Apolipoprotein A-IV Expression in Mouse Liver Enhances
Triglyceride Secretion and Reduces Hepatic Lipid
Content by Promoting Very Low Density Lipoprotein
Particle Expansion
Melissa A. VerHague, Dongmei Cheng, Richard B. Weinberg, Gregory S. Shelness
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Objective—Previous studies demonstrated that apolipoprotein A-IV (apoA-IV) promotes apoB lipoprotein–mediated
triglyceride (TG) secretion in transfected enterocytes and hepatoma cells; however, evidence for a role in lipid transport in
vivo is lacking. Using mouse models, we explored the role of apoA-IV in hepatic very low density lipoprotein–mediated
lipid efflux under conditions that promote hepatic steatosis.
Approach and Results—Hepatic steatosis, induced by either high-fat diet or enhanced de novo lipogenesis caused by transgenic
overexpression of SREBP-1a (SREBP-1aTg), was associated with up to a 43-fold induction of hepatic apoA-IV mRNA and
protein levels. In both models, a positive linear correlation between hepatic TG content and apoA-IV mRNA abundance
was observed (r2=0.8965). To examine whether induction of apoA-IV affected hepatic TG secretion, SREBP-1aTg mice
were crossed with Apoa4 knockout mice. With Triton blockade of peripheral lipolysis, SREBP-1aTg/Apoa4 knockout
mice demonstrated a 24% reduction in hepatic TG secretion rate, relative to SREBP-1aTg controls, but no change in apoB
production. Negative stain electron microscopy revealed a 33% decrease in the abundance of secreted very low density
lipoprotein particles with diameters ≥120 nm. Conversely, mice infected with a recombinant human apoA-IV adenovirus
demonstrated a 52% increase in the hepatic TG secretion rate, relative to controls, a 38% reduction in liver TG content,
and a 43% increase in large diameter (≥120 nm) very low density lipoprotein particles, with no change in apoB secretion.
Conclusions—Hepatic steatosis in mice induces hepatic apoA-IV expression, which in turn promotes lipoprotein particle
expansion and reduces hepatic lipid burden without increasing the number of secreted atherogenic apoB-containing
lipoprotein particles. (Arterioscler Thromb Vasc Biol. 2013;33:2501-2508.)
Key Words: apolipoprotein B ◼ metabolic syndrome ◼ nonalcoholic fatty liver disease ◼ triglyceride ◼ VLDL
T
he epidemic of obesity and associated metabolic syndrome has caused a rapid increase in the incidence of
nonalcoholic fatty liver disease, which now affects approximately one third of adults in developed countries.1,2 A predominant means by which the liver protects itself from excess
lipid accumulation is the assembly and secretion of very low
density lipoproteins (VLDLs).1 Bulk triglyceride (TG) export
from the liver can be increased by 2 nonexclusive mechanisms: assembly and secretion of a greater number of VLDL
particles or secretion of larger VLDL particles containing an
increased amount of core lipid. Although a broad understanding of the VLDL assembly pathway has emerged, the question of how the liver integrates particle number with particle
size to achieve a given rate of hepatic lipid efflux is poorly
understood.3–5
One factor that may play a role in modulating apoB lipoprotein assembly and particle expansion is apolipoprotein
A-IV (apoA-IV). ApoA-IV is a 46-kDa lipid-binding protein,
which is expressed not only in the mammalian intestine but
also in rodent liver. Since its discovery in 1977, apoA-IV has
been ascribed a wide variety of functions in lipid metabolism
and metabolic regulation.6–9 Perhaps the most notable characteristic of apoA-IV is the close association between active
intestinal lipid absorption and the induction of intestinal
apoA-IV gene expression.10 The first direct functional connection between apoA-IV expression and bulk lipid transport was
observed in a cultured pig intestinal epithelial cell model, in
which transfection of apoA-IV constructs strongly enhanced
transcellular TG transport, primarily by promoting lipoprotein
particle expansion.11,12 Similar results were observed in transfected rat hepatoma cells, in which the impact of apoA-IV on
apoB lipoprotein assembly was attributed to its ability to alter
the trafficking kinetics of nascent apoB-containing lipoproteins by interacting with apoB in the secretory pathway.13,14
However, despite the considerable physiological and in vitro
evidence linking apoA-IV and intestinal TG transport, a
Received on: June 3, 2013; final version accepted on: September 4, 2013.
From the Department of Pathology (M.A.V., D.C., G.S.S.), Department of Internal Medicine (R.B.W.), and Department of Physiology & Pharmacology
(R.B.W.), Wake Forest School of Medicine, Winston-Salem, NC.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301948/-/DC1.
Correspondence to Gregory S. Shelness, PhD, Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC 27157. E-mail
[email protected]
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2501
DOI: 10.1161/ATVBAHA.113.301948
2502 Arterioscler Thromb Vasc Biol November 2013
Nonstandard Abbreviations and Acronyms
A4-KO
apo
ER
MTP
Tg
TG
VLDL
WT
Apoa4 knockout
apolipoprotein
endoplasmic reticulum
microsomal triglyceride transfer protein
transgenic
triglyceride
very low density lipoprotein
wild type
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long-standing conundrum has been that no significant impact
of genetic apoA-IV deficiency or transgenic (Tg) overexpression on intestinal lipid absorption and growth was observed in
mouse models in vivo.15,16
In contrast to the intestine—an organ that possesses both
excess absorptive capacity and robust adaptive mechanisms,
which may mask subtle defects in lipid transport17—the
liver is highly sensitive to signals that alter lipid metabolic
pathways, as evidenced by the many genetic, hormonal, and
dietary factors that contribute to nonalcoholic fatty liver disease.1,18 We, therefore, chose to explore the function of apoAIV in the liver by assessing its impact on TG secretion under
conditions of hepatic steatosis, induced by either feeding a
high-fat and high-cholesterol diet or by unregulated de novo
lipogenesis, caused by Tg expression of a constitutively active
form of SREBP-1a.19,20 We found that, unlike in the intestine,
apoA-IV deficiency and overexpression exert a powerful
impact on hepatic TG secretion rate and lipid content, attributable to its ability to promote nascent VLDL particle expansion. These data provide the first evidence for a direct role of
apoA-IV in modulating lipid transport, in vivo, and suggest
that its expression can dramatically enhance VLDL-mediated
hepatic TG efflux by promoting particle expansion without
increasing the number of atherogenic apoB lipoprotein particles secreted by the liver.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Results
Hepatic ApoA-IV Expression Is Increased
in Steatosis
Two models of hepatic steatosis were used to assess the
impact of cellular TG content on hepatic apoA-IV expression.
In the first model, Tg expression of a constitutively active
form of SREBP-1a (SREBP-1aTg) promotes the transcription of genes responsible for hepatic de novo lipogenesis.19,20
Because the transgene is under the control of the PEPCK
promoter, feeding a low-carbohydrate diet further induces
SREBP-1a and lipogenic gene expression, causing massive
TG accumulation.19 As shown in Figure 1A, SREBP-1a Tg
mice displayed a 15-fold increase in liver TG content relative
to wild-type (WT) littermates on the chow diet and a 35-fold
relative increase on the low-carbohydrate diet. Under these
conditions, apoA-IV mRNA abundance increased 18- and
43-fold, respectively (Figure 1B); immunoblot analysis confirmed that this was accompanied by a corresponding increase
in hepatic apoA-IV protein levels (Figure 1C). In the second
model, hepatic steatosis was induced by feeding a high-fat and
high-cholesterol diet to WT C57BL/6 mice. After 16 weeks
on diet, liver TG content had increased 15-fold relative to
chow-fed controls (Figure 1D) and apoA-IV mRNA abundance increased 27-fold (Figure 1E), with a corresponding
increase in apoA-IV protein mass (Figure 1F). Analysis of all
data from both models revealed a strong positive linear correlation (r2=0.8965) between hepatic TG content and apoAIV mRNA abundance (Figure 1G). These data indicate that
hepatic apoA-IV mRNA and protein levels are dramatically
increased by conditions that promote hepatic TG accumulation and suggest that apoA-IV gene expression may be regulated either directly or indirectly by intracellular TG content.
ApoA-IV Deficiency in SREBP-1aTg Mice
Reduces Hepatic TG Secretion
To determine whether the induction of apoA-IV expression
that accompanies hepatic TG accumulation affects TG secretion, we examined the consequences of Apoa4 gene inactivation in SREBP-1aTg mice. For this purpose, SREBP-1aTg
Figure 1. Hepatic apolipoprotein A-IV (apoA-IV)
expression and triglyceride (TG) content in 2 models of steatosis. A to C, SREBP-1aTg (transgenic
[Tg]) and wild-type (WT) littermates were fed either
a chow or low-carbohydrate (Low Carb) diet for 2
weeks. Mice were euthanized after a 4-hour fast,
and liver and blood were collected. A, Hepatic TG
content. B, ApoA-IV mRNA levels normalized to
chow-fed WT littermates. C, Hepatic apoA-IV protein mass detected by immunoblot analysis. D to F,
C57BL/6 mice were fed either chow or high-fat and
high-cholesterol diet (HFCD; 48% calories from fat,
0.2% cholesterol) for 16 weeks. D, Hepatic TG content. E, ApoA-IV mRNA levels normalized to chowfed. F, Hepatic apoA-IV protein mass detected
by immunoblot analysis. G, Correlation between
apoA-IV mRNA abundance and hepatic TG content.
Linear regression and Pearson correlation coefficients are given. Data are mean±SE (n=5 except for
SB-Tg-Low Carb [n=6]). Bars labeled with different
letters are P<0.05 by ANOVA. *P<0.0001; #P<0.005
by unpaired Student t test.
VerHague et al ApoA-IV Promotes Hepatic VLDL Particle Expansion 2503
Figure 2. Apolipoprotein A-IV (apoA-IV) deficiency
inhibits triglyceride (TG) secretion in SREBP-1aTg
mice. SREBP-1aTg (SB-Tg) and Apoa4 knockout
mice (A4-KO) were crossed to produce SB-Tg/
A4-KO mice and wild-type littermates (SB-Tg/
A4-WT). A, Relative apoA-IV mRNA abundance normalized to SB-Tg/A4-WT. B, Pooled liver samples
were subjected to immunoblot analysis in triplicate
to quantify relative apoA-IV abundance. C, Fasting plasma TG levels. D and E, After a 4-hour fast,
mice were injected with 500 mg/kg Triton WR1339
to inhibit peripheral lipolysis and 200 μCi of [35S]
Met and Cys to measure apoB production. Blood
samples were collected at 0, 30, 60, 120, and 180
minutes post-Triton injection. D, Plasma TG concentrations after Triton block. E, Mean secretion
rate calculated from the slopes of individual plots
of TG vs time. F, Hepatic TG content. Data are
expressed as mean±SE. n=8 for A to C and F; n=5
for D and E. *P<0.05; **P<0.01 by unpaired Student
t test. Tg indicates transgenic.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
mice were crossed with Apoa4 knockout (A4-KO) mice
to yield SREBP-1aTg/A4-KO mice and SREBP-1aTg/WT
(SREBP-1aTg/A4-WT) littermates. As shown in Figure 2,
neither apoA-IV mRNA (Figure 2A) nor protein (Figure 2B)
was detected in the livers of SREBP-1aTg/A4-KO mice.
ApoA-IV deficiency produced a small reduction in plasma
TG levels, consistent with previous reports,16 although in
this study the reduction did not reach statistical significance
(Figure 2C). Mice were maintained on the low-carbohydrate
diet for 3 weeks, and then hepatic TG secretion rates were
measured after administration of Triton WR1339 (Triton) to
block peripheral lipolysis. The absence of apoA-IV expression in the SREBP-1aTg/A4-KO mice significantly reduced
plasma TG accumulation at times >30 minutes compared
with the SREBP-1aTg/A4-WT control littermates (Figure 2D)
and reduced the overall hepatic TG secretion rate by 24%
(Figure 2E). Although there was a trend toward increased
hepatic TG content in the SREBP-1aTg/A4-KO mice, this difference was not significant (Figure 2F). These data indicate
that in the absence of apoA-IV, SREBP-1aTg mice were less
efficient in secreting TG from the liver.
To explore the basis for the lower TG secretion rate in
SREBP-1aTg/A4-KO mice, we first considered whether apoB
secretion had been affected. SDS-PAGE and phosphorimager analysis of radiolabeled apoB in plasma VLDL revealed
no differences in the amount of total secreted apoB protein
(Figure 3A and 3B), indicating that apoA-IV gene deletion
did not change the number of secreted apoB-containing
VLDL particles. We next analyzed the size distribution of
VLDL isolated from the Triton block experiments using
negative stain electron microscopy.21 Although the presence
of Triton in the plasma samples might be perturbing, others have assessed particle characteristics in postdetergent
plasma by size exclusion chromatography and negative stain
electron microscopy.22–24 Visual inspection of representative
images suggested that plasma VLDL from the SREBP-1aTg/
A4-WT mice contained a greater proportion of larger lipoprotein particles compared with the SREBP-1aTg/A4-KO
mice (Figure 3C). Indeed, systematic analysis of the particle
size distribution revealed that the percentage of VLDL particles with diameters ≥120 nm was lower in SREBP-1aTg/
A4-KO plasma (Figure 3D and 3E). This finding suggests
that VLDL particle expansion was impaired in the absence of
apoA-IV expression.
Overexpression of Human ApoA-IV Increases
Hepatic TG Secretion in SREBP-1aTg Mice
Because apoA-IV deficiency appeared to decrease the rate
of hepatic TG secretion by reducing VLDL particle size, we
next explored whether overexpression of human apoA-IV
in SREBP-1aTg mouse liver would have the converse effect.
Figure 3. Apolipoprotein A-IV (apoA-IV) deficiency in SREBP-1aTg
mice reduces nascent very low density lipoprotein (VLDL) particle
diameter. Pooled blood samples were collected 180 minutes
after Triton injection, and VLDL was isolated from plasma by
ultracentrifugation, as described under Materials and Methods.
A, Accumulation of 35S-labeled newly synthesized and secreted
apoB in terminal plasma. B, Relative phosphorimager units of
total apoB (apoB100+B48) normalized to SB-Tg/A4-WT. C, VLDL
visualized by negative stain electron microscopy (×49 000).
D, Size distribution of VLDL particles; >500 particles were measured. E, Percentage of total VLDL particles ≥120 nm. Data in B
were analyzed by unpaired Student t test (n=5). A4-KO indicates
Apoa4 knockout; Tg, transgenic; and WT, wild type.
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For this purpose, we generated a recombinant adenovirus
expressing human apoA-IV (ad-huA4) and a control adenovirus expressing bacterial β-galactose (ad-LacZ). Infection
of SREBP-1aTg mice with either ad-huA4 or ad-LacZ caused
abundant hepatic expression of the respective proteins 3 days
postinjection (Figure 4A). Mice receiving ad-huA4 also demonstrated an increase in plasma TG levels to ≈175 mg/dL,
presumably attributable to increased VLDL secretion and/or
inhibition of VLDL clearance. Studies with apoA-IV Tg mice,
with overexpression predominantly in the intestine, also noted
decreased lipolysis, possibly because of displacement of the
lipoprotein lipase coactivator apoC-II from the TG-rich lipoprotein surface by apoA-IV.15,25,26
To explore the impact of apoA-IV overexpression on the
hepatic TG secretion rate, mice treated with ad-huA4 and adLacZ were administered Triton to block peripheral lipolysis
and plasma TG accumulation was measured as a function
of time. In SREBP-1aTg mice treated with ad-huA4, a 52%
increase in the rate of TG secretion was observed relative to
LacZ controls (Figure 4C and 4D). Because the expression of
apoA-IV produced a dramatic increase in the rate and amount
of TG secreted, we examined whether there was a corresponding decrease in hepatic lipid burden. For this purpose, mice
were separately injected with ad-huA4 or ad-LacZ and 3 days
postinjection, animals were euthanized and liver lipids measured. These data revealed a 38% decrease in the hepatic TG
content of mice overexpressing huA4 compared with mice
administered ad-LacZ (Figure 4E). A similar reduction in
hepatic TG content was also observed in the animals that had
Figure 4. Adenoviral overexpression of human apolipoprotein
A-IV (apoA-IV) increases the rate of hepatic triglyceride (TG)
secretion and reduces hepatic lipid content in SREBP-1aTg
mice. SREBP-1aTg (SB-Tg) mice maintained on chow diet were
administered 1.5×109 pfu of either apoA-IV-adenovirus (huA4)
or the control LacZ-adenovirus (LacZ), and experiments were
performed 3 days later. A, LacZ and huA4 protein in liver from 3
separate animals as detected by immunoblot blot analysis.
B, Fasting plasma TG levels. C, Mice were injected with 500
mg/kg Triton WR1339, and TG concentrations were measured
at 0, 30, 60, 120, and 180 minutes postinjection. The basal TG
concentration (t=0) for each time point was subtracted. D, Mean
secretion rate calculated from the slopes of individual plots of
TG versus time. E, Hepatic TG content measured by enzymatic
assay. Data are expressed as mean±SE. For B to D, n=8 for LacZ
and 9 for huA4. For E, n=6, *P<0.01 by unpaired Student t test.
Tg indicates transgenic.
been subjected to the Triton block analysis used in Figure 4C
(data not shown).
To explore the basis of the enhanced TG secretion in the
mice treated with ad-huA4, radiolabeled apoB in VLDL was
isolated from terminal bleeds and quantitated by SDS-PAGE
and phosphorimager analysis. As with the apoA-IV knockout
experiments, no difference in the secretion of radiolabeled
apoB was observed, indicating that overexpression of apoAIV did not increase the number of VLDL particles secreted by
the liver (Figure 5A and 5B). However, negative stain electron
microscopy analysis of VLDL particles revealed that apoAIV overexpression caused a 43% increase in the percentage
of VLDL particles with diameters ≥120 nm (Figure 5C–5E).
These data suggest that the increase in TG secretion and
concomitant decrease in hepatic TG observed in mice overexpressing apoA-IV is because of secretion of larger, more
TG-enriched apoB-containing VLDL, rather than a greater
number of lipoprotein particles. It should be noted that the
lipoprotein diameters observed in the control LacZ mice
were smaller than those in the SB-Tg/A4-WT control mice in
Figure 3. This is likely attributable to the fact that the secretion of very large chylomicron-sized VLDL from SREBP1aTg mice is dependent on dysregulated de novo lipogenesis
and hepatic lipid accretion.20 Because the animals used for
the adenovirus experiments were maintained on a chow diet
Figure 5. Adenoviral overexpression of human apolipoprotein
A-IV (apoA-IV) increases nascent very low density lipoprotein
(VLDL) particle diameter in SREBP-1aTg mice. Blood samples
from the Triton block experiment in Figure 4 were collected at the
180-minute time point. A, VLDL was isolated by ultracentrifugation, followed by immunoprecipitation with anti–apoB antibody,
SDS-PAGE, and phosphorimager analysis. B, Relative phosphorimager units of total apoB (apoB100+B48) normalized to LacZ
(n=4). C, Negative stain electron microscopy (×49 000) of pooled
VLDL. D, Size distribution of VLDL particles; >500 particles were
measured. E, Percentage of total VLDL particles ≥120 nm.
Tg indicates transgenic.
VerHague et al ApoA-IV Promotes Hepatic VLDL Particle Expansion 2505
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(ie, intermediate steatosis), their lipoprotein particles were
likely smaller than those that were maintained on the low-carbohydrate diet, which induces maximal steatosis (Figure 1A).
Recent studies have demonstrated that apoA-IV might
impact apoB lipoprotein particle dynamics by modulating
the expression of the microsomal triglyceride transfer protein
(MTP).27,28 Hence, we specifically examined whether MTP
mRNA or protein mass was affected by apoA-IV. As seen
in Figure 6, neither MTP mRNA nor protein abundance was
affected by apoA-IV deficiency (Figure 6A and 6C) or overexpression (Figure 6B and 6D). We next explored whether other
genes with roles in hepatic lipid metabolism could account for
the altered TG secretion observed in these studies. No changes
in expression of genes responsible for de novo lipogenesis,
fatty acid esterification, intracellular lipolysis, β-oxidation,
or VLDL assembly were observed (Figure III in the onlineonly Data Supplement), with the exception of DGAT1, where
in both the A4-KO and in the ad-huA4 livers we observed a
small (≈1.5- and 2-fold, respectively) but significant increase
in mRNA abundance, relative to controls. Because this pattern of expression could not explain the observed changes in
VLDL-TG secretion, particularly in the knockout situation,
we suggest that this modest change in one lipogenic gene is
not likely to be biologically relevant to the observed effects
of apoA-IV on hepatic TG secretion and instead confirm the
core conclusion that apoA-IV may directly impact the extent
of VLDL lipidation.
Discussion
Although a broad spectrum of physiological functions has
been proposed for apoA-IV,29 a preponderance of evidence
Figure 6. Microsomal triglyceride transfer protein (MTP) abundance is unaltered by apolipoprotein A-IV (apoA-IV) expression.
Relative apoA-IV mRNA and protein levels were measured by
quantitative polymerase chain reaction and immunoblot analysis,
respectively; fold changes are expressed relative to controls.
A, ApoA-IV mRNA abundance in SB-Tg/A4-WT (A4-WT) and
SB-Tg/A4-KO (A4-KO) mice fed a low-carbohydrate diet (n=6 for
A4-WT; n=8 for A4-KO). B, ApoA-IV mRNA abundance in chowfed SB-Tg mice infected with LacZ or huA4 adenoviruses (n=6).
C and D, Pooled liver extracts from A4-WT and A4-KO mice
(C) or LacZ and huA4 adenovirus–infected mice (D) were subjected to immunoblot blot analyses in triplicate using antimouse
MTP antibody. Data in A and B are expressed as mean±SE.
KO indicates knockout; Tg, transgenic; and WT, wild type.
suggests that it plays a role in intestinal lipid absorption
and chylomicron assembly.9,30–33 Indeed, recent studies with
cultured intestinal and hepatoma cells have established that
apoA-IV expression increases bulk TG transport by enabling
secretion of larger TG-rich lipoproteins.12,14 Nonetheless, elucidation of the specific role of apoA-IV in lipid transport in
vivo has remained elusive, for studies with apoA-IV knockout and Tg mice found no gross abnormalities in dietary lipid
absorption.15,16 In part, this may be attributable to the fact that
the intestine possesses a large absorptive reserve capacity and
robust compensatory cellular mechanisms, which can mask
the impact of perturbed apoA-IV expression on lipid transport. However, in rodents, apoA-IV is also expressed in the
liver.34 In this organ, TG uptake, synthesis, and secretion is
controlled by complex metabolic pathways and is regulated
by multiple dietary and hormonal factors, such that even small
changes in the relative rates of TG import and synthesis versus
oxidation and secretion can rapidly lead to intracellular TG
accumulation (steatosis) and inflammation (steatohepatitis).1
We thus reasoned that examining the impact of altered apoAIV expression on hepatic VLDL secretion and TG content
could reveal an unequivocal metabolic phenotype with direct
clinical relevance.
We, therefore, examined the impact of altering hepatic
apoA-IV expression by genetic deletion and adenoviralinduced overexpression on VLDL-TG secretion and cellular
TG content under conditions of hepatic steatosis caused by Tg
expression of the constitutively active form of SREBP-1a.19,20
Using this approach, we not only observed that hepatic apoAIV gene expression and protein levels are increased by conditions that promote hepatic TG accumulation but also, for the
first time, have demonstrated an unequivocal metabolic phenotype directly related to apoA-IV expression: that is, genetic
absence of apoA-IV reduces hepatic TG secretion and VLDL
particle size, whereas adenoviral overexpression of human
apoA-IV enhances VLDL-TG secretion, increases VLDL particle size, raises plasma TG levels, and reduces hepatic TG
content. These data reveal that apoA-IV expression can exert
a powerful impact on hepatic TG export, which is attributable
to its ability to promote VLDL particle expansion within the
secretory pathway.
The parallel increases in hepatic TG and apoA-IV
mRNA content, which we observed in steatosis induced by
increased lipogenesis and a high-fat and high-cholesterol
diet, have been reported in other mouse models of steatosis.
A 100-fold induction of hepatic apoA-IV mRNA was seen
in suckling fatty liver dystrophy (fld) mice, which returned
to baseline when liver TG content rapidly fell upon weaning.35 Williams et al36 also observed that a high-fat diet
induces hepatic apoA-IV expression, although in their studies the effect was strain specific. The strong positive correlation between apoA-IV mRNA abundance (and in the
current study, protein concentration) and hepatic TG content
in all these models of steatosis implies that cellular TG or
fatty acid accumulation may provide a specific signal for
upregulating apoA-IV gene expression. In this regard, the
mechanisms by which lipids regulate hepatic apoA-IV gene
transcription are not well understood.37–39 Recently, it was
discovered that the endoplasmic reticulum (ER)-tethered,
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liver-specific transcription factor cAMP response elementbinding protein H is required for hepatic apoA-IV synthesis.40,41 Activation of cAMP response element-binding
protein H requires translocation from ER to Golgi, proteolytic processing, and release of the active form into the
nucleus. Although the exact metabolic cues that mediate
this activation sequence are unknown,41 cAMP response element-binding protein H expression is enhanced by a number
of metabolic conditions, including fasting, insulin resistance,42 and ER stress,43 all of which also promote VLDL
secretion.44,45 Thus, it is intriguing to consider that the MTPmediated movement of lipid across the ER membrane46–48 or
incorporation into nascent primordial particles in the ER49
integrates apoA-IV gene expression with VLDL assembly,
perhaps via cAMP response element-binding protein H production and/or processing, which then, in turn, serves as the
specific signal that regulates apoA-IV transcription.
A possible issue compromising the interpretation of
results using the A4-KO mouse is the reduction in expression of the neighboring gene, Apoc3.16 Because apoC-III is a
lipase inhibitor, its lower abundance affects TG-rich lipoprotein clearance, as was observed in previous studies16 and in
Figure 2D, in which a nonsignificant decrease in plasma TG
was observed in SREBP-1aTg/A4-KO mice. ApoC-III overexpression, both in hepatoma cells and in vivo, is also associated with enhanced VLDL-TG production.50,51 In contrast,
studies in Apoc3 knockout mice failed to observe reductions
in hepatic TG secretion, even after feeding a high-fat diet.52,53
Hence, it is unlikely that the decreased hepatic VLDL-TG
secretion associated with apoA-IV deficiency observed in
these studies is the result of decreased apoC-III expression.
This conclusion was supported by the 52% increase in TG
secretion observed when apoA-IV was selectively overexpressed using a recombinant adenovirus (Figure 4D).
Although the apoA-IV/C-III effects on lipolysis may also
complicate interpretation of the impact of production versus
clearance on steady-state plasma TG levels, the use of the
Triton block method clearly demonstrates an impact of apoAIV on hepatic VLDL-TG production. Because these changes
were not accompanied by altered apoB secretion, the implication is that modification of particle size and not particle number was the basis for the observed effects. This conclusion
was supported by direct analysis of particle diameters from
post-Triton block plasma in which, based on the changes in
TG values between the 0- and 180-minute time points, ≈90%
to 95% of the TG-rich particles present were produced under
conditions of lipase inhibition.
Two mechanisms have been proposed to explain how
apoA-IV facilitates the assembly of larger lipoprotein particles. Because only a single molecule of apoB is incorporated
into each nascent lipid particle in the first step of TG-rich
lipoprotein assembly, the expansion of the TG core in the
second stage of assembly exposes surface lipids to the aqueous milieu, which decreases their free energy of stabilization.54,55 Because the interfacial properties of apoA-IV are
ideally suited to stabilizing expanding lipid interfaces,56,57 we
have proposed that adsorption of apoA-IV molecules to the
expanding particle surface renders core lipid incorporation
more thermodynamically favorable, which thus facilitates
particle growth.58 However, we have also presented evidence
in cultured rat hepatoma cells that a protein–protein interaction between apoA-IV and a domain in the amino terminus of
apoB delays the secretory trafficking of nascent TG-rich lipoproteins in a manner that enables their cores to become more
fully lipidated before final secretion.14 These mechanisms are
not mutually exclusive.
Another possible explanation for the impact of apoA-IV
on VLDL-TG secretion centers on its ability to modulate
MTP expression or the expression of other genes involved in
hepatic lipid metabolism. Previous studies demonstrated that
apoA-IV stimulates MTP expression in both intestine and
Huh7 hepatoma cells via the regulation of the transcription
factors FoxA2 and FoxO1.27,28 However, in the current study,
we failed to observe a similar relationship between apoA-IV
and MTP expression (Figure 6). A further exploration of genes
involved in lipid metabolism also failed to identify a pattern
of gene expression that could account for the enhanced TG
secretion associated with apoA-IV expression (Figure III in
the online-only Data Supplement). Again, these data suggest
that apoA-IV may play a direct role in the modulation of apoB
lipoprotein particle expansion and TG transport.
In summary, we have demonstrated in 2 models of liver steatosis that hepatic TG accumulation and apoA-IV RNA levels increase in parallel and that adenoviral-mediated hepatic
apoA-IV expression increases the rate of hepatic VLDL-TG
transport and reduces liver TG content by enabling the assembly of larger apoB-containing lipoprotein particles, rather
than by increasing the number of particles that are secreted.
Because efficient TG export is an important means by which
the liver protects itself from toxic accumulation of intracellular lipids, these data suggest that increasing hepatic apoAIV expression by dietary, pharmacological, or biological
approaches could constitute a novel strategy for treating nonalcoholic fatty liver disease, without raising the long-term risk
of atherosclerotic cardiovascular heart disease attributable to
secretion of increased numbers of atherogenic apoB-containing lipoproteins.
Sources of Funding
This work was supported by National Institutes of Health grants
HL-049373 (G.S. Shelness) and HL-30897 (R.B. Weinberg). M.A.
VerHague is supported by an RJR-Leon Golberg Post-doctoral
Fellowship.
Disclosures
None.
References
1. Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions
and new insights. Science. 2011;332:1519–1523.
2. Bellentani S, Marino M. Epidemiology and natural history of non-­alcholoic
fatty liver disease (NAFLD). Ann Hepatol. 2009;8(suppl 1):S4–S8.
3. Fisher EA, Ginsberg HN. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins. J Biol
Chem. 2002;277:17377–17380.
4. Shelness GS, Ledford AS. Evolution and mechanism of apolipoprotein
B-containing lipoprotein assembly. Curr Opin Lipidol. 2005;16:325–332.
5. Sundaram M, Yao Z. Intrahepatic role of exchangeable apolipoproteins
in lipoprotein assembly and secretion. Arterioscler Thromb Vasc Biol.
2012;32:1073–1078.
VerHague et al ApoA-IV Promotes Hepatic VLDL Particle Expansion 2507
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
6. Swaney JB, Braithwaite F, Eder HA. Characterization of the apolipoproteins of rat plasma lipoproteins. Biochemistry. 1977;16:271–278.
7. Weisgraber KH, Bersot TP, Mahley RW. Isolation and characterization of
an apoprotein from the d less than 1.006 lipoproteins of human and canine
lymph homologous with the rat A-IV apoprotein. Biochem Biophys Res
Commun. 1978;85:287–292.
8. Wu AL, Windmueller HG. Identification of circulating apolipoproteins synthesized by rat small intestine in vivo. J Biol Chem. 1978;253:2525–2528.
9.Green PH, Glickman RM, Riley JW, Quinet E. Human apolipoprotein A-IV. Intestinal origin and distribution in plasma. J Clin Invest.
1980;65:911–919.
10. Kalogeris TJ, Rodriguez MD, Tso P. Control of synthesis and secretion of
intestinal apolipoprotein A-IV by lipid. J Nutr. 1997;127:537S–543S.
11. Lu S, Yao Y, Meng S, Cheng X, Black DD. Overexpression of apolipoprotein A-IV enhances lipid transport in newborn swine intestinal epithelial
cells. J Biol Chem. 2002;277:31929–31937.
12.Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW,
Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg
RB, Black DD. Overexpression of apolipoprotein A-IV enhances lipid
secretion in IPEC-1 cells by increasing chylomicron size. J Biol Chem.
2006;281:3473–3483.
13. Gallagher JW, Weinberg RB, Shelness GS. apoA-IV tagged with the ER
retention signal KDEL perturbs the intracellular trafficking and secretion
of apoB. J Lipid Res. 2004;45:1826–1834.
14. Weinberg RB, Gallagher JW, Fabritius MA, Shelness GS. ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich
lipoproteins. J Lipid Res. 2012;53:736–743.
15.Aalto-Setälä K, Bisgaier CL, Ho A, Kieft KA, Traber MG, Kayden
HJ, Ramakrishnan R, Walsh A, Essenburg AD, Breslow JL. Intestinal
expression of human apolipoprotein A-IV in transgenic mice fails to
influence dietary lipid absorption or feeding behavior. J Clin Invest.
1994;93:1776–1786.
16. Weinstock PH, Bisgaier CL, Hayek T, Aalto-Setala K, Sehayek E, Wu L,
Sheiffele P, Merkel M, Essenburg AD, Breslow JL. Decreased HDL cholesterol levels but normal lipid absorption, growth, and feeding behavior
in apolipoprotein A-IV knockout mice. J Lipid Res. 1997;38:1782–1794.
17. Simon T, Cook VR, Rao A, Weinberg RB. Impact of murine intestinal apolipoprotein A-IV expression on regional lipid absorption, gene expression,
and growth. J Lipid Res. 2011;52:1984–1994.
18. Browning JD, Horton JD. Molecular mediators of hepatic steatosis and
liver injury. J Clin Invest. 2004;114:147–152.
19. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein
JL. Overproduction of cholesterol and fatty acids causes massive liver
enlargement in transgenic mice expressing truncated SREBP-1a. J Clin
Invest. 1996;98:1575–1584.
20.Horton JD, Shimano H, Hamilton RL, Brown MS, Goldstein JL.
Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks
hyperlipidemia resulting from production of lipid-rich VLDL. J Clin
Invest. 1999;103:1067–1076.
21. Johnson FL, St Clair RW, Rudel LL. Effects of the degree of saturation of
dietary fat on the hepatic production of lipoproteins in the African green
monkey. J Lipid Res. 1985;26:403–417.
22. Khatun I, Zeissig S, Iqbal J, Wang M, Curiel D, Shelness GS, Blumberg
RS, Hussain MM. Phospholipid transfer activity of microsomal triglyceride transfer protein produces apolipoprotein B and reduces hepatosteatosis while maintaining low plasma lipids in mice. Hepatology.
2012;55:1356–1368.
23. Nassir F, Xie Y, Patterson BW, Luo J, Davidson NO. Hepatic secretion of
small lipoprotein particles in apobec-1-/- mice is regulated by the LDL
receptor. J Lipid Res. 2004;45:1649–1659.
24.Millar JS, Cromley DA, McCoy MG, Rader DJ, Billheimer JT.
Determining hepatic triglyceride production in mice: comparison of
poloxamer 407 with Triton WR-1339. J Lipid Res. 2005;46:2023–2028.
25. Cohen RD, Castellani LW, Qiao JH, Van Lenten BJ, Lusis AJ, Reue K.
Reduced aortic lesions and elevated high density lipoprotein levels in
transgenic mice overexpressing mouse apolipoprotein A-IV. J Clin Invest.
1997;99:1906–1916.
26.Ostos MA, Conconi M, Vergnes L, Baroukh N, Ribalta J, Girona J,
Caillaud JM, Ochoa A, Zakin MM. Antioxidative and antiatherosclerotic
effects of human apolipoprotein A-IV in apolipoprotein E-deficient mice.
Arterioscler Thromb Vasc Biol. 2001;21:1023–1028.
27. Yao Y, Lu S, Huang Y, Beeman-Black CC, Lu R, Pan X, Hussain MM,
Black DD. Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells. Am J Physiol
Gastrointest Liver Physiol. 2011;300:G357–G363.
28.Pan X, Munshi MK, Iqbal J, Queiroz J, Sirwi AA, Shah S, Younus A,
Hussain MM. Circadian regulation of intestinal lipid absorption by apolipoprotein AIV involves forkhead transcription factors A2 and O1 and microsomal triglyceride transfer protein. J Biol Chem. 2013;288:20464–20476.
29.Stan S, Delvin E, Lambert M, Seidman E, Levy E. Apo A-IV: an
update on regulation and physiologic functions. Biochim Biophys Acta.
2003;1631:177–187.
30. Kalogeris TJ, Rodriguez MD, Tso P. Control of synthesis and secretion of
intestinal apolipoprotein A-IV by lipid. J Nutr. 1997;127:537S–543S.
31. Kalogeris TJ, Fukagawa K, Tso P. Synthesis and lymphatic transport of
intestinal apolipoprotein A-IV in response to graded doses of triglyceride.
J Lipid Res. 1994;35:1141–1151.
32.Tso P, Balint JA, Bishop MB, Rodgers JB. Acute inhibition of intestinal lipid transport by Pluronic L-81 in the rat. Am J Physiol.
1981;241:G487–G497.
33. Koga S, Miyata Y, Funakoshi A, Ibayashi H. Plasma apolipoprotein A-IV
levels decrease in patients with chronic pancreatitis and malabsorption
syndrome. Digestion. 1985;32:19–24.
34. Williams SC, Grant SG, Reue K, Carrasquillo B, Lusis AJ, Kinniburgh AJ.
cis-acting determinants of basal and lipid-regulated apolipoprotein A-IV
expression in mice. J Biol Chem. 1989;264:19009–19016.
35.Langner CA, Birkenmeier EH, Ben-Zeev O, Schotz MC, Sweet HO,
Davisson MT, Gordon JI. The fatty liver dystrophy (fld) mutation. A new
mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic
lipase activities. J Biol Chem. 1989;264:7994–8003.
36. Williams SC, Bruckheimer SM, Lusis AJ, LeBoeuf RC, Kinniburgh AJ.
Mouse apolipoprotein A-IV gene: nucleotide sequence and induction by a
high-lipid diet. Mol Cell Biol. 1986;6:3807–3814.
37.Le Beyec J, Chauffeton V, Kan HY, Janvier PL, Cywiner-Golenzer C,
Chatelet FP, Kalopissis AD, Zannis V, Chambaz J, Pinçon-Raymond M,
Cardot P. The -700/-310 fragment of the apolipoprotein A-IV gene combined with the -890/-500 apolipoprotein C-III enhancer is sufficient to
direct a pattern of gene expression similar to that for the endogenous apolipoprotein A-IV gene. J Biol Chem. 1999;274:4954–4961.
38. Archer A, Sauvaget D, Chauffeton V, Bouchet PE, Chambaz J, PinçonRaymond M, Cardot P, Ribeiro A, Lacasa M. Intestinal apolipoprotein
A-IV gene transcription is controlled by two hormone-responsive elements: a role for hepatic nuclear factor-4 isoforms. Mol Endocrinol.
2005;19:2320–2334.
39.Vergnes L, Taniguchi T, Omori K, Zakin MM, Ochoa A. The apolipoprotein A-I/C-III/A-IV gene cluster: ApoC-III and ApoA-IV expression is regulated by two common enhancers. Biochim Biophys Acta.
1997;1348:299–310.
40. Lee JH, Giannikopoulos P, Duncan SA, Wang J, Johansen CT, Brown JD,
Plutzky J, Hegele RA, Glimcher LH, Lee AH. The transcription factor
cyclic AMP-responsive element-binding protein H regulates triglyceride
metabolism. Nat Med. 2011;17:812–815.
41. Lee AH. The role of CREB-H transcription factor in triglyceride metabolism. Curr Opin Lipidol. 2012;23:141–146.
42. Lee MW, Chanda D, Yang J, Oh H, Kim SS, Yoon YS, Hong S, Park KG,
Lee IK, Choi CS, Hanson RW, Choi HS, Koo SH. Regulation of hepatic
gluconeogenesis by an ER-bound transcription factor, CREBH. Cell
Metab. 2010;11:331–339.
43. Zhang K, Shen X, Wu J, Sakaki K, Saunders T, Rutkowski DT, Back SH,
Kaufman RJ. Endoplasmic reticulum stress activates cleavage of CREBH
to induce a systemic inflammatory response. Cell. 2006;124:587–599.
44.Malmström R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki
Järvinen H, Shepherd J, Taskinen MR. Defective regulation of triglyceride metabolism by insulin in the liver in NIDDM. Diabetologia. 1997;40:
454–462.
45. Danno H, Ishii KA, Nakagawa Y, et al. The liver-enriched transcription
factor CREBH is nutritionally regulated and activated by fatty acids and
PPARalpha. Biochem Biophys Res Commun. 2010;391:1222–1227.
46. Wang Y, Tran K, Yao Z. The activity of microsomal triglyceride transfer
protein is essential for accumulation of triglyceride within microsomes in
McA-RH7777 cells. A unified model for the assembly of very low density
lipoproteins. J Biol Chem. 1999;274:27793–27800.
47.Kulinski A, Rustaeus S, Vance JE. Microsomal triacylglycerol transfer
protein is required for lumenal accretion of triacylglycerol not associated with ApoB, as well as for ApoB lipidation. J Biol Chem. 2002;277:
31516–31525.
48. Hamilton RL, Wong JS, Cham CM, Nielsen LB, Young SG. Chylomicronsized lipid particles are formed in the setting of apolipoprotein B deficiency. J Lipid Res. 1998;39:1543–1557.
2508 Arterioscler Thromb Vasc Biol November 2013
49. Hussain MM, Shi J, Dreizen P. Microsomal triglyceride transfer protein
and its role in apoB-lipoprotein assembly. J Lipid Res. 2003;44:22–32.
50. Sundaram M, Zhong S, Bou Khalil M, Links PH, Zhao Y, Iqbal J, Hussain
MM, Parks RJ, Wang Y, Yao Z. Expression of apolipoprotein C-III in
McA-RH7777 cells enhances VLDL assembly and secretion under lipidrich conditions. J Lipid Res. 2010;51:150–161.
51. Aalto-Setälä K, Fisher EA, Chen X, Chajek-Shaul T, Hayek T, Zechner
R, Walsh A, Ramakrishnan R, Ginsberg HN, Breslow JL. Mechanism of
hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice.
Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles. J Clin
Invest. 1992;90:1889–1900.
52. Jong MC, Rensen PC, Dahlmans VE, van der Boom H, van Berkel TJ,
Havekes LM. Apolipoprotein C-III deficiency accelerates triglyceride
hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice.
J Lipid Res. 2001;42:1578–1585.
53. Duivenvoorden I, Teusink B, Rensen PC, Romijn JA, Havekes LM, Voshol
PJ. Apolipoprotein C3 deficiency results in diet-induced obesity and
aggravated insulin resistance in mice. Diabetes. 2005;54:664–671.
54. Alexander CA, Hamilton RL, Havel RJ. Subcellular localization of B apoprotein of plasma lipoproteins in rat liver. J Cell Biol. 1976;69:241–263.
55. Olofsson SO, Asp L, Borén J. The assembly and secretion of apolipoprotein B-containing lipoproteins. Curr Opin Lipidol. 1999;10:341–346.
56. Weinberg RB, Cook VR, DeLozier JA, Shelness GS. Dynamic interfacial
properties of human apolipoproteins A-IV and B-17 at the air/water and
oil/water interface. J Lipid Res. 2000;41:1419–1427.
57. Weinberg RB, Anderson RA, Cook VR, Emmanuel F, Denefle P, Hermann
M, Steinmetz A. Structure and interfacial properties of chicken apolipoprotein A-IV. J Lipid Res. 2000;41:1410–1418.
58. Rosen MJ. Emulsification by surfactants. In: Rosen MJ, ed. Surfactants
and Interfacial Phenomena. New York, NY: John Wiley & Sons, Inc;
2004:303–331.
Significance
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Plasma apolipoprotein B (apoB) lipoprotein particle number may be the best predictor of susceptibility to atherosclerotic cardiovascular disease. Because nascent very low density lipoprotein particles are heterogeneous, an important issue is how the hepatocyte integrates particle
number with particle size to achieve a given rate of hepatic lipid efflux. The current studies revealed that in mouse liver, apoA-IV is acutely
regulated by hepatic triglyceride content and modulates hepatic lipid efflux by promoting nascent very low density lipoprotein particle expansion. Furthermore, human apoA-IV overexpression in steatotic mouse liver both stimulated very low density lipoprotein-triglyceride secretion
and dramatically reduced hepatic lipid content. Defining this previously unexplored role of apoA-IV in hepatic lipid transport, in vivo, has
important translational potential, for if very low density lipoprotein–mediated hepatic lipid efflux could be achieved by a process of particle
expansion, at the expense of particle number, this would simultaneously protect the liver from ectopic lipid accumulation while potentially
generating a less atherogenic lipoprotein profile.
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Apolipoprotein A-IV Expression in Mouse Liver Enhances Triglyceride Secretion and
Reduces Hepatic Lipid Content by Promoting Very Low Density Lipoprotein Particle
Expansion
Melissa A. VerHague, Dongmei Cheng, Richard B. Weinberg and Gregory S. Shelness
Arterioscler Thromb Vasc Biol. 2013;33:2501-2508; originally published online September 12,
2013;
doi: 10.1161/ATVBAHA.113.301948
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Data Supplement
Apolipoprotein A-IV Expression in Mouse Liver Enhances Triglyceride Secretion
and Reduces Hepatic Lipid Content by Promoting VLDL Particle Expansion
Melissa A. VerHague, Dongmei Cheng, Richard B. Weinberg,
and Gregory S. Shelness
Supplementary Figure I. Production and characterization of rabbit polyclonal antiserum against
murine apoA-IV. Full-length mature murine apoA-IV cDNA was cloned to form a fusion protein
with bacterial maltose binding protein in plasmid pMAL-c2X (New England Biolabs). Fusion
protein in bacterial extract was purified by amylose affinity chromatography (New England
Biolabs), as recommended by the supplier. Rabbits were immunized with 0.5 mg of purified
fusion protein on day 1 and 21 and on day 28, serum was obtained and tested. A and B, COS
cells were transfected with either a control protein (human serum albumin; HSA), mouse apoAIV (mA4), or C-terminal FLAG-tagged mouse apoA-IV (mA4-F), as indicated. Cell lysates (50
µg) were fractionated by 12.5% SDS-PAGE, followed by immunoblot analysis with 28 day postimmune serum from rabbit #13477 (Rα-mA4-MBP) (A) or anti-FLAG monoclonal antibody M2
(Sigma) (B). C, 1 µL of plasma from wild-type or apoA-IV knock out (A4-KO) mice and 50 µg
COS cell extract, containing transfected mouse apoA-IV (mA4 in COS), were fractionated by
12.5% SDS-PAGE. D, 50 µg of whole liver protein extract from high fat/cholesterol diet (HFCD)
fed wild-type (WT1 and WT2), A4-KO mice, and 1 µL wild type mouse plasma, were
fractionated by 12.5% SDS-PAGE. For both C and D, immunoblot analyses were performed
with Rα-mA4-MBP. In D, the arrow indicates the position of mouse apoA-IV, which is absent in
the A4-KO livers but appears to have a slightly different gel mobility relative to plasma apoA-IV,
perhaps due to differences in O-linked glycosylation1.
Supplementary Figure II. Production and characterization of rabbit polyclonal antiserum
against human apoA-IV. Full-length mature human apoA-IV cDNA was cloned to form a fusion
protein with bacterial maltose binding protein in plasmid pMAL-c2X (New England Biolabs).
Fusion protein in bacterial extracts was purified by amylose affinity chromatography (New
England Biolabs), as recommended by the supplier. Rabbits were immunized with 0.5 mg each
of purified fusion protein on days 1, 21 and 28 and on day 52 production bleeds were obtained
and tested. A, McA-RH7777 cells were transfected with plasmids expressing either a control
protein (human serum albumin; HSA) or human apoA-IV (hA4), as indicated. Cell lysates (50
µg) were fractionated by 12.5% SDS-PAGE followed by immunoblot analysis with post-immune
serum from rabbit #5344. B and C, Wild-type mice were administered 1.5x109 pfu of either
apoA-IV-adenovirus (huA4) or the control LacZ-adenovirus (LacZ). One µL of plasma (B) or 50
µg of liver protein lysate (C) were fractionated by 12.5% SDS-PAGE, followed by immunoblot
analysis with the same anti-serum used in A.
Supplementary Figure III. Quantitative PCR of hepatic lipid metabolism genes. A, Liver RNA
samples from SREBP-1aTg/A4-WT (A4-WT) and SBEBP-1aTg/A4-KO (A4-KO) mice, maintained
on a low carbohydrate diet, were used for quantitative PCR; data are normalized to A4-WT. B,
Liver RNA samples from SREBP-1a mice maintained on chow diet and infected with Ad-LacZ
(LacZ) or Ad-huA4 (huA4) were used for quantitative PCR; data are normalized to LacZ. Data
are expressed as mean ±SE. For A, n=6 for A4-WT and 8 for A4-KO; For B, n=6 per group. *,
P<0.05 by Student’s unpaired T-test; all other differences were not significant. Quantitative PCR
for LDLr and LRP were performed using pooled RNA samples. ACC-1 and -2, Acetyl-CoA
carboxylase 1 and 2; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase 1; DGAT 1 and
2, diacylglycerol O-acyltransferase 1 and 2; CPT-1, Carnitine palmitoyltransferase 1; LCAD,
Long-chain acyl-coenzyme A dehydrogenase; MCAD, Medium-chain acyl-CoA dehydrogenase;
HADH, hydroxyacyl-CoA dehydrogenase; ACOX, Peroxisomal acyl-coenzyme A oxidase 1;
ATGL, adipocyte triglyceride lipase; CGI-58; comparative gene identification-58; HSL, hormone
sensitive lipase; TGH, triacylglycerol hydrolase, PLTP, phospholipid transfer protein; ApoB,
apolipoprotein B; LDLr, LDL receptor; LRP, LDL receptor related protein 1.
Reference
1.
Weinberg RB, Scanu AM. Isolation and characterization of human apolipoprotein
A-IV from lipoprotein-depleted serum. J.Lipid Res. 1983;24:52-59.
Apolipoprotein A-IV Expression in Mouse Liver Enhances Triglyceride Secretion
and Reduces Hepatic Lipid Content by Promoting VLDL Particle Expansion
Melissa A. VerHague, Dongmei Cheng, Richard B. Weinberg,
and Gregory S. Shelness
Materials and Methods
Animals
All animal procedures were approved by the Wake Forest School of Medicine Animal
Care and Use Committee. C57BL/6 mice (Harlan) were fed either a chow diet or a high
fat and cholesterol diet (HFCD) containing (as percent of calories) 48% fat, 36%
carbohydrate, 16% protein and 0.2% w/w cholesterol for 16 weeks. The diet
composition per 100 grams was as follows: 23.0 g lard, 2.5 g soybean oil, 22.0 g
sucrose, 11.3 g maltodextrin, 8.0 g dextrin, 19.3 g casein, 5.0 g Hegsted salt mixture,
0.35 g L-cystine, 2.5 g vitamin mix, 5.85 g cellulose, and 0.2 g crystalline cholesterol.
SREBP-1aTg mice (The Jackson Laboratory, stock #002840) were crossed with Apoa4
knock out mice1 to generate SREBP-1aTg/A4-KO and SREBP-1aTg/A4-WT littermates.
Transgenic mice were maintained on chow diet throughout the study or at 6–8 weeks of
age were switched to a low carbohydrate, high protein diet (Purina Test Diet #5789) for
2–3 weeks. Mice were euthanized with ketamine and xylazine. Blood was collected via
heart puncture, and organs were perfused with saline. Livers were harvested and snap
frozen in liquid N2.
Recombinant Adenoviruses
Recombinant adenoviruses expressing genes for either human apoA-IV or LacZ, under
control of the human cytomegalovirus promoter, were constructed using the Adeno-X
system (Clontech #631513). Recombinant adenoviruses were expanded in HEK293
cells, purified by cesium chloride gradient ultracentrifugation, and stored in 10% (v/v)
glycerol in phosphate-buffered saline at -80°C, as recommended by the supplier. Retroorbital injection of 1.5 x 109 plaque forming units (pfu) of adenovirus per mouse was
administered under isofluorane sedation. Experiments were performed three days after
injection. The extent of hepatic expression of human apoA-IV is documented in Figure 4
by immunoblot analysis. Although, the absolute level of overexpression cannot be
directly ascertained, because our anti-human and anti-mouse apoA-IV antisera do not
cross-react, it should be noted that the relative band intensities of mouse apoA-IV in low
carbohydrate diet-fed SREBP-1a transgenic mouse liver (~40-fold induction; Figure 1C)
and human apoA-IV adenovirus-infected livers (Figure 4A) is nearly identical. Although
these antisera may not have identical sensitivities, both were raised in rabbits against
full-length mature apoA-IV fused to maltose binding protein. Assuming a similar degree
of sensitivity, this comparison would imply that Ad-A4 achieves a similar ~40-fold level
of overexpression of human apoA-IV as is observed for the TG-mediated induction of
the endogenous gene in mouse.
1
mRNA and Protein Quantification
RNA was extracted from frozen liver samples using TRIzol (Invitrogen). Total RNA (2
µg; 20 µl reaction volume) was reverse transcribed into cDNA with random primers
using the Omniscript RT kit (Qiagen) or qScript cDNA Supermix (Quanta Biosciences).
Quantitative PCR (qPCR) was performed using a 7500 Fast Real Time PCR System
(Applied Biosystems, Foster City, CA). A typical PCR reaction (20 µl) contained 10 µl
2X Fast SYBR Green Master Mix (Applied Biosystems), 1 µl each of 5 µM forward and
reverse primers, and 5 µl of cDNA reaction mixture that was previously diluted 1:20 into
water. Copy numbers were normalized to GAPDH. The following primers were used for
mouse apoA-IV: forward, TTC CTG AAG GCT GCG GTG CTG; reverse, CTG CTG
AGT GAC ATC CGT CTT CTG. For immunoblot analysis ~500 mg of frozen tissue was
homogenized with a Polytron homogenizer in lysis buffer (25 mM Tris HCl pH 7.4, 300
mM NaCl and 1% Triton X-100, 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin).
Protein was separated by SDS-PAGE and then transferred to a PVDF membrane.
ApoA-IV protein was detected using rabbit anti-serum against mouse apoA-IV (1:1000
dilution. Supplementary Figure I) or human apoA-IV (1:2000 dilution) (Supplementary
Figure II). LacZ was detected with a mouse anti-β-galactosidase monoclonal antibody
(Sigma #G-4644). Mouse MTP was detected with a mouse monoclonal antibody (BD
Biosciences #612022).
Analysis of Plasma and Liver Lipids
Following a 4 hour fast, blood was collected by heart puncture and placed into a tube
containing a protease inhibitor cocktail (Sigma #P2714) dissolved in 0.05% EDTA,
0.05% NaN3. Samples were centrifuged at 12,000 x g for 10 min at 4°C, and the plasma
was analyzed for TG concentration using an enzymatic colorimetric assay kit
(Triglycerides/GB kit, Wako). For analysis of liver lipids, ~100 mg of liver was thawed,
minced, and weighed in a glass tube. Lipids were extracted in CHCl3:methanol (2:1)2
and dried down under a stream of nitrogen. Triton X-100 (1%) in CHCl3 was then added
and the solvent was again evaporated under nitrogen3. Deionized water was added and
each tube was vortexed until the solution was clear. Lipids were then quantified by
enzymatic colorimetric assay, as described above for plasma.
Hepatic TG and ApoB Secretion Rates
Following a 4 hour fast, mice were sedated by isofluorane inhalation and then
administered 500 mg/kg Triton WR1339 (Triton; Sigma #T0307-5G) and in some
studies, 200 µCi [35S]Met/Cys (PerkinElmer) by retro-orbital injection. Blood was
collected in heparinized capillary tubes by retro-orbital bleeding at 0 (before injection),
30, 60, 120, and 180 min. TG concentration in plasma samples was measured by
enzymatic assay. TG secretion rates were calculated as the mean of the slopes of linear
regressions of TG concentration versus time for each individual animal, using GraphPad
Prism 5.
To measure secretion of newly synthesized apoB, 30 µl of post-Triton block plasma was
diluted in 2 mL of saline containing 1mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin,
10 mM EDTA and centrifuged at 100,000 rpm in a TL-100 centrifuge for 4 hours at 4°C
using a TLA 100.2 rotor (Beckman). The d<1.006 g/mL (VLDL) fraction was collected
2
from the top 0.5 ml of the tube by tube slicing. The sample was then adjusted to lysis
buffer conditions (above) and 0.2% bovine serum albumin using concentrated stocks.
Samples were immunoprecipitated by addition of 5 µl of rabbit anti-mouse apoB
antibody (Biodesign International [Meridian Life Science] #K23300R). After 18 h of
incubation with rotation at 4° C, 20 µl of protein G-Sepharose (50:50 slurry; Amersham
Biosciences) was added to the samples, followed by an additional 90 min incubation.
Beads were collected by centrifugation at 10,000 rpm for 10 s and washed three times
with lysis buffer. Proteins were eluted from the beads by heating in SDS-PAGE sample
buffer at 100°C for 5 min, and then fractionated by 12.5% SDS-PAGE. Gels were then
dried and visualized with a Fuji BAS5000 PhosphorImager.
Electron Microscopy of Plasma VLDL
Negative stain electron microscopy of VLDL from post-Triton terminal bleeds was
performed as described4. The d<1.006 g/mL (VLDL) fraction obtained as above, was
collected from the top 0.5 ml of the tube by tube slicing. VLDL were absorbed onto
Formvar substrate 200 mesh copper grids stabilized with carbon (Pelco #01801) for 30
seconds. The grid was then stained with 2% phosphotungstic acid (pH 6.6) for 1 minute,
and excess stain was removed with filter paper. Grids were then examined using a FEI
Technai BioTwin 120 keV transmission electron microscope.
Statistics
Results are presented as means ± SE. Data were analyzed using GraphPad Prism by
unpaired Student's t-test or one-way analysis of variance (ANOVA) with Tukey's
multiple comparisons, as indicated. Significant differences are indicated with symbols
defined within the legend of each figure; where no symbol is indicated, differences of
mean values did not reach statistical significance (P>0.05).
References
1.
Simon T, Cook VR, Rao A, Weinberg RB. Impact of murine intestinal
apolipoprotein A-IV expression on regional lipid absorption, gene expression, and
growth. J Lipid Res. 2011;52:1984-1994.
2.
Temel RE, Lee RG, Kelley KL, Davis MA, Shah R, Sawyer JK, Wilson MD, Rudel
LL. Intestinal cholesterol absorption is substantially reduced in mice deficient in
both ABCA1 and ACAT2. J Lipid Res. 2005;46:2423-2431.
3.
Carr TP, Andresen CJ, Rudel LL. Enzymatic determination of triglyceride, free
cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem.
1993;26:39-42.
4.
Johnson FL, St Clair RW, Rudel LL. Effects of the degree of saturation of dietary
fat on the hepatic production of lipoproteins in the African green monkey. J Lipid
Res. 1985;26:403-417.
3