(Received for publication, July 24, 1995; and in revised form, September 27, 1995)
From the
The metabolism of chylomicron remnants in mice deficient in low
density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) was
compared with that of control C57BL/6J mice. Mice were injected
intravenously with chylomicron-like emulsions labeled with radioactive
lipids. Blood samples were taken at fixed time intervals from the
retro-orbital sinus, and clearance rates of the lipoproteins were
assessed from the decline in plasma radioactivities. To follow the
intracellular pathway of remnants in the liver, emulsions labeled with
a fluorescent cholesteryl ester (BODIPY) were injected, and liver
sections were processed and assayed by laser confocal microscopy.
Catabolism of remnant cholesteryl esters was assessed by injecting
emulsions labeled with cholesteryl[1-C]oleate
and measuring the expired CO
from each animal.
In
apoE-deficient mice, remnant removal from plasma was totally impeded,
while the clearance of remnants in LDLr-deficient mice was similar to
that in C57BL/6J control mice. The confocal micrographs of livers 20
min after injection of fluorescent chylomicron-like emulsions showed
evenly distributed fluorescent particles in the hepatocytes from
control mice. In contrast, the fluorescent particles were mainly
located in sinusoidal spaces in LDLr-deficient mice. Three hours after
injection the livers from control mice showed few fluorescent
particles, indicating that remnants have been catabolized, while the
sections from LDLr-deficient mice were still highly fluorescent.
Micrographs from apoE-deficient mice showed no fluorescent particles in
the liver at any time after injection. Measurement of expired
radioactive CO after injection of emulsions labeled in the
fatty acid moiety of cholesteryl oleate indicated that remnant
metabolism was slower in the LDLr-deficient mice and essentially nil in
the apoE-deficient mice. Control mice had expired 50% of the injected
label by 3 h after injection.
We conclude that under normal circumstances, chylomicron remnants are rapidly internalized by LDLr and catabolized in hepatocytes, with a critical requirement for apoE. When LDLr is absent, remnants are taken up by a second apoE-dependent pathway, first to the sinusoidal space of the liver, with subsequent slow endocytosis and slow catabolism. Hepatic clearance via this second pathway is increased by heparin, inhibited by lactoferrin, heparinase, and suramin, and down-regulated by feeding a high fat diet.
Chylomicrons are responsible for transporting most dietary
lipids from the intestinal tract into the bloodstream. Along with the
bulk of lipids in the form of triacylglycerols (triglycerides) and
phospholipids, a small proportion of the total mass of chylomicron is
made up of cholesterol and cholesteryl esters. In the circulation,
chylomicrons are metabolized by a two-stage process. Initially, the
majority of the triglyceride is hydrolyzed by the action of lipoprotein
lipase and taken up by extrahepatic tissues. The resulting smaller
particles, known as chylomicron remnants, contain the residual
triglyceride and all of the cholesterol and cholesteryl ester. The
remnants have acquired apolipoprotein E (apoE) ()and are
removed rapidly from the plasma by the liver into hepatocytes and
delivered to lysosomes(1) . After hydrolysis of the cholesteryl
esters by cholesterol ester hydrolase, the liberated fatty acids enter
the pathways of oxidative metabolism and carbon dioxide is formed. The
exhaled CO
can be quantified (2) to provide a
measure for the internalization and catabolism of remnants.
The
central role played by apoE in the metabolism of remnant lipoproteins
has been well established by several lines of evidence. In type III
hyperlipidemia, the presence of defective apoE, which does not bind to
the liver receptors, leads to an accumulation of -very low density
lipoproteins (
-VLDL)(3) . For mice in which the apoE gene
was targeted and nullified by homologous recombination (apoE
knock-out), remnant lipoproteins accumulated in the plasma, and the
mice developed hypercholesterolemia and premature atherosclerosis even
on a chow (low fat) diet(4) . In contrast, transgenic mice
overexpressing rat apoE (5) or human apoE-3 (6) showed
enhanced remnant clearance and were protected from diet-induced
hypercholesterolemia. Transgenic mice overexpressing human apoE-4 were
less protected against hypercholesterolemia, and chylomicron clearance
was enhanced when mice were fed a low fat diet but not when fed a high
fat diet(7) . These findings have confirmed the critical role
of apoE in directing the clearance of remnants from plasma and show
that remnant clearance is modulated by the isoform of apoE as well as
by the amount of apoE available.
Although the necessity of apoE for hepatic uptake of chylomicron remnants is well established, the putative hepatic receptor that mediates this uptake remains controversial. The frequently cited remnant (apoE) receptor (8) has not been identified. However, it is now well accepted that under normal circumstances the bulk of chylomicron remnants are cleared by the apoB/E receptor, also known as the low density lipoprotein receptor (LDLr)(9, 10) . Apart from LDLr, both the low density lipoprotein receptor-related protein (LRP) (11) and a newly defined lipolysis-stimulated receptor (12) have been proposed to serve as receptors mediating the removal of chylomicron remnants from the circulation. Recently, an important contributory role for heparan sulfate proteoglycans (HSPG) in remnant uptake has been clarified. The LDLr knockout mice provide a unique model for testing the contributions by receptors such as LRP or lipolysis-stimulated receptor and HSPG for remnant clearance in vivo.
Using the H-retinol-fat tolerance test in
apoE-deficient mice, Chang et al.(13) recently
described an apoE-independent pathway for the hepatic clearance of
chylomicron remnants. By 4-6 h after a fat meal containing
H-retinol, only 17-20% of the label remained in
plasma, and up to 53% of the radioactivity was recovered in the liver.
These findings are inconsistent with previous reports of
hypercholesterolemia caused by the accumulation of triglyceride-rich
lipoproteins (d < 1.006 g/ml) and the high incidence of
atherosclerosis found in apoE-deficient mice (4, 14) and also fail to explain the high
concentration of apoB-48 in apoE deficiency shown by Ishibashi et
al.(9) .
In the present investigation, we measured
plasma clearance and liver uptake of injected chylomicron-like
emulsions in apoE-deficient mice and LDLr-deficient mice. Findings were
compared with control C57BL/6J mice. Chylomicron-like emulsions mimic
the metabolism of lymph chylomicrons (7, 15) and were
preferred for this study to avoid injecting exogenous apolipoproteins
that would complicate interpretation of results. To establish the
intracellular pathway of the remnants, emulsions were labeled with a
fluorescent cholesteryl ester (BODIPY). The emulsions were injected
into the knockout and control mice, and the liver sections were
processed for confocal microscopic images. We also traced the
catabolism of the remnants by quantifying the exhaled CO from both LDLr knockout and apoE knockout mice for comparison
with controls, following injection of emulsions labeled with
C on the fatty acid moiety of cholesteryl oleate.
Immunoblots were prepared by electrophoretically transferring proteins from other nonstained SDS-PAGE gels to nitrocellulose membranes. Individual membranes were incubated with a polyclonal antiserum to rat apoE and apoA-I (15) after blocking of the nonspecific binding sites with 10% albumin followed by 10% skim milk solution. The apoE and apoA-I bands were detected on these blots using HRP-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) system (Amersham).
Figure 1:
Plasma clearance and liver uptake of
injected chylomicron-like emulsions in control C57BL/6J,
LDLr-deficient, or apoE-deficient mice. A, emulsion lipids
were labeled with H-cholesteryl oleate to trace the
clearance of the injected particles up to 30 min after injection.
Results are means ± S.E. with n = 5 for
apoE-deficient mice, 9 for control, and 14 for LDLr-deficient mice. Closed circle, control mice; open square,
LDLr-deficient mice; open triangle, apoE-deficient mice. B, radioactivity recovered in the liver of control C57BL/6J,
LDLr-deficient or apoE-deficient mice. Mice were anesthetized with
avertin, and the animals were exsanguinated 30 min after the injection
of radiolabeled emulsions. Hepatic uptake of the radiolabels was
measured as described under ``Experimental Procedures.''
Results are means ± S.E. with n = 5 for
apoE-deficient mice, 24 for control, and 26 for LDLr-deficient
mice.
To confirm the results obtained with chylomicron-like emulsions, clearances of lymph chylomicrons were also measured. Plasma clearances of endogenously labeled rat lymph chylomicrons in control and LDLr-deficient mice were similar to those of chylomicron-like emulsions. Clearance of lymph chylomicrons in apoE-deficient mice was not measured, as the chylomicrons contained endogenous apoE from the donor rats.
Figure 2:
Laser-scanning confocal micrographs of
liver sections from mice. A, control C57BL/6J, LDLr-deficient,
and apoE-deficient mice were injected with 50 µl of
chylomicron-like emulsion labeled with cholesteryl-BODIPY and H-cholesteryl oleate. At various time intervals mice were
anesthetized, the liver was removed, and sections were processed for
confocal microscopy as described under ``Experimental
Procedures.'' B, LDLr-deficient mice were injected with
the fluorescent emulsion and stained with Texas Red. At 20 min after
emulsion injection, mice were anesthetized, and the liver was perfused
through the portal vein with Texas Red in saline (1 mg/ml) for 1 min.
The liver was removed and processed for confocal microscopy as
described under ``Experimental Procedures.'' C,
recovery of liver radioactivity in control C57BL/6J, LDLr-deficient, or
apoE-deficient mice after the injection of the fluorescent radioactive
labeled emulsions. Mice were anesthetized with avertin, and the animals
were exsanguinated at 5 min, 20 min, or 3 h after the injection of
emulsions. Hepatic uptake of the radiolabels was measured as described
under ``Experimental Procedures.''
It was of interest to compare the
hepatic uptake of the fluorescent label with the hepatic uptake of
radioactivity after injection of the emulsion labeled with H-cholesteryl oleate and the fluorescent cholesteryl ester.
The hepatic recovery of
H-cholesteryl oleate after
injection of the double labeled emulsion is shown in Fig. 2C. There is good agreement between the two labels
at all time points. The amounts of radioactivity in the livers of
control and LDLr-deficient mice were similar at 5 and 20 min after
injection. After 3 h, little of the
H-cholesteryl oleate
radioactivity, as with the fluorescent label, remained in the livers of
control mice. Approximately 40% of the injected radioactivity remained
in the livers of LDLr-deficient mice at 3 h. In contrast, the
radioactivity recovered in the liver of apoE-deficient mice was less
than 5% throughout the whole time course of the experiment. Therefore,
the findings with the fluorescent study were confirmed by the
radioactive label.
The catabolism of remnants by the liver was
quantified by measuring the output of CO
in
the expired breath of the control, LDLr-deficient and apoE-deficient
mice (19) . The rate of expiration of
CO
was much slower in the apoE-deficient mice than in the controls (p < 0.0001). The radioactivity in expired breath was also
significantly less in the LDLr-deficient mice compared with controls (p < 0.0001), indicating a defect or delay in the
catabolism of remnants removed from the plasma.
In previous studies lactoferrin has been shown to delay clearance (21) and to inhibit the endocytosis of chylomicron remnants (22) in the rat. Fig. 3A shows that injection of both lactoferrin and heparinase prior to emulsion injection inhibited the clearance of chylomicron remnants similarly in control and LDLr-deficient mice. Heparinase appeared to be a more effective inhibitor of remnant removal than lactoferrin, with about 80% of the injected radioactivity remaining in plasma at 30 min (Fig. 3A). The removal of emulsion triglyceride closely mirrored that of cholesteryl oleate. Triglyceride clearance was fast in both control and LDLr-deficient mice but severely retarded in the presence of lactoferrin or heparinase. Heparinase also appeared to be more potent than lactoferrin in inhibiting triglyceride clearance (Fig. 3B). Consistent with the clearance data, the hepatic recovery of labeled emulsion cholesteryl oleate was reduced from about 60 to 12% (p < 0.0005) in both control and LDLr-deficient mice, in the presence of lactoferrin or heparinase (Fig. 3C). The hepatic recovery of labeled triglyceride was similar with and without the inhibitors.
Figure 3:
Effects of lactoferrin and heparinase on
plasma clearance and liver uptake of chylomicron-like lipid emulsions
in control and LDLr-deficient mice. Emulsion lipids were labeled with H-cholesteryl oleate (CO) and
C-triolein (TO) to trace the remnant clearance
and triglyceride removal of the injected particles. An aliquot of
lactoferrin (2 mg/mouse) and heparinase (30 units/mouse) was injected 1
and 5 min, respectively, before the injection of the emulsion. Closed circle, control mice; open square,
LDLr-deficient mice; closed triangle, control mice with
lactoferrin; open triangle, LDLr-deficient mice with
lactoferrin; diamond, control with heparinase; cross,
LDLr-deficient mice with heparinase. A, plasma clearance of
emulsion
H-cholesteryl oleate; B, plasma clearance
of
C-triolein; C,
H-cholesteryl
oleate recovered in the liver of control C57BL/6J and LDLr-deficient
mice.
In contrast to lactoferrin and heparinase, heparin
increased the rates of lipolysis and remnant clearance of the injected
emulsion in both control and LDLr-deficient mice (Fig. 4A).
Heparin is known to release lipoprotein lipase from the vascular
endothelial surface, and hence an increase in lipolysis is expected. As
shown in Fig. 4A, 10 min after emulsion injection, the
amount of radioactive cholesteryl oleate remaining in the plasma was
about 10% in the presence of heparin, compared with 40% without heparin (p < 0.0005). Similarly, the amount of C-labeled triolein remaining in plasma decreased from
about 25 to about 5%, accompanied by a significant (p <
0.0005) increase in the liver uptake of
C-label (35% with
heparin compared with 25% without) at 30 min after emulsion injection (Fig. 4D). Heparin also increased the uptake of
cholesteryl oleate (Fig. 4C) in the liver of control
and LDLr mice.
Figure 4:
The
effect of heparin on plasma clearance and liver uptake of
chylomicron-like lipid emulsions in control and LDLr-deficient mice.
Emulsion lipids were labeled with H-cholesteryl oleate (CO) and
C-triolein (TO) to trace the
remnant clearance and triglyceride removal of the injected particles.
An aliquot of heparin (5 units/mouse) was injected 1 min before the
injection of emulsions. Closed circle, control mice; open
square, LDLr-deficient mice; diamond, control with
heparin; cross, LDLr-deficient mice with heparin. A,
plasma clearance of emulsion
H-cholesteryl oleate; B, plasma clearance of
C-triolein; C,
H-cholesteryl oleate recovered in the livers; D,
C-triolein recovered in the liver of control C57BL/6J and
LDLr-deficient mice.
The clearance of chylomicron remnants in the presence
of activated -macroglobulin was also compared in
control and LDLr-deficient mice. Consistent with Choi and
Cooper(23) , in control mice unlabeled activated
-macroglobulin slightly inhibited (difference not
significant) the plasma clearance of chylomicron remnants (results not
shown). However, the slight inhibition was not observed in
LDLr-deficient mice.
RAP, the 39-kDa protein that co-purifies with
-macroglobulin, was injected as a fusion protein
conjugated to GST. Because of the fast metabolism of RAP, the clearance
of emulsion was measured at 5, 10, and 20 min. 1 mg/mouse of GST-RAP
injected prior to emulsion injection did not affect the clearance of
chylomicrons in control mice but slightly delayed the remnant clearance
in LDLr-deficient mice at 10 min (p < 0.05) after emulsion
injection (Fig. 5A). The removal of emulsion triglyceride in
the presence of RAP was also delayed in the LDLr-deficient mice (Fig. 5B).
Figure 5:
The effect of RAP on plasma clearance of
chylomicron-like lipid emulsions in control and LDLr-deficient mice.
Emulsion lipids were labeled with H-cholesteryl oleate (CO) and
C-triolein (TO) to trace the
remnant clearance and triglyceride removal of the injected particles.
An aliquot of RAP-conjugated to GST (1 mg/mouse) was injected 1 min
before the injection of emulsions. Closed circle, control
mice; open square, LDLr-deficient mice; closed
triangle, control mice with RAP; open triangle,
LDLr-deficient mice with RAP. A, plasma clearance of emulsion
H-cholesteryl oleate; B, plasma clearance of
C-triolein.
Suramin, a polysulfated compound with a molecular weight of 1429, has been reported to be a potent inhibitor of remnant uptake in fibroblast cells(17) . In our experiments, a low dose of suramin (0.2 mg/mouse) did not affect remnant clearance in the control and LDLr-deficient mice. When the suramin dose was increased to 1 mg/mouse, clearance of emulsion triglyceride and remnants was almost completely abolished in the LDLr-deficient mice and to a lesser extent in control mice (Fig. 6, A and B). The inhibitory effect on emulsion clearance was more profound in the earlier time points, particularly at 5 and 10 min after the injection of emulsion, possibly due to the fast metabolism of suramin in vivo. The effects on emulsion remnant clearance of these competitors and inhibitors in the control and LDLr-deficient mice are summarized in Table 1.
Figure 6:
The effect of suramin on plasma clearance
of chylomicron-like lipid emulsions in control and LDLr-deficient mice.
Emulsion lipids were labeled with H-cholesteryl oleate (CO) and
C-triolein (TO) to trace the
remnant clearance and triglyceride removal of the injected particles.
An aliquot of suramin (1 mg/mouse) was injected 1 min before the
injection of emulsions. Closed circle, control mice; open
square, LDLr-deficient mice; closed triangle, control
mice with suramin; open triangle, LDLr-deficient mice with
suramin. A, plasma clearance of emulsion
H-cholesteryl oleate; B, plasma clearance of
C-triolein.
Figure 7:
The effect of a high fat diet on plasma
clearance and liver uptake of chylomicron-like lipid emulsions in
LDLr-deficient mice. Emulsion lipids were labeled with H-cholesteryl oleate (CO) and
C-triolein (TO) to trace the remnant clearance
and triglyceride removal of the injected particles. LDLr-deficient mice
were fed for 3 weeks with a high fat diet prior to emulsion injection. A, plasma clearance of emulsion
H-cholesteryl
oleate and
C-triolein. Closed square, cholesteryl
oleate clearance in LDLr-deficient mice on chow; open square,
LDLr-deficient mice on a high fat diet; closed triangle,
triolein clearance in LDLr-deficient mice on chow; open
triangle, triolein clearance in LDLr-deficient mice on a high fat
diet. B,
H-cholesteryl oleate recovered in the
liver of mice fed either chow or a high fat
diet.
Table 2summarizes the plasma lipid profile in the LDLr-deficient mice fed the high fat diet. The total plasma cholesterol in these mice increased 5-fold to 1550 mg/dl, while the HDL cholesterol decreased from 112.5 mg/dl to 37.6 mg/dl. Plasma triglyceride in the high fat-fed animals increased 2.5-fold.
The plasma apolipoprotein profile in control, LDLr-deficient, and high fat-fed LDLr-deficient mice was compared by a gradient SDS-PAGE (Fig. 8). LDLr-deficient mice consuming chow (lanes 5-8) contained slightly more apoB-100 and apoE than control mice (lanes 9-11). No accumulation of apoB-48 was observed in the mice. In contrast, the LDLr-deficient mice consuming a high fat diet (lanes 1-4) accumulated high concentrations of apoE, apoB-48, and apoB-100 and contained less apoA1 than mice consuming chow. Immunoblots of similar gels with an polyclonal antibody to rat apoE and A1 confirmed the observations (results not shown).
Figure 8: SDS-PAGE of apolipoproteins from plasma in control C57BL/6 mice fed chow, LDLr-deficient mice fed chow, and LDLr-deficient mice fed a high fat diet. Lanes 1-4, LDLr-deficient mice fed high fat; lanes 5-8, LDLr-deficient mice fed chow, lanes 9-11, control mice fed chow.
In these experiments, we compared the clearance of chylomicron-like lipid emulsions in control C57BL/6J and mice lacking apoE or the LDL receptor (LDLr). The endocytosis and metabolism of remnants derived from the injected emulsions in these mice were visualized by confocal microscopy and quantified using the carbon dioxide breath test(19) . Lipid emulsions were preferred in these experiments because they do not contain any exogenous proteins or apolipoproteins, which may nullify the defects of the knockout mice. The emulsions become associated with the endogenous apoE of the recipient animals after intravenous injection and were metabolized similarly to lymph chylomicrons(24) .
Our results confirm
the critical requirement for apoE in the hepatic clearance of
chylomicron remnants. In mice deficient in apoE, more than 60% of the
injected H-cholesteryl oleate, which traces emulsion
remnants, remained in plasma 30 min after injection, compared with 15%
in the control or LDLr-deficient mice (Fig. 1A).
Moreover, apoE-deficient mice had negligible hepatic recovery of the
labeled cholesteryl oleate from the clearance study (Fig. 1B) and expired little radioactive carbon dioxide
following injection of
C-cholesteryl oleate-labeled
emulsion. The confocal images of liver sections following injection of
fluorescent emulsions confirmed the delayed internalization of remnants
into the hepatocytes. Our findings are contradictory to Chang et
al. who recently reported the fast hepatic clearance of
chylomicron remnants in apoE-deficient mice 4-6 h after a vitamin
fat load labeled with
H-retinol (13) . In our
experiments, chylomicron remnants were not cleared or metabolized in
these mice for up to 3 h. The discrepancy is probably due to different
methods used in the studies. Retinyl palmitate had been shown to clear
differently from triglyceride lipoproteins(25) . Alternatively,
the longer period of experimental time may have resulted in the
lipoprotein particles being depleted of all core triglycerides and
entering the liver via a totally different pathway independent of apoE,
since we know that lipolysis is fast, and lipase activity is intact in
these mice. Moreover, the efficient remnant clearance from plasma in
the apoE-deficient mice shown by Chang et al. is inconsistent
with earlier reports of hypercholesterolemia,
atherogenesis(4, 14) , and increased plasma
concentration of apoB-48 (9) .
The rates of chylomicron
remnant clearance from plasma were similar in the control and
LDLr-deficient mice, with similar liver uptakes. The expired
radioactive carbon dioxide, however, was significantly lower in the
LDLr-deficient mice than in the control animals, suggesting that in the
absence of LDLr, catabolism of the chylomicron remnants is probably
delayed or defective. The slow catabolism is probably due to the slow
internalization of remnants via the LDLr-independent pathway. Results
from the confocal microscopy agree with measurements of the expired CO
. In LDLr-deficient mice, remnants
accumulated in sinusoidal spaces of the liver prior to internalization
into the hepatocytes; these remnants also remained within the
hepatocytes for a longer period (up to 3 or 4 h) in the LDLr-deficient
mice. These findings are consistent with Herz et al.(26) who compared the endosomal uptake of radioactive
labels to show a delay in the endocytosis of chylomicron remnants in
LDLr-deficient mice.
The finding that lactoferrin inhibits remnant
clearance in LDLr-deficient mice (Fig. 3A) is
consistent with the inhibition of apoE-enriched -VLDL uptake by
lactoferrin in human fibroblasts lacking LDLr(27) . Ji and
Mahley (28) showed that lactoferrin bound to HSPG and to LRP in
Chinese hamster ovary cells. Recently, Meilinger et al. found
a stable lactoferrin-LRP complex in isolated endosome
fractions(29) . In the LDLr-deficient mice, it is not known if
the inhibition of remnant clearance is due to the binding of
lactoferrin to cell surface HSPG or to LRP.
Similarly, injection of
heparinase blocked hepatic clearance of remnants from plasma in both
the control and the LDLr-deficient mice (Fig. 3A). Ji et al. recently showed that heparinase decreased the amount of
liver HSPG and inhibited the hepatic clearance of chylomicrons,
apoE-enriched chylomicron remnants, and apoE-enriched -VLDL (30) but not the clearance of LDL and
2-macroglobulin.
This reinforces the importance of HSPG and apoE in hepatic chylomicron
remnant clearance via the receptor pathway. Thus, results from the
lactoferrin and heparinase studies suggest the involvement of HSPG,
apoE, and receptors in the hepatic clearance of chylomicron remnants in
both control and LDLr-deficient mice.
The mechanism for the slower removal of emulsion triglyceride in the presence of lactoferrin and heparinase is unclear (Fig. 3B). Triglyceride removal reflects the sum of two processes, i.e. lipolysis and remnant uptake by the liver. The slower clearance of triglyceride is partly attributable to the delay in the remnant clearance but is more likely to be due to slower lipolysis, as the rates of triglyceride removal in apoE-deficient mice, mice fed high fat diet(7) , and rats fed a high cholesterol diet (21) were all fast and normal. It is possible that the inhibitors also interact with the lipoprotein lipase, reducing the availability of lipases for the hydrolysis of triglycerides in the monolayer.
In contrast, injection of heparin increased the removal of both emulsion triglyceride and remnants (Fig. 4, A and B). The increase in remnant clearance in the presence of heparin is probably due to the faster remnant production; however, the increase in the rate of lipolysis is not enough to account for the increased remnant removal. Heparin has been known to increase the uptake of chylomicron remnants by isolated rat hepatocytes (31) and to increase remnant clearance in streptozotocin diabetic rats (32) and also in rats consuming a high cholesterol diet(21) , possibly by stabilizing hepatic lipase and increasing its secretion. Hepatic lipase has been reported to be involved in the binding of chylomicron remnants to receptors and the endocytosis of the remnants(33, 34) . The alternative explanation is that in the presence of heparin, binding sites in HSPG molecules originally occupied by the lipases are now available for the binding of remnants. The availability of HSPG has been reported to be directly related to the clearance of lipoprotein remnants. For example, in diabetes mellitus it has been reported that a decrease in concentration of HSPG led to poor chylomicron clearance and hypercholesterolemia(35) .
Consistent with the studies of
Mokuno et al.(36) in the rat, we did not find any
significant effect of -macroglobulin in plasma
clearance or the hepatic uptake of chylomicron remnants. In contrast,
while RAP did not affect the remnant clearance from plasma in control
mice, it delayed (p < 0.05) the remnant and triglyceride
clearance in LDLr-deficient mice, suggesting the involvement of LRP.
RAP has been known to block the binding of ligands to LRP but only
interacts weakly with LDLr(36) . In the presence of LDLr, RAP
did not inhibit remnant uptake, as shown by Willnow et
al.(37). Herz et al.(38) reported that in FH
fibroblasts lacking LDLr, RAP inhibited up to 80% of cholesterol
esterification stimulated by the apoE-enriched
-VLDL, but RAP had
no effect on cholesteryl ester formation in normal fibroblasts. The
effect of RAP on remnant clearance is relatively less marked in
vivo, probably because of its short half-life when compared with
remnants. When the concentration of RAP was sustained by gene transfer
using an adenoviral vector to the liver of LDLr-deficient mice, hepatic
remnant uptake was effectively inhibited(39) . Mokuno et
al.(36) also demonstrated that GST-RAP produced a weak
inhibitory effect on the hepatic uptake of apoE-enriched chylomicrons.
Suramin is a highly sulfated compound, structurally unrelated to heparin or dextran sulfate. The inhibition of lipoprotein uptake has been ascribed to its polyanionic nature. Due to its toxicity, suramin was initially tested at a low dose (0.2 mg in each animal) without producing effects on clearance or hepatic uptake. When the dose was increased to 1 mg/animal, suramin effectively inhibited the clearance of both triglyceride and remnants (Fig. 6, A and B) in LDLr mice and, to a lesser extent, in control mice. The mechanism by which suramin inhibits remnant clearance is still unclear but could probably be attributable to its binding to the lipases, the ligands, or the receptors, although the more profound inhibitory effect produced in LDLr-deficient mice cannot be determined without further studies.
A high fat, high cholesterol, atherogenic diet is known to retard chylomicron remnant clearance in normal and apoE-4 transgenic mice, while the removal of emulsion triglyceride was increased due to an increase in lipase concentration(7) . In LDLr-deficient mice consuming a high fat diet, plasma clearance and liver uptake of remnants are greatly retarded (Fig. 7). Lipolysis of emulsion triglyceride increased, but not to the extent observed for the normal and transgenic mice expressing apoE-4(7) . The high fat diet elevated the plasma levels of apoB-48, apoB-100, and apoE, consistent with Ishibashi et al.(9) . In addition, a lower HDL-cholesterol and apoA-I concentration was also observed in the LDLr mice after high fat feeding.
In conclusion, LDLr and apoE are both required for the normal fast internalization and metabolism of chylomicron remnants by the liver. An alternative apoE-dependent pathway operates to internalize chylomicron remnants in the absence of LDLr. The catabolism of remnants via this pathway is significantly slower when compared with the LDLr endocytotic pathway, probably due to the slow internalization of remnants. From the competition studies with lactoferrin, heparinase, heparin, and RAP, it appears that both HSPG and LRP are required in this pathway. Whether or not LRP is involved synergistic by or independent to the HSPG needs further investigation.