Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research
Volume 46, 2005
Triglyceride-rich lipoprotein metabolism in unique VLDL
receptor, LDL receptor, and LRP triple-deficient mice
Sonia M. S. Espirito Santo,*
Netherlands Organization for Applied Scientific Research-Quality of Life,* Gaubius Laboratory, Leiden, The
Netherlands; Departments of General Internal Medicine
University Medical Center, Leiden, The Netherlands; Division of Nutritional Sciences,
Ithaca, NY; Department of Plasma Proteins,** Sanquin Research at Central Laboratory of the Netherlands
Red Cross Blood Transfusion Service, Amsterdam, The Netherlands; and Departments of Cardiology
University Medical Center, Leiden, The Netherlands
Patrick C. N. Rensen,
** Peter J. Voshol,*
Jeltje R. Goudriaan,* André Bensadoun,
Louis M. Havekes,* and Bart J. M. van Vlijmen
and Endocrinology and Metabolism,
low density lipoprotein receptor (LDLR), and low density lipo-
protein receptor-related protein (LRP) are the three main
apolipoprotein E-recognizing endocytic receptors involved
in the clearance of triglyceride (TG)-rich lipoproteins from
plasma. Whereas LDLR deficiency in mice results in the ac-
cumulation of plasma LDL-sized lipoproteins, VLDLR or LRP
deficiency alone only minimally affects plasma lipoproteins.
To investigate the combined effect of the absence of these
receptors on TG-rich lipoprotein levels, we have generated
unique VLDLR, LDLR, and LRP triple-deficient mice. Com-
pared with wild-type mice, these mice markedly accumulated
plasma lipids and lipases. These mice did not show aggra-
vated hyperlipidemia compared with LDLR and LRP dou-
ble-deficient mice, but plasma TG was increased after high-
fat diet feeding. In addition, these mice showed a severely
decreased postprandial TG clearance typical of VLDLR-
deficiency in LRP
hyperlipidemia, these triple-deficient mice represent a unique
model of markedly delayed TG clearance on a hyperlipidemic
—Espirito Santo, S. M. S., P. C. N. Rensen, J. R.
Goudriaan, A. Bensadoun, N. Bovenschen, P. J. Voshol, L. M.
Havekes, and B. J. M. van Vlijmen.
tein metabolism in unique VLDL receptor, LDL receptor,
and LRP triple-deficient mice.
The very low density lipoprotein receptor (VLDLR),
Collectively, although VLDLR
mice does not aggravate
J. Lipid Res.
Supplementary key words
protein receptor-related protein
very low density lipo-
low density lipo-
very low density lipoproteins
low density lipoprotein receptor
The very low density lipoprotein receptor (VLDLR) is
expressed in tissues active in fatty acid metabolism (i.e., heart,
muscle, adipose) and macrophages (1, 2). In vitro studies
show that the VLDLR binds apolipoprotein E (apoE) but
not apoB-100 and that LPL modulates the binding of tri-
glyceride (TG)-rich lipoprotein particles to the VLDLR
and vice versa (3, 4). Frykman et al. (5) generated VLDLR-
) mice and showed that these mice
present normal plasma lipoprotein levels when fed a chow
diet. Interestingly, only when the TG metabolism was stressed
either by a high-fat diet (HFD) or by cross-breeding on a
background of obesity (
receptor (LDLR) deficiency (6, 7) did VLDLR deficiency
result in moderate accumulation of plasma TG-rich lipo-
proteins. In agreement with these observations, we recently
demonstrated that the postprandial TG response after an
oral fat load is strongly increased in VLDLR
Hence, the VLDLR plays a role in TG-rich lipoprotein me-
tabolism that becomes apparent only after severely stress-
ing TG metabolism.
The LDLR and the low density lipoprotein receptor-
related protein (LRP) are two other major members of the
LDLR family that act in concert in the hepatic clearance
of plasma lipoproteins (9). Absence of the LDLR in mice
results in the accumulation of LDL-sized lipoproteins in
plasma, whereas LRP deficiency does not affect plasma
lipoprotein levels (10, 11). Strikingly, LRP deficiency on
background in mice results in aggravated hy-
perlipidemia attributable to the accumulation of TG-rich
lipoprotein remnants (9). This indicates that the role of
) or low density lipoprotein
Abbreviations: apoE, apolipoprotein E; AUC, area under the curve;
HFD, high-fat diet; LDLR, low density lipoprotein receptor; LRP, low
density lipoprotein receptor-related protein; RAP, receptor-associated
protein; TG, triglyceride; VLDLR, very low density lipoprotein receptor.
To whom correspondence should be addressed.
Present address of B. J. M. van Vlijmen: Department of Hematol-
ogy, C2-R, Hemostasis and Thrombosis Research Center, Leiden Uni-
versity Medical Center, Leiden, The Netherlands.
Manuscript received 8 July 2004 and in revised form 17 February 2005.
Published, JLR Papers in Press, March 16, 2005.
1098 Journal of Lipid Research
Volume 46, 2005
LRP in lipoprotein metabolism in vivo is masked by the
LDLR, the presence of which can apparently fully compen-
sate for the absence of the LRP (9). Whether a similar in-
teraction exists between the VLDLR and LDLR and/or
LRP is not yet known.
Because we hypothesized that the addition of the ab-
sence of the VLDLR to the absence of the LDLR and he-
patic LRP would severely aggravate the hyperlipidemic
phenotype of LDLR
VLDLR, LDLR, and conditional LRP triple-deficient mice.
It appeared that VLDLR deficiency in LDLR
mice does not aggravate hyperlipidemia on a chow diet.
However, we do show that the additional absence of the
VLDLR does lead to an aggravated phenotype on stressing
TG metabolism, either by high-fat feeding or by giving an
intragastric olive oil bolus, although these results may be
attributed to the cumulative effects of the phenotypes of
the individual mice (i.e., LRP
In summary, LRP
represent a unique model of markedly delayed TG clear-
ance on a hyperlipidemic background.
mice, we generated unique
, and VLDLR
MATERIALS AND METHODS
Transgenic animals and diet
We cross-bred VLDLR
lacking the LRP on an LDLR
LDLR ; referred to hereafter as LRP
ing a unique triple-knockout mouse model that lacks (condition-
ally) LRP, LDLR, and VLDLR (MX1Cre:LRP
VLDLR; referred to hereafter as LRP
Mice were genotyped by PCR analysis on tail tip DNA for the
presence of the “floxed” LRP allele, the MX1Cre transgene, and
the disrupted LDLR and VLDLR allele as described previously
(7, 10). For experiments, 8–10 week old male VLDLR
LRP deficiency, all mice (including VLDLR
received three intraperitoneal injections of 250
ml solution of polyinosinic:polycytidylic ribonucleic acid (pI:pC;
Sigma, St. Louis, MO) at 2 day intervals as described previously
(10). PCR analysis for the presence of the disrupted LRP allele
and immunoblot analysis with antibodies directed against the 85
kDa subunit of LRP from liver tissue were performed as de-
scribed previously (9, 12). Mice were kept for 12 weeks on a stan-
dard rat/mouse chow diet (SRM-A; Hope Farms, Woerden, The
Netherlands), followed by 10 weeks of HFD containing 24% corn
oil, 24% casein, 18% corn starch, and 6% cellulose (Hope Farms).
Diet and water were given ad libitum to the animals. The institu-
tional committee on animal welfare of Netherlands Organization
for Applied Scientific Research-Quality of Life approved all ani-
mice (6) with mice conditionally
) (10), yield-
10) mice were used. To induce
l of a 1 mg/
Blood was collected by tail bleeding into chilled paraoxon-
coated capillaries (13) 4 weeks after induction of LRP deficiency
either after 4 h of fasting from 7:00 to 11:00 AM or after over-
night fasting from 5:00 PM to 7:00 AM. Plasma was isolated and
assayed for total cholesterol, TG, and glucose levels using enzy-
matic kits C0534, 337-B, and 315-500 (Sigma Diagnostics, Deisen-
hofen, Germany) and for FFA using the enzymatic kit Nefa-C
(Wako Chemicals GmbH, Neuss, Germany). Plasma lipid distri-
bution over lipoproteins was determined by size fractionation us-
ing fast-performance liquid chromatography (12). Plasma con-
centrations of mouse apoB-48 and apoB-100, apoE, and apoA-I
were determined by immunoblotting using mouse apolipopro-
tein-specific polyclonal rabbit antiserum as described previously
(12). Plasma levels of HL activity, LPL activity, and LPL mass were
determined after 4 h of fasting as described previously (9, 14).
Postprandial TG response
Mice were fasted for 4 h. After a basal blood sample was taken
by tail bleeding, the animals received an intragastric load of 200
l of olive oil. Subsequently, blood was drawn at the indicated
times after olive oil administration. Plasma was isolated and TG
levels were determined as described above and are presented as
relative increases from time 0.
All data are presented as means
ing the Mann-Whitney
0.05 was regarded as statisti-
SD. Data were analyzed us-
Treatment of LDLR
VLDLR mice with pI:pC resulted in the presence of the
disrupted LRP allele in tail tip DNA and in the complete
absence of LRP protein in liver membrane extracts (
Upon LRP inactivation, the LRP
mice appeared healthy and displayed no signs of abnormal-
ities, but throughout their life span they had a slightly lower
body weight compared with LRP
1.8 g vs. 22.4
duced body weight was also observed in VLDLR deficiency
only compared with wild-type controls (18.7
2.7 g; P
0.001), which is in agreement with previ-
ous reports (5, 6).
0.02). However, re-
0.9 g vs.
Plasma lipids on a chow diet
On a regular chow diet, single VLDLR deficiency re-
sulted in increased plasma TG levels after an overnight
deficiency. Mice were treated with polyinosinic:polycytidylic ribonu-
cleic acid, and LRP deficiency was assessed by the presence of the
disrupted LRP allele by PCR analysis of tail tip DNA (upper panel)
and by the absence of the 85 kDa subunit of the LRP protein in the
liver (lower panel). LDLR, low density lipoprotein receptor; VLDLR,
very low density lipoprotein receptor.
Low density lipoprotein receptor-related protein (LRP)
Espirito Santo et al.
LDL receptor family members and triglyceride metabolism1099
did not affect plasma cholesterol, FFA, and glucose levels
or plasma lipoprotein distribution (
of both LRP and LDLR (LRP
hyperlipidemia as a result of the accumulation of VLDL/
LDL-sized lipoproteins. Interestingly, VLDLR deficiency
on this LRP
LDLR background did not influence
plasma lipids, glucose (Table 1), and lipoprotein profiles
(Fig. 2). In addition, levels of plasma apoB-100 (90
0.818), apoB-48 (95
0.394), apoE (125
0.100), and apoA-I (102
46% vs. 100
were not altered in LRP
LDLR VLDLR mice, respectively.
We next investigated the effect of VLDLR status in the
presence or absence of the LDLR and LRP on plasma lip-
ids after 4 h of fasting. As shown in
wild-type and LRP
VLDLR deficiency on plasma lipid levels were comparable
). Under these conditions, VLDLR deficiency
). The deletion
) elicited severe
20% vs. 100
12% vs. 100 P
, for both the
backgrounds, the effects of
to those observed for the overnight fasting state. Single
mice showed only modestly increased plasma
TG levels and no effects on plasma cholesterol, FFA, and
glucose levels (Table 2). Again, VLDLR deficiency on an
LDLR background did not affect plasma lipids
and glucose levels.
Plasma HL, LPL activity, and LPL mass
Yagyu et al. (15) reported that the increase in plasma
TG levels in VLDLR
mice is associated with reduced
LPL activity in these mice. As shown in Table 2, single
mice indeed showed a significant 19% de-
crease in LPL activity (
0.02) and a 23% decrease in
LPL mass (
0.03) compared with control VLDLR
mice. Interestingly, LPL activity and mass levels were
fold and 2.4-fold higher for mice on an LRP
background compared with mice on a wild-type back-
ground, respectively. The LRP
also had a significant 30% lower plasma LPL activity (
TABLE 1. Plasma lipid and glucose levels on a chow diet after overnight fasting
Chow Diet High-Fat Diet
Animals CholesterolTGs FFA Glucose CholesterolTGs FFA Glucose
32.4 ? 6.5
34.1 ? 3.6
8.6 ? 1.9
8.9 ? 2.3
1.3 ? 0.2
1.5 ? 0.5
6.0 ? 1.4
5.6 ? 1.6
32.4 ? 6.1
39.4 ? 7.4
6.3 ? 2.5
12.5 ? 5.0a
1.1 ? 0.2
1.5 ? 0.3
5.8 ? 0.9
6.5 ? 0.9
LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; n.d., not determined; TG, triglyceride; VLDLR,
very low density lipoprotein receptor. Values represent means ? SD of 8–10 mice per group.
a P ? 0.05.
(A, E) and VLDLR?/? (B, F), and on a LRP?LDLR?/? background, LRP?LDLR?/?VLDLR?/? (C, G) and
LRP?LDLR?/?VLDLR?/? (D, H), after 4 weeks of inducing LRP deficiency on a chow diet (upper panels)
and after 10 weeks on a high-fat diet (lower panels) with overnight fasting. Lipoproteins in pooled plasma
were size-fractionated by fast-performance liquid chromatography, and the plasma cholesterol (closed cir-
cles) and triglyceride (TG; open circles) contents of the individual fractions were determined.
Lipoprotein distribution. Plasma was obtained from mice on a wild-type background, VLDLR?/?
1100 Journal of Lipid Research
Volume 46, 2005
0.001) and a 16% decrease in plasma LPL mass (P ? 0.04)
compared with control LRP?LDLR?/?VLDLR?/? mice.
HL activity was not affected upon deletion of the VLDLR
on both wild-type and LRP?LDLR?/? backgrounds. How-
ever, as for LPL activity, HL activity levels were increased
?2-fold in mice on an LRP?LDLR?/? background com-
pared with mice on a wild-type background (Table 2).
Postprandial TG response
Stressing TG metabolism by forced feeding through an
intragastric load of olive oil proved to be very effective at
evoking a clear effect of VLDLR deficiency on plasma TG.
Single VLDLR?/? mice had a strong increase in postpran-
dial TG response compared with controls (Fig. 3A), as in-
dicated by a 12-fold increased area under the curve (AUC)
[218 ? 170 mM TG/h vs. 12 ? 5 mM TG/h (P ? 0.01) for
VLDLR?/? and VLDLR?/?, respectively], which is in agree-
ment with our recent report (8). Likewise, LRP?LDLR?/?
VLDLR?/? mice had a strong increase in postprandial TG
response compared with control LRP?LDLR?/?VLDLR?/?
mice (Fig. 3B) [AUC ? 411 ? 107 mM TG/h vs. 163 ? 85
mM TG/h (P ? 0.002) for LRP?LDLR?/?VLDLR?/? and
LRP?LDLR?/?VLDLR?/?, respectively]. The total TG re-
sponse in VLDLR?/? and LRP?LDLR?/?VLDLR?/? mice
was similar, as indicated by a similar mean increase in AUC
compared with their respective controls (206 and 248 mM
TG/h, respectively). However, the TG response in the LRP?
LDLR?/?VLDLR?/? mice was remarkably prolonged.
Whereas TG levels in VLDLR?/? mice reached baseline
TG levels within 24 h after gavage, TG levels were still sig-
nificantly increased at 48 h after gavage in LRP?LDLR?/?
VLDLR?/? mice, which is probably related to the high
remnant levels in plasma that compete for the binding of
the nascent chylomicrons to LPL.
Plasma lipids under HFD
Finally, TG metabolism was assessed by challenge of mice
with HFD (Table 1). As observed on a chow diet, VLDLR?/?
mice had ?3-fold higher plasma TG levels (P ? 0.02) com-
pared with wild-type mice, whereas plasma cholesterol was
not different. Remarkably, on this diet, LRP?LDLR?/?
VLDLR?/? mice also displayed 2-fold higher plasma TG lev-
els (P ? 0.032) but no change in plasma cholesterol, FFA,
and glucose compared with the control LRP?LDLR?/?
VLDLR?/? mice. Thus, under high-fat feeding conditions,
the role of the VLDLR in TG-rich lipoprotein metabolism
becomes evident in the absence of the LRP and LDLR.
Studies in VLDLR?/? mice only revealed a role of this
receptor specifically in postprandial TG-rich lipoprotein
metabolism after severely stressing TG metabolism (5–8).
Interestingly, the role of the LRP in TG-rich lipoprotein
metabolism is fully compensated for by the presence of
the LDLR (9). We wondered whether a prominent role of
the VLDLR would be similarly overtaken by the LDLR or
TABLE 2.Plasma lipids, glucose, and lipolytic enzymes on a chow diet after 4 h of fasting
Animals CholesterolTGsFFAGlucose HL Activity LPL ActivityLPL Mass
2.2 ? 0.3
2.0 ? 0.3
0.6 ? 0.2
1.3 ? 0.6a
8.2 ? 1.3
7.0 ? 0.8
21.0 ? 3.5
17.1 ? 2.1a
76.6 ? 3.1
59.3 ? 7.9a
24.9 ? 5.6
19.8 ? 4.3
6.4 ? 2.4
4.6 ? 1.7
0.8 ? 0.2
0.7 ? 0.1
10.5 ? 1.3
11.1 ? 1.4
14.2 ? 2.2
14.6 ? 1.2
39.5 ? 3.6
27.7 ? 4.1a
180.6 ? 2.8
151.6 ? 10.5a
Values represent means ? SD of 8–10 mice per group.
a P ? 0.05.
squares) mice on a wild-type (A) or LRP?LDLR?/? (B) background were given an intragastric bolus of 200 ?l
of olive oil. Blood samples were drawn at the indicated times after gavage. Plasma TG concentrations were de-
termined and corrected for time 0 values. Values represent means ? SD of eight mice per group. * P ? 0.05.
Postprandial TG response. Overnight fasted VLDLR?/? (closed squares) and VLDLR?/? (open
Espirito Santo et al.
LDL receptor family members and triglyceride metabolism 1101
LRP. Here, we show that the absence of a phenotype for
VLDLR?/? mice with respect to TG-rich lipoprotein rem-
nant levels is not attributable to backup activity of the
LRP/LDLR pathway. Therefore, it seems reasonable to
conclude that the contribution of VLDLR to the clearance of
TG-rich lipoproteins is not rate-limiting under physiological
circumstances but becomes apparent after stressing TG
metabolism by high-fat feeding or giving a large TG bolus.
Previously, we demonstrated that adenovirus-mediated
overexpression of the LDLR family antagonist receptor-
associated protein (RAP) in LRP/LDLR double-deficient
mice elicits marked hyperlipidemia in addition to the pre-
existing hypercholesterolemia in these animals and de-
creases LPL activity (9). Because RAP binds to the VLDLR
with high affinity (16), we speculated that it was possible
that a RAP-mediated inhibition of the VLDLR underlies
the observed impaired LPL-mediated lipolysis and sub-
sequent hypertriglyceridemia. However, because LRP?
LDLR?/?VLDLR?/? and LRP?LDLR?/?VLDLR?/? mice
have comparable hyperlipidemia, we can now conclude
that RAP-induced hypertriglyceridemia does not directly
involve the VLDLR. This is further supported by our ob-
servation that adenovirus-mediated overexpression of RAP
still elicits marked hypertriglyceridemia and decreases LPL
activity in LRP?LDLR?/?VLDLR?/? mice (data not shown).
LRP?LDLR?/?VLDLR?/? and LRP?LDLR?/?VLDLR?/?
mice present higher plasma lipid levels after overnight fast-
ing compared with 4 h of fasting, as observed for LDLR?/?
VLDLR?/? mice (7). This effect is probably caused by an in-
creased hepatic production of VLDL-TG, which is the pri-
mary source of FFA for peripheral tissues in the absence
of chylomicrons in the fasted state.
Yagyu et al. (15) and Goudriaan et al. (8) showed that
impaired TG-rich lipoprotein catabolism in VLDLR?/? mice
is associated with reduced LPL activity. This has been ex-
plained by reduced translocation of LPL over endothelial
cells as related to the chaperone function of the VLDLR
(17). Likewise, we now show that VLDLR deficiency also
reduces LPL protein and activity on an LRP?LDLR?/?
background. In addition, the LRP?LDLR?/? background
resulted in increased LPL and HL activities in postheparin
plasma, irrespective of VLDLR status. LPL and HL are
both well-established ligands for LRP (18), and plasma
LPL mass is increased in LRP? mice (12). However, as
LPL activity levels are not affected in LRP? mice (12), it is
uncertain whether LRP deficiency contributes to increased
LPL protein and activity levels in LRP?LDLR?/? mice.
Most likely, the increase in LPL is a direct consequence of
the severe hyperlipidemia in LRP?LDLR?/? mice, be-
cause both total HDL and LPL are also increased in genet-
ically unrelated severely hypertriglyceridemic mice as a re-
sult of apoC-I expression (19).
The unique triple-deficient mouse model (LRP?LDLR?/?
VLDLR?/?) enabled us to conclude that the LRP/LDLR
pathway does not mask a prominent role for the VLDLR in
TG-rich lipoprotein metabolism. Apart from the LDLR
family members, other mechanisms also have been identi-
fied that contribute to TG-rich lipoprotein uptake and deg-
radation, such as LRP5 (20), apoB-48 receptor (21), LR11
(22), heparan sulfate proteoglycans (23), and scavenger
receptor class B type I (24). Our LRP?LDLR?/?VLDLR?/?
mouse model serves as a unique tool to elucidate the con-
tributions of these pathways in TG-rich lipoprotein clear-
ance in the absence of the three quantitatively important
main apoE-recognizing receptors. This will further advance
our understanding of the mechanisms by which plasma
levels of TG-rich lipoproteins are regulated in vivo.
This research was supported by the Royal Netherlands Acad-
emy of Arts and Sciences (fellowship to B.J.M.v.V.), the Euro-
pean Union (project QLK1-CT-1999-498), the Leiden Univer-
sity Medical Center (Gisela Thier Fellowship to P.C.N.R.), the
Netherlands Organization for Scientific Research (Grant 903-
39-192/194 and Netherlands Organization for Scientific Re-
search-VIDI Grant 917.36.351 to P.C.N.R., Netherlands Organi-
zation for Scientific Research-VENI Grant 916.36.071 to P.J.V.),
and the Netherlands Diabetes Foundation (Grant 96.604). In
addition, the authors thank Marijke Voskuilen (Netherlands
Organization for Applied Scientific Research-Quality of Life,
Leiden, The Netherlands) for excellent technical assistance.
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