Metformin inhibits leptin secretion via a mitogen-activated protein kinase signalling pathway in brown adipocytes Johannes Klein*, Sören Westphal*, Daniel Kraus, Britta Meier, Nina Perwitz, Volker Ott, Mathias Fasshauer1 and H Harald Klein2
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany1Department of Internal Medicine III, University of Leipzig, Germany2Department of Medicine 1, Kliniken Bergmannsheil, Ruhr-University Bochum, Germany(Requests for offprints should be addressed to J Klein; Email: [email protected])
(*J Klein and S Westphal contributed equally to this work)
Metformin is an anti-diabetic drug with anorexigenic
activator of transcription (STAT), and phosphatidylinositol
properties. The precise cellular mechanisms of its action
(PI) 3-kinase signalling pathways such as p38 MAP kinase,
are not entirely understood. Adipose tissue has recently
STAT3, and Akt was unaltered. Furthermore, chronic
been recognized as an important endocrine organ that is
metformin treatment for 12 days dose-dependently inhibi-
pivotal for the regulation of insulin resistance and energy
ted leptin secretion by 35% and 75% at 500 µmol/l and
homeostasis. Due to its thermogenic capacity brown
1 mmol/l metformin respectively (P<0·01). This reduc-
adipose tissue contributes to the regulation of energy
tion was not caused by alterations in adipocyte diﬀeren-
metabolism and is an attractive target tissue for pharma-
tiation. Moreover, the impairment in leptin secretion by
cological approaches to treating insulin resistance and
metformin was reversible within 48 h after removal of the
obesity. Leptin is the prototypic adipocyte-derived hor-
drug. Pharmacological inhibition of p44/p42 MAP kinase
mone inducing a negative energy balance. We investi-
prevented the metformin-induced negative eﬀect on
gated eﬀects of metformin on adipocyte metabolism,
leptin secretion. Taken together, our data demonstrate
signalling, and leptin secretion in a brown adipocyte
direct acute eﬀects of metformin on adipocyte signalling
model. Metformin acutely stimulated p44/p42 mitogen-
and endocrine function with robust inhibition of leptin
activated protein (MAP) kinase in a dose- (3·2-fold at
secretion. They suggest a selective molecular mechanism
1 mmol/l, P<0·05) as well as time-dependent (3·8-fold at
that may contribute to the anorexigenic eﬀect of this
5 min, P<0·05) manner. This stimulation was highly
selective since phosphorylation of intermediates in the
Journal of Endocrinology (2004) 183, 299–307
stress kinase, janus kinase (JAK)–signal transducer and
shown to be increased by chronic metformin treatment. In hepatocytes metformin inhibits gluconeogenesis and
Metformin is a widely used anti-diabetic agent for the
glycogenolysis probably due to a number of mechanisms
treatment of type 2 diabetes. It enhances insulin sensitiv-
such as diminished lactate uptake (Radziuk et al. 1997),
ity. Furthermore, this compound displays the unique
reduction in pyruvate carboxylase–phosphoenolpyruvate
characteristic of promoting weight loss and reducing
carboxykinase activity (Large & Beylot 1999), antagonism
appetite (Bailey & Turner 1996, Matthaei et al. 2000,
to glucagon (Dominguez et al. 1996), enhancement of
Kirpichnikov et al. 2002). Although used as a drug since
insulin action (Wiernsperger & Bailey 1999), and de-
the late 1950s, the mechanisms of action by which
creased concentrations of adenosine triphosphate (Argaud
metformin lowers glucose and lipid levels remain unclear. et al. 1993). In this context, modulation of cellular respir-
Potential direct eﬀects of metformin on signalling path-
ation via unidentified cell-signalling pathways appears to
ways are poorly understood. In muscle, insulin receptor
play a role (Dominguez et al. 1996, Yki-Jarvinen et al.
tyrosine kinase activity (Stith et al. 1996, 1998) and recruit-
1999, Kirpichnikov et al. 2002). Activation of 5 -AMP-
ment of glucose transporter (GLUT) 4 to the plasma mem-
activated protein kinase (AMPK) has been implicated in
brane (Sarabia et al. 1992, Rouru et al. 1995) have been
metformin action in hepatocytes (Zhou et al. 2001). Journal of Endocrinology (2004) 183, 299–307
0022–0795/04/0183–299 2004 Society for Endocrinology Printed in Great Britain
300 J KLEIN, S WESTPHAL and others · Direct effects of metformin on brown adipocytesFigure 1 Metformin acutely activates p44/p42 MAP kinase. Fully differentiated brown adipocytes were stimulated with metformin for the times (1–40 min) (A) and at the concentrations (B) indicated. (A) Cell lysates and immunoblots using phospho-specific antibodies were prepared as described in Materials and Methods. (B) Bar graph analyses with S.E.M. of d6 independent experiments and representative immunoblots are shown. * Denotes statistically significant (P<0·05) differences comparing non-treated (Basal) to metformin-treated cells. Journal of Endocrinology (2004) 183, 299–307 Direct effects of metformin on brown adipocytes · J KLEIN, S WESTPHAL and others 301
By contrast to liver and muscle, relatively little is known
about direct metformin actions in adipocytes. In ratadipose tissue glucose uptake has been found to beenhanced (Matthaei et al. 1991, 1993) whereas in humanadipocytes no change has been described by metformintreatment (Pedersen et al. 1989, Ciaraldi et al. 2002). Recently, there has been a growing appreciation ofadipose tissue as an endocrine organ that is pivotal for thesystemic regulation of insulin action and energy homeo-stasis (Rajala & Scherer 2003). Direct interactions ofmetformin with adipocyte signalling and endocrine func-tion may thus be instrumental for this compound’s eﬀects. Clinical studies with metformin have constantly showneither a decrease in body weight (DeFronzo et al. 1991,DeFronzo & Goodman 1995) or at least a significantly
Figure 2 Metformin does not stimulate p38 MAP kinase, Akt or
smaller increase in body weight compared with other
STAT3 phosphorylation. Adipocytes were stimulated with
forms of treatment (Yki-Jarvinen et al. 1999). The
metformin (1 mM) for the indicated times (30 s and 1, 2, 5 and
adipocyte-derived hormone, leptin, is an essential player in
10 min). Cell lysates and immunoblots using phospho-specific
regulating energy homeostasis (Friedman & Halaas 1998,
antibodies were prepared as described in Materials and Methods. Representative blots of phospho-p38 MAP kinase (upper panel),
Spiegelman & Flier 2001, Friedman 2002). Brown adipose
phospho-Akt (middle panel), and phospho-STAT3 (lower panel) of
tissue importantly contributes to the modulation of energy
d5 independent experiments are shown.
homeostasis in rodents (Lowell & Flier 1997, Lowell &Bachman 2003), has been implicated in human obesity(Fumeron et al. 1996, Oberkofler et al. 1997, Fogelholmet al. 1998, Valve et al. 1998), and is an attractive target
transcription (STAT) 3 (phospho-Tyr705), p44/p42 MAP
tissue for pharmacotherapeutic approaches to obesity
kinase (phospo-Thr202/Tyr204), Akt (phospho-Ser473)
(Danforth & Himms-Hagen 1997, Lowell & Flier 1997,
(Cell Signaling Technology, Inc., Beverly, MA, USA),
Bray & Greenway 1999, Tiraby et al. 2003, Klaus 2004).
Recent studies suggest the existence of a basal brown
some proliferator-activated receptor (PPAR)
adipose phenotype that may be important for the main-
Cruz Biotechnology, Inc., Santa Cruz, CA, USA), uncou-
tenance of normal insulin sensitivity and energy homeo-
pling protein (UCP)-1 (Alpha Diagnostic International,
stasis (Yang et al. 2003). Moreover, transdiﬀerentiation of
San Antonio, TX, USA). The pharmacological MAP
white to brown adipocytes has been demonstrated and
kinase inhibitor, PD98059, was purchased from Cell
may oﬀer interesting new therapeutic perspectives for
Signaling Technology, Inc. Unless stated otherwise, all
treating insulin resistance and energy balance disorders
other chemicals were purchased from Sigma-Aldrich Co.
(Tiraby & Langin 2003, Tiraby et al. 2003). We have
previously demonstrated robust leptin secretion in brownadipocytes (Klein et al. 2002, Kraus et al. 2002). Investi-
gation of direct metformin interaction with adipose tissuemay identify molecular targets and provide insights into
SV40T-immortalized brown adipocytes from the FVB
mechanisms of insulin resistance and energy homeostasis
strain of mice – generated as described elsewhere (Klein
et al. 1999) – were used for all experiments. Preadipocytes
Here, we studied direct metformin eﬀects on adipocyte
were seeded on tissue culture plates (Sarstedt, Nümbrecht,
signalling, diﬀerentiation, and leptin secretion (Klein et al.
Germany) and grown to confluence in culture medium
2002, Kraus et al. 2002). We demonstrate a selective
with Dulbecco’s modified Eagle’s medium (Life Tech-
activation of p44/p42 mitogen-activated protein (MAP)
nologies, Paisley, Strathclyde, UK), supplemented with
kinase by metformin and a diﬀerentiation-independent,
20% fetal bovine serum, 4·5 g/l glucose, 20 nM insulin,
robust reduction in leptin secretion that is prevented by
1 nM triiodothyronine (‘diﬀerentiation medium’), and
pharmacological inhibition of p44/p42 MAP kinase.
penicillin/streptomycin. Adipocyte diﬀerentiation wasinduced by complementing the medium further with250 µM indomethacin, 500 µM isobutylmethylxanthine
Materials and Methods
and 2 µg/ml dexamethasone for 24 h when confluencewas reached. After this induction period, cells were
changed back to diﬀerentiation medium. Cell culture was
Antibodies against the following molecules were employed
continued for 5 more days before cells were starved for
for immunoblotting: signal transducer and activator of
24 h with serum-free medium prior to carrying out the
Journal of Endocrinology (2004) 183, 299–307
302 J KLEIN, S WESTPHAL and others · Direct effects of metformin on brown adipocytes
using whole cell lysis buﬀer containing 2 mM vanadate,10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mMPMSF. Protein content of lysates was determined by theBradford method using the dye from Bio-Rad (Hercules,CA, USA). Lysates were submitted to SDS-PAGE andtransferred to nitrocellulose membranes (Schleicher andSchuell Inc., Keane, NH, USA). Membranes wereblocked with rinsing buﬀer (10 mM Tris, 150 mM NaCl,0·05% Tween, pH 7·2) containing 3% bovine serumalbumin (‘blocking solution’) overnight. Membranes werethen incubated in blocking solution for 1–2 h with theantibodies indicated. Protein bands were visualizedusing the chemiluminescence kit from Roche MolecularBiochemicals (Mannheim, Germany) and enhancedchemiluminescence films (Amersham Pharmacia Biotech,Freiburg, Germany). Figure 3 Chronic metformin treatment dose-dependently inhibits leptin secretion. Cells were chronically exposed to the indicated concentrations of metformin over the entire differentiation course.
Medium was collected every 24 h. Secretion of leptin wasanalysed in the culture medium using a mouse leptin RIA. A line
graph with S.E.M. of d3 independent experiments is shown
(SPSS Science; Chicago, IL, USA) was employed for
comparing untreated cells (Con, d) with 500 M (♦) and 1 mM() metformin treatment. ** Denotes high statistical significance
statistical analysis of all data. Statistical significance was
determined using the unpaired Student’s t-test. P values
<0·05 are considered significant, <0·01 highly significant.
immunoblotting experiments. For leptin secretion experi-ments, cell culture was continued for up to 9 days after
Metformin acutely induces p44/p42 MAP kinase but not p38MAP kinase, Akt and STAT3 phosphorylation
Cells were chronically treated with or without metformin
P44/42 MAP kinase is an important signalling intermedi-
and medium was collected every 24 h from day 4 to day
ate of growth factor signalling pathways and a major
12 of the diﬀerentiation course. Treatment with the
regulator of gene transcription. Treatment of fully diﬀer-
pharmacological MAP kinase inhibitor, PD98059, was
entiated brown adipocytes with metformin resulted in a
begun 30 min prior to adding metformin. The amount of
time- and dose-dependent stimulation of p44/p42 MAP
leptin released into the medium was determined using a
kinase as assessed using phospho-specific antibodies (Fig.
mouse leptin RIA (Linco Research, Inc., St Louis, MO,
1A and B). Metformin-induced activation was most
prominent after 5 min (Fig. 1A) with a maximal 3·5-foldphosphorylation increase at a concentration of 1 mM (Fig.
1B). There was no change in protein amounts of MAPkinase as assessed by immunoblots using p44/p42 MAP
Tissue culture plates were washed twice with PBS and
kinase antibodies (data not shown). Furthermore, met-
fixed with 10% formalin for at least 1 h at room tempera-
formin treatment did not induce significant changes in
ture. Cells were then stained for 1 h at room temperature
phosphorylation of p38 MAP kinase, Akt and STAT3 –
with a filtered Oil Red O solution (0·5 g Oil Red O
key signalling molecules of the stress kinase, phosphati-
in 100 ml isopropyl alcohol). The staining solution was
dylinositol 3-kinase (PI 3-kinase), and janus kinase (JAK)/
washed oﬀ the cells with distilled water twice.
STAT signalling pathways respectively (Fig. 2). Metformin treatment inhibits leptin secretion in a
SV40T-immortalized mouse brown adipocytes were used
between passages 10 and 25. For p44/p42 MAP kinase,Akt, p38 MAP kinase, and STAT3 analysis fully diﬀeren-
When cells were chronically exposed to metformin, there
tiated cells were starved for 24 h in serum-free medium
was a dose-dependent impairment in leptin secretion.
prior to carrying out the experiments. Following treatment
Non-treated control cells displayed a diﬀerentiation-
with metformin as indicated, proteins were isolated
dependent increase in leptin secretion over two orders of
Journal of Endocrinology (2004) 183, 299–307 Direct effects of metformin on brown adipocytes · J KLEIN, S WESTPHAL and others 303
Figure 4 The inhibitory effect of metformin on leptin secretion is not caused by alterations in adipocyte differentiation. (A) Differentiation was assessed in cell lines either non-treated (Con) or chronically exposed to metformin (Met, 1 mM) using the fat-specific Oil Red O staining. (B) Using specific antibodies as applicable, protein expression of the differentiation markers uncoupling protein-1 (UCP-1, upper panel), peroxisome proliferator-activated receptor gamma (PPAR , middle panel) and CCAAT enhancer-binding protein alpha (C/EBP , lower panel) was analysed in immunoblots. Representative blots and staining results of d2 independent experiments are shown.
magnitude with the lowest detectable leptin levels at a
ment. When diﬀerentiating adipocytes were stained with
concentration of 0·2 µg/l rising to the maximum detect-
the fat-specific Oil Red O at days 4, 7, 10 and 13 of the
able level of 20 µg/l during a 12-day-diﬀerentiation course
diﬀerentiation course there was no diﬀerence between
(Fig. 3). Chronic metformin treatment dose-dependently
metformin-treated and non-treated control cells (Fig. 4A).
inhibited this increase in leptin secretion with a maximum
Furthermore, protein expression of early and late adipocyte
reduction of 35% and 75% at the end of the diﬀerentation
diﬀerentiation markers such as C/EBP , PPAR , and
course at concentrations of 500 µM and 1 mM metformin
UCP-1 did not diﬀer between metformin-treated and
respectively. These changes were highly significant (Fig.
non-treated control cells throughout the diﬀerentiation
3). A significant inhibition of leptin secretion was also seen
at 100 µM metformin (data not shown). Furthermore,metformin did not influence glucose utilization and lactate
Subacute metformin treatment induces a reversible impairment
To further define the kinetics of the inhibitory metformin
The inhibitory eﬀect of metformin on leptin secretion is not
eﬀect on leptin secretion, we pretreated adipocytes for
caused by alterations in diﬀerentiation
various periods of time with 1 mM metformin on day 8 of
To separate the impairment in leptin secretion from a
the diﬀerentiation course, collected the medium every
diﬀerentiation-dependent eﬀect, we next investigated
24 h, and continued cell culture for two more days
adipocyte diﬀerentiation under chronic metformin treat-
without metformin exposure. Interestingly, metformin
Journal of Endocrinology (2004) 183, 299–307
304 J KLEIN, S WESTPHAL and others · Direct effects of metformin on brown adipocytes
MAP kinase phosphorylation suggested an involvement ofthis signalling intermediate in the mediation of this eﬀect. Metformin treatment for 24 h again significantly dimin-ished leptin secretion by 30% on the following day ascompared with non-treated control cells (Fig. 5B). How-ever, when cells were pretreated with the p44/p2 MAPkinase inhibitor, PD98059, exposure to metformin failedto significantly inhibit leptin secretion (Fig. 5B). Treat-ment with the pharmacological inhibitor alone did notchange basal leptin secretion (Fig. 5B). Discussion
In this study, we show direct eﬀects of the anorexigenicanti-diabetic drug, metformin, on adipocyte signalling andendocrine function with robust inhibition of leptinsecretion.
Metformin directly induced p44/p42 MAP kinase
activation. To our knowledge, this is the first reportdemonstrating stimulation of this important growth factorsignalling intermediate by metformin. Apart from p44/p42 MAP kinase, only AMPK and p38 MAP kinase havebeen shown to be implicated in intracellular metforminaction so far. Zhou et al. (2001) and Hawley et al. (2002)described activation of AMPK by chronic treatment withmetformin in rat hepatocytes and skeletal muscle. Inskeletal muscle, Kumar & Dey (2002) also found anincrease in p38 MAP kinase activity by metformin. Interestingly, however, p38 stress kinase-, PI 3-kinase-,and JAK/STAT-signalling pathways remained unaﬀectedby metformin treatment in our study using adipocytes. Figure 5 Subacute metformin treatment induces an impairment in
These discrepancies may indicate tissue- and cell-specific
leptin secretion that can be prevented by inhibition of p44/p42
MAP kinase. On day 8 of the differentiation course cells were
Of note, stimulation of p44/p42 MAP kinase occurred
either left untreated (Con) or treated with metformin (Met, 1 mM)for 24 h. Medium was collected 24 h (A, left panel) or 72 h (A,
acutely and was time- and dose-dependent. In concert
right panel) after removal of metformin. (B) The MAP kinase
with the demonstrated selectivity of action, these findings
inhibitor, PD98059 (PD, 50 M), was added 1 h prior to
suggest a receptor-mediated signalling mechanism em-
metformin treatment for 24 h, and the medium was analysed for
ployed by metformin in adipocytes. However, no specific
leptin concentrations 24 h later. A bar graph analysis with S.E.M. of d5 independent experiments is shown. * Denotes statistical
receptor mediating the eﬀects of metformin has been
identified so far. Rather, this lipophilic compound mayexert its eﬀects by alterations of the cellular membrane
treatment for 24 h resulted in a significant 30% reduction
structure (Meuillet et al. 1999).
of leptin secretion within the next 24 h (Fig. 5A, left
Activation of p44/p42 MAP kinase plays an important
panel). This eﬀect was completely reversible 72 h after
role in regulating gene expression, insulin signalling and –
metformin removal from the medium (Fig. 5A, right
specifically in brown adipocytes – thermogenesis (Porras
panel). Furthermore, there was a time-dependent trend
et al. 1998, Klein et al. 2000). Therefore, it appears
towards impaired leptin secretion after 8 and 16 h of
plausible to propose important functional consequences of
metformin treatment whereas shorter periods of time did
metformin-induced acute changes in p44/p42 MAP
not show significant alterations in leptin secretion as
kinase signalling in adipocytes. Indeed, we found that
compared with control cells (data not shown).
metformin directly aﬀected endocrine function and inhib-ited leptin secretion. We used a previously well charac-
Inhibition of p44/p42 MAP kinase prevents the inhibitory
terised adipocyte model (Klein et al. 2002) that displays
strong leptin secretion (Kraus et al. 2002). A decrease
The impairment of leptin secretion by subacute metformin
in leptin levels in metformin-treated individuals has
treatment in concert with the acute induction of p44/p42
been found in several studies (Freemark & Bursey 2001,
Journal of Endocrinology (2004) 183, 299–307 Direct effects of metformin on brown adipocytes · J KLEIN, S WESTPHAL and others 305
Figure 6 Metformin directly modulates adipocyte signalling and endocrine function. Metformin activates p44/p42 MAP kinase and impairs leptin secretion unless p44/p42 MAP kinase is inhibited. This effect is reversible and is not caused by alterations in adipocyte differentiation. Furthermore, it is selective since there is no activation of stress kinase, PI 3-kinase, and JAK/STAT signalling pathways. Modulation of endocrine adipocyte function by metformin may be important in the regulation of energy homeostasis.
Glueck et al. 2001, Fruehwald-Schultes et al. 2002);
ling prevented the metformin-induced reduction in leptin
however, in other studies, no eﬀect on serum leptin was
secretion, thus suggesting an involvement of this important
found (Guler et al. 2000, Mannucci et al. 2001, Uehara
growth factor signalling intermediate in the modulation of
et al. 2001, Ciaraldi et al. 2002, Sivitz et al. 2003). Possible
explanations for these discrepancies may be the length of
In summary, our data show a direct selective interac-
treatment and the study population, with obese people
tion of metformin with adipocyte p44/p42 MAP kinase
showing a decrease in leptin levels after long-term treat-
signalling and leptin secretion. They describe a potential
ment. A negative correlation of the length of metformin
molecular mechanism mediating this anorexigenic com-
therapy with circulating leptin levels in this setting could
pound’s eﬀects on adipose tissue. Selective modulation
possibly be accounted for by a direct subacute eﬀect of this
of adipose tissue function could have important implica-
anti-diabetic drug on adipose tissue as described in this
tions for therapeutic strategies of the insulin resistance
In a previous study in rat white adipocytes, a negative
influence of chronic metformin exposure on leptin secre-
tion has also been reported (Mueller et al. 2000). As weshow here, the direct metformin-induced impairment in
We would like to thank M Schümann for expert help with
leptin secretion is independent of changes in adipocyte
morphology and diﬀerentiation. Furthermore, it is alreadyevident after 24 h of treatment, and it is reversible. As wasthe case with activation of p44/p42 MAP kinase, these
observations point towards a selective signalling mechan-ism mediating these eﬀects. In favour of this assumption,
This study was supported by grants from the Deutsche
we found that inhibition of p44/p42 MAP kinase signal-
Forschungsgemeinschaft (Kl 1131/2-1 and Kl 1131/2-2),
Journal of Endocrinology (2004) 183, 299–307
306 J KLEIN, S WESTPHAL and others · Direct effects of metformin on brown adipocytes
German Diabetes Association, and Faculty grants of the
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