Atherosclerosis 229 (2013) 62e70

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Adiponectin increases macrophages cholesterol efux and suppresses

foam cell formation in patients with type 2 diabetes mellitus
Min Wang 1, Duan Wang 1, Yuhua Zhang, Xiaoming Wang, Yan Liu, Min Xia*
Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Nutrition, School of Public Health,
Sun Yat-sen University (Northern Campus), Guangzhou, Guangdong Province 510080, China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 November 2012
Received in revised form
4 January 2013
Accepted 15 January 2013
Available online 6 February 2013

Objectives: Low levels of blood adiponectin contribute to an increased risk of cardiovascular disease
(CVD) in patients with type 2 diabetes mellitus (T2DM). To determine the mechanism through which
adiponectin deciency mediates accelerated cardiovascular disease in patients with diabetes, we
investigated the effects of adiponectin on macrophage cholesterol deposition.
Methods and results: 35 diabetic patients and 35 nondiabetic healthy subjects were recruited in this
study. Macrophages from patients with diabetes mellitus were cultured in adiponectin-free or
adiponectin-supplemented media and exposed to oxidized low-density lipoprotein cholesterol (OxLDL).
Adiponectin treatment markedly suppressed foam cell formation in OxLDL-treated macrophages from
diabetic subjects only, which was mainly attributed to an increase in cholesterol efux. Adiponectin
treatment signicantly increased ATP-binding cassette transporter (ABC) ABCG1 mRNA and protein
levels but not ABCA1, without affecting protein expression of scavenger receptors, including scavenger
receptor-A (SR-A) and CD36 in diabetics. Pharmacological or genetic inhibition of liver X receptor a
(LXRa) blocks the adiponectin-mediated ABCG1 expression, suggesting that LXRa activation is necessary
for the attenuation of lipid accumulation of macrophages by adiponectin. In addition, deletion of the
adiponectin receptor (adipoR1) in macrophages from diabetic patients accelerated foam cell formation
induced by OxLDL. Finally, a strong positive correlation was noted between decreased serum adiponectin
levels and impaired cholesterol efux capacity both before and after adjustment for HDL-C and ApoAI in
diabetic patients (both P < 0.001).
Conclusions: The present study identies reduced adiopoR signaling as a critical mechanism underlying
increased foam cell formation and accelerated cardiovascular disease in diabetic subjects.
2013 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Adiponectin
Macrophages
Cholesterol efux
Diabetes

1. Introduction
Type 2 diabetes mellitus (T2DM) is a well-known risk factor for
in the initiation and development of atherosclerotic cardiovascular
disease, accounting for a high proportion of disability and deaths
among diabetics [1,2]. Several studies indicate that in poorly controlled diabetes mellitus, altered insulin signaling and/or hyperglycemia promote unbalanced cholesterol metabolism, which
favors oxidized low-density lipoprotein (OxLDL) cholesterol retention and macrophage-derived foam cell formation, a hallmark of the
initiation and development of atherosclerosis [3e5].

* Corresponding author. Tel.: 86 20 87332433; fax: 86 20 87330446.

E-mail address: xiamin@mail.sysu.edu.cn (M. Xia).
1
M.W. and D.W. contributed equally to this work.
0021-9150/$ e see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.atherosclerosis.2013.01.017

Cellular cholesterol levels reect a balance between uptake,

efux, and endogenous synthesis. Under hyperglycemia and/or an
insulin-resistant state, macrophages upregulate the expression of
scavenger receptors (SR-A, and CD36), which have the ability to
take up modied lipoproteins [6,7]. On the contrary, members of
the cholesterol reverse transporter family, such as ATP-binding
cassette (ABC) mainly ABCG1, are also downregulated in response
to high glucose [8,9]. Thus, increased scavenger receptor expression
[10] and decreased ABCG1 expression [11] promote macrophage
foam cell formation and are considered as a link between diabetes
mellitus and atherosclerosis. Therefore, strategies to modulate
macrophage cholesterol deposition could have therapeutic potential for limiting the accelerated vascular disease observed in
patients with T2DM.
Adiponectin (encoded by Adipoq) is an insulin-sensitizing
plasma protein expressed in adipose tissue, and it plays an
important role in insulin-sensitizing, anti-inammatory and anti-

M. Wang et al. / Atherosclerosis 229 (2013) 62e70

atherogenic properties [12,13]. Plasma adiponectin levels are

reduced not only among obese patients [14] but also in disease
states frequently associated with insulin resistance and T2DM [14],
dyslipidemia [15], hypertension [16] and coronary artery disease
(CAD) [17]. The anti-atherogenic effects of adiponectin include the
suppression of adhesion molecule expression on vascular endothelial cells [18] and the inhibition of vascular smooth muscle cell
proliferation and migration [19]. Adiponectin also stimulates the
production of nitric oxide (NO) in endothelial cells [20] and reduces
atherosclerosis by suppressing the endothelial inammatory reaction and macrophage-to-foam cell transformation [21]. Therefore,
understanding the molecular mechanism of the accelerated atherosclerosis induced by hypoadiponectinemia may be crucial for
treating the epidemic of CVD in diabetics.
This study was designed to explore whether hypoadiponectinemia levels contribute to the increase in macrophagemediated cholesterol deposition observed in patients with diabetes
and investigate the effects of adiponectin on macrophage cholesterol
deposition in diabetics and nondiabetic matched controls.
2. Materials and methods
2.1. Materials
The recombinant human and mouse full-length adiponectin
were obtained from Alexis (San Diego, CA). CP113818, an acylcoenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was purchased from Shanghai Ennopharm Co., Ltd (Shanghai, China).
22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5Cholen- 3b-Ol (NBD-cholesterol, Catlog. N-1148), an environmentsensitive probe for investigating lipid transport processes as well
lipideprotein interactions, were purchased from Molecular Probes,
Inc. Eugene, OR). Human recombinant lipid-free apolipoprotein A-I
(ApoAI, Catlog. SRP4693), high density lipoprotein (HDL, L8039)
from human plasma, human recombinant macrophage colonystimulating factor (M-CSF, SRP4237), 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP, C3912) were obtained from Sigmae
Aldrich (St. Louis, MO). 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl
indocarbocyanine percholate (Dil)-labeled OxLDL (Invitrogen,
Grand Island, NY). The primary rabbit polyclonal antibodies antiLXRa, CD36, goat anti-SR-A and horseradish peroxidase (HRP)conjugated anti-rabbit or goat secondary antibody were all
obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA).
Rabbit monoclonal antibody to ABCG1 and mouse monoclonal
antibody to ABCA1 were provided by Abcam.
2.2. Study population
Our study population included 35 adult subjects with type 2
diabetes mellitus on medication, with an age of 62  8 years, body
mass index of 25.50  2.92 kg/m2, diabetes duration of 4  1.4
years, and hemoglobin A1c level of 7.94  0.58%. We excluded
recently diagnosed diabetes mellitus, pregnancy, known coronary
artery disease, and normal adiponectin. This population was compared with 35 normal weight control subjects with no history of
diabetes mellitus or hypertension. Subjects were recruited from the
outpatient clinic at Guangzhou Military General Hospital in
Guangzhou. The study was approved by the ethics committee of
Sun Yat-sen University and was conducted in accordance with
the Declaration of Helsinki. Participation was voluntary, and each
participant provided written informed consent. The ethylenediaminetetraacetic acid samples were immediately centrifuged,
aliquoted, and stored at 80  C until batch analysis. Peripheral
blood samples were collected from healthy subjects and patients
after an overnight fast between 8:00 AM and 10:00 AM.

63

2.3. Biochemical measurements

Serum levels of total cholesterol, HDL cholesterol, triglycerides
and glucose were measured through an enzymatic method
(Wako Pure Chemical Industries) using an automatic analyzer
(Hitachi 747 autoanalyzer, Hitachi Ltd, Tokyo). LDL cholesterol
was calculated according to the Friedewald formula: LDL
cholesterol total (triglycerides/5 HDL cholesterol). Serum levels
of apoAI and apo B were measured by immunonephelometry using a
BN Prospect analyzer (Dade Behring). Plasma insulin levels were
measured with a chemiluminescent enzyme immunoassay (Immulite 1000 Analyzer). The intra-assay and inter-assay coefcients of
variation of all measured biochemical parameters were <5%.
2.4. Human serum cholesterol efux capacity assay
We measured the cholesterol efux capacity of human serum
according to previously established methods [22]. J774 murine
macrophages were rst radiolabeled with 2 mCi/ml 3H-cholesterol.
ABCA1 was upregulated by 6 h incubation with 0.3 mM 8-CPTcAMP. Subsequently, efux media from control healthy subjects or
diabetic patients containing 2.8% apolipoprotein B (ApoB)-depleted
serum were added for another 4 h. All steps were performed in the
presence of 2 mg/ml CP113818. Liquid scintillation counting was
used to quantify the efux of radioactive cholesterol from the cells.
The quantity of radioactive cholesterol incorporated into cellular
lipids was calculated through the isopropanol extraction of control
wells not exposed to patient serum. The percentage efux was
calculated by the following formula: [(microcuries of 3H-cholesterol in media containing 2.8% ApoB-depleted serummicrocuries
of 3H-cholesterol in serum-free media) O microcuries of 3H-cholesterol in cells extracted before the efux]  100. All assays were
performed in duplicate.
2.5. Human serum adiponectin concentrations measurement
Adiponectin levels in serum were determined by a commercially
available competitive ELISA assay kit (Cat No. AG-45A-0002, AdipoGen, Seoul) according to the manufacturers instruction. The assay
used standards in the range of 0.001e1 mg/ml. The intra-assay and
inter-assay coefcients of variation were 4% and 3%, respectively.
The normal blood levels of adiponectin range from 8.3 to 13.9 mg/ml.
2.6. Human monocyte isolation and macrophage differentiation
Human monocyte isolation and macrophage differentiation
were performed as described previously [23]. Peripheral monocytes
were isolated by standard Ficoll isolation techniques according to
the manufacturers protocol and selected by CD14 marker purity
(>90% as assessed by ow cytometry). Monocytes were then plated
in tissue culture medium in DMEM containing 1.5 mg/ml glucose,
100 ng/ml human macrophage colony-stimulating factor (M-CSF),
10% charcoal/dextran-treated fetal bovine serum (FBS, Hyclone),
and 1% antibiotic/antimycotic mixture. Monocytes were allowed to
differentiate into macrophages for 3 d in adiponectin-free media
before being used in further experiments.
2.7. Mouse peritoneal macrophages
Mouse peritoneal macrophages from db/db mice and wild-type
C57BL/6J mice (n 8 per each group) were isolated 3 d after the
intraperitoneal injection of 4% thioglycollate solution. Cholesterol
uptake was evaluated after 6 h of stimulation with Dil-labeled
OxLDL cholesterol in mouse macrophages cultured for 24 h in the
absence or presence of adiponectin treatment.

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M. Wang et al. / Atherosclerosis 229 (2013) 62e70

2.8. Real-time quantitative PCR

Total mRNA was collected from human and mouse macrophages
with TRIZOL Reagent (Invitrogen) according to the manufacturers
protocol. Real-time PCR was performed with the QuantiTect SYBR
Green PCR Kit (Qiagen, Valencia, CA) on the Applied Biosystems
7500 DNA Sequence Detection System (Applied Biosystems). The
reverse transcription of RNA was carried out using random primers
and SuperScript II Reverse Transcriptase (Invitrogen). Real-time
PCR was performed using TaqMan Universal PCR Master Mix or
SYBR Green PCR Master Mix (Applied Biosystems) according to the
manufacturers instructions. Primers for human ABCA1, ABCG1 and
GAPDH were ordered from SABiosciences Co., Ltd. (Shanghai,
China). Samples were normalized to GAPDH using the DCt (cycle
threshold) method [24].
2.9. Western blot
Human monocyte-derived macrophages and mouse peritoneal
macrophages were washed twice with PBS and harvested in lysis
buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5%
Nonidet P-40, 1 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM
phenylmethylsulfonyl uoride) for immunoblotting. Nuclei were
pelleted at 5000 g for 5 min at 4  C, and the resulting supernatants were used as the cytosolic fraction. Nuclei were resuspended in lysis buffer, sheared for 15 s with a microprobe
sonicator and incubated on ice for 5 min. After centrifugation at
12,000 g for 5 min at 4  C, the supernatants were collected as
nuclear extracts. Cellular lysates or nuclear protein extracts
(40 mg) were separated with 10% SDS-PAGE and transferred onto
an Immobilon-P membrane (Millipore). The membranes were
blocked in 5% skim milk in TBS-Tween buffer (0.1% Tween 20, pH
7.4) and incubated with primary antibodies against ABCG1,
ABCA1, CD36, SR-AI and LXRa, followed by secondary antibodies.
The b-actin was used as the loading control. Antigen detection
was performed with an enhanced chemiluminescence detection
system (Pierce) [24].
2.10. Cholesterol loading and efux
Human and mouse macrophages were plated in 12-well plates
and loaded with 1 mg/ml NBD-cholesterol in serum-free medium
containing 0.2% fatty acid-free BSA for 24 h to equilibrate cellular
cholesterol pools. Then, cell layers were rinsed and incubated in the
absence or presence of adiponectin for an additional 6 h. Cholesterol efux proceeded for 6 h at 37  C in medium containing 0.2%
BSA, 0.2% BSA plus 15 mg/ml lipid-free human apoAI, or 0.2% BSA
plus 50 mg/ml of human HDL. At the end of this incubation, the
supernatant was collected and centrifuged at 13,000 rpm for
10 min to remove debris. Cells were lysed with 0.5 ml of 0.1 M
NaOH. The uorescence-labeled cholesterol released from the cells
into the medium was measured with a multilabel counter (PerkinElmer). Cholesterol efux was expressed as the percentage of
uorescence in the medium relative to the total amount of uorescence (cells and medium). The specic efux of apoAI or HDL
was calculated by subtracting non-specic efux in the presence of
0.2% BSA only [25].
2.11. Cholesterol uptake assay
Cholesterol uptake was evaluated as previously described [26].
Human and mouse macrophages (0.5  106 cells/well) in 12-well
plates were cultured in the absence or presence of 20 mg/ml adiponectin for 24 h. After then, the cells were washed and incubated
with 10 mg/ml Dil-labeled OxLDL for another 6 h. Cholesterol uptake

was measured by confocal microscopy and the results were normalized to total cell protein concentrations.
2.12. Cholesteryl ester determination
Human monocytes were isolated and allowed to differentiate
for 3 d in 100 mm dishes. Adiponectin was added at the concentration of 20 mg/ml during the nal 24 h. At the end of this
incubation, the lipids in the cells and media were separately
extracted in chloroform and methanol, the samples were dried
under liquid nitrogen, and free cholesterol and total cholesterol
were measured by gaseliquid chromatography and normalized to
cellular protein as described previously [26]. Esteried cholesterol
was calculated as the difference between total and free cholesterol times 1.67.
2.13. Oil red O staining
Macrophages derived from the healthy and diabetic patients
peripheral blood mononuclear cells were cultured in the absence or
presence of 20 mg/ml adiponectin for 24 h. To assess foam cell
formation, macrophage slides were xed with 4% paraformaldehyde for 15 min and stained with 0.5% Oil red O and hematoxylin.
Images were acquired with the 20 or 40 objective of a microscope (Leica SP5II, Leica) equipped with a digital camera using the
LAS (Leica Application Suite) software program [26].
2.14. Small interfering RNA (siRNA)
Macrophages obtained from diabetic subjects were infected
with lentivirus containing shRNA lentiviral particles against human
LXRa (sc-38828-V), AdipoR1 (sc-60123-V), AdipoR2 (sc-60125-V),
or control shRNA lentiviral particles (sc-108080) for 48 h. After
then, the cells were washed and cultured for 24 h in adiponectinfree or -supplemented media. The efciency of transfection
was >70e80% as determined by the quantitative analysis of green
uorescent protein (GFP) transfection. The effectiveness of the
shRNA treatment was assessed by measuring LXRa, AdipoR1 and
AdipoR2 protein levels by immunoblotting.
2.15. Mouse peritoneal macrophages
Mouse peritoneal macrophages from db/db mice and wild-type
C57BL/6J mice (n 8 per each group) were isolated 3 d after the
intraperitoneal injection of 4% thioglycollate solution. Cholesterol
uptake was evaluated after 6 h of stimulation with Dil-labeled
OxLDL cholesterol in mouse macrophages cultured for 24 h in the
absence or presence of adiponectin (at 20 mg/ml).
2.16. Statistical analysis
Categorical variables are presented as frequencies and percentages, and continuous variables as mean  SD. The signicance of the differences in mean values among two groups
was evaluated by two-tailed unpaired Students t tests. More than
three groups were evaluated by analysis of variance (ANOVA)
followed by post-hoc analysis using Bonferronis Multiple Comparison Test. Logistic regression was used to estimate the association between cholesterol efux capacity and plasma
adiponectin levels after adjustment for age, sex, smoking status,
presence or absence of diabetes, and LDL-C, HDL-C and apoAI
levels were added in subsequent models. Adjusted odds ratios
are reported for a 1-SD increase in efux capacity. P < 0.05 was
considered signicant.

M. Wang et al. / Atherosclerosis 229 (2013) 62e70

3. Results
3.1. Adiponectin prevents macrophage foam cell formation from
diabetic patients
We rst examined the impact of adiponectin on monocytederived macrophage foam cell formation. Macrophages derived
from diabetic patients cultured in media exhibited a signicant
increase in foam cell formation induced by OxLDL compared with
macrophages cultured under adiponectin-supplemented conditions (Fig. 1A). Direct lipid analysis showed that adiponectintreated macrophages exposed to OxLDL had almost 50% less total
cholesterol (Fig. 1B) content and 55% less cholesteryl ester levels
(Fig. 1C) than macrophages maintained in adiponectin-free media
(both P < 0.01). However, in macrophages obtained from nondiabetic controls, OxLDL-induced cholesteryl ester formation was
demonstrated to be reduced in the presence of adiponectin, but to a
less extent (33%, P < 0.05) compared with diabetic macrophages
cultured with adiponectin (Fig. 1D). These data suggest that adiponectin confers a protective role in the formation of macrophage
foam cells from monocytes isolated from diabetic patients.
3.2. Adiponectin enhances cholesterol efux from macrophages
from diabetic patients
To investigate the mechanism underlying the decrease in lipidladen macrophage foam cells induced by adiponectin in diabetics,
we assessed cholesterol uptake and efux in macrophages cultured
in either adiponectin-free or -supplemented media. Confocal
microscopy analysis showed that diabetes-derived macrophages
cultured in adiponectin-supplemented media did not demonstrate
the different levels of Dil-OxLDL cholesterol uptake compared with

65

macrophages cultured in adiponectin-free media (Supplemental

Fig. 1A and B).
We next performed cholesterol efux studies in macrophages
isolated from diabetic patients. Treatment with adiponectin
markedly increased the cholesterol efux to mature HDL in macrophages from diabetic patients compared with control subjects
(Fig. 2A). However, cholesterol efux to lipid-poor ApoAI was
slightly and insignicantly augmented in response to adiponectin
treatment (Fig. 2B). These ndings indicate clear differences
between control subjects and diabetic subjects in adiponectin
regulation of macrophage cholesterol metabolism.
3.3. Adiponectin did not alter the protein expression of lipid
synthesis-related genes
We then investigated the effect of adiponectin on endogenous
lipid synthesis-related gene expression. Our data showed that neither the protein expression of SREBP1-targeted genes (acetyl-CoA
carboxylase and fatty acid synthase) (Supplemental Fig. 2A) nor that
of SREBP2etargeted genes (3-hydroxy-3-methylglutaryl coenzyme
A reductase [HMGCR] and LDL receptor) (Supplemental Fig. 2B) was
inuenced by adiponectin treatment, excluding the possibility that
the adiponectin-induced suppression of intracellular lipid accumulation is not likely attribute to the inhibition of endogenous lipid
synthesis.
3.4. Adiponectin upregulates ABCG1 expression but not SR-A, CD36,
and SR-BI in macrophages
ABCA1 and ABCG1 play critical roles in cholesterol reverse
transport and foam cell formation [27,28]. In diabetes-derived
macrophages cultured in high and normal glucose, macrophages

Fig. 1. Adiponectin prevents macrophage foam cell formation. (A) Oil red O staining of cholesterol accumulation in monocyte-derived macrophages from diabetic patients. Top,
Adiponectin-free cells; bottom, adiponectin-treated cells. Arrowheads indicate foam cells. (B) Cholesterol and (C) Cholesterol ester and contents in macrophages from diabetics
incubated in adiponectin-free or -supplemented media (n 12 per group) (*P < 0.05 vs adiponectin free OxLDL-treated cells). (D) Cholesteryl ester formation in macrophages from
nondiabetics incubated in adiponectin-free or -supplemented media (n 12 per group).

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M. Wang et al. / Atherosclerosis 229 (2013) 62e70

Fig. 2. Cholesterol efux from macrophages of diabetic patients. Peripheral blood monocytes were isolated from control subjects (n 12) and patients with T2DM (n 12) and
differentiated into macrophages with M-CSF for 3 d. Macrophages were then incubated in NBD-cholesterol for 24 h, and cholesterol efux to lipid-free apoAI or HDL was measured
as indicated. (A) Cholesterol efux efciency to HDL. (B) Cholesterol efux efciency to lipid-free apoAI. **, P < 0.01 vs. untreated diabetic macrophages (Students t test).

treated with adiponectin for 24 h showed 5-fold higher ABCG1

mRNA levels (Fig. 3A) after OxLDL stimulation under both glucose
conditions compared with macrophages in adiponectin-free media.
In contrast, adiponectin exposure did not exert signicant effects
on ABCA1 mRNA levels (Fig. 3B). Western blot analysis of ABCG1
protein expression was consistent the changes of mRNA levels. In
addition, adiponectin-treated macrophages showed a 3-fold higher
ABCG1 protein expression than untreated macrophages, but did not
signicantly inuence ABCA1 protein expression (Fig. 3C). The
effects of adiponectin on ABCG1 mRNA and protein were independent of glucose concentrations. Additionally, in macrophages
from nondiabetic controls, adiponectin did not signicantly induce
ABCG1 expression (Supplemental Fig. 3A and B). Furthermore,
adiponectin did not evidently inuence SR-AI (Supplemental

Fig. 4A) or CD36 (Supplemental Fig. 4B) mRNA levels and both
protein expression (Supplemental Fig. 4C) in diabetes-derived
macrophages cultured under high or normal glucose.
3.5. Increase of macrophage cholesterol efux induced by
adiponectin is ABCG1 dependent
To further determine the inuence of adiponectin on cholesterol
accumulation in macrophages under diabetic conditions, we measured cholesterol uptake and cholesterol efux after OxLDL stimulation in peritoneal macrophages from diabetic db/db mice cultured
in the absence or presence of adiponectin. Cholesterol uptake
analysis did not show signicant difference between adiponectintreated and untreated diabetic macrophages (Supplemental

Fig. 3. Adiponectin induces ABCG1 expression in diabetics. Macrophages from diabetic subjects cultured in adiponectin-free or -supplemented media with high glucose (450 mg/dl)
or normal glucose (100 mg/dl) were used to prepare total RNA and protein. After stimulation with oxLDL for another 6 h. (A) ABCG1 and (B) ABCA1 mRNA expression were
determined by quantitative real-time PCR (n 10). *, P < 0.05 vs. adiponectin-free macrophages (High glucose, Students t test). #, P < 0.05 vs. adiponectin-free macrophages
(Normal glucose, Students t test). C, Western blot analysis of ABCG1 and ABCA1 expression normalized to b-actin (n 4).

M. Wang et al. / Atherosclerosis 229 (2013) 62e70

Fig. 5A). On the contrary, adiponectin exposure enhanced cholesterol efux to HDL from OxLDL-pretreated macrophages in a dosedependent fashion compared with adiponectin-free macrophages
(Supplemental Fig. 5B). To clarify the role of ABCG1 and ABCA1 in the
regulation of cholesterol efux after OxLDL stimulation by adiponectin, we transfected macrophages isolated from db/db mice with
ABCG1 siRNA or ABCA1 siRNA and then cultured in the absence or
presence of adiponectin. Notably, the inductive effect of adiponectin
on cholesterol efux was largely blunted by ABCG1 siRNA but not
ABCA1 siRNA in macrophages (Supplemental Fig. 5C). Collectively,
these data suggest that the adiponectin-induced suppression of
intracellular lipid accumulation is likely due to the augment of
cholesterol efux and at least partially mediated by ABCG1.
3.6. LXRa activation mediates the anti-foam cell formation effect of
adiponectin
Because ABCG1 and ABCA1 are regulated by the nuclear receptor
LXRa [29,30], we further examined the nuclear protein level of LXRa
in adiponectin-treated macrophages. In diabetic macrophages cultured in adiponectin-supplemented media, LXRa expression was
increased before and after OxLDL stimulation (Fig. 4A). Furthermore, in this population, to explore the transcriptional

67

regulation of LXRa in adiponectin-treated macrophages, LXRa

activation assays were performed by transfecting cells with
3  LXRE-Luc followed by adiponectin treatment in the absence or
presence of OxLDL. Compared with the untreated group, adiponectin markedly induced LXRE-mediated luciferase activity by 7.2
fold. The increase in LXRE-mediated luciferase activity conrmed
that adiponectin markedly induced LXRa activation before and after
OxLDL stimulation compared with macrophages cultured in
adiponectin-free media (Fig. 4B). No signicant change in LXRa
activation was observed in nondiabetic controls (Supplemental
Fig. 6A and B). Moreover, coincubation with an LXRa inhibitor
(GGPP) abolished the induction of ABCG1 by adiponectin (Fig. 4C),
thus abrogating the inhibitory effect of adiponectin on cholesterol
efux (Fig. 4D) and lipid accumulation (Fig. 4E) in macrophages
from diabetic patients. These results were further conrmed by
genetic inhibition of LXRa by specic siRNA (Supplement Fig. 7AeC).
Since ABCG1 are also regulated by PPARg, we also evaluated the
role of PPARg in the regulation of ABCG1 expression by adiponectin.
However, GW9662 did not exert signicant inuence on ABCG1
protein expression (Supplement Fig. 8A) and macrophages cholesterol efux (Supplement Fig. 8B) by adiponectin from diabetic
patients, excluding the participation of PPARg in the effects of
adiponectin. These data suggest that the adiponectin-mediated

Fig. 4. LXRa activation is required for adiponectin-mediated ABCG1 expression and attenuation of lipid accumulation. Peripheral blood monocytes were isolated from patients with
T2DM (n 12) and differentiated into macrophages with M-CSF for 3 d. (A) Macrophages were incubated with adiponectin (20 mg/ml) for 24 h before and after the pretreatment of
OxLDL (100 mg/ml) for 6 h. Western blot analysis of LXRa protein expression before and after stimulation with oxLDL. (B) Macrophages were transfected with plasmid 3  LXRE-Luc
and incubated with adiponectin (20 mg/ml) for 12 h. Then, cells were lysed for Luc activity assays, and renilla activity was used as an internal control. **P < 0.01 vs. control (Before
OxLDL); ##P < 0.01 vs. control (After OxLDL). (CeE) Macrophages were preincubated with vehicle (methanol) or GGPP (20 mM) for 2 h, followed by adiponectin (20 mg/ml)
treatment for another 24 h in the presence of 100 mg/ml OxLDL. (C) Cellular lysates were subjected to Western blot to determine the protein level of ABCG1 and b-actin. (D)
Cholesterol efux and (E) the intracellular lipid accumulation were measured as indicated. **P < 0.01 vs. adiponectin-treated cells.

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M. Wang et al. / Atherosclerosis 229 (2013) 62e70

upregulation of LXRa is a unifying signaling pathway that induces

cholesterol efux in diabetic patients.
3.7. Activation of adipoR signaling prevents foam cell formation
AdipoR1 and AdipoR2 serve as the major receptors for adiponectin and key physiological regulators of glucose and lipid
metabolism in vitro and in vivo [31]. To identify whether the
adiponectin-induced effects on cholesterol efux were AdipoR
dependent, we analyzed diabetic monocyte-derived macrophages
cultured in adiponectin-supplemented media in the presence of
AdipoR1 siRNA, AdipoR2 siRNA or control siRNA. AdipoR siRNAtransfected macrophages showed an 80% reduction in AdipoR
protein levels compared with control siRNA-transfected macrophages (Supplemental Fig. 9). Oil Red O staining (Fig. 5A) and
cholesterol efux quantication (Fig. 5B) conrmed that adiponectin increased macrophage cholesterol efux and limited cholesterol accumulation from macrophage foam cells only in
macrophages with intact AdipoR signaling pathways; this response
was blunted in macrophages lacking an AdipoR1 signaling pathway
but not AdipoR2. In addition, adiponectin upregulated ABCG1
expression (Fig. 5C) and LXRa protein expression (Fig. 5D) in the
presence of intact AdipoR signaling, but these effects were reduced
in AdipoR1 siRNA-infected macrophages. These data conrm the
importance of the activation of AdipoR signaling in the regulation
of both cholesterol transporters and cell signaling pathways
involved in macrophage foam cell formation.

Patients with diabetes had laboratory proles that were typical of

metabolic syndrome and T2DM, with high levels of triglycerides,
low HDL cholesterol, and elevated fasting glucose and hemoglobin
A1c. The control population was within normal parametric variables for each category. All of the parametric variables tested were
normally distributed (Supplemental Table 1).
Compared with control healthy subjects, patients with diabetes
had signicantly lower levels of not only HDL-C and ApoAI (P < 0.01
for each comparison) but also cholesterol efux capacity (mean
value for diabetic patients, 0.80; mean value for control subjects,
0.96; P 0.006) (Supplemental Table 1). The HDL-C level was the
strongest predictor of efux capacity (P < 0.001) but accounted for
only 21% of the observed variation. Male sex and current smoking
were associated with decreased efux capacity (P 0.025 and
P 0.048, respectively). Subsequent adjustment for HDL-C largely
attenuated the relationship between sex and cholesterol efux
capacity (P 0.114), although smoking remained a signicant
inverse predictor of efux capacity (P 0.002). In the logistic
regression analysis adjusted for age, sex, and traditional cardiovascular risk factors, we found that lower adiponectin levels
were strongly correlated with a decrease in cholesterol efux
capacity. This relationship remained robust after the addition of
HDL-C as a covariate. The results were similar when the ApoAI level
was substituted for the HDL-C level (Supplemental Table 2). Thus,
lower levels serum adiponectin are closely correlated with
impaired cholesterol efux capacity in diabetic subjects.
4. Discussion

3.8. Serum adiponectin levels are strongly associated with

cholesterol efux capacity in diabetic subjects
Finally, we studied 35 adult subjects with T2DM and 35 adult
healthy volunteers as controls. Sixty percent of patients were on
diabetic oral medications. Both groups were similar with respect to
age, gender, total cholesterol, lipid medications, and tobacco use.

In this study, we rst showed that lower plasma adiponectin

levels were strongly correlated with the impairment of cholesterol
efux capacity in patients with diabetes mellitus. Then, we demonstrated that activation of adiponectin receptor (AdipoR) signaling
prevents foam cell formation by inducing cholesterol efux in
monocyte-derived
macrophages
from
diabetics.
human

Fig. 5. Adiponectin activation of AdipoR signaling increases macrophage cholesterol efux in diabetics. Peripheral blood monocytes were isolated from diabetic patients and
differentiated into macrophages using M-CSF for 3 d. Then the cells were transfected with either adipoR1 siRNA, adipoR2 siRNA or control siRNA cultured in adiponectin-free or supplemented media (20 mg/ml) for 24 h. (A) Cholesterol accumulation was assessed by Oil red O staining. (B) HDL-mediated cholesterol efux from oxLDL-pretreated macrophages.
(C and D) Western blot analysis was performed to determine the ABCG1 (C) and LXRa (D) protein expression.

M. Wang et al. / Atherosclerosis 229 (2013) 62e70

Furthermore, adiponectin upregulates the expression of ABCG1, a

critical transporter involved in macrophage cholesterol deposition,
via the induction of LXRa activation. The impairment of AdipoR1
signaling conrmed the increase in foam cell formation in diabetics. Taken together, these results suggest that the modulation of
AdipoR signaling is a potential therapeutic target to prevent vascular disease progression in diabetes.
T2DM signicantly increases the risk for the development of
atherosclerosis, primarily due to the imbalance of cholesterol inux
[7] and efux [11] in macrophages, leading to increased intracellular cholesteryl ester accumulation. We rst examined the
potential role of adiponectin on lipid deposition in monocytederived macrophages from diabetic subjects. Our data showed that
adiponectin indeed ameliorated the OxLDL-induced intracellular
lipid accumulation in human macrophages from diabetic patients
but not normal healthy subjects.
The intracellular lipid homeostasis of foam cells is dynamically
regulated by OxLDL internalization and cholesterol efux. Macrophages from mice lacking SR-A and/or CD36 show reduced OxLDL
internalization and are less prone to foam cell formation in vitro [6].
In addition to the scavenger receptors that regulate the inux of
cholesterol in macrophages, several equally important ABC transporters are involved in the efux of cholesterol from cells: ABCA1,
ABCG1, and ABCG4. Vaughan and Oram [32] have shown that these
ABC transporters act sequentially to eliminate excess cholesterol
from the cell, with ABCA1 acting rst by efuxing cholesterol to
lipid-poor ApoAI. ABCG1 can then efux cholesterol to these newly
made HDL moieties before ABCG4 adds additional cholesterol.
However, macrophages do not express ABCG4, leaving ABCA1 and
ABCG1 as the known primary regulators of cholesterol efux in
macrophages. Previous studies using macrophages from patients
with T2DM [11] and diabetic mice [33] showed that diabetic macrophages have an increased activation of proatherogenic pathways
because of the signicant reductions in ABCG1-specic cholesterol
efux. Therefore, we examined the regulatory role of adiponectin
and showed that adiponectin augmented both mRNA and protein
expression of ABCG1 but not ABCA1, SR-A and CD36 in diabetic
macrophages. We also demonstrated that adiponectin treatment
may affect HDL-mediated cholesterol efux but not ApoAImediated cholesterol efux or OxLDL uptake during the transformation of foam cells. Although adiponectin had been shown to
protect against atherosclerosis by increasing apoAI-mediated cholesterol efux from macrophages through ABCA1-dependent
pathway [34], ABCG1 is the primary protein in macrophages
affected by adiponectin on the setting of diabetes that regulates
cholesterol efux to HDL. Thus, upregulation of ABCG1 in T2DM by
adiponectin most likely will be important for the prevention of
atherosclerosis and diabetic vascular complications.
The liver X receptors (LXRs) are nuclear receptors that play
central roles in the transcriptional control of lipid metabolism [35].
Activation of LXRs in lipid-loaded macrophages leads to induction of
genes involved in the cholesterol efux pathway in an attempt to
reduce the intracellular cholesterol burden [35]. The ABC transporters such as ABCA1 and ABCG1 are downstream target genes of
LXRa and critical for the ability LXRs to enhance efux to cholesterol
acceptors [36]. More important, we also showed that the upregulation of ABCG1 by adiponectin was accompanied by increased
nuclear LXRa levels and enhanced LXRa transcriptional activity.
Moreover, the pharmacological and genetic inhibition of LXRa
activation by GGPP or siRNA abolished the adiponectin-mediated
upregulation of ABCG1. These results suggest that LXRa-mediated
transcriptional regulation is required for the induction of ABCG1 by
adiponectin. Our ndings are in agreement with those of Tian [37],
who found that adiponectin upregulated ABCA1 and ABCG1 and
inhibited the development of atherosclerosis in mice. Despite the

69

unique pathway discovered in this study, the detailed mechanisms

of how adiponectin affects cholesterol efux merit further study.
We nally examined the correlation between adiponectin and
cholesterol efux capacity in diabetic patients in a cross-sectional
study. We used a previously established assay that integrates the
pathways known to mediate cholesterol efux from macrophages
(i.e., ABCA1, ABCG1, scavenger receptor B1, and aqueous diffusion)
[22]. Our ndings demonstrated that only a small part of the
observed relationship between diabetes and cholesterol efux
capacity was explained by variations in plasma HDL-C and ApoAI
levels. Indeed, adiponectin levels served as a stronger predictor of
cholesterol efux capacity. The association between serum adiponectin levels and cholesterol efux capacity remained signicant
after adjustment for the HDL cholesterol level in diabetic patients in
the regression models. Although cholesterol efux from macrophages represents only a small fraction of overall ux through the
reverse cholesterol transport pathway [38], a substantial body of
evidence suggests that cholesterol efux capacity, an integrated
measure of HDL quantity and quality, is most likely the most relevant component of atheroprotection [38e40]. Nevertheless, our
ndings indicate that the impaired cholesterol efux capacity in
patients with T2DM is largely due to the lower levels of
adiponectin.
In conclusion, the present study reveals a direct mechanistic link
between hypoadiponectinemia and the pathophysiology of accelerated atherosclerosis in diabetes. Our ndings suggest that adiponectin may potentially be of therapeutic value in inhibiting the
process of atherosclerosis. Interventional studies are needed to
assess the effects of adiponectin status on CVD in diabetic subjects.
Conicts of interests
The authors have no conicts of interests.
Sources of funding
This work was supported by grants from the National Natural
Science Foundation of China (81172663), Thousand, Hundred and,
Ten project of Guangdong Province, and the Foundation for the
Author of National Excellent Doctoral Dissertation of PR China
(200978).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.atherosclerosis.2013.01.017.
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