Sterol Regulatory ElementBinding Protein-1 Determines

Plasma Remnant Lipoproteins and Accelerates

Atherosclerosis in Low-Density Lipoprotein
ReceptorDeficient Mice
Tadayoshi Karasawa, Akimitsu Takahashi, Ryo Saito, Motohiro Sekiya, Masaki Igarashi,
Hitoshi Iwasaki, Shoko Miyahara, Saori Koyasu, Yoshimi Nakagawa, Kiyoaki Ishii,
Takashi Matsuzaka, Kazuto Kobayashi, Naoya Yahagi, Kazuhiro Takekoshi, Hirohito Sone,
Shigeru Yatoh, Hiroaki Suzuki, Nobuhiro Yamada, Hitoshi Shimano
ObjectiveSterol regulatory element binding protein-1 (SREBP-1) is nutritionally regulated and is known to be a key
transcription factor regulating lipogenic enzymes. The goal of this study was to evaluate the roles of SREBP-1 in
dyslipidemia and atherosclerosis.
Methods and ResultsTransgenic mice that overexpress SREBP-1c in the liver and SREBP-1-deficient mice were crossed
with low-density lipoprotein receptor (LDLR) deficient mice, and the plasma lipids and atherosclerosis were analyzed.
Hepatic SREBP-1c overexpression in LDLR-deficient mice caused postprandial hypertriglyceridemia, increased
very-low-density lipoprotein (VLDL) cholesterol, and decreased high-density lipoprotein cholesterol in plasma, which
resulted in accelerated aortic atheroma formation. Conversely, absence of SREBP-1 suppressed Western dietinduced
hyperlipidemia in LDLR-deficient mice and ameliorated atherosclerosis. In contrast, bone marrow-specific SREBP-1
deficiency did not alter the development of atherosclerosis. The size of nascent VLDL particles secreted from the liver
was increased in SREBP-1c transgenic mice and reduced in SREBP-1-deficient mice, accompanied by upregulation and
downregulation of phospholipid transfer protein expression, respectively.
ConclusionHepatic SREBP-1c determines plasma triglycerides and remnant cholesterol and contributes to atherosclerosis in hyperlipidemic states. Hepatic SREBP-1c also regulates the size of nascent VLDL particles. (Arterioscler
Thromb Vasc Biol. 2011;31:1788-1795.)
Key Words: atherosclerosis hyperlipoproteinemia lipids lipoproteins triglycerides

terol regulatory element binding proteins (SREBPs) are

transcription factors that belong to the basic helix-loophelix leucine zipper family.1,2 Unlike other basic helix-loophelix leucine zipper family transcription factors, SREBPs are
synthesized as membrane-bound precursors and embedded in
the endoplasmic reticulum membrane. There, they form a
complex with SREBP cleavage-activating protein. When
cellular cholesterol levels are depleted, SREBP cleavageactivating protein escorts SREBPs to the Golgi apparatus,
where SREBPs are cleaved by site-1 and site-2 proteases.
After cleavage, SREBPs transfer to the nucleus and activate
enzymes involved in lipid synthesis. The SREBP family
consists of 3 isoforms: SREBP-1a, SREBP-1c, and SREBP-2.
SREBP-1a and SREBP-1c are derived from a single gene
through the use of alternative promoters. SREBP-1a has a

potent transcriptional activity for genes involved in synthesis

of cholesterol, fatty acids, and triglycerides (TGs).3 In contrast, SREBP-1c has a transcriptional activity for genes
involved in fatty acid and TG synthesis.4,5 SREBP-2 selectively activates transcription of genes involved in cholesterol
synthesis.6 Unlike SREBP-1a, SREBP-1c is regulated by
nutritional conditions and therefore plays a central role in
nutritional regulation of lipogenesis as a dominant isoform in
liver and adipose tissues.7 Whereas SREBP-1c levels are low
in fasting states, they are dramatically increased in refed
states in response to higher levels of glucose and insulin.8 10
Owing to its activation by overnutrition, SREBP-1c could be
the cause of several metabolic disorders. Previous studies
revealed that SREBP-1 is involved in hepatic steatosis and
insulin resistance.11,12 Because SREBP-1c is activated in the

Received on: November 6, 2010; final version accepted on: April 6, 2011.
From the Departments of Internal Medicine (Endocrinology and Metabolism) (T.K., A.T., R.S., H.I., S.M., S.K., Y.N., K.I., T.M., K.K., N. Yahagi,
H. Sone, S.Y., H. Suzuki, N. Yamada, H. Shimano) and Clinical Pathology (K.T.), Graduate School of Comprehensive Human Sciences, University of
Tsukuba, Ibaraki, Japan; Department of Internal Medicine (M.S., M.I.), University of Tokyo, Tokyo, Japan.
Drs Karasawa and Takahashi contributed equally to this work.
Correspondence to Hitoshi Shimano, Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human
Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail hshimano@md.tsukuba.ac.jp
2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

DOI: 10.1161/ATVBAHA.110.219659

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Karasawa et al
hyperglycemic or hyperinsulinemic state, it may contribute to
diabetes-associated dyslipidemia and progression of atherosclerosis. Although the impact of SREBP-1c on the expression of lipogenic enzyme genes is well established, how
SREBP-1c affects plasma lipoprotein metabolism is poorly
understood. In addition, the contribution of endogenous
SREBP-1 to progression of dyslipidemia and atherosclerosis
is still unclear. To investigate the role of SREBP-1 in
lipoprotein metabolism and development of atherosclerosis,
we generated low-density lipoprotein receptor-deficient
(LDLR/) mouse models that either overexpress SREBP-1c
in the liver or lack SREBP-1.

Materials and Methods

Mice
This project was approved and performed under the guidelines of the
Animal Care Committee of the University of Tsukuba. Transgenic
mice overexpressing amino acids 1 to 436 of human SREBP-1c
under control of the phosphoenolpyruvate carboxykinase promoter
on a C57BL/6J background (hereafter referred to as TgBP-1c) were
generated as previously described.4 The TgBP-1c mice were crossed
with LDLR/ mice13 to produce TgBP-1cLDLR/ mice. SREBP-1deficient mice (SREBP-1/)14 on a C57BL/6J background were crossed
onto an LDLR/ background to generate SREBP-1/LDLR/ mice.
All mice were maintained on normal rodent chow (MF, Oriental
Yeast Company) for a 14-hour light/10-hour dark cycle. For the
fasting-refeeding experiment, mice were fasted for 24 hours and then
fed chow for 12 hours. Plasma TGs, total cholesterol, and highdensity lipoprotein cholesterol were measured with a Wako
enzymatic kit.

High-Performance Liquid
Chromatography Analysis
For the lipoprotein distribution analysis, pooled plasma samples
from 4 to 5 mice per group were analyzed by an upgraded
high-performance liquid chromatography (HPLC) technique as previously described (Skylight Biotech).15

Isolation of Very-Low-Density Lipoprotein Faction

and Western Blot
Very-low-density lipoprotein (VLDL) (d1.006 g/mL) fraction was
isolated by ultracentrifugation using TLA120.2 rotor (Beckman
Coulter). The VLDL fractions were separated by SDS-PAGE and
subject to Coomassie Brilliant Blue staining or Western blot analysis
using anti-apolipoprotein B (ApoB) antibody (sc-12332, Santa
Cruz). Levels of ApoB100 and ApoB48 were semiquantified using
National Institutes of Health ImageJ software, version 1.41
(http://rsb.info.nih.gov/ij/).

Atherosclerotic Lesion Analysis

LDLR/ and littermate TgBP-1cLDLR/ mice were maintained

on normal rodent chow until 24 weeks of age. Eight-week-old
LDLR/ mice and SREBP-1/LDLR/ mice were fed a Western diet (D12079B [34% sucrose, 21% fat, 0.15% cholesterol],
Research Diet) for 10 weeks. The mice were then euthanized, and
their hearts and aortas were isolated. The hearts were fixed in 4%
formalin for more than 48 hours. The basal half of each heart was
embedded in Tissue-Tek OCT compound (Sakura Finetek). Crosssections were stained with Oil Red O and hematoxylin. The aorta
was cut open along the midline from the iliac arteries to the aortic
root. The aorta was pinned out flat, and the lesions were stained with
Sudan IV for 15 minutes, destained with 70% ethanol, and then fixed
in 4% phosphate-buffered formalin. The atherosclerotic lesions were
quantified using Photoshop CS software (Adobe Systems Inc).

SREBP-1 and Atherosclerosis

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TG Production
Mice fasted for the indicated times were injected with Triton
WR-1339 (0.5 mg/g body weight, Sigma-Aldrich) via the tail veins
to block the clearance of nascent ApoB-containing lipoproteins.
Blood samples were collected at 0, 30, 60, and 120 minutes after the
injection.

Electron Microscopy of Plasma VLDL

The isolated VLDL fraction was negatively stained with phosphotungstic acid (pH 7) for 2 minutes. The specimen was viewed under
a JEM-1400 electron microscope (JEOL). The mean diameters of the
VLDL particles were determined using ImageJ software version
1.41.

Results
Hepatic SREBP-1c Activation Induces Atherogenic
Lipoprotein Profiles in LDLR/ Mice
To investigate whether hepatic SREBP-1c overexpression
alters plasma lipoprotein profiles, we crossed TgBP-1c mice
with LDLR/ mice (TgBP-1cLDLR/). First, the metabolic characteristics were investigated. TgBP-1cLDLR/ mice
exhibited higher plasma cholesterol levels than did LDLR/
mice in both the fasted and refed states (1.3-fold and 1.4-fold,
respectively; Figure 1 A, top). Plasma TG levels were
2.6-fold higher in the refed TgBP-1cLDLR/ mice than in
the refed LDLR/ mice (Figure 1A, bottom). The elevated
plasma cholesterol levels were also observed in the presence
of LDLR (Supplemental Figure IA, available online at
http://atvb.ahajournals.org). Plasma HPLC analysis revealed
that VLDL and remnant cholesterol levels were higher in the
TgBP-1cLDLR/ mice than in the LDLR/ mice (Figure
1B). Interestingly, the marked VLDL-TG elevation and
high-density lipoprotein cholesterol reduction were observed
only in the refed TgBP-1cLDLR/ mice. To validate these
results, the VLDL fraction was isolated by ultracentrifugation. In agreement with the HPLC analysis, VLDL cholesterol levels were higher in both the fasted and refed TgBP1cLDLR/ mice than in the LDLR/ mice (1.9-fold in the
fasted state and 3.1-fold in the refed state; Figure 1C).
Elevation of the VLDL-TG levels in TgBP-1cLDLR/ mice
was prominent in the refed state but not in the fasted state.
The VLDL fraction was analyzed by SDS-PAGE (Figure
1D). ApoB100 protein exhibited similar levels between
TgBP-1cLDLR/ mice and LDLR/ controls in either the
fasted or the refed state, whereas ApoB48 was 2-fold higher
in the refed state. Liver TG and cholesterol contents were
significantly higher in the refed TgBP-1cLDLR/ mice than
in the refed LDLR/ mice (Supplemental Table I). These
data indicated that activation of hepatic SREBP-1c induced
accumulation of postprandial remnant lipoprotein rich in both
the cholesterol and the TGs. Plasma glucose and insulin levels in
the TgBP-1cLDLR/ mice were not significantly changed as
compared with the LDLR/ mice in both the fasted and refed
states. However, TgBP-1c LDLR/ mice exhibited impaired
glucose tolerance and mild insulin resistance compared with
LDLR/ mice (Supplemental Figure II).

Hepatic SREBP-1c Activation Accelerates

Atherosclerosis in LDLR/ Mice
To determine the effect of hepatic SREBP-1c activation on
atherosclerosis in LDLR/ mice, we examined atheroscle-

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Figure 1. Proatherosclerotic plasma lipoprotein profiles in TgBP-1cLDLR/ mice.

Plasma was collected from 8-week-old
male LDLR/ and TgBP-1cLDLR/
mice after they were fasted for 24 hours
or refed for 12 hours (n4 to 5). A,
Plasma total cholesterol and TG levels. B,
HPLC analysis of plasma lipoprotein profiles. Plasma from 4 to 5 mice in each
genotype was pooled and used for HPLC
analysis. C, Quantification of plasma
VLDL-TG, VLDL cholesterol (VLDL-C), and
high-density lipoprotein-cholesterol
(HDL-C) levels in fasted and refed
LDLR/ and TgBP-1cLDLR/ mice. The
VLDL fraction was isolated by ultracentrifugation. D, Determination of VLDL ApoB
levels. VLDL fractions obtained in Figure
1C were subjected to SDS-PAGE.
ApoB100 and ApoB48 were visualized by
Western blot. Each lane represents an
individual mouse. Coomassie Brilliant
Blue staining of ApoB100 and ApoB48
was semiquantified. Values are shown as
meanSEM. *P0.05, ***P0.005 vs
control. Similar results were obtained
from 2 independent experiments.

rotic lesion formation in the proximal aorta of 24-week-old

mice fed normal chow. The lesions stained with Oil Red O
were 2.4-fold higher in the TgBP-1cLDLR/ mice than in
the LDLR/ mice (Figure 2A and 2B).

Systemic SREBP-1 Deficiency Improves Atherogenic

Lipoprotein Profiles in LDLR/ Mice
To investigate whether systemic SREBP-1 deficiency improves
lipoprotein profiles and atherosclerotic lesion formation, we next
generated SREBP-1/LDLR/ mice. In the mice given normal chow, plasma total cholesterol levels did not significantly
change in the male LDLR/ and SREBP-1/LDLR/ mice,
but plasma TG levels were lower in the SREBP-1/LDLR/
mice than in the LDLR/ mice (1277 mg/dL versus 765
mg/dL; Figure 3A). The mice were placed on a Western diet for
4 weeks, and the plasma lipid profiles were then analyzed. In the
LDLR/ mice, the plasma cholesterol levels were elevated to
more than 1000 mg/dL, and the TG levels were also elevated to
590 mg/dL. In the SREBP-1/LDLR/ mice, plasma cholesterol was only 67% that of the LDLR/ mice (Figure 3A).
Furthermore, plasma TG levels were dramatically lower (71%)
in the SREBP-1/LDLR/ mice than in the LDLR/ mice.

Analysis by high-performance liquid chromatography revealed a

marked reduction of cholesterol in the chylomicrons and VLDL
of the SREBP-1/LDLR/ mice compared with that in
the LDLR/ mice (Figure 3B, top). Furthermore, TG levels in
the SREBP-1/LDLR/ mice were lower than those in the
LDLR/ mice in all lipoprotein subclasses (Figure 3B, bottom). Hypolipidemic effects of SREBP-1 deficiency were also
observed in the LDLR/ background (Supplemental Figure
IB). To evaluate VLDL lipid composition and ApoB levels,
plasma lipoprotein was separated by ultracentrifugation. In accordance with the HPLC analysis, VLDL-TG, VLDL cholesterol, and
VLDL-phospholipids in the SREBP-1/LDLR/ mice were
markedly lower (86%, 84%, and 73%, respectively) than those in
the LDLR/ mice (Figure 3C). VLDL ApoB100 levels in the
SREBP-1/LDLR/ mice decreased to 50% of those in the
LDLR/ mice, and ApoB48 levels in the SREBP-1/LDLR/
mice decreased more markedly, to 10% of those in the LDLR/
mice (Figure 3D). Liver TG and cholesterol contents were not
significantly changed in the LDLR/ and SREBP-1/LDLR/
mice (Supplemental Table II). To determine whether hypolipidemic
phenotype in SREBP-1/LDLR/ mice was due to the hepatic
SREBP-1 deficiency, we attempted restoration of SREBP-1c by

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Figure 2. Cross-sectional analysis of aortic roots in LDLR/

mice and TgBP-1cLDLR/ mice. Mice were fed normal chow
for 24 weeks. A, Representative aortic root sections from
LDLR/ and TgBP-1cLDLR/mice. Cross-sections were
stained with Oil Red O and hematoxylin. B, Quantification of
aortic lesion areas (n8 to 10; **P0.01 vs control).

adenovirus-mediated gene transfer to the liver. Injection of

adenovirus encoding SREBP-1c with SREBP-1/LDLR/
mice reverted their plasma lipoprotein profile to LDLR/ mice
(Supplemental Figure IIIA to IIID).

Systemic SREBP-1 Deficiency Reduces

Atherosclerotic Lesion Formation in LDLR/ Mice

LDLR/ and SREBP-1/LDLR/ mice fed a Western diet

for 10 weeks were subjected to atherosclerosis analysis in both
cross-sections of the proximal aorta and pinned-out en face
aorta. Analysis of the proximal aorta revealed that lesion
formation in the male SREBP-1/LDLR/ mice was significantly suppressed by 71% of that of LDLR/ mice (Figure 4A
and 4B). The lesion area in the whole aorta stained with Sudan
IV was 40% lower in the male SREBP-1/LDLR/ mice
than in the male LDLR/ mice (Figure 4C and 4D). For further
investigation, gene expression analysis was also performed to
characterize the aortic lesion. Real-time reverse transcriptionpolymerase chain reaction analysis revealed that interleukin-6 expression in the SREBP-1/LDLR/ mice was lower than in the
LDLR/ mice, whereas CD68 and Emr1 expression was unchanged (Supplemental Figure IV). These results suggest that
atherosclerosis development was attenuated in SREBP-1/
LDLR/ mice.

Macrophage SREBP-1 Deficiency Does Not

Reduce Atherosclerotic Lesion Formation in
LDLR/ Mice
Macrophages play a central role in the initial step of atherosclerotic lesion formation. To evaluate the contribution of
macrophage SREBP-1 deficiency to the antiatherosclerotic
effects of systemic SREBP-1 deficiency in LDLR/ mice,

Figure 3. Improved plasma lipoprotein profile in SREBP-1/

LDLR/ mice fed a Western diet (WTD). Eight-week-old male
LDLR/ and SREBP-1/LDLR/ mice were fed a Western
diet for 4 weeks. Plasma was collected after the mice were
fasted for 5 hours. A, Plasma total cholesterol and TG levels in
LDLR/ and SREBP-1/LDLR/ mice (n13 to 16). B, HPLC
analysis of plasma lipoprotein profiles. Plasma from 4 to 5 mice
in each genotype was pooled and used for HPLC analysis. C,
Quantification of plasma VLDL-TG, VLDL-C, and VLDLphospholipid (PL) in LDLR/ and SREBP-1/LDLR/ mice
(n4 to 5). The VLDL fraction was isolated by ultracentrifugation. D, Determination of VLDL ApoB levels. VLDL fractions
obtained in C were subject to SDS-PAGE. ApoB100 and
ApoB48 were visualized by Western blot. Each lane represents
1 mouse. Coomassie Brilliant Blue staining of ApoB100 and
ApoB48 was semiquantified. Values are shown as meanSEM.
*P0.05, **P0.01, ***P0.005 vs control. CM indicates
chylomicrons.

we performed bone marrow transplantation studies to generate a bone marrow-specific SREBP-1-deficient model. Bone
marrow prepared from SREBP-1/ or SREBP-1/ mice
was transplanted to irradiated LDLR/ mice (BM SREBP1/ or BM SREBP-1/ mice). Lesion areas in the whole
aorta were not different between BM SREBP-1/ and BM
SREBP-1/LDLR/ mice fed a Western diet for 16 weeks
(Supplemental Figure V). The impact of SREBP-1 deficiency
on foam cell formation in vitro was also tested. Mouse
peritoneal macrophages (MPM) from SREBP-1/ and
SREBP-1/ mice were incubated with oxidized low-density
lipoprotein (oxLDL), acetylated low-density lipoprotein
(AcLDL), or VLDL. Cholesterol accumulation was similar
in the SREBP-1/ MPM and SREBP-1/ MPM (Supplemental Figure VIA and VIB). Furthermore, expression levels

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TgBP-1cLDLR/ mice. After being fasted for 24 hours,
LDLR/ and TgBP-1cLDLR/ mice were injected with
Triton WR-1339 to block ApoB lipoprotein clearance. Although TG production rates were similar in the LDLR/ and
TgBP-1cLDLR/ mice (Figure 5A), only plasma from the
TgBP-1cLDLR/ mice became milky. To characterize the
secreted lipoproteins, VLDL fractions were isolated from
plasma after Triton WR-1339 injection and analyzed by
electron microscopy. Larger particles were prominent in the
nascent VLDL fraction from TgBP-1cLDLR/ mice than in
that from LDLR/ mice (Figure 5B). Even on a LDLR/
background, larger particles were observed in the nascent
VLDL fraction from TgBP-1c mice (Figure 5C). Consequently, the average diameter of the nascent VLDL particles
observed at fasting was 1.3-fold higher and the volume of the
particles was 2.9-fold higher in the TgBP-1c mice than in the
wild-type (WT) mice. Because TgBP-1cLDLR/ mice exhibited higher plasma TG levels in the refed state, TG
production after feeding was also examined. TG production
rate after refeeding in TgBP-1cLDLR/ mice was greater
than in LDLR/ mice (Supplemental Figure VIIA). Meanwhile, lipase activity and postprandial plasma TG response
examined by oral fat load test was not changed between
LDLR/ mice and TgBP-1cLDLR/ mice (Supplemental
Figure VIIB and VIIC). These data indicated that production
of large VLDL particles causes atherogenic lipoprotein profiles in TgBP-1cLDLR/ mice.

Hepatic SREBP-1c Activation Increases

Phospholipid Transfer Protein Expression

Figure 4. Atherosclerotic lesion analysis of LDLR/ and

SREBP-1/LDLR/ mice. Eight-week-old male LDLR/ mice
and SREBP-1/LDLR/ mice were fed a Western diet for 10
weeks. A, Representative aortic root sections of LDLR/ and
SREBP-1/LDLR/ mice. Cross-sections were stained with
Oil Red O and hematoxylin. B, Quantification of aortic root
lesion areas (n8 to 9). C, Representative images of Sudan
IVstained entire aorta. D, Quantification of the surface area
occupied by the lesions (n13 to 16). *P0.05, ***P0.005 vs
control.

of genes involving cholesterol efflux or modified lipoprotein

uptake were similar in the SREBP-1/ MPM and SREBP1/ MPM (Supplemental Figure VIC and VID). Collectively, SREBP-1 deficiency in macrophages did not prevent
accumulation of cholesterol either in vivo or in vitro.

Hepatic SREBP-1c Activation Enlarges Nascent

VLDL Particle Size
To investigate how hepatic SREBP-1c regulates plasma
lipoprotein profiles, plasma TG production was evaluated in

The hepatic gene expression profiles demonstrated that in

agreement with the increased nuclear SREBP-1 level, lipogenic genes, such as fatty acid synthase, stearoyl coenzyme
A desaturase-1, and glycerol-3-phosphate acyltransferase mitochondrial, were elevated in TgBP-1cLDLR/ mice (Supplemental Figure VIIIA and VIIIB). To define the genes
responsible for the enlarged nascent VLDLs, genes involved
in TG synthesis and secretion were also examined (Figure
5D). Expressions of diacylglycerol O-acyltransferase 1 and 2
were unchanged in TgBP-1cLDLR/ mice. Furthermore,
neither microsomal TG transfer protein, known to be rate
limiting for VLDL assembly, nor ApoB was increased. On
the other hand, phospholipid transfer protein (PLTP), which
participates in lipid transfer among plasma lipoproteins and
promotes VLDL secretion,16,17 exhibited a 5.9-fold increase
as compared with the LDLR/ mice. Expression of lipoprotein lipase in both the liver and the adipose tissue, as well as
of hepatic lipase, was not decreased (Supplemental Figure
VIIIC).

Systemic SREBP-1 Deficiency Reduces the Size of

Nascent VLDL Particles
Consistent with the marked reduction in plasma TGs, the rates of
TG secretion from SREBP-1/LDLR/ mice were 28%
lower than those in LDLR/ mice at 120 minutes after the
Triton WR-1339 injection (Figure 6A). The size of nascent
VLDL particles secreted from SREBP-1/ mice fed a Western
diet for 4 weeks was apparently smaller than that in WT mice
(Figure 6B). The average diameter and volume of the particles

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Figure 5. Characterization of VLDL production and

lipoprotein metabolism in SREBP-1c transgenic
mice. A, TG production rates in LDLR/ and
TgBP-1cLDLR/ mice. LDLR/ and TgBP1cLDLR/ mice fasted for 24 hours were injected
with Triton WR-1339 via the tail vein (n5). Plasma
was collected at 0, 30, 60, and 120 minutes after
injection with Triton WR-1339. Plasma obtained at
120 minutes was used for ultracentrifugation to
isolate the VLDL fraction. B, Electron microscopic
image of secreted VLDL particles from LDLR/
and TgBP-1cLDLR/ mice. Isolated VLDL was
negatively stained with phosphotungstic acid and
visualized by electron microscopy. C, Particle size
analysis of nascent VLDL from WT and TgBP-1c
mice. WT and TgBP-1c mice fasted for 24 hours
were injected with Triton WR-1339 via the tail vein
(n4). Isolated VLDL particles were visualized by
electron microscopy. Three hundred VLDL particles were randomly selected from each mouse
and the diameters of VLDL particles were measured using ImageJ software. The VLDL particle
volume was calculated on the basis of the particles as entire spheres. D, Gene expression analysis of LDLR/ and TgBP-1cLDLR/ mice. The
livers were collected from 8-week-old LDLR/
and TgBP-1cLDLR/ mice after they were fasted
for 24 hours (n5). Gene expressions were analyzed using reverse transcriptionpolymerase chain
reaction. MTP indicates microsomal TG transfer
protein. Values are shown as meanSEM.
*P0.05, **P0.01, ***P0.005 vs control. Similar
results were obtained from 2 independent experiments. CIDEB indicates cell death-inducing DIVA
fragmentation factor, alpha subunit-like effector B.

were reduced in SREBP-1 / mice. Northern blot analysis

revealed truncated SREBP-1 expression and decreased lipogenic
enzymes, such as fatty acid synthase, stearoyl coenzyme A
desaturase-1, and glycerol-3-phosphate acyltransferase mitochondrial, in the liver of SREBP-1/LDLR/ mice (Supplemental Figure IXA). Real-time polymerase chain reaction analysis revealed that diacylglycerol O-acyltransferase 1 and 2
expressions were unchanged (Figure 6C). In addition, expression
of both ApoB and microsomal TG transfer protein was not
decreased in SREBP-1/LDLR/ mice. Importantly, PLTP
expression was 47% lower in the SREBP-1/LDLR/ mice
than in the LDLR/ mice. In addition, adenoviral-mediated
SREBP-1c transfer caused PLTP expression level in the
SREBP-1/LDLR/ mice to revert to the level in the
LDLR/ mice (Supplemental Figure IIIF). We also examined
the expression of lipases in the liver and white adipose tissue.
Lipoprotein lipase expression was lower in both the liver and the
adipose tissue from SREBP-1/LDLR/ mice (Supplemental
Figure IXB) and plasma lipase activity was lower in the
SREBP-1/LDLR/ mice than in the LDLR/ mice (Supplemental Figure IXC). These data suggest that plasma TG
clearance cannot account for the reduced plasma TGs in
SREBP-1/LDLR/ mice.

Discussion
Our current study clearly demonstrates that hepatic SREBP-1c
plays a determinant role in levels of plasma TGs and remnant
cholesterol and contributes to atherosclerosis in hyperlipidemic
models. Overexpression of hepatic SREBP-1c causes dyslipidemia and accelerates aortic atheroma formation in LDLR/
mice. Dyslipidemia in TgBP-1cLDLR/ mice was characterized by postprandial elevation of remnant cholesterol and TG
and reduction of high-density lipoprotein cholesterol, making
this animal a good atherogenic model of postprandial hyperlipidemia and impaired glucose tolerance. Overproduction of lipidrich large VLDL is a hallmark of dyslipidemia in type 2 diabetes
and insulin resistance,18 and SREBP-1c activation could be
involved in this process. Meanwhile, SREBP-1 deficiency attenuated diet-induced hyperlipidemia and atherosclerosis progression in LDLR/ mice. In contrast, macrophage-specific
SREBP-1 deficiency had no effect on atherosclerosis development in LDLR/ mice. Taken together with the observation
that the hypolipidemic phenotype in SREBP-1/LDLR/
mice was abolished by hepatic SREBP-1c overexpression, these
results suggest that suppression of atherosclerosis in SREBP1/ LDLR/ mice is due mainly to altered plasma lipoprotein
profiles as a consequence of the absence of hepatic SREBP-1

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Figure 6. Characterization of VLDL production and

lipoprotein metabolism in SREBP-1-deficient mice. A,
TG production rates in LDLR/ and SREBP-1/
LDLR/ mice. LDLR/ and SREBP-1/LDLR/
mice fed a Western diet for 4 weeks were injected
with Triton WR-1339 via the tail vein after they were
fasted for 5 hours (n4). Plasma was collected at 0,
30, 60, and 120 minutes after the Triton WR-1339
injection. B, Particle size analysis of nascent VLDL
from WT and SREBP-1-deficient mice. WT and
SREBP-1 / mice fed a Western diet for 4 weeks
were injected with Triton WR-1339 after they were
fasted for 5 hours (n3). Isolated VLDL particles
were visualized by electron microscopy. Three hundred VLDL particles were randomly selected from
each mouse, and the diameters of VLDL particles
were measured using ImageJ software. C, Hepatic
mRNA expression analysis of LDLR/ and SREBP1/LDLR/mice. The livers were collected from
LDLR/ and SREBP-1/ mice (n10 to 12) fed a
Western diet for 10 weeks and then fasted for 5
hours. Gene expressions were analyzed using
reverse transcriptionpolymerase chain reaction.
MTP indicates microsomal TG transfer protein. Values are shown as meanSEM. *P0.05, **P0.01,
***P0.005 vs control.

and that SREBP-1 in macrophages have less influence on the

development of atherosclerosis.
One of the most remarkable changes in altered lipoprotein
profiles by hepatic SREBP-1c activation was increased large
plasma VLDL and remnant lipoproteins. The large VLDL
seemed to be a resource of remnant lipoproteins through the
lipolysis process. These changes were inversely suppressed in
the SREBP-1/LDLR/ mice fed a Western diet. Therefore it
is suggested that SREBP-1 determines plasma remnant lipoprotein level and consequent atherosclerosis development.
In relation to this issue, another novel finding in our
models is that hepatic SREBP-1c controls the size of nascent
VLDL particles. Liver X receptor is also known to be a
transcription factor that regulates SREBP-1c and VLDL
particle size.19 21 A recent report revealed that massive
hyperlipidemia with large VLDL particle formation by pharmacological liver X receptor activation was diminished in
SREBP-1c-deficient LDLR/ mice.22 Moreover, this response to the liver X receptor agonist was partially rescued by
adenoviral PLTP overexpression, indicating the importance
of liver X receptorSREBP-1c-PLTP axis in the VLDL
particle size. It has been shown that PLTP accelerates
atherosclerosis development by promoting ApoB production.17,23 Our data from models with SREBP-1-transgenic

and -deficient mice are also associated with the changes in

PLTP expression consistent with these observations. Although it is currently unknown whether SREBP-1c directly
activates PLTP and accelerates lipidation of VLDL, PLTP
plays a key role in plasma lipoprotein profiles. Meanwhile, it
is also possible that SREBP-1c might activate one or more
other genes involved in lipidation of VLDL particles in the
liver. Recently, it has been recognized that VLDL maturation
in the liver comprises 2 steps: large VLDL, called VLDL1,
seemed to be generated as a consequence of bulk lipidation of
VLDL2.24 26 Although little is known about the genes responsible for VLDL lipidation, it is intriguing to speculate
that SREBP-1 regulates these unknown genes. A previous
study reported that the lipid dropletassociated protein
cell death-inducing DIVA fragmentation factor, alpha
subunit-like effector B promotes VLDL lipidation by interacting with ApoB,27 but in our models, the cell deathinducing DIVA fragmentation factor, alpha subunit-like effector B expression was unchanged.
In summary, we have shown that hepatic SREBP-1c
controls plasma TG-rich lipoproteins and atherosclerosis. The
altered lipoprotein profiles are partially due to production of
large VLDL particles. Because SREBP-1c is elevated in
insulin resistance and type 2 diabetes, it might play a central

Downloaded from http://atvb.ahajournals.org/ by guest on September 14, 2015

Karasawa et al
role in diabetic dyslipidemia and consequent atherosclerosis
development.

Acknowledgments
The authors thank Yoshiki Ohno (University of Tsukuba) for
technical help with electron microscopic analysis, Dr Alyssa H.
Hasty (Vanderbilt University) for critical reading of the manuscript,
and Flaminia Miyamasu (University of Tsukuba) for grammatical
revision.

Sources of Funding
This work was supported by a Japan Heart Foundation/Pfizer Grant
for Research on Hypertension, Hyperlipidemia and Vascular Metabolism (to A.T.) and by a Grant-in-Aid for Scientific Research from
the Ministry of Science, Education, Culture, and Technology of
Japan (to A.T. and H. Shimano).

Disclosures
None.

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9. Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS,
Goldstein JL. Insulin selectively increases SREBP-1c mRNA in the livers
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10. Matsuzaka T, Shimano H, Yahagi N, Amemiya-Kudo M, Okazaki H,
Tamura Y, Iizuka Y, Ohashi K, Tomita S, Sekiya M, Hasty A, Nakagawa
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Amemiya-Kudo M, Tomita S, Okazaki H, Tamura Y, Iizuka Y, Ohashi K,
Osuga J, Harada K, Gotoda T, Nagai R, Ishibashi S, Yamada N. Absence
of sterol regulatory element-binding protein-1 (SREBP-1) ameliorates
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12. Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T,
Nakagawa Y, Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu
A, Yamada N. SREBPs suppress IRS-2-mediated insulin signalling in the
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Supplemental Materials
SREBP-1 Determines Plasma Lipoproteins and Accelerates Atherosclerosis
in LDLR-Deficient Mice
Karasawa et al
Supplemental Methods
Adenovirus Treatment
Adeno virus encoding the active amino- terminal fragment of human SREBP-1c
(amino acids 1 to 436) was generated as previously described.1

Eight-nine weeks

LDLR-/- and SREBP-1-/- LDLR-/- mice were injected with adeno virus (1 x 1011OPU/
mice). After virus treatment, mice were fed western diet for 7 days. After 5 hour
fasting, mice were euthanized and plasma and liver tissue were collected.

Bone Marrow Transplantation

To eliminate the endogenous bone marrow-derived cells, male LDLR-/- mice (aged 8
weeks) were exposed to a single dose of 900-cGy total body irradiation. Irradiated
recipients were transplanted with bone marrow cells isolated from male SREBP-1+/+
or SREBP-1-/- mice. Mice were maintained on a chow diet for the first 4 weeks and

then switched to a Western diet. After 16 weeks on the Western diet, the mice were
euthanized for assessment of their aortic atherosclerosis lesions.

Preparation of Lipoproteins
Blood was collected from normolipidemic volunteers who had fasted overnight to
isolate plasma. LDL (d = 1.019-1.063g/mL) was isolated from the plasma by
sequential density ultracentrifugation as previously described. AcLDL was prepared
by repetitive additions of acetic anhydride to LDL. To prepare ox LDL, LDL was
incubated for 18 hours at 37C with 10 M CuSO4, followed by addition of EDTA.
Beta-VLDL was isolated by sequential ultracentrifugation from overnight-fasted male
New Zealand White rabbits (Kitayama Labes) maintained on a cholesterol-enriched
diet containing 1% (w/w) cholesterol (Oriental Yeast Company).

Isolation of Mouse Peritoneal Macrophages

Thioglycollate-elicited macrophages were isolated from SREBP-1+/+ and SREBP-1-/mice. The mice were intraperitoneally injected with 1 mL of 4% Brewer
Thioglycollate Medium (DIFCO Laboratories), and 4 days later, peritoneal

macrophages were collected. Cells were plated on 24-well plates in RPMI 1640
medium supplemented with 10% FCS. After 2 hours, the nonadherent cells were
washed out and fresh medium was added. On the following day, cells were then
incubated with 50 g/mL of AcLDL, OxLDL, and beta-VLDL for 24 hours.

Determination of Cholesterol Content

Cellular lipids were extracted by hexane/isopropyl alcohol, and cholesterol content
was determined by an enzymatic fluorometric microassay according to the method of
Heider and Boyett, with minor modifications.2,3

Quantitative Real-Time PCR Analysis

Total RNA was isolated using Sepasol RNAI Super G reagent (Nacalai Tesque).
Two micrograms of total RNA was reverse-transcribed using a High Capacity cDNA
Reverse Transcription kit (Applied Biosystems). Quantitative real-time (RT) PCR
analysis was performed using an ABI 7300 RT-PCR system (Applied Biosystems)
with SYBR Premix EX Taq II (Takara). The primer sequences for the quantitative
RT-PCR analysis are provided in Supplemental Table IV.

Northern Blot Analysis

Total RNA was isolated using Sepasol RNAI Super G (Nacalai Tesque). Ten
micrograms of RNA samples equally pooled from each genotype were
electrophoresed in a 1% agarose gel containing formaldehyde and were transferred
to a nylon membranes. The cDNA probe was cloned as described previously.4,5
The membranes were hybridized with probes labeled with [32P] dCTP using the
Rediprime II DNA Labeling System (GE Healthcare) in Rapid-hyb Buffer (GE
Healthcare) and washed in 0.1

SSC, 0.1% SDS at 65. Blots were analyzed

using a bioimaging analyzer (BAS-2500; Fuji Photo Film).

Preparation and Immunoblot Analysis of Liver Nuclear Extracts

Nuclear proteins from mouse liver were extracted as described previously.6

Briefly,

excised livers (0.5 g) were homogenized (Polytron) in 5 mL of buffer A, which

consisted of 10 mM HEPES, pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10%
glycerol, 0.15 mM spermine, and 2 mM spermidine, supplemented with protease
inhibitors (12.5 g/mL N-acetyl-leucyl-leucyl-norleucinal [ALLN; Sigma], 5 g/mL

pepstatin A, 10 g/mL leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 2.5

g/mL aprotinin). Pooled homogenate was homogenized with one stroke of a
Potter-Elvehjem homogenizer, filtered through 2 layers of cheesecloth, and layered
over 15 mL of buffer A. After centrifugation at 24,000 rpm on a SW28 rotor
(BECKMAN) for 1 hour at 4, the resulting nuclear pellet was resuspended in a
buffer containing 10 mM HEPES, pH 7.9, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1
mM dithiothreitol, and 10% glycerol supplemented with protease inhibitors, after
which 0.1 volume of 5 M NaCl was added. Each mixture was agitated gently for 45
minutes at 4 and then centrifuged at 89,000 rpm on a TLA120.2 rotor (BECKMAN)
for 45 minutes at 4. The supernatant was used as the nuclear extract. Aliquots of
nuclear extract (25 g protein) were separated by SDS-PAGE, transferred to
immobilon-P membranes (Millipore), and probed with anti-SREBP-1 (sc-8984; Santa
Cruz).

Statistical Analysis

Values are expressed as the mean standard error of the mean. Differences
between groups were calculated with a t test. A probability value less than 0.05 was
considered statistically significant.

Supplemental References
1.

Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T, Nakagawa Y,

Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu A, Yamada N. SREBPs

suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol. 2004;6:351-357.

2.

Heider JG, Boyett RL. The picomole determination of free and total cholesterol in cells

in culture. J Lipid Res. 1978;19:514-518.

3.

Yagyu H, Kitamine T, Osuga J, Tozawa R, Chen Z, Kaji Y, Oka T, Perrey S, Tamura Y,

Ohashi K, Okazaki H, Yahagi N, Shionoiri F, Iizuka Y, Harada K, Shimano H,

Yamashita H, Gotoda T, Yamada N, Ishibashi S. Absence of ACAT-1 attenuates

atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with

congenital hyperlipidemia. J Biol Chem. 2000;275:21324-21330.

4.

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.

5.

Shimomura I, Shimano H, Korn BS, Bashmakov Y, Horton JD. Nuclear sterol

regulatory element-binding proteins activate genes responsible for the entire program

of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem.

1998;273:35299-35306.

6.

Sheng Z, Otani H, Brown MS, Goldstein JL. Independent regulation of sterol

regulatory element-binding proteins 1 and 2 in hamster liver. Proc Natl Acad Sci USA.

1995;92:935-938.

Supplemental Figure Legends

Supplemental Figure I. Plasma total cholesterol and TG levels in SREBP-1c
transgenic mice and SREBP-1 deficient mice. A Plasma total cholesterol and TG
levels in WT and TgBP-1c mice. Plasma was collected from 8-week-old male WT
and TgBP-1c mice after they were fasted for 24 hours or refed for 12 hours (n = 6-8).
B Plasma total cholesterol and TG levels in WT and SREBP-1-/- mice. Plasma was
collected from WT and SREBP-1-/- mice fed normal chow or mice from fed western
diet for 4weeks (n=3). *P < 0.05, ***P < 0.005 versus control.

Supplemental Figure II. Glucose tolerance tests (GTT) and insulin tolerance tests
(ITT) in LDLR-/- mice and TgBP-1cLDLR-/- mice. A LDLR-/- mice and
TgBP-1cLDLR-/- mice were injected with glucose (0.5g/kgBw) via tail vein after they
were fasted for overnight. Plasma was collected at 0, 5, 15, 30, and 60 minutes after
injection and plasma glucose levels were determined. B ITT in LDLR-/- and
TgBP-1cLDLR-/- mice. LDLR-/- mice and TgBP-1cLDLR-/- mice were injected with
insulin (0.5U/kgBw) intraperitoneally after they were fasted for 4 hour. Plasma was
collected at 0, 15, 30, 60, and 120 minutes after injection and plasma glucose levels

were determined.

Supplemental Figure III. The hypolipidemic effects of systemic SREBP-1 deficiency

was reverted by adenoviral-mediated gene transfer to liver. LDLR-/- mice and
SREBP-1-/-LDLR-/- mice were treated with adeno virus encoding GFP (Ad-GFP) or
SREBP-1c (Ad-SREBP-1c). After virus treatment, mice were fed western diet for 7
days and analyzed after 5 hour fasting. A Northern blot analysis of SREBP-1 in the
liver. B Western blot analysis of nuclear SREBP-1 levels in the liver. C Plasma
cholesterol and TG levels. D HPLC analysis of plasma lipoprotein profiles. Plasma
from 3-5 mice in each group was pooled and used for HPLC analysis. E Northern
blot analysis of lipogenic enzymes. 36B4 was used as a loading control. F Gene
expressions involving VLDL production were analyzed using RT-PCR. Values are
shown as the mean SEM. P < 0.05, P < 0.01, P < 0.005 versus control.

Supplemental Figure IV. Gene expression analysis of aorta in LDLR-/- and

SREBP-1-/-LDLR-/- mice. Eight-week-old male LDLR-/- mice and SREBP-1-/-LDLR-/mice were fed a Western diet for 10 weeks. Gene expression in aorta was analyzed

using RT PCR. Values are shown as the mean SEM. P < 0.05, versus control.

Supplemental Figure V. Effect of bone marrow SREBP-1 deficiency on

atherosclerosis development in LDLR-/- mice. A Representative images of
SUDAN-IV-stained aorta in BM SREBP-1+/+ LDLR-/- and BM SREBP-1-/-LDLR-/- mice.
Irradiated LDLR-/- mice were transplanted with bone marrow cells isolated from
SREBP-1+/+ or SREBP-1-/- mice. After 4 weeks of adaptation, the diet was switched
to a Western diet. After 16 weeks on a Western diet, the mice were euthanized for
assessment of their aortic atherosclerosis lesions. B Quantification of the aortic
surface area occupied by the lesions (n = 12).

Supplemental Figure VI. Effects of SREBP-1 deficiency on cholesterol accumulation

and gene expression in mouse peritoneal macrophages.

Thioglycollate-elicited

peritoneal macrophages were isolated from WT or SREBP-1-/- mice.

Peritoneal

macrophages were incubated with 50g/L OxLDL, AcLDL, or VLDL for 24 hours.
A Cellular-free cholesterol levels, and B cellular esterified cholesterol levels in MPM
were measured using fluorometry. C Gene expression profiles involving cholesterol

efflux, and D gene eexpression profiles involving modified lipoprotein uptake were
analyzed using RT PCR. P<0.05 WT OxLDL vs KO OxLDL.
WT Vehicle vs WT OxLDL.

P<0.05,

P<0.005

P<0.05, P<0.005, KO Vehicle vs KO OxLDL.

Supplemental Figure VII. Plasma lipid metabolism in LDLR-/- and TgBP-1cLDLR-/mice. A TG production rates in LDLR-/- and TgBP-1cLDLR-/- mice after feeding.
LDLR-/- and TgBP-1cLDLR-/- mice were fasted for 24 hours and then fed 0.8g normal
chow. After 2 hour refeeding, mice were injected with Triton WR-1339 via the tail
vein (n = 4-6). Plasma was collected at 0,15, and 30 minutes after injection with
Triton WR-1339. B Postprandial TG response in LDLR-/- and TgBP-1cLDLR-/- mice.
Overnight fasted LDLR-/- and TgBP-1cLDLR-/- mice were given oral fat load. Plasma
was collected after 0, 1, 2, and 3 hour after oral fat load.

C Plasma lipase activity in

LDLR-/- and TgBP-1cLDLR-/- mice. LDLR-/- and TgBP-1cLDLR-/- mice fasted for 24
hours were injected with Heparin Sodium via the tail vein. After 10 minutes, post
heparin plasma was collected and used for measuring lipase activity. LPL Activity
was determined using Roar LPL Activity Assay Kit (Roar Biomedical) according to
manufacturers instruction. P < 0.05 versus control.

Supplemental Figure VIII. Gene expression analysis in LDLR-/- and TgBP-1cLDLR-/mice. Livers and WAT were collected from 8-week-old LDLR-/- and TgBP-1cLDLR-/mice after they were fasted for 24 hours.

A Western blot analysis of nuclear

SREBP-1 levels in LDLR-/- and TgBP-1cLDLR-/- mice.

B Northern blot analysis of

lipogenic enzymes in LDLR-/- and TgBP-1cLDLR-/- mice. 36B4 was used as a loading
control.

C Real- time RT PCR analysis was performed to evaluate genes involved

in plasma lipoprotein catabolism in the liver and the WAT. Values are shown as the
mean SEM. P < 0.005 versus control.

Supplemental Figure IX. Hepatic gene expression analysis in LDLR-/- and

SREBP-1-/-LDLR-/- mice. Livers and WAT were collected from LDLR-/- and
SREBP-1-/- mice fed a Western diet for 10 weeks after they were fasted for 5 hours.
A Northern blot analysis was performed to evaluate lipogenic enzyme expressions in
the liver. 36B4 was used as a loading control. B Real- time RT PCR analysis was
performed to evaluate genes involved in plasma lipoprotein catabolism in the liver
and the WAT. C Plasma lipase activity in LDLR-/- and SREBP-1-/-LDLR-/- mice.

LDLR-/- and SREBP-1-/-LDLR-/- mice fasted for 5 hours were injected with Heparin
Sodium via the tail vein. After 10 minutes, post heparin plasma was collected and
used for measuring lipase activity. Values are shown as the mean SEM. P < 0.05,
P < 0.005, versus control.

Supplemental Table I.
Metabolic characteristics of LDLR-/- and TgBP-1c/LDLR-/- mice
Genotype
Fasted

Refed

LDLR

-/-

TgBP-1cLDLR

-/-

Glucose (mg/dL)

87

12

112

NEFA (mEq/L)

1.0

0.1

1.0

0.1

Insulin (ng/mL)

0.25

0.09

0.42

0.15

Liver weight (g)

0.75

0.02

0.88

0.03

Epididymal WAT weight (g)

0.16

0.02

0.17

0.02

Liver TG (mg/g tissue)

66.1

9.2

83.2

0.8

Liver T-Cho (mg/g tissue)

7.2

10.0

8.9

1.7

Glucose (mg/dL)

241

22

222

14

NEFA (mEq/L)

0.3

0.0

0.3

0.0

Insulin (ng/mL)

0.9

0.2

0.7

0.1

Liver weight (g)

1.44

0.05

1.67

0.06

Epididymal WAT weight (g)

0.13

0.02

0.17

0.01

Liver TG (mg/g tissue)

21.5

2.1

32.9

2.4

2.2

0.1

2.8

0.1

Liver T-Cho (mg/g tissue)

-/-

-/-

Metabolic characteristics were measured form LDLR and TgBP-1cLDLR mice after they
were fasted for 24 hours or refed for 12 hours. Values are expressed as the mean
*

P < 0.05; P < 0.005

SEM.

Supplemental Table II.

Metabolic characteristics of male LDLR-/- and SREBP-1-/-LDLR-/- mice
Genotype
Chow

Western Diet

LDLR

-/-

-/-

SREBP-1 LDLR

-/-

Body weight (g)

23.0

0.3

23.0

0.4

Glucose (mg/dL)

191

11

176

10

NEFA (mEq/L)

0.65

0.07

0.56

0.06

HDL-C (mg/dL)

64.3

3.9

46.9

1.4

Body weight (g)

32.1

0.7

35.9

1.6

Glucose (mg/dL)

233

19

245

21

Insulin (ng/mL)

1.56

0.21

3.34

0.45

Plasma TG (mg/dL)

649

56

228

37

Plasma T-Cho (mg/dL)

1142

78

825

70

NEFA (mEq/L)

1.03

0.10

0.57

0.07

HDL-C (mg/dL)

61

Liver weight (g)

1.33

0.07

1.37

0.10

Epididymal WAT weight (g)

1.08

0.08

1.39

0.09

Liver TG (mg/g tissue)

81.1

8.2

72.5

8.2

Liver T-Cho (mg/g tissue)

16.3

1.8

15.7

1.6

77

Metabolic characteristics of male mice fasted for 5 hours were measured. Mice were fed
normal rodent chow (8 weeks old) or a Western diet for 10 weeks (18 weeks old). Values are
expressed as the mean

SEM. n = 12-16; P < 0.05; P < 0.01; P < 0.005.

Supplemental Table III.

Metabolic characteristics of BM SREBP-1+/+LDLR-/- and BM SREBP-1-/-LDLR-/- mice
BM SREBP-1

+/+

-/-

Body weight (g)

28.3

0.9

27.4

0.8

Glucose (mg/dL)

273

17

246

10

Plasma TG (mg/dL)

807

77

512

54

1430

133

1124

138

NEFA (mEq/L)

2.4

0.2

1.6

0.1

HDL Cholesterol (mg/dL)

87

76

Liver weight (g)

1.39

0.11

1.22

0.09

Epididymal WAT weight (g)

0.90

0.11

0.75

0.09

Plasma T-Cho (mg/dL)

Metabolic characteristics were measured from mice fed a Western diet for 16
weeks. Values are expressed as the mean

SEM. n = 12; P < 0.05; P < 0.01.

Supplemental Table IV.

Primer sequence used for RT-PCR
Gene
Name

Forward primer

Reverse primer

ABCA1

AAAACCGCAGACATCCTTCAG

CATACCGAAACTCGTTCACCC

ABCG1

CCATGAATGCCAGCAGCTACT

CTGTGAAGTTGTTGTCCACCTTCT

ApoB

TCACCCCCGGGATCAAG

TCCAAGGACACAGAGGGCTTT

CCR2

TGGCTGTGTTTGCCTCTCTA

CCTACAGCGAAACAGGGTGT

CD36

CCAAATGAAGATGAGCATAGGACAT

GTTGACCTGCAGTCGTTTTGC

CD68

CCTCCACCCTCGCCTAGTC

TTGGGTATAGGATTCGGATTTGA

Cideb

CCCAAGAGTGGGATGTTGTCA

GCTTGTACACATCGAAGGTGATGCG

Cyclophilin

TGGCTCACAGTTCTTCATAACCA

ATGACATCCTTCAGTGGCTTGTC

DGAT1

CACGGATCATTGAGCGTCTCT

AGTGGAAAAACCAATAGAAGAAGATAAGC

DGAT2

CGTGGTATCCTGAATTGGT

GGCGCTTCTCAATCTGAAAT

Emr1

CTTTGGCTATGGGCTTCCAGTC

GCAAGGAGGACAGAGTTTATCGTG

HL

ACGGGAAGAACAAGATTGGAAG

CGTTCCCTCAAACATAGGGC

ICAM

CCGCAGGTCCAATTCACACT

TCCAGCCGAGGACCATACAG

IL-1

AGTTGACGGACCCCAAAAGAT

GGACAGCCCAGGTCAAAGG

IL-6

TAGTCCTTCCTACCCCAATTTCC

TTGGTCCTTAGCCACTCCTTC

LPL

CTCTGTGTCTAACTGCCACTTCAAC

GACATTGGAGTCAGGTTCTCTCTTG

LRP1

TGTTCTCGAATGGGTTGTCA

CCATCTGTGTTGGTGCAAAG

MCP-1

CCCACTCACCTGCTGCTACT

ATTTGGTTCCGATCCAGGTT

MMP9

CAAGTGGGACCATCATAACATCA

TCTCGCGGCAAGTCTTCAG

MTP

GAGCGGTCTGGATTTACAAC

GTAGGTAGTGACAGATGTGGCTTTTG

p47phox

GATGTTCCCCATTGAGGCCG

GTTTCAGGTCATCAGGCCGC

p67phox

CTGGCTGAGGCCATCAGACT

AGGCCACTGCAGAGTGCTTG

PLTP-

CCGAGTGACCTGGACATGCT

GTCGGACTCAGGAGAACAATGC

SR-A

TTGCTCTCTACCTCCTTGTGTTTG

CCATAGGACCTTGAGATGTGTCACT

SR-BI

TGGTGGACAAATGGAACGG

CATGAAGGGTGCCCACATCT

TNF-

TCGTAGCAAACCACCAAGTG

AGATAGCAAATCGGCTGACG

VCAM

GTGAAGATGGTCGCGGTCTT

GGCCATGGAGTCACCGATT

VLDLR

TTCCTAGCTCATCCTCTTGCAC

CTGACCCAGTGAATTTATTGGC

Supplemental Figure I

120

Plasma T-Cholesterol (mg/dL)

100

200

150

80
60

100

40
20
0

200
150

50

Refed

WT
SREBP-1-/*

50
Chow

Fasted

100

***

0
Fasted

250

P=0.058

250
Plasma TG (mg/dL)

140

WT
TgBP-1c

Plasma TG (mg/dL)

Plasma T-Cholesterol (mg/dL)

WTD

100
90
80
70
60
50
40
30
20
10
0

Refed
P=0.075

Chow

WTD

Plasma Glucose (mg/dL)

Plasma Glucose (mg/dL)

Supplemental Figure II
350

LDLR-/TgBP-1cLDLR-/-

300
250
200
150
100
50
0

15

30
(min)

45

60

250
200
150

100
50
0

30

60
(min)

90

120

Supplemental Figure III

B

A
SREBP-1 +/+
GFP

-/-

SREBP-1 +/+

GFP SREBP-1c

GFP

Endogeneous

SREBP-1

-/GFP SREBP-1c

hSREBP-1

Truncated
hSREBP-1c

1000
800
600
400
200
0

GFP
SREBP-1 +/+

N.S

N.S

800
TG (mg/dL)

Cholesterol (mg/dL)

***

600
400
200
0

GFP
SREBP-1 +/+

GFP SREBP-1c
-/-

P=0.08

GFP SREBP-1c
-/-

SREBP-1-/-LDLR-/SREBP-1-/-LDLR-//SREBP-1c
/GFP
CM
VLDL
LDL
HDL
800
120 CM VLDL LDL HDL
700
100
600
80
500
60
400
300
40
200
20
100
0
0
15
20
25
15
20
25
Elution Time
Elution Time
TG (mV)

Cholesterol (mV)

LDLR-/- /GFP

F
GFP
FAS
SCD1
G-PAT
HMGCR
36B4

-/-

SREBP-1-/-LDLR-//GFP

GFP SREBP-1c
Relative mRNA Levels
(Normalized to Cyclophilin)

SREBP-1 +/+

LDLR-/- /GFP

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

SREBP-1-/-LDLR-//SREBP-1c
***

* ***

PLTP

MTP

ApoB

Supplemental Figure IV

Relative mRNA Levels

(Normalized to Cyclophilin)

1.8

LDLR-/-

1.6

SREBP-1-/-LDLR-/-

1.2

1.4

1.2
1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0
TNFalpha

Relative mRNA Levels

(Normalized to Cyclophilin)

1.4

IL6

IL1beta

MCP-1

Emr1

CCR2

1.4

1.4

1.2

1.2

0.8

0.8

0.6

0.6

0.4

0.4

0.4

0.2

0.2

0.2

p47phox

p67phox

1.2

1
0.8
0.6

VCAM

ICAM

MMP9

CD68

Supplemental Figure V
B

8
Lesion Area (%)

7
6
5
4
3
2
1

Donor: SREBP-1 +/+

-/-

-/-

-/-

Recipient:

LDLR

0
Donor: SREBP-1
Recipient:

LDLR

+/+

-/-

-/-

-/-

Supplemental Figure VI
A

B
300

Esterified Cholesterol
(nmol/mg protein)

250

Free Cholesterol
(nmol/mg protein)

300

WT
SREBP-1-/-

250

200

200

150

150

100
50
0

Relative mRNA Levels

(Normalized to Cyclophilin)

100

OxLDL AcLDL

WT/ Vehicle
SREBP-1-/-/ Vehicle
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

,
N.S

VLDL

WT/ OxLDL
SREBP-1-/-/ OxLDL

,
N.S

2.5

N.S

N.S

1.5

OxLDL AcLDL

D
,
N.S

ABCG1

N.S

0.5
0

ABCA1

2
,

N.S

50

SR-BI

CD36

SRA

VLDL

Supplemental Figure VII

LDLR-/TgBP-1cLDLR-/P=0.05

Plasma TG (mg/dL)

1000

700
TG production (mg/dL)

1200

800
600
*

400
200
0

15
(min)

30

600
500
400
300
200
100
0

15
(min)

D
LDLR-/TgBP-1cLDLR-/-

600

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

250
TG (mg/dL)

500

300

Lipase Activity (mol/ml/h)

700
Plasma TG (mg/dL)

30

200

400

150

300

100

200
100

50

(hour)

(hour)

LDLR-/- TgBP-1c
LDLR-/-

Supplemental Figure VIII

A

B
TgBP-1c
LDLR

-/-

-/-

TgBP-1c
LDLR

SREBP-1

-/-

-/Endogeneous

SREBP-1

Transgene
FAS
SCD1
G-PAT
HMGCR
36B4

Relative mRNA levels

(Normalized to Cyclophilin)

C
2

1.5

Liver
1.5
LDLR
-/TgBP-1cLDLR

WAT

-/-

1
0.5

0.5
0

0
LPL

HL

LRP1

LPL

Supplemental Figure IX
A
SREBP-1
LDLR

+/+
-/-

-/-/-

SREBP-1 +/+
LDLR -/-

SREBP-1

-/-/-

G-PAT

Wild type
Truncated

HMGCR

SCD1

36B4

Relative mRNA levels

(Normalized to Cyclophilin)

-/-

1.4
1.2
1
0.8
0.6
0.4
0.2
0

-/-

SREBP-1 LDLR
LDLR
Liver
1.2

***

-/-

WAT
*

1
0.8
0.6
0.4
0.2

LPL

HTGL

LPL

Lipase Activity (mol/ml/h)

FAS

6
5

***

4
3
2
1
0

LDLR-/- SREBP-1-/LDLR-/-

Sterol Regulatory ElementBinding Protein-1 Determines Plasma Remnant Lipoproteins

and Accelerates Atherosclerosis in Low-Density Lipoprotein ReceptorDeficient Mice
Tadayoshi Karasawa, Akimitsu Takahashi, Ryo Saito, Motohiro Sekiya, Masaki Igarashi,
Hitoshi Iwasaki, Shoko Miyahara, Saori Koyasu, Yoshimi Nakagawa, Kiyoaki Ishii, Takashi
Matsuzaka, Kazuto Kobayashi, Naoya Yahagi, Kazuhiro Takekoshi, Hirohito Sone, Shigeru
Yatoh, Hiroaki Suzuki, Nobuhiro Yamada and Hitoshi Shimano
Arterioscler Thromb Vasc Biol. 2011;31:1788-1795; originally published online May 5, 2011;
doi: 10.1161/ATVBAHA.110.219659
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright 2011 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

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