From the
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
Lecithin cholesterol acyltransferase (LCAT)
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-
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.
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
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.
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.
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
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
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
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.
. 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.
(
)
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) .
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.
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.
-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 ().
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).
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.
-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).
-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.
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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.