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J Biol Chem, Vol. 275, Issue 11, 7515-7520, March 17, 2000
From the Microsomal triglyceride transfer
protein (MTP) transfers lipids to apolipoprotein B (apoB) within the
endoplasmic reticulum, a process that involves direct interactions
between apoB and the large subunit of MTP. Recent studies with
heterozygous MTP knockout mice have suggested that half-normal levels
of MTP in the liver reduce apoB secretion. We hypothesized that reduced
apoB secretion in the setting of half-normal MTP levels might be caused
by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were
true, half-normal levels of MTP might have little impact on lipoprotein
secretion in the setting of half-normal levels of apoB synthesis (since
the ratio of MTP to apoB would not be abnormally low) and might cause
an exaggerated reduction in lipoprotein secretion in the setting of
apoB overexpression (since the MTP:apoB ratio would be even lower). To
test this hypothesis, we examined the effects of heterozygous MTP
deficiency on apoB metabolism in the setting of normal levels of apoB
synthesis, half-normal levels of apoB synthesis (heterozygous
Apob deficiency), and increased levels of apoB synthesis
(transgenic overexpression of human apoB). Contrary to our
expectations, half-normal levels of MTP reduced the plasma apoB100
levels to the same extent (~25-35%) at each level of apoB
synthesis. In addition, apoB secretion from primary hepatocytes was
reduced to a comparable extent at each level of apoB synthesis. Thus,
these results indicate that the concentration of MTP within the
endoplasmic reticulum rather than the MTP:apoB ratio is the critical
determinant of lipoprotein secretion. Finally, we found that
heterozygosity for an apoB knockout mutation lowered plasma apoB100
levels more than heterozygosity for an MTP knockout allele. Consistent
with that result, hepatic triglyceride accumulation was greater in
heterozygous apoB knockout mice than in heterozygous MTP knockout mice.
Microsomal triglyceride transfer protein
(MTP)1 is expressed at high
levels in the absorptive enterocytes of the intestine and hepatocytes,
at moderate levels in the visceral endoderm of the yolk sac during
embryonic development, and at relatively low levels in kidney and
cardiac muscle (1-4). In each of these tissues, MTP plays a key role
in the assembly and secretion of apoB-containing lipoproteins (1, 3,
5). MTP is located within the lumen of the endoplasmic reticulum (ER)
and is thought to transfer lipids to apoB as that molecule is
translated, allowing apoB to assume a proper conformation for forming a
spherical lipoprotein with a neutral lipid core (6). In the absence of
MTP, triglyceride-rich, apoB-containing lipoproteins cannot be
assembled or secreted (3, 6, 7). The mechanism of MTP action has
attracted considerable scrutiny. Of note, several lines of evidence
have suggested that the assembly of lipoproteins involves a direct
interaction between MTP and apoB. First, Wu et al. (8)
demonstrated that apoB can be immunoprecipitated from the microsomes of
cultured hepatoblastoma cell lines with an antiserum against MTP.
Second, the laboratory of Mahmood Hussain has provided biochemical
evidence that MTP binds to apoB (9) and that this interaction depends
on arginine and lysine residues within apoB (10). Third, a pair of
recent studies, each using independent experimental approaches,
identified specific domains within the amino terminus of apoB that bind
to the 97-kDa subunit of MTP (11, 12). It has been suggested that
apoB-MTP complexes could play an important role in the assembly of
apoB-containing lipoproteins (12, 13).
To define the role of MTP in lipoprotein assembly in vivo,
our laboratory recently inactivated the mouse gene for the 97-kDa subunit of MTP (Mttp) (4). Homozygous Mttp
knockout mice died early in development, likely because an absence of
lipoprotein secretion by the yolk sac interferes with the delivery of
lipid nutrients to the developing mouse embryo (4, 14). Heterozygous knockout mice developed normally and manifested a 50% reduction in MTP
activity levels in both the liver and intestine. The half-normal MTP
levels were accompanied by an ~25% reduction in plasma apoB100 levels on both chow and high fat diets.
The finding that half-normal levels of MTP appeared to reduce plasma
apoB100 levels suggested that there is not a great excess of MTP within
the ER of hepatocytes (4). A key goal of the current study was to
further elucidate that finding. In particular, we wanted to determine
whether the effects of half-normal MTP levels in reducing lipoprotein
levels would be equally noticeable at different levels of apoB
expression. We imagined two possible scenarios by which half-normal
levels of MTP might reduce the secretion of apoB-containing
lipoproteins. One possibility was that the half-normal levels of MTP
would reduce the ratio of MTP to apoB within the lumen of the ER,
thereby reducing the number of direct protein-protein interactions
between these molecules. If a normal ratio of MTP to apoB were critical
for lipoprotein assembly, one might reasonably surmise that half-normal
levels of MTP would have little effect on lipoprotein production in the setting of half-normal levels of apoB synthesis, since the MTP:apoB ratio would not be reduced below that in wild-type cells. According to
this scenario, half-normal MTP levels might cause an even greater reduction in lipoprotein synthesis in the setting of increased levels
of apoB synthesis, since the MTP:apoB ratio would be further reduced. A
second possible scenario is that the ratio of MTP to apoB molecules is
irrelevant and that the key consideration is simply the absolute
concentration of MTP within the ER. If the absolute concentration of
MTP were the most important factor, one would surmise that the
consequences of half-normal MTP levels would be independent of the apoB
synthetic rate. We tended to favor the former scenario; thus, our
a priori hypothesis was that the ratio of MTP to apoB would
prove to be the critical factor governing lipoprotein production in the
setting of half-normal MTP levels.
The fact that MTP and apoB bind to each other during lipoprotein
assembly prompted us to consider another hypothesis. We hypothesized that overexpression of apoB might lead to more MTP-apoB interactions within the ER and that these more numerous MTP-apoB interactions might
stabilize MTP, increasing its half-life within the ER. To the extent
that this were the case, MTP levels in liver microsomes might be
increased in the setting of high levels of apoB expression and
decreased at low levels of apoB expression.
A key goal of this project was to address those two hypotheses: that
the ratio of MTP to apoB might be the critical factor governing apoB
production rates, and that changes in apoB expression might affect the
levels of MTP activity within the liver. To address the latter
hypothesis, we simply measured hepatic MTP activity levels in liver
microsomes at three levels of apoB expression: low (heterozygosity for
an Apob knockout mutation) (15), normal (two wild-type
Apob alleles), and high (two wild-type alleles plus a
"high expressing" human APOB transgene) (16). To assess the former hypothesis, that the ratio of MTP to apoB within the ER was
a critical determinant of apoB production, we examined the phenotype of
heterozygous Mttp knockout mice at each of the three
distinct levels of apoB synthesis. For these studies, the metabolic
effects of a single Mttp knockout allele were assessed with
specific mouse apoB100 immunoassays and with metabolic labeling studies
in primary mouse hepatocytes.
In addition to addressing these two hypotheses, we wanted to quantify
and compare the effects of a mutant Apob allele and a mutant
Mttp allele on hepatic lipoprotein secretion. This topic cannot be addressed convincingly with human investigations but can be
addressed definitively in experiments with knockout mice.
Genetically Modified Mice--
Heterozygous Apob
knockout mice (Apob+/ Lipid and Apolipoprotein Measurements--
Plasma lipid levels
were measured on fresh plasma samples (in duplicate) after a 4-h fast.
Cholesterol levels were measured with the Abbott Spectrum kit (Abbott
Laboratories Diagnostics Division, Abbott Park, IL), and triglyceride
levels were determined with the triglyceride/GB kit (Roche Molecular
Biochemicals) (19). The plasma levels of mouse and human apoB100 were
measured with monoclonal antibody-based radioimmunoassays (RIAs) (20,
21).
Assessing the Distribution of Cholesterol within the Plasma
Lipoproteins--
The distribution of cholesterol within the plasma
lipoproteins was assessed by size-fractionating 250 µl of plasma
(pooled from 7 female mice after a 4-h fast) by fast protein liquid
chromatography (FPLC) on a Superose 6B 10/50 column (Amersham Pharmacia
Biotech) (19).
MTP Activity Assay and MTP Western Blots--
Mouse livers were
dissected and homogenized, and MTP activity levels were performed
according to the procedures described by Wetterau et al. (1,
5). For several groups of mice, the amount of the 97-kDa subunit of MTP
was assessed on Western blots of sodium dodecyl sulfate-polyacrylamide
gels using a rabbit antiserum against bovine MTP (provided by Dr. John
Wetterau, Bristol-Myer Squibb, Princeton, NJ).
Metabolic Labeling of Primary Hepatocytes--
Primary mouse
hepatocytes were prepared and cultured as described previously (22).
The cells were allowed to attach for 90 min, and the medium was removed
and replaced with fresh medium containing 50 µl of
[35S]methionine/cysteine (Pro-mix, 530 Mbq/ml, Amersham
Pharmacia Biotech) per ml of medium. After a 3-h incubation, a sample
of the medium (40 µl) was size-fractionated on a 4-15%
polyacrylamide-sodium dodecyl sulfate gel. The gel was dried, and the
incorporation of 35S into apoB48 and apoB100 was assessed
with a Fuji Bio-Imaging Analyzer (Fiji Medical Systems, Stamford, CT).
For each well of primary hepatocytes, the amount of 35S
incorporation into the apoB proteins was normalized to the amount of
35S incorporation into characteristic triplet bands
(secreted non-apoB proteins) that migrated immediately below the apoB48
band (see Fig. 4E below).
Measurement of Liver Triglyceride Stores--
Liver samples from
each animal were disrupted with a homogenizer (Ultra-Turbax T8, VWR)
for 2 min in 1.0 ml of a 2:1 mixture of chloroform and methanol. The
homogenizer probe was rinsed twice with 1.0 ml of the same
chloroform/methanol mixture, bringing the total volume of the mixture
to 3 ml. Next, 1.0 ml of water and 0.576 mg of tripentadecanoin (an
internal standard dissolved in hexane) were added. After vortexing for
2 min, the sample was centrifuged for 10 min at 900 × g. The bottom layer was transferred to a new tube and dried
under a stream of nitrogen at 30 °C. Each sample was resuspended in
40 µl of hexane. The plate was sprayed with a fixative (10 mg of
8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium in 50 ml of methanol),
and the triglyceride band was visualized with a hand-held UV lamp. The
silica containing the triglycerides was scraped off and placed in a new
tube and transmethylated with 1.0 ml of 3 N methanolic HCl.
The samples were vortexed, incubated at room temperature overnight, and
centrifuged for 3 min at 900 × g. The liquid portion
was transferred to a glass tube and dried under nitrogen. The samples
were then resuspended in 1.0 ml of heptane, and the fatty acid methyl
esters were quantified by gas chromatography (23). After normalization
of the data to the C-15 internal standard, triglyceride measurements
were calculated as µM of triglyceride/g of liver tissue.
Statistical Analysis--
Mean levels of apoB, plasma lipids, or
liver triglyceride stores are reported for each group of mice along
with standard deviations or standard errors of the means. Statistical
differences were modeled by analysis of variance with genotype being an
intergroup factor.
MTP Activity and Protein Levels in Liver Extracts from Different
Groups of Mice--
We found that hepatic MTP activity levels were
49.7% lower in Mttp+/ Decreased Plasma ApoB100 and Lipid Levels in Mttp+/
We also assessed the effects of the Mttp knockout mutation
on the plasma levels of human apoB100 in HuBTg+/o and
HuBTg+/oMttp+/
Measurements of total plasma cholesterol or triglycerides were poor
surrogates for the apoB100 measurements, at least from the perspective
of identifying metabolic perturbations resulting from half-normal MTP
activity levels. Inactivating one of the two Mttp alleles
had no effect on plasma triglyceride levels. The total plasma
cholesterol level was significantly lower in Mttp+/ Decreased ApoB Secretion in the Setting of a Single Mttp Knockout
Allele--
To investigate the mechanism for the reduced plasma apoB
levels in the setting of half-normal levels of MTP, we performed metabolic labeling experiments on the primary hepatocytes of different groups of mice (Fig. 4). The amount of
apoB100 in the medium from Mttp+/ The Effect of a Single Mttp Knockout Allele on Hepatic
Triglyceride Stores--
We determined whether the reduced apoB
secretion in the setting of the Mttp and Apob
mutations would be accompanied by an accumulation of triglycerides
within the liver. Pairwise comparisons of littermates revealed a
tendency for higher liver triglyceride stores in the setting of the
Mttp mutation, but none of these differences achieved
statistical significance (Fig. 5).
Interestingly, the hepatic triglyceride stores were greater in
heterozygous Apob knockout mice than in heterozygous
Mttp knockout mice (Fig. 5).
In our initial characterization of Mttp knockout mice
(4), we noted that the Mttp+/ In the current study, we hypothesized that the ratio of MTP to apoB
might be a key determinant of lipoprotein secretion. Thus, we suspected
that half-normal MTP levels might have little effect on lipoprotein
production when apoB synthesis was also reduced by 50% (since the
ratio of MTP to apoB within the ER would not be abnormally low) and
might have an exaggerated effect in the setting of apoB overexpression
(since the ratio of MTP to apoB would be further reduced). We further
hypothesized that increased levels of apoB synthesis could affect the
stability of the MTP protein and, hence, the amount of MTP activity in
liver microsomes. Each of these two hypotheses seemed plausible in view
of evidence indicating the existence of a direct MTP-apoB interaction
within the ER (8, 9, 11, 12) and suggestions that an MTP-apoB complex
might play a key role in lipoprotein assembly (12, 13). Our data,
however, provided no support for either hypothesis. Hepatic MTP
activity levels were completely unaffected by either apoB
overexpression (a human APOB transgene) or by apoB
underexpression (heterozygosity for an Apob knockout
mutation). Also, half-normal levels of MTP reduced apoB100 production
rates to a similar extent at each level of apoB synthesis.
To the best of our knowledge, this is the only study to quantify the
relative impact of half-normal levels of MTP and apoB on lipoprotein
assembly and secretion. Our experiments revealed that both mutations
reduced hepatic lipoprotein secretion, but the effect of the
Apob mutation was much greater. Of course, this conclusion
should not be generalized past the specific experimental model that we
examined. We examined hepatic secretion in animals fed a chow diet, and
no conclusions can or should be drawn regarding the relative importance
of these two gene products on intestinal lipoprotein secretion or on
hepatic lipoprotein secretion in the setting of a high fat diet.
The fact that the effects of half-normal MTP levels were similar at
each level of apoB synthesis indicates that the absolute ratio of MTP
to apoB is not an important determinant of lipoprotein secretion, at
least within the range of apoB synthesis rates that we examined.
Instead, it appears that the most important factor is the absolute
amount or concentration of MTP within the ER. We emphasize that our
experiments did not attempt to study MTP-apoB interactions directly nor
do the results of our experiments necessarily cast doubt on the
potential importance of these direct interactions during lipoprotein
assembly. Our data simply show that half-normal levels of MTP influence
lipoprotein secretion similarly at different levels of apoB synthesis.
MTP is not the only factor that can affect lipoprotein production. Our
findings with the Apob knockout mice reveal that reduced amounts of apoB synthesis can also limit hepatic lipoprotein
production. Other investigations have documented that the levels and
intracellular availability of lipids can limit cellular lipoprotein
secretion (27-32). Thus, lipoprotein assembly and secretion require
adequate levels of apoB, MTP, and lipids, and reductions in any one of these can limit the output of lipoproteins from cells. Reductions in
two of these factors can result in additive decreases in lipoprotein production, as demonstrated in the current studies by the low plasma
apoB100 levels and low apoB100 production rates in
Apob+/ In this study, we relied mainly on apoB metabolic labeling studies and
monoclonal antibody-based apoB RIAs to investigate the metabolic
effects of half-normal MTP activity levels. Both of these strategies
revealed reproducible and concordant effects of the Mttp
knockout mutation on apoB metabolism. It is interesting that the plasma
lipid levels were poor surrogates for the apoB measurements. The total
plasma cholesterol levels were quite insensitive to the effects of the
Mttp knockout mutation, although the FPLC fractionation
studies did uncover ~20% reductions in low density lipoprotein
cholesterol levels at each level of apoB synthesis. A single copy of
the Mttp knockout allele had no detectable effect on plasma
triglyceride levels, a somewhat surprising result in light of recent
studies that have shown that hepatic MTP plays a crucial role in
triglyceride secretion (7, 33). We do not understand why the
Mttp mutation failed to reduce the plasma triglyceride levels. It seems possible that the variability in plasma triglyceride levels made it difficult to identify such an effect or perhaps that
changes in the activity of triglyceride removal pathways masked any
changes in hepatic triglyceride secretion rates.
The metabolic labeling studies revealed that half-normal MTP levels
reduced apoB100 secretion more than they reduced apoB48 secretion. This
result is consistent with recent findings with MTP inhibitor compounds
in cultured cells (29, 34-36) and also with recent findings from
liver-specific Mttp knockout mice (7). We used
Cre/loxP recombination techniques to produce liver-specific Mttp knockout mice and documented a >95% reduction in both
hepatic MTP activity levels and plasma apoB100 levels (7).
Interestingly, the effects of the liver-specific knockout on plasma
apoB48 levels were modest, and primary hepatocytes from those mice
secreted substantial amounts of the apoB48 protein.
In this study, a defective Apob allele was more potent in
reducing plasma apoB levels than a defective Mttp allele.
Equally intriguing, however, were the relative effects of the
Mttp and Apob mutations on hepatic stores of
triglycerides. Given the obligatory role of MTP in the entry of
triglycerides into the secretory organelles of hepatocytes (7), one
might have reasonably hypothesized that half-normal MTP levels would
significantly increase liver triglyceride stores. Moreover, it would
have been reasonable to hypothesize that the normal MTP activity levels
in Apob+/ The results of our current experiments, that a 50% reduction in MTP
levels reduces plasma lipoprotein levels with only a modest effect on
liver triglyceride stores, will likely be heartening to pharmaceutical
company investigators who are developing MTP inhibitor drugs to treat
human hyperlipidemias. Although the safety profile of these drugs in
humans remains to be established, a study of an MTP inhibitor drug in
rabbits and hamsters was recently reported (37). The MTP inhibitor
compound reduced plasma lipoprotein levels while increasing liver
triglyceride stores only modestly. Those results appeared to be
consistent with our genetic studies.
The finding of increased triglyceride stores in the
Apob+/ We thank S. Ordway and G. Howard for
editorial assistance and J. Carroll for graphics. We thank Dr.
John Wetterau, Bristol-Myer Squibb, Princeton, NJ for helpful
discussions and for the rabbit antiserum against bovine MTP.
*
This work was supported in part by National Institutes of
Health Grant (NIH) HL47660, an NIH-supported Cardiovascular Research Institute Molecular/Cellular Cardiology training grant position (to
G. L.), an Howard Hughes Medical Institute Postdoctoral Fellowship for
Physicians (to G.L.), and grants from the University of California Tobacco-related Disease Research Program (to M. M. V. and
S. G. Y.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
Current address: Bayer AG, Cardiovascular Research Institute, 42096 Wuppertal, Germany.
§§
To whom correspondence should be addressed: Gladstone Institute
of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA
94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail: syoung@gladstone.ucsf.edu.
The abbreviations used are:
MTP, microsomal
triglyceride transfer protein;
apo, apolipoprotein;
Mttp, the mouse gene for the large subunit of microsomal triglyceride
transfer protein;
ER, endoplasmic reticulum;
FPLC, fast protein liquid
chromatography;
RIA, radioimmunoassay.
A Deficiency of Microsomal Triglyceride Transfer Protein Reduces
Apolipoprotein B Secretion*
§¶
,§,,,§**,§,,, and§¶§§
Gladstone Institute of Cardiovascular
Disease, § Cardiovascular Research Institute, and
¶ Department of Medicine, University of California, San Francisco,
California 94141-9100 and Department of
Nutritional Sciences, University of California,
Berkeley, California 94720-3104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), heterozygous
Mttp knockout mice (Mttp+/), and
high expressing hemizygous human APOB transgenic mice
(HuBTg+/o) have been previously described (4, 15-17). In
high expressing human apoB transgenic mice on a chow diet, we have
documented human apoB100 levels of ~40-50 mg/dl (18), and the total
amount of apoB secretion from these cells is approximately twice as
much as from wild-type mice (14). The HuBTg+/o,
Apob+/, and Mttp+/
mice were intercrossed to produce human apoB transgenic mice that were
heterozygous for the Mttp knockout mutation
(HuBTg+/oMttp+/) and mice that
were heterozygous for both the Apob and Mttp
mutations (Apob+/Mttp+/). We
compared groups of mice with a single copy of an Mttp
knockout allele with control mice with two normal Mttp
alleles. For each of these comparisons, the control mice were
invariably littermates. All mice were female and had a mixed genetic
background (~75% C57BL/6 and 25% 129/Sv). The mice were housed in a
full-barrier facility and were fed a chow diet containing 4.5% fat
(PicoLab Mouse Chow 20, No. 5058, Ralston Purina, St. Louis, MO). Blood was sampled for lipid and apolipoprotein measurements when the mice
were greater than 2 months old. The mice sacrificed for measurement of
liver triglyceride stores were more than 4 months old.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mice than in wild-type
mice (p < 0.001) (Fig.
1A). Recent studies have
suggested that apoB and MTP bind to each other during lipoprotein assembly (8, 11, 12). We hypothesized that overexpression of human apoB
might conceivably lead to an increased number of those interactions,
potentially stabilizing MTP within the ER and leading to increased
amounts of MTP activity in hepatic microsomes. Our data, however, did
not support this hypothesis. The hepatic MTP activity levels were no
higher in HuBTg+/o mice than in wild-type mice and were
50% lower in HuBTg+/oMttp+/
mice than in HuBTg+/o mice (p < 0.001)
(Fig. 1A). A single copy of an Apob knockout allele had no significant effect on hepatic MTP activity levels (Fig.
1A). As judged by Western blots, the half-normal MTP
activity levels in heterozygous Mttp knockout mice were due
to half-normal levels of the 97-kDa subunit of MTP (Fig.
1B).
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Fig. 1.
MTP activity and protein levels in liver
extracts from different groups of mice. A, bar
graph illustrating MTP activity levels in the livers of mice with
normal levels of apoB expression (wild-type apoB mice), high levels of
apoB expression (human apoB transgenic mice), and low levels of apoB100
expression (Apob+/ mice) (n = 6 in each group). Each bar shows mean ± S.D.
B, Western blot analysis of MTP protein levels in
Mttp+/ mice and wild-type mice. The antiserum
detects the 97-kDa subunit of MTP as well as a 14-kDa protein (whose
identity is unknown). TG, triglyceride.
Mice--
We sought to assess the metabolic effects of half-normal MTP
levels at several different levels of apoB synthesis. Initially, we
performed RIAs on a series of dilutions of pooled plasma from female
mice of each of the six genotypes (wild-type,
Mttp+/, HuBTg+/o,
HuBTg+/oMttp+/,
Apob+/, and
Apob+/Mttp+/) (Fig.
2A-C). These studies
suggested that the Mttp knockout mutation reduced mouse
apoB100 levels to a similar extent at each level of apoB synthesis. To
further test this idea, we measured plasma apoB100 levels for each
mouse in each of the six groups (Table
I). Three features of those data are
noteworthy. First, the Mttp knockout allele reduced plasma
apoB100 levels to a similar extent at each level of apoB synthesis
(~25-35%). The effect of half-normal MTP activity levels on apoB100
levels was neither diminished at reduced levels of apoB synthesis nor
exaggerated by increased levels of apoB expression. Second, the
Apob knockout allele reduced plasma apoB levels far more
(>50%) than the Mttp knockout allele (~25%). Third,
human APOB expression increased the plasma levels of mouse
apoB100. Increased plasma levels of mouse apoB100 in the setting
of human apoB overexpression were not unexpected, as we had encountered
the same finding in a recent study of atherogenesis in human apoB
transgenic mice (24).
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Fig. 2.
Sandwich radioimmunoassays illustrating
relative amounts of mouse apoB100 and human apoB100 in the plasma of
different groups of mice. A, RIA illustrating reduced
amounts of mouse apoB100 in the pooled plasma of
Mttp+/ mice (n = 17) compared
with the pooled plasma from littermate wild-type mice
(n = 39). B, RIA illustrating reduced
amounts of mouse apoB100 in the pooled plasma of
HuBTg+/oMttp+/ mice
(n = 18) compared with littermate HuBTg+/o
mice (n = 13). C, RIA illustrating reduced
amounts of mouse apoB100 in the pooled plasma from
Apob+/Mttp+/ mice
(n = 8) compared with the plasma from
Apob+/ mice (n = 10).
D, RIA illustrating reduced amounts of human apoB100 in the
pooled plasma of HuBTg+/oMttp+/
mice (n = 18) compared with the plasma from littermate
HuBTg+/o mice (n = 13).
Human and mouse apoB100 levels in six groups of female mice
mice. As shown in
Table I and Fig. 2, B and D, the defective Mttp allele resulted in very similar reductions in the
levels of mouse and human apoB100 (29 and 32%, respectively).
mice than in the wild-type mice, but no
significant effect on cholesterol levels was noted in the setting of
the human apoB transgene or the Apob knockout mutation
(Table II). The FPLC studies revealed
that a single copy of the Mttp mutation reduced the levels of cholesterol in the intermediate and low density lipoproteins by
~20% at each level of apoB synthesis (Fig.
3). However, on a chow diet, the
quantitative significance of this reduction was modest and in some
cases was offset by increases in high density lipoprotein cholesterol
levels.
Total plasma cholesterol and triglyceride concentrations in six
groups of female mice
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Fig. 3.
Distribution of cholesterol within the plasma
lipoproteins of different groups of mice. Plasma (250 µl) was
pooled from 7 female mice after a 4-h fast and fractionated on an FPLC
column. A, wild-type versus Mttp+/
mice. B, HuBTg+/o versus
HuBTg+/oMttp+/ mice. C,
Apob+/ versus
Apob+/Mttp+/ mice. Fractions
14-20 represent very low density lipoprotein fractions;
21-26, intermediate and low density lipoprotein fractions;
27-34, high density lipoprotein fractions. The total amount
of cholesterol in fractions 21-26 (intermediate and low density
lipoprotein) was calculated. There was a 21% reduction in
intermediate/low density lipoprotein cholesterol in
Mttp+/ mice compared with wild-type mice, a
20% reduction in HuBTg+/oMttp+/
mice versus HuBTg+/o mice, and a 21% reduction
in Apob+/Mttp+/ mice
versus Apob+/ mice.
hepatocytes
was 27% lower than in wild-type mice (p = 0.026 and 0.037 in two independent experiments) (Fig. 4, A and
B). The Mttp mutation lowered apoB100 secretion
by 28% in the setting of the human apoB transgene (p = 0.002 and 0.015 in two experiments) and by 21% in the setting of an
Apob knockout allele (p = 0.016 and 0.014 in
two experiments) (Fig. 4, A and B). The effect of the Mttp mutation on apoB48 secretion was modest. ApoB48
secretion from Mttp+/ hepatocytes was 15%
lower than from wild-type hepatocytes (p = 0.125 and
0.048 in two experiments) (Fig. 4, C and D). The
Mttp mutation lowered apoB48 secretion by 18%
(p = 0.036 and 0.049 in two experiments) in the setting
of the human apoB transgene and by 15% in the setting of an
Apob knockout allele (p = 0.027 and 0.034 in
two experiments) (Fig. 4, C and D). Fig.
4E shows an autoradiogram of the media from
HuBTg+/o and
HuBTg+/oMttp+/ primary
hepatocytes; a reduction in apoB100 in the medium from HuBTg+/oMttp+/ primary hepatocytes
was visually apparent.
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Fig. 4.
Accumulation of apoB100 and apoB48 in the
medium of primary hepatocytes prepared from the six groups of mice
(n = 4). A, quantification of apoB100
secretion in wild-type, Mttp+/ ,
HuBTg+/o,
HuBTg+/oMttp+/,
Apob+/, and
Apob+/Mttp+/ primary
hepatocytes. B, quantification of apoB100 secretion in a
second experiment. C, quantification of apoB48 secretion in
wild-type, Mttp+/, HuBTg+/o,
HuBTg+/o Mttp+/,
Apob+/,
Apob+/Mttp+/ primary
hepatocytes. D, quantification of apoB48 secretion in a
second experiment. Data represent mean ± S.D. The amounts of
35S-labeled apoB48 and apoB100 in the medium were
quantified with a PhosphorImager and normalized to the amount of
35S incorporation into characteristic triplet bands
(secreted non-apoB proteins) located immediately below the apoB48 band
(see panel E). E, an autoradiogram of the media
samples (duplicate lanes) from primary hepatocytes prepared
from a HuBTg+/o mouse and a
HuBTg+/oMttp+/ mouse. A
reduction in apoB100 secretion from
HuBTg+/oMttp+/ primary hepatocytes
is visually apparent. *, p < 0.05; **,
p < 0.01.
View larger version (12K):
[in a new window]
Fig. 5.
Liver triglyceride stores in six different
groups of mice. This bar graph shows comparisons of
three groups of mice (means ± S.E.): wild-type versus
Mttp+/ (p = 0.24),
HuBTg+/o versus
HuBTg+/oMttp+/(p = 0.34), and Apob+/ versus
Apob+/Mttp+/ (p = 0.78). The levels of triglycerides in the
Apob+/ and
Apob+/Mttp+/ livers
were greater than in the wild-type mice (p = 0.004 and
0.001, respectively), Mttp+/ mice
(p = 0.005 and 0.003, respectively), and
HuBTg+/o mice (p = 0.009 and 0.008, respectively). A nonparametric statistical analysis revealed virtually
identical levels of statistical significance.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mice displayed
half-normal MTP activity levels and an ~25% reduction in plasma
apoB100 levels. The latter finding was somewhat surprising, given the
prevailing view that heterozygosity for MTP deficiency in humans has no
effect on plasma lipid or lipoprotein profiles (25, 26).
Mttp+/ mice.
mice would completely prevent an
accumulation of triglycerides in the liver simply by packing each
nascent apoB-containing lipoprotein with a double dose of
triglycerides. Our current data appear to be inconsistent with both of
these hypotheses. Liver triglyceride stores were increased only
modestly by the Mttp mutation, and the increase was not
statistically significant. In addition, the Apob mutation
appeared to have a much greater effect on liver triglyceride stores
than the Mttp mutation, just as the Apob mutation had a greater effect in reducing the secretion of apoB-containing lipoproteins. These findings, as far as we are aware, are novel, as
there have been no prior studies of the effects of heterozygous apoB or
MTP deficiency on hepatic triglyceride stores in either mice or humans.
mice should also be of interest to the
pharmaceutical industry, particularly to investigators who are seeking
new therapeutic strategies for treating hyperlipidemias. Our results
suggest that pharmaceutical strategies designed to reduce the
transcription of the apoB gene or any other strategy to reduce the
number of apoB transcripts may not be free of the hepatic steatosis
issue. The issue of increased triglyceride stores may be shared by any
strategy that reduces the secretion of apoB-containing lipoproteins.
ACKNOWLEDGEMENTS
FOOTNOTES
The first two authors contributed equally to this project.
ABBREVIATIONS
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DISCUSSION
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