Biomedical subjects

D Sviridov

Publications and source records attributed to D Sviridov.

17 recordsLinked to original sources

PubMed2001

Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae).

We have studied the effect of lipid-free human plasma apolipoprotein A-I (apoA-I) on the transport of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which in human skin fibroblasts are mainly represented by caveolae. Changes in transport of newly synthesized cholesterol were assessed after labelling cells with [(14)C]acetate at 15 degrees C and warming cells to permit the transfer of cholesterol, followed by the selective oxidation of cholesterol in cholesterol-rich domains (caveolae) in the plasma membrane before their partial purification. ApoA-I, but not BSA added in an equimolar concentration, enhanced the transport of cholesterol to the caveolae up to 5-fold in a dose- and time-dependent manner. The effect of apoA-I on cholesterol transport exceeded its effect on cholesterol efflux, resulting in an accumulation of intracellular cholesterol in caveolae. Methyl-beta-cyclodextrin, added at a concentration promoting cholesterol efflux to the same extent as apoA-I, also stimulated cholesterol trafficking, but was 3-fold less effective than apoA-I. Progesterone inhibited the transport of newly synthesized cholesterol to the caveolae. Treatment of cells with apoA-I stimulated the expression of caveolin, increasing the amount of caveolin protein and mRNA by approx. 2-fold. We conclude that apoA-I induces the transport of intracellular cholesterol to cell-surface caveolae, possibly in part through the stimulation of caveolin expression.

Apolipoprotein A-I↗

PubMed2001

Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model.

The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were investigated in an in vitro model. ProapoA-I, mutants and wild type, were expressed in the PGEX/E. coli expression system as fusion proteins with glutathione S-transferase (GST). Use of GST-proapoA-I and truncated forms of proapoA-I enabled quantitation of the amount of GST and apoA-I formed as a result of cleavage following incubation with human serum. Deletion of the pro-peptide (GST-apoA-I) resulted in complete inhibition of the reaction. Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction. Substitution of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide. The reaction was inhibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta-mercaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. Calcium was essential for the activation of the cleavage enzyme, but it had a biphasic effect on the cleavage, activating it at concentrations below 1.5 mM and inhibiting at concentrations above 1.75 mM. Manganese alone was not essential for activation of the enzyme nor did it modify the effect of low concentration of calcium. However, a high concentration of manganese partially reverted the inhibitory effect of a high calcium concentration. Thus, residues within -2 to +4 are involved in forming the cleavage site for the maturation enzyme. The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.

Apolipoprotein A-I↗

PubMed2001

Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein.

In the present study apoA-I (Lys 107del), a naturally occurring human apoA-I variant with a deletion of Lys 107, was expressed in E. coli to examine the effect of this mutation on lipid binding, cholesterol efflux and lecithin:cholesterol acyltranferase (LCAT) activation. Dimyristoyl phosphatidylcholine (DMPC) binding studies revealed slow interaction of proapoA-I(Lys107del) with DMPC relative to normal proapoA-I. After preincubation with human plasma lipoprotein (d<1.225 g/ml) for 1 h at 37 degrees C, 125I-labeled normal proapoA-I chromatographed as a single peak with the high density lipoprotein (HDL) fraction, whereas 125I-labeled proapoA-I(Lys107del) chromatographed with both HDL and free proapoA-I (26% of the radioactivity). Circular dichroism measurements showed that the alpha-helical content of lipid-bound proapoA-I (Lys107del) was reduced to 64 versus 73% of normal proapoA-I. Non-denaturing gradient gel electrophoresis of reconstituted HDL assembled with either proapoA-I(Lys107del) or normal proapoA-I showed that the mutation led to the formation of a second population of smaller rHDL particles. DMPC/proapoA-I(Lys107del) and normal DMPC/proapoA-I complexes exhibited a similar capacity to promote cholesterol efflux from fibroblasts. ProapoA-I (Lys107del) also activated LCAT similar to wild type proapoA-I and human plasma apoA-I. We conclude that deletion of Lys 107 substantially alters the lipid binding properties of the protein, which correlated with reduced binding to plasma HDL in vitro, but did not affect the capacity of the mutant/lipid complex to promote cholesterol efflux or activate LCAT.

Apolipoprotein A-I↗

PubMed2000

Identification of a sequence of apolipoprotein A-I associated with the activation of Lecithin:Cholesterol acyltransferase.

We aimed to distinguish between the effects of mutations in apoA-I on the requirements for the secondary structure and a specific amino acid sequence for lecithin:cholesterol acyltransferase (LCAT) activation. Several mutants were constructed targeting region 140-150: (i) two mutations affecting alpha-helical structure, deletion of amino acids 140-150 and substitution of Ala(143) for proline; (ii) two mutations not affecting alpha-helical structure, substitution of Val(149) for arginine and substitution of amino acids 63-73 for sequence 140-150; and (iii) a mutation in a similar region away from the target area, deletion of amino acids 63-73. All mutations affecting region 140-150 resulted in a 4-42-fold reduction in LCAT activation. Three mutations, apoA-I(Delta140-150), apoA-I(P143A), and apoA-I(140-150 --> 63-73), affected both the apparent V(max) and K(m), whereas the mutation apoA-I(R149V) affected only the V(max). The mutation apoA-I(Delta63-73) caused only a 5-fold increase in the K(m). All mutants, except apoA-I(P143A) and apoA-I(Delta63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(Delta63-73) caused a significant reduction in the stability of apoA-I.phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depends on the sequence that includes amino acids 140-150.

Amino Acid Sequence↗

PubMed2000

Effect of weight reduction on the distribution of apolipoprotein A-I in high-density lipoprotein subfractions in obese non-insulin-dependent diabetic subjects.

High-density lipoprotein (HDL) plays an important role in the process of reverse cholesterol transport, which may become suboptimal with increasing body fatness. HDL cholesterol that is reduced in obese subjects paradoxically decreases during weight reduction. To determine how weight reduction affects HDL subclasses that are involved in reverse cholesterol transport, we studied HDL from obese diabetic subjects before and after energy restriction within background diets high in either carbohydrate or monounsaturated fatty acids (MUFAs). Body weight, blood glucose, total cholesterol, and LDL cholesterol decreased after 8 and 12 weeks of weight reduction. With the very-low-fat diet, HDL cholesterol decreased significantly at 8 weeks, but recovered to initial levels after 12 weeks as body weight began to stabilize. Plasma apolipoprotein A-I (apo A-I) decreased substantially and significantly at 8 and 12 weeks with both diets, and was reflected in the reduction of apo A-I in HDL subclasses alpha1, alpha2, pre-beta1, and pre-beta2 + pre-beta3. The calculation of the percentage distribution of apo A-I among HDL species showed that only the proportion of pre-beta1-HDL decreased, whereas alpha2-HDL increased. This led to a significant increase in the alpha1 + alpha2/pre-beta ratio, ie, the ratio of the large cholesterol "storage" or "sink" HDL to the HDL "shuttle" fraction considered to be the initial acceptor of cell cholesterol. These data suggest that despite the reduction in HDL cholesterol and apo A-I, the redistribution of apo A-I in pre-beta1-HDL and alpha-HDL observed with weight reduction appears to revert to the pattern that we have previously reported in lean as opposed to overweight subjects.

Apolipoprotein A-I↗

PubMed2000

Deletion of the propeptide of apolipoprotein A-I reduces protein expression but stimulates effective conversion of prebeta-high density lipoprotein to alpha-high density lipoprotein.

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.

Animals↗

PubMed1999

Effectivity of expression of mature forms of mutant human apolipoprotein A-I.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.

Amino Acid Motifs↗

PubMed1999

Intracellular cholesterol trafficking.

The overall picture of intracellular cholesterol trafficking is very complex. The transfer of cholesterol within the cell depends on the contribution of several trafficking mechanisms. The known elements of cholesterol trafficking machinery include clathrin-coated pits, scavenger receptor type B1, caveolae, phospholipid rafts, Niemann-Pick C disease protein, sterol carrier protein 2, multidrug resistance protein, microsomal triglyceride transfer protein and steroidogenic acute regulation protein. Several pathways of intracellular cholesterol trafficking, for example retroendocytosis and cholesterol absorption in the intestine, are yet to be connected to specific structural elements. The contribution of different pathways depends on cell type, the source and destination of cholesterol and cellular cholesterol content and requirements. Some pathways are found in most, if not all, cell types, while others are associated with the specialized function of a particular cell type, for example, lipoprotein assembly in the liver or intestine and steroid hormone synthesis in steroidogenic tissue. Certain routes of intracellular cholesterol trafficking are heavily backed up by several auxiliary pathways, others entirely depend on a single functional element. In this review we describe the intracellular machinery involved in the intracellular transfer of cholesterol and give an overview of both the general and specialized pathways of intracellular cholesterol trafficking known to date.

Animals↗

PubMed1998

Cholesterol transport between cells and high density lipoprotein subfractions from obese and lean subjects.

We studied the pathway of cholesterol efflux from fibroblasts by testing plasma samples from obese and lean subjects. Plasma samples were incubated with [3H]cholesterol-labeled human skin fibroblasts for 1 h to ensure uniform labeling of all of the high density lipoprotein (HDL) subfractions. Supernatants were then transferred to unlabeled cells and the displacement of labeled cholesterol within HDL subfractions by unlabeled cellular cholesterol was analyzed in short-term experiments. Plasma samples of obese subjects were characterized by a lower content of total apolipoprotein A-I (apoA-I) and alpha1-HDL and a lower overall capacity to take up labeled cholesterol. In plasma of lean subjects, pre beta2-HDL and alpha1-HDL appeared to be the most active particles in the initial uptake of unlabeled cellular cholesterol. By contrast, in plasmas of obese subjects, the pre beta1-HDL appeared to be most active in taking up unlabeled cellular cholesterol and transferring [3H]cholesterol. There were negative correlations between body mass index (BMI) and apoA-I and alpha1-HDL concentrations, and with the apparent increments of cellular cholesterol uptake within pre beta2-HDL and alpha1-HDL, as well as with the overall capacity to promote cholesterol efflux. By contrast, BMI was positively correlated with the apparent increment in cellular cholesterol within pre beta1-HDL. While cholesterol efflux was correlated with total plasma apoA-1, there were no such correlations with the concentration of any individual HDL subfraction. We conclude that the pattern of cholesterol transfer between fibroblasts and high density lipoprotein particles is influenced by body fatness and may be a factor in the abnormal metabolism of HDL in obesity.

Adult↗

PubMed1997

Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.

To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus-insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus-insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.

Amino Acid Sequence↗

PubMed1997

Antibodies against high-density lipoprotein binding proteins enhance high-density lipoprotein uptake but do not affect cholesterol efflux from rat hepatoma cells.

High-density lipoprotein plays a key role in the reverse cholesterol transport pathway as well as in the delivery of cholesterol to the liver and steroidogenic tissues. Metabolism of high-density lipoprotein is determined by one of its apolipoproteins, apolipoprotein A-I; however, the identity and function of cellular protein which binds high-density lipoprotein remains unclear. The effect of antibodies against rat high-density lipoprotein binding proteins, HB1 and HB2, on high-density lipoprotein metabolism in a rat hepatoma cell line were studied. Cells were preincubated with the antibodies and 125I-labeled high-density lipoprotein binding and uptake as well as cholesterol biosynthesis and cholesterol efflux to human plasma or isolated high-density lipoprotein were studied. Both antibodies reacted specifically with HB1 and HB2 on the ligand and Western blots, but their binding was not blocked by high-density lipoprotein. Both antibodies inhibited 125I-labeled high-density lipoprotein binding to cells by 20-40%, but stimulated 125I-labeled high-density lipoprotein uptake by up to 2.5-fold. The antibodies had no effect on cholesterol efflux or on cholesterol synthesis. It is concluded that high-density lipoprotein binding proteins, HB1 and HB2, may be involved in high-density lipoprotein uptake in the liver rather than in mediating cholesterol efflux.

Animals↗

PubMed1997

Altered properties of high density lipoprotein subfractions in obese subjects.

Human HDL are heterogeneous in their metabolism and comprise small, nascent pre-beta-HDL and more mature alpha-HDL. Evidence exists that pre-beta 1-HDL is the initial acceptor of cellular free cholesterol, which then transfers sequentially to other pre-beta species and then, after esterification, into alpha-HDL. As HDL particles are themselves transformed during this process, we postulated that in disorders in which HDL-cholesterol is low, such as obesity, the distribution of HDL particles may be disturbed. In this study, we analyzed the HDL profile in 23 obese and 18 lean subjects, and further investigated the effects of dietary change in 15 obese subjects. HDL were separated by two-dimensional nondenaturing electrophoresis and the apoA-I content in each fraction was quantified. alpha 1-HDL in obese subjects was significantly lower (P < 0.001) and alpha 2-, alpha 3-, and pre-beta 1-HDL were significantly higher (P < 0.05 for alpha 2-HDL, P < 0.001 for alpha 3- and pre-beta 1-HDL) than in lean subjects. On stepwise regression analysis, body mass index accounted for 52% (negatively) of the variance in alpha 1-HDL and for 16% and 33% (positively) for the variances in alpha 3- and pre-beta 1-HDL, respectively. alpha 1- and pre-beta 3-HDL increased significantly after low-fat, oleic acid-rich, or alpha-linolenic acid-rich diets. The profile of alpha-HDL particles and also of pre-beta-HDL particles therefore shifted to smaller species in obese subjects, and this was influenced by dietary fat. Increased pre-beta 1-HDL-apoA-I in obese subjects is likely to derive from increased HDL catabolism but may also reflect diminished transformation of pre-beta 1- to pre-beta 2-HDL which might reduce capacity for reverse cholesterol transport and partly explain lower HDL-cholesterol levels.

Adolescent↗

PubMed1996

Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(-6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(-6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(-6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(-6-222)) and 210 (apoA-I-(-6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(-6-150)) and 135 (apoA-I-(-6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(-6-243), apoA-I-(-6-222), and apoA-I-(-6-210), but slightly higher for apoA-I-(-6-150) and apoA-I-(-6-135). When complexed with phospholipid, all forms except apoA-I-(-6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent.

Apolipoprotein A-I↗

PubMed1996

Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles.

The effect of monoclonal antibodies against apolipoprotein A-I (apoA-I) on the efflux of intracellular and plasma membrane cholesterol from HepG2 cells to human serum, high-density lipoprotein (HDL), apoA-I, and apoA-I/phosphatidylcholine complex (apoA-I/PC) was studied. Fab fragments of two monoclonal antibodies, AI-3 (residues 140-147) and Al-4.2 (residues 149-150), inhibited the efflux of intracellular cholesterol to serum in a dose-dependent manner. In combination, these antibodies were twice as effective than when used alone. None of the antibodies tested inhibited efflux of the plasma membrane cholesterol. When different types of acceptors were compared for their ability to promote intracellular cholesterol efflux, they were effective in the following order: serum > HDL > apoA-I/PC > pure apoA-I. Antibody AI-3 inhibited efflux of intracellular cholesterol to serum, HDL, and pure apoA-I, but not to apoA-I/PC. Antibody AI-4.2 inhibited efflux to serum, apoA-I/PC, and pure apoA-I, but not to HDL. An explanation for this is that antibody AI-4.2 reacts poorly with isolated alpha-HDL in an immunoprecipitation assay and has higher affinity for pre beta 2-HDL and pre beta 3-HDL particles than antibody AI-3 in nondenaturing two-dimensional electrophoresis. In conclusion, we have demonstrated that a region of apoA-I within or adjacent to residues 140-150 determines the ability of apoA-I to promote intracellular cholesterol efflux.

Amino Acid Sequence↗

PubMed1995

Pathway of cholesterol efflux from human hepatoma cells.

Studies have been carried out with HepG2 cells, as a model for human hepatocytes, to explore a novel proposition that the liver contributes free cholesterol to the plasma lipoproteins which participate in the process of reverse-cholesterol transport. Specifically, we compared efflux of cholesterol from HepG2 cells and human fibroblasts (a model for extrahepatic tissues) after labeling cells with [14C]cholesterol. Incubation of both types of cells with human serum resulted in the efflux of [14C]cholesterol and net cholesterol flux from the cells to the medium and its subsequent esterification. Rates of cholesterol efflux from HepG2 cells and fibroblasts were similar. Nondenaturing two-dimensional gel electrophoresis showed that about 10% of the cell-derived [14C]cholesterol moves rapidly through pre beta 1-. pre beta 3- and pre beta 2-HDL particles into alpha HDL and LDL, although the majority moves directly to alpha HDL and LDL, with most of [14C]cholesterol and cholesterol mass accumulating in LDL. When cells were incubated with equivalent concentrations of isolated lipoproteins, HDL was much more effective in promoting [14C]cholesterol efflux than LDL, suggesting that unesterified cholesterol is initially transferred to HDL and then to LDL. Incubation with whole serum in contrast to isolated lipoproteins did not enhance cholesterol efflux despite a 3-fold higher esterification rate. We also investigated the identity of newly secreted lipoproteins following the labeling of HepG2 cells with [14C]cholesterol: 72% of labeled cholesterol was released as LDL, 20% was released as pre beta 2-HDL and 8% as small alpha HDL particles. Novel apo A-I rich but [14C]cholesterol-deficient pre beta 1-HDL particles were also secreted by HepG2 cells.

Carcinoma, Hepatocellular↗

PubMed1995

Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I.

We have compared the efflux of cholesterol from different cellular pools of human hepatoma cells HepG2 using intact cells or isolated membrane fractions. To label different pools, cells were incubated with either unesterified [14C]cholesterol that had been incorporated into high density lipoproteins ([14C]FC-HDL), low density lipoproteins ([14C]FC-LDL), or phosphatidylcholine liposomes ([14C]FC-PC), or with [14C]acetate. Cell fractionation revealed that labeling of cells with [14C]FC-PC resulted in the incorporation of [14C]cholesterol almost exclusively into the plasma membrane (PM), while incubation with [14C]FC-HDL resulted in the majority of [14C]cholesterol incorporation into the PM, but with a smaller component associated with lysosomes. Labeling with [14C]FC-LDL or [14C]acetate led to an accumulation of [14C]cholesterol predominantly in lysosomes or the endoplasmic reticulum (ER), respectively. When the kinetics of [14C]cholesterol efflux was analyzed after pulse-labeling of different cellular pools, half-times of cholesterol efflux from lysosomes and ER were significantly longer than that from PM. In another set of experiments, when both labeling and efflux times varied, efflux of [14C]cholesterol from the PM to human serum after 1.5 h pulse and chase incubations was double that from lysosomes and 8-fold that from ER. Extension of the incubation times from 1.5 to 3 h diminished the difference in cholesterol efflux from different membranes. Further incubation to 6 h almost abolished the different responses. Cell-free preparations of membranes, obtained from cells labeled with [14C]cholesterol, showed no differences in cholesterol efflux. No differences in the distribution of [14C]cholesterol released into serum among lipoprotein subfractions was observed. Pretreatment of the serum with Fab fragments of polyclonal rabbit anti-human apolipoprotein A-I antibodies reduced its ability to promote efflux of cholesterol from the ER by 77%, but had no effect on cholesterol efflux from the PM. Fab fragments of non-immune IgG had no effect on the efflux of both ER and PM cholesterol. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is strongly influenced by its subcellular location, and is regulated by apolipoprotein A-I.

Apolipoprotein A-I↗

PubMed1991

Effect of lovastatin on the interaction between high density lipoprotein and cultured rat adrenocortical cells.

Preincubation of rat adrenocortical cells with lovastatin inhibits high density lipoprotein (HDL3) interaction with rat adrenocortical cells in a dose- and time-dependent fashion. Lovastatin causes a decrease in the number of binding sites and a moderate increase in the affinity of HDL binding to cells. Lovastatin also produced a dose- and time-dependent inhibition of cholesterol synthesis in these cells. Dose-dependence, but not time-dependence, of the inhibition of cholesterol synthesis, correlates with that of HDL binding (r = 0.91, P less than 0.004). Incubation of cells with lovastatin for up to 24 h does not change their free or total cholesterol content. The inhibitory effect was apparently not due to a modification of HDL by lovastatin. These results indicate that lovastatin causes a down-regulation of HDL binding sites on cultured rat adrenocortical cells.

Adrenal Cortex↗