Using a chitosan nanolayer as an efficient pH buffer to protect pH-sensitive supramolecular assemblies.

It is attractive to control the properties of macro objects and films by employing simple nanolayer composites, as in the case of nanoarchitectured Layer-by-Layer (LbL) coating. In this paper, we use chitosan as a surface-based pH buffer to protect adsorbed supramolecular fibres from pH-mediated disassembly. Protons are generated on a titania surface under illumination at 405 nm leading to an appreciable pH change on the surface. We find that supramolecular polymers that are highly sensitive to pH change will not disassemble after irradiation if a nanolayer of chitosan is present. We propose that chitosan can be used as an efficient pH-responsive protective layer for pH sensitive soft materials.

Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter.

ApoA-I mimetics are short synthetic peptides that contain an amphipathic α-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenyl group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop-Prog-Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-helical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p < 0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides.

Advanced glycation end-products (AGEs) and functionality of reverse cholesterol transport in patients with type 2 diabetes and in mouse models.

AIMS/HYPOTHESIS: We investigated the contribution of AGEs to the impairment of reverse cholesterol transport (RCT) variables in diabetic individuals and in two animal models of diabetic obesity and of renal impairment. METHODS: The capacity of plasma and HDL from 26 individuals with moderately controlled type 2 diabetes to support cholesterol efflux was compared with 26 age- and sex-matched individuals without diabetes. We also compared the rates of RCT in vivo in two animal models: db/db mice and mice with chronic renal failure. RESULTS: Diabetic individuals had characteristic dyslipidaemia and higher levels of plasma AGEs. The capacity of whole plasma, ApoB-depleted plasma and isolated HDL to support cholesterol efflux was greater for diabetic patients compared with controls despite their lower HDL-cholesterol levels. The capacity of plasma to support cholesterol efflux correlated with plasma levels of cholesteryl ester transfer protein and levels of ApoB, but not with levels of AGE. RCT was severely impaired in db/db mice despite elevated HDL-cholesterol levels and no change in AGE concentration, whereas RCT in uraemic mice was unaffected despite elevated AGE levels. CONCLUSIONS/INTERPRETATION: AGEs are unlikely to contribute significantly to the impairment of RCT in type 2 diabetes.

Cholesterol efflux from macrophages is influenced differentially by plasmas from overweight insulin-sensitive and -resistant subjects.

OBJECTIVE: In vitro measurements of cholesterol efflux from macrophages have recently been shown to associate with cardiovascular risk. We investigated whether cholesterol efflux from macrophages incubated with plasmas from overweight/obese subjects with metabolic syndrome was influenced by the presence of insulin resistance. METHODS: Plasmas were obtained from 47 men and women with metabolic syndrome, of whom 25 were found to be insulin resistant (IR) and 22 insulin sensitive (IS) (Matsuda, De Fronzo equation based on oral glucose tolerance test). Activated human macrophage THP-1 cells in which cholesterol had been radiolabelled were incubated with the subjects' plasmas to allow calculation of % cholesterol efflux. RESULTS: Body mass index and waist measurements, as well as plasma lipid levels, did not differ between the two groups. Homeostatic model assessment-insulin resistance value as well as plasma insulin and leptin concentrations were higher in IR subjects. Cholesterol efflux was found to be significantly greater with plasmas from IR subjects (9.1%) than from IS subjects (6.7%) (P=0.005). Further, cholesterol efflux was significantly inversely associated with insulin sensitivity index (P<0.001), directly with arterial insulin concentration (P<0.001) and directly with cholesteryl ester transfer protein (CETP) mass (P=0.044). CONCLUSION: Plasmas from overweight subjects with insulin resistance induced greater in vitro cholesterol efflux compared with IS subjects. Efflux inversely correlated with insulin sensitivity suggesting an increase in reverse cholesterol transport in the IR state that may lead to greater transfer of cholesterol to apoB lipoproteins from high-density lipoproteins via CETP as a factor in the association between IR and atherosclerosis.

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter.

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

HDL therapy: two kinds of right?

Cardiovascular disease remains the leading cause of morbidity and mortality globally. The disease is largely controlled with interventions managing atherogenic lipids including LDL and triglycerides. However a number of studies have shown that increasing HDL levels is likely to provide better outcomes for patients suffering from this disease. There has been an extensive research effort into understanding how HDL levels are regulated in the body and which pathways can be targeted therapeutically. The HDL metabolic pathway is however overwhelmingly complex. This has provided only limited success in trialing drugs designed to raise HDL. To add to the complexity HDL itself is a heterogeneous population of particles and there is controversy surrounding which HDL particle is the most cardio-protective. In addition there is varying opinions on which of the HDL cellular receptors are more important in humans (as opposed to what has been discovered in mice) in regulating these effects. In this article we explore the evidence for and against using the currently suggested methods of raising HDL and provide some evidence for how the adverse effects of these drugs could be corrected.

The anti inflammatory effects of high density lipoproteins.

It is well recognised that increased levels of high density lipoprotein (HDL) protect against atherosclerosis and correlate with improved prognosis for vascular disease associated events. While many of the atheroprotective effects of HDL are ascribed to the ability to remove cholesterol from the vasculature through the reverse cholesterol transport system, recent work has shown that HDL may be atheroprotective through its other functions, such as regulation of endothelial adhesion molecule expression, stimulation of endothelial nitric oxide synthase and inhibition of the damaging effects of oxidised low density lipoproteins. Recently, HDL has also been described to interact with circulating cells inhibiting both leukocyte and platelet activation, therefore having further systemic anti-inflammatory functions. This review summarises the studies and models used to examine the anti-inflammatory effects of HDL and details data describing the ability to inhibit leukocyte activation, contributing to the hypothesis that raised HDL is beneficial in the context of inflammation in atherosclerosis. Further, HDL modification in disease and current therapeutic strategies such as reconstituted HDL particles and apoA I mimetic peptides is discussed to provide insights to the potential applicability of raising HDL to regress cardiovascular disease.

Advanced glycation of apolipoprotein A-I impairs its anti-atherogenic properties.

AIMS/HYPOTHESIS: AGE contribute to the pathogenesis of diabetic complications, including dyslipidaemia and atherosclerosis. However, the precise mechanisms remain to be established. In the present study, we examined whether AGE modification of apolipoprotein A-I (apoA-I) affects its functionality, thus altering its cardioprotective profile. MATERIALS AND METHODS: The ability of AGE-modified apoA-I to facilitate cholesterol and phospholipid efflux, stabilise ATP-binding cassette transporter A1 (ABCA1) and inhibit expression of adhesion molecules in human macrophages and monocytes was studied. RESULTS: The ability of AGE-modified apoA-I to promote cholesterol efflux from THP-1 macrophages, isolated human monocytes and from ABCA1-transfected HeLa cells was significantly reduced (>70%) compared with unmodified apoA-I. This effect was reversed by preventing AGE formation with aminoguanidine or reversing AGE modification using the cross-link breaker alagebrium chloride. AGE-modification of HDL also reduced its capacity to promote cholesterol efflux. AGE-apoA-I was also less effective than apoA-I in stabilising ABCA1 in THP-1 cells as well as in inhibiting expression of CD11b in human monocytes. CONCLUSIONS/INTERPRETATION: AGE modification of apoA-I considerably impairs its cardioprotective, antiatherogenic properties, including the ability to promote cholesterol efflux, stabilise ABCA1 and inhibit the expression of adhesion molecules. These findings provide a rationale for targeting AGE in the management of diabetic dyslipidaemia.

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 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.

Initiation and inhibition of free radical processes in H2O2-metmyoglobin (methemoglobin)-2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) systems.

Rates of free radical initiation were determined at 20 degrees C in 10 mM phosphate buffer (pH 7.4) in the systems metmyoglobin (methemoglobin)-H2O2 using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the diammonium salt (ABTS). The catalytic activity of MetMb was 2-3-fold higher than that of MetHb. The process can be described by the Michaelis-Menten equation, from which effective values of Km and Vmax were calculated. Comparative kinetic studies on the inhibition of ABTS oxidation were carried out using Trolox, propylgallate (PG), polydisulfide of gallic acid (poly(DSG)), polydisulfide of (2-amino-4-nitrophenol) (poly(ADSNP)), and its conjugate with human serum albumin (HSA-poly(ADSNP)). The inhibitors were characterized by inhibition constants Ki and stoichiometric inhibition coefficients f (the number of radicals terminated by a single molecule of inhibitor). The minimum Ki and the maximum f values were obtained for poly(DSG), and in the system of MetHb-H2O2-ABTS they were 0.08 microM and 27.5, respectively. According to their antiradical activities, the inhibitors can be arranged as follows: poly(DSG) > poly(ADSNP) > PG > Trolox. PG, poly(DSG), poly (ADSNP), and its conjugate with HSA are suggested as "calibrators", i.e., inhibition standards for evaluation of antioxidant status of biological fluids in possible test systems based on the free radical-generating pair MetMb-H2O2 with ABTS as the acceptor of the active radicals.

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.

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.

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.

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.

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.

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.

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.

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.

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.