Reverse cholesterol transport and hepatic osteodystrophy.

In this issue of Cell Metabolism, Lu et al. show that chronic liver disease increases the expression and activity of PP2Ac, a phosphatase that downregulates the excretion of lecithin-cholesterol aceyltransferase (LCAT). LCAT, a liver-derived enzyme, protects bone and prevents bone loss, and its lowered levels in progressive liver injury cause hepatic osteodystrophy (HOD) and worsen liver fibrosis. These discoveries open the possibility that recombinant LCAT may be a treatment for both HOD and liver fibrosis.

Cholesterol: the good, the bad, and the ugly - therapeutic targets for the treatment of dyslipidemia.

Maintaining cholesterol and triglyceride (TG) levels within healthy limits is critical for decreasing the risk of heart disease. Dyslipidemia refers to the abnormal levels of lipids in the blood, including low high-density lipoprotein cholesterol (HDL-C), also known as good cholesterol, high low-density lipoprotein cholesterol (LDL-C), also known as bad cholesterol, and/or high TG levels that contribute to the development and progression of atherosclerosis. In this article we reviewed some of the current therapeutic targets for the treatment of dyslipidemia, with a primary focus on endothelial lipase and lecithin cholesterol acyl transferase for raising HDL-C, and the proprotein convertase subtilisin-like kexin type 9 (PCSK9), microsomal triglyceride transfer protein, and the messenger RNA of apolipoprotein B for lowering LDL-C. In addition, we reviewed the role of apolipoprotein AI (apoAI) in raising HDL-C, where we discuss three apoAI-based drugs under development. These are its mutated dimer (apoAI-Milano), a complex with phospholipids, and a mimetic peptide. Atherosclerosis, mainly because of dyslipidemia, is a leading cause of cardiovascular disease. Regarding the title of this article, the 'good' refers to HDL-C, the 'bad' refers to LDL-C, and the 'ugly' refers to atherosclerosis.

Role of plasma and liver cholesterol- and lipoprotein-metabolism determinants in LpX formation in the mouse.

Cholestasis is characterized by hypercholesterolemia and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. We used ATP-binding cassette A1 (ABCA1)-/- and scavenger receptor class B type I (SR-BI)-/- mice to test the hypothesis that hepatic sinusoidal cholesterol transporters contribute to LpX formation and hypercholesterolemia during cholestasis. Bile-duct ligation (BDL) of both ABCA1-/- and SR-BI-/- mice, as well as their respective controls, induced a dramatic increase in plasma cholesterol and phospholipid concentrations. Plasma fractionation revealed the presence of LpX in plasma of cholestatic mice, irrespective of their genetic background. We observed that the presence of HDL before cholestasis, a decrease in the activity of LCAT, and an increase in VLDL synthesis were not required for hypercholesterolemia and lipoprotein modifications induced by obstructive cholestasis in mice. In addition, murine cholestasis resulted in increased hepatic cholesterol synthesis that may contribute to the higher plasma free cholesterol levels found during the early hours after BDL. Together these findings indicate that hypercholesterolemia and LpX formation associated with obstructive cholestasis are correlated with an increase in hepatic cholesterol synthesis and are independent of plasma HDL levels, LCAT activity, VLDL synthesis, and ABCA1 and SR-BI expression.

Fractal binding and dissociation kinetics of heart-related compounds on biosensor surfaces.

A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (1), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (2), and TRITC-labeled HDL in solution to a bare optical fiber surface (3). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (4). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.

[Expression of lecithin cholesterol acyltransferase and/or apoA-I mediated by recombinant adeno-associated virus in myogenic cells].

Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10(14)/L (rAAVAIL) and 1 x 10(14)/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source.

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.

Construction and characterization of polycistronic retrovirus vectors for sustained and high-level co-expression of apolipoprotein A-I and lecithin-cholesterol acyltransferase.

Apolipoprotein A-I (apo A-I) and lecithin-cholesterol acyltransferase (LCAT) are constituents of circulating high-density lipoprotein (HDL) particles and play an important role in 'reverse cholesterol transport', the process by which cholesterol in peripheral tissues is transferred to the liver for excretion. Enhancing levels of apo A-I, as well as LCAT, in plasma may promote the removal of excess cholesterol from the arterial wall and thus reduce the formation of atherosclerotic lesions. Indeed, both apo A-I and LCAT genes have been identified as therapeutic targets to prevent or limit atherogenesis. Here, we have constructed two retroviral vectors, one containing LCAT cDNA and the neomycin phosphotransferase (NEO) gene (pLLEN), the other apo A-I cDNA, LCAT cDNA and the NEO gene (pLAPLEN) linked by internal ribosome entry sites (IRES). Both bi- and tricistronic retroviral vectors efficiently co-expressed their two or three genes when transfected into cultured mouse C2C12 muscle cells or human 293 cells. After 30 days, the retroviral vector sequences were retained by the host cells, whereas those of a conventional plasmid vector were lost. Moreover, transduced C2C12 mouse myoblasts maintained the ability for heterologous expression of human LCAT and apo A-I even after differentiation into myotubes. Stably-transduced clones of C2C12 cells were selected by neomycin (G418) resistance and continued to efficiently express human LCAT for 60 days. These findings indicate that the use of polycistronic retrovirus vectors to genetically modify myoblasts, which can be transplanted back into skeletal muscle, might be a safe and feasible strategy to express human apo A-I and LCAT and hence have therapeutic potential to regress atherosclerotic lesions.

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn-2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure;-function studies, LCAT containing a carboxy-terminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ( approximately 15 mg L(-1)) was achieved over a 72-h period using 10 mm sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( approximately 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 +/- 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 +/- 6 and 26 +/- 3 nmol CE formed microg(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at approximately 2 microm substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.

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.

High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase.

A subset of patients with high plasma HDL concentrations have enhanced rather than reduced atherosclerosis. We have developed a new transgenic mouse model overexpressing human lecithin-cholesteryl acyltransferase (LCAT) that has elevated HDL and increased diet-induced atherosclerosis. LCAT transgenic mouse HDLs are abnormal in both composition and function. Liver uptake of [3H]cholesteryl ether incorporated in transgenic mouse HDL was reduced by 41% compared with control HDL, indicating ineffective transport of HDL-cholesterol to the liver and impaired reverse cholesterol transport. Analysis of this LCAT-transgenic mouse model provides in vivo evidence for dysfunctional HDL as a potential mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels.

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase.

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.

Influence of vesicle surface composition on the interfacial binding of lecithin:cholesterol acyltransferase and apolipoprotein A-I.

Interfacial binding affinities and capacities of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-I) for surfaces of different phosphatidylcholine (PC) composition, cholesterol content, and apolipoprotein content were measured with a vesicle model system. Native polyacrylamide gel electrophoresis was used to separate free protein from vesicle-bound protein. ApoA-I was isolated from human plasma and radiolabeled with iodine, whereas radiolabeled LCAT was purified from the media of Chinese hamster ovary cells that were transfected with human LCAT cDNA and incubated in the presence of [35S] cysteine and methionine. Bound and free radiolabeled LCAT and apoA-I were quantified by phosphorimage analysis. ApoA-I binding was not influenced by cholesterol content (14 mole%) but was influenced by the PC fatty acyl composition of the vesicle. PC species containing long chain, polyunsaturated fatty acids (PUFA) in the sn-2 position resulted in increased binding affinity (Kd = 75-177 nM) but reduced capacity (0.1-0.3 apoA-I/ 1000 PC) in comparison to sn-1 palmitoyl, sn-2 oleoyl PC (POPC, 750 nM and 1.4 apoA-I/1000 PC). LCAT binding affinity to POPC (2190 nM) was stronger in the presence of cholesterol (530 nM), and LCAT binding capacity was reduced (2.63 and 0.6 molecules LCAT/1000 PC, respectively). In comparison to POPC, LCAT binding affinity to sn-1 palmitoyl, sn-2 arachidonyl PC was stronger (611 nM) and binding capacity was reduced (0.7 LCAT/1000 PC). LCAT binding affinity and capacity to sn-1 palmitoyl, sn-2 eicosapentaneoyl PC (2041 nM, and 2.5 LCAT/1000 PC) were similar to those observed for POPC. We conclude that vesicle surface PC fatty acyl composition and cholesterol content significantly influence LCAT and apoA-I interfacial binding and therefore may alter LCAT enzymatic activity.

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.

Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.

An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease).

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence. Three sisters were shown to suffer from fish-eye disease (FED), a disorder which is caused by mutations in the gene coding for lecithin:cholesterol acyltransferase (LCAT). Sequencing of the LCAT gene of all three probands revealed compound heterozygosity for a missense mutation in exon 4 which is reported to underlie the FED phenotype, and a point mutation located in intron 4 (IVS4:T-22C). By performing in vitro expression of LCAT minigenes and reverse transcriptase PCR on mRNA isolated from leukocytes of the patient, this gene defect was shown to cause a null allele as the result of complete intron retention. In conclusion, we demonstrated that a point mutation in a lariat branchpoint consensus sequence causes a null allele in a patient with FED. In addition, our finding illustrates the importance of this sequence for normal human mRNA processing. Finally, this report provides a widely applicable strategy which ensures fast and effective screening for intronic defects that underlie differential gene expression.

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.

Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits. In vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner.

Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT-induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration.

Lecithin:cholesterol acyltransferase overexpression generates hyperalpha-lipoproteinemia and a nonatherogenic lipoprotein pattern in transgenic rabbits.

Cholesterol esterification within plasma lipoprotein particles is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact of the overexpression of this enzyme on plasma concentrations of the different plasma lipoproteins in an animal model expressing cholesteryl ester transfer protein was evaluated by generating rabbits expressing human LCAT. A 6.2-kilobase human genomic DNA construct was injected into the pronuclei of rabbit embryos. Of the 1002 embryos that were injected, 3 founder rabbits were characterized that expressed the human LCAT gene. As in mice and humans, the principal sites of mRNA expression in these rabbits is in the liver and brain, indicating that the regulatory elements required for tissue-specific expression among these species are similar. The alpha-LCAT activity correlated with the number of copies of LCAT that integrated into the rabbit DNA. Compared with controls, the high expressor LCAT-transgenic rabbits total and high density lipoprotein (HDL) cholesterol concentrations were increased 1.5-2.5-fold with a 3.1-fold increase in the plasma cholesterol esterification rate. Analysis of the plasma lipoproteins by fast protein liquid chromatography indicates that these changes reflected an increased concentration of apolipoprotein E-enriched, HDL1-sized particles, whereas atherogenic apolipoprotein B particles disappeared from the plasma. The concentrations of plasma HDL cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT activity (r = 0.94; p < 0.001) in the transgenic rabbits. These results indicate that overexpression of LCAT in the presence of cholesteryl ester transfer protein leads to both hyperalpha-lipoproteinemia and reduced concentrations of atherogenic lipoproteins.

Chicken lecithin-cholesterol acyltransferase. Molecular characterization reveals unusual structure and expression pattern.

Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.V., Sanders, E.J., Steyrer, E., Nimpf, J., and Schneider, W.J. (1995) J. Lipid Res. 36, 601-610), pointing to the importance of this process in nonplacental reproduction. We have begun to delineate the details of this unique transport pathway branch by molecular characterization of the first nonmammalian lecithin-cholesterol acyltransferase (LCAT), the enzyme that catalyzes an early step in RCT. The biological significance of the enzyme is underscored by the high degree of protein sequence identity (73%) maintained from chicken to man. Interestingly, the conservation extends much less to the cysteine residues; in fact, two of the cysteines thought to be important in mammalian enzymes (residues 31 and 184 in man) are absent from the chicken enzyme, providing proof of their dispensability for enzymatic activity. Antibodies prepared against a chicken LCAT fusion protein cross-react with human LCAT and identify a 64-kDa protein present in enzymatically active fractions obtained by hydrophobic chromatography of chicken serum. The developmental and tissue distribution pattern of LCAT in females is striking; during embryogenesis and adolescence, LCAT expression is extremely high in liver but undetectable in brain. Upon onset of laying, however, brain LCAT mRNA increases suddenly and is maintained at levels 5 times higher than in liver, in stark contrast to most mammals. In adult roosters, the levels of LCAT transcripts in brain are lower than in liver. Together with the molecular characterization of chicken LCAT, these newly discovered developmental changes and gender differences in its expression establish the avian oocyte/liver system as a powerful model to delineate in vivo regulatory elements of RCT.

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.