Fu5AH Rat Hepatoma Cells Research Articles

Impact of serum amyloid A on cellular cholesterol efflux to serum in type 2 diabetes mellitus

ObjectiveSerum amyloid A (SAA) is an acute phase response protein and has apolipoprotein properties. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective of this study is to investigate the changes in SAA level in type 2 diabetic patients and to evaluate the relationship between SAA and the capacity of serum to induce cellular cholesterol efflux via the two known cholesterol transporters, scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G1 (ABCG1). Methods264 patients with type 2 diabetes mellitus (42% with normoalbuminuria, 30% microalbuminuria, and 28% proteinuria) and 275 non-diabetic controls were recruited. SAA was measured by ELISA. SR-BI and ABCG1-mediated cholesterol efflux to serum were determined by measuring the transfer of [3H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from human ABCG1-transfected CHO-K1 cells to the medium containing the tested serum respectively. ResultsSAA was significantly increased in diabetic patients with incipient or overt nephropathy. Both SR-BI and ABCG1-mediated cholesterol efflux to serum were significantly impaired in all three groups of diabetic patients (p < 0.01). SAA inversely correlated with SR-BI-mediated cholesterol efflux (r = −0.36, p < 0.01) but did not correlate with ABCG1-mediated cholesterol efflux. Stepwise linear regression analysis showed that HDL, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum. ConclusionSAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum.

Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter

ObjectiveThe goal of this study was to examine the effects of thyroid hormone status on the ability of serum to accept cellular cholesterol. Methods and resultsSera from hypophysectomized rats treated ±T3 was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T3 treated rats had increased ApoA-I, ApoA-IV and fetuin A levels with decreased Apo E levels. Microarray and real-time RT-PCR analysis of rat liver revealed large increases in ApoA-I, ApoA-IV, ABCG5, and ABCG8 in response to T3. J774 macrophages, BHK cells, and Fu5AH rat hepatoma cells were used to measure cholesterol efflux mediated by ABCA1, ABCG1 transporters or SR-BI. Sera from T3-treated rats stimulated efflux via ABCA1 but not by ABCG1 or SR-BI. Gel filtration chromatography revealed that T3 treatment caused a decrease in HDL particle size accompanied by higher levels of lipid-poor ApoA-I. ConclusionsThyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter. This effect is most likely attributable to increases in small HDL and lipid poor ApoA-I in response to T3.

High-density lipoprotein-associated 17β-estradiol fatty acyl ester uptake by Fu5AH hepatoma cells: Implications of the roles of scavenger receptor class B, type I and the low-density lipoprotein receptor

17β-Estradiol (E 2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E 2 cellular uptake and to determine the intracellular distribution of E 2 and its fatty acyl esters (E 2-FAE) after uptake. [ 3H]E 2 or [ 3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [ 3H]E 2-FAE or [ 3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[ 3H]cholesterol, cellular uptake of HDL-[ 3H]E 2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E 2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E 2 uptake. HDL-E 2-FAE was hydrolyzed into free E 2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E 2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E 2-FAE into free E 2 ensues.

Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity

Fu5AH rat hepatoma cells and cAMP (cyclic AMP)-pretreated J774 mouse macrophages are commonly used as models for SR-BI (scavenger receptor class B type I) and ABCA1 (ATP binding cassette transporter 1)-mediated free cholesterol efflux to whole serum, respectively. However, the responsiveness of Fu5AH, control or cAMP pretreated J774 cells to the various lipids and HDL (high-density lipoprotein)-parameters from both normo- and dyslipidaemic subjects has never been compared within the same study. Fifty-eight men were classified into four groups: type IIa hypercholesterolaemic (n = 12), type IIb dyslipidaemic (n = 13), type IV hypertriglyceridaemic (n = 18) and normolipidaemic (n = 15) were recruited. A complete lipid profile including prebeta-HDL was performed. Cholesterol efflux from Fu5AH cells as well as from control or cAMP pretreated J774 cells were measured; the difference between these two latter values being taken as the ABCA1-mediated efflux. The Fu5AH and the control J774 cells delivered cholesterol to mature HDLs, especially to phospholipid (PL)-rich HDL. Using cAMP pretreated cells, the ABCA1-dependent efflux was highly sensitive to prebeta-HDL, which appeared to be a factor in determining the efflux. Consistent with the dependence of the SR-BI-mediated efflux on HDL-PL levels, which are not different between groups, all sera displayed similar efflux capacities from the Fu5AH cells. Conversely, in accordance with their high prebeta-HDL levels, the ABCA1-dependent efflux highlighted the efficiency of type IV sera. Two complementary cellular models providing SR-BI and ABCA1-dependent efflux should be used to measure the capacity of a biological fluid which contains a wide variety of components to promote cholesterol efflux.

Measurement of cholesterol bidirectional flux between cells and lipoproteins

We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.

A low‐saturated‐fat, low‐cholesterol diet decreases plasma CETP activity and pre β‐HDL formation but does not affect cellular cholesterol efflux to plasma from type 1 diabetic patients

The aim of this study was to evaluate the effect of a low‐saturated‐fat, low‐cholesterol diet on plasma lipopoproteins, pre β‐high density lipoprotein (HDL) formation, lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activities, as well as on the ability of plasma to stimulate cellular cholesterol efflux. Twelve male type 1 diabetic patients with plasma cholesterol >5.0 mmol/L were studied while consuming their usual diet and after 6 weeks of a low‐fat, low‐cholesterol diet. Pre β‐HDL formation was measured using crossed immuno‐electrophoresis. Plasma LCAT, CETP and PLTP activities were assayed by exogenous substrate methods. The ability of plasma to promote cellular cholesterol efflux out of Fu5AH rat hepatoma cells and out of human skin fibroblasts was also determined. Saturated fat intake was lowered (p = 0.001) due to replacement with carbohydrates, while mono‐ and polyunsaturated fat intake remained unchanged. Cholesterol intake decreased as well (p = 0.003). The changes in plasma total cholesterol, very low and low‐density lipoprotein (VLDL+LDL) cholesterol, HDL cholesterol, HDL phospholipids, apolipoprotein (apo) A‐I, plasma LCAT activity and PLTP activity were not significant. Plasma CETP activity (p = 0.008) and pre β‐HDL formation (p = 0.008) decreased. The ability of plasma to promote cholesterol efflux out of fibroblasts and Fu5AH cells remained unchanged. Reduction in dietary saturated fat and cholesterol intake does not adversely affect cellular cholesterol efflux to plasma from type 1 diabetic patients, despite a drop in pre β‐HDL formation.

Pitavastatin increases ABCA1-mediated lipid efflux from Fu5AH rat hepatoma cells

ATP binding cassette A1 (ABCA1) is responsible in vivo for the formation of HDL by promoting the lipidation of apoprotein A-I (apoA-I) via cholesterol and phospholipid efflux from the liver. Treatment of patients with statins produces an increase in HDL plasma level, but the underlying mechanism is not completely understood. In this work we investigated the ability of pitavastatin to modulate ABCA1-mediated efflux from Fu5AH rat hepatoma cells, that here we demonstrate to express functional ABCA1 upon treatment with 22OH/cRA. In both basal and ABCA1 expressing cells pitavastatin 0.1–50 μM induced a dose-dependent increase in cholesterol efflux to apoA-I; this effect was reversed by mevalonate or geranyl geraniol. A stimulatory effect was also observed on phospholipid efflux. Similar results were obtained with compactin, suggesting a class-related effect of statins. These results indicate a potential mechanism for the improvement in HDL plasma profile observed in patients treated with statins.

W13.316 Cholesterol efflux potential of sera from subjects with LCAT deficiency

17β-Estradiol (E2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E2 cellular uptake and to determine the intracellular distribution of E2 and its fatty acyl esters (E2-FAE) after uptake. [3H]E2 or [3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [3H]E2-FAE or [3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[3H]cholesterol, cellular uptake of HDL-[3H]E2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E2 uptake. HDL-E2-FAE was hydrolyzed into free E2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E2-FAE into free E2 ensues.

Depletion of Pre-β-high Density Lipoprotein by Human Chymase Impairs ATP-binding Cassette Transporter A1- but Not Scavenger Receptor Class B Type I-mediated Lipid Efflux to High Density Lipoprotein

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.

Analysis of the relationship between triglyceridemia and HDL-phospholipid concentrations: consequences on the efflux capacity of serum in the Fu5AH system

The high triglyceride/low HDL-cholesterol trait is a common finding in the general population. The aim of the present study was to analyze and interpret the relationships between triglycerides (TG), HDL-related parameters and serum cholesterol efflux potential in an asymptomatic population including both normo- and hyperlipidemic individuals. In a large sample ( n=1143) of this population, there was a negative correlation between TG and HDL-cholesterol (HDL-C) ( r=−0.49, P<0.0001) whereas the negative correlation between TG and HDL-phospholipid (HDL-PL) ( r=−0.29, P<0.0001) was weaker, leading to a strong positive correlation between TG and HDL-PL/C ratio ( r=0.58, P<0.0001). Thus, increased TG concentrations were associated with an enrichment of HDL with PL. Since we have demonstrated previously that HDL-PL is the major determinant for cholesterol efflux potential from Fu5AH rat hepatoma cells, we determined the effect of the variations in HDL lipid composition on the cholesterol efflux capacity in a subsample of 198 subjects. Compared with normolipidemic subjects (NLP) (TG≤1.7 mmol/l; LDL-C≤4.1 mmol/l, n=58), hypertriglyceridemic subjects (HTG) (TG>1.7 mmol/l, n=63) exhibited lower HDL-C levels (1.08±0.21 vs. 1.25±0.32, P=0.0003) whereas they showed similar HDL-PL concentrations (1.25±0.21 vs. 1.25±2.7) and, thus, higher HDL-PL/C ratio (1.17±0.15 vs. 1.02±0.14, P=0.0001). The relative efflux capacity of serum measured in the Fu5AH system (5% serum, 4 h incubation at 37°C) was on average identical in the HTG and NLP groups. Thus, this study provides evidence that despite decreased HDL concentrations, as determined routinely by the HDL-C assay, some HTG subjects maintained serum cholesterol efflux capacity thanks to the enrichment of HDL with PL.

Cholesterol Efflux Capacity In Vitro Predicts the Severity and Extent of Coronary Artery Disease in Patients with and without Type 2 Diabetes

Objective - To investigate the relation between severity and extent of coronary artery disease (CAD) and in vitro cholesterol efflux capacity. Design - This study consisted of 46 type 2 diabetic, and 42 nondiabetic men undergoing coronary angiography. Quantitative coronary angiography was used to estimate the severity, extent, and overall "atheroma burden" of CAD. The capacity of patient plasma to induce cholesterol efflux from cultured Fu5AH rat hepatoma cells was measured in vitro. Results - In the combined study population (n = 88), there was a significant inverse correlation between efflux and global atheroma burden (r = -0.23, p < 0.05). In the diabetic group, the global atheroma burden index was independently associated both with cholesterol efflux and with LpA-I levels. However, in the nondiabetic CAD group this association was lost when efflux and LpA-I levels were included in the same model. Conclusion - The present study demonstrated that efflux capacity was inversely associated with the severity and extent of CAD. In the diabetic group this association was independent of LpA-I levels, suggesting impaired antiatherogenic potential of these particles in type 2 diabetic patients.

Cholesterol efflux from Fu5AH cells to the serum of patients with Alagille syndrome: importance of the HDL-phospholipids/free cholesterol ratio and of the HDL size distribution

We have previously described the lipoprotein abnormalities in cholestatic children with paucity of interlobular bile ducts (PILBD), and we have shown that two different profiles emerged among these patients, depending on the level of lecithin:cholesterol acyltransferase (LCAT) activity. Reduced LCAT activity was associated with hypo-α-lipoproteinemia (group I) whereas normal LCAT activity was associated with hyper-α-lipoproteinemia (group II). In both groups, high density lipoproteins (HDL) were enriched with phospholipids and LpA-I particles were pre-dominant. Here, we have investigated the ability of serum and of isolated HDL, obtained from PILBD and control subjects, to promote cellular cholesterol efflux, from Fu5AH rat hepatoma cells. The mean fractional efflux to 5% serum in each group was, on average, following the differences in HDL concentrations (control: 30.1 ± 4.2%; group I: 23.7 ± 7.9%, ns; group II: 44.2 ± 6.5%, P < 0.001). The variations in efflux values in group II were positively correlated to the variations in HDL-PL concentrations (P < 0.0001) and in HDL-PL to serum apo-AI ratio (P < 0.003). By contrast, the variation in efflux in group I was only positively related to the large range of HDL-PL to free cholesterol (FC) ratio values (P < 0.0004). Fractional efflux to isolated HDL, measured at a constant HDL-PL amount, confirmed this relationship (P < 0.0001). Two-dimensional gel electrophoresis of the HDL size and apo A-I distribution in serum, revealed that small size HDL3 and pre-β HDL were predominant in the serum of patients from group I, especially those exhibiting low HDL-PL to FC ratio, whereas in the serum of patients from group II, both small HDL3 and large HDL2 were present. These results suggest that a combination of an imbalance between phospholipids and free cholesterol in the HDL particles and a deficit in large acceptors of cholesterol will be responsible for an impairment of cellular cholesterol efflux in PILBD patients with reduced lecithin:cholesterol acyltransferase activity.—Davit-Spraul, A., V. Atger, M. L. Pourci, M. Hadchouel, A. Legrand, and N. Moatti. Cholesterol efflux from Fu5AH cells in the serum of patients with Alagille syndrome: importance of the HDL-phospholipids/free cholesterol ratio and of the HDL size distribution. J. Lipid Res. 1999. 40: 328–335.

Abnormal capacity to induce cholesterol efflux and a new LpA-I pre-β particle in type 2 diabetic patients

In this study, we first characterized the lipoprotein components of serum samples obtained from a group of well-controlled diabetic patients and from healthy subjects in fasting and postprandial states. We then explored some aspects of reverse cholesterol transport in the same population. Patients showed high levels of fasting triglycerides, postprandial triglyceride responses and LpC-III levels (3.18±0.86 vs 2.17±0.54 mg/dl, P<0.001). There were also positive correlations between LpC-III and fasting triglycerides ( r=0.82, P<0.001), total triglyceride area ( r=0.75, P<0.001) and incremental triglyceride area ( r=0.54, P<0.001). HDL-C and apo A-I were significantly decreased in diabetic patients due to a selective reduction in LpA-I subfraction, whose antiatherogenic role is generally accepted (37.4±8.0 vs 49.2±12.5 mg/dl, P<0.001). In addition, HDL from patients proved to be triglyceride enriched and cholesteryl ester depleted, alterations which were further amplified in the postprandial state. The molar ratio HDL-C/apo A-I+apo A-II, already defined as a predictor of apo A-I fractional catabolic rate, was significantly diminished in the patient group (15.1±2.2 vs 20.8±3.3, P<0.001), thus suggesting an accelerated catabolism of apo A-I. For the first time, we describe here the presence of a small apo A-I-containing particle, isolated by two-dimensional electrophoresis and characterized by immunoblotting, only in samples from diabetic patients. This particle that we named pre-β 0, has an apparent molecular weight of 40 kDa. As regards the capacity of serum samples to promote cholesterol efflux from [ 3H]cholesterol-labeled Fu5AH rat hepatoma cells, patient samples were found to induce significantly lower cholesterol efflux than controls only in the postprandial state (21.2±3.3 vs 23.8±1.8%, P=0.012). The presence of pre-β 0 in samples from diabetic patients might therefore be associated to an altered capacity of these serum samples to promote cellular cholesterol efflux. Overall, these abnormalities may contribute to a delay in the reverse cholesterol transport pathway in type 2 diabetic patients.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins

This study investigates the suitability of the trimeric apolipoprotein (apo) AI(145–183) peptide that we recently described, to serve as a model to probe the relationship between apo AI structure and function. Three copies of the apoAI(145–183) unit, composed each of two amphipatic α-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102–140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)–apoAI complexes. The DMPC–trimeric-apoAI(145–183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149–186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145–183) bind specifically to HeLa cells with comparable affinity to the DMPC–apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145–183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145–183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102–140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102–140) construct had low affinity for lipid compared with the (145–183) construct. After association with lipids, it was a poor competitor of DMPC–apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure–function relationship of apoAI.

Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice.

The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. The contribution of specific lipoprotein components to this process remains to be established. In this study, the role of human apolipoproteins (apo) A-I and A-II in the efflux of cellular cholesterol was investigated in transgenic mouse models in which the expression of murine apoA-I was abolished due to gene targeting (A-IKO). Serum from A-IKO mice and from mice expressing human apoA-I and/or human apoA-II was incubated with [3H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours at 37 degrees C. The cholesterol efflux to the serum of A-IKO mice was markedly lower than that to the serum of mice transgenic for human apoA-I (5.0 +/- 1.5% versus 25.0 +/- 4.0%). Expression of human apoA-II alone did not modify the cholesterol efflux capacity of A-IKO mouse serum. Cholesterol efflux to serum of mice expressing human apoA-II together with human apoA-I was significantly lower than that to human apoA-I mouse serum (20.0 +/- 2.3% versus 25.0 +/- 4.0%). Regression analysis of cholesterol efflux versus the lipid/apolipoprotein concentrations of mouse serum suggested that 3 independent factors contribute to determine the cholesterol efflux potential of serum: the apolipoprotein composition of HDL, the serum concentration of HDL phospholipids, and the presence of a small fraction of particles containing apoA-I.

Use of cyclodextrins for manipulating cellular cholesterol content

Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.

Subclasses of LpA‐I in coronary artery disease: distribution and cholesterol efflux ability

We analysed the distribution of LpA-I particles according to their molecular weight in 34 men with symptomatic coronary artery disease (CAD) and 11 men with no symptoms of CAD (control group). Using an original rapid and reproducible gradient gel electrophoresis technique, three LpA-I subclasses were defined: large (L-LpA-I), intermediate (I-LpA-I) and small LpA-I (S-LpA-I). The proportion of L-LpA-I was significantly lower in the CAD group (37.5 +/- 18.5%) than in the control group (58.9 +/- 15.0%) (P < 0.01). Conversely, a significantly (P < 0.05) higher proportion of I-LpA-I (31.9 +/- 20.7%) was observed in the CAD group compared with the control group (14.2 +/- 8.2%). Also, in the CAD group, the proportion of L-LpA-I was positively associated with the plasma level of LpA-I (P < 0.05) and, conversely, the proportion of S-LpA-I was negatively associated with LpA-I levels (P < 0.01). L-LpA-I and I-LpA-I from CAD patients and from control subjects were most effective in promoting cholesterol efflux from Fu5AH rat hepatoma cells, whereas S-LpA-I was ineffective in this regard. In conclusion, the decreased ratio in CAD patients of L-LpA-I, lipoprotein subspecies that are required for cholesterol efflux from cells, suggests a potential anti-atherogenic effect of these particles associated with the larger LpA-I subfractions.

Remodeling and Shuttling

In normal physiology, cells are exposed to cholesterol acceptors of different sizes simultaneously. The current study examined the possible interactions between two different classes of acceptors, one large (large unilamellar phospholipid vesicles, LUVs) and one small (HDL or other small acceptors), added separately or in combination to Fu5AH rat hepatoma cells. During a 24-hour incubation, LUVs of palmitoyl-oleoyl phosphatidylcholine at 1 mg phospholipid (PL) per milliliter extracted approximately 20% of cellular unesterified cholesterol (UC) label and mass in a slow, continuous fashion (half-time [t1/2] for UC efflux was approximately 50 hours) and human HDL3 at 25 micrograms PL per milliliter extracted approximately 15% cellular UC label with no change in cellular cholesterol mass (t1/2 of approximately 8 hours). In contrast, the combination of LUVs and HDL3 extracted over 90% of UC label (t1/2 of approximately 4 hours) and approximately 50% of the UC mass, indicating synergy. To explain this synergy, specific particle interactions were examined, namely, remodeling, in which the two acceptors alter each other's composition and thus the ability to mobilize cellular cholesterol, and shuttling, in which the small acceptor ferries cholesterol from cells to the large acceptor. To examine remodeling, LUVs and HDL were coincubated and reisolated before application to cells. This HDL became UC depleted, PL enriched, and lost a small amount of apolipoprotein A-I. Compared with equivalent numbers of control HDL particles; remodeled HDL caused faster efflux (t1/2 approximately 4 hours) and exhibited a greater capacity to sequester cellular cholesterol over 24 hours (approximately 38% versus approximately 15% for control HDL), consistent with their enrichment in PL. Remodeled LUVs still extracted approximately 20% of cellular UC. Thus, remodeling accounted for some but not all of the synergy between LUVs and HDL. To examine shuttling, several approaches were used. First, reisolation of particles after an 8-hour exposure to cells revealed that HDL contained very little of the cellular UC label. The label was found almost entirely with the LUVs, suggesting that LUVs continuously stripped the HDL of cellular UC. Second, bidirectional flux studies demonstrated that LUVs blocked the influx of HDL UC label into cells, while the rate of efflux of cellular UC was maintained. These kinetic effects explained the massive net loss of cellular UC to LUVs with HDL. Third, cyclodextrin, an artificial small acceptor that does not acquire PL and hence does not become remodeled, exhibited substantial synergy with LUVs, supporting shuttling. Thus, the presence of large and small acceptors together can overcome intrinsic deficiencies in each. Small acceptors are efficient at extracting cellular cholesterol because they approach cell surfaces easily but have a low capacity, whereas large acceptors are inefficient but have a high capacity. When present simultaneously, where the small acceptor can transfer cholesterol quickly to the large acceptor, high efficiency and high capacity are achieved. The processes responsible for this synergy, namely, remodeling and shuttling, may be general phenomena allowing cooperation both during normal physiology and after therapeutic administration of acceptors to accelerate tissue cholesterol efflux in vivo.

Cholesterol efflux from Fu5AH hepatoma cells induced by plasma of subjects with or without coronary artery disease and non-insulin-dependent diabetes: importance of LpA-I:A-II particles and phospholipid transfer protein

We measured the capacity of human plasma to induce cholesterol efflux from Fu5AH rat hepatoma cells in four groups of men with or without non-insulin-dependent diabetes mellitus (NIDDM) and coronary artery disease (CAD). Plasma from men with both NIDDM and CAD ( n = 47) had the lowest efflux capacity (17.3 ± 3.6%) whereas healthy control subjects with neither diabetes nor CAD ( n = 25) had the highest capacity (19.8 ± 3.4%). The groups with CAD but no diabetes ( n = 44) and with NIDDM but no CAD ( n = 35) had intermediate efflux values (18.5 ± 3.8 and 18.5 ± 3.9%, respectively). In a 2 × 2 factorial ANOVA, the differences were significant with respect to the presence of CAD ( P = 0.038) and NIDDM ( P = 0.041), with no interaction between the factors. The concentration of HDL particles containing apolipoprotein (apo) A-I but no apo A-II (LpA-I) was not related to efflux capacity in univariate or multivariate analyses. A multivariate regression analysis showed that when controlled for the presence of NIDDM and CAD, the concentration of particles containing both apo A-I and apo A-II (LpA-I:A-II) and plasma phospholipid transfer protein activity were both positively, independently, and significantly ( P < 0.001) related to cholesterol efflux capacity.

High-density lipoprotein 3 physicochemical modifications induced by interaction with human polymorphonuclear leucocytes affect their ability to remove cholesterol from cells.

1. We have recently reported that a short incubation (60 min) in vitro of high-density lipoprotein (HDL) 3 with human polymorphonuclear leucocytes (PMNs) leads to a proteolytic cleavage of apolipoprotein (apo) AII and to a change in the distribution of apo AI isoforms [Cogny, Paul, Atger, Soni and Moatti (1994) Eur. J. Biochem. 222, 965-973]. Since PMNs have been observed to be present in the earliest atherosclerotic lesions for a number of days, we investigated the HDL3 physiochemical modifications induced by in vitro interaction for a long period of time (24 h) with PMNs and the consequences of the changes on the ability of HDL3 to remove cholesterol from cells. 2. The stimulated PMN modification of HDL3 over 24 h resulted in a partial loss of protein with no variation in lipid molar ratio and a loss of 50% of HDL alpha-tocopherol content. The decrease in total protein was due first to a complete degradation of apo AII, and secondly to a partial loss of apo AI. The apo AI remaining on the particles was in part hydrolysed and the apo AI-1 isoform was completely shifted to the apo AI-2 isoform. These apo changes were accompanied by a displacement of the native HDL3 apparent size toward predominantly larger particles. 3. The ability of PMN-modified HDL3 to remove 3H-labelled free cholesterol from cells was measured in two cell lines: Fu5AH rat hepatoma cells and J774 mouse macrophages. HDL3 which had only a limited contact with PMNs (60 min) showed only a small non-significant reduction in the efficiency of cholesterol efflux. On the other hand, compared with native HDL3, HDL3 modified by PMNs for 24 h had a markedly reduced ability to remove cholesterol from cells, regardless of the type of cell. 4. The results suggest that PMN-modified HDL3, if occurring in vivo, could contribute to acceleration of the atherogenic process by decreasing the cholesterol efflux from cells.