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
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) Abstract
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 Introduction
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 adipocytes Figure 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 p38 MAP 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).
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- Acknowledgements
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 Hawley SA, Gadalla AE, Olsen GS & Hardie DG 2002 The antidiabetic drug metformin activates the AMP-activated protein
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Pharmacokinetic parameters of Gtfx after oral administration in dogs( n = 4) Concentration - time curves of Gtfx after oral administration in dogs serum( n = 4) Perry CM , Balfour JAB , Lamb HM. Catifloxacin[J ] . Drugs ,1999 ,58Stahlberg HJ , Cohler K , Cuillane M , et al . Effeets of gatifloxacin me2sylate ( GTFX) on the pharmacokinetics of theophyline in heathly youngvolun

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