Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher (125)I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71-144% increase in (125)I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%-73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%-66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.     

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.     

Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein

Cholesteryl ester (CE) accumulation in arterial wall macrophages (foam cells), mediated by the intracellular enzyme acyl coenzyme A:cholesterol acyl transferase (ACAT), is a prominent feature of atherosclerotic lesions. However, native low density lipoprotein (LDL) does not cause activation of ACAT or CE accumulation in cultured mouse peritoneal macrophages despite both substantial LDL uptake and degradation and the presence of ACAT in these cells. We now report that when protein synthesis is inhibited in mouse peritoneal macrophages by treatment with cycloheximide, puromycin, or actinomycin D, native LDL-induced whole-cell ACAT activity and CE accumulation is 10-fold higher than that seen in LDL-treated control cells. The enhancement of ACAT activity was seen 4 h after the addition of cycloheximide, and ACAT activity returned to control values 4 h after the withdrawal of cycloheximide. Postnuclear supernatants and microsomes from cycloheximide-treated mouse peritoneal macrophages also had higher ACAT activity than microsomes from control cells, but the relative enhancement (maximum 3.3-fold) was less than that seen when ACAT was assayed in the intact cell. In contrast to the situation with mouse peritoneal macrophages, cycloheximide treatment of J774 macrophages, which under normal conditions display high ACAT activity and CE accumulation in the presence of native LDL, did not result in further enhancement of either ACAT activity or LDL-induced CE accumulation. From these data we postulate that mouse peritoneal macrophages have a short-lived protein that inhibits ACAT-mediated cholesterol esterification which is responsible for their lack of ACAT response and CE accumulation in the presence of native LDL. The explanation for high ACAT activity and LDL-induced CE accumulation in J774 macrophages may be that these cells lack the putative mouse peritoneal macrophage cholesterol esterification inhibitor.     

Differential abilities of mouse liver parenchymal and nonparenchymal cells in HDL and LDL (native and oxidized) association and cholesterol efflux.

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.     

The role of human and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake of low-density lipoprotein-cholesteryl esters

Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.     

Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.

Scavenger receptor, class B, type I (SR-BI) is a cell-surface glycoprotein that mediates selective uptake of high density lipoprotein cholesteryl ester (CE) without the concomitant uptake and degradation of the particle. We have investigated the endocytic and selective uptake of low density lipoprotein (LDL)-CE by SR-BI using COS-7 cells transiently transfected with mouse SR-BI. Analysis of lipoprotein uptake data showed a concentration-dependent LDL-CE-selective uptake when doubly labeled LDL particles were incubated with SR-BI-expressing COS-7 cells. In contrast to vector-transfected cells, SR-BI-expressing COS-7 cells showed marked increases in LDL cell association and CE uptake by the selective uptake pathway, but only a modest increase in CE uptake by the endocytic pathway. SR-BI-mediated LDL-CE-selective uptake exceeded LDL endocytic uptake by 50-100-fold. SR-BI-mediated LDL-CE-selective uptake was not inhibited by the proteoglycan synthesis inhibitor, p-nitrophenyl-beta-D-xylopyranoside or by the sulfation inhibitor sodium chlorate, indicating that SR-BI-mediated LDL-CE uptake occurs independently of LDL interaction with cell-surface proteoglycan. Analyses with subclones of Y1 adrenocortical cells showed that LDL-CE-selective uptake was proportional to the level of SR-BI expression. Furthermore, antibody directed to the extracellular domain of SR-BI blocked LDL-CE-selective uptake in adrenocortical cells. Thus, in cells that normally express SR-BI and in transfected COS-7 cells SR-BI mediates the efficient uptake of LDL-CE via the selective uptake mechanism. These results suggest that SR-BI may influence the metabolism of apoB-containing lipoproteins in vivo by mediating LDL-CE uptake into SR-BI-expressing cells.     

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoproteins C-II and C-III inhibit selective uptake of low- and high-density lipoprotein cholesteryl esters in HepG2 cells

Plasma low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors and are either totally degraded or their cholesteryl esters (CE) are selectively delivered to cells by receptors such as the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-II and apoC-III on the uptake of LDL and HDL by HepG2 cells. Stable transformants were obtained with sense or antisense strategies that secrete 47-294% the normal level of apoC-II or 60-200% that of apoC-III. Different levels of secreted apoC-II or apoC-III had little effect on LDL and HDL protein degradation by HepG2 cells. However, compared to controls, cells under-expressing apoC-II showed a 160% higher capacity to selectively take up HDL-CE, while cells under-expressing apoC-III demonstrated 70 and 160% higher capacity to take up CE from LDL and HDL, respectively. In experiments conducted with exogenously added apoC-II or apoC-III, no significant effect was observed on lipoprotein-protein association/degradation; however, LDL-CE and HDL-CE selective uptake was significantly reduced in a dose-dependent manner. These results indicate that apoC-II and apoC-III inhibit CE-selective uptake.     

Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP

The citrus flavonoids, naringenin and hesperetin, lower plasma cholesterol in vivo. However, the underlying mechanisms are not fully understood. The ability of these flavonoids to modulate apolipoprotein B (apoB) secretion and cellular cholesterol homeostasis was determined in the human hepatoma cell line, HepG2. apoB accumulation in the media decreased in a dose-dependent manner following 24-h incubations with naringenin (up to 82%, P < 0.00001) or hesperetin (up to 74%, P < 0.002). Decreased apoB secretion was associated with reduced cellular cholesteryl ester mass. Cholesterol esterification was decreased, dose-dependently, up to 84% (P < 0.0001) at flavonoid concentrations of 200 microM. Neither flavonoid demonstrated selective inhibition of either form of acyl CoA:cholesterol acyltransferase (ACAT) as determined using CHO cells stably transfected with either ACAT1 or ACAT2. However, in HepG2 cells, ACAT2 mRNA was selectively decreased (- 50%, P < 0.001) by both flavonoids, whereas ACAT1 mRNA was unaffected. In addition, naringenin and hesperetin decreased both the activity (- 20% to - 40%, P < 0.00004) and expression (- 30% to - 40%, P < 0.02) of microsomal triglyceride transfer protein (MTP). Both flavonoids caused a 5- to 7-fold increase (P < 0.02) in low density lipoprotein (LDL) receptor mRNA, which resulted in a 1.5- to 2-fold increase in uptake and degradation of (125)I-LDL. We conclude that both naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by 1) reduced activities of ACAT1 and ACAT2, 2) a selective decrease in ACAT2 expression, and 3) reduced MTP activity. Together with an enhanced expression of the LDL receptor, these mechanisms may explain the hypocholesterolemic properties of the citrus flavonoids.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

Apolipoprotein C-I reduces cholesteryl esters selective uptake from LDL and HDL by binding to HepG2 cells and lipoproteins

Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL₃ by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein–protein association and degradation; however, LDL- and HDL₃-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL₃. Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL₃-CE selective uptake and pre-incubation of LDL and HDL₃ with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.     

Urotensin II differentially regulates macrophage and hepatic cholesterol homeostasis

Urotensin II (UII) is a vasoactive peptide with pleotropic activity. Interestingly, UII levels are elevated in hyperlipidemic patients, and UII induces lipase activity in some species. However, the exact role UII plays in cholesterol homeostasis remains to be elucidated. UII knockout (UII KO) mice were generated and a plasma lipoprotein profile, and hepatocytes and macrophages cholesterol uptake, storage and synthesis was determined. UII KO had a decreased LDL cholesterol profile and liver steatosis compared to wildtype mice (WT). UII KO macrophages demonstrated enhanced ACAT activity and LDL uptake in the short term (up to 4 h), of which more LDL-delivered exogenously derived cholesterol was incorporated into cholesteryl ester (CE) than the WT macrophages. UII KO macrophages generated more than two times the amount of de novo endogenously synthesized cholesterol, and of this cholesterol more than two times the relative amount was esterified to CE. In comparison, results in hepatocytes demonstrated that far more exogenously derived cholesterol was incorporated into CE in the WT cells, generating almost ten times the amount of CE than UII KO. WT cells synthesize de novo almost ten times the amount of cholesterol than UIIKO, and of that cholesterol, almost two times the amount of CE in WT than UII KO hepatocytes. In addition, more ApoB lipoproteins were secreted from WT than UII KO hepatocytes. These results demonstrate a fundamental difference between macrophages and hepatocytes in terms of cholesterol homeostasis, and suggest an important role for UII in modulating cholesterol regulation.     

Selective uptake from LDL is stimulated by unsaturated fatty acids and modulated by cholesterol content in the plasma membrane: role of plasma membrane composition in regulating non-SR-BI-mediated selective lipid transfer

We previously reported that unsaturated fatty acids stimulated low-density lipoprotein (LDL) particle uptake in J774 macrophages by increasing LDL receptor activity. Since free fatty acids (FFA) also change plasma membrane properties, a putative cholesteryl ester (CE) acceptor for selective uptake (SU), we questioned the ability of FFA to modulate SU from LDL. Using [(3)H]cholesteryl ether/(125)I-LDL to trace CE core and whole particle uptake, we found that oleic acid and eicosapentaenoic acid, but not saturated stearic acid, increased SU by 30% over control levels. An ACAT inhibitor, Dup128, abolished FFA effects on SU, indicating that increased SU by FFA was secondary to changes in cell-free cholesterol (FC). Consistent with these observations, ACAT inhibition increased cell FC and reduced LDL SU by half. The important role of plasma membrane composition was further demonstrated in that beta-cyclodextrin- (beta-CD-) mediated FC removal from the plasma membrane increased SU from LDL and was further stimulated by U18666A, a compound that inhibits FC transport between lysosomes and the plasma membrane. In contrast, cholesterol-saturated beta-CD markedly reduced LDL SU. In contrast to LDL SU, oleic acid, ACAT inhibition, U18666A, or beta-CD had no effects on HDL SU. Moreover, HDL SU was inhibited by antimouse SR-BI antibody by more than 50% but had little effect on LDL SU. In C57BL/6 mice fed a high fat diet, plasma FFA levels increased, and SU accounted for an almost 4-fold increased proportion of total cholesterol delivery to the arterial wall. Taken together, these data suggest that LDL SU is mediated by pathways independent of SR-BI and is influenced by plasma membrane FC content. Moreover, in conditions where elevated plasma FFA occur, SU from LDL can be an important mechanism for cholesterol delivery in vivo.     

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.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.     

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.