©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression of Human Lecithin Cholesterol Acyltransferase Leads to Hyperalphalipoproteinemia in Transgenic Mice (*)

Boris L. Vaisman , Hanns-Georg Klein , Mustapha Rouis , Annie M. Bérard , Marie R. Kindt , Glenda D. Talley , Susan M. Meyn , Robert F. HoytJr. (1), Santica M. Marcovina (2), John J. Albers (2), Jeffrey M. Hoeg , H. Bryan BrewerJr. , Silvia Santamarina-Fojo (§)

From the (1) Molecular Disease Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, the Laboratory of Animal Medicine & Surgery, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, and the (2) Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, School of Medicine, Seattle, Washington 98103

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme which catalyzes the esterification of free cholesterol present in plasma lipoproteins. In order to evaluate the role of LCAT in HDL metabolism, a 6.2-kilobase (kb) fragment consisting of 0.851 and 1.134 kb of the 5`- and 3`-flanking regions, as well as the entire human LCAT gene, was utilized to develop transgenic mice. Three different transgenic mouse lines overexpressing human LCAT at plasma levels 11-, 14-, and 109-fold higher than non-transgenic mice were established. Northern blot hybridization analysis demonstrated that the injected 6.2-kb fragment contained the necessary DNA sequences to direct tissue specific expression of the human LCAT gene in mouse liver. Compared to age- and sex-matched controls, total cholesterol and HDL cholesterol levels were increased in all 3 transgenic mice lines by 124-218 and 123-194%, respectively, while plasma triglyceride concentrations remained similar to that of control animals. Fast protein liquid chromatography analysis of transgenic mouse plasma revealed marked increases in high density liposportin (HDL)-cholesteryl ester and phospholipid as well as the formation of larger size HDL. Thus, the majority of the increase in transgenic plasma cholesterol concentrations was due to accumulation of cholesteryl ester in HDL consistent with enhanced esterification of free cholesterol in mouse HDL by human LCAT. Plasma concentrations of apoA-I, apoA-II, and apoE were increased in high expressor homozygote mice who also demonstrated an accumulation of an apoE-rich HDL. Like the mouse enzyme, human LCAT was found to be primarily associated with mouse HDL. Our studies demonstrate a high correlation between plasma LCAT activity and total as well as HDL cholesterol levels establishing that in mice LCAT modulates plasma HDL concentrations. Overexpression of LCAT in mice leads to HDL elevation as well as increased heterogeneity of the HDL lipoprotein particles, indicating that high levels of plasma LCAT activity may be associated with hyperalphalipoproteinemia and enhanced reverse cholesterol transport.


INTRODUCTION

Lecithin cholesterol acyltransferase (LCAT)() is an important enzyme that plays a major role in extracellular cholesterol metabolism. LCAT catalyzes the synthesis of cholesteryl esters and lysolecithin from phosphatidylcholine and unesterified cholesterol present in plasma lipoproteins (1, 2) . The cholesteryl esters either become part of the lipoprotein core or are transferred by cholesteryl ester transfer protein (CETP) to other lipoproteins. Although LCAT preferentially esterifies free cholesterol present in small HDL particles that contain apolipoprotein A-I, the most potent activator of the enzyme (2, 3) , it also exerts its action on apoB containing lipoproteins. In normolipidemic subjects, the majority of LCAT is associated with apoA containing lipoproteins, especially with the apoA-I (without apoA-II) HDL particle subclass (4) .

In the past several years the identification of individuals with LCAT gene defects have provided new insights into the role that LCAT plays in normal HDL metabolism. Patients with either partial or total deficiency of plasma LCAT activity present with two clinically distinct syndromes, Fish Eye Disease (FED) and classic LCAT deficiency (CLD), respectively. Both FED and CLD are characterized by the development of corneal opacities and marked hypoalphalipoproteinemia (HDL < 15 mg/dl) (1). However, patients with CLD but not FED often present with anemia and glomerulosclerosis. Recent studies have demonstrated significant overlap in the biochemical features of both syndromes (5) . Characterization of the lipoprotein particles isolated from the plasma of patients with LCAT deficiency reveals the presence of abnormal small spherical as well as aggregated discoidal particles establishing the importance of LCAT for the synthesis, maturation as well as metabolism of HDL (6, 7, 8) .

Because of its unique function as the key enzyme involved in the esterification of intravascular free cholesterol, LCAT may not only modulate HDL concentrations in plasma but also, play an important role in the processes of reverse cholesterol transport and atherosclerosis (9). Thus, LCAT has been proposed to facilitate the uptake of cholesterol into HDL particles by maintaining a concentration gradient for the efflux of free cholesterol from peripheral tissues (10) . In addition, esterification of free cholesterol on nascent pre- HDL particles by LCAT appears to be essential for the maturation of the pre- particles to the particles present in HDL and HDL(11, 12) . Cholesteryl esters in HDL and HDL may then be transported directly to the liver or alternatively, transferred to apoB-containing lipoproteins by the action of CETP (13) for ultimate hepatic uptake by LDL and remnant receptor mediated pathways (14, 15, 16) . Enhanced LCAT function may be anticipated to increase reverse cholesterol transport and protect against the development of premature cardiovascular disease. However, at present it is not known if increased plasma LCAT activity results in elevated HDL cholesterol levels or protection from atherosclerosis.

In order to gain a better understanding of LCAT function and determine the consequences of elevating plasma LCAT concentrations on HDL metabolism and atherosclerosis, we have produced several different lines of transgenic mice which express high levels of human LCAT in plasma. Our findings indicate that overexpression of human LCAT leads to alterations in the levels, composition, and size of plasma HDL consistent with a central role for this enzyme in HDL cholesterol esterification. These studies, thus, establish that plasma LCAT activity modulates HDL concentrations in vivo and indicates that elevations of plasma LCAT may be associated with hyperalphalipoproteinemia in humans.


MATERIALS AND METHODS

Animals

Fertilized eggs used in the microinjection experiments were isolated from 6-week-old C57BL/6N females after mating with C57BL/6N male mice (Charles River, Wilmington, MA). Transgenic and control animals used in the study included 2-month-old male and female mice.

Production of Transgenic Mice

A 6.2-kb genomic fragment consisting of the entire LCAT gene, including 0.851 kb of the 5`-flanking and 1.134 kb of the 3`-flanking region was generated from of an LCAT clone isolated from a human genomic library (kindly provided by Dr. Neil Epstein, NIH, Bethesda MD). For microinjection, the isolated 6.2-kb DNA fragment was purified on CsCl density gradient as described (17) . Approximately 740 copies (5 10 pg) of the human LCAT gene construct were injected into the male pronucleus of fertilized eggs from superovulated C57BL/6N females. Eggs were then reimplanted into pseudo-pregnant CB6/FI females (Charles River, Wilmington, MA) by oviduct transfer (18) . Integration of the human LCAT gene in newborn mice was determined by Southern blot hybridization analysis of 10 µg of mice tail DNA (19) after digestion with PstI. Hybridization was performed with a 446-base pair polymerase chain reaction-amplified DNA fragment spanning the 3` end of introns I through exon 4 of the human LCAT gene (Fig. 1). Out of 48 mice screened, 5 founder transgenic mice were identified and the 3 highest expressors were utilized for this study.


Figure 1: Schematic representation of the 6235-kb SmaI LCAT gene fragment utilized to generate transgenic mice. The location of the six exons (solid bars), PstI restriction enzyme sites (P), and the DNA fragment utilized as a probe (hatched bar) are indicated. bp, base pair.



Northern Blot Hybridization Analysis

To study the specificity of human LCAT mRNA expression, total RNA was isolated from a variety of different tissues (brain, heart, lung, kidney, liver, and testis) from 4-month-old mice and their age- and sex-matched control siblings using the guanidinium thiocyanate method (20) . Analysis of tissue RNA was performed by fractionation on a 0.7% formaldehyde-agarose gel, transfer to a nylon membrane, and hybridization with the same LCAT probe utilized for Southern blotting, as described (17) .

Determination of LCAT Mass and Activity in Mouse Plasma

-LCAT activity was assayed as described previously (21) using 10 µl of mouse plasma. Cholesterol esterification rate (CER) was quantitated by determining the rate of esterification of [C]cholesterol using 125 µl of pooled plasma from at least 5 different controls or transgenic mice described (22) . LCAT mass was quantitated by radioimmunoassay using a polyclonal antibody generated against human LCAT as well as purified human LCAT standards and I-human LCAT as described previously (23) .

Analysis of Plasma Lipids, Lipoproteins, and Apolipoproteins

Blood from study animals was collected after a 4-h fast into tubes containing 2 mM EDTA and centrifuged for 15 min at 4°C. Plasma samples were separated, aliquoted, and stored frozen prior to analysis. Total cholesterol and triglyceride concentrations in plasma were determined by enzymic methods using commercial kits (Sigma) as were free cholesterol (FC) and phospholipid (PL) concentrations (Wako Chemicals USA, Inc., Richmond, Va). Cholesteryl ester (CE) values were calculated by subtracting free cholesterol from total plasma cholesterol concentrations. HDL cholesterol was quantitated in heparin-calcium treated plasma. Ten µl of mouse plasma was incubated with 100 µl of heparin-calcium (0.1%, 0.03 M, v/v) for 20 min at room temperature followed by centrifugation at 4 °C (24) . Cholesterol concentrations in the supernatant were determined spectrophotometrically at 490 nm. Non-HDL cholesterol was calculated by subtraction of HDL cholesterol from total cholesterol concentrations. Relative concentrations of apoA-I, apoA-II, and apoE in mouse plasma were determined by densitometric scanning of immunoblots as previously reported (25) .

Gel Filtration Chromatography

Fifty-100 µl of fresh plasma was applied to an FPLC system with two Superose 6 columns connected in series (Pharmacia LKB Biotechn, Piscataway, NJ). Lipoproteins were eluted at 0.3 ml/min with phosphate-buffered saline buffer containing 1 mM EDTA and 0.02% sodium azide. After the first 10 ml were eluted, 60 (0.5 ml) fractions were collected. Total cholesterol, triglycerides, free cholesterol, phospholipids, and cholesteryl ester were determined in each fraction as described above. The presence of apoA-I, apoA-II, and apoE in each lipoprotein peak was determined by immunoblotting (25) after delipidation of 200 of eluted pooled fractions with ethanol/ether.

RESULTS

Several different lines of transgenic mice containing multiple copies of the human LCAT gene were generated by injection of fertilized eggs with a construct containing the native human LCAT gene illustrated in Fig. 1. This DNA fragment consisted of the entire LCAT gene and included 0.851 and 1.134 kb of the 5`- and 3`-flanking regions, respectively. Integration of the human transgene in several founders was established by Southern blot hybridization analysis which demonstrated the presence of 2-120 copies of the human gene in the mouse genome (data not shown). Three different transgenic mouse lines were established from the founders with the highest LCAT expression levels and utilized in the present study.

Human LCAT is expressed primarily in liver (26) . However, in rats, there is evidence for expression in brain, testis, as well as liver (27). In order to evaluate whether the integrated 6.2-kb human LCAT DNA fragment was able to direct the expression of the human LCAT gene in a tissue-specific manner, total RNA was isolated from different transgenic mouse tissues and analyzed by Northern blot hybridization. LCAT mRNA was present primarily in the liver of transgenic mice (data not shown) although upon prolonged exposure, small amounts of LCAT message was also detected in the testis, brain, heart, and lung of these animals (data not shown).

summarizes the LCAT mass and activity measured in the plasma of the 3 different transgenic mouse lines (T-1, T-2, and T-3). The number of integrated copies of the transgene was estimated by Southern blot hybridization to range from 15 to 120 and correlated with human LCAT concentrations (r = 0.997) in mouse plasma (data not shown). Despite the use of antibodies generated against human LCAT (23), our immunoassay detected mouse LCAT in non-transgenic animals. Thus, the LCAT concentrations for transgenic mice listed in refer to the sum of both mouse and human enzymes. However, compared to the low levels of expression observed in control nontransgenic animals (1 µg/ml), plasma LCAT concentrations in all 3 transgenic lines were markedly increased and correlated with the plasma -LCAT activity. In the highest expressor line, T-3, the LCAT mass, like the -LCAT activity, was approximately 100-fold greater than that of non-transgenic animals, respectively (). Assuming that the contribution of the mouse enzyme to the total plasma LCAT concentrations in the high expressor line, T-3, is insignificant, the calculated specific activity of the human enzyme in transgenic mice is similar to that of human LCAT (19.5 ± 1.8 nmol/µg/h) (5) , indicating expression of a fully functional enzyme. Interestingly, compared to in vitro measurements of LCAT mass and -LCAT activity, the endogenous plasma LCAT activity (CER) was increased only 1.5-2-fold in transgenic mice compared to control animals, suggesting that in vivo other factors may limit full activation of human LCAT in mice ().

summarizes the plasma lipids and lipoproteins of heterozygote mice overexpressing of the human LCAT gene. Compared to age- and sex-matched controls, all 3 transgenic lines exhibited significant increases in fasting TC as well as CE which paralled the fold increase in endogenous LCAT activity (CER). Statistically significant differences in plasma PL and FC were observed primarily in the highest expressor line, T-3, while fasting plasma triglycerides in all 3 transgenic lines were similar to that of controls. Thus, overexpression of human LCAT in mice results in enhanced accumulation of plasma CE leading to significant increases in the CE/FC ratio. Heparin calcium precipitation of mouse plasma indicates that most of the cholesterol increase in transgenic animals was associated with mouse HDL (). Interestingly, analysis of all 3 transgenic lines revealed a significant correlation between the level of plasma cholesterol as well as HDL cholesterol and plasma LCAT activity (Fig. 2). The correlation was most significant in male transgenic animals which for the same concentrations of human LCAT, exhibited greater changes in plasma lipids than transgenic female mice.


Figure 2: Correlation between plasma levels of LCAT activity and TC as well as HDL-C concentrations in control and heterozygous transgenic (T-1, T-2, T-3) mice. The values for the correlation coefficient are indicated.



I compares the changes in the plasma lipids, lipoproteins, apolipoproteins, as well as LCAT activity between heterozygous and homozygous male mice for the T-3 line and control animals. Compared to heterozygotes, homozygotes had further increases in plasma concentrations of TC, PL, CE, and HDL-C as well as -LCAT activity (p < 0.05). Measurement of plasma apoA-I, apoA-II, and apoE by immunoassay or Western blot analysis demonstrated that, despite a greater than 2-fold increase in HDL levels, there were no significant difference in the apoE plasma concentration of apoA-I, apoA-II between heterozygote transgenic and control animals. However, compared to age- and sex-matched non-transgenic siblings, homozygous transgenic mice had significantly higher plasma concentrations of apoA-I, apoA-II, and especially apoE (I).

Fig. 3 illustrates the FPLC profile of fasting plasma from heterozygote mice (T-1 and T-3 lines), as well as a non-transgenic sex-matched sibling (T-3 line). In agreement with prior plasma lipid and lipoprotein determinations, FPLC analysis revealed accumulation of cholesterol (Fig. 3A) in HDL-sized particles, which were also enriched in CE and PL (Fig. 3, B and C). HDL in mice overexpressing human LCAT eluted in an FPLC fraction containing a larger apparent particle size than that of control mice (Fig. 3, A-C). Analysis of the elution of profile of human LCAT on FPLC revealed that the distribution of the human enzyme on the lipoproteins in transgenic plasma was similar to that of mouse LCAT (Fig. 3D). Both mouse and human LCAT associated primarily with HDL sized particles.


Figure 3: Elution profile of control and transgenic (T-1, T-3) mouse plasma on FPLC is illustrated. The relative positions of normal mice and human very LDL, LDL, and HDL on FPLC were determined after the ultracentrifuged fractions were individually chromatographed on Sepharose-6B and are indicated by the bars. HDL-sized particles present in transgenic mouse plasma had a relative enrichment in TC, CE, and PL as well as an increase in particle size (Panels A-C). In Panel C, the phospholipid concentration for fractions 10-26 ml as well as 31-40 ml were below the levels of detection of the assay. Panel D illustrates the relative distribution of LCAT in control and transgenic mouse lipoproteins.



The plasma lipoprotein profile of homozygous animals for the T-3 line, which had marked overexpression of human LCAT in plasma, is illustrated in Fig. 4. Unlike the heterozygous animals, the plasma profile of homozygous mice on FPLC demonstrated two distinct particles of apparent HDL(28) and HDL size (Fig. 4, A-C). Like heterozygotes, human LCAT in homozygote plasma was found to be primarily associated with the major HDL particle (Fig. 4D). Characterization of the apolipoprotein content of these two fractions by immunoblot analysis (Fig. 5) revealed that the larger particles (peak I) contained predominantly apoE as well as apoA-I and the smaller particles (peak II) contained apoA-I and apoA-II. Together with the increased plasma apoE concentrations observed in homozygous mice (I) these findings suggest enhanced synthesis of apoE-enriched HDL with preferential accumulation of newly synthesized CE in apoE containing particles. Minimal amounts of apoB could be detected in FPLC fractions 22-24 and 26-30 ml by immunoblotting (data not shown) indicating that the major lipoproteins present in elution fractions 22-30 ml were HDL.


Figure 4: Analysis of heterozygote and homozygote mouse plasma (T-3 line) on FPLC. In homozygous animals, a second distinct HDL-lipoprotein peak (fraction 22-24 ml) is apparent. Panel D illustrates the relative distribution of LCAT in transgenic mouse lipoproteins.




Figure 5: Immunoblot analysis of transgenic mouse plasma (P) from a control and homozygote mice (T-3) as well as pooled FPLC fractions containing the larger (peak I, fractions 22-24 ml) and smaller (peak II, fractions 26-30 ml) lipoproteins is illustrated. ApoA-I, apoA-II, and apoE standards are shown. Markers (M) are shown on the left.



DISCUSSION

HDL synthesis, maturation, and catabolism are complex processes which require the function of several different enzymes, receptors as well as transfer proteins. Thus, hepatic lipase, CETP, and LCAT as well as the proposed HDL receptor appear to play key roles in a complex metabolic cascade which ultimately may result in the delivery of cholesterol from peripheral cells to the liver via the process of reverse cholesterol transport. The metabolic consequences of hepatic lipase, CETP, and LCAT enzyme deficiencies have been well characterized (29-38) and include abnormalities in the composition and concentration of HDL as well as alterations in HDL particle size indicating marked derangements of HDL metabolism. Patients with LCAT deficiency (CLD or FED) present with profound hypoalphalipoproteinemia (1) . Characterization of plasma lipoproteins isolated from these affected individuals reveals the presence of abnormal small spherical HDL particles in the small HDL to d > 1.21 g/1-ml range as well as of large, aggregated discoidal HDL particles in the HDL density range (6, 7) . Thus, LCAT function is necessary for HDL particle maturation as well as the maintenance of HDL plasma concentrations. Detailed kinetic studies of the two major lipoprotein particles in HDL, LpA-I and LpA-I:A-II, in patients with CLD and FED revealed increased catabolism of both LpA-I and LpA-I:A-II with LpA-I:A-II catabolism being greater than LpA-I resulting in a relative increase in the plasma concentration of LpA-I (8) . These findings may explain at least in part why, despite the very low plasma HDL levels, patients with LCAT deficiency are not at an increased risk for the development of premature cardiovascular disease. Thus, the pre- HDL particles present in LCAT-deficient patients may still effectively facilitate efflux of cholesterol from cells. Despite these observations, the role that LCAT plays in modulating HDL plasma levels, as well as reverse cholesterol transport and atherosclerosis remains unclear.

In order to gain a better understanding of LCAT function in HDL metabolism we have introduced a 6.2-kb fragment of the human LCAT gene in C57BL/6 mice to generate different transgenic mouse lines overexpressing the human LCAT gene. Previous studies have suggested that in vitro, maximal expression of the reporter gene, CAT, can be achieved under the control of 71 base pairs of the 5`-flanking region of the human LCAT gene (39) . However, no data is available regarding cis-acting elements in the 5`-flanking region of the human LCAT gene that are necessary for directing its tissue-specific expression in liver. The DNA fragment utilized to generate our transgenic animals contained 851 and 1,134 base pairs of the 5`- and 3`-flanking region of the human LCAT gene, respectively. Our studies demonstrate that the 6.2-kb LCAT gene fragment is sufficient to direct very high levels of expression in mouse liver, thus indicating that transcriptional elements necessary for tissue-specific expression are located in this region of the human LCAT gene. Interestingly, analysis of the 5`-flanking sequences reveals the presence of 4 different potential binding sites for the hepatic transcription factor LF-AI (40) at position -54, -145, -591, and -602 relative to the transcriptional start site. These sequences could play a functional role in mediating the tissue-specific expression of LCAT in man. Analysis of the expression of human LCAT in transgenic animals demonstrated that the plasma -LCAT activities, measured by using a proteoliposome substrate, varied considerably in different lines, ranging from 19- to 109-fold higher than the background enzyme activity found in normal mice. Assuming that most of the plasma LCAT concentrations in the high expressor T-3 line represented human LCAT, the -LCAT specific activity of these transgenic animals was similar to that of controls, indicating expression of a fully functional enzyme. Compared to the marked increases in -LCAT activity, the endogenous plasma LCAT activity (CER) in LCAT transgenic mice was only 1.5-2-fold greater than that of control mice, suggesting that in vivo, other factors may limit full activation of the human enzyme in mice. Potential explanations may include differences in the size and composition of mouse versus human HDL, the different specificities of the 2 enzymes for available esters as well as limited availability of FC as substrate as LCAT overexpression leads to enhanced CE accumulation altering the composition and conformation of HDL particles. Although differences in mouse and human apoA-I (41, 42, 43) , could lead to reduced activation of human LCAT by the mouse cofactor, separate in vitro studies have demonstrated no difference in the -LCAT activity of transgenic mouse plasma when proteoliposomes containing mouse versus human apoA-I were utilized (data not shown).

Overexpression of human LCAT in mice resulted in significant increases in plasma total as well as HDL cholesterol concentrations. Consistent with LCAT's enzymic function, most of this increase represented an accumulation of cholesterol in the form of CE. Mice with very high plasma LCAT concentrations (109 µg/ml), also demonstrated elevations in plasma FC and PL concentrations with no major changes in plasma triglycerides. Plasma total and HDL cholesterol levels in transgenic mice were significantly correlated with both LCAT concentration and activity. Despite the fact that the levels of human LCAT expression in the plasma of different transgenic animals differed by at least 10-fold, the maximal increase achieved in total and HDL cholesterol in transgenic males from the T-3 line were 2.3- and 2.1-fold, respectively, indicating that LCAT is only one of several important factors modulating HDL plasma concentrations. Interestingly, these changes in plasma HDL-C concentrations paralled the increase in endogenous (CER) plasma LCAT activity (1-5-2-fold) rather than the changes observed in in vitro -LCAT activity (19-109-fold) indicating that, in these animals, CER is the more physiologically relevant measurement of enzyme function. Thus, a 2-fold increase in the endogenous LCAT activity (CER) results in a similar doubling of plasma total and HDL cholesterol concentration in transgenic mice.

Further characterization of the plasma lipoproteins in heterozygous transgenic mice demonstrated alterations in HDL composition as well as particle size. In addition to an enrichment in TC and CE, the size of the HDL particles present in the plasma of transgenic mice was significantly increased. HDL particle size appeared to correlate with LCAT plasma concentrations with the largest HDL detected in the plasma of transgenic animals with highest expression levels. Since the concentration of plasma apoA-I, apoA-II, and apoE was normal, LCAT overexpression in these animals appears to primarily increase plasma HDL-C levels by increasing the size and not the number of HDL particles. Thus, in heterozygote mice LCAT overexpression leads to the formation of larger CE and PL enriched HDL consistent with esterification of FC in mouse HDL by the human enzyme.

In homozygous animals with marked overexpression of human LCAT, the plasma profile on FPLC demonstrated the presence of two distinct particles which exhibited major differences in apolipoprotein composition. The larger HDL fraction (peak I) contained primarily apoE as well as apoA-I, whereas the smaller HDL particle contained primarily apoA-I as well as apoA-II. These two particles are similar in both lipid and apolipoprotein composition to HDL and HDL present in human plasma as well as normal dogs, rats, mice, and rabbits (28, 44) . Previous studies have suggested that in vitro, the formation of HDL from HDL requires LCAT activity as well as the presence of apoE (44). The identification of HDL particles similar to HDL in homozygote mice, especially in the context of increased plasma concentrations of mouse apoE indicate that together with apoE, LCAT plays an important role in the formation of large apoE-enriched HDL particles from smaller HDL in vivo. In homozygous mice, apoE is present primarily in the larger apoE-enriched HDL particles, suggesting either increased synthesis of HDL and/or preferential accumulation of the newly synthesized CE in apoE containing HDL. Particles containing apoE may be necessary for the formation of an expanded CE core (44, 45) . In fact, the increase in plasma PL observed primarily in high expressor animals may reflect a requirement for additional PL as well as apoE to be added to the surface layer of the lipoprotein to accommodate an expanding CE core. In this context, it is possible that the availability of either PL or apoE could limit further cholesterol esterification by the human enzyme. Thus, the pool of apoE-enriched HDL particles available to accommodate increasing amounts of newly generated CE may be limited, preventing full expression of LCAT function in transgenic mice. Our studies indicate that marked LCAT overexpression leads to both elevation in HDL-C concentrations as well as increased heterogeneity of the HDL lipoprotein particles present in mouse plasma in a pattern reminiscent of human HDL.

The enhanced accumulation of apoE containing HDL particles in transgenic mice may have important physiological consequences. The ability of apoE-rich HDL to interact with the LDL receptor in vitro has been previously demonstrated (46) . Thus, in LCAT transgenic mice the increase in apoE containing HDL particles may mediate the delivery of CE to the liver via apoE-dependent receptor-mediated mechanisms, potentially enhancing reverse cholesterol transport and ultimately, protecting these animals against the development of diet-induced lesions. Although, in mice LCAT overexpression may be beneficial, the consequence of raising plasma LCAT levels in an animal model which unlike the mouse expresses CETP may be quite different. In the presence of CETP increased LCAT activity could result in enhanced exchanged of newly generated CE into apoB-containing lipoproteins ultimately leading to marked differences in lesion development and atherosclerosis.

In the present study we have characterized 3 different transgenic mouse lines overexpressing human LCAT by 9-109-fold. Our studies indicate that LCAT modulates plasma total and HDL cholesterol concentrations as well as HDL particle heterogeneity. Thus, overexpression of LCAT in mice leads to significant elevation of TC and HDL, suggesting that high plasma LCAT concentrations may, in humans, be responsible for hyperalphalipoproteinemia. Previous studies (47) have demonstrated that overexpression of human apoA-I in C57BL/6N mice results in increasing mouse plasma HDL concentration as well as protection against the development of diet-induced fatty lesions. The availability of transgenic mice overexpressing human LCAT in C57BL/6N mice will permit the evaluation of the potential role of LCAT in both reverse cholesterol transport and atherosclerosis.

  
Table: LCAT concentration and activity in the plasma of control and transgenic mice


  
Table: Plasma lipids and lipoproteins in the plasma of transgenic mice


  
Table: Plasma lipid, lipoprotein, and apolipoprotein concentrations as well as plasma LCAT activities in the high expressor (T-3) transgenic male mice



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL30086. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: National Institutes of Health, National Heart, Lung and Blood Institute, Molecular Disease Branch, Bldg. 10, Rm. 7N115, 10 Center Dr. MSC 1666, Bethesda, MD 20892-1666. Tel.: 301-496-5095; Fax: 301-402-0190.

The abbreviations used are: LCAT, lecithin cholesterol acyltransferase; HDL, high density lipoproteins; FPLC, fast protein liquid chromatography; LDL, low density lipoproteins; apoA-I, apolipoprotein A-I; apoA-II, apolipoprotein A-II; TC, total cholesterol; CE, cholesteryl ester; FC, free cholesterol; PL, phospholipids; CER, cholesterol esterification rate; FDC, Fish Eye Disease; CLD, classic LCAT deficiency; kb, kilobase(s); CETP, cholesteryl ester transfer protein.


REFERENCES
  1. Norum, K. R., Gjone, E., and Glomset, J. A.(1989) in The Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 1181-1194, McGraw-Hill, New York
  2. Jonas, A.(1991) Biochim. Biophys. Acta 1084, 205-220 [Medline] [Order article via Infotrieve]
  3. Fielding, C. J., Shore, V. G., and Fielding, P. E.(1972) Biochem. Biophys. Res. Commun. 46, 1493-1498 [Medline] [Order article via Infotrieve]
  4. Duverger, N., Rader, D. J., Duchateau, P., Fruchart, J. C., Castro, G., and Brewer, H. B., Jr(1993) Biochemistry 32, 12373-12379
  5. Klein, H.-G., Santamarina-Fojo, S., Duverger, N., Clerc, M., Dumon, M.-F., Albers, J. J., Marcovina, S., and Brewer, H. B., Jr.(1993) J. Clin. Invest. 92, 479-485 [Medline] [Order article via Infotrieve]
  6. Forte, T., Norum, K. R., Glomset, J. A., and Nichols, A. V.(1971) J. Clin. Invest. 50, 1141-1148 [Medline] [Order article via Infotrieve]
  7. Forte, T. M., and Carlson, L. A.(1984) Arteriosclerosis 4, 130-137 [Abstract]
  8. Rader, D. J., Ikewaki, K., Duverger, N., Schmidt, H., Pritchard, H., Frohlich, J., Clerc, M., Dumon, M.-F., Fairwell, T., Zech, L., Santamarina-Fojo, S., and Brewer, H. B., Jr.(1994) J. Clin. Invest. 93, 321-330 [Medline] [Order article via Infotrieve]
  9. Glomset, J. A., Janssen, E. T., Kennedy, R., and Dobbins, J.(1966) J. Lipid Res. 7, 638-648 [Medline] [Order article via Infotrieve]
  10. Fielding, C. J.(1984) J. Lipid Res. 25, 1624-1628 [Medline] [Order article via Infotrieve]
  11. Castro, G. R., and Fielding, C. J.(1988) Biochemistry 27, 25-29 [Medline] [Order article via Infotrieve]
  12. Fielding, P. E., Kawano, M., Catapano, A. L., Zoppo, A., Marcovina, S., and Fielding, C. J.(1994) Biochemistry 33, 6981-6985 [Medline] [Order article via Infotrieve]
  13. Tall, A. R.(1993) J. Lipid Res. 34, 1255-1274 [Medline] [Order article via Infotrieve]
  14. Goldstein, J. L., Brown, M. S., Anderson, R. G., Russell, D. W., and Schneider, W. J.(1985) Annu. Rev. Cell Biol. 1, 1-39 [CrossRef]
  15. Herz, J., Hamann, U., Rogne, S., Myklebos, O., Gausepohl, H., and Stanley, K. K.(1988) EMBO J. 7, 4119-4127 [Abstract]
  16. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S.(1990) J. Biol. Chem. 265, 17401-17404 [Abstract/Free Full Text]
  17. Fojo, S. S., Law, S. W., Sprecher, D. L., Gregg, R. E., Baggio, G., and Brewer, H. B., Jr.(1984) Biochem. Biophys. Res. Commun. 124, 308-313 [Medline] [Order article via Infotrieve]
  18. Hogan, B., Costantini, F., and Lacy, E.(1986) Manipulating the Mouse Embryo: A Laboratory Manual, 256th Ed., pp. 91-149, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Chirgwin, J. A., Pryzbyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  21. Klein, H.-G., Lohse, P., Duverger, N., Albers, J. J., Rader, D. J., Zech, L. A., Santamarina-Fojo, S., and Brewer, H. B., Jr.(1993) J. Lipid Res. 34, 49-58 [Abstract]
  22. Stokke, K. T., and Norum, K. R.(1971) Scand. J. Clin. Lab. Invest. 27, 21-27 [Medline] [Order article via Infotrieve]
  23. Albers, J. J., Adolphson, J. L., and Chen, C.-H.(1981) J. Clin. Invest. 67, 141-148 [Medline] [Order article via Infotrieve]
  24. Burstein, M., Scholnick, H. R., and Morfin, R.(1970) J. Lipid Res. 11, 583-95 [Abstract/Free Full Text]
  25. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  26. McLean, J., Wion, K., Drayna, D., Fielding, C., and Lawn, R.(1986) Nucleic Acids Res. 14, 9397-9406 [Abstract]
  27. Warden, C. H., Langner, C. A., Gordon, J. I., Taylor, B. A., McLean, J. W., and Lusis, A. J.(1989) J. Biol. Chem. 264, 21573-21581 [Abstract/Free Full Text]
  28. De Silva, H. V., Mas-Oliva, J., Taylor, J. M., and Mahley, R. W.(1994) J. Lipid Res. 35, 1297-1310 [Abstract]
  29. Yamashita, S., Hui, D. Y., Wetterau, J. R., Sprecher, D. L., Harmony, J. A. K., Sakai, N., Matsuzawa, Y., and Tarui, S.(1991) Metab. Clin. Exp. 7, 756-763
  30. Inazu, A., Brown, M. L., Hesler, C. B., Agellon, L. B., Koizumi, J., Takata, K., Maruhama, Y., Mabuchi, H., and Tall, A. R.(1990) N. Engl. J. Med. 323, 1234-1238 [Abstract]
  31. Takegoshi, T., Haba, T., Kitoh, C., Tokuda, T., and Mabuchi, H.(1988) Jpn. J. Med. 27, 295-299 [Medline] [Order article via Infotrieve]
  32. Brown, M. L., Inazu, A., Hesler, C. B., Agellon, L. B., Mann, C., Whitlock, M. E., Marcel, Y. L., Milne, R. W., Koizumi, J., Mabuchi, H., Takeda, R., and Tall, A. R.(1989) Nature 342, 448-451 [CrossRef][Medline] [Order article via Infotrieve]
  33. Breckenridge, W. C., Little, J. A., Alaupovic, P., Wang, C. S., Kuksis, A., Kakis, G., Lindgren, F., and Gardiner, G.(1982) Atherosclerosis 45, 161-179 [Medline] [Order article via Infotrieve]
  34. Carlson, L. A., Holmquist, L., and Nilsson-Ehle, P.(1986) Acta Med. Scand. 219, 435-447 [Medline] [Order article via Infotrieve]
  35. Connelly, P. W., Maguire, G. F., Lee, M., and Little, J. A.(1980) Atherosclerosis 36, 40-48
  36. Auwerx, J. H., Marzetta, C. A., Hokanson, J. E., and Brunzell, J. D. (1989) Arteriosclerosis 9, 319-325 [Abstract]
  37. Connelly, P. W., Maguire, G. F., Lee, M., and Little, J. A.(1990) Am. J. Hum. Genet. 46, 470-477 [Medline] [Order article via Infotrieve]
  38. Demant, T., Carlson, L. A., Holmquist, L., Karpe, F., Nilsson-Ehle, P., Packard, C. J., and Shepherd, J.(1988) J. Lipid Res. 29, 1603-1611 [Abstract]
  39. Meroni, G., Malgaretti, N., Pontoglio, M., Ottolenghi, S., and Taramelli, R.(1991) Biochem. Biophys. Res. Commun. 180, 1469-1475 [Medline] [Order article via Infotrieve]
  40. Ramji, D. P., Tadros, M. H., Hardon, E. M., and Cortese, R.(1991) Nucleic Acids Res. 19, 1139-1146 [Abstract]
  41. Karathanasis, S. K., Zannis, V. I., and Breslow, J. L.(1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6147-6151 [Abstract]
  42. Stoffel, W., Muller, R., Binczek, E., and Hofmann, K.(1992) Biol. Chem. Hoppe-Seyler 373, 187-193 [Medline] [Order article via Infotrieve]
  43. Boyle, T. P., and Marotti, K. R.(1992) Gene (Amst.) 117, 243-247 [Medline] [Order article via Infotrieve]
  44. Gordon, V., Innerarity, T. L., and Mahley, R. W.(1983) J. Biol. Chem. 258, 6202-6212 [Abstract/Free Full Text]
  45. Tall, A. R., Atkinson, D., Small, D. M., and Mahley, R. W.(1977) J. Biol. Chem. 252, 7288-93 [Medline] [Order article via Infotrieve]
  46. Koo, C., Innerarity, T. L., and Mahley, R. W.(1985) J. Biol. Chem. 260, 11934-11943 [Abstract/Free Full Text]
  47. Rubin, E. M., Krauss, R. M., Spangler, E. A., Verstuyft, J. G., and Clift, S. M.(1991) Nature 353, 265-267 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.