Troglitazone Inhibits Atherosclerosis in Apolipoprotein
E–Knockout Mice
Pleiotropic Effects on CD36 Expression and HDL
Zhong Chen, Shun Ishibashi, Stéphane Perrey, Jun-ichi Osuga, Takanari Gotoda, Tetsuya Kitamine,
Yoshiaki Tamura, Hiroaki Okazaki, Naoya Yahagi, Yoko Iizuka, Futoshi Shionoiri, Ken Ohashi,
Kenji Harada, Hitoshi Shimano, Ryozo Nagai, Nobuhiro Yamada
Abstract—Atherosclerotic coronary heart disease is a common complication of the insulin resistance syndrome that canoccur with or without diabetes mellitus. Thiazolidinediones (TZDs), which are insulin-sensitizing antidiabetic agents,
can modulate the development of atherosclerosis not only by changing the systemic metabolic conditions associatedwith insulin resistance but also by exerting direct effects on vascular wall cells that express peroxisome proliferator–
activated receptor-␥ (PPAR-␥), a nuclear receptor for TZDs. Here we show that troglitazone, a TZD, significantly
inhibited fatty streak lesion formation in apolipoprotein E– knockout mice fed a high-fat diet (en face aortic surface
lesion areas were 6.9⫾2.5% vs 12.7⫾4.7%, P⬍0.05; cross-sectional lesion areas were 191 974⫾102 911 ␮m2 vs
351 738⫾175 597 ␮m2, P⬍0.05; n⫽10). Troglitazone attenuated hyperinsulinemic hyperglycemia and increased highdensity lipoprotein cholesterol levels. In the aorta, troglitazone markedly increased the mRNA levels of CD36, a
scavenger receptor for oxidized low density lipoprotein, presumably by upregulating its expression, at least in part, inthe macrophage foam cells. These results indicate that troglitazone potently inhibits fatty streak lesion formation bymodulating both metabolic extracellular environments and arterial wall cell functions. (Arterioscler Thromb Vasc Biol.
2001;21:372-377.)
Key Words: atherosclerosis 䡲 thiazolidinediones 䡲 lipoproteins 䡲 CD36 䡲
peroxisome proliferator–activated receptorT

ype 2 diabetes mellitus is a major risk factor contributing
to coronary heart disease (CHD), the leading cause ofmorbidity and mortality in developed countries.1 The mechanisms by which diabetes promotes atherosclerosis remain
obscure. Evidence suggests that the metabolic derangement
frequently associated with type 2 diabetes and the insulin
resistance syndrome, such as hypertension and lipoprotein
abnormalities, are responsible for the increased incidence of
CHD in diabetic patients.2,3 Prospective studies have shownthat hyperinsulinemia is an independent predictor of thedevelopment not only of type 2 diabetes but also of CHD.4
Stout et al5 proposed that hyperinsulinemia directly promotes
the development of atherosclerosis. However, overwhelming
evidence supports the idea that hyperinsulinemia is merely aconsequence of insulin resistance, which directly or indirectly
enhances the development of atherosclerosis in type 2
diabetes.6
Thiazolidinediones (TZDs), which are novel, insulinsensitizing agents, are increasingly used as oral hypoglycemic
agents.7 They improve insulin sensitivity and thereby reduce

hyperinsulinemia as well as hyperglycemia.8,9 Furthermore,
troglitazone, a TZD, improves hypertension and hypertriglyceridemia, both of which represent constellations of the insulin
resistance syndrome.10,11
In addition to these potential indirect effects of TZDs on
the risk factors of atherosclerosis, increasing evidence suggests that TZDs exert potent, direct effects on arterial wallcells. Recently, Sinohara12 et al reported that the developmentof intimal lesions after balloon catheter injury in the rat aorta
was significantly inhibited by troglitazone. They argued that
this inhibition was mediated by troglitazone’s antimitogenic
effects on vascular smooth muscle cells.13 It has also beenreported that TZDs inhibit monocyte inflammatory cytokines,14 macrophage activation,15 and the expression of celladhesion molecules expressed by vascular endothelial
cells.16,17 Peroxisome proliferator–activated receptor-␥(PPAR-␥), the receptor for TZDs,18 is expressed in arterial
wall cells such as vascular smooth muscle cells and macrophages.19 PPAR-␥ may be involved in regulation of theuptake of oxidized LDL by modulating CD36, a scavenger

Received January 13, 2000; revision accepted November 8, 2000.
From the Departments of Metabolic Diseases (Z.C., S.I., S.P., J.-i.O., T.G., T.K., Y.T., H.O., N. Yahagi, Y.I., F.S., K.O., K.H., H.S., N. Yamada) andCardiovascular Medicine (R.N.), Faculty of Medicine, University of Tokyo, Tokyo, Japan. Dr Yamada and Dr Shimano are presently at the Metabolism,
Endocrinology and Atherosclerosis Section, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan.
Correspondence to Shun Ishibashi, MD, PhD, Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan. E-mail ishibash-tky@umin.u-tokyo.ac.jp© 2001 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

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Effect of Troglitazone on Body Weight, Blood Glucose, Plasma Insulin, Plasma Free Fatty Acid (FFA) Levels, and Plasma Lipids
Body Weight, g

Blood Glucose, mg/dL

Plasma Insulin, pg/mL

Plasma FFA, ␮Eg/L

TC, mg/dL

TG, mg/dL

HDL-C, mg/dL

Control (n⫽20)
Before18.8⫾3.4

128.4⫾34.0

641.5

935⫾12909⫾415

67⫾37

21⫾6

After

30.5⫾4.5‡

139.0⫾18.9

1366.91319⫾257‡

1746⫾598‡

236⫾115‡

21⫾10

Troglitazone (n⫽15)
Before21.0⫾1.2

141.6⫾26.3

590.7

976⫾901043⫾403

83⫾26

26⫾3

After

30.4⫾3.8‡

112.8⫾40.9*†658.41210⫾3312‡

1651⫾655†

319⫾181‡36⫾11*†

TC indicates total cholesterol; TG, triglycerides; and HDL-C, HDL cholesterol.
*P⬍0.05 vs control group; †P⬍0.05 vs before; ‡P⬍0.01 vs before by ANOVA.

receptor, in macrophages.20,21 On the basis of these results,
TZDs may promote foam cell formation. However, it remains
unknown whether TZDs have antiatherogenic effects.
To investigate the effects of troglitazone on fatty streak
formation, the initial step of atherosclerosis, we treatedapolipoprotein (apo) E– knockout mice, an animal model ofatherosclerosis,22 with troglitazone and examined the resultant atherosclerotic lesions.

Methods
Animals
ApoE-knockout mice were a gift from Dr N. Maeda (University ofNorth Carolina, Chapel Hill).22 These mice were back-crossed to
C57BL6 mice 9 times at our facility to produce congenic animals.
They were maintained on a 12-hour dark/12-hour light cycle andallowed ad libitum access to food and water. The diets used were (1)a Western-type diet (containing 0.15% cholesterol, 15% unsalted
butter) and (2) a Western-type diet supplemented with 0.1% (wt/wt)
troglitazone, which was a gift of Sankyo Pharmaceutical Ltd, Tokyo,
Japan. Thirty-five male animals aged 5 weeks (20 as controls and 15
for the troglitazone experiments) were fed the diets for 2 months.
C57BL6 mice were used for preparation of peritoneal macrophages.

Plasma Lipids, Glucose, and Insulin
After a 6-hour fast, blood was collected from the retro-orbital venous
plexus.23 Blood glucose levels were immediately measured on an
Antsense II (Daikin). Plasma levels of total cholesterol (TC),triglyceride (TG), and free fatty acids (FFAs) were measured
enzymatically by using Determiner TC 555, Determiner TG 555, andDeterminer NEFA methods (Kyowa Medex), respectively. Pooled
plasma was used for determining insulin levels with an insulin assay
kit from Morinaga. Lipoprotein profiles were analyzed by high-performance liquid chromatography (HPLC) essentially as described,
with some modifications.23 In brief, 5 ␮L of plasma was applied to
a combined column system composed of 2 TSK gel Lipopropacks(Tosoh) connected in tandem and eluted with the supplied buffer(TSK eluent LP-1, Tosoh) at a rate of 0.6 mL/min. TC concentrationsin the effluents were monitored by using a kit (Determiner LTC).
Areas under the elution curves for HDL peaks were determined.
These values and plasma TC levels were used to calculate HDLcholesterol concentrations.

Measurement of Atherosclerotic Lesion Size
Mice were killed by cervical dislocation after blood collection. The
en face surface and cross-sectional lesion areas in the aorta were
determined as described previously.23

Cell Culture
Peritoneal macrophages were prepared as described previously.23 In
brief, 1 mL of thioglycolate broth was injected into the peritoneal
cavities of mice aged 3 months. After 4 days, the peritoneal cavities
were lavaged with 10 mL of ice-cold saline. The cells were washed
3 times with PBS and resuspended in high-glucose Dulbecco’s
modified Eagle’s medium (HG-DMEM), and 106 cells were plated in
60-mm dishes (Corning). After incubation at 37°C for 2 hours,

nonadherent cells were removed by washing 3 times with prewarmed
PBS. The adherent cells were incubated with HG-DMEM supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS,JRL Biosciences) at 37°C in an atmosphere of 5% CO2 and 95% air.
After 16 hours, the media were replaced with fresh media containing
10% (vol/vol) FCS supplemented with the indicated concentrationsof troglitazone. Troglitazone was solubilized in dimethylsulfoxide(DMSO), and equivalent concentrations of DMSO were used forcontrols. The final concentrations of DMSO were kept at 0.1%
(vol/vol).

Northern Blot Analysis
Total RNA was prepared from the aortas by use of Trizol reagent(Life Technologies, GIBCO-BRL). Twenty micrograms of RNA was
subjected to electrophoresis in a 1.0% (wt/vol) agarose gel containing formalin. After transfer, the nylon membrane (Hybond N,
Amersham Pharmacia Biotech) was hybridized with 32P-labeledprobes for mouse CD36, lipoprotein lipase (LPL), PPAR-␥, and␤-actin. cDNA polymerase chain reaction fragments were used asprobes for CD36 and PPAR-␥. Human LPL cDNA probes were aspreviously described.23

Statistics
All experimental data are expressed as mean⫾SD. Mean values werecompared by Student’s t test and ANOVA. Spearman’s coefficientsof correlation were calculated.

Results
Plasma Glucose and Lipoproteins
Table 1 compares the body weight, blood glucose, andplasma concentrations of insulin, FFAs, and lipoprotein
profiles. Fasting blood glucose levels were significantly
decreased by 18.8% in the troglitazone-treated group compared with controls (P⬍0.05). The plasma insulin level was
decreased by 52% in the troglitazone-treated group. On theother hand, there were no significant differences in either
body weight or plasma FFA levels between the 2 groups.
Similarly, no significant differences in plasma TC and TG
levels were observed between the 2 groups. HPLC analyses
revealed a selective increase in cholesterol content in a peakcorresponding to HDL (Figure 1). On average, HDL cholesterol levels were increased by 74% in the troglitazone-treatedgroup compared with controls (36⫾11 vs 21⫾10 mg/dL,
P⬍0.05).

Atherosclerotic Lesions in the Aorta andAortic Sinus
En face surface atherosclerotic lesions of the troglitazonetreated group were grossly smaller than those of the control
group (Figures 2A and 2B). These lesions were mainly
distributed on the aortic arches and the areas surrounding thebranching points of the arteries. The en face surface aortic

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Arterioscler Thromb Vasc Biol.

March 2001

Induction of CD36 Expression in the Aorta
Figure 4 shows the results of Northern blot analysis of CD36,
PPAR-␥, and ␤-actin in the aorta. The mRNA levels of CD36
were markedly increased in the troglitazone-treated groupcompared with the control group (7-fold), while those of
PPAR␥ were not significantly changed.

Induction of CD36 Expression in Macrophages
in Culture

Figure 1. Comparison of plasma lipoprotein profiles between
control (A) and troglitazone-treated (B) apoE-knockout mice.
ApoE-knockout mice were fed a Western-type diet with or without 0.1% troglitazone for 2 months. Pooled plasma was subjected to HPLC. Cholesterol contents in the eluates were monitored. Lipoproteins isolated by standard ultracentrifugation wereused to calibrate the elution positions of chylomicron (CM),VLDL, IDL/LDL, and HDL, as indicated above the elution pattern. Note that the HDL peak was significantly elevated in thetreated group compared with the control group without changing the retention time.

To determine whether troglitazone induced the expression of
CD36 in vitro, peritoneal macrophages were incubated withvarious concentrations of troglitazone for 12 hours, and thenthe mRNA expression levels of CD36, LPL, PPAR-␥, and␤-actin were estimated by Northern blot analysis (Figure 5).
Incubation with troglitazone induced a 2.5-fold increase in
CD36 mRNA levels in a dose-dependent manner, whereas itincreased the LPL mRNA levels by only 50% at 12 hours.
Later, at 48 hours, the mRNA levels of LPL and PPAR-␥
were also significantly induced by 20 ␮mol/L troglitazone(Figure 6).

lesion area of the troglitazone group was decreased by 45%
compared with the control group (6.9% vs 12.7%, P⬍0.05)(Figure 3A).
Microscopic examination determined that the lesions inboth groups were mainly of the intermediate type, consistingof several layers of foam cells with some intracellular lipid
accumulation but without a typical lipid core (Figures 2C and
2D). Typical atheromas with well-developed, lipid-rich coresand foam cell infiltration but without fibrous tissue proliferation were observed in some sections of the control group.
The cross-sectional lesion areas of the troglitazone-treatedgroup were significantly smaller (by 45%) than those of thecontrol group (191 974⫾102 911 vs 351 738⫾175 597 ␮m2,
P⬍0.05) (Figure 3B).
To determine the factors mediating the antiatherosclerotic
effects of troglitazone, we performed correlational analyses
between lesion sizes and plasma lipid levels. When analyzed
as a whole, only HDL cholesterol levels were negatively
correlated with the en face lesion sizes (R⫽⫺0.54, P⬍0.05).
When analyzed in each group, however, there was nocorrelation between them.

In the present study, we have demonstrated that troglitazone
significantly inhibits the development of atherosclerotic foamcell lesions in apoE-knockout mice fed a high-fat diet. The
antiatherogenic effects of troglitazone appear to be mediated,
at least in part, by favorable changes in the extracellularenvironment: suppression of hyperinsulinemic hyperglycemia and an increase in HDL cholesterol levels. Troglitazone
also strongly increased the mRNA expression of CD36 in theaorta, which is conceivably proatherogenic, according toreported observations that targeted disruption of the CD36
gene was protective against atherosclerosis.24 Therefore, theCD36-increasing effects of troglitazone might be overwhelmed by the antiatherogenic effects of other factors, suchas glucose and lipoproteins metabolism. The possibility
remains that the other effects, which include antiinflammation, inhibition of cell adhesion, and antioxidation,accounted for the antiatherogenic effects of troglitazone.
Troglitazone increased HDL cholesterol levels in apoEknockout mice fed a high-fat diet (Table 1), an effect that is
likely antiatherogenic. It is well established that HDL cholesterol is negatively correlated with CHD in humans.25 In

Discussion

Figure 2. Morphology of en face surface
lesions (A, B) and cross-sectional lesions(C, D) in control (A, C) and toglitazonetreated (B, D) apoE-knockout mice. ApoEknockout mice were fed a Western-type
diet with or without 0.1% troglitazone for 2
months. A representative aorta was stained
for lipids with Sudan IV. Cross sections of
the aortic sinus were stained with oil red O
and counterstained with hematoxylin (⫻20).

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Antiatherogenic Effects of Troglitazone

375

Figure 5. The mRNA expression of CD36, LPL, and PPAR-␥ inperitoneal macrophages incubated with increasing concentrations of troglitazone. Peritoneal macrophages were harvested
after thioglycolate elicitation. Sixteen hours after preparation, thecells were incubated with HG-DMEM containing 10% (vol/vol)FCS supplemented with the indicated concentrations of troglitazone for 12 hours. Twenty micrograms total RNA as subjected
to Northern blot analysis. ␤-Actin was used as a loading control.

apoE-knockout mice that have extremely low levels of HDLcholesterol, subtle changes in this lipoprotein fraction may
have profound effects on the development of atherosclerosis.
For example, apoE-knockout mice that overexpress apo A-I,
which results in increased HDL levels, are resistant toatherosclerosis.26 Because the apo A-I gene contains a peroxisome proliferator–responsive element, it is possible thatthe apo A-I gene is positively regulated by PPAR-␥ inaddition to PPAR-␣ agonists.27 Furthermore, activation of
PPAR-␥ upregulates LPL gene expression.28,29 The increased
LPL may stimulate lipolysis of TG-rich lipoproteins, thereby
contributing to the elevation of plasma HDL cholesterollevels in apoE-knockout mice, as is the case with LPLtransgenic mice.23

In addition to favorable changes in plasma lipoprotein
metabolism, attenuation of hyperinsulinemic hyperglycemia,
a hallmark of the insulin resistance syndrome, may underlie
the antiatherogenic effects of troglitazone. Indeed, insulin has
mitogenic effects on vascular smooth muscle cells.30 However, it also causes vascular dilation31 and inhibition ofplatelet aggregation.32 Therefore, it is reasonable to conclude
that the effects of insulin on atherosclerosis are neutral as awhole.
In contrast to changes in the extracellular environment,
upregulation of CD36 mRNA in the aorta might promoteatherosclerosis. CD36 is a membrane-bound molecule withdiverse functions.33 In addition to functioning as a receptor
for fatty acids, CD36 mediates the uptake of oxidized LDL bymacrophages as a sort of scavenger receptor.34,35 Althoughlesion size was markedly reduced in troglitazone-treatedmice, the overall mRNA levels of CD36 were increased in
response to treatment with troglitazone (Figure 4). Accordingly, CD36 expression in each cell should be significantly
enhanced by treatment with troglitazone. In the current study,
we did not precisely define which cells expressed CD36.
Macrophage foam cells and endothelial cells are likely
candidates. In support of this possibility, troglitazone markedly increased the mRNA expression of CD36 of macrophages in culture (Figure 5). Because CD36 is highly ex-

Figure 4. Northern blot analysis of CD36 and PPAR-␥ in theaorta. Total RNA was extracted from the aorta. Twenty micrograms of pooled samples was subjected to Northern blot analysis. ␤-Actin was used as a loading control. Relative intensities ofthe bands for CD36 or PPAR-␥ to those for ␤-actin are indicated below each panel. CD36 expression was elevated by
7-fold, whereas the PPAR-␥ expression was slightly elevated by
1.4-fold in response to treatment with troglitazone.

Figure 6. Time courses of mRNA expression of CD36, LPL, and
PPAR-␥ in peritoneal macrophages incubated with 20 ␮mol/L
troglitazone. Peritoneal macrophages were harvested after thioglycolate elicitation. Sixteen hours after preparation, the cells
were incubated with HG-DMEM containing 10% (vol/vol) FCS
supplemented with 20 ␮mol/L troglitazone for the indicated periods. Twenty micrograms of total RNA was subjected to Northern blot analysis. ␤-Actin was used as a loading control.

Figure 3. Comparison of the size of en face surface (A) andcross-sectional (B) lesions between the control and troglitazonetreated groups. ApoE-knockout mice were fed a Western-type
diet with or without 0.1% troglitazone for 2 months (n⫽10).
Short lines indicate the mean⫾SD * P⬍0.05 by Student’s t test.

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March 2001

pressed in human atherosclerotic lesions36 and its expression
is enhanced by high cholesterol levels,37 it has been speculated that CD36 is atherogenic. Indeed, disruption of CD36 isreportedly protective against atherosclerosis under conditionsof apoE deficiency.24 Therefore, we may speculate that theincreased expression of CD36 stimulates the uptake ofoxidized LDL, thereby promoting foam cell formation andatherosclerosis. However, troglitazone exerted apparently
contradictory effects in the current experiments: protection
against atherosclerosis and enhanced expression of CD36. It
is possible that the antiatherogenic effects of troglitazone onother factors, such as induction of LPL and cell adhesion,
overcome the proatherogenic CD36-inducing effects. Alternatively, the induction of CD36 may be protective against
atherosclerosis under certain conditions. For example, it isplausible that efficient removal of denatured lipoproteinsdeposited in the extracellular matrixes reduces atherogenic
inflammatory reactions. Further studies are needed to clarify
this issue.
Currently, it is widely accepted that PPAR agonists haveanti-inflammatory effects.38 In particular, it has been reported
that TZDs decrease the expression of cell adhesion molecules
expressed by vascular endothelial cells, such as vascular celladhesion molecule-1 and intercellular adhesion molecule-1,
thereby suppressing the homing of inflammatory leukocytes,
including monocytes.16,17 Therefore, it is conceivable thatthese anti-inflammatory effects of TZDs predominate overthe induced expression of CD36 in macrophage foam cells.
Furthermore, it is also possible that the antiatherosclerotic
effects of troglitazone are, at least in part, accounted for by
the effects of antioxidation due to its structural similarity to␣-tocopherol.39
In conclusion, TZDs were shown to have protective effects
against atherosclerosis in a mouse model of atherosclerosis.
These effects may be mediated by the pleiotropic actions oftroglitazone. Thus, TZDs are promising therapeutic agentsthat can be used for the prevention and/or treatment of
atherosclerosis.

Acknowledgments
This work was supported by a grant-in-aid for scientific researchfrom the Ministry of Education, Science and Culture; the Promotionof Fundamental Studies in Health Science of the Organization for
Pharmaceutical Safety and Research; health sciences research grants
from the Ministry of Health and Welfare; the Takeda Medical
Research Foundation; the Yamanouchi Foundation for Research on
Metabolic Disorders; Suzuken Memorial Foundation; and the Asahi
Life Foundation. We would like to thank M. Amemiya-Kudo, T.
Yoshikawa, and A.H. Hasty for comments and discussion.

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