Cholesteryl Ester Uptake Research Articles

Functionality of postprandial larger HDL2 particles is enhanced following CETP inhibition therapy

ObjectiveTo evaluate the impact of CETP inhibition on the capacity of individual postprandial HDL subspecies to promote key steps of the reverse cholesterol transport pathway. MethodsThe capacity of HDL particles to mediate cellular free cholesterol efflux and selective hepatic uptake of cholesteryl esters was evaluated throughout postprandial phase (0–8h) following consumption of a standardised mixed meal before and after treatment for 6 weeks with atorvastatin alone (10mg/d) and subsequently with combination torcetrapib/atorvastatin (60/10mg/d) in 16 patients displaying low HDL-C levels (<40mg/dl). ResultsThe larger HDL2b and HDL2a subfraction displayed a superior capacity to mediate cellular free cholesterol efflux via both SR-BI and ABCG1-dependent pathways than smaller HDL3 subspecies. CETP inhibition specifically enhanced the capacity of HDL2b subfraction for both SR-BI and ABCG1 dependent efflux. However, only the SR-BI-dependent efflux to HDL2b subspecies can be further enhanced during postprandial lipemia following CETP inhibition. Concomitantly, postprandial lipemia was associated with a reduced capacity of total HDL particles to deliver cholesteryl esters to hepatic cells in a drug independent manner. ConclusionCETP inhibition specifically improves postprandial SR-BI and ABCG1-dependent efflux to larger HDL2b subspecies. In addition, CETP inhibition improves HDL-CE delivery to hepatic cells and maintains an efficient direct return of cholesteryl esters to the liver during postprandial lipemia.

A lipoprotein source of cholesteryl esters is essential for proliferation of CEM-CCRF lymphoblastic cell line

Tumour are characterised by a high content of cholesteryl esters (CEs) stored in lipid droplets purported to be due to a high rate of intracellular esterification of cholesterol. To verify whether and which pathways involved in CE accumulation are essential in tumour proliferation, the effect of CE deprivation, from both exogenous and endogenous sources, on CEM-CCRF cells was investigated. Cholesterol synthesis, esterification and content, low-density lipoprotein (LDL) binding and high-density lipoprotein (HDL)-CE uptake were evaluated in cultured in both conventional and delipidated bovine serum with or without oleic or linoleic acids, cholesteryl oleate, LDL and HDL. High content of CEs in lipid droplets in this cell line was due to esterification of both newly synthesised cholesterol and that obtained from hydrolysis of LDL; moreover, a significant amount of CE was derived from HDL-CE uptake. Cell proliferation was slightly affected by either acute or chronic treatment up to 400 μM with Sz-58035, an acyl-cholesteryl cholesterol esterification inhibitor (ACAT); although when the enzyme activity was continuously inhibited, CE content in lipid droplets was significantly higher than those in control cells. In these cells, analysis of intracellular and medium CEs revealed a profile reflecting the characteristics of bovine serum, suggesting a plasma origin of CE molecules. Cell proliferation arrest in delipidated medium was almost completely prevented in the first 72 h by LDL or HDL, although in subsequent cultures with LDL, it manifested an increasing mortality rate. This study suggests that high content of CEs in CEM-CCRF is mainly derived from plasma lipoproteins and that part of CEs stored in lipid droplets are obtained after being taken up from HDL. This route appears to be up-regulated according to cell requirements and involved in low levels of c-HDL during cancer. Moreover, the dependence of tumour cells on a source of lipoprotein provides a novel impetus in developing therapeutic strategies for use in the treatment of some tumours.

C323 of SR-BI is required for SR-BI-mediated HDL binding and cholesteryl ester uptake

Scavenger receptor BI (SR-BI) is an HDL receptor. It binds HDL and mediates the uptake of cholesteryl ester from HDL. Early studies have pointed out that the extracellular domain of SR-BI is critical for SR-BI-mediated cholesteryl ester uptake. However, the extracellular loop of SR-BI is large: it contains 403 amino acids. The HDL binding site and the modulation of SR-BI-mediated cholesteryl ester uptake remain to be identified. In this study, using C323G mutant SR-BI, we showed that C323G mutant SR-BI lost its HDL binding and cholesteryl ester uptake activity, indicating that the highly conserved C323 is required for SR-BI-mediated HDL binding and cholesteryl ester uptake. Using a blocking antibody against C323 region, we demonstrated that C323 is directly involved in HDL binding and likely an HDL binding site. Using C323G mutant transgenic mouse model, we further demonstrated that C323 of SR-BI is required for regulating plasma cholesterol levels in vivo. Using redox reagents, we showed that physiological relevant levels of H(2)O(2) upregulated the SR-BI-mediated cholesteryl ester uptake activity by 65%, whereas GSH or DTT significantly downregulated SR-BI-mediated cholesteryl ester uptake activity by 45%. C323 of SR-BI is critical for SR-BI-mediated HDL binding and cholesteryl ester uptake, and changes in redox status may be a regulatory factor modulating SR-BI-mediated cholesterol transport.

Differential Roles of Cysteine Residues in the Cellular Trafficking, Dimerization, and Function of the High-Density Lipoprotein Receptor, SR-BI

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

Scavenger receptor class B type I: a multifunctional receptor

The scavenger receptor class B type I (SR-B1) plays an important role in meditating the uptake of HDL-derived cholesterol and cholesteryl ester in the liver and steroidogenic tissues. In addition to being ubiquitous, SR-B1 is a high-density lipoprotein (HDL) receptor in many tissues, though the mechanism by which SR-B1 does this is unclear. Other than its role as an HDL receptor, SR-B1 is also involved in pathogen recognition; its expression can be modulated by lipopolysaccharide and oxidative stress; and it plays a significant role in the uptake of lipid soluble vitamins, such as vitamin E and carotenoids. In this short review, we have summarized the biological aspects to which SR-B1 has been thus far associated.

Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes.

Extracellular Disulfide Bonds Support Scavenger Receptor Class B Type I-Mediated Cholesterol Transport

Scavenger receptor class B type I (SR-BI) binds high-density lipoprotein (HDL) and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesized that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite for SR-BI-mediated cholesterol transport. Thus, single-Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S-, and C384S-SR-BI), as well as Cys-less SR-BI, a mutant SR-BI receptor void of all Cys residues, were created, and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, and C334S-SR-BI and Cys-less SR-BI mutant receptors displayed weakened HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI's ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.

Glycine Dimerization Motif in the N-terminal Transmembrane Domain of the High Density Lipoprotein Receptor SR-BI Required for Normal Receptor Oligomerization and Lipid Transport

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (∼ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).

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

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

Validation of the reconstituted high-density lipoprotein (rHDL) drug delivery platform using dilauryl fluorescein (DLF).

Dilauryl fluorescein (DLF) is a lipid soluble molecule that becomes fluorescent when lauric acid is removed by hydrolysis The purpose of these studies was to evaluate DLF as a potential probe for the function of reconstituted high-density lipoproteins (rHDL) as hydrophobic drug transport vehicles. The DLF containing rHDL nanoparticles were characterized regarding their physical/chemical properties, including molecular diameter, molecular weight, chemical composition, and buoyant density. We investigated the uptake of DLF from rHDL in cells that overexpress the scavenger receptor (SR-B1), known to facilitate the selective cellular uptake of cholesteryl esters from HDL. These studies show that DLF can be incorporated into rHDL and redistributed in the plasma compartment. In addition, these studies demonstrated an enhanced uptake and hydrolysis of DLF from rHDL by cells that overexpress the SR-B1 receptor, suggesting the involvement of a receptor mediated mechanism. The incorporation of DLF into the rHDL nanoparticles appear to protect against hydrolysis in the systemic circulation based on the lower rate of rHDL/DLF hydrolysis compared with the free DLF during incubation with human plasma. DLF may thus be used as a probe to track the movement and metabolism of HDL core constituents, including cancer chemotherapeutic agents.

Expression of the Scavenger Receptor Class B type I (SR-BI) family in Drosophila melanogaster

In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Scavenger receptor BI: a multi-purpose player in cholesterol and steroid metabolism.

Scavenger receptor class B type I (SR-BI) is an important member of the scavenger receptor family of integral membrane glycoproteins. This review highlights studies in SR-BI knockout mice, which concern the role of SR-BI in cholesterol and steroid metabolism. SR-BI in hepatocytes is the sole molecule involved in selective uptake of cholesteryl esters from high-density lipoprotein (HDL). SR-BI plays a physiological role in binding and uptake of native apolipoprotein B (apoB)-containing lipoproteins by hepatocytes, which identifies SR-BI as a multi-purpose player in lipid uptake from the blood circulation into hepatocytes in mice. In adrenocortical cells, SR-BI mediates the selective uptake of HDL-cholesteryl esters, which is efficiently coupled to the synthesis of glucocorticoids (i.e. corticosterone). SR-BI knockout mice suffer from adrenal glucocorticoid insufficiency, which suggests that functional SR-BI protein is necessary for optimal adrenal steroidogenesis in mice. SR-BI in macrophages plays a dual role in cholesterol metabolism as it is able to take up cholesterol associated with HDL and apoB-containing lipoproteins and can possibly facilitate cholesterol efflux to HDL. Absence of SR-BI is associated with thrombocytopenia and altered thrombosis susceptibility, which suggests a novel role for SR-BI in regulating platelet number and function in mice. Transgenic expression of cholesteryl ester transfer protein in humanized SR-BI knockout mice normalizes hepatic delivery of HDL-cholesteryl esters. However, other pathologies associated with SR-BI deficiency, i.e. increased atherosclerosis susceptibility, adrenal glucocorticoid insufficiency, and impaired platelet function are not normalized, which suggests an important role for SR-BI in cholesterol and steroid metabolism in man. In conclusion, generation of SR-BI knockout mice has significantly contributed to our knowledge of the physiological role of SR-BI. Studies using these mice have identified SR-BI as a multi-purpose player in cholesterol and steroid metabolism because it has distinct roles in reverse cholesterol transport, adrenal steroidogenesis, and platelet function.

Biliary Sterol Secretion Is Required for Functional In Vivo Reverse Cholesterol Transport in Mice

High-density lipoproteins (HDLs) protect against atherosclerotic cardiovascular disease, mainly by promoting reverse cholesterol transport (RCT). Biliary sterol secretion supposedly represents the final step in RCT, but the relevance of this pathway has not been explored. We tested the dependency of RCT on functional biliary sterol secretion. Macrophage-to-feces RCT was studied in mice with abolished (bile duct ligation) or decreased biliary sterol secretion (adenosine triphosphate binding cassette transporter B4 (Abcb4)-/- mice, with and without administration of a liver X receptor [LXR] agonist) after intraperitoneal injection of (3)H-cholesterol-loaded primary macrophage foam cells from mice. Fecal tracer excretion and also fecal mass sterol excretion were measured. Metabolism and tissue uptake of HDL cholesteryl ester was assessed with HDL kinetic studies. Bile-duct ligation completely abolished RCT from (3)H-cholesterol-loaded macrophages to feces (P < .001). In Abcb4-/- mice lacking biliary cholesterol secretion, RCT was decreased markedly; fecal (3)H-tracer excretion was almost absent within neutral sterols (P < .001) and reduced within bile acids (P < .05). LXR activation stimulated RCT in wild-type (5.5-fold; P < .001) but not Abcb4-/- mice, whereas mass fecal sterol excretion increased similarly in both models (P < .05). Kinetic studies revealed minimal uptake of HDL cholesteryl ester by the intestine, which decreased on LXR activation (P < .05). Functional RCT depends on biliary sterol secretion; there is no compensatory increase in RCT via bile acids. The stimulating effect of LXR agonists on RCT requires biliary cholesterol secretion. These results have implications for therapies against atherosclerotic cardiovascular disease targeting the RCT pathway.

Deletion of the High-Density Lipoprotein Receptor Scavenger Receptor BI in Mice Modulates Thrombosis Susceptibility and Indirectly Affects Platelet Function by Elevation of Plasma Free Cholesterol

Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) by the liver. In mice, SR-BI deficiency results in increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. The aim of this study was to investigate the role of SR-BI deficiency on platelet function. SR-BI-deficient mice were thrombocytopenic, and their platelets were abnormally large, probably because of an increased cholesterol content. The FeCl(3) acute injury model to study arterial thrombosis susceptibility showed that SR-BI wild-type mice developed total arterial occlusion after 24±2 minutes. In SR-BI-deficient mice, however, the time to occlusion was reduced to 13±1 minutes (P=0.02). Correspondingly, in SR-BI-deficient mice, platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. In contrast, platelet-specific disruption of SR-BI by bone marrow transplantation in wild-type mice did not alter plasma cholesterol levels or affect platelet count, size, cholesterol content, or reactivity, suggesting that changes in plasma cholesterol levels were responsible for the altered responsiveness of platelets in SR-BI-deficient mice. The function of SR-BI in HDL cholesterol homeostasis and prevention of atherosclerosis is indirectly also essential for maintaining normal platelet function and prevention of thrombosis.

Postprandial lipemia enhances the capacity of large HDL2 particles to mediate free cholesterol efflux via SR-BI and ABCG1 pathways in type IIB hyperlipidemia

Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (-17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver.

Plasma lipoproteins are required for both basal and stress-induced adrenal glucocorticoid synthesis and protection against endotoxemia in mice

Lipoprotein-associated cholesterol has been suggested to make a significant contribution to adrenal steroidogenesis in vivo. To determine whether lipoproteins indeed contribute to optimal adrenal steroidogenesis in mice, in the current study we have determined the effect of relative lipoprotein deficiency on adrenal steroidogenesis in C57BL/6 wild-type mice. Feeding C57BL/6 mice the lipid-lowering drug probucol (0.25% wt/wt) for 2 wk induced a 90% decrease in plasma high-density lipoprotein (HDL) cholesterol levels and a 77% reduction in low-density lipoprotein (LDL) cholesterol levels. Neutral lipid stores were depleted upon probucol treatment specifically in the glucocorticoid-producing zona fasciculata of the adrenal, leading to a 44% decreased plasma corticosterone level under basal conditions. Exposure to lipopolysaccharide (LPS) induced a 37% increase in the adrenal uptake of HDL cholesteryl esters. Probucol-treated mice could induce only a relatively minor corticosterone response upon a LPS challenge compared with controls, which coincided with an approximately twofold increased hepatic expression level of interleukin-6 and tumor necrosis factor (TNF)α and an 89% higher TNFα response in plasma. Furthermore, a compensatory two- to fivefold upregulation of LDL receptor (cholesterol uptake) and HMG-CoA reductase (cholesterol synthesis) expression was noticed in the adrenals of probucol-treated mice. In conclusion, we have shown that lipoprotein deficiency in mice as a result of probucol feeding is associated with decreased adrenal cortex cholesterol levels, a lower basal and stress-induced plasma glucocorticoid level, and an increased susceptibility to LPS-induced inflammation. Therefore, it is suggested that plasma lipoproteins are required for optimal adrenal steroidogenesis and protection against endotoxemia in mice.

The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model

BackgroundThe interaction between Mycobacterium tuberculosis (Mtb) and host cells is complex and far from being understood. The role of the different receptor(s) implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.Methods and FindingsTo identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1) as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1−/− or wild type mice in vitro. Moreover, when wild type and SR-B1−/− animals were infected with a low dose of Mtb (100 CFU/mouse) there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNγ, and IL10 were observed in SR-B1−/− mice following infection with a high dose of Mtb (1000 CFU/mouse), which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.ConclusionSR-B1 is involved in mycobacterial recognition, but this receptor plays only a minor role in anti-mycobacterial immunity in vivo. Like many other receptors for these pathogens, the loss of SR-B1 can be functionally compensated for under normal conditions.

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)-mediated stimulation of HDL cholesterol uptake on liver lipid metabolism and biliary cholesterol secretion in wild-type, SR-BI knockout, and SR-BI overexpressing mice. In each model, injection of an EL expressing adenovirus decreased plasma HDL cholesterol (P < 0.001) whereas hepatic cholesterol content increased (P < 0.05), translating into decreased expression of sterol-regulatory element binding protein 2 (SREBP2) and its target genes HMG-CoA reductase and LDL receptor (each P < 0.01). Biliary cholesterol secretion was dependent on hepatic SR-BI expression, being decreased in SR-BI knockouts (P < 0.001) and increased following hepatic SR-BI overexpression (P < 0.001). However, in each model, biliary secretion of cholesterol, bile acids, and phospholipids as well as fecal bile acid and neutral sterol content, remained unchanged in response to EL overexpression. Importantly, hepatic ABCG5/G8 expression did not correlate with biliary cholesterol secretion rates under these conditions. These results demonstrate that an acute decrease of plasma HDL cholesterol levels by overexpressing EL increases hepatic cholesterol content but leaves biliary sterol secretion unaltered. Instead, biliary cholesterol secretion rates are related to the hepatic expression level of SR-BI. These data stress the importance of SR-BI for biliary cholesterol secretion and might have relevance for concepts of reverse cholesterol transport.

Effects of cholesteryl ester transfer protein inhibition on apolipoprotein A-II-containing HDL subspecies and apolipoprotein A-II metabolism

This study was designed to establish the mechanism responsible for the increased apolipoprotein (apo) A-II levels caused by the cholesteryl ester transfer protein inhibitor torcetrapib. Nineteen subjects with low HDL cholesterol (<40 mg/dl), nine of whom were also treated with 20 mg of atorvastatin daily, received placebo for 4 weeks, followed by 120 mg of torcetrapib daily for the next 4 weeks. Six subjects in the nonatorvastatin cohort participated in a third phase, in which they received 120 mg of torcetrapib twice daily for 4 weeks. At the end of each phase, subjects underwent a primed-constant infusion of [5,5,5-(2)H(3)]L-leucine to determine the kinetics of HDL apoA-II. Relative to placebo, torcetrapib significantly increased apoA-II concentrations by reducing HDL apoA-II catabolism in the atorvastatin (-9.4%, P < 0.003) and nonatorvastatin once- (-9.9%, P = 0.02) and twice- (-13.2%, P = 0.02) daily cohorts. Torcetrapib significantly increased the amount of apoA-II in the alpha-2-migrating subpopulation of HDL when given as monotherapy (27%, P < 0.02; 57%, P < 0.003) or on a background of atorvastatin (28%, P < 0.01). In contrast, torcetrapib reduced concentrations of apoA-II in alpha-3-migrating HDL, with mean reductions of -14% (P = 0.23), -18% (P < 0.02), and -18% (P < 0.01) noted during the atorvastatin and nonatorvastatin 120 mg once- and twice-daily phases, respectively. Our findings indicate that CETP inhibition increases plasma concentrations of apoA-II by delaying HDL apoA-II catabolism and significantly alters the remodeling of apoA-II-containing HDL subpopulations.