Pharmacokinetics, disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid, a potent and subtype-selective peroxisome proliferator-activated receptor alpha agonist in preclinical species and human.

5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-alpha). In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague-Dawley rats (3.2 +/- 1.4 ml min(-1) kg(-1)) and cynomolgus monkeys (6.1 +/- 1.6 ml min(-1) kg(-1)) resulting in plasma half-lives of 7.1 +/- 0.7 h and 9.4 +/- 0.8 h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50 mg kg(-1)). In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50's > 30 microM) was discerned with 1. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels.

Excess accumulation of cholesterol in macrophages results in foam cell production and lesion development. Recent studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) is highly regulated in macrophages and mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for HDL formation. The goal of this study was to determine the contribution of monocyte/macrophage ABCA1 to HDL formation in vivo. We generated mice expressing ABCA1 in macrophages and mice with selected inactivation of ABCA1 in macrophages by bone marrow transplantation in ABCA1-deficient (ABC1(-/-)) and wild-type (WT) mice. At all times, the level of HDL in ABC1(-/-) recipient mice remained low relative to WT recipient mice irrespective of the genotype of the donor macrophage ABCA1 or high-fat feeding. Expression of WT macrophage ABCA1 in ABC1(-/-) mice resulted in a small but significant increase in apoA-I levels starting 2 weeks after transplantation. No further increase in apoAI was observed up to 14 weeks after transplantation. The increase in apoAI was accompanied by a small but significant increase in HDL cholesterol 6 weeks after transplantation. The HDL formed as a consequence of the expression of WT macrophage ABCA1 migrated to the alpha position in a two-dimensional gel electrophoresis. These results demonstrate that monocyte/macrophage ABCA1 contributes to HDL formation; however, the contribution to the overall plasma HDL levels is minimal.

Alteration of plasma HDL cholesteryl ester composition with transgenic expression of a point mutation (E149A) of human LCAT.

We have previously identified a single amino acid mutation (hE149A) in human LCAT that increases its in vitro reactivity with phosphatidylcholine species containing sn-2 arachidonate (Wang et al. 1997. J. Biol. Chem. 272: 280-286). The purpose of the present study was to determine whether in vivo overexpression of hE149A compared with human wild-type LCAT (hLCAT-wt) would be sufficient to enrich the steady state composition of plasma HDL cholesteryl esters (CE) with long chain (>18 carbon) polyunsaturated fatty acyl species. Transgenic lines with 20-fold overexpression of hLCAT were created and studied between 12 and 16 weeks of age while consuming a chow diet. Transgenic overexpression of hE149A compared with hLCAT-wt significantly enriched HDL with CE species containing 20:4 (45%) and 22:6 n-3 (108%), at the expense of those containing 18:2, without a significant change in the plasma HDL concentration, particle size, or phospholipid fatty acyl composition. Removing the contribution of endogenous mouse LCAT by crossing the transgenic mice into the mouse LCAT knockout background resulted in even greater changes in HDL CE composition, with a 2.4-, 5-, and 5-fold increase in 20:4, 20:5 n-3, and 22:6 n-3 cholesteryl esters in the hE149A mice compared with hLCAT-wt Tg mice, respectively. Our results demonstrate that in vivo expression of hE149A significantly enriches HDL cholesteryl esters in 20- and 22-carbon fatty acyl species without affecting HDL concentration or size. Furthermore, the data suggest that endogenous mouse LCAT in hLCAT transgenic mice contributes to the plasma HDL CE pool out of proportion to its mass, presumably because the hLCAT transgene is poorly activated by mouse apolipoprotein A-I.

The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

Relationship between expression levels and atherogenesis in scavenger receptor class B, type I transgenics.

Both in vitro and in vivo studies of scavenger receptor class B type I (SR-BI) have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate the effect of SR-BI on atherogenesis, we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) SR-BI expression in an inbred mouse background hemizygous for a human apolipoprotein (apo) B transgene. Unlike non-HDL cholesterol levels that minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol, whereas the high expression SR-BI transgene was associated with 2-fold decreases in HDL cholesterol as well as dramatic alterations in HDL composition and size including the near absence of alpha-migrating particles as determined by two-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a 2-fold decrease in the development of diet-induced fatty streak lesions compared with the apo B transgenics (4448 +/- 1908 micrometer(2)/aorta to 10133 +/- 4035 micrometer (2)/aorta; p < 0.001), whereas the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692 +/- 7238 micrometer(2)/aorta) but 3-fold greater than the low SR-BI/apo B mice (p < 0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences, illustrating the complexity of the relationship between SR-BI and atherogenesis.

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI. These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.

Secretion of lecithin:cholesterol acyltransferase by brain neuroglial cell lines.

The ability of different human and rat brain cell lines (neuronal and gliomal) to secrete lecithin:cholesterol acyltransferase (LCAT) was examined. Of these, the strongly secreting human gliomal (U343 and U251) cell lines were selected for a detailed study of enzymatic and structural properties of the secreted LCAT. Both plasma- and brain-derived enzymes are inhibited by DTNB (90%) and are activated by apolipoprotein A-I. LCAT mRNA was measured in these cell lines at levels similar to that found in HepG2 cells. In contrast, apoA-I, apoE, and apoD mRNAs were undetectable in these cell lines. The presence of functional LCAT secreted by cultured nerve cells provides an in vitro model to study the expression and function of LCAT in the absence of others factors of plasma cholesterol metabolism.

Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice.

Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.

Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.

It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP-/- mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP-/- mice. On a chow diet, PLTP-/- mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol- and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP-/- mice on a high-fat diet could influence the development of atherosclerosis.

Binding and functional effects of transcription factors Sp1 and Sp3 on the proximal human lecithin:cholesterol acyltransferase promoter.

Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5' variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA-protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of transactivating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Modification of the cholesterol efflux properties of human serum by enrichment with phospholipid.

To investigate the importance of phospholipid in promoting cholesterol efflux from cells, phospholipid multilamellar vesicles were incubated with normal human serum and the efflux ability of these lipid-modified sera was tested. When incubated under appropriate conditions, both dimyristoylphosphatidylcholine (DMPC) and bovine brain sphingomyelin (BBSM) were shown to combine with components of human serum to form new protein:lipid complexes and to markedly enhance the ability of serum to promote efflux of cholesterol from Fu5AH cells. In particular, the high density lipoprotein (HDL) particles were altered in their composition and electrophoretic properties and the alpha-migrating species, which were reactive with antibodies to apo-A-I, were converted to larger, pre-beta-migrating particles, similar in electrophoretic properties to pre beta(2)-HDL. DMPC, but not BBSM, also generated particles with mobility similar to pre beta(2)-HDL; These species were demonstrably different from the discoidal complexes formed by reaction of DMPC with purified apoA-I. However, no change in cholesterol efflux potential was observed when serum was mixed with phospholipids that failed to interact or when cells were incubated with phospholipid multilamellar vesicles alone. To further identify the components of serum that become altered in their efflux potential after reaction with phospholipid, isolated lipoprotein fractions were incubated with DMPC or BBSM and it was found that only interaction with HDL caused enhancement of cholesterol efflux. In summary, cholesterol removal from the Fu5AH cells by serum can be promoted by adding phospholipid under conditions where new HDL-like complexes can be formed between the phospholipid and serum components, most notably apolipoprotein A-I.

Expression of human lecithin:cholesterol acyltransferase in transgenic mice: effects on cholesterol efflux, esterification, and transport.

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol and is postulated to participate in a physiologic process called reverse cholesterol transport. We have used transgenic mice expressing the human LCAT transgene to study the effect of increased plasma levels of LCAT in each of the proposed steps involved in the reverse cholesterol transport pathway. High density lipoprotein (HDL) from LCAT transgenic mice was 44% more efficient than control mouse HDL in the efflux of cholesterol from human skin fibroblasts. Esterification of cell-derived cholesterol was also markedly increased in mice expressing the human LCAT transgene. The rate of plasma clearance of HDL cholesteryl ester was virtually the same in both types of animals whereas the HDL cholesteryl ester transport rate was significantly increased in mice expressing the human LCAT transgene (152.3 +/- 16.9 micrograms/h vs. 203.1 +/- 30.9 micrograms/h in control and transgenic mice, respectively). Liver cholesteryl ester uptake was significantly increased in mice expressing human LCAT (58.0 +/- 1.4 micrograms/h/g liver vs. 77.9 +/- 1.7 micrograms/h/g liver in control and transgenic mice, respectively). These studies indicate that LCAT modulates the rate by which cholesterol is effluxed from cell membranes onto HDL, esterified, and transported to the liver.

A mouse model with features of familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) is a common inherited lipid disorder, affecting 1 to 2 percent of the population in Westernized societies. Individuals with FCHL have large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) and develop premature coronary heart disease. A mouse model displaying some of the features of FCHL was created by crossing mice carrying the human apolipoprotein C-III (APOC3) transgene with mice deficient in the LDL receptor. A synergistic interaction between the apolipoprotein C-III and the LDL receptor defects produced large quantities of VLDL and LDL and enhanced the development of atherosclerosis. This mouse model may provide clues to the origin of human FCHL.

Increased prebeta-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes.

Human plasma phospholipid transfer protein (PLTP) circulates bound to high density lipoprotein (HDL) and mediates both net transfer and exchange of phospholipids between different lipoproteins. However, its overall function in lipoprotein metabolism is unknown. To assess the effects of increased plasma levels of PLTP, human PLTP transgenic mice were established using the human PLTP gene driven by its natural promoter. One line of PLTP transgenic mice with moderate expression of PLTP mRNA and protein was obtained. The order of human PLTP mRNA expression in tissues was: liver, kidney, brain, small intestine > lung > spleen > heart, adipose tissue. Western blotting using a human PLTP monoclonal antibody revealed authentic human PLTP (Mr 80 kD) in plasma. Plasma PLTP activity was increased by 29% in PLTP transgenic mice. However, plasma lipoprotein analysis, comparing PLTP transgenic mice to control littermates, revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins. Since previous studies have shown that human cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase only function optimally in human apoAI transgenic mice, the human PLTP transgenic mice were cross-bred with human apoAI transgenic mice. In the human apoAI transgenic background, PLTP expression resulted in increased PLTP activity (47%), HDL phospholipid (26%), cholesteryl ester (24%), free cholesterol (37%), and apoAI (22%). There was a major increase of apoAI in prebeta-HDL (56%) and a small increase in alpha-HDL (14%). The size distribution of HDL particles within alpha- and prebeta-migrating species was not changed. The results suggest that PLTP increases the influx of phospholipid and secondarily cholesterol into HDL, leading to an increase in potentially antiatherogenic prebeta-HDL particles.

Effects of carboxy-terminal truncation on human lecithin:cholesterol acyltransferase activity.

Mutagenesis was carried out in human lecithin:cholesterol acyltransferase (LCAT) to generate mutants with stop codons at positions corresponding to amino acids 315, 341, 359, 375, 388, 394, and 398 of the 416-amino acid sequence of the mature enzyme protein. Deletion of the 18 terminal amino acids of the protein was without effect on LCAT phospholipase or acyltransferase activity, or the stability of the protein to denaturation at 37 degrees C. Further deletion led to loss of most of the activity, associated with a 10-fold increase in the rate of denaturation at 37 degrees C. These data indicate that the proline-rich C-terminus of LCAT is not required for effective enzyme activity. The loss of activity that accompanied deletion of residues 394-398 suggests a structural role for these residues, part of a series of predicted beta-sheet sequences in the C-terminal third of the LCAT primary sequence.

Increased prebeta-HDL levels, cholesterol efflux, and LCAT-mediated esterification in mice expressing the human cholesteryl ester transfer protein (CETP) and human apolipoprotein A-I (apoA-I) transgenes.

The effects of cholesteryl ester transfer protein (CETP) on the distribution of apolipoprotein (apo) A-I between high density lipoprotein (HDL) subspecies and its impact on efflux and esterification of cell-derived cholesterol was studied in transgenic mice expressing either the human apoA-I (HuAITg) or both the human apoA-I and CETP (HuAICETPTg) transgenes. The simultaneous expression of the human CETP and apoA-I transgenes induced a 2-fold increase in the proportion of human apoA-I in the prebeta-HDL fraction and 1.4- and 2.2-fold increases in the HDL3a and HDL3c fractions, respectively, at the expense of the larger HDL2b population. HuAICETPTg mouse plasma has a greater ability to cause efflux of cholesterol from 3H-labeled fibroblasts than plasma from HuAITg mice. Furthermore, the LCAT-mediated esterification of cell-derived cholesterol is increased 1.7-fold in mice expressing the human apoA-I and CETP transgenes compared to HuAITg mouse plasma. LCAT activity (measured with an exogenous substrate) was increased 1.4-fold and LCAT mRNA levels were increased 1.3-fold as a result of CETP expression. Taken together, these data indicate that in vivo, the expression of CETP resulted in an increase in the proportion of apoA-I in the prebeta-HDL fraction and a stimulation of the efflux and esterification of cell-derived cholesterol.

Cholesterol efflux potential of sera from mice expressing human cholesteryl ester transfer protein and/or human apolipoprotein AI.

The ability of whole serum to promote cell cholesterol efflux and the relationships between apoprotein and lipoprotein components of human serum efflux have been investigated previously (de la Llera Moya, M., V. Atger, J.L. Paul, N. Fournier, N. Moatti, P. Giral, K.E. Friday, and G.H. Rothblat. 1994. Arterioscler. Thromb. 14:1056-1065). We have now used this experimental system to study the selective effects of two human lipoprotein-related proteins, apoprotein AI (apo AI) and cholesteryl ester transfer protein (CETP) on cell cholesterol efflux, when these proteins are expressed in transgenic mice. The percent efflux values for cholesterol released in 4 h from Fu5AH donor cells to 5% sera from the different groups of mice were in the order: background = human apo AI transgenic (HuAITg) > human CETP transgenic (HuCETPTg) > human apo AI and CETP transgenic (HuAICETPTg) >> apo AI knockout mice. In each group of mice a strong, positive correlation (r2 ranging from 0.64 to 0.76) was found between efflux and HDL cholesterol concentrations. The slopes of these regression lines differed between groups of mice, indicating that the cholesterol acceptor efficiencies of the sera differed among groups. These differences in relative efficiencies can explain why cholesterol efflux was not proportional to the different HDL levels in the various groups of mice. We can conclude that: (a) HDL particles from HuAITg mice are less efficient as cholesterol acceptors than HDL from the background mice; (b) despite a lower average efflux due to lower HDL cholesterol concentrations, HDL particles are more efficient in the HuCETPTg mice than in the background mice; and (c) the coexpression of both human apo AI and CETP improves the efficiency of HDL particles in the HuAICETPTg mice when compared with the HuAITg mice. We also demonstrated that the esterification of the free cholesterol released from the cells by lecithin cholesterol acyltransferase in the serum was reduced in the HuAITg and AI knockout mice, whereas it was not different from background values in the two groups of mice expressing human CETP.

Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism.

Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.