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: firstname.lastname@example.org)
(*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
Hawley SA, Gadalla AE, Olsen GS & Hardie DG 2002 The
antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes 51 2420–2425. References
Kirpichnikov D, McFarlane SI & Sowers JR 2002 Metformin: an
update. Annals of Internal Medicine 137 25–33.
Klaus S 2004 Adipose tissue as a regulator of energy balance. Current
Argaud D, Roth H, Wiernsperger N & Leverve XM 1993 Metformin
Drug Targets 5 241–250.
decreases gluconeogenesis by enhancing the pyruvate kinase flux in
Klein J, Fasshauer M, Ito M, Lowell BB, Benito M & Kahn CR 1999
isolated rat hepatocytes. European Journal of Biochemistry 213
Beta(3)-adrenergic stimulation diﬀerentially inhibits insulin
signalling and decreases insulin-induced glucose uptake in brown
Bailey CJ & Turner RC 1996 Metformin. New England Journal of
adipocytes. Journal of Biological Chemistry 274 34795–34802. Medicine 334 574–579.
Klein J, Fasshauer M, Benito M & Kahn CR 2000 Insulin and the
Bray GA & Greenway FL 1999 Current and potential drugs for
beta3-adrenoceptor diﬀerentially regulate uncoupling protein-1
treatment of obesity. Endocrine Reviews 20 805–875.
expression. Molecular Endocrinology 14 764–773.
Ciaraldi TP, Kong AP, Chu NV, Kim DD, Baxi S, Loviscach M,
Klein J, Fasshauer M, Klein HH, Benito M & Kahn CR 2002 Novel
Plodkowski R, Reitz R, Caulfield M, Mudaliar S & Henry RR
adipocyte lines from brown fat: a model system for the study of
2002 Regulation of glucose transport and insulin signalling by
diﬀerentiation, energy metabolism, and insulin action. Bioessays 24
troglitazone or metformin in adipose tissue of type 2 diabetic
subjects. Diabetes 51 30–36.
Kraus D, Fasshauer M, Ott V, Meier B, Jost M, Klein HH & Klein J
Danforth E Jr & Himms-Hagen JH 1997 Obesity and diabetes and the
2002 Leptin secretion and negative autocrine crosstalk with insulin
beta-3 adrenergic receptor. European Journal of Endocrinology 136
in brown adipocytes. Journal of Endocrinology 175 185–191.
Kumar N & Dey CS 2002 Metformin enhances insulin signalling in
DeFronzo RA & Goodman AM 1995 Eﬃcacy of metformin in
insulin-dependent and -independent pathways in insulin resistant
patients with non-insulin-dependent diabetes mellitus. The
muscle cells. British Journal of Pharmacology 137 329–336.
Multicenter Metformin Study Group. New England Journal of
Large V & Beylot M 1999 Modifications of citric acid cycle activity
Medicine 333 541–549.
and gluconeogenesis in streptozotocin-induced diabetes and eﬀects
DeFronzo RA, Barzilai N & Simonson DC 1991 Mechanism of
of metformin. Diabetes 48 1251–1257.
metformin action in obese and lean noninsulin-dependent diabetic
Lowell BB & Flier JS 1997 Brown adipose tissue, beta 3-adrenergic
subjects. Journal of Clinical Endocrinology and Metabolism 73 1294–1301.
receptors, and obesity. Annual Review of Medicine 48 307–316.
Dominguez LJ, Davidoﬀ AJ, Srinivas PR, Standley PR, Walsh MF &
Lowell BB & Bachman ES 2003 Beta-adrenergic receptors,
Sowers JR 1996 Eﬀects of metformin on tyrosine kinase activity,
diet-induced thermogenesis, and obesity. Journal of Biological
glucose transport, and intracellular calcium in rat vascular smooth
Chemistry 278 29385–29388.
muscle. Endocrinology 137 113–121.
Mannucci E, Ognibene A, Cremasco F, Bardini G, Mencucci A,
Fogelholm M, Valve R, Kukkonen-Harjula K, Nenonen A,
Pierazzuoli E, Ciani S, Messeri G & Rotella CM 2001 Eﬀect of
Hakkarainen V, Laakso M & Uusitupa M 1998 Additive eﬀects of
metformin on glucagon-like peptide 1 (GLP-1) and leptin levels in
the mutations in the beta3-adrenergic receptor and uncoupling
obese nondiabetic subjects. Diabetes Care 24 489–494.
protein-1 genes on weight loss and weight maintenance in Finnish
Matthaei S, Hamann A, Klein HH, Benecke H, Kreymann G, Flier JS
women. Journal of Clinical Endocrinology and Metabolism 83 4246–4250.
& Greten H 1991 Association of metformin’s eﬀect to increase
Freemark M & Bursey D 2001 The eﬀects of metformin on body
insulin-stimulated glucose transport with potentiation of
mass index and glucose tolerance in obese adolescents with fasting
insulin-induced translocation of glucose transporters from
hyperinsulinemia and a family history of type 2 diabetes. Pediatrics
intracellular pool to plasma membrane in rat adipocytes. Diabetes 40
Friedman JM 2002 The function of leptin in nutrition, weight, and
physiology. Nutrition Reviews 60 S1–S14
Matthaei S, Reibold JP, Hamann A, Benecke H, Haring HU, Greten
Friedman JM & Halaas JL 1998 Leptin and the regulation of body
H & Klein HH 1993 In vivo metformin treatment ameliorates
weight in mammals. Nature 395 763–770.
insulin resistance: evidence for potentiation of insulin-induced
Fruehwald-Schultes B, Oltmanns KM, Toschek B, Sopke S, Kern W,
translocation and increased functional activity of glucose transporters
Born J, Fehm HL & Peters A 2002 Short-term treatment with
in obese (fa/fa) Zucker rat adipocytes. Endocrinology 133 304–311.
metformin decreases serum leptin concentration without aﬀecting
Matthaei S, Stumvoll M, Kellerer M & Haring HU 2000
body weight and body fat content in normal-weight healthy men.
Pathophysiology and pharmacological treatment of insulin resistance. Metabolism 51 531–536. Endocrine Reviews 21 585–618.
Fumeron F, Durack-Bown I, Betoulle D, Cassard-Doulcier AM,
Meuillet EJ, Wiernsperger N, Mania-Farnell B, Hubert P & Cremel
Tuzet S, Bouillaud F, Melchior JC, Ricquier D & Apfelbaum M
G 1999 Metformin modulates insulin receptor signalling in normal
1996 Polymorphisms of uncoupling protein (UCP) and beta 3
and cholesterol-treated human hepatoma cells (HepG2). European
adrenoreceptor genes in obese people submitted to a low calorie
Journal of Pharmacology 377 241–252.
diet. International Journal of Obesity and Related Metabolic Disorders 20
Mueller WM, Stanhope KL, Gregoire F, Evans JL & Havel PJ 2000
Eﬀects of metformin and vanadium on leptin secretion from
Glueck CJ, Fontaine RN, Wang P, Subbiah MT, Weber K, Illig E,
cultured rat adipocytes. Obesity Research 8 530–539.
Streicher P, Sieve-Smith L, Tracy TM, Lang JE & McCullough P
Oberkofler H, Dallinger G, Liu YM, Hell E, Krempler F & Patsch W
2001 Metformin reduces weight, centripetal obesity, insulin, leptin,
1997 Uncoupling protein gene: quantification of expression levels in
and low-density lipoprotein cholesterol in nondiabetic, morbidly
adipose tissues of obese and non-obese humans. Journal of Lipid
obese subjects with body mass index greater than 30. Metabolism 50 Research 38 2125–2133.
Pedersen O, Nielsen O, Bak J, Richelsen B, Beck-Nielsen H &
Guler S, Cakir B, Demirbas B, Gursoy G, Serter R & Aral Y 2000
Sorensen N 1989 The eﬀects of metformin on adipocyte insulin
Leptin concentrations are related to glycaemic control, but do not
action and metabolic control in obese subjects with type 2 diabetes.
change with short-term oral antidiabetic therapy in female patients
Diabetic Medicine 6 249–256.
with type 2 diabetes mellitus. Diabetes, Obesity and Metabolism 2
Porras A, Alvarez AM, Valladares A & Benito M 1998 p42/p44
mitogen-activated protein kinases activation is required for the
Journal of Endocrinology (2004) 183, 299–307 Direct effects of metformin on brown adipocytes · J KLEIN, S WESTPHAL and others 307
insulin-like growth factor-I/insulin induced proliferation, but
Tiraby C, Tavernier G, Lefort C, Larrouy D, Bouillaud F, Ricquier D
inhibits diﬀerentiation, in rat fetal brown adipocytes. Molecular
& Langin D 2003 Acquirement of brown fat cell features by human
Endocrinology 12 825–834.
white adipocytes. Journal of Biological Chemistry 278 33370–33376.
Radziuk J, Zhang Z, Wiernsperger N & Pye S 1997 Eﬀects of
Uehara MH, Kohlmann NE, Zanella MT & Ferreira SR 2001
metformin on lactate uptake and gluconeogenesis in the perfused rat
Metabolic and haemodynamic eﬀects of metformin in patients with
liver. Diabetes 46 1406–1413.
type 2 diabetes mellitus and hypertension. Diabetes, Obesity and
Rajala MW & Scherer PE 2003 Minireview: The adipocyte - at the
Metabolism 3 319–325.
crossroads of energy homeostasis, inflammation, and atherosclerosis.
Valve R, Heikkinen S, Rissanen A, Laakso M & Uusitupa M 1998
Endocrinology 144 3765–3773.
Synergistic eﬀect of polymorphisms in uncoupling protein 1 and
Rouru J, Koulu M, Peltonen J, Santti E, Hanninen V, Pesonen U &
beta3-adrenergic receptor genes on basal metabolic rate in obese
Huupponen R 1995 Eﬀects of metformin treatment on glucose
Finns [see comments]. Diabetologia 41 357–361.
transporter proteins in subcellular fractions of skeletal muscle in
Wiernsperger NF & Bailey CJ 1999 The antihyperglycaemic eﬀect of
(fa/fa) Zucker rats. British Journal of Pharmacology 115 1182–1187.
metformin: therapeutic and cellular mechanisms. Drugs 58 (Suppl 1)
Sarabia V, Lam L, Burdett E, Leiter LA & Klip A 1992 Glucose
transport in human skeletal muscle cells in culture. Stimulation by
Yang X, Enerback S & Smith U 2003 Reduced expression of
insulin and metformin. Journal of Clinical Investigation 90 1386–1395.
FOXC2 and brown adipogenic genes in human subjects with
Sivitz WI, Wayson SM, Bayless ML, Larson LF, Sinkey C, Bar RS &
insulin resistance. Obesity Research 11 1182–1191.
Haynes WG 2003 Leptin and body fat in type 2 diabetes and
Yki-Jarvinen H, Nikkila K & Makimattila S 1999 Metformin prevents
monodrug therapy. Journal of Clinical Endocrinology and Metabolism 88
weight gain by reducing dietary intake during insulin therapy in
patients with type 2 diabetes mellitus. Drugs 58 (Suppl 1) 53–54;
Spiegelman BM & Flier JS 2001 Obesity and the regulation of energy
balance. Cell 104 531–543.
Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M,
Stith BJ, Goalstone ML, Espinoza R, Mossel C, Roberts D &
Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear
Wiernsperger N 1996 The antidiabetic drug metformin elevates
LJ & Moller DE 2001 Role of AMP-activated protein kinase in
receptor tyrosine kinase activity and inositol 1,4,5-trisphosphate
mechanism of metformin action. Journal of Clinical Investigation 108
mass in Xenopus oocytes. Endocrinology 137 2990–2999.
Stith BJ, Woronoﬀ K & Wiernsperger N 1998 Stimulation of the
intracellular portion of the human insulin receptor by the
antidiabetic drug metformin. Biochemical Pharmacology 55 533–536.
Tiraby C & Langin D 2003 Conversion from white to brown
adipocytes: a strategy for the control of fat mass? Trends in
Made available online as an Accepted Preprint
Endocrinology and Metabolism 14 439–441. Journal of Endocrinology (2004) 183, 299–307
Drugs on Tap: What’s In Our Tap Water? Here’s a question to ponder. What happens to the hundreds of millions of prescription drugs and the over-the-counter medications that are swallowed daily? The answer: they go out through the plumbing. Being flushed down the toilet and into the sewage system, 90 per cent of every drug swallowed is either excreted, totally unchanged, or is