Biochemical and Biophysical Research Communications 472 (2016) 68e74

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1

preadipocytes by reducing the expression of glucocorticoid receptor
gene
Weiwei Chu a, Wei Wei a, Shigang Yu a, Haiyin Han a, Xiaoli Shi a, Wenxing Sun a, b,
Ying Gao c, Lifan Zhang a, Jie Chen a, *
a
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
b
College of Public Health, Nantong University, Nantong 226019, PR China
c
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and
Received 9 February 2016 metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the inter-
Accepted 15 February 2016 action of muscle and adipose development. Previous related studies mainly focus on the effects of adi-
Available online 17 February 2016
pocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development
remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment
Keywords:
showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and
Co-culture
the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 pre-
Adipocyte
Myocyte
adipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis
Glucocorticoid receptor factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that
Sensitivity C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1
preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we
conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by
reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may
be a promising therapy for treating patients with obesity or diabetes.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Obesity is a global health disorder in the modern world. The
excess body fat is usually associated with insulin resistance,
inflammation, type 2 diabetes and metabolic syndrome [1]. Adi-
pocytes and myocytes have been confirmed both deriving from
mesoderm cells which is tightly connected by autocrine, paracrine
and endocrine during the development of animals [2]. Now skeletal
muscles are well recognized as large endocrine organs in the body
[3], and myokines appear to be involved in local autocrine/para-
crine interactions within adipose tissue [4]. Recent studies of
myocytes and adipocytes cross-talk could help to explore the
function and mechanism of cytokines which regulate fat deposi-
tion. The co-culture system denotes the growth of two cell types in
* Corresponding author.
E-mail address: jiechen@njau.edu.cn (J. Chen).
a shared medium, where the physical contact might have in-
fluences on the cellular function [5]. It is an advanced model to
study the cross talk of two cell types. Previous studies were mainly
focused on the effects of adipocytes on the myocytes, and indicated
that adipocytes modulate the development of insulin resistance in
skeletal muscle [6]. Only a few studies aim to uncover the effects of
this co-culture system on the adipocytes by altering the mRNA
expression of its adipogenic marker genes. For example, C2C12
myotubes upregulated adipogenic marker gene mRNA expression
in differentiated 3T3-L1 cells [7]. Co-culture of bovine muscle sat-
ellite cells with preadipocytes increased C/EBPb and GPR43 gene
expression in adipocytes [8]. Besides, the differentiation of porcine
preadipocytes was inhibited in the co-cultured experiment, and the
expression of marker genes in earlier stage of adipocytes differ-
entiation was lower than those in the solo-cultured cells [2]. These
paradoxical results produced from those studies might be caused
by the different co-culture methods and the detection of different
marker genes, and the effects of myocyte on adipocyte might vary

http://dx.doi.org/10.1016/j.bbrc.2016.02.063
0006-291X/© 2016 Elsevier Inc. All rights reserved.
 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74
in different stages of adipocyte differentiation. However, the effects
of myocytes on adipocytes are still not confirmed, and the under-
lying mechanism is unclear.
The development process in muscle starts earlier than fat for the
fetal, postnatal, and adult animals. The developing fat cells closed to
muscle undergo all growth and differentiation phases in close
proximity similar to mature, multinucleated, and functional skel-
etal muscle [5]. Hence, in this study, we co-cultured 3T3-L1 pre-
adipocytes with differentiated C2C12 myotubes, and examined the
comprehensive impacts of C2C12 myotubes on the proliferation,
differentiation, cell cycle and apoptosis of co-cultured 3T3-L1 pre-
adipocytes. What's more, the sensitivity alteration of 3T3-L1 pre-
adipocytes to glucocorticoids (GCs), which is well known as the
regulator of cell proliferation [9], differentiation [10] and anti-
apoptosis [11], was analyzed to elucidate the underlying
mechanism.
2. Materials and methods
2.1. Co-culture of C2C12 myotubes and 3T3-L1 preadipocytes
Mouse C2C12 myoblasts were seeded independently on the
trans-well inserts with a 1 mm porous membrane, incubated and
grown to confluence in growth medium at 37
C in 5% CO2 incu-
bator. The growth medium was Dulbecco's Modified Eagle's Me-
dium (DMEM) (Hyclone, Beijing, China) containing 10% fetal bovine
serum and 1% antibiotics. To induce C2C12 cells differentiation,
growth medium was switched to DMEM with 2% horse serum and
changed every day to get the myotubes. 3T3-L1 preadipocytes
(1  105 per well) were placed in 6-well plates and cultured for 2
days. Then trans-well inserts containing differentiated C2C12
myotubes were transferred to adipocyte plates. For the control
group, the inserted trans-well did not contain any cells. After cell
grew to confluence, 3T3-L1 preadipocytes in control and co-culture
groups were induced to differentiate with DMEM medium sup-
plemented with 1 mM dexamethasone, 10 mg/ml insulin, and
500 nM 3-isobutyl-1-methylxanthine. After induced for 3 days, the
differentiation medium was replaced to growth medium for
another 12 days and freshened every 2 days.
2.2. Cell proliferation and viability assay
EdU (5-Ethynyl-2'-deoxyuridine) assay and cell counting
method were carried out to determine the effect of myotubes on
the 3T3-L1 preadipocytes proliferation. The 3T3-L1 preadipocytes
were plated at a density of 1  105 cells per well in 6-well culture
plates, and grew to 50% confluence, then added 0.2‰ EdU in the
medium cultured for 12 h in both group. EdU staining was con-
ducted using Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit
(RiboBio, Guangzhou, China) according to the manufacturer's pro-
tocol. Then the EdU-labeled cells were imaged with confocal mi-
croscope (Zeiss 710, Germany). After co-cultured with myotubes or
nothing for 3 days, 3T3-L1 preadipocytes were trypsinized and
stained by 2% trypan blue, then the cells were imaged and counted
by the automated cell counter (Invitrogen, USA).
The cell viability was determined using the Cell Counting Kit-8
(CCK-8) (Vazyme, Jiangsu, China) assay. The reagent 2-(2-
Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)
-2H-tetrazolium Sodium Salt (WST-8) in this kit can be deoxidized
to the hydrosoluble formazan dye by mitochondrial dehydrogenase
in living cells, therefore indicates the relative cell numbers. The
cells in control group and co-culture group were both treated with
different concentrations of dexamethasone (0, 10, 100, 1000 nM) for
3 days, and then were treated with 10 ml CCK-8 solutions for 4 h at
37
C. The absorbance was measured using an automated
69
microplate reader (Bio-Rad, Japan) at 450 nm. Results were
expressed as percentages of the controls, which were arbitrarily
assigned with 100% viability.
2.3. Apoptosis and cell cycles assay
After co-cultured for 72 h, 3T3-L1 preadipocytes were harvested
for the following assays. Annexin V-FITC/PI Apoptosis Detection Kit
(Vazyme, Jiangsu, China) was used for the apoptosis assay in our
study. The cells were stained with 5 ml of Annexin V and 5 ml of
propidium iodide (PI) in 100 ml loading binding buffer for 15 min at
room temperature in the dark. Cell Cycle Assay Kit (Vazyme,
Jiangsu, China) was used for cell cycle assay, and the cells were
stained with 5 ml of PI. The cells fluorescent signal was measured by
flow cytometer (FACS Calibur; BectoneDickinson, Franklin Lakes,
NJ, USA). Total of 20 000 cells were examined in each data.
2.4. Oil red O staining
Differentiated adipocytes were washed 3 times with PBS, and
then fixed with 10% paraformaldehyde for 15 min. After fixation,
the cells were washed with PBS for 3 times and stained with oil red
O for 20 min. Subsequently, the cells were washed with 60% iso-
propanol for 20 s, and then imaged with inverted microscope.
2.5. RNA extraction, cDNA synthesis and RT-qPCR
Total RNA isolation was performed using TRI reagent (Invi-
trogen, USA) according to the manufacturer's protocol. RNA con-
centration was measured with NanoDrop1000 Spectrophotometer
(Thermo Scientific, USA), and integrity was checked on 1% dena-
turing agarose gels. First-strand cDNA was synthesized using Pro-
toScript M-MuLV (NEB, USA) in a reaction containing 2 mg RNA and
2 mL gene special primer. Real-time PCR was performed using
TransStart Green qPCR SuperMix (Transgen Biotech, Beijing, China)
on ABI StepOne Plus™ Real-Time PCR System (Applied Biosystems,
USA). The sequence of PCR primers were obtained from PrimerBank
[12] and previous study [13]. Data were analyzed by using 2DDCt
method and are referred to the control treatment using GAPDH as a
reference gene. Sequences of the primers used are as follows: Cyclin
D forward, 50 -GCGTACCCTGACACCAATCTC-30 ; reverse, 50 -
CTCCTCTTCGCACTTCTGCTC-30 . Cyclin E forward, 50 -GTGGCTCC
GACCTTTCAGTC-30 ; reverse, 50 -CACAGTCTTGTCAATCTTGGCA-30 .
PPARg forward, 50 -GGAAGACCACTCGCATTCCTT-30 ; reverse, 50 -
GTAATCAGCAACCATTGGGTCA-30 . FASN forward, 50 - GGAGGTGGT-
GATAGCCGGTAT-30 ; reverse, 50 -TGGGTAATCCATAGAGCCCAG-30 .
FAS forward, 50 -GAACACTGTGACCCTTGCACCAAA-30 ; reverse, 50 -
CTCTTTGCACTTGGTGTTGCTGGT-30 . Common GR forward, 50 -AAA-
GAGCTAGGAAAAGCCATTGTC-30 ; GRa reverse, 50 -TCAGCTAA-
CATCTCTGGGAATTCA-30 . GRb reverse, 50 -
CTGTCTTTGGGCTTTTGAGATAGG-30 .
2.6. Caspase 3 activity assay
Caspase 3 activity was measured with Caspase 3 Activity Assay
Kit (Beyotime, Jiangsu, China), determined by a colorimetric assay,
based on the ability of Caspase-3 to change acetylAsp-Glu-Val-Asp
p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product p-
nitroaniline (pNA). An increase in absorbance at 405 nm was used
to quantify the activation of caspases activities and expressed by
pNA concentrations.
2.7. Western blot
Total protein extracts from fat tissues and cells were prepared
70 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74

with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China), and
protein quantities were measured by the BCA Protein Assay kit
(Beyotime Biotechnology, Jiangsu, China). Antibodies against GR
(sc-393232, Santa Cruz, USA) and tubulin (sc-5274, Santa Cruz,
USA) were used in western blot analysis. Images were captured
with VersaDoc 4000 MP system (Bio-Rad). Tubulin was used as
loading control in western blotting analysis.
2.8. Statistical analysis
All the results are presented as mean ± SEM. Statistically sig-
nificant differences were determined using unpaired t-test or one-
way ANOVA of variance. P-value less than 0.05 was considered
significant.
3. Results
3.1. C2C12 myotubes inhibited proliferation and differentiation of
3T3-L1 preadipocytes
Co-culturing with differentiated C2C12 myotubes significantly
reduced the confluent of 3T3-L1 preadipocytes, and more cell
debris were observed in the co-culture group (Fig. 1A left). Cell
counting result revealed that the numbers of total, live and dead
cells were significantly different (P < 0.01) between the control and
co-culture group. The total cells number of 3T3-L1 preadipocytes in
co-culture group was 33% less than that of the control group, and
the number of live cells decreased by 47%, whereas the number of
dead cells increased approximately 100% (Fig. 1A right). The pro-
portion of EdU negative cells increased to 8.8% in co-culture group
compare to 4.4% in control group (P < 0.01), which confirmed the
negative effect of C2C12 myotubes on the 3T3-L1 preadipocytes
proliferation (Fig. 1B).
Next the influence of co-culture on the differentiation of 3T3-L1
preadipocytes was measured and the results were shown in Fig. 1C.
After co-cultured with C2C12 myotubes for 3 days, 3T3-L1 pre-
adipocytes were long spindle shape, while in control groups 3T3-L1
preadipocytes were in round shape. On the day 4 of differentiation,
some lipid droplets began to gather in the control group, but few
lipid drops could be observed in the co-culture group. The oil red
staining and triglyceride accumulation results revealed that co-
culture treatment decreased the accumulation of lipid droplets in
3T3-L1 adipocytes on the day 15.
3.2. C2C12 myotubes affected the cell cycle of 3T3-L1 preadipocytes
After co-cultured with C2C12 for 3 days, more 3T3-L1 cells were
arrested in G0/G1 phase compared with the control group. The
percentage of cells in G0/G1 stage was increased from 62% to 67%,
whereas the cell population at the S phase decreased from 15% to

Fig. 1. C2C12 myotubes inhibited proliferation and differentiation of 3T3-L1 preadipocytes. 3T3-L1 cells were co-cultured with differentiated C2C12 for 3 days. (A) The 3T3-L1 cells
were stained by trypan blue, imaged and counted by automated cell counter. Bright cycles were live cells, and black were cell debris. (B) 3T3-L1 cells were stained with EdU, and the
EdU-negative cells were indicated by white arrows. (C) After co-cultured, 3T3-L1 preadipocytes were induced to differentiation, and the inverted microscope images of day 2, day
4, and day15 (oil red O-stained) were shown, and the cell triglyceride accumulation was detected. Magnification, 400  . Data are shown as the mean ± SEM, n ¼ 3, **P < 0.01,
***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74
12%, there was no significant difference between groups for cell
population at G2/M stage (Fig. 2A,B). Next we examined the alter-
ation of mRNA levels of Cyclin D and Cyclin E (responsible for cell
cycle G1/S transition). The RT-qPCR results showed that the mRNA
levels of Cyclin D and Cyclin E were significantly reduced in co-
culture group compared with control group (Fig. 2C). Therefore,
C2C12 myotubes prevented 3T3-L1 preadipocytes transiting to S
phase from G0/G1 phase.
3.3. C2C12 myotubes induced apoptosis of 3T3-L1 preadipocytes
According to our results, co-culturing with differentiated C2C12
myoblasts induced obvious increasing apoptosis of 3T3-L1 pre-
adipocytes. Compared to the control group, the early and late
apoptosis percentages raised from 3.9% to 14.2% (P < 0.01), and
5.8%e8.3% (P < 0.01), respectively (Fig. 3A, B). Correspondingly, the
activity of caspase 3 increased (P < 0.01) in 3T3-L1 cells when co-
cultured with differentiated C2C12 myotubes for 3 days (Fig. 3C).
3.4. C2C12 myotubes decreased glucocorticoids sensitivity of 3T3-
L1 preadipocytes
After confirming the effects of C2C12 myotubes on pre-
adipocytes proliferation, differentiation and apoptosis, we designed
experiments to discover the underlying mechanism. Firstly, the
sensitivity of 3T3-L1 preadipocytes to glucocorticoids, the key
regulator for cell proliferation, differentiation and apoptosis, was
detected to confirm whether C2C12 myotubes influence pre-
adipocytes activity through altering GCs signal. The 3T3-L1 pre-
adipocytes in both control group and co-culture group were treated
with different concentrations of dexamethasone (Dex, the mimics
of glucocorticoids) for 3 days, then we found that Dex (0, 10, 100,
1000 nM) increased cell viability of 3T3-L1 preadipocytes; more-
over, the cell viabilities were significant higher in control group
than co-culture group when cells were treated with 100 nM Dex
Fig. 2. C2C12 myotubes altered cell cycle of 3T3-L1 preadipocytes. (A) 3T3-L1 cells were co-cultured with differentiated C2C12 for 3 days, stained with propidium iodine, then cell
phases was examined by flow cytometry. (B) Statistics of cell proportions in the different phases of control and co-culture group. (C) mRNA expression of cyclins in control and co-
culture group. Data are shown as the mean ± SEM, n ¼ 3, **P < 0.01, ***P < 0.001.
71
(P < 0.05) (Fig. 4A).
Treating with 100 nM Dex for 3 days elevated the mRNA levels of
cyclin D compared with untreated counterpart, but the Dex treat-
ment couldn0 t completely complement the cyclin D expression
reduction in the co-culture system compared with the corre-
sponding Dex treated control group (Fig. 4B). The expression of
preadipocytes differentiation associated genes, peroxisome
proliferator-activated receptor-g (PPARg) and fatty acid synthase
(FASN), followed the same pattern as cyclin D. Factor associated
suicide (FAS) is a transmembrane molecule that induces apoptosis
of many cell types [14]. The mRNA levels of FAS in preadipocytes
were increased after co-cultured with myotubes, and were then
reduced after treated with 100 nM Dex. However, the FAS mRNA
levels in co-culture group was significantly higher than control
group (P < 0.05) even if both treated with Dex (Fig. 4B).
These results revealed that C2C12 myotubes decreased gluco-
corticoids sensitivity of 3T3-L1 preadipocytes. Then we detected
the expression of glucocorticoid receptor (GR), and the results
showed that the mRNA levels of GRa and GRb were lower in the co-
culture group (Fig. 4C). Western blot results also demonstrated that
C2C12 myotubes reduced total GR protein in preadipocytes
(Fig. 4D).
4. Discussion
Obesity is a global health disorder and attracts the growing
concern of world. Because of the same derive and the close location,
the studies about the influence of myocytes on adipocytes could
help to explore the function and mechanism of cytokines that
regulate fat deposition. Several studies have showed that myocytes
suppressed differentiation [15], but the others indicated that
myocytes elevated adipogenic marker gene expression of mature
adipocytes [7]. Jun Yan et al. mixed porcine preadipocytes and
muscle satellite cells in a ratio of 1:10, and found that the number of
total cells in the mixture co-cultured system was increased
72 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74

Fig. 3. C2C12 myotubes induced apoptosis of 3T3-L1 preadipocytes. (A) 3T3-L1 cells were co-cultured with differentiated C2C12 for 3 days, and stained with propidium iodine and
Annexin V, then the cell apoptosis was analyzed by flow cytometry. (B) Statistics of apoptosis in control and co-culture group. (C) Activity of caspase 3 was indicated by the pNA
concentrations. **P < 0.01, ***P < 0.001.

Fig. 4. GCs sensitivity of 3T3-L1 preadipocytes in control and co-culture group. (A) After co-cultured, 3T3-L1 preadipocytes in control and co-culture groups were treated with
100 nM dex for another 3 days, then mRNA expression of Cyclin D, PPARg，FASN and FAS was determined by RT-qPCR. (B) 3T3-L1 cells were treated with different concentrations of
dexamethasone (0, 10, 100, 1000 nM) for 3 days, cell viability were determined by CCK-8. (C) GRa、GRb mRNA expression in control and co-culture groups. (D) Western blot result of
total GR (left) and its quantification (right) in control and co-culture group. *P < 0.05, **P < 0.01, ***P < 0.001.

compared with mono-preadipocytes, meanwhile, the differentia- preadipocytes, at the same time, the proliferation of 3T3-L1 pre-
tion of preadipocytes was inhibited [2]. Our present study revealed adipocytes was also suppressed. Imaging with inverted microscope,
that C2C12 myotubes inhibited differentiation of 3T3-L1 cell counting and EdU staining methods were used to confirm this
 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74
results. In contrast to the previous mixture co-culture system [2],
only 3T3-L1 preadipocytes were included in our proliferation assay,
which was more reliable for data analysis.
Besides, other studies showed that the muscle environment also
affected the cellular morphology of 3T3-L1 preadipocytes. The
adipogenic cells showed an elongated spindle shape when
distributed in the narrow space between regenerating myofibres,
whereas most of them showed an enlarged rounded morphology
when distributed in the extended interstitial space between
degenerating myofibres [16]. The similar results obtained in our
studies suggested that mono-cultured 3T3-L1 preadipocytes were
round, while C2C12 myotubes co-cultured preadipocytes were
fibrotic, long spindle appearance, supporting the previous studies.
Next, the experiments were performed to uncover the molec-
ular basis of these effects on 3T3-L1 preadipocytes. The cell cycle is
mainly driven by cyclin-dependant kinases (CDKs), which are
activated by forming specific complexes with cyclins in different
cell cycle phases [17]. Cyclin D usually functions as a regulatory
subunit of CDK4 or CDK6, whose activity is required for cell cycle
G1/S transition [18]. In this study, we found that 3T3-L1 cell cycle
were arrested at G0/G1 phase by C2C12 myotubes, at the same time
the mRNA levels of Cyclin D and Cyclin E were significantly reduced,
leading to the inhibition of cell proliferation. FAS is the cell death
receptor [14], and caspase 3 has been identified as a key protease in
the execution of apoptosis [19]. Our results indicated that C2C12
myotubes increased the mRNA expression of FAS and the activity of
caspase 3, therefore induced apoptosis.
Moreover, the potential signaling pathway was detected to
study the underlying mechanism. GCs are important regulators
involved in cell proliferation, differentiation and apoptosis, so the
GCs sensitivity of 3T3-L1 preadipocytes in control and co-culture
groups were analyzed. The cell viability, the mRNA expression
pattern of proliferation marker Cyclin D, adipose differentiation
marker PPARg [20] and FASN [21], and apoptosis marker FAS, indi-
cated that the proliferation, differentiation and apoptosis were
regulated by GCs mimics Dex. But these effects were inhibited
when co-cultured with C2C12 myotubes, indicating the GCs
sensitivity was decreased in 3T3-L1 preadipocytes. As we knew that
physiological effects of circulating glucocorticoids are mainly
mediated by the GR. GRa is a hormone-activated transcription
factor, while GRb is transcriptionally inactive and usually acts as a
potential inhibitor of activated GRa [22]. The amount of GR usually
determines GCs sensitivity, so the reduction of GR accompanied
GCs resistance [23], while the increase of GR increased GCs sensi-
tivity [24]. Hereby we hypothesized that myocytes might influence
GCs activity by altering adipocytes GR expression levels. As ex-
pected, co-culture system reduced GR mRNA and protein, sup-
porting our hypothesis.
Muscles are large endocrine organs, producing many cytokines.
A number of studies found that many pro-inflammatory cytokines
including interleukin-1a, interleukin-2, interleukin-4, tumor ne-
crosis factor-alpha, interferon-alpha and nuclear factor kappa-B
decreased the expression of cytosolic GR protein in many cell
types [25]. Interleukin-1a suppresses GR translocation and function
[26]. Tumor necrosis factor-alpha was reported to decrease the GR
concentration in a human monocytic cell line. Nuclear factor
kappa-B, a transcription regulator, was found to directly interact
with GR in the nucleus through physical association, causing
mutual repression of GR [27]. Besides, some myokines which
inhibit adipogenesis may have the ability to regulate expression of
GR. For instance, interleukin-6 is the first identified myokine, many
studies showed that interleukin-6 induced lipolysis and fat oxida-
tion [28]. Interleukin-15 is an anabolic factor in muscle growth, also
presents a negative effect to the fat mass [29]. Myostatin is a muscle
secreted growth differentiation factor, it regulated DNA
73
methylation levels in adipose-derived stem cells, and inhibits the
proliferation and differentiation of adipocytes [30,31]. All these
cytokines can be produced by muscles, may have the potential
ability to regulate the GR expression in the adipose close-by.
In summary, we found that co-culture with differentiated C2C12
myotubes reduced the GR expression and GCs sensitivity of 3T3-L1
preadipocytes, therefore arrested the proliferation, differentiation
and induced apoptosis of 3T3-L1 preadipocytes. Our study provides
myokines which could be used as the effective anti-obesity medi-
cine by suppressing GR expression.
Conflict of interest
The authors have declared that no conflict of interest exists.
Acknowledgments
This study was supported by National Natural Science Founda-
tion of China (No. 31272423), and National Key Technology Support
Program (No. 2015BAD03B01).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2016.02.063
References
[1] M. Alvehus, J. Buren, M. Sjostrom, J. Goedecke, T. Olsson, The human visceral
fat depot has a unique inflammatory profile, Obesity 18 (2010) 879e883,
http://dx.doi.org/10.1038/oby.2010.22.
[2] J. Yan, L. Gan, H. Yang, C. Sun, The proliferation and differentiation charac-
teristics of co-cultured porcine preadipocytes and muscle satellite cells
in vitro, . Biol. Rep. 40 (2013) 3197e3202, http://dx.doi.org/10.1007/s11033-
012-2395-0.
[3] B.K. Pedersen, M.A. Febbraio, Muscles, exercise and obesity: skeletal muscle as
a secretory organ, Nat. Rev. Endocrinol. 8 (2012) 457e465, http://dx.doi.org/
10.1038/nrendo.2012.49.
[4] P. Trayhurn, C.A. Drevon, J. Eckel, Secreted proteins from adipose tissue and
skeletal muscle e adipokines, myokines and adipose/muscle cross-talk, Arch.
Physiol. Biochem. 117 (2011) 47e56, http://dx.doi.org/10.3109/
13813455.2010.535835.
[5] M. Pandurangan, I. Hwang, Application of cell co-culture system to study fat
and muscle cells, Appl. Microbiol. Biotechnol. 98 (2014) 7359e7364, http://
dx.doi.org/10.1007/s00253-014-5935-9.
[6] B. Havekes, H.P. Sauerwein, Adipocyte-myocyte crosstalk in skeletal muscle
insulin resistance; is there a role for thyroid hormone? Curr. Opin. Clin. Nutr.
Metab. Care 13 (2010) 641e646, http://dx.doi.org/10.1097/
MCO.0b013e32833e341d.
[7] P. Muthuraman, Effect of coculturing on the myogenic and adipogenic marker
gene expression, Appl. Biochem. Biotechnol. 173 (2014) 571e578, http://
dx.doi.org/10.1007/s12010-014-0866-6.
[8] S.H. Choi, K.Y. Chung, B.J. Johnson, G.W. Go, K.H. Kim, C.W. Choi, S.B. Smith, Co-
culture of bovine muscle satellite cells with preadipocytes increases PPAR-
gamma and C/EBPbeta gene expression in differentiated myoblasts and in-
creases GPR43 gene expression in adipocytes, J. Nutr. Biochem. 24 (2013)
539e543, http://dx.doi.org/10.1016/j.jnutbio.2012.01.015.
[9] X. Sun, K.L. Morris, M.B. Zemel, Role of calcitriol and cortisol on human
adipocyte proliferation and oxidative and inflammatory stress: a microarray
study, J. Nutrigenetics Nutrigenomics 1 (2008) 30e48.
[10] M.-J. Lee, P. Pramyothin, K. Karastergiou, S.K. Fried, Deconstructing the roles of
glucocorticoids in adipose tissue biology and the development of central
obesity, Biochim. Biophys. Acta (BBA) Mol. Basis Dis. 1842 (2014) 473e481.
[11] I. Herr, N. Gassler, H. Friess, M.W. Büchler, Regulation of differential pro-and
anti-apoptotic signaling by glucocorticoids, Apoptosis 12 (2007) 271e291.
[12] X. Wang, B. Seed, A PCR primer bank for quantitative gene expression anal-
ysis, Nucl. Acids Res. 31 (2003) e154.
[13] C. Zou, J. Chen, K. Chen, S. Wang, Y. Cao, J. Zhang, Y. Sheng, A. Huang, H. Tang,
Functional analysis of miR-181a and Fas involved in hepatitis B virus-related
hepatocellular carcinoma pathogenesis, Exp. Cell Res. 331 (2015) 352e361,
http://dx.doi.org/10.1016/j.yexcr.2014.11.007.
[14] U. Dianzani, M. Bragardo, D. DiFranco, C. Alliaudi, P. Scagni, D. Buonfiglio,
V. Redoglia, S. Bonissoni, A. Correra, I. Dianzani, U. Ramenghi, Deficiency of the
Fas apoptosis pathway without Fas gene mutations in pediatric patients with
autoimmunity/lymphoproliferation, Blood 89 (1997) 2871e2879.
[15] D. Dietze, M. Koenen, K. Rohrig, H. Horikoshi, H. Hauner, J. Eckel, Impairment
74 W. Chu et al. / Biochemical and Biophysical Research Communications 472 (2016) 68e74
of insulin signaling in human skeletal muscle cells by co-culture with human
adipocytes, Diabetes 51 (2002) 2369e2376, http://dx.doi.org/10.2337/
diabetes.51.8.2369.
[16] A. Uezumi, S. Fukada, N. Yamamoto, S. Takeda, K. Tsuchida, Mesenchymal
progenitors distinct from satellite cells contribute to ectopic fat cell formation
in skeletal muscle, Nat. Cell Biol. 12 (2010) 143e152, http://dx.doi.org/
10.1038/ncb2014.
[17] M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: a changing paradigm,
Nat. Rev. Cancer 9 (2009) 153e166, http://dx.doi.org/10.1038/nrc2602.
[18] X. Li, X. Gong, J. Chen, J. Zhang, J. Sun, M. Guo, miR-340 inhibits glioblastoma
cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2, Biochem.
Biophys. Res. Commun. 460 (2015) 670e677, http://dx.doi.org/10.1016/
j.bbrc.2015.03.088.
[19] K. Blomgren, C. Zhu, X. Wang, J.O. Karlsson, A.L. Leverin, B.A. Bahr, C. Mallard,
H. Hagberg, Synergistic activation of caspase-3 by m-calpain after neonatal
hypoxia-ischemia: a mechanism of "pathological apoptosis, J. Biol. Chem. 276
(2001) 10191e10198, http://dx.doi.org/10.1074/jbc.M007807200.
[20] E.D. Rosen, P. Sarraf, A.E. Troy, G. Bradwin, K. Moore, D.S. Milstone,
B.M. Spiegelman, R.M. Mortensen, PPAR gamma is required for the differen-
tiation of adipose tissue in vivo and in vitro, Mol. Cell 4 (1999) 611e617,
http://dx.doi.org/10.1016/S1097-2765(00)80211-7.
[21] B. Schmid, J.F. Rippmann, M. Tadayyon, B.S. Hamilton, Inhibition of fatty acid
synthase prevents preadipocyte differentiation, Biochem. Biophys. Res.
Commun. 328 (2005) 1073e1082.
[22] H. Shahidi, A. Vottero, C.A. Stratakis, S.E. Taymans, M. Karl, C.A. Longui,
G.P. Chrousos, W.H. Daughaday, S.A. Gregory, J.M. Plate, Imbalanced expres-
sion of the glucocorticoid receptor isoforms in cultured lymphocytes from a
patient with systemic glucocorticoid resistance and chronic lymphocytic
leukemia, Biochem. Biophys. Res. Commun. 254 (1999) 559e565.
[23] N.N. Ansari, E. Spilioti, V. Kalotychou, G. Dalagiorgou, P. Moutsatsou,
A. Papavassiliou, E. Kassi, Vitamin D Decreases in Vitro Glucocorticoid Sensi-
tivity via Down Regulation of Glucocorticoid Receptor Expression, 2015.
[24] X.-J. Sun, Z.-H. Li, Y. Zhang, G. Zhou, J.-Q. Zhang, J.-M. Deng, J. Bai, G.-N. Liu, M.-
H. Li, W. MacNee, Combination of erythromycin and dexamethasone im-
proves corticosteroid sensitivity induced by CSE through inhibiting PI3K-d/Akt
pathway and increasing GR protein, Am. J. Physiology-Lung Cell. Mol. Physiol.
309 (2) (2015) L139eL146.
[25] T.W.W. Pace, H. Fang, A.H. Miller, Cytokine-effects on glucocorticoid receptor
function: Relevance to glucocorticoid resistance and the pathophysiology and
treatment of major depression, Brain Behav. Immun. 21 (2007) 9e19.
[26] C.M. Pariante, B.D. Pearce, T.L. Pisell, C.I. Sanchez, C. Po, C. Su, A.H. Miller, The
Proinflammatory cytokine, interleukin-1a, reduces glucocorticoid receptor
translocation and function 1, Endocrinology 140 (1999) 4359e4366.
[27] L.I. Mckay, Molecular control of immune/inflammatory responses: In-
teractions between nuclear factor- B and steroid receptor-signaling pathways,
Endocr. Rev. 20 (1999) 435e459.
[28] B.K. Pedersen, M.A. Febbraio, Muscle as an endocrine organ: focus on muscle-
derived interleukin-6, Physiol. Rev. 88 (2008) 1379e1406.
[29] L.S. Quinn, B.G. Anderson, L. Strait-Bodey, A.M. Stroud, J.M. Argiles, Over-
secretion of interleukin-15 from skeletal muscle reduces adiposity, Am. J.
Physiol. Endocrinol. Metab. 296 (2009) E191eE202, http://dx.doi.org/10.1152/
ajpendo.90506.2008.
[30] W. Sun, M. Dodson, Z. Jiang, S. Yu, W. Chu, J. Chen, Myostatin inhibits porcine
intramuscular preadipocyte differentiation in vitro, Domest. Anim. Endo-
crinol. 55 (2016) 25e31.
[31] F. Zhang, B. Deng, J. Wen, K. Chen, W. Liu, S. Ye, H. Huang, S. Jiang, Y. Xiong,
PPARg and MyoD are differentially regulated by myostatin in adipose-derived
stem cells and muscle satellite cells, Biochem. Biophys. Res. Commun. 458
(2015) 375e380.