arterioscler thromb vasc biol 2011 karasawa 1788 95

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Sterol Regulatory ElementBinding Protein-1 DeterminesPlasma 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 elementbinding 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 atheroscle-

rosis 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

S terol regulatory elementbinding proteins (SREBPs) aretranscription factors that belong to the basic helix-loop-helix leucine zipper family.1,2 Unlike other basic helix-loop-

helix 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 cleavage-

activating 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 activateenzymes 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 con-

trast, SREBP-1c has a transcriptional activity for genes

involved in fatty acid and TG synthesis.4,5 SREBP-2 selec-

tively 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.810

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 [email protected]

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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hyperglycemic or hyperinsulinemic state, it may contribute to

diabetes-associated dyslipidemia and progression of athero-

sclerosis. Although the impact of SREBP-1c on the expres-

sion 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

MiceThis 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) weregenerated as previously described.4 The TgBP-1c mice were crossed

with LDLR/ mice13 to produce TgBP-1cLDLR/ mice. SREBP-1-

deficient 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 high-

density lipoprotein cholesterol were measured with a Wako

enzymatic kit.

High-Performance LiquidChromatography Analysis

For the lipoprotein distribution analysis, pooled plasma samplesfrom 4 to 5 mice per group were analyzed by an upgraded

high-performance liquid chromatography (HPLC) technique as pre-

viously described (Skylight Biotech).15

Isolation of Very-Low-Density Lipoprotein Factionand Western BlotVery-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 AnalysisLDLR/ 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 West-

ern 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). Cross-

sections 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 werequantified using Photoshop CS software (Adobe Systems Inc).

TG ProductionMice fasted for the indicated times were injected with TritonWR-1339 (0.5 mg/g body weight, Sigma-Aldrich) via the tail veinsto block the clearance of nascent ApoB-containing lipoproteins.Blood samples were collected at 0, 30, 60, and 120 minutes after theinjection.

Electron Microscopy of Plasma VLDLThe isolated VLDL fraction was negatively stained with phospho-tungstic acid (pH 7) for 2 minutes. The specimen was viewed undera JEM-1400 electron microscope (JEOL). The mean diameters of theVLDL particles were determined using ImageJ software version1.41.

Results

Hepatic SREBP-1c Activation Induces AtherogenicLipoprotein Profiles in LDLR/ MiceTo investigate whether hepatic SREBP-1c overexpression

alters plasma lipoprotein profiles, we crossed TgBP-1c mice

with LDLR/ mice (TgBP-1cLDLR/). First, the metabol-

ic 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 ultracentrifuga-

tion. In agreement with the HPLC analysis, VLDL cholester-

ol levels were higher in both the fasted and refed TgBP-

1cLDLR/ 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 thanin 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 AcceleratesAtherosclerosis in LDLR/ Mice

To determine the effect of hepatic SREBP-1c activation onatherosclerosis in LDLR/ mice, we examined atheroscle-

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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 AtherogenicLipoprotein Profiles in LDLR/ MiceTo investigate whether systemic SREBP-1 deficiency improves

lipoprotein profiles and atherosclerotic lesion formation, we next

generated SREBP-1/LDLR/ mice. In the mice given nor-

mal 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 cho-

lesterol 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, bot-

tom). 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 accor-

dance 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 hepaticSREBP-1 deficiency, we attempted restoration of SREBP-1c by

Figure 1. Proatherosclerotic plasma lipo-protein profiles in TgBP-1cLDLR/ mice.Plasma was collected from 8-week-oldmale LDLR/ and TgBP-1cLDLR/

mice after they were fasted for 24 hoursor refed for 12 hours (n4 to 5). A,Plasma total cholesterol and TG levels. B,HPLC analysis of plasma lipoprotein pro-files. Plasma from 4 to 5 mice in eachgenotype was pooled and used for HPLCanalysis. C, Quantification of plasma

VLDL-TG, VLDL cholesterol (VLDL-C), andhigh-density lipoprotein-cholesterol(HDL-C) levels in fasted and refedLDLR/ and TgBP-1cLDLR/ mice. The

VLDL fraction was isolated by ultracentrif-ugation. D, Determination of VLDL ApoBlevels. VLDL fractions obtained in Figure1C were subjected to SDS-PAGE.

ApoB100 and ApoB48 were visualized byWestern blot. Each lane represents anindividual mouse. Coomassie BrilliantBlue staining of ApoB100 and ApoB48was semiquantified. Values are shown asmeanSEM. *P0.05, ***P0.005 vscontrol. Similar results were obtainedfrom 2 independent experiments.

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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 ReducesAtherosclerotic Lesion Formation in LDLR/ MiceLDLR/ 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 signif-

icantly 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 transcriptionpoly-

merase chain reaction analysis revealed that interleukin-6 expres-

sion in the SREBP-1/LDLR/ mice was lower than in the

LDLR/ mice, whereas CD68 and Emr1 expression was un-

changed (Supplemental Figure IV). These results suggest that

atherosclerosis development was attenuated in SREBP-1/

LDLR/ mice.

Macrophage SREBP-1 Deficiency Does NotReduce Atherosclerotic Lesion Formation inLDLR/ MiceMacrophages play a central role in the initial step of athero-

sclerotic lesion formation. To evaluate the contribution of

macrophage SREBP-1 deficiency to the antiatheroscleroticeffects of systemic SREBP-1 deficiency in LDLR/ mice,

we performed bone marrow transplantation studies to gener-

ate a bone marrow-specific SREBP-1-deficient model. Bonemarrow prepared from SREBP-1/ or SREBP-1/ mice

was transplanted to irradiated LDLR/ mice (BM SREBP-

1/ 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 (Supple-mental Figure VIA and VIB). Furthermore, expression levels

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

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

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

LDLR/ mice fed a Western diet (WTD). Eight-week-old maleLDLR/ and SREBP-1/LDLR/ mice were fed a Westerndiet for 4 weeks. Plasma was collected after the mice werefasted for 5 hours. A, Plasma total cholesterol and TG levels in

LDLR/ and SREBP-1/LDLR/ mice (n13 to 16). B, HPLCanalysis of plasma lipoprotein profiles. Plasma from 4 to 5 micein each genotype was pooled and used for HPLC analysis. C,Quantification of plasma VLDL-TG, VLDL-C, and VLDL-phospholipid (PL) in LDLR/ and SREBP-1/LDLR/ mice(n4 to 5). The VLDL fraction was isolated by ultracentrifuga-tion. D, Determination of VLDL ApoB levels. VLDL fractionsobtained in C were subject to SDS-PAGE. ApoB100 and

ApoB48 were visualized by Western blot. Each lane represents1 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 indicateschylomicrons.




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of genes involving cholesterol efflux or modified lipoprotein

uptake were similar in the SREBP-1/ MPM and SREBP-

1/ MPM (Supplemental Figure VIC and VID). Collec-

tively, SREBP-1 deficiency in macrophages did not prevent

accumulation of cholesterol either in vivo or in vitro.

Hepatic SREBP-1c Activation Enlarges NascentVLDL Particle Size

To investigate how hepatic SREBP-1c regulates plasmalipoprotein profiles, plasma TG production was evaluated in

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. Al-

though 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). Conse-

quently, 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 ex-

hibited 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 greaterthan in LDLR/ mice (Supplemental Figure VIIA). Mean-

while, 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 pro-

files in TgBP-1cLDLR/ mice.

Hepatic SREBP-1c Activation IncreasesPhospholipid Transfer Protein ExpressionThe hepatic gene expression profiles demonstrated that in

agreement with the increased nuclear SREBP-1 level, lipo-

genic genes, such as fatty acid synthase, stearoylcoenzymeA desaturase-1, and glycerol-3-phosphate acyltransferase mi-

tochondrial, were elevated in TgBP-1cLDLR/ mice (Sup-

plemental 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 diacylglycerolO-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 lipopro-

tein 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 ofNascent VLDL ParticlesConsistent 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

Figure 4. Atherosclerotic lesion analysis of LDLR/ andSREBP-1/LDLR/ mice. Eight-week-old male LDLR/ miceand SREBP-1/LDLR/ mice were fed a Western diet for 10weeks. A, Representative aortic root sections of LDLR/ andSREBP-1/LDLR/ mice. Cross-sections were stained withOil Red O and hematoxylin. B, Quantification of aortic root

lesion areas (n8 to 9). C, Representative images of SudanIVstained entire aorta. D, Quantification of the surface areaoccupied by the lesions (n13 to 16). *P0.05, ***P0.005 vscontrol.




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

revealed truncated SREBP-1 expression and decreased lipogenic

enzymes, such as fatty acid synthase, stearoylcoenzyme A

desaturase-1, and glycerol-3-phosphate acyltransferase mito-

chondrial, in the liver of SREBP-1/LDLR/ mice (Supple-

mental Figure IXA). Real-time polymerase chain reaction anal-

ysis 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, PLTPexpression 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 (Sup-

plemental Figure IXC). These data suggest that plasma TG

clearance cannot account for the reduced plasma TGs inSREBP-1/LDLR/ mice.

DiscussionOur 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 dyslipid-

emia and accelerates aortic atheroma formation in LDLR/

mice. Dyslipidemia in TgBP-1cLDLR/ mice was character-

ized by postprandial elevation of remnant cholesterol and TG

and reduction of high-density lipoprotein cholesterol, making

this animal a good atherogenic model of postprandial hyperlip-idemia and impaired glucose tolerance. Overproduction of lipid-

rich 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 atten-

uated diet-induced hyperlipidemia and atherosclerosis progres-

sion in LDLR/ mice. In contrast, macrophage-specific

SREBP-1 deficiency had no effect on atherosclerosis develop-

ment 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 SREBP-

1/

LDLR/

mice is due mainly to altered plasma lipoproteinprofiles as a consequence of the absence of hepatic SREBP-1

Figure 5. Characterization of VLDL production andlipoprotein metabolism in SREBP-1c transgenicmice. A, TG production rates in LDLR/ andTgBP-1cLDLR/ mice. LDLR/ and TgBP-1cLDLR/ mice fasted for 24 hours were injected

with Triton WR-1339 via the tail vein (n5). Plasmawas collected at 0, 30, 60, and 120 minutes afterinjection with Triton WR-1339. Plasma obtained at120 minutes was used for ultracentrifugation toisolate the VLDL fraction. B, Electron microscopicimage of secreted VLDL particles from LDLR/

and TgBP-1cLDLR/ mice. Isolated VLDL wasnegatively stained with phosphotungstic acid andvisualized by electron microscopy. C, Particle sizeanalysis of nascent VLDL from WT and TgBP-1cmice. WT and TgBP-1c mice fasted for 24 hourswere injected with Triton WR-1339 via the tail vein(n4). Isolated VLDL particles were visualized byelectron microscopy. Three hundred VLDL parti-cles were randomly selected from each mouseand the diameters of VLDL particles were mea-

sured using ImageJ software. The VLDL particlevolume was calculated on the basis of the parti-cles as entire spheres. D, Gene expression analy-sis of LDLR/ and TgBP-1cLDLR/ mice. Thelivers were collected from 8-week-old LDLR/

and TgBP-1cLDLR/ mice after they were fastedfor 24 hours (n5). Gene expressions were ana-lyzed using reverse transcriptionpolymerase chainreaction. MTP indicates microsomal TG transferprotein. Values are shown as meanSEM.*P0.05, **P0.01, ***P0.005 vs control. Similarresults were obtained from 2 independent experi-ments. CIDEB indicates cell death-inducing DIVAfragmentation factor, alpha subunit-like effector B.




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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 lipopro-

tein level and consequent atherosclerosis development.

In relation to this issue, another novel finding in ourmodels 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.1921 A recent report revealed that massive

hyperlipidemia with large VLDL particle formation by phar-

macological liver X receptor activation was diminished in

SREBP-1c-deficient LDLR/ mice.22 Moreover, this re-

sponse 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 produc-tion.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. Al-

though 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

VLDL2.2426 Although little is known about the genes re-sponsible 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 inter-

acting with ApoB,27 but in our models, the cell death-

inducing DIVA fragmentation factor, alpha subunit-like ef-

fector 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 ininsulin resistance and type 2 diabetes, it might play a central

Figure 6. Characterization of VLDL production andlipoprotein 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 injectedwith Triton WR-1339 via the tail vein after they werefasted for 5 hours (n4). Plasma was collected at 0,30, 60, and 120 minutes after the Triton WR-1339injection. B, Particle size analysis of nascent VLDLfrom WT and SREBP-1-deficient mice. WT andSREBP-1 / mice fed a Western diet for 4 weekswere injected with Triton WR-1339 after they werefasted for 5 hours (n3). Isolated VLDL particleswere visualized by electron microscopy. Three hun-dred VLDL particles were randomly selected fromeach mouse, and the diameters of VLDL particleswere measured using ImageJ software. C, HepaticmRNA expression analysis of LDLR/ and SREBP-1/LDLR/mice. The livers were collected fromLDLR/ and SREBP-1/ mice (n10 to 12) fed aWestern diet for 10 weeks and then fasted for 5hours. Gene expressions were analyzed usingreverse transcriptionpolymerase chain reaction.MTP indicates microsomal TG transfer protein. Val-ues are shown as meanSEM. *P0.05, **P0.01,***P0.005 vs control.




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role in diabetic dyslipidemia and consequent atherosclerosis

development.

AcknowledgmentsThe authors thank Yoshiki Ohno (University of Tsukuba) fortechnical help with electron microscopic analysis, Dr Alyssa H.Hasty (Vanderbilt University) for critical reading of the manuscript,

and Flaminia Miyamasu (University of Tsukuba) for grammaticalrevision.

Sources of FundingThis work was supported by a Japan Heart Foundation/Pfizer Grantfor Research on Hypertension, Hyperlipidemia and Vascular Metab-olism (to A.T.) and by a Grant-in-Aid for Scientific Research fromthe Ministry of Science, Education, Culture, and Technology ofJapan (to A.T. and H. Shimano).

DisclosuresNone.

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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,

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Y, Sone H, Toyoshima H, Ishibashi S, Osuga J, Yamada N. Insulin-

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Osuga J, Harada K, Gotoda T, Nagai R, Ishibashi S, Yamada N. Absence

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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


10/35

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


11/35

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.


12/35

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 . 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


13/35

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 , 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 and then centrifuged at 89,000 rpm on a TLA120.2 rotor (BECKMAN)

for 45 minutes at . 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


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Values are expressed as the mean standard error of the mean. Differences

between groups were calculated with a ttest. 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.


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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.


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Supplemental Figure Legends

Supplemental Figure I. Plasma total cholesterol and TG levels in SREBP-1c

transgenic mice and SREBP-1 deficient mice. APlasma 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).

BPlasma 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. ALDLR-/-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. BITT 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


17/35

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. ANorthern blot analysis of SREBP-1 in the

liver. BWestern blot analysis of nuclear SREBP-1 levels in the liver. CPlasma

cholesterol and TG levels. DHPLC analysis of plasma lipoprotein profiles. Plasma

from 3-5 mice in each group was pooled and used for HPLC analysis. ENorthern

blot analysis of lipogenic enzymes. 36B4 was used as a loading control. FGene

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


18/35

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. ARepresentative 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. BQuantification of the aortic

surface area occupied by the lesions (n = 12).

Supplemental FigureVI.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.

ACellular-free cholesterol levels, and Bcellular esterified cholesterol levels in MPM

were measured using fluorometry. C Gene expression profiles involving cholesterol


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efflux, and D gene eexpression profiles involving modified lipoprotein uptake were

analyzed using RT PCR. #P


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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. AWestern blot analysis of nuclear

SREBP-1 levels in LDLR-/-and TgBP-1cLDLR-/-mice. BNorthern blot analysis of

lipogenic enzymes in LDLR

-/-

and TgBP-1cLDLR

-/-

mice. 36B4 was used as a loading

control. CReal- 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.

ANorthern blot analysis was performed to evaluate lipogenic enzyme expressions in

the liver. 36B4 was used as a loading control. BReal- 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.


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22/35

Supplemental Table I.

Metabolic characteristics of LDLR-/-

and TgBP-1c/LDLR-/-

mice

Genotype LDLR-/-

TgBP-1cLDLR-/-

Fasted Glucose (mg/dL) 87 12 112 7

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

Refed 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*




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 SEM.

*P< 0.05;

P < 0.005


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Supplemental Table II.

Metabolic characteristics of male LDLR-/-

and SREBP-1-/-

LDLR-/-

mice

Genotype LDLR-/-

SREBP-1-/-

LDLR-/-

Chow 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

Western Diet 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 6 77 5

Liver weight (g) 1.33 0.07 1.37 0.10

Epididymal WAT weight (g) 1.08 0.08 1.39 0.09*



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.


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Metabolic characteristics were measured from mice fed a Western diet for 16

weeks. Values are expressed as the . n = 12;*P< 0.05; P < 0.01.

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*

Plasma T-Cho (mg/dL) 1430 133 1124 138

NEFA (mEq/L) 2.4 0.2 1.6 0.1

HDL Cholesterol (mg/dL) 87 7 76 8

Liver weight (g) 1.39 0.11 1.22 0.09



25/35

Supplemental Table IV.

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


26/35

0

50

100

150

200

250

Fasted Refed

0

50

100

150

200

250

Chow WTD

0

10

20

30

40

50

60

70

80

90

100

Chow WTD

0

20

40

60

80

100120

140

Fasted Refed

PlasmaTG(

mg/dL)

PlasmaTG(

mg/dL)

PlasmaT-Cholesterol(mg/dL)

Pla

smaT-Cholesterol(mg/dL)

WT

TgBP-1c

WT

SREBP-1-/-

***

*

*

*

*

*

P=0.058

P=0.075

Supplemental Figure I

A

B


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LDLR-/-

TgBP-1cLDLR-/-

A

B

0

50

100

150

200

250

300

350

0 15 30 45 60

0

50

100

150

200

250

0 30 60 90 120

PlasmaGlucose(mg/dL)

PlasmaGlucose(mg/dL)

(min)

(min)

Supplemental Figure II


28/35

Cholesterol(m

V)

hSREBP-1SREBP-1 Endogeneous

Truncated

hSREBP-1c

A B

C

D

E F

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

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

Elution Time

0100200300400500600700

800

15 20 25

CMVLDL LDL HDL

TG(mV)

Elution Time

0

20

40

60

80100

120

15 20 25

CM VLDL LDL HDL

FAS

SCD1

G-PAT

HMGCR

36B4

+/+ -/-GFP GFP SREBP-1c

SREBP-1

00.2

0.40.60.8

11.21.41.61.8

PLTP MTP ApoB

* ***

***

LDLR-/-/GFP

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

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

Re

lativemRNALevels

(Norm

alizedtoCyclophilin)

+/+ -/-

0

200

400

600

800

000

GFP GFP SREBP-1cCholesterol(mg/dL)

*

SREBP-1

0

200

400

600

800

+/+ -/-

GFP GFPSREBP-1c

TG(

mg/dL) ***

P=0.08

SREBP-1

Supplemental Figure III

+/+ -/-

GFP GFP SREBP-1c

SREBP-1 +/+ -/-

GFP GFP SREBP-1c

SREBP-1


29/35

0

0.2

0.4

0.6

0.8

1

1.2

1.4

p47phox p67phox

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

TNFalpha IL6 IL1beta MCP-1 CCR2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

VCAM ICAM

RelativemRNA

Levels

(NormalizedtoC

yclophilin)

R

elativemRNALevels

(NormalizedtoCyclophilin)

0

0.2

0.4

0.6

0.8

1

1.2

MMP9

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

Supplemental Figure IV

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Emr1 CD68


30/35

0

1

2

3

4

5

6

7

8

SREBP-1 +/+ +/+

ASupplemental Figure V

B

LesionArea(%)

-/- -/- -/--/-

-/--/-

Recipient:

Donor:

LDLR

SREBP-1

Recipient:

Donor:

LDLR


31/35

0

50

100

150

200

250

300

- OxLDL AcLDL VLDL0

50

100

150

200

250

300

- OxLDL AcLDL VLDL

FreeCholeste

rol

(nmol/mgprote

in)

EsterifiedCholes

terol

(nmol/mgprote

in)

00.5

11.5

22.5

33.5

44.5

5

ABCA1 ABCG1 SR-BI

0

0.5

1

1.5

2

2.5

CD36 SRA

RelativemRNALevels


WT/ Vehicle

SREBP-1-/-/ Vehicle

WT/ OxLDL

SREBP-1-/-/ OxLDL

N.S

N.S

N.S

N.S

N.S

N.S

N.S

, ,

,

,

Supplemental Figure VI

A B

C D

WT

SREBP-1-/-


32/35

(min)

TGp

roduction

(mg/dL)

LDLR-/-

TgBP-1cLDLR-/-

0

100

200

300

400

500

600

700

0 15 30

00.2

0.40.60.8

11.21.41.61.8

2

Li

aseActivit

mol/ml/h

LDLR-/- TgBP-1c

LDLR-/-

0

100

200

300

400

500

600

700

0 1 2 3

PlasmaTG(

mg/dL)

0

50

100

150

200

250

0 1 2 3

TG(

mg/dL)

A

C D

LDLR-/-

TgBP-1cLDLR-/-

(hour) (hour)

Supplemental Figure VII

PlasmaTG(m

g/dL)

(min)

*

P=0.05

0

200

400

600

800

1000

1200

0 15 30

*


33/35

Supplemental Figure VIII

SREBP-1

FAS

SCD1

G-PAT

HMGCR

36B4

SREBP-1

A B

Endogeneous

Transgene

0

0.5

1

1.5

LPL

Relativ

emRNAlevels

(Normaliz

edtoCyclophilin)

C

Liver WAT

0

0.5

1

1.5

2

LPL HL LRP1

TgBP-1cLDLR-/-LDLR-/-

TgBP-1c +-

-/- -/-LDLR

TgBP-1c +-

-/- -/-LDLR


34/35

Supplemental Figure IX

SREBP-1 Wild type

Truncated

36B4

FAS

G-PAT

HMGCR

SCD1

0

1

2

3

4

5

6

Lipase

Activity(mol/ml/h)

***

LDLR-/- SREBP-1-/-

LDLR-/-

0

0.2

0.4

0.6

0.8

1

1.2

.

LPL HTGL

0

0.2

0.4

0.6

0.8

1

1.2

LPL

Liver WAT

Rela

tivemRNAlevels


*** *

A

SREBP-1 +/+ -/-

-/- -/-LDLR

SREBP-1 +/+ -/-

-/- -/-LDLR

B CLDLR

-/- SREBP-1-/-

LDLR-/-


35/35

Yatoh, Hiroaki Suzuki, Nobuhiro Yamada and Hitoshi ShimanoMatsuzaka, Kazuto Kobayashi, Naoya Yahagi, Kazuhiro Takekoshi, Hirohito Sone, ShigeruHitoshi Iwasaki, Shoko Miyahara, Saori Koyasu, Yoshimi Nakagawa, Kiyoaki Ishii, Takashi

Tadayoshi Karasawa, Akimitsu Takahashi, Ryo Saito, Motohiro Sekiya, Masaki Igarashi,Deficient Miceand Accelerates Atherosclerosis in Low-Density Lipoprotein Receptor

Binding Protein-1 Determines Plasma Remnant LipoproteinsSterol Regulatory Element

Print ISSN: 1079-5642. Online ISSN: 1524-4636Copyright 2011 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.110.2196592011;31:1788-1795; originally published online May 5, 2011;Arterioscler Thromb Vasc Biol.

http://atvb.ahajournals.org/content/31/8/1788

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