Molecular belt models for the apolipoprotein A-I Paris and Milano mutations

Models for the binding of the 200-residue carboxy-terminal domain of two mutants of apolipoprotein A-I (apo A-I), apo A-I(R173C)(Milano) and apo A-I(R151C)(Paris), to lipid in discoidal high-density lipoprotein (HDL) particles are presented. In both models, two monomers of the mutant apo A-I molecule bind to lipid in an antiparallel manner, with the long axes of their helical repeats running perpendicular to the normal of the lipid bilayer to form a single disulfide-linked homodimer. The overall structures of the models of these two mutants are very similar, differing only in helix-helix registration. Thus these models are consistent with experimental observations that reconstituted HDL particles containing apo A-I(Milano) and apo A-I(Paris) are very similar in diameter to reconstituted HDL particles containing wild-type apo A-I, and they support the belief that apo A-I binds to lipid in discoidal HDL particles via the belt conformation.     

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.     

Human apolipoprotein A-I and A-I mimetic peptides: potential for atherosclerosis reversal

PURPOSE OF REVIEW: Recent publications related to the potential use of apolipoprotein (apo)A-I and apoA-I mimetic peptides in the treatment of atherosclerosis are reviewed. RECENT FINDINGS: A preliminary report indicating that infusion of apoA-IMilano into humans once weekly for 5 weeks caused a significant decrease in coronary artery atheroma volume has sparked great interest in the potential therapeutic use of apoA-I. Recent studies have revealed that HDL quality (e.g. HDL apolipoprotein and lipid content, including oxidized lipids, particle size and electrophoretic mobility, associated enzymatic activities, inflammatory/anti-inflammatory properties, and ability to promote cholesterol efflux) may be more important than HDL-cholesterol levels. Therefore, when developing new strategies to raise HDL-cholesterol concentrations by interfering with HDL metabolism, one must consider the quality of the resulting HDL. In animal models, raising HDL-cholesterol levels by administering oral phospholipids improved both the quantity and quality of HDL and was associated with lesion regression. An apoA-I mimetic peptide, namely 4F synthesized from D-amino acids (D-4F), administered orally to mice did not raise HDL-cholesterol concentrations but promoted the formation of pre-beta HDL containing increased paraoxonase activity, resulting in significant improvements in HDL's anti-inflammatory properties and ability to promote cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol efflux from macrophages in vivo. SUMMARY: The quality of HDL may be more important than HDL-cholesterol levels. ApoA-I and apoA-I mimetic peptides appear to have significant therapeutic potential in atherosclerosis.     

Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.     

Expressed protein ligation-mediated template protein extension

Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I(Milano)(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I(Milano). The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.     

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.     

A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux

Carriers of the apolipoprotein A-I(Milano) (A-I(M)) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-I(M) carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-I(M) than control sera (3.4 +/- 1.0% versus 2.3 +/- 1.0% in macrophages; 5.2 +/- 2.4% versus 1.9 +/- 0.1% in fibroblasts). A-I(M) and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-I(M) sera contain normal amounts of apoA-I-containing prebeta-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-I(M) dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-I(M) serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing prebeta-HDL, together with a unique protease-sensitive, small HDL particle containing the A-I(M) dimer, both effective in removing cell cholesterol via ABCA1.     

Effects of probucol on plasma lipids, lipoproteins and parameters of high density lipoprotein metabolism.

Eight patients with primary hypercholesterolemia were treated with probucol for 17 weeks. Plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol decreased by 16.6, 15.0 and 25.7%, respectively, in response to probucol treatment. Plasma levels of apolipoprotein B and apolipoprotein A-I also decreased, while apolipoprotein A-II concentrations were unchanged. The decrease in HDL-cholesterol levels was associated with a reduction in HDL particle size. No changes in the plasma lecithin:cholesterol acyltransferase activity or mass occurred in response to probucol treatment. In contrast, a significant 25% increase in plasma cholesteryl ester and triglyceride transfer activity occurred following probucol treatment. There was a positive correlation (R = 0.94) between cholesterol ester and triglyceride transfer. We propose that the increase in lipid transfer activity may in part explain the changes in HDL concentration and size, as well as the previously reported effect probucol has on reducing atherosclerosis in animal models.     

Recent studies of lipoprotein kinetics in the metabolic syndrome and related disorders

PURPOSE OF REVIEW: Dyslipoproteinemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. Recent in-vivo kinetic studies of dyslipidemia in the metabolic syndrome are reviewed here. RECENT FINDINGS: The dysregulation of lipoprotein metabolism may be caused by a combination of overproduction of VLDL apolipoprotein B-100, decreased catabolism of apolipoprotein B-containing particles, and increased catabolism of HDL apolipoprotein A-I particles. Nutritional modifications and increased physical exercise may favourably alter lipoprotein transport by collectively decreasing the hepatic secretion of VLDL apolipoprotein B and the catabolism of HDL apolipoprotein A-I, as well as by increasing the clearance of LDL apolipoprotein B. Conventional and new pharmacological treatments, such as statins, fibrates and cholesteryl ester transfer protein inhibitors, can also correct dyslipidemia by several mechanisms, including decreased secretion and increased catabolism of apolipoprotein B, as well as increased secretion and decreased catabolism of apolipoprotein A-I. SUMMARY: Kinetic studies provide a mechanistic insight into the dysregulation and therapy of lipid and lipoprotein disorders. Future research mandates the development of new tracer methodologies with practicable in-vivo protocols for investigating fatty acid turnover, macrophage reverse cholesterol transport, cholesterol transport in plasma, corporeal cholesterol balance, and the turnover of several subpopulations of HDL particles.     

Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans

Amino acid precursors labelled with stable isotopes have been successfully used to explore the metabolism of the apolipoproteins of HDL. Some methodological and mathematical modelling problems remain, mainly related to amino acid recycling in a plasma protein such as apolipoprotein A-I with a long residence time (the reciprocal of the fractional catabolic rate) of 4-5 days. Apolipoprotein A-I, apolipoprotein E, and apolipoprotein A-IV in triglyceride-rich lipoproteins (containing chylomicrons, VLDL, and remnants) exhibit more complex kinetics. The small amounts of apolipoprotein A-I and of apolipoprotein A-IV in the triglyceride-rich lipoproteins have a residence time similar to that of the apolipoprotein A-I of HDL. In contrast, the apolipoprotein E in triglyceride-rich lipoproteins has been found to have an average residence time of 0.11 days. Diets low in saturated fat and cholesterol, which lower HDL levels, do so by decreasing the secretion of apolipoprotein A-I, with apolipoprotein A-II kinetics unaffected. Individuals with impaired glucose tolerance have a decreased residence time of apolipoprotein A-I but no change in secretion rate or in apolipoprotein A-II kinetics. This suggests a link between insulin resistance and the risk of atherosclerosis. In heterozygous familial hypercholesterolemia, both the fractional catabolic rate and the secretion rate of apolipoprotein A-I are increased, resulting in no change in the plasma level. Stable isotope studies have strengthened the evidence that triglyceride enrichment of HDL increases its catabolism Laboratory.     

Orp8 Deficiency in Bone Marrow-Derived Cells Reduces Atherosclerotic Lesion Progression in LDL Receptor Knockout Mice

Introduction

Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity.

Conclusions

Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.     

Interconversion of prebeta-migrating lipoproteins containing apolipoprotein A-I and HDL

Mouse plasma from strains C57BL/6J and C3H/HeJ includes a high density lipoprotein (HDL) fraction containing apolipoprotein A-I which migrates in the prebeta region upon agarose gel electrophoresis, similar to the prebeta HDL previously reported in humans. This prebeta A-I lipoprotein species has a buoyant density of 1.080-1.210 g/ml and has two molecular weight species, 65,000 and 71,000. It is lipid-poor and deficient in apolipoprotein E. When mice are fed a high fat and high cholesterol diet, the quantity of prebeta A-I increases in both strains as determined by quantitative densitometry of agarose gel immunoblots. Prebeta A-I species are highly unstable in plasma at 37 degrees C. Initially (0-1 h) levels decreased and with further incubation (1-8 h) levels increased. Nondenaturing polyacrylamide gel electrophoresis (PAGE) demonstrated that the prebeta HDL formed during prolonged incubation (1-8 h) was identical in size to HDL in unincubated samples. The initial decrease of prebeta HDL observed during the first hour of incubation, phase I, was inhibited by DTNB, suggesting that phase I is dependent on lecithin:cholesterol acyltransferase (LCAT); however, the subsequent increase, phase II, was unaffected by DTNB and appears LCAT-independent. The prebeta A-I species formed in plasma containing DTNB after a 4-h incubation resulted in a polydisperse particle size distribution. The two strains, the atherosclerosis-susceptible C57BL/6 and -resistant C3H, displayed a similar elevation and induction of prebeta HDL during a dietary switch from laboratory chow to an atherogenic diet with a transient peak occurring at 7 days even when total HDL in the susceptible strain was greatly reduced.     

Cholesterol transport via ABCA1: New insights from solid-phase binding assay

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).     

Cholesterol mobilization by free and lipid-bound apoAI(Milano) and apoAI(Milano)-apoAII heterodimers

Despite very low plasma levels of HDL, carriers of the apolipoprotein AI Arg173 --> Cys mutation apoAI(Milano) (AIM) have no apparent increase in risk for atherosclerotic vascular disease. HDL apolipoprotein species in AIM carriers include apoAI-AII heterodimers, previously found to confer the enhanced ability of tyrosyl radical-oxidized HDL to mobilize cholesterol for removal from cultured cells. To determine whether enhanced mobilization of cholesterol by apoprotein species in AIM explains a cardioprotective action of this mutation, we examined the ability of lipid-free and lipid-bound AIM and AIM-AII heterodimers to deplete cholesterol from cultured cells. Free AIM and AIM-AII heterodimers showed a decreased capacity to act as acceptors of cholesterol from cholesterol-loaded human fibroblasts compared with native apoAI but similar capacities to deplete fibroblasts of the pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Discoidal reconstituted HDL (rHDL) containing apoAI depleted both of these cholesterol pools more readily than AIM-containing rHDL when compared at equivalent rHDL protein levels, but similar abilities of these rHDL to deplete cell cholesterol were seen when compared at equivalent phospholipid levels. Spherical rHDL generated using the whole lipid fraction of HDL and apoAI or AIM showed similar capacities to deplete total and ACAT-accessible cell cholesterol when compared at similar protein levels, but an increased capacity of AIM-containing particles was seen when compared at equivalent phospholipid levels. Unlike the apoAI-AII heterodimer in tyrosylated HDL, AIM-AII heterodimer-containing spherical rHDL showed no increased capacity to deplete either of these pools of cholesterol. These results suggest a similar or better capacity of native apoAI in lipid-free or lipid-bound form in discoidal rHDL to enhance the mobilization of cellular cholesterol when compared to AIM in its free or lipid-bound forms. Any increase in depletion of cellular cholesterol by lipid-bound AIM in spherical rHDL appears related to altered phospholipid-binding rather than intrinsic cholesterol-mobilizing characteristics of this protein compared to native apoAI. The lack of major differences in these studies in cholesterol mobilization by native apoAI and AIM, or by apoAIM-AII heterodimers, suggests that any protection against atherosclerosis conferred by this mutation is likely related to other beneficial vascular effects of AIM.     

Metabolism of high density lipoprotein subfractions.

Over the past few years, new experimental approaches have reinforced the awareness among investigators that the heterogeneity of HDL particles indicates significant differences in production and catabolism of HDL particles. Recent kinetic studies have suggested that small HDL, containing two apolipoprotein A-I molecules per particle, are converted in a unidirectional manner to medium HDL or large HDL, containing three or four apolipoprotein A-I molecules per particle, respectively. Conversion appears to occur in close physical proximity with cells and not while HDL particles circulate in plasma. The medium and large HDL are terminal particles in HDL metabolism with large HDL, and perhaps medium HDL, being catabolized primarily by the liver. These novel kinetic studies of HDL subfraction metabolism are compelling in-vivo data that are consistent with the proposed role of HDL in reverse cholesterol transport.     

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

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

Uptake of endogenous cholesterol by a synthetic lipoprotein

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Characterization of A-I-containing lipoproteins in subjects with A-I Milano variant

The A-I Milano variant of apolipoprotein A-I (A-IM), by virtue of its Arg-173----Cys substitution, is capable of forming a disulfide bond with the 77-amino-acid apolipoprotein A-II polypeptide (A-IIS) as well as with itself to produce dimers, A-IM/A-IIS and A-IM/A-IM, respectively. A-I-containing lipoproteins (Lp): particles with A-II (Lp(A-I with A-11)) and particles without A-II (Lp(A-I without A-II)) in the plasma of two nonhyperlipidemic A-IM carriers were investigated to determine the effect of A-IM on these lipoproteins. Despite the existence of abnormal apolipoprotein dimers and the unusually low HDL cholesterol (17 and 14 mg/dl), A-I (67 and 75 mg/dl), and A-II (18 and 18 mg/dl) levels in the two carriers, the plasma A-I of the carriers was distributed between Lp(A-I with A-II) and Lp(A-I without A-II) in a proportion comparable to that observed in normals. As expected, A-IM/A-IIS mixed dimer was found in carrier Lp(A-I with A-II). However, A-IM/A-IM dimer was located almost exclusively in carrier Lp(A-I without A-II). Chemical (dimethylsuberimidate) crosslinking of the protein moieties of the major subpopulations of Lp(A-I with A-II) and Lp(A-I without A-II) of normal and A-IM carriers showed that Lp(A-I with A-II), which is located predominantly in the 7.8-9.7 nm interval ((HDL2a + 3a + 3b)gge), had an apparent protein molecular weight equivalent to two molecules of A-I and one to two molecules of A-II per particle. Most of the Lp(A-I without A-II) particles, located predominantly in the size intervals of 9.7-12.9 nm (designated (HDL2b)gge) and 8.2-8.8 nm (HDL3a)gge) had protein moieties exhibiting a molecular weight equivalence predominantly of four and three molecules of A-I, respectively. A small quantity of particles with apparent protein content of two molecules of A-I in the 7.2-8.2 nm interval ((HDL3b + 3c)gge) was also detected. These studies showed that in nonhyperlipidemic A-IM carriers, the occurrence of apolipoprotein dimers had not markedly affected the protein stoichiometry of Lp(A-I with A-II) and Lp(A-I without A-II).     

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.