The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration.

Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1.

We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies.

Infusion of reconstituted HDL (rHDL) leads to changes in HDL metabolism as well as to an increased capacity of plasma to support cholesterol efflux providing an opportunity to investigate mechanisms linking cholesterol efflux to changes in plasma HDL.

Patient plasmas after infusion of rHDL were tested ex vivo for their capacity to stimulate cholesterol efflux. Reconstituted HDL enhanced mobilization of cholesterol from tissues in vivo as shown by rising HDL cholesterol concentrations over the infusion period. Infusion of rHDL in vivo led to increased cholesterol efflux ex vivo; surprisingly, removing apoB-containing lipoproteins while preserving all HDL subfractions eliminated this increase. Infusion of rHDL led to the remodelling of plasma HDL; however, the capacity of plasma to support cholesterol efflux did not correlate with changes in the concentrations of any of HDL subfractions. Unmodified rHDL accounted for only a proportion of the increment in cholesterol efflux capacity. Furthermore, studies using HeLa and BHK cells overexpressing ABCA1, ABCG1, and SR-B1 showed that the contribution of these cellular mediators of cholesterol efflux to the enhanced capacity of plasma for the efflux was minimal.

Enhanced cholesterol efflux from tissues requires the presence of apoB-containing lipoproteins and may involve enhanced flow of cholesterol through multiple components of the reverse cholesterol transport pathway rather than being determined by a specific HDL subfraction.

Neutrophils play a key role in the immune response but can undesirably exacerbate inflammation. High-density lipoproteins (HDL) are antiinflammatory particles, exerting beneficial cardiovascular influences. We determined whether HDL exerts antiinflammatory effects on neutrophils and explored the mechanisms by which these occur.

CD11b on activated human neutrophils was significantly attenuated by apolipoprotein A-I (apoA-I) and HDL. The effects of apoA-I were mediated via ABCA1, whereas the effects of HDL were via scavenger receptor BI. Both were associated with a reduction in the abundance of lipid rafts, and a strong correlation between raft abundance and CD11b activation was observed. ApoA-I and HDL reduced neutrophil adhesion to a platelet monolayer under shear flow, as well as neutrophil spreading and migration. ApoA-I also inhibited leukocyte recruitment to the endothelium in an acute in vivo model of inflammation. Finally, infusion of reconstituted HDL in patients with peripheral vascular disease was demonstrated to significantly attenuate neutrophil activation.

We describe here a novel role for HDL and apoA-I in regulating neutrophil activation using in vitro, in vivo, and clinical approaches. We also show that these effects of HDL and apoA-I involve a mechanism requiring changes in membrane domain content rather than in cholesterol efflux per se.

The effects of the statin, rosuvastatin on indices of reverse cholesterol transport were studied in a randomized, placebo-controlled, cross-over trial in 25 overweight subjects with defined metabolic syndrome.

Four weeks' treatment with 40 mg/day rosuvastatin significantly reduced levels of plasma cholesterol (44%), LDL cholesterol (60%) and triglyceride (38%). HDL cholesterol (mean [S.D.]) rose (0.97[0.17] to 1.05[0.17]mmol/L; P<0.05) and the LpA-I component of HDL from 39[7] to 45[9]mg/dL (P<0.05). LCAT activity fell (0.55[0.13] to 0.35[0.07]nmol/mL/h; P<0.05); CETP activity and mass fell from 89[13] to 80[11]nmol//L/h and from 1.66[0.57] to 1.28[0.41]mug/mL respectively, (P<0.05). Cholesterol efflux in vitro (to plasmas from THP-1 activated cells) fell from 7.1[1.8]% (placebo) to 6.2[1.7]% (rosuvastatin); P<0.05, but when plasmas depleted of apoB lipoproteins were studied, the difference in efflux was no longer statistically significant. During placebo efflux was paradoxically inversely correlated with HDL-C (P=0.016) and LpA-I (P=0.035) concentrations but these correlations were absent after rosuvastatin.

The data suggest possible HDL dysfunctionality in subjects with metabolic syndrome. The reduced capacity of plasmas following statin treatment to stimulate cholesterol efflux in vitro occurred in association with reduction in apoB lipoproteins and reduced activities of CETP and LCAT, and despite increased levels of HDL cholesterol.

Liver is one of the most important organs in energy metabolism. Most plasma apolipoproteins and endogenous lipids and lipoproteins are synthesized in the liver. It depends on the integrity of liver cellular function, which ensures homeostasis of lipid and lipoprotein metabolism. When liver cancer occurs, these processes are impaired and the plasma lipid and lipoprotein patterns may be changed. Liver cancer is the fifth common malignant tumor worldwide, and is closely related to the infections of hepatitis B virus (HBV) and hepatitis C virus (HCV). HBV and HCV infections are quite common in China and other Southeast Asian countries. In addition, liver cancer is often followed by a procession of chronic hepatitis or cirrhosis, so that hepatic function is damaged obviously on these bases, which may significantly influence lipid and lipoprotein metabolism in vivo. In this review we summarize the clinical significance of lipid and lipoprotein metabolism under liver cancer.

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).

We have established an immunoassay for pre beta 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre beta 1-HDL is unstable during storage, fresh plasma must be used for pre beta 1-HDL measurements. In this study, we describe a method of stabilizing pre beta 1-HDL, and evaluate the analytical performance of the immunoassay for pre beta 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre beta 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre beta 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre beta 1-HDL was more stable at 0 degrees C than at room temperature, it increased from 30.2 +/- 8.5 (SE) to 56.5 +/- 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 +/- 1.2 to 37.9 +/- 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at -80 degrees C, pre beta 1-HDL increased from 29.0 +/- 4.0 to 38.0 +/- 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, pre beta 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre beta 1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of pre beta 1-HDL.

The role of pre-beta1-high density lipoprotein (pre-beta1-HDL) in cholesterol efflux was investigated by separating human plasma into purified pre-beta1-HDL and pre-beta1-HDL-deficient plasma by using a monoclonal antibody specifically reacting with pre-beta1-HDL.

When compared with whole plasma, pre-beta1-HDL-deficient plasma was equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human macrophage cells. When added at the same apolipoprotein A-I concentration, pre-beta1-HDL was less effective than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective in promoting cholesterol efflux from THP-1 cells. However, pre-beta1-HDL-deficient plasma reconstituted with 16% pre-beta1-HDL was more active than whole plasma, demonstrating that pre-beta1-HDL does promote cholesterol efflux actively. The amount of cellular cholesterol present in reisolated pre-beta1-HDL was 1.5- to 2-fold greater after incubation of the cells with whole plasma than after incubation of the cells with pre-beta1-HDL-deficient plasma or plasma treated with the anti-pre-beta1-HDL antibody. However, the anti-pre-beta1-HDL antibody did not inhibit cholesterol efflux.

We conclude that whereas pre-beta1-HDL is capable of taking up cellular cholesterol, its presence in plasma is not essential for cholesterol efflux, at least in vitro. Instead, pre-beta1-HDL may be the first product of apolipoprotein A-I lipidation during the formation of HDL but may not play a major role in transferring cellular cholesterol to HDL.

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.

Prebeta1-high density lipoprotein (prebeta1-HDL), the initial acceptor of cell-derived cholesterol, can be generated from HDL(2) by hepatic lipase. Because bezafibrate elevates lipase activity, it may increase prebeta1-HDL at the expense of HDL(2). To answer this question, we determined the apolipoprotein A-I (apoA-I) distribution in 20 hypertriglyceridemics (triglycerides>2.26 mmol/L) and 20 sex-matched normolipidemics by native 2-dimensional gel electrophoresis. At baseline, prebeta1-HDL was 70% higher in hypertriglyceridemics than in normolipidemics (123.5+/-49.9 versus 72.5+/-34.1 mg/L apoA-I, P<0.01). Prebeta1-HDL was positively correlated with triglyceride (r=0.624, P<0.0001). A 4-week bezafibrate treatment (400 mg daily) increased prebeta1-HDL by 30% (160.2+/-64.5 mg/L apoA-I, P<0.05) but decreased HDL(2b) by 31% (from 188.8+/-94.9 to 129.3+/-78.7 mg/L apoA-I, P<0.05). Hepatic lipase activity increased by 24% (P<0.005). Prebeta1-HDL was generated either from ultracentrifugally isolated HDL(2) or from plasma during incubation with triglyceride lipase. In conclusion, bezafibrate increases prebeta1-HDL at the expense of HDL(2). We speculate that such an effect might partly contribute to the antiatherogenic action of bezafibrate.

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.

Evidence that the high density lipoproteins (HDL) in human plasma are antiatherogenic has stimulated considerable interest in the factors which regulate their structure and function. Plasma HDL consist of a number of subpopulations of particles of varying size, density and composition. This structural heterogeneity is caused by the continual remodelling of individual HDL subpopulations by various plasma factors. One of the consequences of this remodelling is that the HDL subpopulations in plasma are functionally diverse, particularly in terms of their antiatherogenic properties. This review documents what is currently known about the interaction of HDL with plasma factors and presents an overview of the remodelling of HDL which occurs as a consequence of those interactions.

We have previously shown that human skin fibroblasts exposed to preformed low density lipoprotein (LDL)-thyroxine (T4) complexes internalize more T4 than they do when exposed to T4 alone. The system is set to function when the LDL receptor is up-regulated by reducing the intracellular concentration of cholesterol, and the LDL concentration outside the cell is in the range of the kDa of the receptor. High density lipoproteins (HDL), albumin (HSA), transthyretin (TTR), and thyroxine-binding globulin (TBG) interfere with, rather than facilitate, T4 entry. Of the three classes of lipoproteins (VLDL, LDL, and HDL), HDL is the major carrier of thyroid hormones. While LDL delivers cholesterol (and T4) to cells, HDL is the scavenger of cholesterol. We thus hypothesized that HDL could also facilitate thyroid hormone exit from cells. This hypothesis was tested on two human cell lines: skin fibroblasts and hepatocytes (Hep G2), using physiological concentrations of HDL or, as control, physiological concentrations of LDL, HSA, TTR, and TBG or buffer. Because cell surface receptors for HDL are regulated by intracellular cholesterol in a manner opposite to that of LDL receptors, we evaluated the effect of HDL (and other proteins) in three states: normal, high, and low intracellular cholesterol content (i.e. normal, high, and low expression of HDL receptors). In both cell lines and with either T4 or T3, we found that: 1) HDL as well as the other proteins tested increased the efflux and augmented both the initial rate of exit and the equilibrium value. 2) The efflux did not saturate over a wide range of protein concentrations. 3) The effect of HDL, LDL, and the other proteins on the fractional efflux rate of thyroid hormones remained the same irrespective of the intracellular cholesterol content (and, therefore, irrespective of the expression of either LDL or HDL receptors). 4) HSA, TTR, and TBG were, on a mass basis, equipotent and more efficient than lipoproteins. However, the effect of lipoproteins--whose Ka for T4 is comparable to that of HSA--was disproportionately high. On a molar basis, LDL (about 80% of the weight being accounted for by lipids) was more effective than HDL2 (about 60% lipids) and HDL2 was more effective than HDL3 (about 40% lipids), suggesting that the disproportionate effect of lipoproteins was due to transfer of the lypophylic thyroid hormones to the lipid moiety of lipoproteins. 5. A mixture of HDL and LDL gave the same efflux rate as a mixture of HSA, TTR, and TBG. The data indicate that the efflux of T4 and T3 from cells is rapid and appears not to be mediated by a particular lipoprotein. The disproportionately large effect of lipoproteins, which are low affinity thyroid hormone carriers, compared with nonlipoprotein carriers, and the greater effect of LDL compared with HDL, might indicate that the lipoproteins establish a nonspecific physical contact with the plasma membrane and that their hydrophobic nature favors the release of the similarly hydrophobic thyroid hormones.

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.

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.

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(-6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(-6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(-6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(-6-222)) and 210 (apoA-I-(-6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(-6-150)) and 135 (apoA-I-(-6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(-6-243), apoA-I-(-6-222), and apoA-I-(-6-210), but slightly higher for apoA-I-(-6-150) and apoA-I-(-6-135). When complexed with phospholipid, all forms except apoA-I-(-6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent.