PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI.

PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/(3)H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change (3)H-tracer plasma appearance, but surprisingly decreased fecal (3)H-free sterol excretion by 43% (P<0.01) over 48h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with (3)H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived (3)H-CE uptake by adipose tissue (P<0.005) with concomitant 22% decrease in HDL derived (3)H-CE uptake by the liver (P<0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived (3)H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.

Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.

OBJECTIVE: Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro. METHODS AND RESULTS: Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001). CONCLUSIONS: The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

Lecithin: cholesterol acyltransferase expression has minimal effects on macrophage reverse cholesterol transport in vivo.

BACKGROUND: Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high-density lipoprotein metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT). METHODS AND RESULTS: The role of LCAT in RCT from macrophages was quantified with a validated assay involving intraperitoneal injection in mice of (3)H-cholesterol-labeled J774 macrophages and monitoring the appearance of tracer in plasma, liver, bile, and feces. Human LCAT overexpression in human apolipoprotein A-I transgenic mice substantially increased plasma high-density lipoprotein cholesterol levels but surprisingly did not increase macrophage RCT. Even in the setting of coexpression of scavenger receptor BI or cholesteryl ester transfer protein, both of which promoted the transfer of LCAT-derived high-density lipoprotein cholesterol ester to the liver, LCAT overexpression still had no effect on RCT. Serum from LCAT-overexpressing mice had reduced ability to promote cholesterol efflux from macrophages ex vivo via ABCA1. To determine the effect of LCAT deficiency on macrophage RCT, LCAT(-/-) and LCAT(+/-) mice were compared with wild-type mice. Despite extremely low plasma levels of high-density lipoprotein cholesterol, LCAT-deficient mice had only a 50% reduction in RCT. LCAT(+/-) mice had normal RCT despite a significant reduction in high-density lipoprotein cholesterol. Serum from LCAT-deficient mice had increased ability to promote ABCA1-mediated cholesterol efflux from macrophages ex vivo. CONCLUSIONS: These results demonstrate that LCAT overexpression does not promote an increased rate of macrophage RCT. Although LCAT activity does become rate limiting in the context of complete LCAT deficiency, RCT is reduced by only 50% even in the absence of LCAT. These data suggest that macrophage RCT may not be as dependent on LCAT activity as has previously been believed.

A naturally occurring variant of endothelial lipase associated with elevated HDL exhibits impaired synthesis.

Human endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels.

Both the peroxisome proliferator-activated receptor delta agonist, GW0742, and ezetimibe promote reverse cholesterol transport in mice by reducing intestinal reabsorption of HDL-derived cholesterol.

Peroxisome proliferator-activated receptor delta (PPARdelta) agonism increases HDL cholesterol and has therefore the potential to stimulate macrophage-to-feces reverse cholesterol transport (RCT). To test whether PPARdelta activation promotes RCT in mice, in vivo macrophage RCT was assessed using cholesterol-loaded/3H-cholesterol-labeled macrophages injected intraperitoneally. PPARdelta agonist GW0742 (10 mg/kg per day) did not change 3H-tracer plasma appearance, but increased fecal 3H-free sterols excretion by 103% ( p < 0.005) over 48 hours. Total free cholesterol efflux from macrophages to serum (collected from both control and GW0742 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW0742. The metabolic fate of HDL labeled with 3H- cholesteryl ether or 3H-cholesteryl oleate was also measured. While 3H-cholesteryl ether tissue uptake was unchanged, the 3H-tracer recovered in fecal free sterol fraction after 3H-cholesteryl oleate injection increased by 88% with GW0742 ( p < 0.0005). This was associated with a lower Niemann-Pick C1 like 1 (NPC1L1) mRNA expression in the small intestine ( p < 0.05). The same experiments in mice treated with ezetimibe, which blocks NPC1L1, showed a similar 2-fold increase in fecal free sterol excretion after labeled macrophages orHDL injection. In conclusion, PPARdelta activation enhances excretion of macrophage or HDL-derived cholesterol in feces through reduced NPC1L1 expression in mice, comparable to the effect of ezetimibe.

Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo.

OBJECTIVE: Apolipoprotein F (ApoF) is a protein component of several lipoprotein classes including HDL. It is also known as lipid transfer inhibitor protein (LTIP) based on its ability to inhibit lipid transfer between lipoproteins ex vivo. We sought to investigate the role of ApoF in HDL metabolism. METHODS AND RESULTS: Adeno-associated viruses (AAV) based on serotype 8, were used to overexpress either murine or human ApoF in mice. Overexpression of murine ApoF significantly reduced total cholesterol levels by 28% (P<0.001), HDL by 27% (P<0.001), and phospholipid levels by 19% (P<0.001). Overexpression of human ApoF had similar effects. Human ApoF was nearly exclusively HDL-associated in mice. In agreement with this finding, greater than 90% of the ApoF in human plasma was found on HDL(3), with only a small amount on LDL. Overexpression of mouse ApoF accelerated the plasma clearance of [(3)H]-cholesteryl ether labeled HDL. Plasma from mice overexpressing ApoF showed improved macrophage cholesterol efflux on a per HDL-C basis. CONCLUSIONS: ApoF overexpression reduces HDL cholesterol levels in mice by increasing clearance of HDL-CE. ApoF may be an important determinant of HDL metabolism and reverse cholesterol transport.

Potent and selective PPAR-alpha agonist LY518674 upregulates both ApoA-I production and catabolism in human subjects with the metabolic syndrome.

OBJECTIVE: The study of PPAR-alpha activation on apoA-I production in humans has been limited to fibrates, relatively weak PPAR-alpha agonists that may have other molecular effects. We sought to determine the effect of a potent and highly specific PPAR-alpha agonist, LY518674, on apoA-I, apoA-II, and apoB-100 kinetics in humans with metabolic syndrome and low levels of HDL cholesterol (C). METHODS AND RESULTS: Subjects were randomized to receive LY518674 (100 microg) once daily (n=13) or placebo (n=15) for 8 weeks. Subjects underwent a kinetic study using a deuterated leucine tracer to measure apolipoprotein production and fractional catabolic rates (FCR) at baseline and after treatment. LY518674 significantly reduced VLDL-C (-38%, P=0.002) and triglyceride (-23%, P=0.002) levels whereas LDL-C and HDL-C levels were unchanged. LY518674 significantly reduced VLDL apoB-100 (-12%, P=0.01) levels, attributable to an increased VLDL apoB-100 FCR with no change in VLDL apoB-100 production. IDL and LDL apoB-100 kinetics were unchanged. LY518674 significantly increased the apoA-I production rate by 31% (P<0.0001), but this was accompanied by a 33% increase in the apoA-I FCR (P=0.002), resulting in no change in plasma apoA-I. There was a 71% increase in the apoA-II production rate (P<0.0001) accompanied by a 25% increase in the FCR (P<0.0001), resulting in a significant increase in plasma apoA-II. CONCLUSIONS: Activation of PPAR-alpha with LY518674 (100 microg) in subjects with metabolic syndrome and low HDL-C increased the VLDL apoB-100 FCR consistent with enhanced lipolysis of plasma triglyceride. Significant increases in the apoA-I and apoA-II production rates were accompanied by increased FCRs resulting in no change in HDL-C levels. These data indicate a major effect of LY518674 on the production and clearance of apoA-I and HDL despite no change in the plasma concentration. The effect of these changes on reverse cholesterol transport remains to be determined.

Effects of nevirapine and efavirenz on HDL cholesterol levels and reverse cholesterol transport in mice.

OBJECTIVE: The mechanism by which non-nucleoside reverse transcriptase inhibitors (NNRTIs) increase HDL cholesterol (HDL-C) in HIV+ patients and the benefits of this with respect to cardiovascular risk are not known. Studies were conducted to test the hypothesis that NNRTIs have a beneficial effect on HDL-C and reverse cholesterol transport (RCT). METHODS: LDLr-/- and hA-I transgenic mice were fed a Western diet containing either nevirapine (20mg/kg per day), efavirenz (10mg/kg per day), or diet alone. hA-I transgenic mice underwent a study to measure RCT (measured by excretion of macrophage [(3)H]-cholesterol into HDL and feces) at 8 weeks. RESULTS: LDLr-/- and hA-I transgenic mice treated with nevirapine and efavirenz had a significant increase in HDL-C level (up to 23% in hA-I transgenic) at 4 weeks. However, there was no difference in HDL levels beyond 4 weeks of treatment. At 4 weeks, the FPLC profile of hA-I transgenic mice showed an increase in large HDL. hApoA-I transgenic mice treated with efavirenz for 4 weeks had increased expression of human apoA-I in liver and an increased human apoA-I production rate. Incubation of plasma from hA-I transgenic mice treated for 4 weeks with [(3)H]-cholesterol-labeled macrophages revealed increased cholesterol efflux to plasma from mice treated with efavirenz and nevirapine. Following injection of hA-I transgenic mice treated for 8 weeks with [(3)H]-cholesterol-labeled macrophages, RCT was increased in the efavirenz (p=0.01) group and trended towards an increase in the nevirapine (p=0.15) group. CONCLUSION: Nevirapine and efavirenz transiently increased HDL-C in LDLr-/- and hA-I transgenic mice fed a Western diet that was associated with increased apoA-I production. An increase in RCT in hA-I transgenic mice at 8 weeks despite no difference in HDL levels indicates that these drugs affect additional factors in the RCT pathway that enhance cholesterol efflux from the macrophage and peripheral tissues to plasma and delivery to liver for excretion. These results suggest that treatment with NNRTIs has a beneficial effect on cholesterol efflux and RCT.

Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo.

Macrophage ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and ABCG1 have been shown to promote cholesterol efflux to extracellular acceptors in vitro and influence atherosclerosis in mice, but their roles in mediating reverse cholesterol transport (RCT) from macrophages in vivo are unknown. Using an assay of macrophage RCT in mice, we found that primary macrophages lacking ABCA1 had a significant reduction in macrophage RCT in vivo, demonstrating the importance of ABCA1 in promoting macrophage RCT, however substantial residual RCT exists in the absence of macrophage ABCA1. Using primary macrophages deficient in SR-BI expression, we found that macrophage SR-BI, which was shown to promote cholesterol efflux in vitro, does not contribute to macrophage RCT in vivo. To investigate whether macrophage ABCG1 is involved in macrophage RCT in vivo, we used ABCG1-overexpressing, -knockdown, and -knockout macrophages. We show that increased macrophage ABCG1 expression significantly promoted while knockdown or knockout of macrophage ABCG1 expression significantly reduced macrophage RCT in vivo. Finally, we show that there was a greater decrease in macrophage RCT from cells where both ABCA1 and ABCG1 expression were knocked down than from ABCG1-knockdown cells. These results demonstrate that ABCA1 and ABCG1, but not SR-BI, promote macrophage RCT in vivo and are additive in their effects.

Endothelial lipase is less effective at influencing HDL metabolism in vivo in mice expressing apoA-II.

Endothelial lipase (EL) plays an important physiological role in modulating HDL metabolism. Data suggest that plasma contains an inhibitor of EL, and previous studies have suggested that apolipoprotein A-II (apoA-II) inhibits the activity of several enzymes involved in HDL metabolism. Therefore, we hypothesized that apoA-II may reduce the ability of EL to influence HDL metabolism. To test this hypothesis, we determined the effect of EL expression on plasma phospholipase activity and HDL metabolism in human apoA-I and human apoA-I/A-II transgenic mice. Expression of EL in vivo resulted in lower plasma phospholipase activity and significantly less reduction of HDL-cholesterol, phospholipid, and apoA-I levels in apoA-I/A-II double transgenic mice compared with apoA-I single transgenic mice. We conclude that the presence of apoA-II on HDL particles inhibits the ability of EL to influence the metabolism of HDL in vivo.

Adipocyte enhancer-binding protein 1 is a potential novel atherogenic factor involved in macrophage cholesterol homeostasis and inflammation.

Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) and liver X receptor alpha (LXRalpha) play pivotal roles in macrophage cholesterol homeostasis and inflammation, key biological processes in atherogenesis. Herein we identify adipocyte enhancer-binding protein 1 (AEBP1) as a transcriptional repressor that impedes macrophage cholesterol efflux, promoting foam cell formation, via PPARgamma1 and LXRalpha down-regulation. Contrary to AEBP1 deficiency, AEBP1 overexpression in macrophages is accompanied by decreased expression of PPARgamma1, LXRalpha, and their target genes ATP-binding cassette A1, ATP-binding cassette G1, apolipoprotein E, and CD36, with concomitant elevation in IL-6, TNF-alpha, monocyte chemoattractant protein 1, and inducible NO synthase levels. AEBP1, but not the C-terminally truncated DNA-binding domain mutant (AEBP1DeltaSty), represses PPARgamma1 and LXRalpha in vitro. Expectedly, AEBP1-overexpressing transgenic (AEBP1TG) macrophages accumulate considerable amounts of lipids compared with AEBP1 nontransgenic macrophages, making them precursors for foam cells. Indeed, AEBP1-overexpressing transgenic macrophages exhibit diminished cholesterol efflux compared with AEBP1 nontransgenic macrophages, whereas AEBP1-knockout (AEBP1-/-) macrophages exhibit enhanced cholesterol efflux compared with wild-type (AEBP1+/+) macrophages. Our in vitro and ex vivo experimental data strongly suggest that AEBP1 plays critical regulatory roles in macrophage cholesterol homeostasis, foam cell formation, and proinflammation. Thereby, we speculate that AEBP1 may be critically implicated in the development of atherosclerosis, and it may serve as a molecular target toward developing antiinflammatory, antiatherogenic therapeutic approaches.

Proprotein convertases [corrected] are responsible for proteolysis and inactivation of endothelial lipase.

Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases.

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.

Role of N-linked glycosylation in the secretion and activity of endothelial lipase.

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.

Endothelial lipase promotes the catabolism of ApoB-containing lipoproteins.

Endothelial lipase (EL) has been found to be a key enzyme in high-density lipoprotein (HDL) metabolism in mice, leading to the concept that inhibition of EL could be a novel strategy for raising HDL cholesterol levels. However, mice are "HDL animals" and the effect of EL on atherogenic apoB-containing lipoproteins has not been elucidated. We previously found that EL is capable of hydrolyzing very low-density lipoprotein (VLDL) and LDL lipids ex vivo. To investigate the role of EL in the metabolism of apoB-containing lipoproteins in vivo, we expressed human EL in three mouse models of elevated apoB-containing lipoproteins: apoE-deficient, LDL receptor-deficient, and human apoB transgenic mice. Unexpectedly, hepatic expression of EL resulted in markedly decreased levels of VLDL/LDL cholesterol, phospholipid, and apoB accompanied by significantly increased LDL apolipoprotein and phospholipid catabolism. To determine whether lipolytic activity is required for this effect, we also expressed a catalytically inactive form of human EL (ELS149A); unexpectedly, expression of ELS149A did not lower and in fact increased plasma lipids. Coexpression and coimmunoprecipitation studies suggested that catalytically inactive ELS149A inhibits endogenous mouse EL, accounting for the increased lipid levels. We conclude that (1) in addition to its known effects on HDL metabolism, EL influences the metabolism of apoB-containing particles; (2) catalytic activity of EL is required for its effects on apoB-containing lipoproteins; and (3) overexpressed catalytically inactive EL inhibits endogenous mouse EL, resulting in increased levels of plasma lipids. In light of these results, inhibition of EL has the potential to raise levels of atherogenic lipoproteins in addition to HDL-C levels.

Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.

HDL and its associated apo, APOE, inhibit S-phase entry of murine aortic smooth muscle cells. We report here that the antimitogenic effect of APOE maps to the N-terminal receptor-binding domain, that APOE and its N-terminal domain inhibit activation of the cyclin A promoter, and that these effects involve both pocket protein-dependent and independent pathways. These antimitogenic effects closely resemble those seen in response to activation of the prostacyclin receptor IP. Indeed, we found that HDL and APOE suppress aortic smooth muscle cell cycle progression by stimulating Cox-2 expression, leading to prostacyclin synthesis and an IP-dependent inhibition of the cyclin A gene. Similar results were detected in human aortic smooth muscle cells and in vivo using mice overexpressing APOE. Our results identify the Cox-2 gene as a target of APOE signaling, link HDL and APOE to IP action, and describe a potential new basis for the cardioprotective effect of HDL and APOE.

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway.

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.

Normal production rate of apolipoprotein B in LDL receptor-deficient mice.

The low density lipoprotein (LDL) receptor is well known for its role in mediating the removal of apolipoprotein B (apoB)-containing lipoproteins from plasma. Results from in vitro studies in primary mouse hepatocytes suggest that the LDL receptor may also have a role in the regulation of very low density lipoprotein (VLDL) production. We conducted in vivo experiments using LDLR-/-, LDLR+/-, and wild-type mice (LDLR indicates LDL receptor gene) in which the production rate of VLDL was measured after the injection of [35S]methionine and the lipase inhibitor Triton WR1339. Despite the fact that LDLR-/- mice had a 3.7-fold higher total cholesterol level and a 2.1-fold higher triglyceride level than those of the wild-type mice, there was no difference in the production rate of VLDL triglyceride or VLDL apoB between these groups of animals. Experiments were also conducted in apobec1-/- mice, which make only apoB-100, the form of apoB that binds to the LDL receptor. Interestingly, the apobec1-/- mice had a significantly higher production rate of apoB than did the wild-type mice. However, despite significant differences in total cholesterol and triglyceride levels, there was no difference in the production rate of total or VLDL triglyceride or VLDL apoB between LDLR-/- and LDLR+/- mice on an apobec1-/- background. These results indicate that the LDL receptor has no effect on the production rate of VLDL triglyceride or apoB in vivo in mice.