From the
The plasma clearance of radiolabeled chylomicrons was compared
in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic
(WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but
was significantly retarded in cholesterol-fed and WHHL rabbits. At 40
min after the injection of chylomicrons, 14-17% of the injected
dose remained in the plasma of normal rabbits, whereas
Chylomicrons are the major lipoproteins responsible for the
transport of dietary lipids to various tissues. Lipoprotein lipase
hydrolyzes triglycerides present in the core of chylomicrons,
converting them to remnants. Chylomicron remnants are rapidly cleared
from the plasma, and this process is mediated primarily by
apolipoprotein E (apoE)
It has been postulated that several steps are involved
in chylomicron remnant clearance from the plasma and ultimate uptake of
these lipoproteins by hepatocytes
(1, 3, 5) .
The initial process responsible for the normal rapid clearance of
remnants from the plasma is thought to involve a sequestration of the
particles in the space of Disse. This process may involve the binding
of apoE on the remnants to heparan sulfate proteoglycans (HSPG) and the
interaction of remnants with hepatic lipase
(6, 7) .
Several lines of evidence have now established that remnant
lipoproteins bind initially to cell-surface HSPG and that, in the
absence of HSPG, remnant binding and uptake are decreased even in the
presence of a functional low density lipoprotein (LDL) receptor-related
protein (LRP)
(8, 9) . Presumably, remnant binding to
HSPG is necessary for the LRP to mediate uptake. Further lipolytic
processing in the space of Disse may occur before recognition of the
lipoprotein particles by the lipoprotein receptors. It has been shown
that lipoprotein lipase facilitates the binding of remnants to the LRP
(10) and that hepatic lipase can stimulate the binding of
remnants to HSPG and facilitate uptake of these particles presumably
through the LRP
(11) . Finally, uptake of the remnants via an
apoE-mediated process appears to involve both the LDL receptor and the
LRP
(1, 2, 3, 4, 5) .
The LDL
receptor has been shown in a variety of in vitro systems to
mediate the uptake of apoE-containing remnant lipoproteins. Choi et
al. (12) have inhibited uptake of chylomicrons using
antibodies that block lipoprotein uptake by LDL receptors. Recently,
several lines of evidence have demonstrated that the LRP also can be
involved in chylomicron remnant clearance
(13, 14, 15, 16, 17, 18, 19, 20) .
The LRP has been shown to bind remnants and remnant-like lipoproteins
containing excess added apoE. The space of Disse is an environment rich
in this apolipoprotein
(21) . Thus, remnants could acquire
excess apoE and then be recognized by the LRP. Physiological evidence
that the LRP is involved in chylomicron remnant clearance in vivo was suggested by the partial inhibition of the clearance of
radioactive chylomicrons from the plasma of mice that had been injected
with activated
The present studies of chylomicron metabolism were
conducted in rabbits, since this species has been used extensively in
lipoprotein studies. It was previously shown that the liver is
responsible for about two-thirds of the uptake of intravenously
injected chylomicrons and that the bone marrow also takes up
significant amounts
(22, 23) . Furthermore, chylomicron
catabolism by the liver increased in rabbits when the chylomicrons were
incubated with apoE
(22) . In addition, plasma cholesterol
levels decreased when apoE was infused into cholesterol-fed and
Watanabe heritable hyperlipidemic (WHHL) rabbits
(24) .
The
hyperlipidemia of cholesterol-fed rabbits is quite different from that
of WHHL rabbits. High cholesterol diets cause a marked accumulation of
There are conflicting reports concerning the effect of
cholesterol-induced hyperlipidemia on chylomicron catabolism in
different species. Chylomicron clearance has been shown to be retarded
in cholesterol-fed rabbits and hypothyroid rats
(32, 33, 37, 38, 39) but not in
cholesterol-fed rats
(32) and dogs
(40, 41) . It
has been reported that chylomicron clearance was normal in WHHL rabbits
(42, 43) . However, other studies suggest that
chylomicron clearance is retarded in these rabbits
(44, 45) .
The present study employs the novel
technique of cross-circulation of plasma lipoproteins either between
normal and WHHL rabbits or normal and cholesterol-fed rabbits to
examine the steps involved in the remnant pathway and to understand the
mechanism involved in the development of hyperlipidemia in these
rabbits. Cross-circulation eliminates the potential problem of
denaturing the fragile chylomicrons and their subsequent uptake by
liver Kupffer cells
(46) . We demonstrate that chylomicron
clearance is impaired in WHHL rabbits and in cholesterol-fed rabbits.
In addition, using this technique, we demonstrate that lipoproteins
present in the plasma of hyperlipidemic rabbits impair chylomicron
clearance. The impairment was due, in part, to competition with
endogenously derived lipoproteins containing apoE. We postulate that
these lipoproteins inhibit the initial sequestration step in remnant
clearance, i.e. the binding of chylomicron remnants to hepatic
HSPG.
For chylomicron clearance studies, the
[
The amount of isolated
lipoproteins to be injected was calculated to elevate the plasma
cholesterol levels of the normal rabbits from a base line of
30-80 mg/dl to
To determine the tissue uptake of the
monoclonal antibody
These studies
demonstrated that cholesterol-fed rabbits expressed lower amounts of
LDL receptor activity than did normal rabbits and that WHHL rabbits
expressed significantly lower amounts of LDL receptor activity compared
with normal and cholesterol-fed rabbits. Even though cholesterol-fed
and WHHL rabbits expressed different amounts of LDL receptor activity,
they catabolized the radiolabeled chylomicrons to a similar extent,
i.e.
In
contrast, LDL from cholesterol-fed rabbits ( d =
1.02-1.063 g/ml) and from humans ( d =
1.02-1.05 g/ml) did not significantly inhibit the uptake of
chylomicrons by the liver. Similarly, human and cholesterol-fed rabbit
HDL do not appear to compete with chylomicrons for plasma clearance
(), suggesting that the competition was specific for the
apoE-enriched HDL
The ease of induction of hypercholesterolemia in rabbits by
dietary fat and cholesterol suggests that the remnant lipoprotein
clearance pathway in this species may be very sensitive to elevated
lipoprotein levels. Previously, we demonstrated that the level of apoE
may be rate limiting under certain conditions in the rabbit
(24) , and clearly apoE is a critical determinant of normal
remnant clearance
(1, 2, 3) . In addition,
hepatic lipase has been shown to be involved in remnant lipoprotein
binding to cells in culture
(11) and in plasma clearance
(6, 7) , and it has been established that the rabbit is
deficient in this enzyme
(54, 55) . Recently, Fan et
al. (56, 57) overexpressed human hepatic lipase in
transgenic rabbits and demonstrated a reduced plasma cholesterol level
in these rabbits on a normal diet and a reduced tendency to develop
diet-induced hypercholesterolemia. Thus, impaired clearance of remnants
may reflect abnormalities at one of several points in the pathway,
including sequestration in the space of Disse, further lipolytic
processing, or receptor-mediated uptake. Other species, such as rats
(32) , dogs
(40, 41) , and humans
(58) ,
may have remnant pathways that are less sensitive to dietary
perturbations, and care must be exercised in trying to extend results
obtained in rabbits to other species, including humans.
The studies
reported in this paper demonstrate that chylomicron clearance is
retarded in both cholesterol-fed and WHHL rabbits. Experiments were
performed to define the mechanism(s) responsible for the impaired
clearance of remnant lipoproteins. Several possibilities to explain the
impaired clearance of chylomicrons were considered.
It was
demonstrated that the retarded clearance did not appear to be due
primarily to the exchange or transfer of the
[
The
level of apoE on the chylomicrons did not appear to be rate limiting.
The addition of excess apoE to the chylomicrons did not result in an
accelerated clearance of these lipoproteins from the plasma of the
hyperlipidemic animals. As will be discussed later, it appears that the
apoE-mediated clearance process is saturated in the hyperlipidemic
animals.
The retarded clearance in hyperlipidemic rabbits was not
correlated with the expression of hepatic receptors. No significant
difference was observed among the rabbits in the level of LRP activity.
However, the level of LDL receptor expression differed among the
rabbits. The cholesterol-fed rabbits had levels of LDL receptor
activity that were lower than those in normal rabbits, but they
expressed considerably more LDL receptor activity than was found in the
WHHL rabbits. It is reasonable to conclude that the reduced expression
of LDL receptors in hyperlipidemic rabbits could result in a reduced
capacity of the hepatocytes to take up the remnant lipoproteins.
However, since the cholesterol-fed and WHHL rabbits had similar
impaired chylomicron clearance but very different levels of LDL
receptor activity, the reduced expression of hepatic LDL receptors did
not appear to be the primary rate-limiting factor in the reduced
clearance of chylomicron remnants in these rabbits.
Consistent with
data from the present study is the conclusion that lipoproteins that
accumulate in the plasma of cholesterol-fed and WHHL rabbits retard
remnant clearance by interfering with an initial rapid phase of
clearance. Cross-circulation of normal and cholesterol-fed or normal
and WHHL rabbits allowed the rapid infusion of cholesterol-induced or
WHHL plasma lipoproteins into the circulation of normal rabbits and
allowed the determination of the acute effects of plasma lipoproteins
on liver uptake. This obviated criticism of the effects of isolation on
lipoprotein composition and function. Furthermore, these studies were
completed within 1 h, a short time interval during which major changes
in LDL receptor activity would not be expected to play a major role.
The rapid influx of the hyperlipidemic plasma during cross-circulation
from cholesterol-fed and WHHL rabbits retarded the liver uptake of
chylomicrons in normal rabbits. We interpret these results as
indicating that the competing lipoproteins saturate the early steps
involved in the clearance of chylomicrons ( i.e. sequestration
in the space of Disse). It is reasonable to speculate that the
apoE-containing remnants rapidly interact with the HSPG in the space of
Disse and saturate the initial step in the remnant clearance pathway.
We have established that HSPG-remnant interaction is essential for the
binding of remnant lipoproteins and facilitates the actual uptake of
the lipoproteins, presumably in association with the LRP
(8, 9) . This process may be facilitated by the
enrichment of the particles with apoE
(8, 9) or by the
interaction of the remnants with hepatic lipase
(6, 7, 11) in the space of Disse.
Canine HDL
In contrast to the
inhibition seen with remnant lipoproteins and HDL
In summary, chylomicron
catabolism was markedly retarded in cholesterol-fed and WHHL rabbits.
The primary factor for the retarded clearance appears to be competition
of the specific plasma lipoproteins that interfere with the clearance
step involved in remnant uptake, i.e. saturation of
sequestration in the space of Disse. Enrichment of the remnants with
apoE may not have enhanced remnant clearance because of impaired access
of these lipoproteins to the already saturated binding sites involved
in the sequestration phase of clearance.
Isolated lipoproteins were
infused intravenously into normal rabbits for a period of 5-8
min. Control animals received a saline infusion for 5-8 min. At
20 min after the start of infusion, chylomicrons were injected into
these rabbits, and blood samples were collected for an additional 40
min, at which time the animals were euthanized, and tissue samples were
obtained (see ``Materials and Methods'').
We gratefully acknowledge Dr. Robert C. Kowal of
Brigham and Women's Hospital in Boston for the monoclonal
antibody LRP-515; Dr. Karl H. Weisgraber for rabbit apoE; BioTechnology
General in Israel for human recombinant apoE; R. Dennis Miranda, Peter
A. Lindquist, and Marilyn Hathaway for technical support; Sylvia
Richmond for manuscript preparation; Charles Benedict and Tom Rolain
for graphics; and Dawn Levy and Lewis DeSimone for editorial
assistance. We appreciate the skilled surgical expertise of Peter A.
Lindquist, who performed the surgeries for the cross-circulation
studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
40-50% of the injected dose remained in the plasma of
cholesterol-fed and WHHL rabbits. The differences were reflected in the
reduced plasma clearance by the liver and bone marrow of the
cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed
normal levels of low density lipoprotein (LDL) receptor-related
protein/
-macroglobulin receptor in the liver. In
contrast, the hepatic levels of LDL receptors were lower in
hyperlipidemic rabbits; as expected, they were significantly lower in
WHHL rabbits compared with normal and cholesterol-fed rabbits.
Furthermore, it was demonstrated that lipoproteins accumulating in the
plasma of the hyperlipidemic rabbits competed for and retarded the
clearance of chylomicrons from the plasma. Competition was demonstrated
by cross-circulation of normal and cholesterol-fed or normal and WHHL
rabbits, in which the rapid influx of plasma containing the accumulated
plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to
impair the uptake of chylomicrons by the liver and bone marrow of
normal rabbits. These observations were extended by infusing isolated
lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml
(remnant) fraction and the canine cholesterol-rich high density
lipoproteins (HDL) with apolipoprotein E (HDL
) inhibited
chylomicron clearance, whereas human LDL and HDL from humans and
rabbits did not. We conclude that the low LDL receptor activity in the
cholesterol-fed and WHHL rabbits may contribute, at least in part, to
the impaired clearance by decreasing remnant uptake and causing the
accumulation of chylomicron and/or very low density lipoprotein
remnants. The accumulated remnant lipoproteins then compete for and
saturate the mechanism responsible for the initial rapid clearance of
chylomicrons from the plasma. We speculate that saturation of the
initial rapid clearance may occur at the sequestration step, which
involves the binding of remnants to heparan sulfate proteoglycans in
the space of Disse.
(
)
(for review, see Refs.
1-5).
-macroglobulin
(16, 19) . Although both the LDL receptor and the LRP
have been implicated in the uptake of chylomicron remnants, it has been
difficult to determine which, if either, plays the dominant role in
this process.
-migrating very low density lipoproteins (
-VLDL), which
consist of VLDL remnants and chylomicron remnants
(25, 26) , and a down-regulation of LDL receptors
(27, 28, 29, 30, 31) . The
hyperlipidemia induced by dietary cholesterol is due to overproduction
of lipoproteins and impaired plasma clearance secondary to the
down-regulation of LDL receptor expression
(27, 32, 33, 34) . In contrast, a
genetic deficiency of LDL receptors in WHHL rabbits causes the
accumulation of LDL and lesser amounts of VLDL and intermediate density
lipoprotein remnant particles (for review, see Refs. 35 and 36).
Animals and Diets
Male New Zealand
White rabbits (Animal West, Soquel, CA or Nitabell, Hayward, CA) were
maintained on a normal chow diet (Purina Mills, St. Louis, MO). The
plasma cholesterol levels in these rabbits ranged between 30 and 80
mg/dl. Rabbits kept on a 0.5% cholesterol diet (Zeigler Brothers,
Gardners, PA) for 2-3 weeks prior to use
(24, 47) showed hypercholesterolemia that ranged between 220 and 830
mg/dl. The homozygous WHHL rabbits used in the study were maintained on
a normal chow diet (Purina Mills) and had cholesterol levels between
200 and 450 mg/dl. The weights of normal, cholesterol-fed, and WHHL
rabbits ranged from 2 to 3.5 kg. All animals were fasted overnight.
Preparation of
Lipoproteins
[H]Retinol- and
[
C]cholesterol-labeled chylomicrons were
obtained from the thoracic duct of fat-fed dogs
(22, 23) , and all lipoproteins used for infusion were
isolated by ultracentrifugation as described
(48) . The
cholesterol-rich high density lipoproteins with apoE (HDL
)
from cholesterol-fed dogs were isolated by Pevikon-block
electrophoresis
(49) .
Iodination of 9D9 and LRP-515
Immunoglobulins
Monoclonal antibodies 9D9 and LRP-515
recognize the rabbit LDL receptor and the LRP, respectively, and have
been shown to be useful in determining the activity of cell-surface
receptors expressed in vivo (18, 50) . The 9D9
and LRP-515 immunoglobulins were purified from ascites fluid using
protein A-Sepharose (Pharmacia Biotech, Inc.) column chromatography.
Purified 9D9 immunoglobulin or LRP-515 (1 mg of protein) was
radiolabeled using Iodobeads (Pierce) with I (0.5 mCi) in
1 ml of phosphate-buffered saline, and the radiolabeling was terminated
by the addition of KI to a final concentration of 0.01
M. The
I-labeled proteins were separated from the free
I by Sephadex G10 (Pharmacia Biotech, Inc.) column
chromatography. The specific activity of the radiolabeled monoclonal
antibodies ranged from 20 to 731 cpm/ng of protein.
Plasma Clearance and Tissue Uptake of Radiolabeled
Chylomicrons and
These studies were
performed using one of two experimental protocols that serve as
controls for the cross-circulation and lipoprotein infusion studies
described below. The anesthetized rabbits were either infused with
saline over a period of 5-8 min or subjected to an
arterial-venous (A-V) shunt for 20 min. Chylomicrons and/or 9D9 were
injected 20 min after the start of either of these procedures. Blood
samples were obtained at designated intervals for an additional 40 min,
at which time the animals were euthanized and tissue samples obtained.
There was no significant difference between the results obtained by
either protocol; therefore, the results were combined (Figs. 2, 3, and
5).
I-Labeled 9D9 in Normal,
Cholesterol-fed, and WHHL Rabbits
H]retinol- and
[
C]cholesterol-labeled chylomicrons were
injected into the animals at a dose of 75 mg of triglyceride/kg of body
weight. Blood samples and liver biopsies were obtained at designated
intervals. The rabbits were euthanized at 40 min by intravenous
injection of euthanasia solution (Anthony Products Co., Arcadia, CA,
200 mg of sodium pentobarbital/kg of body weight) followed by bilateral
thoracotomy or perfusion fixation
(22, 23) . Tissues
(liver, bone marrow, spleen, kidney, adrenals, heart, lung, and
perinephric fat) were obtained. To determine receptor levels,
monoclonal antibodies were injected into the three groups of rabbits at
a dose of 25 µg of protein/kg of body weight, and experiments were
performed as described above for chylomicrons.
Plasma Clearance and Tissue Uptake in
Cross-circulated Rabbits
To perform cross-circulation
between two rabbits, the femoral artery and vein were exposed and
catheterized (16 gauge Medicut, Sherwood Medical, Tullamore, Ireland).
As shown in Fig. 1, the femoral artery from one rabbit was connected to
the femoral vein of a second rabbit using silastic tubing (⅛"
internal diameter, Dow Corning Corp., Midland, MI). Reciprocally, the
femoral artery of the second rabbit was connected to the femoral vein
of the first rabbit. The silastic tubing was pretreated with heparin
(5% tridodecylmethylammonium chloride heparin complex, Polysciences,
Inc., Warrington, PA). To determine plasma lipid levels, blood samples
(1.0 ml) were collected at 5-min intervals from stopcocks in the
arterial port. At 20 min after initiation of the cross-circulation,
each rabbit was simultaneously injected with radiolabeled lipoproteins
through the stopcocks into the femoral vein, and metabolic studies were
performed as described above. As a control for the cross-circulation
studies, normal, cholesterol-fed, and WHHL rabbits were subjected to
A-V shunts. The catheterized artery and vein of the rabbit were
connected by silastic tubing.
Plasma Clearance and Tissue Uptake of Lipoproteins in
Infusion Studies
Lipoproteins (1-2 ml/kg of body
weight) were infused into rabbits through the ear vein over a period of
5-8 min. At 20 min after the start of the infusion, radiolabeled
chylomicrons were injected into these animals. Blood samples were
obtained from the vernicular artery of the contralateral ear. The
experiments were terminated 40 min after the injection of chylomicrons
as described above. As a control for lipoprotein infusion, normal
rabbits were infused with saline.
200 mg/dl. This value was chosen because
levels in this range were typically obtained in the cross-circulated
rabbits. The plasma cholesterol levels were determined just before the
injection of the chylomicrons and 20 min after the beginning of the
lipoprotein infusion. It was possible in most cases to obtain these
levels using the d <1.21 and d <1.02 g/ml fractions and LDL. However, much lower levels were
obtained with HDL. Plasma Clearance of Chylomicrons from the d < 1.006
g/mlFraction-To determine the plasma clearance of
chylomicrons from the d <1.006 g/ml fraction,
plasma samples (0.2-0.5 ml) were overlaid with 0.5-0.8 ml
of saline and centrifuged (Beckman tabletop ultracentrifuge, TLA100.2
rotor, 100,000 rpm, 2.5 h, 4 °C), and the top
200 µl and
bottom fractions were removed and counted. The density distribution of
the radiolabeled chylomicrons mixed with plasma (0.2 ml) was determined
by centrifugation in parallel with samples obtained from the rabbits.
Effect of ApoE on Chylomicron Catabolism in
Cholesterol-fed Rabbits and in Cross-circulated Pairs of
Rabbits
To study the effect of apoE on chylomicrons,
chylomicrons (75 mg of triglyceride/kg) were incubated with apoE
(10-12 mg of protein/kg) for 1 h at 37 °C and injected into
the rabbits for metabolic studies. The apoE used in these studies was
either human recombinant apoE or rabbit apoE purified from
cholesterol-fed rabbits.
Analysis of Plasma and Tissues
To
determine the amount of radioactivity present in plasma, blood samples
(3.0 ml) were collected as described
(22, 23) . In those
cases where I-labeled 9D9 was injected with the
radiolabeled chylomicrons, 0.5 ml of plasma was mixed with 0.5 ml of
100% ethanol, vortexed, and counted in a
counter for 5-10
min. After counting, [
H]retinol and
[
C]cholesterol were extracted twice into hexane,
which was evaporated, and the sample was counted in the presence of 0.5
ml of 100% ethanol and 10 ml of Beckman scintillation mixture. In those
cases where only radiolabeled chylomicrons were injected, the samples
were subjected to direct liquid scintillation counting
(22, 23) .
I-9D9 and radiolabeled chylomicrons,
samples of different tissues were obtained in duplicate, weighed, and
counted for
I in a
counter. The tissues were then
digested with 0.5 ml of 6
N KOH, and lipids were extracted
into ethanol and hexane as described earlier
(22, 23) .
In those cases where chylomicrons were injected alone, the tissues were
digested and extracted as described
(22, 23) .
Determination of Plasma Volume and Tissue
Weights
To determine plasma volume
(51) , rabbit
albumin (fraction V, Sigma) was iodinated using Iodobeads as described
earlier for monoclonal antibodies. The I-labeled rabbit
albumin was injected into the ear vein of unanesthetized normal,
cholesterol-fed, and WHHL rabbits. Blood samples (2.0 ml each) were
collected at designated time points for 1 h. Plasma volume was deduced
by extrapolating the albumin die-away curve to 0 min. Determination of
plasma volume in normal and cholesterol-fed rabbits revealed no
differences in plasma volume (3.5 ± 0.4% ( n =
11) and 3.6 ± 0.3% ( n = 13) of body weight,
respectively). In contrast, WHHL rabbits had significantly lower ( p < 0.001) plasma volumes, i.e. 2.7 ± 0.1% of
body weight ( n = 7). To determine tissue weights,
tissues were rinsed with ice-cold saline, blot dried, and weighed.
Results were expressed as percent of body weight. The weight of the
liver of cholesterol-fed rabbits (3.3 ± 0.5%, n = 8) was significantly greater ( p < 0.05) than
the weight of the liver of normal (2.9 ± 0.3%, n = 18) and WHHL (2.5 ± 0.3%, n = 3)
rabbits. Other tissues had similar weights when expressed as a percent
of body weight. No significant differences were observed between the
weights of different tissues in normal and WHHL rabbits.
Other Analyses
Lipid (total cholesterol,
triglyceride, free cholesterol, phospholipid) analyses were performed
using a Spectrum lipid analyzer (Abbott Laboratories, North Chicago,
IL). Protein concentrations were determined by the method of Lowry
et al. (52) . All the data are presented as mean
± S.D. Data were evaluated by unpaired Student's t test using an IBM PC program called ``Primer'' (written
by Dr. Stanton Glantz, University of California, San Francisco)
(McGraw-Hill, Inc., New York, NY).
Chylomicron Metabolism in Normal, Cholesterol-fed,
and WHHL Rabbits
Previously, we demonstrated that when
[H]retinol- and
[
C]cholesterol-labeled chylomicrons were
injected intravenously into normal rabbits, the chylomicrons were
cleared from the plasma very rapidly, mainly by the liver
(22, 23) . In the present study, at 40 min,
14% of
[
H]retinol- and
17% of
[
C]cholesterol-labeled chylomicrons remained in
the plasma of normal rabbits, i.e.
85% of the
chylomicrons were removed from the plasma (Fig. 2, A and
C). In contrast, chylomicron clearance in cholesterol-fed and
WHHL rabbits was significantly slower. At 40 min, 40-50% of the
radiolabeled chylomicrons remained in the plasma of the WHHL and
cholesterol-fed rabbits. Analysis of chylomicron uptake by the liver,
during the course of in vivo studies, showed that the livers
of normal rabbits accumulated greater amounts of chylomicrons compared
with the livers of cholesterol-fed and WHHL rabbits (Figs. 2, B and D, and 3). For example, the liver of the normal
rabbit took up
44% of the injected dose of
[
C]cholesterol-labeled chylomicrons, whereas the
livers of cholesterol-fed and WHHL rabbits took up 21 and 22% of the
[
C]cholesterol, respectively
(Fig. 2 D).
Figure 2:
Chylomicron metabolism in normal,
cholesterol-fed, and WHHL rabbits. The metabolism studies were
performed as described under ``Materials and Methods.'' The
[H]retinol- and
[
C]cholesterol-labeled chylomicrons (75 mg of
triglyceride/kg) were injected into the ear or femoral vein. Blood
samples and liver biopsies were obtained at designated times after the
injection of chylomicrons. At 40 min after the injection of
chylomicrons, rabbits were euthanized, and plasma and tissue samples
were analyzed (see ``Materials and Methods''). Panels A and C show the plasma clearance of
[
H]retinol- and
[
C]cholesterol-labeled chylomicrons in normal,
cholesterol-fed, and WHHL rabbits. Plasma clearance of chylomicrons in
cholesterol-fed and WHHL rabbits was compared with plasma clearance in
normal rabbits and evaluated using the Student's t test.
The plasma clearance of chylomicrons was significantly slower ( p < 0.001) in the cholesterol-fed and WHHL rabbits at 30 and 40
min compared with normal rabbits. Panels B and D show
uptake by the liver of [
H]retinol- and
[
C]cholesterol-labeled chylomicrons in normal,
cholesterol-fed, and WHHL rabbits.
Uptake of the
[C]cholesterol-labeled chylomicrons by various
tissues at 40 min is summarized in Fig. 3. A significant decrease
in chylomicron uptake is seen in both the liver and bone marrow of
cholesterol-fed and WHHL rabbits. Bone marrow uptake was decreased from
24% of the injected dose in normal rabbits to 10-11% in
cholesterol-fed and WHHL rabbits. Other tissues studied, i.e. spleen, kidney, lung, adrenal, and heart, each contained less than
3% of the injected chylomicrons. These results indicate that
chylomicron clearance from the plasma of cholesterol-fed and WHHL
rabbits was significantly slower, as both the liver and bone marrow
uptake was decreased by
50%.
Figure 3:
Chylomicron uptake by tissues of normal,
cholesterol-fed, and WHHL rabbits. Chylomicron metabolism studies were
performed in normal, cholesterol-fed, and WHHL rabbits as described
under ``Materials and Methods'' and Fig. 2. Rabbits were
euthanized at 40 min, and tissues were collected. Small portions
(0.1-0.2 g) of each tissue were digested, extracted, and
counted as described under ``Materials and Methods.''
Differences in the uptake of
[
C]cholesterol-labeled chylomicrons were
evaluated by Student's t test. The uptake by
cholesterol-fed ( p < 0.001) and WHHL ( p < 0.05)
rabbit liver and by cholesterol-fed ( p < 0.001) and WHHL
( p < 0.05) rabbit bone marrow were significantly lower, as
compared with uptake in the normal rabbit.
The clearance of the
[C]cholesterol and
[
H]retinol from the plasma (Fig. 2) was
compared with the clearance of radiolabeled chylomicrons specifically
from the d <1.006 g/ml ultracentrifugal fraction
in normal, cholesterol-fed, and WHHL rabbits (Fig. 4). As contrasted
with the normal rabbits, clearance of the radiolabeled chylomicrons
from the d <1.006 g/ml fraction was retarded in
the cholesterol-fed and WHHL rabbits. A comparison of Figs. 2 and 4
suggests that at each time point some of the
[
C]cholesterol and
[
H]retinol occurred in the d > 1.006
g/ml fraction. Nevertheless, the clearance of radiolabeled chylomicrons
from whole plasma was followed in subsequent studies since it is
impossible to ascertain whether the label in the d > 1.006
g/ml fraction represents metabolic conversion or exchange or an
artifact of ultracentrifugation.
Effect of ApoE on Chylomicron Clearance in
Cholesterol-fed Rabbits
The possibility existed that the
amount of apoE associated with the chylomicrons could be rate limiting
for clearance in the cholesterol-fed rabbits. Chylomicrons were
preincubated with saline or apoE for 1 h at 37 °C as described
under ``Materials and Methods'' and injected into
cholesterol-fed rabbits. Previously, it was demonstrated that apoE
enrichment resulted in enhanced initial rates of clearance in normal
rabbits
(16, 22) . In three cholesterol-fed rabbits
injected with saline-incubated chylomicrons, 46-47% of the
injected dose remained in the plasma, whereas in two cholesterol-fed
rabbits injected with apoE-enriched chylomicrons, 40 and 60% of the
injected dose remained in the plasma at 40 min. The liver and bone
marrow of the three rabbits injected with chylomicrons alone took up
15-19% (17 ± 2) and 15-18% (17 ± 2) of the
injected dose, respectively. The percentages of injected apoE-enriched
chylomicrons recovered from the liver (17 and 20%) and bone marrow (15
and 18%) of two rabbits were similar to the values obtained in rabbits
injected with chylomicrons without added apoE. These studies
demonstrated that added apoE did not increase chylomicron clearance in
cholesterol-fed rabbits, and thus the amount of apoE on the
chylomicrons does not appear to be rate limiting.
LDL Receptor and LRP Expression in Normal,
Cholesterol-fed, and WHHL Rabbits
The levels of LDL
receptor and LRP expressed in these rabbits were investigated using
monoclonal antibodies against these receptors. The expression of LDL
receptors was studied using the monoclonal antibody 9D9, as described
by Huettinger et al. (50) . During in vivo studies, I-labeled 9D9 immunoglobulin was cleared
more rapidly from the plasma of normal rabbits compared with
cholesterol-fed and WHHL rabbits (Fig. 5 A). At 40 min, about
49% of injected
I-labeled 9D9 remained in the plasma of
normal rabbits, i.e.
51% had been cleared. In contrast,
about 62 and 74% of the injected dose of the radiolabeled 9D9 remained
in the plasma of cholesterol-fed and WHHL rabbits, respectively.
Analysis of 9D9 uptake by the liver during the in vivo studies
demonstrated that, compared with the livers of WHHL rabbits, the livers
of normal rabbits accumulated greater amounts of the antibodies
(Fig. 5 B). Cholesterol-fed rabbits also demonstrated
significantly lower amounts of
I-labeled 9D9 in the liver
than did the normal rabbits, but their livers still expressed higher
levels of functional LDL receptors than those of the WHHL rabbits. At
40 min,
37% of the injected dose was present in the livers of
normal rabbits, whereas the livers of cholesterol-fed rabbits contained
27% of the injected dose ( p < 0.02). In contrast, the
livers of WHHL rabbits contained
10% of the injected dose, and
this value was significantly ( p < 0.005) lower than the
values seen in the livers of normal and cholesterol-fed rabbits. Other
tissues (bone marrow, spleen, kidney, lung, adrenals, and heart) took
up less than 5% of the injected antibodies (data not shown).
Figure 5:I-Labeled 9D9 and LRP-515
monoclonal antibody metabolism in normal, cholesterol-fed, and WHHL
rabbits.
I-Labeled monoclonal antibodies (25 µg/kg)
were injected into animals with or without chylomicrons (as described
in Fig. 2 and under ``Materials and Methods''). Blood samples
and liver biopsies were obtained at designated time points. At 40 min
after the injection of chylomicrons, rabbits were euthanized, and
plasma and tissue samples were analyzed (see ``Materials and
Methods''). Panel A shows the plasma clearance of
I-labeled 9D9 in normal, cholesterol-fed, and WHHL
rabbits. Plasma clearance of 9D9 in these rabbits was evaluated using
the Student's t test. The plasma clearance of 9D9 was
significantly slower in the cholesterol-fed and WHHL rabbits at 2 min
( p < 0.05) and at 10-40 min ( p < 0.001)
as compared with normal rabbits. Moreover, the plasma clearance of 9D9
was significantly slower in WHHL rabbits at 30 min ( p <
0.05) and 40 min ( p < 0.005) as compared with
cholesterol-fed rabbits. Panel B shows the uptake of 9D9 by
the liver in normal, cholesterol-fed, and WHHL rabbits. The uptake of
9D9 by cholesterol-fed ( p < 0.01) and WHHL ( p <
0.001) rabbit liver was significantly lower than the uptake by the
normal rabbit liver. Moreover, the uptake of 9D9 by the liver in WHHL
rabbits was significantly lower ( p < 0.02) than the uptake
by the liver in cholesterol-fed rabbits. Panels C and D show the plasma clearance and liver uptake of
I-labeled LRP-515 in normal, cholesterol-fed, and WHHL
rabbits.
The
expression of the LRP was studied using monoclonal antibody LRP-515 as
described by Herz et al. (18) . The
I-LRP-515 was cleared rapidly from the plasma of normal,
cholesterol-fed, and WHHL rabbits (Fig. 5 C). No
differences in the rate of clearance of this antibody were observed.
The clearance from the plasma was due primarily to uptake by the liver
(Fig. 5 D). The uptake of LRP-515 by the livers of normal,
cholesterol-fed, and WHHL rabbits was similar. Other tissues took up
less than 5% of the injected antibodies. In all animals, the rate of
uptake increased for the first 20 min, and thereafter the amount of
radioactivity decreased in the liver. These studies indicate that the
amount of LRP expressed in these animals is similar.
50% of the injected dose. This finding suggests that
the reduced chylomicron catabolism in these rabbits could only be
explained in part by a decreased expression of LDL receptors.
Inhibition of Chylomicron Uptake by Accumulated
Plasma Lipoproteins
Consideration was given to the
possibility that the decreased chylomicron clearance in cholesterol-fed
and WHHL rabbits might be due to the accumulation of lipoproteins that
compete with chylomicrons for the uptake process. Two protocols were
developed to test for competing lipoproteins in these rabbits that
might impair or saturate the removal process. In the first protocol,
normal and cholesterol-fed or normal and WHHL rabbits were
cross-circulated as a means of rapidly introducing a large amount of
accumulated lipoproteins into the circulation of the normal rabbit and
to reduce the lipoprotein concentrations in the circulation of the
cholesterol-fed and WHHL rabbits. In the second protocol, lipoproteins
were isolated from cholesterol-fed and WHHL rabbits and infused into
normal rabbits to study the effect of these lipoproteins on chylomicron
catabolism.
Cross-circulation of the Blood of Normal and
Cholesterol-fed or Normal and WHHL Rabbits
A normal and a
cholesterol-fed rabbit or a normal and a WHHL rabbit were
cross-circulated by connecting the pair via an arterial-venous
anastomosis (Fig. 1). This A-V shunt allowed the rapid infusion
of the cholesterol-induced or WHHL plasma lipoproteins into the
circulation of the normal rabbit and created a new circulating level of
plasma lipoproteins in common between the two rabbits. As shown in Fig.
6, the plasma cholesterol and triglyceride levels equilibrated in the
cross-circulated rabbits in approximately 10 min and remained similar
in both rabbits. At 20 min after initiating cross-circulation,
radiolabeled chylomicrons were injected simultaneously into each
rabbit, and their plasma clearance and tissue uptake determined over a
period of 40 min. The blood in these rabbits was cross-circulated
throughout the experiment.
Figure 1:
Schematic diagram of
cross-circulation of blood between two rabbits. Two rabbits were
anesthetized; then, the femoral vein and artery of each were exposed,
and an arterial-venous shunt between these rabbits was established as
described under ``Materials and Methods.'' The blood in both
rabbits was allowed to circulate for 20 min. Blood samples were
obtained 5, 10, 15, and 20 min after the start of cross-circulation. At
20 min, radiolabeled chylomicrons with or without
I-labeled 9D9 were injected into the venous circulation
of both rabbits simultaneously. Blood samples and liver biopsies were
obtained at designated times to study the metabolism of chylomicrons
and 9D9.
Chylomicron Metabolism in Cross-circulated Normal and
Cholesterol-fed Rabbits
Data from a single pair of rabbits
are shown in Fig. 7, and all the data for all rabbit pairs are
tabulated in Table I. At 20 min after the start of cross-circulation,
chylomicrons were injected into the rabbits. At 40 min, 41% of
[C]cholesterol-labeled chylomicrons remained in
the plasma of the cross-circulated pair, i.e. only 59% of the
injected dose was cleared (Fig. 7 A). The clearance was
significantly slower than that observed in normal rabbits (compare with
Fig. 2C) but was similar to that observed in
cholesterol-fed rabbits (compare with Fig. 2 C). As shown in
, the uptake by the normal liver of all cross-circulated
pairs was less than that observed for the liver of the A-V shunt
control rabbits and was similar to that observed in cholesterol-fed
rabbits subjected to A-V shunt. This finding suggested that the
cholesterol-induced lipoproteins infused into the normal rabbit (by
cross-circulation) competed for the uptake of chylomicrons by the
normal liver and that the system was readily saturated by competing
diet-induced lipoproteins. Similarly, the cholesterol-induced
lipoproteins competed with the uptake of chylomicrons by the bone
marrow in the normal cross-circulated rabbit. Thus, the competing
lipoproteins reduced chylomicron catabolism by
34-66% in
normal rabbits (). In contrast, the decrease of the plasma
cholesterol and competing lipoproteins in the cholesterol-fed rabbit
after cross-circulation was not sufficient to allow for significantly
increased chylomicron catabolism by the liver of the cholesterol-fed
rabbit.
Chylomicron Metabolism in Cross-circulated Normal and
WHHL Rabbits
Data from a single pair of rabbits are shown
in Fig. 8, and all the data for all rabbit pairs are tabulated in
. The plasma clearance of
[C]cholesterol-labeled chylomicrons was slow in
the cross-circulating pair (Fig. 8 A); At 40 min,
58% of the chylomicrons remained in the plasma, i.e. only
42% of the chylomicrons were cleared. The slow clearance of
chylomicrons was due to less uptake of chylomicrons by the liver and
bone marrow of the normal cross-circulated rabbit, i.e. the
normal animal took up approximately 27% of the injected dose
() as compared with approximately 50% in the normal A-V
shunt control (). The uptake by the liver of the WHHL
rabbit (cross-circulated) was unchanged compared to that of the WHHL
control. As summarized in , the introduction of the WHHL
lipoproteins into the normal rabbit by cross-circulation decreased the
liver clearance of the radiolabeled chylomicrons. Uptake by the bone
marrow may or may not be affected in the cross-circulated rabbits.
Thus, these studies suggest that lipoproteins in the plasma of WHHL
rabbits compete with the uptake of chylomicrons by the liver of normal
rabbits. These lipoproteins reduce the uptake of chylomicrons by the
liver of normal rabbits 30-46% as compared with the rabbits
receiving saline infusion. As a result of cross-circulation, there was
a 50% reduction in the levels of circulating, competing lipoproteins.
This did not increase chylomicron uptake by the livers of WHHL rabbits.
Figure 8:
Chylomicron metabolism in cross-circulated
normal and WHHL rabbits. Normal and WHHL rabbits were cross-circulated
for 20 min as described in Fig. 1 and under ``Materials and
Methods.'' The plasma cholesterol values equilibrated at 230
mg/dl. At 20 min, chylomicrons (75 mg of triglyceride/kg of body
weight) were injected into both rabbits. Plasma samples were obtained
at the designated times. After euthanasia, both rabbits were
perfusion-fixed, and tissues were obtained for analysis. Panel
A, plasma clearance of radiolabeled chylomicrons; panel
B, tissue uptake of [C]cholesterol-labeled
chylomicrons.
Identification and Characterization of Lipoproteins
That Compete with Chylomicron Clearance
The
cross-circulation studies demonstrated that there were, in fact,
competing lipoproteins in the plasma of cholesterol-fed and WHHL
rabbits. To identify the fraction of lipoproteins that competed with
the liver uptake of chylomicrons and to study the extent of their
inhibition, lipoproteins isolated from the plasma of cholesterol-fed
and WHHL rabbits, cholesterol-fed dogs, and humans were used. Table II
presents data comparing the uptake of chylomicrons by the liver and
bone marrow following the infusion of the various classes of
lipoproteins. Infusions of d <l.21 g/ml
lipoproteins from cholesterol-fed and WHHL rabbits significantly
retarded (33-45% inhibition) the uptake of chylomicrons by
the liver (). For example, the liver of the normal rabbit
infused with saline took up
45% of the injected dose of
[
C]cholesterol-labeled chylomicrons as compared
with an uptake of 25 and 30% by the livers of normal rabbits infused
with d <1.21 g/ml lipoproteins from
cholesterol-fed or WHHL rabbits, respectively. This inhibition of
chylomicron uptake by the livers of normal rabbits could be attributed
to the lipoproteins of d <1.02 g/ml, which are
primarily VLDL and chylomicron remnants (). The d <1.006 g/ml lipoproteins from cholesterol-fed rabbits
and dogs and d <1.02 g/ml lipoproteins from
cholesterol-fed rabbits decreased the liver uptake of chylomicrons to
approximately 20-30% of the injected dose compared with 45% in
the control animals. In addition, the HDL
, which are
apoE-containing lipoproteins from cholesterol-fed dogs
(49) ,
competed with the uptake of chylomicrons by the liver ().
Previously, it was shown that canine HDL
was a competitive
inhibitor of chylomicron remnants for uptake by the perfused rat liver
(53) , and in addition to their ability to bind with high
affinity to the LDL receptor
(1, 49) , HDL
have now been demonstrated to bind to cell-surface HSPG in
cell-culture studies
(8, 9) . These data suggest that
lipoprotein fractions that are enriched in VLDL and chylomicron
remnants (and apoE-containing HDL
) compete for chylomicron
catabolism. However, the uptake of chylomicrons by the bone marrow was
not significantly inhibited by the infused lipoproteins. A protein
necessary for the recognition of lipoproteins by the bone marrow may
have been lost during the isolation of individual lipoproteins.
and d <1.02 g/ml
lipoproteins present in the plasma of the hyperlipidemic rabbits.
Therefore, the results obtained both for cross-circulated pairs or
lipoprotein-infused rabbits suggest that there are competing
lipoproteins in the plasma of hyperlipidemic rabbits and that these
lipoproteins rapidly saturate and inhibit remnant clearance from the
plasma.
C]cholesterol or
[
H]retinol from the chylomicrons to higher
density lipoproteins that are cleared more slowly. Although there was a
difference in the die away of the radiolabeled chylomicrons when the
whole plasma clearance was compared with that of the d <
1.006 g/ml fraction, the interpretations were similar after the initial
time points. It is appreciated that exchange or transfer of these
radiolabeled moieties occurs
(42, 43) ; however, whole
plasma clearance of chylomicrons is considered an appropriate measure
of the metabolic activity of these lipoproteins since ultracentrifugal
manipulation and time delays necessitated by lipoprotein fractionation
can introduce artifacts. Clearly, the whole plasma die away reflected
the trends seen with the d < 1.006 g/ml fraction.
and
remnant lipoproteins competed for chylomicron clearance. However, when
comparing the effects of different lipoproteins, it is difficult to
know how to calculate the amount to be infused. The ideal approach
would be to infuse similar numbers of particles, but this is impossible
when comparing very heterogeneous lipoprotein classes such as VLDL or
remnants. Therefore, we decided to inject sufficient d <1.21 g/ml, d <1.02 g/ml, and
LDL to elevate the plasma cholesterol from a normal level of
30-50 mg/dl to
200 mg/dl. The protocol was designed to mimic
the cross-circulation studies ( i.e. infusion of the isolated
lipoproteins, a 12-15-min period of equilibration, and completion
of the study in 1 h). As shown, the d <1.02 g/ml
lipoproteins (remnants and intermediate density lipoproteins) and
cholesterol-induced canine lipoproteins containing primarily apoE
(HDL
) inhibited chylomicron clearance ().
These in vivo data confirm previous results obtained in the
perfused rat liver, demonstrating that HDL
inhibit
chylomicron remnant clearance
(53) .
, LDL did
not appear to inhibit liver uptake. Likewise, HDL did not appear to
inhibit chylomicron uptake. However, since HDL are less
cholesterol-rich lipoproteins, it was difficult to obtain sufficient
HDL to raise the plasma cholesterol to 200 mg/dl. Nevertheless,
tremendous amounts of HDL were infused and did not appear to affect
chylomicron clearance. These results indicate that specific
lipoproteins, e.g. apoE-containing and remnant lipoproteins,
compete for lipoprotein clearance.
Table: Liver and bone marrow uptake of
[C]cholesterol-labeled chylomicrons in the
cross-circulated pairs of rabbits
Table: Effect of isolated lipoproteins on
[C]cholesterol-labeled chylomicron uptake by the
liver and bone marrow of normal rabbits
, cholesterol-induced
canine lipoproteins containing primarily apoE; HSPG, heparan sulfate
proteoglycans; LDL, low density lipoproteins; LRP, LDL receptor-related
protein; VLDL, very low density lipoproteins; WHHL, Watanabe heritable
hyperlipidemic.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.