Uptake Of High-density Lipoprotein Research Articles

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200%) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.

Ezetimibe Inhibits Hepatic Niemann-Pick C1-Like 1 to Facilitate Macrophage Reverse Cholesterol Transport in Mice

Controversies have arisen from recent mouse studies about the essential role of biliary sterol secretion in reverse cholesterol transport (RCT). The objective of this study was to examine the role of biliary cholesterol secretion in modulating macrophage RCT in Niemann-Pick C1-Like 1 (NPC1L1) liver only (L1(LivOnly)) mice, an animal model that is defective in both biliary sterol secretion and intestinal sterol absorption, and determine whether NPC1L1 inhibitor ezetimibe facilitates macrophage RCT by inhibiting hepatic NPC1L1. L1(LivOnly) mice were generated by crossing NPC1L1 knockout (L1-KO) mice with transgenic mice overexpressing human NPC1L1 specifically in liver. Macrophage-to-feces RCT was assayed in L1-KO and L1(LivOnly) mice injected intraperitoneally with [(3)H]-cholesterol-labeled peritoneal macrophages isolated from C57BL/6 mice. Inhibition of biliary sterol secretion by hepatic overexpression of NPC1L1 substantially reduced transport of [(3)H]-cholesterol from primary peritoneal macrophages to the neutral sterol fraction in bile and feces in L1(LivOnly) mice without affecting tracer excretion in the bile acid fraction. Ezetimibe treatment for 2 weeks completely restored both biliary and fecal excretion of [(3)H]-tracer in the neutral sterol fraction in L1(LivOnly) mice. High-density lipoprotein kinetic studies showed that L1(LivOnly) mice compared with L1-KO mice had a significantly reduced fractional catabolic rate without altered hepatic and intestinal uptake of high-density lipoprotein-cholesterol ether. In mice lacking intestinal cholesterol absorption, macrophage-to-feces RCT depends on efficient biliary sterol secretion, and ezetimibe promotes macrophage RCT by inhibiting hepatic NPC1L1 function.

High-density lipoprotein endocytosis in endothelial cells

To describe the way stations of high-density lipoprotein (HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescence microscopy. HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type I mediated selective uptake without concomitant HDL endocytosis. HDL endocytosis occurs via clathrin-coated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: Evidence from cultured explants

It is well established that inactivation of the central endocannabinoid system (ECS) through antagonism of cannabinoid receptor 1 (CB1R) reduces food intake and improves several pathological features associated with obesity, such as dyslipidemia and liver steatosis. Nevertheless, recent data indicate that inactivation of peripheral CB1R could also be directly involved in the control of lipid metabolism independently of central CB1R. To further investigate this notion, we tested the direct effect of the specific CB1R antagonist, SR141716, on hepatic carbohydrate and lipid metabolism using cultured liver slices. CB1R messenger RNA expression was strongly decreased by SR141716, whereas it was increased by the CB1R agonist, arachidonic acid N-hydroxyethylamide (AEA), indicating the effectiveness of treatments in modulating ECS activity in liver explants both from lean or ob/ob mice. The measurement of O(2) consumption revealed that SR141716 increased carbohydrate or fatty acid utilization, according to the cellular hormonal environment. In line with this, SR141716 stimulated ß-oxidation activity, and the role of CB1R in regulating this pathway was particularly emphasized when ECS was hyperactivated by AEA and in ob/ob tissue. SR141716 also improved carbohydrate and lipid metabolism, blunting the AEA-induced increase in gene expression of proteins related to lipogenesis. In addition, we showed that SR141716 induced cholesterol de novo synthesis and high-density lipoprotein uptake, revealing a relationship between CB1R and cholesterol metabolism. These data suggest that blocking hepatic CB1R improves both carbohydrate and lipid metabolism and confirm that peripheral CB1R should be considered as a promising target to reduce cardiometabolic risk in obesity.

Mitochondrial Inhibitory Factor 1 (IF1) Is Present in Human Serum and Is Positively Correlated with HDL-Cholesterol

BackgroundMitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in High Density Lipoproteins (HDL). On hepatocytes, apoA-I binds to cell surface ATP synthase (namely ecto-F1-ATPase) and stimulates its ATPase activity, generating extracellular ADP. This production of extracellular ADP activates a P2Y13-mediated HDL endocytosis pathway. Conversely, exogenous IF1, classically known as a natural mitochondrial specific inhibitor of F1-ATPase activity, inhibits ecto-F1-ATPase activity and decreases HDL endocytosis by both human hepatocytes and perfused rat liver.Methodology/Principal FindingsSince recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 is present in the systemic circulation in humans. We first unambiguously detected IF1 in human serum by immunoprecipitation and mass spectrometry. We then set up a competitive ELISA assay in order to quantify its level in human serum. Analyses of IF1 levels in 100 normolipemic male subjects evidenced a normal distribution, with a median value of 0.49 µg/mL and a 95% confidence interval of 0.22–0.82 µg/mL. Correlations between IF1 levels and serum lipid levels demonstrated that serum IF1 levels are positively correlated with HDL-cholesterol and negatively with triglycerides (TG).Conclusions/SignificanceAltogether, these data support the view that, in humans, circulating IF1 might affect HDL levels by inhibiting hepatic HDL uptake and also impact TG metabolism.

High Density Lipoprotein (HDL) Promotes Glucose Uptake in Adipocytes and Glycogen Synthesis in Muscle Cells

BackgroundHigh density lipoprotein (HDL) was reported to decrease plasma glucose and promote insulin secretion in type 2 diabetes patients. This investigation was designed to determine the effects and mechanisms of HDL on glucose uptake in adipocytes and glycogen synthesis in muscle cells.Methods and ResultsActions of HDL on glucose uptake and GLUT4 translocation were assessed with 1-[3H]-2-deoxyglucose and plasma membrane lawn, respectively, in 3T3-L1 adipocytes. Glycogen analysis was performed with amyloglucosidase and glucose oxidase-peroxidase methods in normal and palmitate-treated L6 cells. Small interfering RNA was used to observe role of scavenger receptor type I (SR-BI) in glucose uptake of HDL. Corresponding signaling molecules were detected by immunoblotting. HDL stimulated glucose uptake in a time- and concentration-dependent manner in 3T3-L1 adipocytes. GLUT4 translocation was significantly increased by HDL. Glycogen deposition got enhanced in L6 muscle cells paralleling with elevated glycogen synthase kinase3 (GSK3) phosphorylation. Meanwhile, increased phosphorylations of Akt-Ser473 and AMP activated protein kinase (AMPK) α were detected in 3T3-L1 adipocytes. Glucose uptake and Akt-Ser473 activation but not AMPK-α were diminished in SR-BI knock-down 3T3-L1 cells.ConclusionsHDL stimulates glucose uptake in 3T3-L1 adipocytes through enhancing GLUT4 translocation by mechanisms involving PI3K/Akt via SR-BI and AMPK signaling pathways, and increases glycogen deposition in L6 muscle cells through promoting GSK3 phosphorylation.

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (-33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (-29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.

Ecto-F₁-ATPase: a moonlighting protein complex and an unexpected apoA-I receptor.

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F₁-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F₁-ATPase. Particularly, the role of the F₁-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions.

P2Y13 Receptor Is Critical for Reverse Cholesterol Transport

A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y(13) (purinergic receptor P2Y, G protein-coupled, 13) activation is essential for HDL uptake but the potential of P2Y(13) as a target to promote RCT has not been documented. Here, we show that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y(13)-deficient mice. Furthermore, cangrelor, a partial agonist of P2Y(13), stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI-knockout(liver)) but had no effect in P2Y(13) knockout mice, which indicate that P2Y(13)-mediated HDL uptake pathway is independent of SR-BI-mediated HDL selective cholesteryl ester uptake. These results establish P2Y(13) as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT.

High density lipoprotein inhibits the activation of sterol regulatory element-binding protein-1 in cultured cells

A link between cellular uptake of high density lipoprotein (HDL) and regulation of sterol regulatory element-binding protein-1 (SREBP-1) was investigated in vitro. HDL decreased nuclear SREBP-1 levels as well as SREBP-1 target gene expression in HepG2 and HEK293 cells. However, HDL did not repress an exogenously expressed, constitutively active form of SREBP-1. HDL increased cellular cholesterol levels, and cellular cholesterol depletion by methyl-β-cyclodextrin abolished the effects of HDL. These results suggest that HDL inhibits the activation of SREBP-1 through a cholesterol-dependent mechanism, which may play an important role in regulating lipid synthetic pathways mediated by SREBP-1.

Paraoxonase 1 deficiency in mice is associated with reduced steroid biosynthesis: Effects on HDL binding, cholesteryl ester accumulation and scavenger receptor type BI expression

ObjectiveSelective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis. Methods and resultsPON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthesis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demonstrated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice. ConclusionPON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.

Atherogenic Lipids Induce High-Density Lipoprotein Uptake and Cholesterol Efflux in Human Macrophages by Up-Regulating Transmembrane Chemokine CXCL16 without Engaging CXCL16-Dependent Cell Adhesion

Atherosclerosis is a complex pathologic process in which chemokine-mediated leukocyte accumulation in arterial walls is thought to be an important mechanism of pathogenesis. An interesting exception to this paradigm is the chemokine CXCL16, also known as the scavenger receptor for phosphatidylserine and oxidized low density lipoprotein, which is highly expressed in mouse and human atherosclerotic lesions, yet appears to be atheroprotective. In this study, we address potential mechanisms responsible for this activity. Consistent with its presence in atherosclerotic plaque, we found that atherogenic lipids up-regulated CXCL16 in primary human monocyte-derived macrophages. However, the same lipids down-regulated the CXCL16-targeted protease ADAM10, resulting in preferential expression of CXCL16 as the transmembrane form, not the shed form. Although transmembrane CXCL16 is known to mediate cell-cell adhesion by binding its receptor CXCR6, and atherogenic lipids are known to stimulate macrophage adhesion to coronary artery smooth muscle cells, we found that heterotypic adhesion of these cell types occurred in a CXCL16-independent manner. Instead we found that in macrophages, CXCL16 promoted internalization of both oxidized low density lipoprotein and high density lipoprotein, as well as release of cholesterol. Moreover, CXCL16 deficiency in macrophages interfered with oxidized low density lipoprotein-induced up-regulation of atheroprotective genes: adenosine triphosphate-binding cassette transporter A1 and G1 as well as apolipoprotein E. Thus, our findings support the hypothesis that CXCL16 mediates atheroprotection through its scavenger role in macrophages and not by cell-cell adhesion.

Scavenger Receptor BI-mediated Selective Uptake Is Required for the Remodeling of High Density Lipoprotein by Endothelial Lipase

Endothelial lipase (EL) is a negative regulator of high density lipoprotein (HDL) cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodeling of HDL. The present study investigates the requirement of SR-BI for the effects of EL-mediated phospholipid hydrolysis on HDL metabolism in vivo. In vitro, selective uptake from EL-modified HDL was 129% higher than selective uptake from control HDL in SR-BI-overexpressing cells (p=0.01). In vivo overexpression of human EL by means of recombinant adenovirus decreased HDL plasma levels significantly (p<0.01). Fast protein liquid chromatography analysis and agarose gel electrophoresis revealed that EL expression resulted in the generation of small pre-beta HDL particles in wild-type mice, whereas in SR-BI-/- mice small HDL were preferentially removed. In kinetic experiments the fractional catabolic rate (FCR) of HDL cholesteryl ester increased by 110% (p<0.001), and the FCR of HDL apolipoproteins increased by 64% (p<0.001) in response to EL overexpression in wild-type mice. In SR-BI-/- mice a similar increase in the HDL apolipoprotein FCR occurred (p<0.001); however, there was no further increase in HDL cholesteryl ester catabolism. The apparent whole body selective uptake was increased 3-fold by EL in wild-type mice (p<0.001), whereas there was no selective uptake in SR-BI knock-out mice. EL overexpression increased hepatic selective uptake as well as holoparticle uptake (each p<0.01) in wild-type mice, whereas in SR-BI knock-out mice only holoparticle uptake increased (p<0.01). Our results indicate that SR-BI-mediated selective uptake of HDL cholesteryl ester is essential for the remodeling of large alpha-migrating HDL particles by EL.

Characterization of the Isoflavone Pratensein as a Novel Transcriptional Up-Regulator of Scavenger Receptor Class B Type I in HepG2 Cells

Scavenger receptor class B type I (SR-BI), as well as its human homologue CLA-1, plays an important role in reverse cholesterol transport (RCT) as high density lipoprotein (HDL) receptor. Using a previously developed cell-based screening model for CLA-1 up-regulators, pratensein, was shown to present activity in elevating CLA-1 transcriptional level. In this study, three other isoflavones including formononetin, genistein and daidzein were also shown to up-regulate CLA-1 transcriptional activity in the cell-based reporter assay. The effects of pratensein on up-regulating CLA-1 expression were demonstrated at both mRNA and protein levels, and validated by its increasing of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled (DiI)-HDL uptake in HepG2 cells. Furthermore, the cis-elements responsible for the pratensein up-regulatory effects were mapped to the -1055/-182 bp fragment of CLA-1 promoter in HepG2 cells. These findings might provide a new molecular mechanism by which isoflavones potentially prevent atherosclerosis.

Abstract 5478: P2Y 13 ADP-Receptor Regulates Plasma HDL Clearance, Biliary Cholesterol and Bile Acids Secretions In Vivo

Plasma high density lipoprotein (HDL) protects against atherosclerosis, in part through the removal of cholesterol from peripheral tissues and its delivery to the liver for metabolism and bile excretion. Thus understanding of the molecular event implicated in HDL-cholesterol uptake by the liver is critical to the development of therapeutic strategies to enhance HDL catabolism. The scavenger receptor (SR)-BI modulates plasma HDL level and contributes to cholesteryl ester uptake from HDL in mice but a distinct pathway for holo-HDL particle uptake (protein + lipid) has been recently identified in human hepatocytes. This pathway which strictly depends on extracellular ADP level involves different cell surface partners: ADP generated from the activation by apo AI (the main HDL apolipoprotein) of a cell surface F 1 -ATPase, activates the P2Y 13 ADP-receptor and subsequent HDL endocytosis through an unknown low-affinity HDL receptor. To evaluate the physiological relevance of this P2Y 13 -mediated HDL endocytosis pathway in vivo in mice, P2Y 13 receptor was activated by treating C57BL/6 and liver specific SR-BI-KO mice (SR-BI-KO liver ) with the specific agonist (AR-C69931MX, 8nmol/kg, intra-orbital injection). Expression of P2Y 13 protein in liver was also disrupted by siRNA adenovirus (10 9 vp/mice, intra-orbital injection) and in P2Y 13 KO mice. Plasma clearance of 125 I-labeled HDL was rapidly enhanced under AR-C69931MX treatment in SR-BI-KO liver mouse. Conversely, HDL clearance was decreased in P2Y 13 KO mice but no modification was observed in plasma lipids profile versus control mice, suggesting a mechanism of modulation of plasma HDL turnover rather than a simply disruption of HDL uptake. Furthermore, biliary lipids content was decreased under P2Y 13 extinction in mice (bile acids: −60%, cholesterol: −30%), whereas it was dramatically increased under stimulation of P2Y 13 by AR-C69931MX treatment in C57BL/6 mice (bile acids: +85%, cholesterol: +50%,). These results indicate that P2Y 13 has an important role in hepatic HDL metabolism in controlling circulating HDL turnover and biliary lipids elimination, and thus may have an impact on the development of atherosclerosis.

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

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

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

A novel compound, R-138329, increases plasma HDL cholesterol via inhibition of scavenger receptor BI-mediated selective lipid uptake

High-density lipoprotein (HDL) has a protective effect against atherosclerosis. Therefore, a compound that elevates the plasma HDL cholesterol (HDL-C) levels is expected to be a promising anti-atherosclerotic agent. We discovered a novel compound, R-138329, that increased HDL-C by 41% in normolipidemic hamsters at a dose of 100 mg/kg. To investigate the mechanism of action of R-138329, we examined the effect of R-138329 on the clearance of [ 3H]cholesterol ether ([ 3H]COE)-labeled and [ 125I]-labeled HDL in mice. R-138329 delayed the clearance of [ 3H]COE-labeled HDL and reduced accumulation of tracer HDL in the liver, whereas the clearance of [ 125I]-labeled HDL particles was unaffected by the compound. In vitro analysis showed that R-154716, a metabolite of R-138329, dramatically inhibited the uptake of [ 3H]COE-labeled HDL in McA-RH 7777 rat hepatoma cells. Furthermore, 100 nM of R-154716 completely inhibited [ 3H]COE-labeled HDL uptake induced by overexpression of scavenger receptor BI (SR-BI) in HEK293 cells. Taken together, these findings suggest that the mechanism by which R-138329 elevates HDL-C in vivo is principally involved in the inhibition of SR-BI-mediated selective lipid uptake in the liver.