Vol.3, No.4, 227-235 (2013) Journal of Diabetes Mellitus
Adipocytes modulate vascular smooth muscle cells
migration potential through their secretions*
Souhad El Akoum1,2, Isabelle Cloutier1, Jean-François Tanguay1,2#
1Montreal Heart Institute, Montréal, Canada; #Corresponding Author: jean-francois.tanguay@icm-mhi.org
2Département de Sciences Biomédicales, Faculté de Médecine, Université de Montréal, Montréal , Canada
Received 3 May 2013; revised 3 June 2013; accepted 12 June 2013
Copyright © 2013 Souhad El Akoum et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Impairment of vascular smooth muscle cells
(VSMC) is recognized as a predisposition factor
for atherosclerosis development. We hypothe-
size that the metabolic syndrome has a direct
impact on VSMC migration and phenotypic swit-
ching, which may increase the inciden ce of athe-
rosclerotic events. Aortic VSMC were extracted
from 10 weeks old C57BL6 mice and incubated
for 24 hr in adipocytes conditioned cell culture
medium. Adipocytes were extracted from dia-
betic C57BL6 male mice fed with either a vegetal
or an animal High-Fat-Diet (HFD) for 20 weeks.
Migration of VSMC in response to conditioned
media stimulations was significantly modulated
compared to control. The most extended effects
on VSMC were triggered by adipocytes from mice
fed with animal HFD. These effects were con-
current with increased leptin con centrations and
decreased adiponectin levels in conditioned me-
dia. A significant up-regulation of CD36 mRNA
level was found in VSMC treated with adipocytes
from HFD-fed mice. In conclusion, we have shown
that the development of adipocyte-induced VSMC
alterations is linked to diet fatty acid composi-
tion and the degree of metabolic alterations. The
modulation of adipokine secretions in the adi-
pose tissue that is linked to metabolic altera-
tions may alter the physiolo gy of VSMC and thus
accelerate the development of metabolic-related
vascular diseases.
Keywords: Adipocytes; Atherosclerosis; Type 2
Diabetes; Vascular Smooth Muscle Cells; Migratio n
Obesity and type 2 diabetes (T2D) are recognized as
predisposition factors for atherosclerosis development.
Endothelial cells (EC) and vascular smooth muscle cells
(VSMC) are sensitive to obesity-linked increased fatty
acids (FA) and T2D-linked hyperglycæmia that triggers
vascular alterations and leads to atherogenesis [1-3]. A
lack in insulin sensitivity lowers intracellular glucose
availability forcing arterial cells to use FA as an alterna-
tive energy source. Thus, in atheroprone regions suscep-
tible to plaque formation, EC and VSMC are subjected to
metabolic modifications that lead to the accumulation of
oxidized LDL (oxLDL) within the intima [4] and the
progression of vascular diseases.
At the early stage of atherogenesis, the oxLDL-ex-
posed endothelium becomes activated and up-regulates
the expression of adhesion molecules (ICAM, VCAM),
thereby allowing monocyte recruitment and initiation of
the inflammatory process [4]. For their part, VSMC un-
dergo “phenotypic switching”, which is characterized by
decreased contractibility and increased proliferation and
migration toward the intima. These processes then con-
tribute to intima thickness and atherosclerotic plaque
formation [5,6]. With time, VSMC may undergo choles-
terol-induced trans-differentiation into foam cells [7].
These cells have a macrophage like phenotype and ex-
press increased levels of the oxLDL scavenger receptor
CD36, which favors lipid accumulation and contributes
to the atherogenic process [8].
The specific factors implicated in VSMC alterations
linked to obesity and T2D remain incompletely under-
stood. However, the migration process of these cells
seems to play a pivotal role in the development of
atherosclerosis and its complications. The secretions of
adipokines from the adipose tissue, which regulate vari-
ous sets of metabolic and physiologic processes, could
*Funding: This study was financed by a grant from the foundation des
maladies du Coeur du Québec and the Montreal Heart Institute founda-
Competing of interest: Neither of the authors has any potential benefits
or conflicts of interest to disclose.
Copyright © 2013 SciRes. OPEN ACCESS
S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5
not be discarded [9,10]. Indeed, little is known about the
effect of these adipokines on the VSMC migration proc-
ess. Thus, since obesity-increased FA is an important in-
ducer of metabolic disorders and vascular pathogenesis,
and regulates adipose tissue activity, we hypothesized that
adipocytes through adipokine secretions play an impor-
tant role in VSMC alterations leading to atherosclerosis.
Therefore, the present study is aimed at determining
the effect of factors secreted by adipocytes isolated from
mice fed with High-Fat-Diet (HFD) on VSMC migration.
We focused our investigation on isolated adipocytes to
exclude the impact of other cell types found in the adi-
pose tissue such as macrophages.
2.1. Experimental Protocol
Adipocytes were obtained from C57BL/6J male mice
fed on a HFD for 20 weeks [11]. Mice were fed one of
two low cholesterol HFD (34.9% fat, 26.3% carbohy-
drate, cholesterol <0.03%; ResearchDiet): a vegetal HFD
(VD) composed of soy and cotton oil and an animal HFD
(AD) composed of lard. The common standard diet (SD)
was used as control (6% fat, 57% carbohydrate; Harlan-
Teklad). The animal protocol was approved by the Ani-
mal Care and Use Committee of the Montreal Heart In-
2.2. Adipocyte Culture
Mature adipocytes were isolated from visceral-ab-
dominal adipose tissue of each mouse at sacrifice. Briefly,
adipose tissue was finely minced and enzymatically di-
gested in Dulbecco’s-Modified-Eagle-Medium (DMEM)
containing 2 mg/ml type I collagenase (37˚C; 40 min).
Samples were then centrifuged (300 × g; 7 min) and the
top white supernatant passed through 100 µm filter and
washed twice in DMEM.
Adipocytes (104 cells/ml) were maintained in serum
free low glucose DMEM supplemented or not with insu-
lin (100 µU/ml), glucose (25mM) or insulin + glucose
for 24 hr. Adipocytes supernatants (AdS) were collected
and immediately stored at 20˚C. At the moment of ex-
perimentation samples were thawed and added in VSMC
To simplify annotations, supernatants from unstimu-
lated adipocytes were noted as AdS-U, supernatants from
insulin-stimulated adipocytes as AdS-I, supernatant from
glucose-stimulated adipocytes as AdS-G and supernatant
from (insulin + glucose)-stimulated adipocytes as AdS-IG.
2.3. VSMC Culture
VSMC were isolated from 10 weeks old C57BL/6J
mice as previously described [12]. Briefly, dissected aor-
tas were discarded of its adventitia, cut in 2 mm square
pieces and incubated in 1.5 mg/ml collagenase type II
solution (37˚C; 5% CO2; 5 hr). Dissociated cells were
suspended in 5 ml of DMEM and centrifuged at 300 × g
for 5 min. The pellet was resuspended in 700 μl DMEM
supplemented with 10% serum and transferred to a single
well of a 48-well plate and left untreated for 5 days.
VSMC purity was assessed by confocal microscopy upon
4 amplification passages [12] before treatment with AdS.
To separate the effects of insulin and glucose from the
effects of secreted factors from adipocytes on VSMC in
AdS, insulin and glucose stimulations on VSMC were
used as controls for each experiment. Each condition was
tested in duplicate and expressed as percentage compared
to control.
2.4. Cell Migration Assay
Confluent VSMC were serum starved overnight before
a 24 hr treatment with AdS or appropriate controls. Cells
(5 × 103) were loaded into the upper chamber of a 24-
transwell plate with an 8 μm pore membrane. Fibroblast
growth factor (bFGF), which was used as the chemo-
attractant, was added into the lower chambers at 10 ng/
ml. Following 5 h of incubation (37˚C; 5% CO2), the
membrane was fixed with paraformaldehyde, stained
with 4’,6’-diamidino-2-phenylindole and scanned on a
microscope. The number of migrated cells was counted
in five random fields for each sample.
2.5. Characterization of VSMC
Cells were cultured and allowed to grow near conflu-
ence on 8 mm glass lamella. Following two washes in
DMEM, cells were fixed in 4% paraformaldehyde and
permeabilized with 0.1% Triton X-100. The cells were
then washed and subsequently blocked for 1 hr in 2%
normal goat serum. After washing with PBS BSA1%,
cells were incubated with an anti-α smooth muscle actin
antibody (NeoMarker, USA) over night at 4˚C. After
incubation, the cells were washed twice in PBS BSA 1%
and further incubated with an affinity purified Alexa 488-
conjugated anti-rabbit antibody (Invitrogen, USA). Fluo-
rescent specimens were visualized under a microscope
and photographed.
FA incorporation in AdS-treated VSMC was evaluated
by Oil-Red-O staining. Cells were fixed in 10% formalin
for 15 minutes. 60% isopropanol was then added before
completely drying wells. A stock of 0.5% ORO staining
solution was prepared in 60% triethyl phosphate (TEP,
Sigma) and filtered. A working solution was prepared by
mixing 30 ml of ORO stock solution with 50 ml of PBS,
filtered through a 1 µm filter and added to the dried wells.
Following a 1-hour incubation period, the ORO solution
was removed and cells were washed in PBS prior to pic-
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S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5
Copyright © 2013 SciRes. OPEN ACCESS
ture acquisition using a light microscope equipped with a
video camera.
2.6. Quantification of mRNA Level
Total RNA was isolated using Qiazol reagent accord-
ing to the manufacture’s instructions (Qiagen, Toronto,
ON, Canada). Single-strand cDNA was synthesized ac-
cording to the procedure in the iScript cDNA Synthesis
Kit manual (Bio-Rad Laboratories, Montreal, QC, Can-
ada). Q-PCR reactions were carried out using the Bril-
liant-II SYBR® Green Master-Mix (Stratagene, Missis-
sauga, ON, Canada) and specific primers for CD36 sca-
venger receptor (Fwd: 5’-GCC-AAG-CTA-TTG-CGA-CAT-
3’) and insulin receptor (Fwd: 5’-CAG-AGA-AGG-TCT-
GCC-GC-3’). The mRNA levels were normalized to
Cyclophilin-A expression levels (Fwd: 5’-CCG-ATG-
GCC-ATT-ATG-3’). The targeted and reference genes
were amplified in duplicates using the Mx3000P Q-PCR
System (Stratagene). The relative quantification of target
genes was determined using the MxProTM Q-PCR soft-
ware version 3.00 (Strategene) as previously described
2.7. Biochemical Assay
Adiponectin and leptin concentrations within AdS
were measured using a mouse ELISA kit according to the
manufacturer’s protocol (ALPCO). Prior to analysis,
samples were thawed on ice and rapidly used.
2.8. Statistical Analysis
All statistical analyses were performed separately for
males and females. Data are presented as mean ± stan-
dard deviation for continuous variables.
Repeated measures analysis of variance (ANOVA)
models were used to compare adiponectin and leptin
levels within supernatants from adipocytes between
groups (SD, VD and AD groups). Cell migration data
and mRNA levels were compared between groups (SD,
VD and AD groups) using an ANOVA model.
In addition, the relationships among adiponectin and
leptin levels, and mRNA levels and migration parameters
were investigated using Pearson or Spearman correla-
tions according to the nature of the distribution data.
All analyses were done using SAS version 9.1 (SAS
Institute Inc., Cary, NC, USA) and data 0.05 were con-
sidered significance.
HFD-triggered obesity in corresponding mouse groups
correlated with increased leptin levels in systemic circu-
lation (Table 1) [11]. The development of T2D in HFD
groups within male mice was also linked to decreased
adiponectin levels in sera. In the AD group, mice also
demonstrated hyperinsulinemia. The blood profile of
adipokines was diet-dependant, as HFD-fed mice showed
decreased adiponectin and increased leptin mRNA levels
within the abdominal visceral white adipose tissue, in
comparison to SD-fed mice. To characterize the effect of
these modulations on VSMC, abdominal adipocytes were
isolated from each animal group and used to condition
culture media.
3.1. Biochemical Characteristics
To determine whether the differential effects of the
AdS subtypes were attributable to variation in production
of adipokines, we examined their leptin and adiponectin
contents. These two adipokines are known for their im-
portant role in cellular glucose and lipid homeostasis.
Table 1. In vivo mice parameter after 20 weeks of diet. Weight gain, glycemic parameters and adipokines secretion profile evaluated
in fasted sera mice. mRNA levels of leptin and adiponectin in visceral abdominal adipose tissue were also evaluated at the sacrifice.
*P < 0.05, **P < 0.01, ***P < 0.001 vs. SD; #P < 0.05, ##P < 0.01 vs. VD.
Diet groups
Weight gain (g) 11.8 ± 0.9 28.1 ± 1.3*** 23.9 ± 1.6***
Fasting glycæmia (mM) 4.0 ± 0.2 9.0 ± 0.6*** 8.3 ± 0.6***
Fasting insulinæmia (μg/ml) 0.3 ± 0.1 0.4 ± 0.1 0.7 ± 0.1** #
Fasting leptin level (μg/ml) 0.06 ± 0.013 0.58 ± 0.05*** 0.8 ± 0.07*** ##
Fasting adiponectin level (μg/ml) 1.97 ± 0.15 1.40 ± 0.12** 1.24 ± 0.15***
Fasting FA level (μM) 0.51 ± 0.12 1.13 ± 0.04* 1.18 ± 0.22*
Leptin mRNA level in adipose tissue 0.78 ± 0.18 3.93 ± 0.26*** 5.39 ± 0.64***
Adiponectin mRNA level in adipose tissue 1.24 ± 0.08 0.53 ± 0.06*** 0.61 ± 0.06***
S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5
Their inverse interactions are linked to metabolic disor-
ders and vascular alterations.
Leptinemia was increased by 4 fold in AdS-U from
HFD groups, as compared to the SD group (Table 2).
Hyper-leptinemia was further increased in response to
insulin and/or glucose stimulations in HFD groups, com-
pared to unstimulated cells where maximum levels were
reached with AdS-G.
On the other hand, adiponectin levels were decreased
as expected in AdS-U of adipocytes from HFD-fed mice,
in comparison to the SD group (Table 2). In the SD
group, adiponectin concentrations were increased by
30% in AdS-I, 70% in AdS-IG, and 96% in AdS-G, as
compared to AdS-U. The effect of insulin adiponectin
secretions was not significant in AD and VD group,
compared to unstimulated cells. However, it was in-
creased by the addition of glucose (AdS-IG: 140% for
VD and 127% for AD) or glucose alone (AdS-G: 350%
for VD and 222% for AD), compared to unstimulated fat
cells. Despite these differences, adiponectin concentra-
tions in HFD-derived AdS remained 30% to 70% higher
in the corresponding controls from the SD group (Table
3.2. Effects of AdS on VSMC Migration
The migration process of VSMC is critical for vascu-
logenesis and vascular repair. Insulin and glucose treat-
ments significantly increased VSMC migration by 10 to
33%, compared to untreated cells (Figure 1(B)). To
characterize the impact of AdS on VSMC physiology, we
evaluated VSMC migration capabilities after 24 hr of
treatment with AdS. All AdS from SD-derived adipo-
cytes reduced migration of VSMC; albeit to a lesser ex-
tend in the case of AdS-G (Figure 1(A)). In contrast, all
AdS from HFD-fed mice showed a strong pro-migratory
effect on VSMC (65% - 70% increase). This diet-induced
effect was more prominent in HFD groups with AdS-I
(200%), AdS-IG (230%) and AdS-G (260%), compared
to AdS-U.
The impact of AdS on VSMC migration was also re-
flected by their cytoskeleton arrangement. While freshly
extracted VSMC showed a twisted form with parallel
α-actin filaments [12], this process was absent in AdS-U
from SD mice, while light disorganization of the actin
filaments was noted (Figure S1(A)). Conversely, in the
AdS-U-VD group, VSMC showed disorganized parallel
actin filament structures with some migratory extensions
(Figure S1(B)), while supernatants from AD groups il-
lustrated increased fibers’ disorder (Figure S1(C)).
These effects were not associated with parallel α-actin
filaments and microscopic snapshots showed blurring
and dot staining, reporting most probably actin polym-
erization that lead to extension of the leading edge.
3.3. Insulin Receptor Gene Modulation
Since the metabolism of VSMC depends upon glucose,
we evaluated the gene expression profile of InsR, which
is responsible for glucose uptake. In control VSMC, only
insulin and glucose stimulations significantly reduced
InsR expression levels (0.55 ± 0.02), compared to un-
Table 2. Adiponectin and leptin levels in adipocytes’ supernatants (AdS). Adiponectin and leptin levels were evaluated in AdS of
untreated (AdS-U), insulin-treated (AdS-I), glucose-treated (AdS-G) and insulin and glucose treated (AdS-IG) mature adipocytes
extracted from visceral abdominal adipose pad of each mice group. Treatments were maintained for 24h. For a same diet: *P < 0.05
vs AdS-U; **P < 0.05 vs. AdS-I; ***P < 0.05 vs. AdS-IG. For a same stimulation: P < 0.05 vs. SD; ††P < 0.05 vs. VD.
Diet groups Adipocytes’ supernatants Adiponectin level (ng/ml) ± SEM Leptin level (ng/ml) ± SEM
AdS-U 2.75 ± 0.46 0.33 ± 0.03
AdS-I 3.61 ± 0.21* 0.47 ± 0.04
AdS-G 4.69 ± 0.04* ** 0.55 ± 0.03*
AdS-IG 5.40 ± 0.51*** 0.77 ± 0.05* ** ***
AdS-U 0.75 ± 0.11 1.20 ± 0.09
AdS-I 0.82 ± 0.27 1.78 ± 0.05* †
AdS-G 1.79 ± 0.29* ** † 2.25 ± 0.16* ** †
AdS-IG 3.36 ± 0.62* ** *** † 2.74 ± 0.09* ** *** †
AdS-U 1.00 ± 0.22 1.42 ± 0.09† ††
AdS-I 1.43 ± 0.14† †† 1.81 ± 0.03* †
AdS-G 2.27 ± 0.39* ** † 2.18 ± 0.07* ** †
AdS-IG 3.22 ± 0.38* ** *** † 2.65 ± 0.04* ** *** †
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S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5 231
Figure 1. (A) VSMC migration rate after 24 hr of treatment
with AdS of male mice. These results are expressed as per-
centage of proliferation rate of unstimulated cells; (B) Incu-
bation of VSMC with insulin or glucose for 24 hours was
used as control compared to unstimulated cells. For a same
diet: *P < 0.05 vs AdS-U; **P < 0.05 vs. AdS-I; ***P < 0.05 vs.
AdS-IG. For a same stimulation: #P < 0.05 vs. SD; ##P < 0.05
vs. VD. In control treatments: +P < 0.05 vs unstimulated cells
(Ø); ++P < 0.05 vs insulin stimulated cells.
stimulated (1.01 ± 0.11) and insulin (0.85 ±0.11) or glu-
cose (1.22 ± 0.25) stimulated cells (Figure 2(B)). In
AdS-treated cells, unstimulated adipocytes did not in-
duce any significant modulation of InsR mRNA in
VSMC compared to control (Figure 2(A)). Stimulation
of adipocytes from the SD group with insulin or glucose
significantly decreased InsR expression levels by 30 and
75% respectively, compared to unstimulated cells. In
contrast, adipocytes from VD and AD groups showed no
effect on InsR gene expression in VSMC compared to
unstimulated condition. Moreover, insulin + glucose sti-
mulation was without any effect on InsR gene expression
in SD groups, compared to unstimulated adipocytes. Fur-
thermore, while adipocytes from AD groups slightly in-
creased InsR mRNA levels in VSMC compared to un-
stimulated cells, those from VD groups markedly in-
creased InsR levels in VSMC (1.25 ± 0.11) in response
to insulin + glucose stimulation compared to the other
culture conditions and to SD derived AdS.
Figure 2. (A) InsR mRNA expression level in VSMC is up-
regulated with AdS stimulation extracted from AD-fed group;
(B) The basic InsR VSMC expression level (Ø) is reported as
100% expression gene level. Insulin + glucose stimulation de-
crease InsR mRNA level without any significant modulation of
this parameter after insulin or glucose VSMC treatment (B).
For a same diet group: *P < 0.05 vs AdS-U; **P < 0.05 vs.
AdS-I; ***P < 0.05 vs. AdS-IG. For a same stimulation: #P <
0.05 vs. SD; ##P < 0.05 vs. VD. In control treatments: +P < 0.05
vs unstimulated cells (Ø), ++P < 0.01 vs insulin-stimulated cells,
+++P < 0.01 vs insulin + glucose (Ins/Glu)-stimulated cells.
3.4. VSMC Fatty Acids Accumulation
The impact of AdS on VSMC physiology could also
be linked to FA released by adipocytes. Modulation of
FA receptors may be an indicator of their implication in
VSMC alterations. We evaluated CD36 mRNA expres-
sion levels in VSMC, a scavenger receptor recognized
for its role in the atherogenesis process [8]. Insulin
stimulation increased CD36 mRNA expression levels by
2 fold in VSMC, compared to unstimulated and glucose
stimulated cells (Figure 3(B)). In addition, glucose alone
increased CD36 mRNA levels by 30% (Figure 3(B)). In
AdS treated cells, when VSMC were exposed to AdS-U
from HFD groups, CD36 mRNA levels were signifi-
cantly enhanced (0.48 ± 0.05 for VD; 0.88 ± 0.55 for
AD), compared to SD groups (0.02 ± 0.13) (Figure
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S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5
Figure 3. (A) CD36 scavenger receptor mRNA expression
level in AdS-treated VSMC in different mouse groups is modu-
lated depending on the type of treatment; (B) Incubation of
VSMC with insulin or glucose for 24 hours was used as control
compared to unstimulated cells. For a same diet: *P < 0.05 vs
AdS-U; **P < 0.05 vs. AdS-I; ***P < 0.05 vs. AdS-IG. For a
same stimulation: #P < 0.05 vs. SD; ##P < 0.05 vs. VD. In con-
trol treatments: +P < 0.05 vs unstimulated cells (Ø); ++P < 0.05
vs insulin stimulated cells; +++P < 0.05 vs insulin/glucose sti-
mulated cells.
3(A)). AdS-I of all diet groups increased CD36 mRNA
expression levels in VSMC with still higher levels in VD
(1.84 ± 0.09) and AD (2.10 ± 0.02) groups, compared to
the SD group (1.03 ± 0.11). AdS-G increased scavenger
receptor mRNA expression in VSMC in SD (0.43 ± 0.15),
VD (0.90 ± 0.03) and AD (1.07 ± 0.02) groups following
a similar pattern but at a lesser extent than AdS-I. Finally,
a cumulative impact was observed in AdS-IG stimulation
in SD (1.99 ± 0.14) and AD (2.67 ± 0.02) groups, com-
pared to AdS-I and AdS-G; an effect absent in the VD
group (1.68 ± 0.21).
On the other hand, Oil-Red-O staining was done to re-
late FA incorporation after supernatant treatment to
CD36 increased levels. In SD groups, AdS-U showed no
increase of FA incorporation compared to untreated cells.
In HFD groups, supernatants increased FA incorporation
in VSMC when stimulated with AdS-U (Figure S2),
with a more marked coloration in AD groups compared
to VD groups.
The physiologic alterations of VSMC triggered by
adipocyte conditioned media was recently addressed in
humans [13]. The novelty of this study is the evaluation
of the impact of adipocyte-conditioned media on VSMC
in a model of obesity and T2D.
Adipocytes were isolated from abdominal adipose tis-
sues of male mice at different stages of metabolic altera-
tions [11] to condition VSMC culture medium. The pro-
tocol was set to reproduce a culture environment that
mimics in vivo hyperglycemic and/or hyperinsulinic con-
ditions. Adipocytes were stimulated with glucose, insulin
or both. Their impact on VSMC was compared to un-
stimulated cells.
Obesity-linked adipocyte alterations modulate VSMC
migration, an important cellular process in atherogenesis.
We previously demonstrated that AdS decreases VSMC
proliferation and correlates with adiponectin and leptin
unbalance [12].
In the present study, we showed that VSMC migration
was statistically correlated with adiponectin and leptin
changes in AdS. Indeed, recent observations have shown
that adiponectin deprivation results in neointima hyper-
plasia, while leptin promotes VSMC migration [14,15].
Equivalent numbers of cultured adipocytes are not trans-
posed in the same adipokine secretion profile in all diet
groups. A sharp decrease in adiponectin levels, coupled
to hyper-leptinemia, was correlated with increased VSMC
migration in HFD groups. Insulin ± glucose treatments
further increased the migratory potential of VSMC, pos-
sibly due to higher secreted levels of leptin in AdS. De-
creased adiponectin levels also contribute to the increase
VSMC migration potential, since this adipokine is known
to inhibit the migratory process [16]. These modulations
were also reflected by the arrangement of the α-actin
filaments, as reported by confocal microscopy. We have
shown different stages of filament disorganizations and
correlated them with an increased level of migration in
AdS extracted from HFD mice groups. These disorders
in the cytoskeleton are most probably due to increased
actin dis-polymerization and re-polymerization events,
given stimulation of the migratory mechanism [17,18].
Dot staining shown in AD groups relates to a higher level
of soluble actin monomers needed to polymerize and
form filaments [18,19]. This will lead to this snapshot
showing the formation of actin filaments that will push
the leading cell front forward and catalyzing VSMC mi-
gration [18,20].
In accordance with the above-discussed hypothesis,
released FA could impact glucose cell homeostasis and
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S. El Akoum et al. / Journal of Diabetes Mellitus 3 (2013) 227-23 5 233
response to insulin. InsR gene expression levels in VSMC
were decreased in the presence of insulin + glucose,
compared to unstimulated VSMC. This level was in-
creased when cells were stimulated with glucose alone,
compared to insulin + glucose stimulated cells. In the
presence of AdS treatment, InsR mRNA levels increased
in AdS-I and AdS-G from AD groups compared to both
SD and VD groups. Saturated FA present at high concen-
trations in AD is known to block insulin activation [21]
and induce hyperleptinemia, which has been reported to
impair insulin signaling pathways [22]. This was noticed
in this study with the lack of insulin-induced adiponectin
release in AdS in HFD groups compared to SD. To coun-
teract the effect of reduced glucose uptake, VSMC in-
crease their InsR expression levels. In contrast, VD im-
proves lipogenesis and lowers FA in AdS through its high
level of oleic acid, which has been documented to trigger
insulin sensitivity [23] and lead to a lower InsR mRNA
Adipocytes used in this protocol were conditioned by
food diets during 20 weeks. Mice of HFD groups were
hyperglycemic; a status usually associated with elevated
advanced glycation end products (A-GEP). We hypothe-
sized that A-GEP present in these adipocytes attenuate
their insulin sensitivity and abolish insulin-activated li-
pogenesis [24]. In this situation, adipocytes from HFD-
groups had increased lipolysis activity, which leads to FA
release increase and VSMC migration [3,25].
These mice had an increased serum concentration of
FA compared to SD; a profile directly linked to the activ-
ity of abdominal adipocytes. FA rich AdS from HFD-
mice can stimulate PPARγ in VSMC, triggering CD36
gene expression [8,26]. CD36 plays a key role in FA acid
uptake and regulation in vivo [27].
Adipokines were also reported to regulate CD36 ex-
pression [28] and therefore FA accumulation in VSMC
and morphologic modulations. While leptin increases
CD36 expression in VSMC, adiponectin prevents it [29,
30]. Thus, VSMC treated with AdS from HFD groups
modulate their CD36 expression in correlation with lep-
tin and adiponectin levels contained in the culture media.
CD36 mRNA upregulation levels are correlated with in-
creased Oil-Red-O cell staining in corresponding diet
groups, indicative of FA accumulation in VSMC cyto-
plasm [31]. This accumulation does not seem to influence
migratory potentials, even though we have shown that
AdS from HFD groups increases apoptotic factors [12].
In fact, it has been reported that caveolin contained in
muscle cells sequester FA on the cytoplasmic membrane
[32]. This could protect the actin fluency and migration
potential even if the apoptosis process is triggered.
Further studies will be needed to further characterize
this aspect of morphologic alteration and FA accumula-
All together, these data indicate that the development
of adipocyte-induced VSMC alterations is linked to the
FA diet composition and the degree of metabolic altera-
tions. Such alterations trigger adipokine unbalance from
adipocytes, defining VSMC migration and FA cytoplas-
mic accumulation. Thus, adipocyte alterations would
directly influence VSMC transmigration towards the
arterial media and thereby inducing, at the same time,
apoptosis and foam cell formation. Simultaneously, these
events may trigger atherogenesis by the migration of
VSMC-derived foam cells in the intima and stimulate
inflammatory cell recruitment into the arterial wall.
These results may contribute to better understand the
complex interplay between adipocytes and vascular cells
that lead to increased atherosclerotic events in obe-
sity-linked metabolic syndromes.
The current study appears to be compelling, and
makes a significant contribution to the field of athero-
genesis process as well as the understanding of the im-
pact of the adipose tissue on VSMC. It reflects the direct
impact of adipose tissue secretion cocktail on the VSMC
migratory potential; and it’s also a main characteristic
that affects atherogenesis. Further, the originality of this
study is the evaluation of these secretions of adipocytes
provided from mice groups showing different levels of
metabolic alterations.
In spite of this, the current research acknowledges a
few limitations that should be noted to help interpret the
results, and be added to the data on the relationships be-
tween adipose tissue and vascular wall cells physiology.
In fact, due to material limitations, we have used adipo-
cytes extracted from abdominal white adipose tissue in-
stead of those of perivascular adipose tissue that are in
direct contact with the vascular wall. Even if these two
types of tissue are comparable, their localization in the
body could affect their physiology [33]. The amount of
perivascular tissue is very limited in our protocol mice
and the quantity couldn’t cover all our stimulation condi-
tions. However, we can speculate that the effect would be
very close between these two types of white adipose tis-
The authors gratefully thank Louis Villeneuve from the Montreal
Heart Institute for immunostaining and confocal microscopy advices
and Mariève Cossette from the Montreal Heart Institute Coordinating
Centre for the statistical analyses.
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Figure S2. FA accumulation reflected by Oil Red O staining in
native VSMC (A) and in VSMC after a 24 h treatment with
AdS from SD (B); VD (C) or AD (D) groups. FA accumulation
was increased with AdS treatment at different level depending
on the diet nature group.
Figure S1. Immunocytochemical characterization of VSMC as
observed on confocal microscopy after a 24 h treatment with
AdS from SD (A); VD (B) or AD (C) groups. α-actin filament
disorganization level was increased depending on the diet na-
ture group.