Remodeling of the HDL in NIDDM: a fundamental role for
cholesteryl ester transfer protein
Christine K.
Castle1,
Susan L.
Kuiper1,
William L.
Blake1,
Beverly
Paigen2,
Keith R.
Marotti1, and
George W.
Melchior1
1 Pharmacia and Upjohn, Inc., Kalamazoo,
Michigan 49001; and 2 Jackson Laboratories, Bar
Harbor, Maine 04609
 |
ABSTRACT |
When the
Ay gene is expressed in KK mice,
the yellow offspring (KKAy mice)
become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process
catalyzed in humans by cholesteryl ester transfer protein (CETP). To
test the hypothesis that it is the absence of CETP that allows these
hypertriglyceridemic mice to maintain high plasma HDL levels, simian
CETP was expressed in the KKAy
mouse. The KKAy-CETP mice retained
the principal characteristics of
KKAy mice except that their plasma
HDL levels were reduced (from 159 ± 25 to 25 ± 6 mg/dl) and
their free apolipoprotein A-I concentrations increased (from 7 ± 3 to 22 ± 6 mg/dl). These changes appeared to result from a
CETP-induced enrichment of the HDL with triglyceride (from 6 ± 2 to
60 ± 18 mol of triglyceride/mol of HDL), an alteration that renders
HDL susceptible to destruction by lipases. These data support the
premise that CETP-mediated remodeling of the HDL is responsible for the
low levels of that lipoprotein that accompany hypertriglyceridemic
non-insulin-dependent diabetes mellitus.
free apolipoprotein A-I; pre-
apolipoprotein A-I; high-density
lipoprotein lipase; mouse plasma lipase; KKAy mouse; mouse model of
non-insulin-dependent diabetes mellitus
 |
INTRODUCTION |
NON-INSULIN-DEPENDENT diabetes mellitus (NIDDM) is
associated with obesity, hyperinsulinemia, hypertriglyceridemia, and
low high-density lipoprotein (HDL) levels. The mechanism by which NIDDM
leads to low HDL levels is not known, but it has often been linked to
the hypertriglyceridemia. There are several rodent models of NIDDM (5,
7, 19, 23, 30), and all have one or more of the characteristics of the
human disease with one key exception: none have low HDL levels, even
when severely hypertriglyceridemic. It has been suggested (7, 29) that
rodents are able to maintain high plasma HDL levels despite the severe
hypertriglyceridemia because they lack the ability to redistribute
cholesteryl esters among circulating lipoproteins, a process catalyzed
in humans by cholesteryl ester transfer protein (CETP) (36). That
hypothesis was based on the results of previous studies in which CETP
was expressed in C57BL/6 mice (27) and on the premise that the
hypertriglyceridemia would augment the enrichment of HDL with
triglyceride and thereby render that lipoprotein more vulnerable to
destruction by a lipase (3, 11, 12).
The purpose of the experiments described here was to test that
hypothesis. To accomplish that, simian CETP was expressed in the
KKAy mouse, a rodent model of
NIDDM with extraordinarily high HDL levels. Simian CETP was chosen as
the source of CETP for these studies because it is virtually identical
in structure to human CETP (32) and because that genetic construct has
been shown previously to work exceptionally well in mice (24, 25, 27, 29).
 |
METHODS |
Animals and diets.
KKAy-CETP mice were produced as
follows: C57BL/6J mice expressing the lethal yellow gene
(C57BL/6J-Ay) were crossed with
C57BL/6 mice expressing simian CETP to produce C57BL/6J-Ay-CETP mice. These mice
are referred to hereafter as
"B6-Ay-CETP mice." The
B6-Ay-CETP mice were produced at
Jackson Laboratories with the use of
C57BL/6J-Ay mice from their colony
and UCTP-20 mice (25, 27) provided by Pharmacia and Upjohn. A colony of
B6-Ay-CETP mice was subsequently
established at Pharmacia and Upjohn. To produce
KKAy-CETP mice,
B6-Ay-CETP mice were crossed with
KK mice (22) and yellow offspring expressing CETP were recrossed with
KK mice until that process had been repeated for six generations. At
that point, the mice were designated
"KKAy-CETP." The mice used
for these studies were obtained from that colony. All were between 5 and 6 mo of age at the time of death and had consumed a custom-made
natural-products rodent diet (Upjohn Irradiated Laboratory Rodent Diet
5011-3; PMI Feeds, Richmond, IN) from the time that they were weaned.
The diet contained (by weight) 20.7% protein, 6.5% fat, 52.0%
carbohydrate, 4.6% crude fiber, and adequate vitamins and minerals.
Fat made up 16.8% of calories, and the cholesterol content was 17.4 parts per million (0.002% by weight). The diet contained 3.13 cal of
metabolizable energy per gram.
Fifty mice were used for these studies: 22 KKAy mice (13 females and 9 males)
and 28 KKAy-CETP mice (16 females
and 12 males). On the day of death, food was removed from the cages at
7:00 AM, and at 1:00 PM, the mice were weighed,
anesthetized with ether, and exsanguinated. The blood was heparinized
and pooled for analysis. Seven
KKAy pools were created that
consisted of blood from three females, four females, four females, two
females, and three pools each containing the blood from three males.
Likewise, eight KKAy-CETP pools
were created that consisted of blood from four females, four females,
five females, three females, and four pools each containing the blood
from three males. All procedures in this study are in compliance with
the Animal Welfare Act regulations and with the National Research
Council Guide for the Care and Use of Laboratory
Animals.
Analytic measurements.
Mouse plasma has exceptionally high lipase activity (14, 15, 33). The
lipase activity in KKAy and
KKAy-CETP mouse plasma was
measured using human HDL that contained [9,10-3H]triolein (Du
Pont-NEN, Boston, MA) as substrate. The HDL was radiolabeled with
[9,10-3H]triolein
exactly as described previously (27). Ten microliters of the HDL
solution (23 µg of HDL cholesterol, 4.03 × 105 dpm of
[9,10-3H]triolein) and
27 µl of the mouse plasma were diluted to a final volume of 200 µl
with PBS and incubated for 5 h at 37°C. The reaction was stopped,
the free fatty acids were extracted as described by Belfrage and
Vaughan (4), and the associated radioactivity was quantified by liquid
scintillation counting. Less than 0.5% of the radioactivity was
extractable from the HDL substrate in the absence of a source of
lipase.
Blood glucose and plasma insulin, cholesterol, triglyceride,
phospholipid, and apolipoprotein (apo) A-I levels were all measured as
described previously (25, 27, 28), as was the CETP activity and the HDL
composition. All samples were placed in ice immediately after they were
obtained, and the plasma glycerol and total and HDL-associated
triglyceride concentrations were determined within 1 h thereafter. The
free glycerol concentrations of plasma were determined using an
enzymatic assay (Sigma, St. Louis, MO). Those values averaged 32 ± 9 mg/dl in KKAy mice and 36 ± 2 mg/dl in KKAy-CETP mice and were
subtracted from the total glycerol concentrations to obtain the
triglyceride concentrations. The concentration of HDL-associated
triglycerides and HDL-associated cholesterol in the plasma was
determined after polyethylene glycol precipitation as
described previously (7). The cholesterol-to-triglyceride ratio
determined by polyethylene glycol precipitation was then used to
correct for loss of HDL triglyceride that occurred before and during
the isolation of HDL by ultracentrifugation.
HDL size.
The Stokes diameter of some HDL was measured by nondenaturing gradient
gel electrophoresis. A Hoefer SE600 gel casting assembly and
electrophoresis unit (Hoefer Scientific, San Francisco, CA) was used to
make the gel and to perform the electrophoresis. A concave, exponential
gradient of polyacrylamide ranging from 5.4 to 36% was used to form
the gel. To produce the gradient, stock acrylamide solutions identical
to those described by Asztalos et al. (2) were used. Glass plates, 16 × 18 cm and separated by a 3-mm spacer, were used to cast the
gel. The acrylamide was pumped into the space between the plates until
it had reached a height of 14 cm and was then allowed to polymerize. A
5% acrylamide solution was then layered over the polymerized gel, a 5- or 10-well comb (3 mm thick) was inserted, and the upper gel was
allowed to polymerize for at least 4 h. Just before the electrophoresis was started, the combs were removed and the samples (25-50 µg of
HDL protein dissolved in electrophoresis buffer) or standards of known
Stokes diameter (high-molecular-weight electrophoresis calibrators plus
ovalbumin; Pharmacia Biotech, Piscataway, NJ) were added to the wells.
The power supply was set at 220 V, and the electrophoresis was started
by applying a 25-mA current for 30 min (to allow the protein to
penetrate the top of the gel). The amperage was then increased until
the voltage became the limiting factor, and those conditions were
maintained for the duration of the run (40 h). The finished
gels were removed from the apparatus and stained with Coomassie blue
R-250 for 3 h. The mean Stokes diameter of a given HDL was determined
by comparing its migration distance with that of the
standards.
HDL structure.
The dimensions of HDL were also determined from the chemical
composition by use of the model of HDL structure proposed by Shen,
Scanu, and Kézdy (SSK model; Ref. 35) as follows.
With the assumption of ideal mixing of the components, the density of a
lipoprotein particle, d, is calculated
from the weight percent composition data by the equation
|
(1)
|
where
the symbols %aa,
%pl,
%ch,
%tg, and
%ce stand for the weight percents
of protein, phospholipid, free cholesterol, triglyceride, and
cholesteryl ester, respectively. The density of a spherical particle of
molecular weight (mol wt) and radius (in cm)
rcm is given by
|
(2)
|
where
N is Avogadro's Number. By solving this equation for
molecular weight and converting the radius from centimeters to ångströms
(rÅ),
we obtain
|
(3)
|
In
accordance with the SSK model, the outer surface of the particle is
defined solely by the number of amino acid residues, naa, and by the
number of phospholipid residues,
npl
|
(4)
|
The
number of residues of amino acids is calculated from the molecular
weight of the particle, the weight percentage of protein, and the
average residue molecular weight (mol
wtaa, estimated to be 112) by
|
(5)
|
The
number of other component molecules per particle is calculated in the
same manner, using the average molecular weights: 775 for
phospholipids, 387 for cholesterol, 850 for triglycerides, and 650 for
cholesteryl esters. Thus the number of phospholipid molecules per
particle, npl, is
|
(6)
|
By
substituting naa
and npl into
Eq. 4, solving it for molecular
weight, and equating it to the expression of the molecular weight from
Eq. 3, we obtain
|
(7)
|
This
equation then yields the radius of the particle as
|
(8)
|
With
this value of
rÅ, the
molecular weight is calculated from Eq. 3, and then the number of individual component
molecules is calculated just as
naa is calculated
from Eq. 5.
The SSK model posits that the volume of the spherical core of the
lipoprotein is the sum of the volumes of the constituent triglyceride
and cholesteryl ester molecules. The radius of the core is then
given by
|
(9)
|
where
ntg and
nce stand for the
number of triglycerides and cholesteryl esters, respectively. The
thickness of the surface monomolecular layer is
rÅ
rcore. Finally, if the surface of the core is covered exclusively by the
hydrophobic tails of the phospholipid and cholesterol molecules, then
the area occupied by one phospholipid tail is
|
(10)
|
where
nch is the number
of free cholesterols.
Statistics.
Differences in the means were tested using two-way ANOVA, with
the factors being CETP and gender. Individual group comparisons were
tested for significance using the Student-Newman-Keuls multiple comparison method (17). The null hypothesis was rejected if the
probability of obtaining a larger t
value simply due to chance was <1 in 20.
 |
RESULTS |
Mice have high levels of a triglyceride lipase in their circulation
(14, 15, 33). To confirm that that lipase was present in the plasma of
KKAy mice and to gain some insight
into which lipoproteins were substrates for that lipase, human HDL
radiolabeled with
[9,10-3H]triolein was
incubated with human plasma, mouse plasma, mouse plasma supplemented
with mouse very low density lipoprotein (VLDL), and mouse plasma
supplemented with mouse HDL. The results of that experiment are shown
in Table 1. Note that the production of [9,10-3H]oleic acid
was markedly higher when the enzyme source was
KKAy mouse plasma than when it was
human plasma. This shows that KKAy
mice also contain high levels of this lipase in their circulation. Note
also that addition of VLDL had no effect on the rate of production of
the radiolabeled oleic acid, whereas addition of mouse HDL reduced
recovery of the labeled free fatty acid almost 20%. Thus HDL
triglycerides appear to be substrates of the lipase in mouse plasma but
not VLDL triglycerides. Consequently, we hereafter refer to that lipase
as "HDL lipase."
Table 2 shows the mean body weight, blood
glucose concentration, plasma insulin concentration, HDL lipase
activity and cholesteryl ester transfer activity in C57BL/6 mice,
KKAy mice, and
KKAy-CETP mice. The
KKAy mice are obese,
hyperglycemic, and severely hyperinsulinemic relative to the C57BL/6
mice, and expression of CETP had no effect on those characteristics.
Thus the KKAy-CETP mice do, as
intended, express fully those genetic traits associated with NIDDM.
Expression of CETP was, however, associated with a substantial lowering
of the HDL lipase activity.
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Table 2.
Body weights, blood glucose and plasma insulin, HDL lipase, and CETP
activity of C57BL/6, KKAy, and KKAy-CETP
transgenic mice
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|
Table 3 shows a comparison of the plasma
lipid concentrations between KKAy
and KKAy-CETP mice. Both strains
of mouse are hypertriglyceridemic relative to the C57BL/6 mouse, in
which the plasma triglycerides average 46 ± 2 mg/dl in males and 36 ± 18 mg/dl in females, and, in agreement with our previous
observations (7), the KKAy mice
have extraordinarily high plasma HDL levels whether measured as HDL
cholesterol, HDL phospholipid, or HDL protein (Table
4). Thus hypertriglyceridemia per se does
not cause "low" HDL levels, at least not in the mouse. By
contrast, expression of CETP in the
KKAy mouse had a severe effect on
the HDL levels: HDL cholesterol concentrations decreased by more than
6-fold, HDL phospholipids decreased by 3.7-fold, HDL protein decreased
by 3.1-fold, and HDL triglyceride increased by 2.8-fold.
Table 4 shows a comparison of the HDL protein and apo A-I distribution
in the two strains of mouse. There was a marked effect of CETP
expression on both of those parameters. Total HDL protein levels were
reduced by ~70% and HDL-associated apo A-I levels by ~50% in
KKAy-CETP mice, relative to the
KKAy mice. This decrease in
HDL-associated apo A-I was accompanied by an increase in the levels of
non-HDL-associated apo A-I (apo A-I that did not migrate with HDL
during agarose electrophoresis) in the plasma from mice expressing
CETP. Comparison of agarose electrophoresis immunoblots with agarose
electrophoresis strips stained with oil red O (Fig.
1) showed that there was little or no lipid
staining in the area of the non-
-migrating apo A-I. As a result,
that apo A-I is referred to as "free" apo A-I in this report.
Quantification of the free apo A-I by agarose electrophoresis immunoblotting (8, 26, 27) indicated that ~5% of the apo A-I in the
circulation of either C57BL/6 or
KKAy plasma is free, whereas
~25% of the apo A-I in the circulation of
KKAy-CETP plasma is free. Note
that free apo A-I has been reported to inhibit lipase activity (10).
The fact that KKAy-CETP mice have
such high levels of free apo A-I may account for the reduced lipase
activity levels observed in these animals (Table 2).

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Fig. 1.
Agarose electrophoresis of plasma from a
KKAy-cholesteryl ester transfer
protein mouse pool. A: immunoblot
stained with anti-mouse apolipoprotein A-I (apo A-I).
B: same plasma stained with lipid
stain oil red O. Arrow, what is referred to in text as "free" apo
A-I. Note that there was little or no oil red O staining in region of
free apo A-I.
|
|
The apo A-I content of KKAy-CETP
HDL (calculated from the HDL-associated apo A-I concentration and the
HDL molecular weight; Table 5) was
significantly higher than that of
KKAy HDL (3.0 ± 0.4 mol of apo
A-I/mol of KKAy-CETP HDL vs. 2.1 ± 0.1 mol of apo A-I/mol of
KKAy HDL;
P = 0.0047). The higher apo A-I
content of KKAy-CETP HDL was
probably a result of the increased levels of free apo A-I in those
mice.
Table 5 compares selected physical characteristics of the HDL from
KKAy mice and from
KKAy-CETP mice. Those
characteristics were calculated from the HDL composition using the SSK
model. The SSK model envisages HDL as a spherical particle with a
hydrophobic core surrounded by a monolayer of phospholipids and free
cholesterol. Previous studies with C57BL/6 mice (25) showed that the
HDLs were monodisperse with regard to size and that the Stokes radius
calculated using the SSK model was in very good agreement with that
measured by nondenaturing gradient gel electrophoresis. To confirm that
the same was true of KKAy-CETP
mice, HDLs isolated from two pools of
KKAy-CETP plasma were analyzed by
both methods. Nondenaturing gradient gel electrophoresis showed that
the HDLs from the KKAy-CETP mice
were monodisperse, with mean Stokes radii of 52.2 and 49.2 Å.
The corresponding Stokes radii calculated using the SSK model were 52.2 and 50.7 Å. Thus the HDLs from
KKAy-CETP mice are monodisperse,
and the SSK model does, in fact, accurately predict its Stokes radius.
That being the case, the data in Table 5 indicate that CETP expression
did not have a significant effect on the density or size of the HDL but
did affect the surface thickness and the area occupied by the nonpolar
portion of the phospholipids (termed the phospholipid tail area). The data indicate that the hydrophobic fatty acyl chains of the
phospholipids are spread apart to a greater extent in
KKAy-CETP HDL than in
KKAy HDL, an effect that is
probably largely due to the absence of free cholesterol, i.e., the
fatty acyl chains that are present have to cover a greater area than
they would with free cholesterol present in that layer.
Table 6 shows the effect of CETP expression
on the composition of the HDL. There was no difference in the amount of
protein (expressed as amino acids) associated with the particles, but there was a statistically significant decrease in the number of molecules of phospholipid, free cholesterol (free cholesterol was not
detected in the HDL of KKAy-CETP
mice), and cholesteryl ester per molecule of HDL and a marked increase
in the number of molecules of triglyceride per molecule of HDL. Thus
CETP expression had a profound effect on the composition of the HDL
core. If one assumes a mean molecular volume of 1,090 Å3 for cholesteryl
esters and 1,600 Å3
for triglycerides, then triglycerides make up, on average,
only 4.3% by volume of the KKAy
HDL core, whereas they make up 48% by volume of the
KKAy-CETP HDL core. In view of the
higher triglyceride content and looser packing of the phospholipids at
the core surface, the KKAy-CETP
HDL should be substantially more vulnerable to the HDL lipase than
KKAy HDL.
Although the purpose of these studies was to evaluate the effects of
CETP expression in these spontaneously hypertriglyceridemic mice, some
gender and gender X gene interactions are noteworthy. The plasma HDL
levels of females tended to be higher than those of males, whether
measured as HDL cholesterol, triglyceride, or phospholipid (Table 3).
The differences in HDL-associated phospholipid and triglyceride levels
in the plasma were statistically significant, and the difference for
HDL-associated cholesterol was of borderline significance. This
observation (that the HDL levels are higher in female
KKAy mice) is in agreement with
reports for other mouse strains (31). There were also gender
differences in the composition of the HDL (Tables 4-6). Females
had higher levels of both HDL-associated and free apo A-I (Table 4),
their HDL tended to be larger and less dense than that of males (Table
5), and the phospholipid and cholesteryl ester contents of the HDL were
higher than those of males (Table 6). Finally, there were five
instances of significant gene × gender interactions: plasma
HDL-associated triglyceride (P = 0.0410), plasma apo A-I levels (P = 0.0066), pre-
apo A-I levels
(P = 0.0118), HDL-associated apo
A-I levels (P = 0.0210), and
HDL phospholipid content (P = 0.0569).
 |
DISCUSSION |
The purpose of the studies described here was to test the hypothesis
that hypertriglyceridemic KKAy
mice are able to maintain exceptionally high HDL levels because they
lack the ability to redistribute cholesteryl esters and triglycerides between VLDL and HDL. It was reasoned that the absence of a mechanism for the redistribution of those lipids between VLDL and HDL in the
wild-type KKAy mice essentially
segregated the HDL, thereby protecting those lipoproteins from any
effects that expansion of the VLDL triglyceride pool might have, and
that introduction of such a mechanism would reverse that segregation,
allow the HDLs to accumulate triglycerides, and thereby render them
susceptible to lipases. It was further reasoned that the larger the
VLDL triglyceride pool, the more rapidly those triglycerides would
equilibrate with the HDL, and thus the greater the effect on the HDL.
The results support that hypothesis: expression of CETP in the
KKAy mouse caused a 17-fold
increase in the triglyceride-to-cholesteryl ester ratio in the HDL core
and a severe reduction in the plasma HDL concentrations to levels
typical of hypertriglyceridemic patients with NIDDM (Table 3). In
addition, free apo A-I accumulated in the plasma of the
KKAy-CETP mice, suggesting that
the triglyceride-enriched HDLs were indeed vulnerable to the action of
lipases. Comparable expression of simian CETP in C57BL/6 mice with
normal triglyceride concentrations (46 ± 2 mg/dl) resulted in only
a twofold decrease in HDL cholesterol levels (from 82 ± 4 to 39 ± 3 mg/dl) and no accumulation of free apo A-I in the plasma (27).
Thus the hypertriglyceridemia appears to exacerbate the HDL-lowering
effect of CETP by accelerating the enrichment of the HDL with a lipid
that is susceptible to destruction by lipases at the expense of one
that is not.
The levels of hypertriglyceridemia in the
KKAy mice used in the present
study are somewhat lower than those reported previously (7) and are
significantly lower than those produced in
KKAy-CETP mice (Table 3). That was
entirely due to the low plasma triglyceride concentrations in the
KKAy females, as the plasma
triglyceride concentrations of the
KKAy males were not significantly
different from those of KKAy-CETP
males when tested by the Student-Newman-Keuls multiple comparison method. We have no explanation for the low triglyceride levels in
KKAy females other than that
day-to-day fluctuations of the plasma triglyceride concentrations in
these females can be quite large, and perhaps we unwittingly selected
animals in which the plasma triglyceride concentration was lower than
normal at the time that they were bled. As a result, the observation
that CETP expression enhanced the severity of the hypertriglyceridemia
(Table 3) may be fortuitous. To exclude the possibility that the
changes observed in the HDL levels in this study were due to the more
severe hypertriglyceridemia in
KKAy-CETP mice rather than to the
expression of CETP, we compared the levels and composition of HDL
isolated from pools of plasma with the same triglyceride concentrations
(650 ± 30 vs. 652 ± 211 mg/dl,
KKAy vs.
KKAy-CETP, respectively). The HDL
cholesterol levels in these KKAy
plasma pools averaged 139 ± 3 mg/dl; those in the
KKAy-CETP plasma averaged 18 ± 3 mg/dl. The triglyceride-to-cholesteryl ester molar ratio in the core
of these KKAy HDLs averaged 0.0459 ± 0.0262, that in the
KKAy-CETP HDLs averaged 0.728 ± 0.217, a 16-fold difference. These results fully support the
conclusion that CETP is the agent affecting HDL levels and that in the
absence of CETP, KKAy mice can
develop a severe hypertriglyceridemia with no detectable effect on the
HDL levels.
Expression of CETP in mice lowers their plasma HDL level (1, 25, 27),
and the observation that hypertriglyceridemia exacerbates that effect
has been reported previously (18), although the changes observed by
those investigators were much smaller than those reported here. In
addition, the inferences regarding the mechanism by which the
hypertriglyceridemia mediated that lowering are also different. The
data presented here suggest that it is the remodeling of the HDL by
CETP that accounts for its loss from the plasma. That remodeling
consists of an enrichment of the HDL core with triglyceride and the
loss of cholesteryl ester. The data further indicate that as the pool
of triglycerides available for transfer increases, that process is
stimulated and the effects on HDL are exacerbated. This interpretation
is at variance with that of Hayek et al. (18), who found minimal change in the HDL triglyceride content but a marked reduction in the size of
the HDL from human apo CIII-CETP transgenic mice and
argued that it was a CETP-induced increase in the cholesteryl ester and apo A-I fractional catabolic rates that were responsible for the loss
of HDL. How hypertriglyceridemia would accelerate the cholesteryl ester
and apo A-I fractional catabolic rates, other than through the
mechanism proposed above, is not clear. Rather, one could argue that the same process was operative in the human apo CIII-CETP mice used by Hayek et al. (18) as was operating in the
KKAy-CETP mice described here and
that the decrease in HDL size and the increased apo A-I and cholesteryl
ester fractional catabolic rates observed by those investigators were
secondary effects due to the enrichment with and subsequent hydrolysis
of HDL core triglycerides.
The accumulation of relatively large amounts of free apo A-I in the
plasma of the KKAy-CETP mice was
somewhat unexpected, but two aspects of the metabolic state of that
animal, HDL with an elevated triglyceride content and kidneys with
thickened glomerular capillary membranes, working in conjunction could
account for that occurrence. Note that triglycerides constitute ~50%
by volume of the KKAy-CETP HDL
core. We have shown previously (27), using mouse HDL in which
triglyceride constituted only one-third of the core volume, that
hydrolysis of those triglycerides in vitro resulted in collapse of the
HDL particle and release of free apo A-I and other surface constituents
into the plasma. That occurred because in vitro there is no source of
cholesterol to replace the triglyceride lost from the HDL. The same
process may occur in the KKAy-CETP
mouse, and, although there is a source of cholesterol to replace the
triglyceride, the rate of replacement is inadequate, i.e., the process
of cholesterol uptake and esterification is too slow to compensate for
triglyceride hydrolysis. Thus there is net loss of core component that
results, with time, in collapse of the particle and release of free apo
A-I. In addition, because the KKAy
mice have thickened glomerular capillary basement membranes (13), plasma clearance of that free apo A-I is retarded. The net effect of
these processes is a low HDL level, increased concentrations of free
apo A-I, and apo A-I-rich HDL. The latter phenomenon is presumably a
result of the high concentrations of free apo A-I, i.e., the high
levels of the free protein saturate the HDL with apo A-I, probably by
displacing other apoproteins, creating the apo A-I-rich particle.
It has been reported previously (10) that free apo A-I inhibits a
lipase in humans that may be analogous to what we refer to as the HDL lipase in these mice. The
KKAy-CETP mice clearly had
increased levels of free apo A-I in their plasma, and the activity of
the HDL lipase in that same plasma was severely reduced. That the lower
lipase activity was due to the higher levels of free apo A-I in these
mice cannot be determined from these experiments, but that is a
reasonable deduction. Note, however, that this process tends to
preserve rather than destroy the HDL, since hydrolysis of the
triglycerides is the key step in destruction of the particle. That may
explain why complete loss of HDL is prevented in these mice and why the
HDLs that remain are triglyceride rich.
One additional observation of interest was the virtual absence of free
cholesterol in the surface of the
KKAy-CETP HDL. Normally, ~25%
of the cholesterol molecules in the HDL of mice are nonesterified and
are located at the surface of the lipoprotein (27). Once esterified,
that cholesterol relocates to the core of the HDL, leaving space in the
surface for additional free cholesterol. The rate-limiting step in this
cholesterol uptake-esterification-relocation process appears to be
esterification, for significant amounts of free
cholesterol are normally present in the lipoprotein. The fact that the
free cholesterol levels were so low in the
KKAy-CETP HDL indicates that
either the esterification rate is increased and therefore no longer
rate limiting in these mice or uptake of free cholesterol by these apo
A-I-rich, triglyceride-rich HDLs is impaired. We cannot at this point
distinguish between these two possibilities, but regardless of which
process has been altered, it has had a significant effect on the
structure of the HDL. It remains to be determined what, if any, effect
these alterations have on HDL function.
One noteworthy difference between the HDL of the
KKAy-CETP mice and that of humans
with NIDDM is the size range of those lipoproteins. The HDLs in humans
with hypertriglyceridemic NIDDM are polydisperse with regard to size,
and those HDLs are generally smaller than normal. That was not the case
with KKAy-CETP HDL. Although the
plasma levels of HDL in the
KKAy-CETP mice were quite similar
to those of humans with hypertriglyceridemic NIDDM, those HDLs were
monodisperse with respect to size, and their Stokes diameters were not
different from those of KKAy
HDLs. What accounts for that difference in size range
between the two species is not known, although it has been suggested
that differences in the structure between mouse and human apo A-I are somehow responsible (9, 34). What is clear from the studies reported
here is that CETP does not contribute to the size polydispersity, and
thus this rodent model of human NIDDM is inexact in this regard.
The results of the studies reported here establish that the creation of
a pathway for movement of cholesteryl esters and triglycerides between
lipoproteins can, in the presence of a large pool of triglycerides, produce an NIDDM-like lipoprotein profile in the
KKAy mouse and imply that it is
the enrichment of the HDL core with triglyceride that accounts for the
HDL-lowering effect. The question then becomes whether this is the
mechanism responsible for the low HDL levels in hypertriglyceridemic
humans. Three observations suggest that it is. First, individuals with
NIDDM can have all of the components necessary for destruction of the
HDL by this pathway: active CETP, triglyceride-rich HDL (6), and an
active HDL lipase (16). Second, it has been shown that incubation of a
mixture of human VLDL, HDL, CETP, and hepatic lipase in the test tube
does, in fact, disrupt human HDL (11, 12). Finally, humans with
severely reduced CETP levels have very high HDL concentrations, and the
composition of that HDL is quite similar to the HDL of KKAy mice (20, 21, 37). Thus, by
expressing the CETP gene in these mice, we have created a condition
indistinguishable from that in hypertriglyceridemic humans: a pathway
for neutral lipid redistribution in the plasma that, in the presence of
a large pool of triglyceride-rich VLDLs, renders the HDLs vulnerable to premature destruction. If this conclusion is correct, then these data
explain why hypertriglyceridemia is often associated with low HDL
levels in humans but not in rodents and why drugs that lower the plasma
triglyceride concentrations tend to raise the HDL levels in humans.
Finally, these data strongly imply that inhibition of CETP activity in
hypertriglyceridemic humans would increase their plasma HDL levels
substantially, an effect that might well slow the progression of the
vascular disease so prevalent in individuals with this disorder.
 |
ACKNOWLEDGEMENTS |
We thank Ferenc J. Kézdy for critical discussions during both
the course of these experiments and the preparation of the manuscript.
 |
FOOTNOTES |
Address for reprint requests: G. W. Melchior, Cell and Molecular
Biology, 7252-209-4, Pharmacia and Upjohn, Inc., Kalamazoo, MI 49001.
Received 20 October 1997; accepted in final form 3 March 1998.
 |
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