Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.     

The role of high-density lipoprotein cholesterol in the prevention and possible treatment of cardiovascular diseases

Despite significant progress in the management of atherosclerosis and its resultant complications, cardiovascular disease remains the principal cause of death in the world. The National Cholesterol Education Project Adult Treatment Panel III (NCEP ATP III) recognizes low levels of high-density lipoprotein cholesterol (HDL) as a risk factor for coronary heart disease (CHD) and high levels of HDL as a risk-reducing factor; however, the elevation of HDL as a specific therapeutic target for the prevention and treatment of CHD has yet to be accepted on the same level as low-density lipoprotein (LDL)-reducing therapies. Current HDL elevators including nicotinic acid, fibric acid derivatives, peroxisome proliferator activated receptor (PPAR) agonists and statins also affect other lipid constituents which make interpretation of the clinical trials of these drugs difficult in teasing out the independent effect of HDL elevation. Ample laboratory investigation suggests that HDL elevation would reduce atherosclerotic burden through multiple independent mechanisms. In this review, we explore HDL biology, its potential mechanisms in the treatment of atherosclerotic disease, and promising new drugs with HDL-raising activity.     

Obesity-related changes in high-density lipoprotein metabolism

Obesity is associated with a 3-or-more-fold increase in the risk of fatal and nonfatal myocardial infarction (1,2,3,4,5,6). The American Heart Association has reclassified obesity as a major, modifiable risk factor for coronary heart disease (7). The increased prevalence of premature coronary heart disease in obesity is attributed to multiple factors (8,9,10). A principal contributor to this serious morbidity is the alterations in plasma lipid and lipoprotein levels. The dyslipidemia of obesity is commonly manifested as high plasma triglyceride levels, low high-density lipoprotein cholesterol (HDLc), and normal low-density lipoprotein cholesterol (LDLc) with preponderance of small dense LDL particles (7,8,9,10). However, there is a considerable heterogeneity of plasma lipid profile in overweight and obese people. The precise cause of this heterogeneity is not entirely clear but has been partly attributed to the degree of visceral adiposity and insulin resistance. The emergence of glucose intolerance or a genetic predisposition to familial combined hyperlipidemia will further modify the plasma lipid phenotype in obese people (11,12,13,14,15).     

Differential roles of cysteine residues in the cellular trafficking, dimerization, and function of the high-density lipoprotein receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.     

Effect of obesity on high-density lipoprotein metabolism

Reduced levels of high-density lipoproteins (HDL) in non-obese and obese states are associated with increased risk for the development of coronary artery disease. Therefore, it is imperative to determine the mechanisms responsible for reduced HDL in obese states and, conversely, to examine therapies aimed at increasing HDL levels in these individuals. This paper examines the multiple causes for reduced HDL in obese states and the effect of exercise and diet--two non-pharmacologic therapies--on HDL metabolism in humans. In general, the concentration of HDL-cholesterol is adversely altered in obesity, with HDL-cholesterol levels associated with both the degree and distribution of obesity. More specifically, intra-abdominal visceral fat deposition is an important negative correlate of HDL-cholesterol. The specific subfractions of HDL that are altered in obese states include the HDL2, apolipoprotein A-I, and pre-beta1 subfractions. Decreased HDL levels in obesity have been attributed to both an enhancement in the uptake of HDL2 by adipocytes and an increase in the catabolism of apolipoprotein A-I on HDL particles. In addition, there is a decrease in the conversion of the pre-beta1 subfraction, the initial acceptor of cholesterol from peripheral cells, to pre-beta2 particles. Conversely, as a means of reversing the decrease in HDL levels in obesity, sustained weight loss is an effective method. More specifically, weight loss achieved through exercise is more effective at raising HDL levels than dieting. Exercise mediates positive effects on HDL levels at least partly through changes in enzymes of HDL metabolism. Increased lipid transfer to HDL by lipoprotein lipase and reduced HDL clearance by hepatic triglyceride lipase as a result of endurance training are two important mechanisms for increases in HDL observed from exercise.     

Cubilin, a high-density lipoprotein receptor

The metabolism of HDL particles is a complex biological process involving various regulating factors in plasma and different cellular receptors. In addition to the well-established scavenger receptor BI-mediated selective HDL-cholesteryl ester uptake in liver and steroidogenic tissues, evidence has been provided that HDL also undergoes holoparticle endocytosis in different tissues. Recently, a novel receptor expressed in various absorptive epithelia was disclosed as a high affinity receptor for endocytosis of HDL and lipid-poor apolipoprotein AI. This receptor, designated cubilin, may play an important role in the renal clearance of filterable apolipoprotein AI/HDL and in the maternal-fetal transport of cholesterol.     

Genome-wide linkage analysis of population variation in high-density lipoprotein cholesterol

Lower plasma levels of high-density lipoprotein cholesterol (HDL-C) are associated with the metabolic syndrome (insulin resistance, obesity, hypertension) and higher cardiovascular risk. Recent association studies have suggested rare alleles responsible for very low HDL-C levels. However, for individual cardiovascular risk factors, the majority of population-attributable deaths are associated with average rather than extreme levels. Therefore, genetic factors that determine the population variation of HDL-C are particularly relevant. We undertook genome-wide and fine mapping to identify linkage to HDL-C in healthy adult nuclear families from the Victorian Family Heart Study. In 274 adult sibling pairs (average age 24 years, average plasma HDL-C 1.4 mmol/l), genome-wide mapping revealed suggestive evidence for linkage on chromosome 4 (Z score=3.5, 170 cM) and nominal evidence for linkage on chromosomes 1 (Z=2.1, 176 cM) and 6 (Z=2.6, 29 cM). Using genotypes and phenotypes from 932 subjects (233 of the sibling pairs and their parents), finer mapping of the locus on chromosome 4 strengthened our findings with a peak probability (Z score=3.9) at 169 cM. Our linkage data suggest that chromosome 4q32.3 is linked with normal population variation in HDL-C. This region coincides with previous reports of linkage to apolipoprotein AII (a major component of HDL) and encompasses the gene encoding the carboxypeptidase E, relevant to the metabolic syndrome and HDL-C. These findings are relevant for further understanding of the genetic determinants of cardiovascular risk at a population level.     

Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [3H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma.     

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.     

Cholesteryl ester transfer protein expression partially attenuates the adverse effects of SR-BI receptor deficiency on cholesterol metabolism and atherosclerosis

Scavenger receptor SR-BI significantly contributes to HDL cholesterol metabolism and atherogenesis in mice. However, the role of SR-BI may not be as pronounced in humans due to cholesteryl ester transfer protein (CETP) activity. To address the impact of CETP expression on the adverse effects associated with SR-BI deficiency, we cross-bred our SR-BI conditional knock-out mouse model with CETP transgenic mice. CETP almost completely restored the abnormal HDL-C distribution in SR-BI-deficient mice. However, it did not normalize the elevated plasma free to total cholesterol ratio characteristic of hepatic SR-BI deficiency. Red blood cell and platelet count abnormalities observed in mice liver deficient for SR-BI were partially restored by CETP, but the elevated erythrocyte cholesterol to phospholipid ratio remained unchanged. Complete deletion of SR-BI was associated with diminished adrenal cholesterol stores, whereas hepatic SR-BI deficiency resulted in a significant increase in adrenal gland cholesterol content. In both mouse models, CETP had no impact on adrenal cholesterol metabolism. In diet-induced atherosclerosis studies, hepatic SR-BI deficiency accelerated aortic lipid lesion formation in both CETP-expressing (4-fold) and non-CETP-expressing (8-fold) mice when compared with controls. Impaired macrophage to feces reverse cholesterol transport in mice deficient for SR-BI in liver, which was not corrected by CETP, most likely contributed by such an increase in atherosclerosis susceptibility. Finally, comparison of the atherosclerosis burden in SR-BI liver-deficient and fully deficient mice demonstrated that SR-BI exerted an atheroprotective activity in extra-hepatic tissues whether CETP was present or not. These findings support the contention that the SR-BI pathway contributes in unique ways to cholesterol metabolism and atherosclerosis susceptibility even in the presence of CETP.     

Acute changes in high-density lipoprotein cholesterol with exercise of different intensities

The purpose of this study was to investigate the acute effects of exercise on plasma high-density lipoprotein cholesterol (HDL-C) and to determine whether the magnitude of this response would be affected by the intensity of the exercise. Twelve men (19-41 yr) ran an equivalent distance (9-12 km) on a treadmill on two separate occasions. On one occasion the exercise was performed at a speed that elicited 60% of the subject's maximal O2 uptake (VO2max), and on the other occasion exercise was performed at a speed that elicited 90% of VO2max. Changes in total cholesterol, triglycerides (TG), HDL-C, HDL apoprotein A (HDL-A), HDL saturation, lactate (LA), and free fatty acids (FFA) were measured during the course of each run, and all values were corrected for changes in plasma volume as indicated by hematocrit. There were significant increases (P less than 0.01) in HDL-C, HDL-A, and HDL saturation with exercise at both intensities, but greater increases in HDL-C (25 vs. 14%) and HDL-A (18 vs. 8%) were observed with the higher intensity exercise. Plasma FFA and TG did not differ between conditions, but LA concentrations rose significantly during the high-intensity exercise. These results indicate that increases in HDL components can occur with a relatively moderate exercise session and that the magnitude of these increases are directly related to the exercise intensity.     

Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis

In the past several years significant advances have been made in our understanding of lecithin-cholesterol acyltransferase (LCAT) function. LCAT beneficially alters the plasma concentrations of apolipoprotein B-containing lipoproteins, as well as HDL. In addition, its proposed role in facilitating reverse cholesterol transport and modulating atherosclerosis has been demonstrated in vivo. Analysis of LCAT transgenic animals has established the importance of evaluating HDL function, as well as HDL plasma levels, to predict atherogenic risk.     

Linkage of the apo CIII microsatellite with isolated low high-density lipoprotein cholesterol

To evaluate whether a highly polymorphic mic-rosatellite region within intron 3 of the apolipoprotein (apo) CIII gene is linked to the isolated low HDL-C phenotype, we studied eight unrelated probands (mean HDL-C=10 ±5mg/dl) and 157 biological family members. After PCR amplification of genomic DNA and denaturing polyacrylamide gel electrophoresis, 26 alleles were identified in this microsatellite including 9 alleles heretofore unreported. Quantitative sib-pair linkage analysis demonstrated strong evidence of linkage between the isolated low HDL-C phenotype and the apo CIII microsatellite region (P=0.007). The microsatellite was also linked to apo AI (P=0.001), the primary apolipoprotein of HDL-C. Therefore, this highly polymorphic microsatellite region is a potentially important marker in the genetic evaluation of the isolated low HDL-C phenotype. Received: 25 October 1996 / Accepted: 29 October 1997     

Immunochromatographic membrane strip assay system for a single-class plasma lipoprotein cholesterol, exemplified by high-density lipoprotein cholesterol measurement

In assessing risk factors of coronary heart disease, a membrane immunochromatographic system that minimizes requirements of instrument and reagent handling was investigated by utilizing high-density lipoprotein (HDL) cholesterol (HDL-C) as model analyte. The system is composed of four functional membrane strip pads connected in sequence as follows (from the bottom): immunoseparation based on the biotin-streptavidin reaction; catalytic conversion of cholesterol to hydrogen peroxide; production of a colorimetric signal; and induction of a continuous wicking of medium. For immunochromatography, a monoclonal antibody, specific to apolipoprotein B100 that is present on the surfaces of low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL), with a high binding constant (5 x 10(10) L/mol), was raised and chemically conjugated to streptavidin. The conjugate was first reacted with lipoprotein particles, and this mixture was absorbed by the capillary action into the biotin pad of the system. After being transferred by medium, immunocapture of LDL and VLDL particles onto the biotin pad took place, and in situ generation of a colorimetric signal in proportion to HDL-C occurred consecutively. The capture was selective as well as effective (minimum 88% of LDL and VLDL in clinical concentration ranges), and the detection limit of the HDL-C was far lower than 20 mg per 100 mL. The same concept may also be applicable to LDL cholesterol measurement provided suitable antibodies specific to HDL and VLDL are available.     

Apolipoprotein M--a novel player in high-density lipoprotein metabolism and atherosclerosis

PURPOSE OF REVIEW: Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. RECENT FINDINGS: Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand-binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein-receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. SUMMARY: Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis.     

Total and high-density lipoprotein cholesterol measurements. Hazards in clinical interpretation.

Duplicate plasma specimens from 24 persons were sent to a community hospital, a commercial laboratory, and a university lipid research laboratory at two separate times to assess the intralaboratory and interlaboratory variations of total and high-density cholesterol measurements. For all three laboratories, the 95% confidence limits for the reproducibility of total cholesterol levels are about +/- 5.5%. For high-density lipoprotein (HDL) values, they are +/- 7%, +/- 14.45%, and +/- 9%. Interlaboratory differences for total cholesterol at the 95% confidence level are +/- 7.7% and for HDL +/- 18.4%. Construction of a "cardiac risk ratio" of total to HDL cholesterol levels is subject to confusion because small errors in HDL cholesterol levels produce large errors in the ratio.