasts and that this process shows many differences compared to the efflux induced by apolipoprotein A-I (apoA-I)-containing lipoproteins. The present study was undertaken to investigate the BSP-mediated efflux mechanism. Compared to the slow and constant rate of cholesterol efflux induced by apoA-I-containing lipoproteins, the BSP proteins stimulated a rapid efflux that gradually reached a plateau. The addition of purified BSP proteins after the establishment of the plateau resulted in a further cholesterol efflux indicating that cellular cholesterol was still available for efflux. Incubation of unlabeled fibroblast culture with the spent medium containing BSP-generated lipid ([(3)H]cholesterol) particles obtained after the establishment of the plateau did not result in any cholesterol influx. Therefore, the plateau did not correspond to an equilibrium of the radiolabel between the medium and the cells but rather to a saturation of the efflux particles with cholesterol. Numerous studies have indicated that the cholesterol efflux induced by apoA-I-containing lipoproteins involves cell-surface receptor, caveolae and intracellular cholesterol mobilization. Therefore, we investigated these characteristics for the BSP-mediated cholesterol efflux. Binding of BSP proteins to cells (evaluated by immunoblotting) reached saturation rapidly and remained constant thereafter. However, after several washings the cell-bound BSP proteins were unable to promote significant cholesterol efflux. Both results indicate no correlation of cholesterol efflux with cell binding. Moreover, in comparison to apoA-I-mediated cholesterol efflux, BSP-mediated efflux was not abolished at temperatures below 22 degrees C indicating that the BSP-induced cholesterol efflux does not involve intracellular cholesterol mobilization. High-density lipoprotein- and apoA-I-mediated cholesterol efflux was inhibited by preincubating fibroblasts with progesterone, whereas the cholesterol efflux by BSP proteins was not, indicating that cell-surface caveolae do not participate in BSP-mediated cholesterol efflux. Our results indicate that the mechanism of cholesterol efflux by BSP proteins is unidirectional and is strikingly different from that mediated by apoA-I-containing lipoproteins.     

Specificity of ABCA7-mediated cell lipid efflux

Adenosine triphosphate-binding cassette transporter subfamily A member 7 (ABCA7) performs incompletely understood biochemical functions that affect pathogenesis of Alzheimer's disease. ABCA7 is most similar in primary structure to ABCA1, the protein that mediates cell lipid efflux and formation of high-density lipoprotein (HDL). Lipid metabolic labeling/tracer efflux assays were employed to investigate lipid efflux in BHK-ABCA7(low expression), BHK-ABCA7(high expression) and BHK-ABCA1 cells. Shotgun lipid mass spectrometry was used to determine lipid composition of HDL synthesized by BHK-ABCA7 and BHK-ABCA1 cells. BHK-ABCA7(low) cells exhibited significant efflux only of choline-phospholipid and phosphatidylinositol. BHK-ABCA7(high) cells had significant cholesterol and choline-phospholipid efflux to apolipoprotein (apo) A-I, apo E, the 18A peptide, HDL, plasma and cerebrospinal fluid and significant efflux of sphingosine-lipid, serine-lipid (which is composed of phosphatidylserine and phosphatidylethanolamine in BHK cells) and phosphatidylinositol to apo A-I. In efflux assays to apo A-I, after adjustment to choline-phospholipid, ABCA7-mediated efflux removed ~4 times more serine-lipid and phosphatidylinositol than ABCA1-mediated efflux, while ABCA1-mediated efflux removed ~3 times more cholesterol than ABCA7-mediated efflux. Shotgun lipidomic analysis revealed that ABCA7-HDL had ~20 mol% less phosphatidylcholine and 3–5 times more serine-lipid and phosphatidylinositol than ABCA1-HDL, while ABCA1-HDL contained only ~6 mol% (or ~1.1 times) more cholesterol than ABCA7-HDL. The discrepancy between the tracer efflux assays and shotgun lipidomics with respect to cholesterol may be explained by an underestimate of ABCA7-mediated cholesterol efflux in the former approach. Overall, these results suggest that ABCA7 lacks specificity for phosphatidylcholine and releases significantly but not dramatically less cholesterol in comparison with ABCA1.