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SHOR T REPOR T Open Access
Targeting AMP-activated protein kinase in
adipocytes to modulate obesity-related adipokine
production associated with insulin resistance and
breast cancer cell proliferation
Jean Grisouard
1*
, Kaethi Dembinski
1
, Doris Mayer
2
, Ulrich Keller
1,3
, Beat Müller
4
and Mirjam Christ-Crain
1,3
Abstract
Background: Adipokines, e.g. TNFa, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia
influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from
adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another
adipokine is protective against breast cancer cell proliferation and insulin resistance.
AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-
resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance.
Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an
alternative way to treat both, insulin resistance and breast cancer.
Methods: Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into
adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h,
1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-
adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation.
Results: Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes,
increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01),
while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1
receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA
expression (p < 0.05).
Conclusions: Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin
improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to
improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast
cancer development and progression.
Introduction
Breast cancer is the most frequent cancer type among
women worldwide, and 21% of all breast cancer deaths
worldwide are estimated to be attributable to obesity
and physical inactivity [1,2]. The incidence of type 2 dia-
betes mellitus (T2DM) is presumed to be a direct result
of the obesity epidemic [3]. The breast cancer risk is
increased in diabetic women independently from obesity
[4,5]. Thus, both, obesity and diabetes are risk factors
for breast cancer and the prevalence of each of these
diseases will continue to rise worldwide [6].
Adipokines are polypeptides produced and secreted by
the adipose tissue and dysregulation of their production
and secretion in the obese state leads to obesity-related
complications [7,8]. Invasive breast tumours break
through the basement membrane and infiltrate fibrous
* Correspondence: jean.grisouard@unibas.ch
1
Department of Biomedicine, University Hospital Basel, Basel, CH-4031,
Switzerland
Full list of author information is available at the end of the article
Grisouard et al.Diabetology & Metabolic Syndrome 2011, 3:16
http://www.dmsjournal.com/content/3/1/16 METABOLIC SYNDROME
DIABETOLOGY &
© 2011 Grisouard et al; licensee BioMed Cen tral Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attri bution License (http://creativecommons.org /licenses/by/2.0), which permits unrestricted use, distribution, and
reproductio n in any medium, provided the original work is properly cited.
tissue barriers, resulting in an immediate juxtaposition
of adipocytes and breast cancer cells, thus allowing para-
crine interactions between the two cell types [9]. Insulin
resistance and consequent hyperinsulinaemia may be a
common factor linking T2DM and cancer [10]. Beside
its stimulating effects on breast cancer cell proliferation
[11], insulin may influence adipokine production from
adipose tissue and adipocyte stromal cells and may
indirectly reinforce cancer cell growth. Similarly, insulin
resistance-induced hyperglycaemia and hyperlipidaemia
may alter adipokine production in adipocytes and
enhance breast cancer development [12].
Chronic low grade systemic inflammation is associated
with obesity and insulin resistance. Lipopolysaccharide
(LPS) from the gut microbiota is a triggering factor link-
ing inflammation to high fat diet-induced metabolic syn-
drome [13,14]. Metformin is the first line oral
antidiabetic drug for patients with T2DM [15]. AMP-
activated protein kinase (AMPK) targets cytokine secre-
tion from the adipose tissue and the activation of this
kinase by metformin could explain the beneficial effects
of this drug on inflammation [16].
In view of these relationships, we aimed to assess the
effect of low grade inflammation induced by LPS and of
metformin on adipokine production in human adipo-
cytes and to observe the stimulatory effect of adipocyte-
secreted factors on breast cancer cell proliferation.
Materials and methods
The study was approved by the local Ethics Committee
and informed consent was obtained from patients. Sub-
cutaneous fat tissue samples were obtained from obese
donors (BMI > 30 kg/m
2
, males and females, mean age
47 years) during elective abdominal surgery performed
for various non-malignant conditions.
Preadipocytes were isolated, expanded in vitro until
confluence and subjected to adipogenic differentiation
medium for 14 days as previously described [17,18].
After differentiation, adipocytes were washed twice with
warm phosphate buffered saline (PBS) and were kept for
48 h in low glucose (5 mM) medium. Then, adipocytes
were pre-incubated with 1 mM metformin (Sigma-
Aldrich, Buchs, Switzerland) for 1 h followed by treat-
ment with 100 ng/ml LPS (Sigma-Aldrich). AMPKa1
silencing was performed as previously described [18].
Low glucose DMEM medium with 2% dextran-coated
charcoal-treated heat-inactivated FCS (DCC-FCS, Lubio
Science, Luzern, Switzerland) was added to confluent
preadipocytes or preadipocyte-derived adipocytes. After
24 h in culture, the supernatant or conditioned medium
was collected, filtered with a 0.2 μm syringe filter and
stored at -20°C.
MCF-7 breast cancer cells were maintained in DMEM
(25 mM glucose) containing 10% FCS (Lubio Science).
Then, cells were grown in medium supplemented with
10% DCC-FCS for 72 h. 1 × 10
4
cancer cells/well were
plated in a 96-well plate in medium containing 2%
DCC-FCS for 24 h and stimulated every 24 h for a total
of 72 h with low glucose DMEM + 2% DCC-FCS or
conditioned medium. At the end of incubation time,
cells were washed twice with PBS, fixed for 5 min with
100 μl of 3% paraformaldehyde and stained for 10 min
with 100 μl of 1% crystal violet dye dissolved in 10%
ethanol. Plates were extensively washed with water to
remove traces of unbound crystal violet dye. After air
drying, the bound dye was dissolved in 100 μl of 10%
acetic acid. Optical density was read at 595 nm using a
plate reader (Bucher biotec, Basel, CH) [11].
For quantitative analysis of adipokine expression, RNA
was isolated and 1 μg total RNA was subjected to
reverse transcription-PCR. cDNA was subjected to
quantitative real-time PCR analysis using the power
Sybr
®
-Green PCR master mix (Applied Biosystems) and
the ABI 7500 Sequence detection system [19]. Primers
were designed as follows: IL-6 sense primer, 5’-
TCTTCAGAACGAATTGACAAACAAA-3’, IL-6 anti-
sense primer, 5’-GCTGCTTTCACACATGTTACTC
TTG-3’, IL-8 sense primer, 5’-GCCATAAAGT-
CAAATTTAGCTGGAA-3’, IL-8 antisense primer, 5’-
GTGCTTCCACATGTCCTCACA-3’; interleukin 1
receptor antagonist (IL-1RA) sense primer, 5’-TGC
CTGTCCTGTGTCAAGTC-3’and IL-1RA antisense
primer, 5’-TCTCGCTCAGGTCAGTGATG-3’.Hypox-
anthine-guanine phosphoribosyltransferase (HPRT) pri-
mers were used as loading control (HPRT sense primer,
5’-TCAGGCAGTATAATCCAAAGATGGT-3’and
HPRT antisense primer, 5’-AGTCTGGCTTATATC-
CAACACTTC-3’.
Data are presented as mean ± standard deviation (SD)
from a minimum of three independent experiments.
One-way analysis of variance was performed and the
Tukey’s posthoc multiple comparison test was applied.
Overall, a Pvalue < 0.05 was considered significant.
Results
Conditioned medium (CM) from preadipocytes and pre-
adipocyte-derived adipocytes (14 days after differentia-
tion) was collected and breast cancer MCF-7 cell
proliferation was evaluated after 72 h in these media (5
mM glucose) (Figure 1). DMEM medium (5 mM glu-
cose) was used as basal. CM from preadipocytes did not
increase MCF-7 cell proliferation (p = n.s.). However,
CM from preadipocyte-derived adipocytes increased
MCF-7 cell proliferation to 1.28 ± 0.02 fold (p < 0.001
vs. basal and p < 0.01 vs. CM preadipocytes).
Incubation of preadipocyte-derived adipocytes with
100 ng/ml LPS increased IL-6 mRNA expression to 10.5
± 1.9 fold (p < 0.001 vs. basal)(Figure 2A) and induction
Grisouard et al.Diabetology & Metabolic Syndrome 2011, 3:16
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Page 2 of 7
of IL-6 mRNA by LPS was significantly reduced by 1
mM metformin to 5.3 ± 0.8 fold (p < 0.01 vs. LPS).
Additionally, 1 mM metformin enhanced LPS-induced
mRNA expression of IL-1RA, a naturally occurring anti-
inflammatory cytokine (p < 0.01 vs. LPS) (Figure 2B).
We silenced AMPKa1 subunit in preadipocyte-derived
adipocytes to check its involvement in the effects of LPS
on adipocytokine mRNA expression. Using siRNA pool
specifically targeting AMPKa1, AMPKa1 mRNA expres-
sion and protein content were decreased to 0.28 ± 0.03
and 0.50 ± 0.08 fold, respectively (p < 0.001 vs. control
siRNA)(data not shown). AMPKa1 silencing enhanced
LPS-induced IL-6 and IL-8 mRNA expression (Figure 3,
p < 0.05 vs. LPS in control siRNA cells).
Discussion
Decreased AMPK activity has been found in visceral
adipose tissue of patients with central obesity due to
Cushing’s syndrome [20] and of obese insulin-resistant
individuals [21]. This suggests a central role for this
enzyme in obesity and related insulin resistance. We
therefore aimed to investigate how AMPK modulates
adipokine production triggered by obesity- and type 2
diabetes mellitus (T2DM)-related factors and how such
a modulation may prevent insulin resistance and breast
tumour cell proliferation. In turn, adipokines alter
AMPK activity and might play a crucial role in adipo-
kine-altered insulin sensitivity and breast tumour cell
growth (Figure 4). Our findings suggest that conditioned
medium from human adipocytes increase breast cancer
cell proliferation. Our data also show that metformin
and AMPK alter LPS-induced adipokine expression,
favoring the anti-inflammatory adipokine expression and
decreasing pro-inflammatory adipokine expression in
human adipocytes.
Interestingly, CM obtained from murine 3T3-L1 fully
differentiated adipocytes induced growth of MCF-7 cells
Figure 1 Effect of adipocyte conditioned medium (CM) on
breast cancer cell proliferation. MCF-7 breast cancer cells were
incubated for 72 h with low glucose DMEM medium (basal), low
glucose DMEM conditioned medium from preadipocytes (CM
Preadipocytes) or low glucose DMEM conditioned medium from
preadipocyte-derived adipoytes (CM Adipocytes). Then, proliferation
of MCF-7 cells was measured by colorimetric method using crystal
violet dye. Results are expressed as fold of basal (**, p < 0.01 vs. CM
Preadipocytes; ***, p < 0.001 vs. basal; n.s., not significant).
Figure 2 Metformin modulates LPS-induced adipocytokine expression. Human subcutaneous preadipocyte-derived adipocytes were pre-
incubated for 1 h with 1 mM metformin followed by stimulation with 100 ng/ml LPS for 24 h. mRNA expression of IL-6 (A) and interleukin-1
receptor antagonist (IL1RA) (B) were analysed by quantitative real-time PCR. (*, p < 0.05 vs. basal; **, p < 0.01 vs. LPS; ***, p < 0.001 vs. basal; n.s.,
not significant).
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Figure 3 AMPKalpha1 prevents the LPS-induced IL-6 and IL-8 adipocytokine expression. Human subcutaneous preadipocyte-derived
adipocytes were transfected for 72 h with control non-targeting siRNA pool (Control siRNA) or siRNA pool specifically targeting AMPKalpha1
(AMPKa1 siRNA) and incubated for 24 h with 100 ng/ml LPS. mRNA expression of IL-6 (A) and IL-8 (B) was analysed by quantitative real-time
PCR. (*, p < 0.05 vs. LPS in control siRNA cells; ***, p < 0.001 vs. basal in control siRNA cells).
Figure 4 Summary of our research aims and hypotheses. Our preliminary results suggest that AMPK modulates the adipokine expression
and secretion profiles from human adipocytes upon stimulation by inflammatory and/or metabolic factors. Imbalance in the adipokine profile
triggers insulin resistance and breast cancer cell growth. Moreover, adipokines alter AMPK activity and modulation of this activity might inhibit
the downstream effects of pro-inflammatory and pro-proliferative adipokines (TNFa, IL-6, IL-8, leptin) and improve the effects of anti-
inflammatory and anti-proliferative adipokines (adiponectin, IL1-RA) in insulin resistant tissues and breast tumours. AMPK: AMP-dependent protein
kinase, LPS: lipopolysaccharides, FFA: free fatty acids, TNFa: tumour necrosis factor a, IL-6: interleukin 6, IL-8: interleukin-8, IL1RA: interleukin-1
receptor antagonist).
Grisouard et al.Diabetology & Metabolic Syndrome 2011, 3:16
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and such changes were attributed to reduced apoptosis
rather than increased proliferation [22]. In our study,
apoptosis was not analysed by specific assays. However,
floating dead cells were removed by washing of the cell
layer with PBS to eliminate most of the dead cells before
fixation and staining. Nevertheless, we cannot exclude
that apoptotic cells still attached to the culture dish
were also stained by crystal violet under our experimen-
tal conditions. Further experiments will be needed to
expand our findings in other parameters which are
important for tumour progression such as apoptosis,
invasion and migration.
Additionally, this doctoral thesis reported that condi-
tioned media from murine preadipocytes was less effi-
cient on MCF-7 cell proliferation and that low glucose
medium reduced the effect of murine adipocyte condi-
tioned media on MCF7 growth [22]. Since incubation
with low glucose medium reduced IGF1mRNA levels in
adipocytes and since adipocyte conditioned media prolif-
erative effect was reverted by inhibiting the IGF-1 path-
way, the author suggests that adipocyte-produced IGF1
has a crucial role in promoting cancer cell growth [22].
Similar results were obtained regarding human preadi-
pocyte conditioned media in our study. To avoid the
possible effects of high glucose concentration on adipo-
cyte secretion such as IGF in the culture supernatant,
our human adipocytes were incubated in low glucose (5
mM) medium before collecting the conditioned media.
Low glucose medium might prevent the IGF release and
this suggests that other adipokines are involved in breast
cancer cell growth.
To obtain our data, both male and female adipose tis-
sues were considered since the number of tissue avail-
able from females was not sufficient. The small size of
the studied cohort of patients does not allow drawing
conclusions about any gender specific differences in pro-
liferation. Further work should be performed and adi-
pose tissues from females are certainly more relevant
regarding breast cancer proliferation. Ultimately, differ-
ence in a variety of parameters between fat depots is
important and fat tissue isolated from the breast should
be considered. Also, discrepancies between adipocytes
from different donors were observed and the results
were not consistent for both LPS-induced IL-1RA
mRNA expression after AMPKa1-silencing and LPS-
induced IL-8 mRNA expression upon metformin incu-
bation (data not shown). This suggests that metformin
and AMPK might alter the expression of some adipo-
kines (e.g. IL-6) in a similar way and expression of some
other adipokines in a different manner. Therefore, met-
formin might have some AMPK-dependent and -inde-
pendent effects, which could be unraveled if AMPK
expression is silenced upon metformin incubation.
Further experiments also need to be performed to check
the effects of metformin and AMPK modulation of adi-
pokine expression on breast cancer cell proliferation.
Recent studies demonstrated that metformin treat-
ment increased adiponectin plasma levels in women
with polycystic ovary syndrome (PCOS) [23] and T2DM
patients [24] while metformin treatment tends to
decrease IL-6 plasma levels [23] as well as circulating
levels of leptin [25] in PCOS patients, and significantly
reduces resistin and TNF-aplasma levels [24] in T2DM
patients. Moreover, metformin inhibition of TNF-a-
induced IL-6 secretion was abolished after silencing of
AMPKa1 in human umbilical vein endothelial cells [26].
Together with our data, this suggests that metformin,
via AMPK activation, improves the adipokine profile
from human adipocytes in overweight and obese
patients. This might contribute to a faster and better
improvement of the chronic low grade inflammatory
state and therefore insulin resistance in these patients.
In turn, adiponectin-induced AMPK activity in skeletal
muscles is involved in the regulation of mitochondrial
function and oxidative stress, glucose and lipid metabo-
lism, and exercise endurance [27]. The same study
further suggests that agonism of adiponectin signaling in
muscles provides a new treatment modality for insulin
resistance.
In patients with breast cancer, the effects of metfor-
min and AMPK on the adipokine imbalance might
diminish tumor progression since the present study
showed that adipocyte conditioned medium promoted
breast cancer cell proliferation. Decreasing pro-inflam-
matory adipokine secretion from adipocyte stromal cells
will reduce breast cancer cell growth and proliferation.
Increasing anti-inflammatory adipokines such as adipo-
nectin will inhibit invasion and migration of breast can-
cer cells. Indeed, adiponectin targets AMPK activity in
breast cancer cells [28]. This leads to an inhibition of
the mammalian target of rapamycin (mTOR) pathway
and a decrease in protein synthesis, inhibition of adhe-
sion, migration and invasion of breast cancer cells.
These results correlate with the activation of AMPK by
metformin in breast cancer cells [29]. However and
unlike adiponectin, other adipokines (e.g. leptin) might
differently alter AMPK activity in various tissues [30].
Therefore, it will be crucial to study the effect of each
adipokine on AMPK activity in breast cancer cells.
In conclusion, the present study shows some prelimin-
ary but encouraging data to further unravel the specific
role of AMPK and adipokines to alleviate obesity-related
insulin resistance and breast cancer complications. Acti-
vation of AMPK might improve adipokine production
triggered by obesity- and diabetes-related factors in
human adipocytes. In turn, some adipokines might acti-
vate AMPK and increase insulin sensitivity and inhibits
breast tumor development.
Grisouard et al.Diabetology & Metabolic Syndrome 2011, 3:16
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Acknowledgements and funding
This study was supported by grants from the University
of Basel (Forschungsfonds-Förderung für Nachwuchs-
forschende der Universität Basel) and the Stiftung der
Diabetes-Gesellschaft Region Basel to Jean Grisouard, by
grants from the Novartis Stiftung für Medizinisch-Biolo-
gische Forschung and the Swiss National Research
Foundation (PP00P3_123346/1) to Mirjam Christ-Crain
and a grant from Sanofi-Aventis to Beat Müller.
We thank Dr. Daniel M. Frey (Department of Surgery,
Div. of General Surgery and Surgical Research, Univer-
sity Hospital Basel) and Dr. Ralph Peterli (Department
of Surgery, Claraspital Basel) for providing us with
human adipose tissue samples.
Author details
1
Department of Biomedicine, University Hospital Basel, Basel, CH-4031,
Switzerland.
2
Hormones and Signal Transduction, German Cancer Research
Centre, DKFZ-ZMBH Alliance, Heidelberg, D-69120, Germany.
3
Division of
Endocrinology, Diabetes and Clinical Nutrition, University Hospital Basel,
Basel, CH-4031, Switzerland.
4
Medical University Clinic, Kantonsspital Aarau,
Aarau, CH-5001, Switzerland.
Authors’contributions
JG conceived of the study, participated in its design and coordination,
carried out the breast cancer cell proliferation assays and the AMPKalpha1
silencing of the adipocytes, analysed the results and drafted the manuscript.
KD carried out the adipocyte and breast cancer cell culture work and the
mRNA analyses. DM, UK, BM and MCC participated in the design of the
study and coordination, and helped to draft the manuscript. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 19 January 2011 Accepted: 20 July 2011
Published: 20 July 2011
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doi:10.1186/1758-5996-3-16
Cite this article as: Grisouard et al.: Targeting AMP-activated protein
kinase in adipocytes to modulate obesity-related adipokine production
associated with insulin resistance and breast cancer cell proliferation.
Diabetology & Metabolic Syndrome 2011 3:16.
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