Biomedical subjects

N Fidge

Publications and source records attributed to N Fidge.

At least 19 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

Lack of effect of serum amyloid A (SAA) on the ability of high-density lipoproteins to inhibit endothelial cell adhesion molecule expression.

Studies have been conducted to determine whether the ability of high density lipoproteins (HDL) to inhibit the cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells is altered by the presence in HDL of the acute phase reactant, serum amyloid-A (SAA). Preparations of HDL(3) were isolated on two separate occasions from the plasma of each of 19 patients: the first was collected before and the second 3 days after undergoing coronary artery bypass graft surgery. Whereas the preoperative HDL(3) sample contained no SAA, in the postoperative sample SAA accounted for an average of 42% of the HDL(3) protein. The preoperative HDL(3) and postoperative, SAA-enriched HDL(3) were identical in terms of their ability to inhibit the tumour necrosis factor-alpha (TNF-alpha)-induced expression of VCAM-1 in human umbilical vein endothelial cells (HUVECs). To assess the effect of having an even greater SAA enrichment of HDL(3), samples of HDL(3) were incubated with purified SAA, which displaced almost all of the apoAI and about 40% of the apoAII from the HDL(3). This in vitro SAA-enriched HDL(3) inhibited the TNF-alpha-induced expression of VCAM-1 in HUVECs in a concentration dependent manner, which was identical to that of the unmodified HDL(3). The presence of SAA did not alter the cell-surface binding of HDL(3) to endothelial cells. It has been concluded that the presence of SAA in HDL has no effect on the ability of these lipoproteins either to inhibit the expression of VCAM-1 in endothelial cells or to bind to proteins on the endothelial cell surface.

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↗

PubMed2001

Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids.

We established a strategy to directly measure cholesterol and triglyceride levels of each lipoprotein fraction using a combination of agarose gel electrophoresis and differential staining. The cholesterol and triglyceride levels determined by electrophoresis correlated significantly with those of ultracentrifugation. The correlation coefficients between these methods were, for cholesterol levels 0.975(very low density lipoproteins, VLDL), 0.986(low density lipoproteins, LDL) and 0.965(high density lipoproteins, HDL) and for triglyceride levels 0.994(VLDL), 0.963(LDL) and 0.959(HDL) respectively. Both intra-and inter-assays showed low values of coefficients of variation (CV) (less than 3.57%). We observed a strong linearity between staining and triglyceride concentration. An increased VLDL-cholesterol was observed in type III subjects, a result which enabled distinction between type III and type IIb or type V lipoproteinemia. The method revealed lipoprotein patterns in some samples otherwise unexpected from their corresponding serum lipid parameters. Analyses of these electrophoretic patterns thus provide an effective technique to classify types of hyperlipidemia defined by the WHO. Furthermore, quantitative measurement of chylomicrons, usually difficult, proved to be achievable, providing an additional analysis of postprandial hyperlipidemia and the exact measurement of LDL-cholesterol after diet. Consequently, we recommend this simple and easy method for clinical evaluation of abnormalities in lipoprotein profiles.

Chylomicrons↗

PubMed2000

Regulation of hepatic high density lipoprotein binding protein, HB2, expression after administration of simvastatin to rabbits.

HB2 a candidate HDL receptor, is quite distinct from other HDL receptors in its structure. However, while changes in cellular cholesterol content, or a reduction in cholesterol biosynthesis accompany corresponding changes in HB2 expression, the level at which these changes occur have not been determined and the regulation and the function of HB2 remain uncertain. In order to further investigate the regulation of HB2, we administered simvastatin to rabbits to reduce cholesterol biosynthesis and follow changes in HB2 mRNA in various tissues. Six rabbits were given 15 mg/kg of simvastatin by oral administration daily and another six rabbits were given the same volume of saline as a control, for 21 days. They were then sacrificed to obtain samples of blood, liver, lung, jejunum and brain. Simvastatin reduced plasma total cholesterol by 47% and free cholesterol concentrations in liver and lung by 25 and 10%, respectively. Northern blot analysis showed that simvastatin lowered the expression of HB2 significantly in the liver and lung by 54% and 42% respectively but not in the jejunum or brain. These results support the findings of a previous study showing that HDL binding activity of both HB1 and HB2, which was determined by ligand blotting using HDL3 as a ligand, were reduced after administering cholesterol lowering agents. (Arteriosclerosis, 10: 1045-1050,1990). The present study suggests that simvastatin down-regulated HB2 at the transcriptional stage. Although the complete physiological function of HB2 is unclear, it appears to play some role in the cholesterol metabolism, warranting further studies to elucidate the nature of this interaction.

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

The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand.

We previously reported the identity and purification of two HDL3-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled human HDL3, fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.

Binding Sites↗

PubMed1998

A candidate high density lipoprotein (HDL) receptor, HB2, with possible multiple functions shows sequence homology with adhesion molecules.

High density lipoprotein (HDL), an antiatherogenic lipoprotein comprises several subclasses differing in composition and metabolic function. This physiological complexity appears to be matched with the growing number of candidate HDL receptors, since several HDL binding proteins have recently been identified in various tissues. Because these putative receptors may signal different pathways it is important to identify their structure and potential role in HDL metabolism. We review recent progress in the cloning and characterization of HB2, one of a pair of HDL binding proteins (HB1 and HB2) first purified from rat liver. The structure of HB2 is consistent with a receptor role for this membrane protein and when expressed in cells, increases HDL binding 2 fold. HB2, minimally present in THP-1 cells, is substantially upregulated in macrophages and appears sensitive to cholesterol loading of these cells. The protein shows high homology with adhesion molecules ALCAM and BEN and the possibility that HDL by binding to HB2, reduces adhesion induced arterial wall injury, is discussed in the context of the known protective role of HDL against atherosclerosis. The possible functions of HB2 are also compared with other recently cloned HDL receptors.

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

Cloning and characterization of HB2, a candidate high density lipoprotein receptor. Sequence homology with members of the immunoglobulin superfamily of membrane proteins.

The protection against coronary artery disease attributed to high density lipoprotein (HDL) may be associated with several functions, including its central role in reverse cholesterol transport, possible antioxidant and antithrombotic properties and others not yet identified which may depend on specific interactions between HDL and cell receptors. Several HDL-binding proteins have been identified including two candidate liver HDL receptors, HB1 and HB2 recently purified in this laboratory. We now report the cloning, sequencing, and some properties of HB2, the most abundant of the pair. It shows significant homology with the adhesion molecules ALCAM and BEN of the immunoglobulin superfamily and the cDNA, when transfected into HepG2 or COS cells, caused specific HDL3 binding to increase by 80-100%. Further, ligand blotting of glycoproteins isolated from phorbol 12-myristate 13-acetate-treated THP-1 cells or from transfected HepG2 and Chinese hamster ovary cells also provided evidence of increased binding of HDL3 to HB2. Differentiation of THP-1 cells into macrophages resulted in a striking increase in HB2 mRNA which was attenuated if cells were cholesterol-loaded by incubation with acetylated low density lipoprotein. If the interaction between HDL and HB2 reduces the adhesion-induced inflammatory cellular events that characterize arterial wall injury, thereby achieving the protection associated with higher plasma levels of HDL, these findings may provide a clue to one mitigating effect of HDL in heart disease.

Activated-Leukocyte Cell Adhesion Molecule↗

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↗

PubMed1996

Determination of apolipoprotein mRNA levels by ribonuclease protection assay.

The ribonuclease protection assay procedure described enables the relative quantitation of either single mRNAs or multiple mRNA species simultaneously in a sample of total RNA and demonstrates its applicability to two systems of relevance to the study of apolipoproteins, namely, liver tissue and liver-derived cell lines in culture. The main requirements of the method are the availability of cDNA cloned into a vector that directs the transcription of antisense RNA for the preparation of radioactive probes, and choice of suitable restriction endonuclease sites for linearizing the cDNA so that the final protected products of the various mRNA species are sufficiently different in size to allow their separation. For moderately abundant apolipoprotein mRNAs in rat liver, the method is sensitive down to 1 microgram total RNA. Other experimental sources of RNA or the assay of less abundant mRNA species may require a larger amount of starting material. These aspects of the assay need to be determined for each probe/ tissue system to be studied. The need for adjustments to the specific radioactivity of the probes will depend on the relative abundance of the target mRNA molecules and can be readily determined empirically. The rationale for varying the specific radioactivities to facilitate multiple assays as suggested here is both simple and effective. A preliminary assay provides information on the relative levels of the mRNA species of interest, and the step of preparing, in parallel, a sample of unlabeled antisense RNA provides the means for quantitative dilution of specific radioactivity of the 32P-labeled RNA probe where required.

Animals↗

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↗

PubMed1995

Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.

Studies of the structure and function of apolipoprotein A-I (apoA-I) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoA-I from other plasma apolipoproteins. To reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoA-I. The recombinant baculovirus containing full-length human apoA-I cDNA, when introduced into Spodoptera frugiperda, directs the synthesis of preproapoA-I, which is subsequently secreted into the growth medium as proapoA-I, indicating correct processing of the signal peptide during secretion. To prevent the extensive degradation of secreted proapoA-I, leupeptin and pepstatin A were added to the serum free cell culture medium. The protein was simply purified by filtration of the medium, which contained up to 80 mg/l proapoA-I, followed by chromatography on phenyl-sepharose CL-4B. The resultant proapoA-I was found to bind lipid and to activate lecithin:cholesterol acyltransferase as effectively as apoA-I from human plasma. The advantage of this expression system is the ease of purification of intact, biologically active apoA-I in high yield.

Animals↗

PubMed1994

Distribution of high density lipoprotein binding proteins among various tissues in the rat.

Levels of high density lipoprotein (HDL) in the blood are negatively correlated with heart disease and factors which regulate the levels and metabolism of this important lipoprotein are being investigated by many laboratories. Following the identification in liver of two candidate HDL receptors, HB1 and HB2, we undertook in the present studies, an examination of various other rat tissues for the identification of similar proteins. Using specific antibodies against each of the binding proteins, we identified both HB1 and HB2 in several other tissues (intestine, lung, kidney, ovary ...) and further, revealed relative differences in the expression of HB1 and HB2 between various tissues. A most interesting observation was the presence of HB1 but not HB2 in the spleen. Since the two proteins are not represented equally in all tissues, it is conceivable that these putative receptors have different functions in HDL metabolism or in other biological processes yet to be determined.

Animals↗