Abstract
Metformin is an anti-diabetic drug with anorexigenic properties. The precise cellular mechanisms of its action are not entirely understood. Adipose tissue has recently been recognized as an important endocrine organ that is pivotal for the regulation of insulin resistance and energy homeostasis. Due to its thermogenic capacity brown adipose tissue contributes to the regulation of energy metabolism and is an attractive target tissue for pharmacological approaches to treating insulin resistance and obesity. Leptin is the prototypic adipocyte-derived hormone inducing a negative energy balance. We investigated effects of metformin on adipocyte metabolism, signalling, and leptin secretion in a brown adipocyte model. Metformin acutely stimulated p44/p42 mitogen-activated protein (MAP) kinase in a dose- (3.2-fold at 1 mmol/l, P< 0.05) as well as time-dependent (3.8-fold at 5 min, P< 0.05) manner. This stimulation was highly selective since phosphorylation of intermediates in the stress kinase, janus kinase (JAK)–signal transducer and activator of transcription (STAT), and phosphatidylinositol (PI) 3-kinase signalling pathways such as p38 MAP kinase, STAT3, and Akt was unaltered. Furthermore, chronic metformin treatment for 12 days dose-dependently inhibited leptin secretion by 35% and 75% at 500 μmol/l and 1 mmol/l metformin respectively (P< 0.01). This reduction was not caused by alterations in adipocyte differentiation. Moreover, the impairment in leptin secretion by metformin was reversible within 48 h after removal of the drug. Pharmacological inhibition of p44/p42 MAP kinase prevented the metformin-induced negative effect on leptin secretion. Taken together, our data demonstrate direct acute effects of metformin on adipocyte signalling and endocrine function with robust inhibition of leptin secretion. They suggest a selective molecular mechanism that may contribute to the anorexigenic effect of this antidiabetic compound.
Introduction
Metformin is a widely used anti-diabetic agent for the treatment of type 2 diabetes. It enhances insulin sensitivity. Furthermore, this compound displays the unique characteristic of promoting weight loss and reducing appetite (Bailey & Turner 1996, Matthaei et al. 2000, Kirpichnikov et al. 2002). Although used as a drug since the late 1950s, the mechanisms of action by which metformin lowers glucose and lipid levels remain unclear.
Potential direct effects of metformin on signalling pathways are poorly understood. In muscle, insulin receptor tyrosine kinase activity (Stith et al. 1996, 1998) and recruitment of glucose transporter (GLUT) 4 to the plasma membrane (Sarabia et al. 1992, Rouru et al. 1995) have been shown to be increased by chronic metformin treatment. In hepatocytes metformin inhibits gluconeogenesis and glycogenolysis probably due to a number of mechanisms such as diminished lactate uptake (Radziuk et al. 1997), reduction in pyruvate carboxylase–phosphoenolpyruvate carboxykinase activity (Large & Beylot 1999), antagonism to glucagon (Dominguez et al. 1996), enhancement of insulin action (Wiernsperger & Bailey 1999), and decreased concentrations of adenosine triphosphate (Argaud et al. 1993). In this context, modulation of cellular respiration via unidentified cell-signalling pathways appears to play a role (Dominguez et al. 1996, Yki-Jarvinen et al. 1999, Kirpichnikov et al. 2002). Activation of 5′-AMP-activated protein kinase (AMPK) has been implicated in metformin action in hepatocytes (Zhou et al. 2001).
By contrast to liver and muscle, relatively little is known about direct metformin actions in adipocytes. In rat adipose tissue glucose uptake has been found to be enhanced (Matthaei et al. 1991, 1993) whereas in human adipocytes no change has been described by metformin treatment (Pedersen et al. 1989, Ciaraldi et al. 2002). Recently, there has been a growing appreciation of adipose tissue as an endocrine organ that is pivotal for the systemic regulation of insulin action and energy homeostasis (Rajala & Scherer 2003). Direct interactions of metformin with adipocyte signalling and endocrine function may thus be instrumental for this compound’s effects. Clinical studies with metformin have constantly shown either a decrease in body weight (DeFronzo et al. 1991, DeFronzo & Goodman 1995) or at least a significantly smaller increase in body weight compared with other forms of treatment (Yki-Jarvinen et al. 1999). The adipocyte-derived hormone, leptin, is an essential player in regulating energy homeostasis (Friedman & Halaas 1998, Spiegelman & Flier 2001, Friedman 2002). Brown adipose tissue importantly contributes to the modulation of energy homeostasis in rodents (Lowell & Flier 1997, Lowell & Bachman 2003), has been implicated in human obesity (Fumeron et al. 1996, Oberkofler et al. 1997, Fogelholm et al. 1998, Valve et al. 1998), and is an attractive target tissue for pharmacotherapeutic approaches to obesity (Danforth & Himms-Hagen 1997, Lowell & Flier 1997, Bray & Greenway 1999, Tiraby et al. 2003, Klaus 2004). Recent studies suggest the existence of a basal brown adipose phenotype that may be important for the maintenance of normal insulin sensitivity and energy homeostasis (Yang et al. 2003). Moreover, transdifferentiation of white to brown adipocytes has been demonstrated and may offer interesting new therapeutic perspectives for treating insulin resistance and energy balance disorders (Tiraby & Langin 2003, Tiraby et al. 2003). We have previously demonstrated robust leptin secretion in brown adipocytes (Klein et al. 2002, Kraus et al. 2002). Investigation of direct metformin interaction with adipose tissue may identify molecular targets and provide insights into mechanisms of insulin resistance and energy homeostasis regulation.
Here, we studied direct metformin effects on adipocyte signalling, differentiation, and leptin secretion (Klein et al. 2002, Kraus et al. 2002). We demonstrate a selective activation of p44/p42 mitogen-activated protein (MAP) kinase by metformin and a differentiation-independent, robust reduction in leptin secretion that is prevented by pharmacological inhibition of p44/p42 MAP kinase.
Materials and Methods
Materials
Antibodies against the following molecules were employed for immunoblotting: signal transducer and activator of transcription (STAT) 3 (phospho-Tyr705), p44/p42 MAP kinase (phospo-Thr202/Tyr204), Akt (phospho-Ser473) (Cell Signaling Technology, Inc., Beverly, MA, USA), CCAAT enhancer binding protein (C/EBP) α, peroxisome proliferator-activated receptor (PPAR) γ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), uncoupling protein (UCP)-1 (Alpha Diagnostic International, San Antonio, TX, USA). The pharmacological MAP kinase inhibitor, PD98059, was purchased from Cell Signaling Technology, Inc. Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO, USA).
Cell culture
SV40T-immortalized brown adipocytes from the FVB strain of mice – generated as described elsewhere (Klein et al. 1999) – were used for all experiments. Preadipocytes were seeded on tissue culture plates (Sarstedt, Nümbrecht, Germany) and grown to confluence in culture medium with Dulbecco’s modified Eagle’s medium (Life Technologies, Paisley, Strathclyde, UK), supplemented with 20% fetal bovine serum, 4.5 g/l glucose, 20 nM insulin, 1 nM triiodothyronine (‘differentiation medium’), and penicillin/streptomycin. Adipocyte differentiation was induced by complementing the medium further with 250 μM indomethacin, 500 μM isobutylmethylxanthine and 2 μg/ml dexamethasone for 24 h when confluence was reached. After this induction period, cells were changed back to differentiation medium. Cell culture was continued for 5 more days before cells were starved for 24 h with serum-free medium prior to carrying out the immunoblotting experiments. For leptin secretion experiments, cell culture was continued for up to 9 days after induction of differentiation.
Determination of leptin secretion
Cells were chronically treated with or without metformin and medium was collected every 24 h from day 4 to day 12 of the differentiation course. Treatment with the pharmacological MAP kinase inhibitor, PD98059, was begun 30 min prior to adding metformin. The amount of leptin released into the medium was determined using a mouse leptin RIA (Linco Research, Inc., St Louis, MO, USA).
Oil Red O staining
Tissue culture plates were washed twice with PBS and fixed with 10% formalin for at least 1 h at room temperature. Cells were then stained for 1 h at room temperature with a filtered Oil Red O solution (0.5 g Oil Red O in 100 ml isopropyl alcohol). The staining solution was washed off the cells with distilled water twice.
Western blotting
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 differentiated cells were starved for 24 h in serum-free medium prior to carrying out the experiments. Following treatment with metformin as indicated, proteins were isolated using whole cell lysis buffer containing 2 mM vanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mM PMSF. Protein content of lysates was determined by the Bradford method using the dye from Bio-Rad (Hercules, CA, USA). Lysates were submitted to SDS-PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell Inc., Keane, NH, USA). Membranes were blocked with rinsing buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween, pH 7.2) containing 3% bovine serum albumin (‘blocking solution’) overnight. Membranes were then incubated in blocking solution for 1–2 h with the antibodies indicated. Protein bands were visualized using the chemiluminescence kit from Roche Molecular Biochemicals (Mannheim, Germany) and enhanced chemiluminescence films (Amersham Pharmacia Biotech, Freiburg, Germany).
Statistical analysis
Data are presented as means±s.e.m. Sigma Plot software (SPSS Science; Chicago, IL, USA) was employed for 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.
Results
Metformin acutely induces p44/p42 MAP kinase but not p38 MAP kinase, Akt and STAT3 phosphorylation
P44/42 MAP kinase is an important signalling intermediate of growth factor signalling pathways and a major regulator of gene transcription. Treatment of fully differentiated brown adipocytes with metformin resulted in a time- and dose-dependent stimulation of p44/p42 MAP kinase as assessed using phospho-specific antibodies (Fig. 1A and B). Metformin-induced activation was most prominent after 5 min (Fig. 1A) with a maximal 3.5-fold phosphorylation increase at a concentration of 1 mM (Fig. 1B). There was no change in protein amounts of MAP kinase as assessed by immunoblots using p44/p42 MAP kinase antibodies (data not shown). Furthermore, met-formin treatment did not induce significant changes in phosphorylation of p38 MAP kinase, Akt and STAT3 –key signalling molecules of the stress kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and janus kinase (JAK)/ STAT signalling pathways respectively (Fig. 2).
Metformin treatment inhibits leptin secretion in a dose-dependent manner
When cells were chronically exposed to metformin, there was a dose-dependent impairment in leptin secretion. Non-treated control cells displayed a differentiation-dependent increase in leptin secretion over two orders of magnitude with the lowest detectable leptin levels at a concentration of 0.2 μg/l rising to the maximum detectable level of 20 μg/l during a 12-day-differentiation course (Fig. 3). Chronic metformin treatment dose-dependently inhibited this increase in leptin secretion with a maximum reduction of 35% and 75% at the end of the differentation course at concentrations of 500 μM and 1 mM metformin respectively. These changes were highly significant (Fig. 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 production (data not shown).
The inhibitory effect of metformin on leptin secretion is not caused by alterations in differentiation
To separate the impairment in leptin secretion from a differentiation-dependent effect, we next investigated adipocyte differentiation under chronic metformin treatment. When differentiating adipocytes were stained with the fat-specific Oil Red O at days 4, 7, 10 and 13 of the differentiation course there was no difference between metformin-treated and non-treated control cells (Fig. 4A). Furthermore, protein expression of early and late adipocyte differentiation markers such as C/EBPα, PPARγ, and UCP-1 did not differ between metformin-treated and non-treated control cells throughout the differentiation course (Fig. 4B).
Subacute metformin treatment induces a reversible impairment in leptin secretion
To further define the kinetics of the inhibitory metformin effect on leptin secretion, we pretreated adipocytes for various periods of time with 1 mM metformin on day 8 of the differentiation course, collected the medium every 24 h, and continued cell culture for two more days without metformin exposure. Interestingly, metformin treatment for 24 h resulted in a significant 30% reduction of leptin secretion within the next 24 h (Fig. 5A, left panel). This effect was completely reversible 72 h after metformin removal from the medium (Fig. 5A, right panel). Furthermore, there was a time-dependent trend towards impaired leptin secretion after 8 and 16 h of metformin treatment whereas shorter periods of time did not show significant alterations in leptin secretion as compared with control cells (data not shown).
Inhibition of p44/p42 MAP kinase prevents the inhibitory metformin effect
The impairment of leptin secretion by subacute metformin treatment in concert with the acute induction of p44/p42 MAP kinase phosphorylation suggested an involvement of this signalling intermediate in the mediation of this effect. Metformin treatment for 24 h again significantly diminished leptin secretion by 30% on the following day as compared with non-treated control cells (Fig. 5B). However, when cells were pretreated with the p44/p2 MAP kinase inhibitor, PD98059, exposure to metformin failed to significantly inhibit leptin secretion (Fig. 5B). Treatment with the pharmacological inhibitor alone did not change basal leptin secretion (Fig. 5B).
Discussion
In this study, we show direct effects of the anorexigenic anti-diabetic drug, metformin, on adipocyte signalling and endocrine function with robust inhibition of leptin secretion.
Metformin directly induced p44/p42 MAP kinase activation. To our knowledge, this is the first report demonstrating stimulation of this important growth factor signalling intermediate by metformin. Apart from p44/ p42 MAP kinase, only AMPK and p38 MAP kinase have been shown to be implicated in intracellular metformin action so far. Zhou et al.(2001) and Hawley et al.(2002) described activation of AMPK by chronic treatment with metformin in rat hepatocytes and skeletal muscle. In skeletal muscle, Kumar & Dey (2002) also found an increase in p38 MAP kinase activity by metformin. Interestingly, however, p38 stress kinase-, PI 3-kinase-, and JAK/STAT-signalling pathways remained unaffected by metformin treatment in our study using adipocytes. These discrepancies may indicate tissue- and cell-specific effects of metformin.
Of note, stimulation of p44/p42 MAP kinase occurred acutely and was time- and dose-dependent. In concert with the demonstrated selectivity of action, these findings suggest a receptor-mediated signalling mechanism employed by metformin in adipocytes. However, no specific receptor mediating the effects of metformin has been identified so far. Rather, this lipophilic compound may exert its effects by alterations of the cellular membrane structure (Meuillet et al. 1999).
Activation of p44/p42 MAP kinase plays an important role in regulating gene expression, insulin signalling and –specifically in brown adipocytes – thermogenesis (Porras et al. 1998, Klein et al. 2000). Therefore, it appears plausible to propose important functional consequences of metformin-induced acute changes in p44/p42 MAP kinase signalling in adipocytes. Indeed, we found that metformin directly affected endocrine function and inhibited leptin secretion. We used a previously well characterised adipocyte model (Klein et al. 2002) that displays strong leptin secretion (Kraus et al. 2002). A decrease in leptin levels in metformin-treated individuals has been found in several studies (Freemark & Bursey 2001, Glueck et al. 2001, Fruehwald-Schultes et al. 2002); however, in other studies, no effect on serum leptin was found (Guler et al. 2000, Mannucci et al. 2001, Uehara et al. 2001, Ciaraldi et al. 2002, Sivitz et al. 2003). Possible explanations for these discrepancies may be the length of treatment and the study population, with obese people showing a decrease in leptin levels after long-term treatment. A negative correlation of the length of metformin therapy with circulating leptin levels in this setting could possibly be accounted for by a direct subacute effect of this anti-diabetic drug on adipose tissue as described in this study.
In a previous study in rat white adipocytes, a negative influence of chronic metformin exposure on leptin secretion has also been reported (Mueller et al. 2000). As we show here, the direct metformin-induced impairment in leptin secretion is independent of changes in adipocyte morphology and differentiation. Furthermore, it is already evident after 24 h of treatment, and it is reversible. As was the case with activation of p44/p42 MAP kinase, these observations point towards a selective signalling mechanism mediating these effects. In favour of this assumption, we found that inhibition of p44/p42 MAP kinase signalling prevented the metformin-induced reduction in leptin secretion, thus suggesting an involvement of this important growth factor signalling intermediate in the modulation of endocrine adipocyte function.
In summary, our data show a direct selective interaction of metformin with adipocyte p44/p42 MAP kinase signalling and leptin secretion. They describe a potential molecular mechanism mediating this anorexigenic compound’s effects on adipose tissue. Selective modulation of adipose tissue function could have important implications for therapeutic strategies of the insulin resistance syndrome.
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 ≥6 independent experiments and representative immunoblots are shown. * Denotes statistically significant (P<0.05) differences comparing non-treated (Basal) to metformin-treated cells.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
Metformin does not stimulate p38 MAP kinase, Akt or STAT3 phosphorylation. Adipocytes were stimulated with metformin (1 mM) for the indicated times (30 s and 1, 2, 5 and 10 min). Cell lysates and immunoblots using phospho-specific antibodies were prepared as described in Materials and Methods. Representative blots of phospho-p38 MAP kinase (upper panel), phospho-Akt (middle panel), and phospho-STAT3 (lower panel) of ≥5 independent experiments are shown.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
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 was analysed in the culture medium using a mouse leptin RIA. A line graph with s.e.m. of ≥3 independent experiments is shown comparing untreated cells (Con, •) with 500 μM (♦) and 1 mM (▪) metformin treatment. ** Denotes high statistical significance (P<0.01).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
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 (PPARγ, middle panel) and CCAAT enhancer-binding protein alpha (C/EBPα, lower panel) was analysed in immunoblots. Representative blots and staining results of ≥2 independent experiments are shown.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
Subacute metformin treatment induces an impairment in leptin secretion that can be prevented by inhibition of p44/p42 MAP kinase. On day 8 of the differentiation course cells were 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, right panel) after removal of metformin. (B) The MAP kinase inhibitor, PD98059 (PD, 50 μM), was added 1 h prior to metformin treatment for 24 h, and the medium was analysed for leptin concentrations 24 h later. A bar graph analysis with s.e.m. of ≥5 independent experiments is shown. * Denotes statistical significance (P<0.05).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
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.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05646
(J Klein and S Westphal contributed equally to this work)
We would like to thank M Schümann for expert help with the leptin radioimmunoassay.
Funding
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Kl 1131/2-1 and Kl 1131/2–2), German Diabetes Association, and Faculty grants of the University of Lübeck to J K.
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