A normal rate of cellular cholesterol removal can be mediated by plasma from a patient with familial lecithin-cholesterol acyltransferase (LCAT) deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme involved in the esterification of cholesterol in circulating plasma lipoproteins. In the present study, we describe the molecular defects in the LCAT gene and in lipoprotein metabolism of a 34-year-old patient presenting with features of classic familial LCAT deficiency. DNA sequencing revealed two separate point mutations in exon 3 of the patient's LCAT gene: a C to A substitution converting Tyr(83) to a Stop and a C to T transition converting an Arg(99) to a Cys. Digestion of patient PCR-amplified DNA with the restriction enzymes AccI and AciI established that the patient was a compound heterozygote for both mutations. In vitro expression of LCAT (Arg(99)-->Cys) in human embryonic kidney-293 cells demonstrated reduced expression, as well as reduced secretion and/or increased intracellular degradation of the mutant enzyme with significantly decreased alpha-LCAT specific activity, thus, establishing the functional significance of the LCAT (Arg(99)-->Cys) mutation. The plasma cholesterol esterification rate (CER, 2+/-0.3 nmol/ml/h), alpha-LCAT activity (2.9+/-0.1 nmol/ml/h) and LCAT concentration (0.3+/-0.1 microg/ml) were 2.9%, 2.3% and 6.1% that of normal subjects, respectively. Analysis of the patient's plasma lipid profile revealed reduced plasma concentrations of total cholesterol (111+/-0.5 mg/dl), HDL cholesterol (1.6+/-0.2 mg/dl), apolipoprotein (apo) A-I (52+/-4 mg/dl) and apo A-II (11+/-0.5 mg/dl). Nevertheless, for the first time, we demonstrate that the LCAT-deficient plasma is as efficient as control plasma in cholesterol efflux experiments performed with [(3)H]-cholesterol loaded fibroblasts. This result could explain the absence of premature atherosclerosis in this LCAT-deficient patient.

Regulation and intracellular trafficking of the ABCA1 transporter.

The discovery of the role of the ATP-binding cassette transporter A1 (ABCA1) in mediating apolipoprotein A-I-mediated efflux has led to a dramatic increase in our knowledge of the molecular mechanisms involved in cholesterol efflux and cellular metabolism. In this review, we discuss several aspects of ABCA1 regulation including i) transcriptional regulation, ii) substrate specificity and availability, iii) accessory proteins, iv) acceptor specificity and availability, and v) protein trafficking. The majority of studies of ABCA1 regulation to date have focused on the identification of promoter elements that determine ABCA1 gene transcription. Here we also review the potential functional role of ABCA1 in reverse cholesterol transport. Given the key role that ABCA1 plays in cholesterol homeostasis, it is likely that there are multiple mechanisms for controlling the overall transporter activity of ABCA1.

ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice.

The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.

Cellular localization and trafficking of the human ABCA1 transporter.

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.

Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter.

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.

Role of hepatic and lipoprotein lipase in lipoprotein metabolism and atherosclerosis: studies in transgenic and knockout animal models and somatic gene transfer.

Hepatic lipase (HL) and lipoprotein lipase (LPL) are the two major lipolytic enzymes responsible for the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Both lipases are attached to the vascular endothelium via cell surface proteoglycans. HL is primarily involved in the metabolism of chylomicron remnants, intermediate density lipoproteins and high-density lipoproteins whereas LPL catalyzes the hydrolysis of triglycerides from chylomicrons and very low-density lipoproteins. In addition to their traditional function as lipolytic enzymes, HL and LPL appear to serve as ligands that mediate the interaction of lipoproteins to cell surface receptors and/or proteoglycans. Over the past several years significant advances have been made in our understanding of new, alternative mechanisms by which HL and LPL modulate lipoprotein metabolism and the development of atherosclerosis in vivo. This review will summarize some of the new insights generated from the study of transgenic and knockout HL and LPL animal models as well as somatic gene transfer of these two lipases.

Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis.

In the past several years significant advances have been made in our understanding of lecithin-cholesterol acyltransferase (LCAT) function. LCAT beneficially alters the plasma concentrations of apolipoprotein B-containing lipoproteins, as well as HDL. In addition, its proposed role in facilitating reverse cholesterol transport and modulating atherosclerosis has been demonstrated in vivo. Analysis of LCAT transgenic animals has established the importance of evaluating HDL function, as well as HDL plasma levels, to predict atherogenic risk.

Hepatic lipase deficiency decreases the selective uptake of HDL-cholesteryl esters in vivo.

Recent in vitro studies have provided evidence that hepatic lipase (HL) facilitates the selective uptake of HDL cholesteryl esters (CE), but the in vivo physiological relevance of this process has not been demonstrated. To evaluate the role that HL plays in facilitating the selective uptake of HDL-CE in vivo, we studied the metabolism of [(3)H]CEt, (125)I-labeled apolipoprotein (apo) A-I, and (131)I-labeled apoA-II-labeled HDL in HL-deficient mice. Kinetic analysis revealed similar catabolism of (125)I-labeled apoA-I (as well as (131)I-labeled apoA-II) in C57BL controls and HL deficient mice, with fractional catabolic rates (FCR) of 2.17 +/- 0.15 and 2.16 +/- 0.11 d(-)(1) (2.59 +/- 0.14 and 2.67 +/- 0.13 d(-)(1), respectively). In contrast, despite similar hepatic scavenger receptor BI expression, HL-deficient mice had delayed clearance of [(3)H]CEt compared to controls (FCR = 3.66 +/- 0.29 and 4.41 +/- 0.18 d(-)(1), P < 0.05). The hepatic accumulation of [(3)H]CEt in HL-deficient mice (62.3 +/- 2.1% of total) was significantly less than in controls (72.7 +/- 3.0%), while the [(3)H]CEt remaining in the plasma compartment increased (20.7 +/- 1.8% and 12.6 +/- 0.5%) (P < 0.05, all). In summary, HL deficiency does not alter the catabolism of apoA-I and apoA-II but decreases the hepatic uptake and the plasma clearance of HDL-CE. These data establish for the first time an important role for HL in facilitating the selective uptake of HDL-CE in vivo.

In vivo evidence for both lipolytic and nonlipolytic function of hepatic lipase in the metabolism of HDL.

To investigate the in vivo role that hepatic lipase (HL) plays in HDL metabolism independently of its lipolytic function, recombinant adenovirus (rAdV) expressing native HL, catalytically inactive HL (HL-145G), and luciferase control was injected in HL-deficient mice. At day 4 after infusion of 2 x 10(8) plaque-forming units of rHL-AdV and rHL-145G-AdV, similar plasma concentrations were detected in postheparin plasma (HL=8.4+/-0.8 microg/mL and HL-145G=8.3+/-0.8 microg/mL). Mice expressing HL had significant reductions of cholesterol (-76%), phospholipids (PL; -68%), HDL cholesterol (-79%), apolipoprotein (apo) A-I (-45%), and apoA-II (-59%; P<0.05 for all), whereas mice expressing HL-145G decreased their cholesterol (-49%), PL (-40%), HDL cholesterol (-42%), and apoA-II (-89%; P<0.005 for all) but had no changes in apoA-I. The plasma kinetics of (125)I-labeled apoA-I HDL, (131)I-labeled apoA-II HDL, and [(3)H]cholesteryl ester (CE) HDL revealed that compared with mice expressing luciferase control (fractional catabolic rate [FCR] in d(-1): apoA-I HDL=1.3+/-0.1; apoA-II HDL=2.1+/-0; CE HDL=4.1+/-0.7), both HL and HL-145G enhanced the plasma clearance of CEs and apoA-II present in HDL (apoA-II HDL=5.6+/-0.5 and 4.4+/-0.2; CE HDL=9.3+/-0. 0 and 8.3+/-1.1, respectively), whereas the clearance of apoA-I HDL was enhanced in mice expressing HL (FCR=4.6+/-0.3) but not HL-145G (FCR=1.4+/-0.4). These combined findings demonstrate that both lipolytic and nonlipolytic functions of HL are important for HDL metabolism in vivo. Our study provides, for the first time, in vivo evidence for a role of HL in HDL metabolism independent of lipolysis and provides new insights into the role of HL in facilitating distinct metabolic pathways involved in the catabolism of apoA-I- versus apoA-II-containing HDL.

LCAT modulates atherogenic plasma lipoproteins and the extent of atherosclerosis only in the presence of normal LDL receptors in transgenic rabbits.

Elevated low density lipoprotein cholesterol (LDL-C) and reduced high density lipoprotein cholesterol (HDL-C) concentrations are independent risk factors for coronary heart disease. We have previously demonstrated that overexpression of an enzyme with a well established role in HDL metabolism, lecithin:cholesterol acyltransferase (LCAT), in New Zealand White rabbits not only raises HDL-C concentrations but reduces those of LDL-C as well, ultimately preventing diet-induced atherosclerosis. In the present study, the human LCAT gene (hLCAT) was introduced into LDL receptor (LDLr)-deficient (Watanabe heritable hyperlipidemic) rabbits to (1) investigate the role of the LDLr pathway in the hLCAT-mediated reductions of LDL-C and (2) determine the influence of hLCAT overexpression on atherosclerosis susceptibility in an animal model of familial hypercholesterolemia. Heterozygosity or homozygosity for the LDLr defect was determined by polymerase chain reaction, and 3 groups of hLCAT-transgenic (hLCAT+) rabbits that differed in LDLr status were established: (1) LDLr wild-type (LDLr+/+), (2) LDLr heterozygotes (LDLr+/-), and (3) LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared with those of nontransgenic (hLCAT-) rabbits of the same LDLr status. Plasma HDL-C concentrations were significantly elevated in the hLCAT+ animals of each LDLr status. However, LDL-C levels were significantly reduced only in hLCAT+/LDLr+/+ and hLCAT+/LDLr+/- rabbits but not in hLCAT+/LDLr-/- rabbits (405+/-14 versus 392+/-31 mg/dL). Metabolic studies revealed that the fractional catabolic rate (FCR, d(-1)) of LDL apolipoprotein (apo) B-100 was increased in hLCAT+/LDLr+/+ (26+/-4 versus 5+/-0) and hLCAT+/LDLr+/- (4+/-1 versus 1+/-0) rabbits, whereas the FCR of LDL apoB-100 in both groups of LDLr-/- rabbits was nearly identical (0.16+/-0.02 versus 0.15+/-0.02). Consistently, neither aortic lipid concentrations nor the extent of aortic atherosclerosis was significantly different between hLCAT+/LDLr-/- and hLCAT-/LDLr-/- rabbits. Significant correlations were observed between the percent of aortic atherosclerosis and both LDL-C (r=0.985) and LDL apoB-100 FCR (-0.745), as well as between LDL-C and LDL apoB-100 FCR (-0.866). These data are the first to establish that LCAT modulates LDL metabolism via the LDLr pathway, ultimately influencing atherosclerosis susceptibility. Moreover, LCAT's antiatherogenic effect requires only a single functional LDLr allele, identifying LCAT as an attractive gene therapy candidate for the majority of dyslipoproteinemic patients.

Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice.

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.

Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of approximately 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST. These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity.

Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1.

Hepatic lipase (HL) plays a major role in high-density lipoprotein (HDL) metabolism both as a lipolytic enzyme and as a ligand. To investigate whether HL enhances the uptake of HDL-cholesteryl ester (CE) via the newly described scavenger receptor BI (SR-BI), we measured the effects of expressing HL and SR-BI on HDL-cell association as well as uptake of 125I-labeled apoA-I and [3H]CE-HDL, by embryonal kidney 293 cells. As expected, HDL cell association and CE selective uptake were increased in SR-BI transfected cells by 2- and 4-fold, respectively, compared to controls (P < 0.001). Cells transfected with HL alone or in combination with SR-BI expressed similar amounts of HL, 20% of which was bound to cell surface proteoglycans. HL alone increased HDL cell association by 2-fold but had no effect on HDL-CE uptake in 293 cells. However, in cells expressing SR-BI, HL further enhanced the selective uptake of CE from HDL by 3-fold (P < 0.001). To determine whether the lipolytic and/or ligand function of HL are required in this process, we generated a catalytically inactive form of HL (HL-145G). Cells co-transfected with HL-145G and SR-BI increased their HDL cell association and HDL-CE selective uptake by 1.4-fold compared to cells expressing SR-BI only (P < 0.03). Heparin abolished the effect of HL-145G on SR-BI-mediated HDL-CE selective uptake.Thus, the enhanced uptake of HDL-CE by HL is mediated by both its ligand role, which requires interaction with proteoglycans, and by lipolysis with subsequent HDL particle remodeling. These results establish HL as a major modulator of SR-BI mediated selective uptake of HDL-CE.

In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess apoE.

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe -/-. Ldlr -/-) using adenovirus-mediated gene transfer. We found that a low level of APOE expression, that was capable of reducing the hyperlipidemia in Apoe -/- mice, did not result in a reduction of the hyperlipidemia in Apoe -/-. Ldlr -/- mice. Surpisingly, a very high level of APOE expression also did not result in a reduction of hypercholesterolemia in Apoe -/-. Ldlr -/- mice, despite very high levels of circulating apoE (>160 mg/dl). Only a moderately high level of APOE expression resulted in a reduction of serum cholesterol level (from 35.2 +/- 6.7 to 14.6 +/- 2.3 mmol/l) and the disappearance of VLDL from the serum. Moreover, the very high level of APOE expression resulted in a severe hypertriglyceridemia in Apoe -/-. Ldlr -/- mice and not Apoe -/- mice (25.7 +/- 8.9 and 2.2 +/- 1.8 mmol/l, respectively). This hypertriglyceridemia was associated with an APOE-induced increase in the VLDL triglyceride production rate and an inhibition of VLDL-triglyceride lipolysis. We conclude from these data that, for efficient clearance, the non-LDL receptor-mediated pathway requires a higher level of APOE expression as compared to the LDL receptor, but is more sensitive to an APOE-induced increase in VLDL production and inhibition of VLDL-triglyceride lipolysis.-van Dijk, K. W., B. J. M. Van Vlijmen, H. B. van't Hof, A. van der Zee, S. Santamarina-Fojo, T. J. C. van Berkel, L. M. Havekes, and M. H. Hofker. In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess APOE.

Hepatic lipase facilitates the selective uptake of cholesteryl esters from remnant lipoproteins in apoE-deficient mice.

We have investigated the role of hepatic lipase (HL) in remnant lipoprotein metabolism independent of lipolysis by using recombinant adenovirus to express native and catalytically inactive HL (HL-145G) in apolipoprotein (apo)E-deficient mice characterized by increased plasma concentrations of apoB-48-containing remnants. In the absence of apoE, the mechanisms by which apoB-48-containing remnants are taken up by either low density lipoprotein (LDL)-receptor or LDL-receptor-related protein (LRP) remain unclear. Overexpression of either native or catalytically inactive HL in apoE-deficient mice led to similar reductions (P > 0.5) in the plasma concentrations of cholesterol (41% and 53%) and non high density lipoprotein (HDL)-cholesterol (41% and 56%) indicating that even in the absence of lipolysis, HL can partially compensate for the absence of apoE in this animal model. Although the clearance of [3H]cholesteryl ether from VLDL was significantly increased (approximately 2-fold; P < 0. 02) in mice expressing native or inactive HL compared to luciferase controls, the fractional catabolic rates (FCR) of [125I-labeled] apoB- very low density lipoprotein (VLDL) in all three groups of mice were similar (P > 0.4, all) indicating selective cholesterol uptake. Hepatic uptake of [3H]cholesteryl ether from VLDL was greater in mice expressing either native HL (87%) or inactive HL-145G (72%) compared to luciferase controls (56%). Our combined findings are consistent with a role for HL in mediating the selective uptake of cholesterol from remnant lipoproteins in apoE-deficient mice, independent of lipolysis. These studies support the concept that hepatic lipase (HL) may serve as a ligand that mediates the interaction between remnant lipoproteins and cell surface receptors and/or proteoglycans. We hypothesize that one of these pathways may involve the interaction of HL with cell surface receptors, such as scavenger receptor (SR)-BI, that mediate the selective uptake of cholesteryl esters.

The familial chylomicronemia syndrome.

The chylomicronemia syndrome is a disorder characterized by severe hypertriglyceridemia and fasting chylomicronemia. Genetic causes of the syndrome are rare and include deficiency of lipoprotein lipase (LPL), apolipoprotein C-II, and familial inhibitor of LPL. Patients with familial forms of hypertriglyceridemia in combination with secondary acquired disorders account for most individuals presenting with chylomicronemia. The clinical manifestations--lipid and other biochemical abnormalities--as well as treatment options for chylomicronemic patients are discussed.

Correction of hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:cholesterol acyltransferase.

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/-) and 2) hLCAT+/LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT-) rabbits of the same LDLr status. In LDLr+/- rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 +/- 8, 59 +/- 4) relative to hLCAT- rabbits (21 +/- 1, 26 +/- 2). This was, likewise, the case when hLCAT+/ LDLr-/- (27 +/- 2, 19 +/- 6) and hLCAT-/LDLr-/- (5 +/- 1, 6 +/- 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d(-1)) of apoA-I was substantially delayed in hLCAT+ (0.376 +/- 0.025) versus hLCAT- (0.588) LDLr+/- rabbits, as well as in hLCAT+ (0.666 +/- 0.033) versus hLCAT- (1.194 +/- 0.138) LDLr-/- rabbits. ApoA-I production rate (PR, mg x kg x d(-1)) was greater in both hLCAT+/LDLr+/- (10 +/- 2 vs. 6) and hLCAT+/LDLr-/- (9 +/- 1 vs. 4 +/- 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = -0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (-0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.