High-density Lipoprotein Lipid Research Articles

Alcohol consumption is associated with enrichment of high-density lipoprotein particles in polyunsaturated lipids and increased cholesterol esterification rate.

Alcohol consumption is associated with high levels of high-density lipoproteins (HDLs). Moreover, changes in the fatty acid patterns of red blood cell phospholipids and plasma lipids have been observed in drinkers. The objectives of this study were to characterize the composition of HDL particles with respect to lipid molecular species in regular wine drinkers and to assess the functional properties of those HDLs as regards key steps of reverse cholesterol transport. Forty-six subjects were recruited in the frame of a population study performed in Toulouse, southern France, and a nutritional investigation, including daily alcohol consumption, was performed. Subjects were sorted according to their daily alcohol intake (0, < or =35, and >35 g/day), mostly as red wine. The plasma HDL fraction was isolated, and neutral lipid molecular lipids and phospholipid fatty acids were analyzed by gas liquid chromatography. Efflux of cellular cholesterol and rates of cholesterol esterification and cholesteryl ester transfers between lipoproteins were assayed in a cell-plasma incubation system. Wine drinking, at 47 g/day, was associated with an increase in HDL cholesterol and apolipoprotein A-I, but not with triglycerides. Isolated HDL displayed a 27% increase in all cholesteryl ester molecular species. The particles were also enriched in unsaturated phospholipids and, particularly, in those containing arachidonic (+30%) and eicosapentaenoic (+90%) acids. The plasma cholesterol esterification rate, reflecting lecithin cholesterol acyl transferase activity on HDL, was found to be higher (+27%) in drinkers than in nondrinkers, whereas the rate of cellular cholesterol efflux to plasma was identical. Regular wine consumption is associated with high levels of polyunsaturated lipids in HDL and with increases in the cholesterol esterification rate.

A Liver-High-Density Lipoprotein-Ovarian Axis of Fertility

Human plasma transports about 90% of its cholesterol on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles. LDL delivers its cholesterol to cells primarily through receptor-mediated endocytosis via the LDL receptorpathwaydiscoveredbyGoldsteinandBrown(1)more than 30 yr ago. HDL is somewhat more complicated because cholesterol traffic between HDL and cells occurs in a twoway direction (2, 3). Moreover, lipoproteins carry two forms of cholesterol—cholesterol itself and cholesterol esters. These two molecules have distinct solubility properties and are therefore transported through different mechanisms. Unlike LDL, HDL delivers cholesterol and cholesterol esters to cells through a process not involving classical receptor-mediated endocytosis. Rather, certain cells (hepatocytes and steroidogenic cells) take up cholesterol esters from HDL particles without taking in the entire particle (4, 5). This selective uptake pathway, first identified by the Pittman and Stein groups (4, 6), was shown by Krieger (7) to be mediated by the scavenger receptor B-1 (SR-BI). In contrast to cholesterol ester traffic, cholesterol traffic between cells and HDL is bidirectional and largely depends on the magnitude of the concentration gradient. Hence, a cholesterol-loaded cell will spontaneously release cholesterol to an HDL particle. This process is in part mediated by SR-BI (8, 9). Early studies on dynamics of HDL-mediated cholesterol delivery in vivo showed that steroidogenic tissues take up substantial amounts of cholesterol from HDL, leading to the inference that this cholesterol is necessary for steroid production (10). This was further supported by the findings that SR-BI is regulated in steroidogenic tissues (11, 12), cholesteroldeliveredviaSR-BIisasubstrateforsteroidogenesis(11, 12), and steroidogenic tissues are cholesterol ester depleted in the SR-BI/ mice (12, 13). Thus, the discovery by Krieger’s team that SR-BI-deficient female mice are not fertile suggested that they might not be able to supply themselves with sufficient cholesterol ester. But, Krieger’s group (14) showed with ovary transplant experiments that in fact the ovaries function normally when placed in wild-type mice. Hence, SR-BI deficiency in a tissue other than the ovary had to be the culprit. In this issue, Yesilaltay et al. (15) show that adenovirusmediated or stable transgenic expression of SR-BI in the livers of SR-BI knockout mice is sufficient to restore fertility. SR-BI deficiency leads to an accumulation of abnormally large HDL particles with an unusually high content of unesterified cholesterol, apparently because these particles are poor substrates for the plasma enzyme responsible for esterifying cholesterol, lecithin:cholesterol acyl transferase. The rescue by ectopic SR-BI normalizes the HDL size and composition. A mutant form of SR-BI that interacts with LDL but not HDL, normalizes the abnormal HDL of the SR-BI knockout mice and restores fertility, pointing to the abnormal HDL (or the absence of normal HDL) as the basis for infertility. HDL levels are affected by the ability of cells to secrete its protein components, primarily apolipoprotein A1. Its stability requires a requisite supply of phospholipids and cholesterol, which are delivered by ABC transporters. In the bloodstream, HDL lipids and proteins exchange with other lipoproteins and are modified through the action of lipases, esterases, and lecithin:cholesterol acyl transferase (16). Krieger hypothesizes that there are specific structural requirements for HDL to properly support ovarian function. GiventherangeofdietaryandgeneticfactorsthataffectHDL structure, this hypothesis supplies several new categories of candidates to explain the high frequency of human female infertility. In addition to the major phospholipids and neutral lipids, HDL transports many other hydrophobic molecules; e.g. fatsoluble vitamins, dolichol, and other isoprenoids. Thus, it is possible that the abnormal lipoproteins in the SR-BI knockout mice carry inappropriate amounts of essential molecules for proper oocyte maturation or an excess of detrimental molecules. New analytical methods may be able to sort this out.

Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells

Class B scavenger receptor type I (SR-BI) is a multiligand membrane protein expressed in a variety of cell types. This receptor is responsible for the incorporation of lipids from high density lipoprotein (HDL) by steroidogenic cells, as well as for the phosphatidylserine (PS)-mediated phagocytosis of apoptotic cells by some phagocytic cell types, such as testicular Sertoli cells. Although SR-BI directly binds to PS present on the surface of apoptotic cells, as to whether SR-BI transmits signals to induce engulfment has not been clear. In the present study, we examined this issue using a monoclonal antibody that neutralizes SR-BI activity and a chemical known to be an inhibitor of the SR-BI-mediated incorporation of HDL lipids. The chemical compound inhibited the incorporation of HDL lipids and PS-containing liposomes by an SR-BI-expressing culture cell line, with no effect on the binding of these targets. Similarly, the phagocytosis of PS-exposing apoptotic cells by primary cultured rat Sertoli cells was inhibited in the presence of either reagent, not at the recognition but at the engulfment step. The addition of apoptotic cells or PS-containing liposomes caused a temporal increment of the phosphorylation of all three mitogen-activated protein kinases, p38, extracellular-signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), in Sertoli cells. The increase of phosphorylated p38 and ERK, but not of phosphorylated JNK, was cancelled in the presence of the monoclonal antibody. Furthermore, the level of Sertoli cell phagocytosis of PS-exposing apoptotic cells, as well as that of PS-containing liposomes, was reduced only when the actions of p38 and ERK were simultaneously repressed. In conclusion, these results indicate that SR-BI, when it binds to PS, transmits signals to activate the mitogen-activated protein kinase pathway, which leads to the induction of the engulfment of PS-exposing apoptotic cells by phagocytic cells.

The Catalytic Histidine Dyad of High Density Lipoprotein-associated Serum Paraoxonase-1 (PON1) Is Essential for PON1-mediated Inhibition of Low Density Lipoprotein Oxidation and Stimulation of Macrophage Cholesterol Efflux

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.

Serum Opacity Factor, a Streptococcal Virulence Factor That Binds to Apolipoproteins A-I and A-II and Disrupts High Density Lipoprotein Structure

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.

Rev-erbβ Regulates the Expression of Genes Involved in Lipid Absorption in Skeletal Muscle Cells: EVIDENCE FOR CROSS-TALK BETWEEN ORPHAN NUCLEAR RECEPTORS AND MYOKINES

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for >30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an "exercise-induced myokine" that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.

Severe Hypoalphalipoproteinemia in Mice Expressing Human Hepatic Lipase Deficient in Binding to Heparan Sulfate Proteoglycan

Unlike human hepatic lipase (hHL) that is mainly cell surface-anchored via binding to heparan sulfate proteoglycans (HSPG), mouse HL (mHL) has a low affinity to HSPG and thus is largely blood-borne. The reduced HSPG binding of mHL is attributable to the C-terminal amino acids. To determine the functions of HSPG binding of hHL in vivo, we created adenovirus vectors encoding hHL or a chimeric protein (designated hHLmt) in which the C-terminal HSPG-binding sequences were replaced with the corresponding mouse sequences. Injecting hHLmt-expressing virus into C57BL/6J mice (1.8 x 10(10) virus particles/mouse) resulted in a 3-fold increase in pre-heparin HL activity, whereas infection with an identical dose of hHL virus did not change pre-heparin HL activity. In hHLmt-expressing mice, the concentration of total cholesterol and phospholipids was inversely related to the hHL activity in pre-heparin plasma in a dose- and time-dependent manner, and the decrease was mainly attributable to high density lipoproteins (HDL) cholesterol and HDL phospholipids. The expression of hHL exhibited no change in plasma total cholesterol or phospholipid levels as compared with control mice infected with luciferase or injected with saline. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apolipoprotein (apo) A-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I was decreased by 2-3-fold as compared with hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Infection of primary hepatocytes with hHLmt virus ex vivo also resulted in reduced apoA-I secretion but had no effect on cell-associated apoA-I. These results suggest that expression of HSPG binding-deficient hHL has a profound HDL-lowering effect.

Structural basis of endothelial lipase tropism for HDL.

Lipoprotein lipase (LPL) and endothelial lipase (EL), the most closely related enzymes among the members of the triglyceride lipase gene family with regard to primary sequence, have distinct lipolytic properties (triglyceride lipase vs. phospholipase) as well as different preferences for specific types of lipoproteins [triglyceride-rich lipoproteins vs. high density lipoprotein (HDL)] Lipid substrate specificity is believed to be conferred by the lid region located in the amino-terminal domain of the enzymes, whereas surprisingly little work has been done to identify the region mediating lipoprotein substrate specificity. To determine the domain responsible for lipoprotein preference within each enzyme, we generated the domain chimeric enzyme LPL-EL. The heterologous carboxy-terminal (C terminal) domain did not change lipid substrate preference (triglyceride vs. phospholipase) as determined by using artificial substrates. The EL C-terminal domain, however, enabled LPL-EL to bridge HDL particles like wild-type EL, whereas LPL only mediated binding of very low density lipoprotein. Unlike wild-type LPL, LPL-EL had substantial ability to hydrolyze HDL lipids similar to that of wild-type EL. Overexpression of LPL-EL in wild-type mice resulted in significantly reduced levels of HDL cholesterol and phospholipids by 93 and 85%, respectively, similar to the extent seen in EL-expressing mice, whereas no reduction of these parameters was observed in LPL-expressing mice. We conclude that the C-terminal domain of EL is crucial for the ability of EL to bind and to hydrolyze HDL and converts LPL to an enzyme fully capable of hydrolyzing HDL, highlighting the importance of the C-terminal lipase domain in lipoprotein substrate preference.

Scavenger receptor BI: a scavenger receptor with a mission to transport high density lipoprotein lipids.

This review will survey recent findings on the cholesterol transport and scavenger functions of scavenger receptor BI. Although scavenger receptor BI and CD36 bind many of the same ligands, these two receptors have very specific lipid transport functions: CD36 facilitates the uptake of long chain fatty acids and SR-BI mediates the transport of cholesterol and cholesteryl ester from HDL particles. Scavenger receptor BI is a physiologically relevant HDL receptor that, along with HDL, is protective against cardiovascular disease. Its atheroprotective role has been hypothesized to be due to its function in the reverse cholesterol transport pathway. Recent studies suggest that scavenger receptor BI function is not only crucial for cholesterol delivery to the liver but is also important for cholesterol efflux at the vessel wall. Therefore, the receptor acts at both ends of the reverse cholesterol transport pathway. In addition, it stimulates nitric oxide production in endothelial cells, which may also contribute to its positive influence on the vasculature. Lastly, the glycoprotein was cloned as a scavenger receptor and in some cases is still thought to operate in this fashion. It will be interesting to follow future research on scavenger receptor BI that will delineate its functions in cholesterol transport as well as its scavenger functions. Additionally, we are only beginning to learn of the glycoprotein's effects on disease states besides atherosclerosis and cardiovascular disease.

Lipoprotein Compositional Changes With Combination Hormone Therapy (Conjugated Estrogen And Medroxyprogesterone) in African American Women

To determine whether combination hormone therapy (HT) significantly alters lipoprotein composition in healthy African American women. Postmenopausal African American women, 45 to 65 years old, were randomly assigned to receive daily HT (conjugated equine estrogen, 0.625 mg, and medroxyprogesterone, 2.5 mg) or placebo (treated, 44; placebo, 16) for 12 weeks. Lipoproteins were separated by gradient ultracentrifugation into very-low-density, intermediate-density, and low-density lipoproteins (VLDL, IDL, and LDL) and 3 high-density lipoprotein (HDL) subfractions (light, medium, and dense--L, M, and D). Apolipoprotein (apo) A-I, A-II, C-III, C-II, and C-I were measured by high-performance liquid chromatography. Apo B, phospholipids, triglycerides, cholesterol, and free cholesterol were measured by standard assays. Total plasma cholesterol, triglycerides, LDL apo B, and total apo B did not change during HT. A small, transient reduction occurred in LDL cholesterol, and a persistent reduction was noted in VLDL apo B, apo C-II, and apo C-III. Total HDL phospholipids, cholesterol, apo A-I, and apo A-II increased, whereas the LDL/HDL ratio and the apo B/apo A-I ratio decreased. Cholesterol ester increased in both HDL-L and HDL-M, but only a transient increase occurred in HDL-L phospholipids. Apo A-I increased in HDL-L, HDL-M, and HDL-D; however, a similar increase occurred in HDL-D apo A-I in the control subjects. Moreover, an increase occurred in apo A-II in HDL-M. A reduction in the apo A-II:A-I ratio in HDL-L but not in HDL-M indicated an increase in HDL-L LpA-I particles. The increase in HDL particles in HDL-M was entirely due to an increase in LpA-I:A-II particles. Apo C-III increased in both HDL-L and HDL-M. The absence of changes in the HDL lipid ratios indicated an unaltered lipid composition of these particles. No changes were found in IDL compositional measurements. In 12 treated patients and 4 control subjects, Lp(a) was detected by ultracentrifugation; no changes were noted in Lp(a) composition or quantity with HT. Total Lp(a) measured by enzyme immunosorbent assay showed a trend toward an increase in treated patients after 12 weeks of HT. African American women had a beneficial response to HT by increasing the number of LpA-I particles in HDL-L and LpA-I:A-II particles in HDL-M as well as cholesterol esters in both. There was a small reduction in VLDL apo B (and thus particle number) but only a transient reduction in LDL cholesterol. A shift of apo C-III from VLDL to HDL was noted. No detrimental changes occurred in any lipoprotein subfraction (specifically, no increase in triglycerides).

High Density Lipoprotein Uptake by Scavenger Receptor SR-BII

Scavenger receptor class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) lipids. It is unclear whether this process occurs at the cell membrane or via endocytosis. Our group previously identified an alternative mRNA splicing variant of SR-BI, named SR-BII, with an entirely different, yet highly conserved cytoplasmic C terminus. In this study we aimed to compare HDL uptake by both isoforms. Whereas SR-BI was mainly ( approximately 70%) localized on the surface of transfected Chinese hamster ovary cells, as determined by biotinylation, HDL binding at 4 degrees C, and studies of enhanced green fluorescent protein-tagged SR-BI/II fusion proteins, the majority of SR-BII ( approximately 80-90%) was expressed intracellularly. The cellular distribution of SR-BI was not affected by deletion of the C terminus, which suggests that the distinct C terminus of SR-BII is responsible for its intracellular expression. Pulse-chase experiments showed that SR-BII rapidly internalized HDL protein, whereas in the case of SR-BI most HDL protein remained surface bound. Like its ligand, SR-BII was more rapidly endocytosed compared with SR-BI. Despite more rapid HDL uptake by SR-BII than SR-BI, selective cholesteryl ether uptake was significantly lower. Relative to their levels of expression at the cell surface, however, both isoforms mediated selective uptake with similar efficiency. HDL protein that was internalized by SR-BII largely co-localized with transferrin in the endosomal recycling compartment. Within the endosomal recycling compartment of SR-BII cells, there was extensive co-localization of internalized HDL lipid and protein. These results do not support a model that selective lipid uptake by SR-BI requires receptor/ligand recycling within the cell. We conclude that SR-BII may influence cellular cholesterol trafficking and homeostasis in a manner that is distinct from SR-BI.

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

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

Experimental and interventional dietary study in humans on the role of HDL fatty acid composition in PGI2 release and Cox-2 expression by VSMC.

High-density lipoproteins (HDLs) induce prostacyclin (PGI2) release in vascular smooth muscle cells (VSMCs) by up-regulation of cyclooxygenase-2 (Cox-2). Our goal was to analyze the role of human HDL lipid moiety on Cox-2-dependent PGI2 synthesis in human VSMCs and to assess the impact that the intake of diets with different fatty acid composition exert on HDL-induced PGI2 release. Human VSMCs were treated with HDL or fatty acids in the presence or absence of different cell signalling inhibitors and PGI2 (by enzyme immunoassay) and Cox-2 protein levels (by Western blot) were analyzed. High-density lipoproteins were obtained from a plasma pool or from plasma of 12 volunteers subjected to a longitudinal dietary interventional study of three consecutive diets periods enriched in monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids n-6 (PUFA n-6) or n-3 (PUFA n-3). High-density lipoprotein delipidation attenuated the effect of HDL on both PGI2 synthesis and Cox-2 up-regulation, while arachidonic acid (AA) but not other fatty acids mimicked the effects of HDL. Arachidonic acid induced PGI2 synthesis and Cox-2 expression through similar mechanisms to those activated by HDL [pertussis toxin-sensitive G proteins, p42/44 mitogen-activated protein kinase (MAPK), p38MAPK, and c-Jun N-terminal kinase-1 (JNK-1) pathways]. Finally, we observed that HDL from the PUFA n-3 dietary period induced lower PGI2 release than that from the PUFA n-6 period (64% vs. 100%). Our results suggest that lipid moiety modulates HDL-induced PGI2 release/Cox-2 up-regulation in human VSMCs, and that changes in fatty acids as accomplished with the diet can modulate vascular PGI2 homeostasis.

Formation of apolipoprotein AI-phosphatidylcholine core aldehyde Schiff base adducts promotes uptake by THP-1 macrophages.

High-density lipoprotein (HDL) is believed to protect against development of atherosclerosis by inhibiting the accumulation of oxidized lipids in low-density lipoprotein (LDL). Paradoxically, HDL lipid is more susceptible to oxidation than LDL lipid. In the present study, we examined the effect of oxidized phospholipids on the uptake of HDL by macrophages. Oxidation of HDL increased formation of phosphatidylcholine core aldehydes that was paralleled by increased covalent binding of phospholipids to HDL protein from 0.96+/-0.44 to 8.5+/-1.76 phosphorus/HDL protein (mol/mol). Incubation of apolipoprotein AI with synthetically prepared phosphatidylcholine core aldehydes, 1-palmitoyl-2-[5-oxo]valeroyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-[9-oxo] nonanoyl-sn-glycero-3-phosphocholine, significantly increased the phosphorus:apolipoprotein AI ratio from 1.1+/-0.5 to 7.2+/-2.0 and from 0.9+/-0.6 to 8.5+/-0.8, respectively. The binding and uptake of phosphatidylcholine core aldehyde-apolipoprotein AI proteoliposomes, by THP-1 macrophages, was similar to that observed for oxidized HDL and oxidized LDL. We conclude that oxidation of HDL increased formation of phosphatidylcholine core aldehyde-apolipoprotein AI Schiff base adducts and enhanced uptake of oxidized HDL by THP-1 macrophages.

Identification of the N-Linked Glycosylation Sites on the High Density Lipoprotein (HDL) Receptor SR-BI and Assessment of Their Effects on HDL Binding and Selective Lipid Uptake

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.

Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.

The high-density lipoprotein (HDL) receptor, scavenger receptor, class B, type I (SR-BI), mediates both the selective uptake of lipids, mainly cholesterol esters, from HDL to cells and the efflux of cholesterol from cells to lipoproteins. The mechanism underlying these lipid transfers is distinct from classic receptor-mediated endocytosis, but it remains poorly understood. To investigate SR-BI's mechanism of action and in vivo function, we developed a high-throughput screen to identify small molecule inhibitors of SR-BI-mediated lipid transfer in intact cells. We identified five compounds that in the low nanomolar to micromolar range block lipid transport (BLTs), both selective uptake and efflux. The effects of these compounds were highly specific to the SR-BI pathway, because they didn't interfere with receptor-mediated endocytosis or with other forms of intracellular vesicular traffic. Surprisingly, all five BLTs enhanced, rather than inhibited, HDL binding by increasing SR-BI's binding affinity for HDL (decreased dissociation rates). Thus, the BLTs provide strong evidence for a mechanistic coupling between HDL binding and lipid transport and may serve as a starting point for the development of pharmacologically useful modifiers of SR-BI activity and, thus, HDL metabolism.

Effects of dietary fish oil on the fatty acid composition of the main lipid classes of chick plasma lipoproteins.

We have studied the effects of diet supplementation with 10% fish oil on fatty acid composition of the main lipid classes of chick plasma lipoproteins bearing in mind the relationship between platelet aggregation and eicosanoid production from arachidonic acid. Fish oil drastically increased the percentages of 20:5 n-3 and 22:6 n-3 acids in the high density lipoprotein lipids. The 20:5/22:6 ratio increased in triacylglycerol fraction whereas in phospholipids and cholesterol esters both 20:5 and 22:6 acids increased in a similar proportion. The percentage of arachidonic acid was higher in phospholipids than in the other lipid classes from this lipoprotein fraction and was significantly reduced by fish oil feeding. Linoleic acid, which was the most abundant fatty acid in cholesterol esters, strongly decreased after fish oil consumption. Changes induced in low- and very low density lipoproteins were similar to that observed in the high density lipoproteins. However, in the very low density lipoproteins, the 20:5/22:6 ratio was not increased in triacylglycerols, in contrast to that found in the high- and low density fractions. Our results suggest that decreases observed by fish oil feeding in the percentages of arachidonic acid in phospholipids and linoleic acid in cholesterol esters in the three lipoprotein fractions may be of importance to explain some pharmacological effects of n-3 PUFA with regard to vascular diseases.

Alcohol Consumption Is Associated With Enrichment of High‐Density Lipoprotein Particles in Polyunsaturated Lipids and Increased Cholesterol Esterification Rate

Background Alcohol consumption is associated with high levels of high‐density lipoproteins (HDLs). Moreover, changes in the fatty acid patterns of red blood cell phospholipids and plasma lipids have been observed in drinkers. The objectives of this study were to characterize the composition of HDL particles with respect to lipid molecular species in regular wine drinkers and to assess the functional properties of those HDLs as regards key steps of reverse cholesterol transport.Methods Forty‐six subjects were recruited in the frame of a population study performed in Toulouse, southern France, and a nutritional investigation, including daily alcohol consumption, was performed. Subjects were sorted according to their daily alcohol intake (0, ≤35, and &gt;35 g/day), mostly as red wine. The plasma HDL fraction was isolated, and neutral lipid molecular lipids and phospholipid fatty acids were analyzed by gas liquid chromatography. Efflux of cellular cholesterol and rates of cholesterol esterification and cholesteryl ester transfers between lipoproteins were assayed in a cell‐plasma incubation system.Results Wine drinking, at 47 g/day, was associated with an increase in HDL cholesterol and apolipoprotein A‐I, but not with triglycerides. Isolated HDL displayed a 27% increase in all cholesteryl ester molecular species. The particles were also enriched in unsaturated phospholipids and, particularly, in those containing arachidonic (+30%) and eicosapentaenoic (+90%) acids. The plasma cholesterol esterification rate, reflecting lecithin cholesterol acyl transferase activity on HDL, was found to be higher (+27%) in drinkers than in nondrinkers, whereas the rate of cellular cholesterol efflux to plasma was identical.Conclusions Regular wine consumption is associated with high levels of polyunsaturated lipids in HDL and with increases in the cholesterol esterification rate.

Alcohol Consumption Is Associated With Enrichment of High-Density Lipoprotein Particles in Polyunsaturated Lipids and Increased Cholesterol Esterification Rate

Background Alcohol consumption is associated with high levels of high-density lipoproteins (HDLs). Moreover, changes in the fatty acid patterns of red blood cell phospholipids and plasma lipids have been observed in drinkers. The objectives of this study were to characterize the composition of HDL particles with respect to lipid molecular species in regular wine drinkers and to assess the functional properties of those HDLs as regards key steps of reverse cholesterol transport. Methods Forty-six subjects were recruited in the frame of a population study performed in Toulouse, southern France, and a nutritional investigation, including daily alcohol consumption, was performed. Subjects were sorted according to their daily alcohol intake (0, ≤35, and >35 g/day), mostly as red wine. The plasma HDL fraction was isolated, and neutral lipid molecular lipids and phospholipid fatty acids were analyzed by gas liquid chromatography. Efflux of cellular cholesterol and rates of cholesterol esterification and cholesteryl ester transfers between lipoproteins were assayed in a cell-plasma incubation system. Results Wine drinking, at 47 g/day, was associated with an increase in HDL cholesterol and apolipoprotein A-I, but not with triglycerides. Isolated HDL displayed a 27% increase in all cholesteryl ester molecular species. The particles were also enriched in unsaturated phospholipids and, particularly, in those containing arachidonic (+30%) and eicosapentaenoic (+90%) acids. The plasma cholesterol esterification rate, reflecting lecithin cholesterol acyl transferase activity on HDL, was found to be higher (+27%) in drinkers than in nondrinkers, whereas the rate of cellular cholesterol efflux to plasma was identical. Conclusions Regular wine consumption is associated with high levels of polyunsaturated lipids in HDL and with increases in the cholesterol esterification rate.