Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by approximately 50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of (125)I-[(14)C]cholesteryl oleate-labeled HDL(3) was decreased. Cholesterol efflux to apoA-I and HDL(3) was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.     

Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).     

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.     

High density lipoprotein (HDL) particle uptake mediated by scavenger receptor class B type 1 results in selective sorting of HDL cholesterol from protein and polarized cholesterol secretion

High density lipoprotein (HDL) mediates reverse transport of cholesterol from atheroma foam cells to the liver, but the mechanisms of hepatic uptake and trafficking of HDL particles are poorly understood. In contrast to its accepted role as a cell surface receptor, scavenger receptor class B type 1 (SR-BI) is shown to be an endocytic receptor that mediates HDL particle uptake and recycling, but not degradation, in both transfected Chinese hamster ovary cells and hepatocytes. Confocal microscopy of polarized primary hepatocytes shows that HDL particles enter both the endocytic recycling compartment and the apical canalicular region paralleling the movement of SR-BI. In polarized epithelial cells (Madin-Darby canine kidney) expressing SR-BI, HDL protein and cholesterol undergo selective sorting with recycling of HDL protein from the basolateral membrane and secretion of HDL-derived cholesterol through the apical membrane. Thus, HDL particles, internalized via SR-BI, undergo a novel process of selective transcytosis, leading to polarized cholesterol transport. A distinct process not mediated by SR-BI is involved in uptake and degradation of apoE-free HDL in hepatocytes.     

LDL particle subspecies are distinct in their capacity to mediate free cholesterol efflux via the SR-BI/Cla-1 receptor

The human scavenger receptor SR-BI/Cla-1 promotes efflux of free cholesterol from cells to both high-density and low-density lipoproteins (HDL, LDL). SR-BI/Cla-1-mediated cholesterol efflux to HDL is dependent on particle size, lipid content and apolipoprotein conformation; in contrast, the capacity of LDL subspecies to accept cellular cholesterol via this receptor is indeterminate. Cholesterol efflux assays were performed with CHO cells stably transfected with Cla-1 cDNA. Expression of Cla-1 in CHO cells induced elevation in total cholesterol efflux to plasma, LDL and HDL. Such Cla-1-specific efflux was abrogated by addition of anti-Cla-1 antibody. LDL were fractionated into five subspecies either on the basis of hydrated density or size. Among LDL subfractions, small dense LDL (sdLDL) were 1.5-to 3-fold less active acceptors for Cla-1-mediated cellular cholesterol efflux. Equally, sdLDL markedly reduced Cla-1-specific cholesterol efflux to large buoyant LDL in a dose-dependent manner. Conversely, sdLDL did not influence efflux to HDL(2). These findings provide evidence that LDL particles are heterogeneous in their capacity to promote Cla-1-mediated cholesterol efflux. Relative to HDL(2), large buoyant LDL may constitute physiologically-relevant acceptors for cholesterol efflux via Cla-1.     

Scavenger receptor-BI inhibits ATP-binding cassette transporter 1- mediated cholesterol efflux in macrophages

Scavenger receptor BI (SR-BI) facilitates the efflux of cellular cholesterol to plasma high density lipoprotein (HDL). Recently, the ATP-binding cassette transporter 1 (ABC1) was identified as a key mediator of cholesterol efflux to apolipoproteins and HDL. The goal of the present study was to determine a possible interaction between the SR-BI and ABC1 cholesterol efflux pathways in macrophages. Free cholesterol efflux to HDL was increased ( approximately 2.2-fold) in SR-BI transfected RAW macrophages in association with increased SR-BI protein levels. Treatment of macrophages with 8-bromo-cAMP (cAMP) resulted in a 4.1-fold increase in ABC1 mRNA level and also increased cholesterol efflux to HDL (2.2-fold) and apoA-I (5.5-fold). However, in SR-BI transfected RAW cells, cAMP treatment produced a much smaller increment in cholesterol efflux to HDL (1.1-fold) or apoA-I (3.3-fold) compared with control cells. In macrophages loaded with cholesterol by acetyl-LDL treatment, SR-BI overexpression did not increase cholesterol efflux to HDL but did inhibit cAMP-mediated cholesterol efflux to apoA-I or HDL. SR-BI neutralizing antibody led to a dose- and time-dependent increase of cAMP-mediated cholesterol efflux in both SR-BI transfected and control cells, indicating that SR-BI inhibits ABC1-mediated cholesterol efflux even at low SR-BI expression level. Transfection of a murine ABC1 cDNA into 293 cells led to a 2.3-fold increase of cholesterol efflux to apoA-I, whereas co-transfection of SR-BI with ABC1 blocked this increase in cholesterol efflux. SR-BI and ABC1 appear to have distinct and competing roles in mediating cholesterol flux between HDL and macrophages. In nonpolarized cells, SR-BI promotes the reuptake of cholesterol actively effluxed by ABC1, creating a futile cycle.     

Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages

By lowering high density lipoprotein (HDL) cholesterol, testosterone contributes to the gender difference in HDL cholesterol and has been accused to be pro-atherogenic. The mechanism by which testosterone influences HDL cholesterol is little understood. We therefore investigated the effect of testosterone on the gene expression of apolipoprotein A-I (apoA-I), hepatic lipase (HL), scavenger receptor B1 (SR-BI), and the ATP binding cassette transporter A1 (ABCA1), all of which are important regulators of HDL metabolism. In both cultivated HepG2 hepatocytes and primary human monocyte-derived macrophages, testosterone led to a dose-dependent up-regulation of SR-BI, which was assessed on both the mRNA and the protein levels. As a functional consequence, we observed an increased HDL(3)-induced cholesterol efflux from macrophages. At supraphysiological dosages, testosterone also increased the expression of HL in HepG2 cells. Testosterone had no effect on the expression of apoA-I in HepG2 cells and ABCA1 in either HepG2 cells or macrophages. These data suggest that testosterone, despite lowering HDL cholesterol, intensifies reverse cholesterol transport and thereby exerts an anti-atherogenic rather than a pro-atherogenic effect.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

High glucose stimulates macrophage SR-BI expression and induces a switch in its activity from cholesterol efflux to cholesterol influx

Aims

Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions.

Methods and results

ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5 mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI.

Conclusions

Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.     

Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines

Multiple changes in HDL metabolism occur during infection and inflammation that could potentially impair the antiatherogenic functions of HDL. Scavenger receptor class B type I (SR-BI) promotes cholesterol efflux from peripheral cells and mediates selective uptake of cholesteryl ester into hepatocytes, thereby playing a pivotal role in reverse cholesterol transport. We studied the effect of endotoxin (lipopolysaccharide, LPS) and cytokines [tumor necrosis factor (TNF) and interleukin 1 (IL-1)] on hepatic SR-BI mRNA and protein levels in Syrian hamsters. LPS significantly decreased SR-BI mRNA levels in hamster liver. This effect was rapid and sustained, and was associated with a decrease in hepatic SR-BI protein levels. High cholesterol diet did not change hepatic SR-BI mRNA levels, and LPS was able to decrease SR-BI mRNA levels during high cholesterol feeding. TNF and IL-1 decreased SR-BI mRNA levels in the liver, and the effects of TNF and IL-1 were additive. TNF and IL-1 also decreased SR-BI levels in Hep3B hepatoma cells. More importantly, TNF and IL-1 decreased the uptake of HDL cholesteryl ester into Hep3B cells. In addition, we studied the effect of LPS on SR-BI mRNA in RAW 264.7 cells, a macrophage cell line. LPS rapidly decreased SR-BI mRNA levels in RAW 264.7 cells, but the effect was not sustained and did not lead to a reduction in SR-BI protein levels. Our results suggest that the decrease in hepatic SR-BI levels due to LPS and cytokines during infection and inflammation may decrease selective uptake of cholesteryl ester into the liver and result in impaired reverse cholesterol transport.     

Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages.

Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nm phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation up-regulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% +/- 1.9% to 38% +/- 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

Regulatory effects of synthetic liver X receptor- and peroxisome-proliferator activated receptor agonists on sterol transport pathways in polarized cerebrovascular endothelial cells

The blood-brain barrier contributes to maintain brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. Brain capillary endothelial cells, representing a physiological barrier to the central nervous system, express apolipoprotein A-I (apoA-I, the major high-density lipoprotein (HDL)-associated apolipoprotein), ATP-binding cassette transporter A1 (ABCA1), and scavenger receptor, class B, type I (SR-BI), proteins that promote cellular cholesterol mobilization. Liver X receptors (LXRs) and peroxisome-proliferator activated receptors (PPARs) are regulators of cholesterol transport, and activation of LXRs and PPARs has potential therapeutic implications for lipid-related neurodegenerative diseases. To clarify the functional impact of LXR/PPAR activation, sterol transport along the: (i) ABCA1/apoA-I and (ii) SR-BI/HDL pathway was investigated in primary, polarized brain capillary endothelial cells, an in vitro model of the blood-brain barrier. Activation of LXR (24(S)OH-cholesterol, TO901317), PPARalpha (bezafibrate, fenofibrate), and PPARgamma (troglitazone, pioglitazone) modulated expression of apoA-I, ABCA1, and SR-BI on mRNA and/or protein levels without compromising transendothelial electrical resistance or tight junction protein expression. LXR-agonists and troglitazone enhanced basolateral-to-apical cholesterol mobilization in the absence of exogenous sterol acceptors. Along with the induction of cell surface-located ABCA1, several agonists enhanced cholesterol mobilization in the presence of exogenous apoA-I, while efflux of 24(S)OH-cholesterol (the major brain cholesterol metabolite) in the presence of exogenous HDL remained unaffected. Summarizing, in cerebrovascular endothelial cells apoA-I, ABCA1, and SR-BI represent drug targets for LXR and PPAR-agonists to interfere with cholesterol homeostasis at the periphery of the central nervous system.     

Serum amyloid A promotes cholesterol efflux mediated by scavenger receptor B-I

Serum amyloid A (SAA) is an acute phase protein whose expression is markedly up-regulated during inflammation and infection. The physiological function of SAA is unclear. In this study, we reported that SAA promotes cellular cholesterol efflux mediated by scavenger receptor B-I (SR-BI). In Chinese hamster ovary cells, SAA promoted cellular cholesterol efflux in an SR-BI-dependent manner, whereas apoA-I did not. Similarly, SAA, but not apoA-I, promoted cholesterol efflux from HepG2 cells in an SR-BI-dependent manner as shown by using the SR-BI inhibitor BLT-1. When SAA was overexpressed in HepG2 cells using adenovirus-mediated gene transfer, the endogenously expressed SAA promoted SR-BI-dependent efflux. To assess the effect of SAA on SR-BI-mediated efflux to high density lipoprotein (HDL), we compared normal HDL, acute phase HDL (AP-HDL, prepared from mice injected with lipopolysaccharide), and AdSAA-HDL (HDL prepared from mice overexpressing SAA). Both AP-HDL and AdSAA-HDL promoted 2-fold greater cholesterol efflux than normal HDL. Lipid-free SAA was shown to also stimulate ABCA1-dependent cholesterol efflux in fibroblasts, in line with an earlier report (Stonik, J. A., Remaley, A. T., Demosky, S. J., Neufeld, E. B., Bocharov, A., and Brewer, H. B. (2004) Biochem. Biophys. Res. Commun. 321, 936-941). When added to cells together, SAA and HDL exerted a synergistic effect in promoting ABCA1-dependent efflux, suggesting that SAA may remodel HDL in a manner that releases apoA-I or other efficient ABCA1 ligands from HDL. SAA also facilitated efflux by a process that was independent of SR-BI and ABCA1. We conclude that the acute phase protein SAA plays an important role in HDL cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways.     

Phosphatidylcholine transfer protein promotes apolipoprotein A-I-mediated lipid efflux in Chinese hamster ovary cells.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein of unknown function that catalyzes intermembrane transfer of phosphatidylcholines in vitro. Using stably transfected CHO cells, we explored the influence of PC-TP on apolipoprotein A-I- and high density lipoprotein 3 (HDL(3))-mediated lipid efflux. In proportion to its cellular level of expression, PC-TP accelerated apolipoprotein A-I-mediated phospholipid and cholesterol efflux as pre-beta-HDL particles. PC-TP increased rates of efflux of both lipids by >2-fold but did not affect mRNA levels or the activity of ATP-binding cassette A1, a plasma membrane protein that regulates apolipoprotein A-I-mediated lipid efflux. Overexpression of PC-TP was associated with only slight increases in HDL(3)-mediated phospholipid efflux and no changes in cholesterol efflux. In scavenger receptor BI-overexpressing cells, PC-TP expression minimally influenced apolipoprotein A-I- or HDL(3)-mediated lipid efflux. PC-TP did not affect cellular phospholipid compositions, phosphatidylcholine contents, or phosphatidylcholine synthetic rates. These findings suggest that a physiological function of PC-TP is to replenish the plasma membrane with phosphatidylcholines that are removed during pre-beta-HDL particle formation due to the activity of ATP-binding cassette A1.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.