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research letter

Diabetes, Obesity and Metabolism 15: 276–279, 2013.


© 2012 Blackwell Publishing Ltd

Metformin directly inhibits ghrelin secretion through


AMP-activated protein kinase in rat primary gastric cells

The antidiabetic drug Metformin causes weight loss in both diabetic and non-diabetic individuals. Metformin treatment is also
associated with lower circulating levels of the orexigenic hormone ghrelin. To test whether Metformin directly affects ghrelin cells,
research
letter

rat primary stomach cells were treated with Metformin and the levels of ghrelin secretion, proghrelin gene expression and activation
of adenosine monophosphate-activated protein kinase (AMPK) were examined. Metformin significantly reduced ghrelin secretion
and proghrelin mRNA production and both these effects were blocked by co-incubation with the AMPK inhibitor compound C.
Furthermore, the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) significantly inhibited ghrelin secretion.
Additionally, ghrelin cells were shown to express AMPK. Finally, Metformin treatment caused a significant increase in the level
of phosphorylated (active) AMPK. Our results show that Metformin directly inhibits stomach ghrelin production and secretion
through AMPK. This reduction in ghrelin secretion may be one of the key components in Metformin’s mechanism of weight
loss.
Keywords: antidiabetic drug, appetite control, biguanides, cellular pharmacology, drug mechanism, metformin

Date submitted 10 August 2012; date of first decision 11 September 2012; date of final acceptance 1 October 2012

Introduction Materials and Methods


The antidiabetic biguanide Metformin is one of the Animals
most prescribed, first-line medications in the treatment of All animal studies were approved by the Dalhousie University
type 2 diabetes mellitus. In contrast to some antidiabetics animal care committee and strictly adhere to the policies of the
(sulphonylureas and insulin), Metformin does not cause weight Canadian Council for Animal Care. Pregnant Sprague Dawley
gain and can lead to significant weight loss [1]. One of the rats were purchased from Charles River (Wilmington, MA,
known targets of Metformin action is the intracellular signalling USA).
enzyme, adenosine monophosphate-activated protein kinase
(AMPK). In the liver and muscle, AMPK activation reduces Primary Culture
hepatic gluconeogenesis and promotes fatty acid oxidation, Primary rat stomach culture was prepared as previously
respectively (see review in Ref [2]). In addition to these effects, described in detail [8]. Briefly, 8-day-old rat pup stomachs
Metformin treatment also appears to affect the levels of the were dispersed using type 1A collagenase and seeded in
orexigenic hormone, ghrelin. Ghrelin is produced primarily in 25 mM glucose, 10% foetal bovine serum (FBS) Dulbecco’s
the X/A-like endocrine cells of the stomach and acts to increase modified Eagle’s medium (DMEM) into six-well plates (1 × 106
appetite and promote energy storage [3]. Accordingly, ghrelin cells/well) and 10 cm plates (5 × 106 cells) for secretion
levels rise before a meal and drop postprandial [4]. Studies or RNA/protein experiments, respectively. All media and
in humans have shown that Metformin treatment prolongs compounds unless otherwise stated were purchased from Sigma
the postprandial or postglucose drop in circulating ghrelin Aldrich (Oakville, ON, Canada).
levels [5,6]. However, other studies found no difference in the
postprandial drop in ghrelin in Metformin-treated individuals Ghrelin Secretion
[7]. As ghrelin secretion is known to be altered by hormones
After 24 h in culture, cells were incubated in media
and nutrients including insulin [8] and glucose [9], it is difficult supplemented with 10 μM octanoic acid for an additional
to conclude from in vivo studies if Metformin has a direct role 24 h. Cells were rinsed with phosphate buffered saline and
on ghrelin secretion. In this study, we used primary culture of given fresh DMEM with 5 mM glucose and 0.5% FBS
rat gastric cells to show a direct effect of Metformin on ghrelin containing treatments for 4 h. Both media and cell lysates were
secretion. acidified and concentrated using reverse phase SepPak column
(C-18 SepPak cartridges, Waters, Milford, MA) according
Correspondence to: Dr Younes Anini, Dalhousie University, IWK Health Centre, 7th Floor
Women’s Site. Rm# 7043, P.O. Box 9700, Halifax NS B3K 6R8, Canada. to the manufacturer’s instructions. Acylated ghrelin levels
E-mail: Younes.anini@dal.ca were determined using active ghrelin enzyme immunoassay
DIABETES, OBESITY AND METABOLISM research letter
A B

cretion relative to control

control
1.5
1.5

pl13a relative to c
1.0 1.0
* **
percentage sec 0.5
** 0.5

proghrelin/rp
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0.0 0.0

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Figure 1. Metformin inhibits ghrelin secretion and mRNA expression. Acyl ghrelin secretion was determined after 4-h incubation of the rat gastric
primary culture with Metformin (0.1–100 mM) (A). Proghrelin/Rpl13a mRNA expression was examined by quantitative RT-PCR after 24-h incubation
of the rat gastric primary culture with Metformin (10 mM) (B). Results are expressed as mean ± s.e.m. (n = 6). *p < 0.05 and **p < 0.01 vs. control.

(Cayman Chemical, Ann Arbor, MI, USA) as per the secretion at 1 mM (0.632 ± 0.071 fold of control, p > 0.05),
manufacturer’s guidelines. Ghrelin secretion was normalized 10 mM (0.617 ± 0.060 fold of control, p < 0.01) and 100 mM
to total ghrelin content of each well [media/(media + lysate)] (0.365 ± 0.042 fold of control, p < 0.01) (figure 1A). To
to control for changes in the proportion of ghrelin cells in each examine if Metformin treatment could alter proghrelin gene
experiment. expression, quantitative RT-PCR was completed. Metformin
treatment (10 mM) caused a strong reduction in proghrelin
Proghrelin mRNA Expression gene expression (0.106 ± 0.019 fold of control, p < 0.001,
After 48 h in culture, cells were incubated in DMEM with 5 mM figure 1B).
glucose and 0.5% FBS containing treatments for 24 h. Relative As Metformin has been shown to activate AMPK, we tested if
proghrelin/RPL13a mRNA expression was determined using the AMPK agonists, AICAR could also reduce ghrelin secretion.
standard curve method with the following primers: proghrelin Cells treated with 0.5 mM of AICAR had a significant reduction
(forward: TGG CAT CAA GCT GTC AGG AGC and reverse: in ghrelin secretion (0.453 ± 0.063 fold of control, p < 0.01,
AGC TGG CGC CTC TTT GAC CT) and Rpl13a (forward: figure 2A). To further investigate the role of AMPK, cells
ATG GCG GAG GGG CAG GTT CT and reverse: CCA CCA were incubated with Metformin (10 mM) in the presence
CCT TTC GGC CCA GC). of the AMPK inhibitor, compound C (10 μM). Inhibition
of AMPK completely blocked the effect of Metformin on
AMPK phosphorylation ghrelin secretion (figure 2B). Similarly, the inhibitory effect of
Metformin on proghrelin mRNA expression was also blocked
After 48 h in culture, cells were stabilized in 0.5% FBS DMEM
by co-incubation with the AMPK inhibitor, Compound C
with 5 mM glucose for 2 h and then given 10 mM Metformin
(figure 2C). To confirm that ghrelin cells express the AMPK
in fresh media for 4 h. Lysates were separated in 8% acrylamide
protein, double immunofluorescence using antighrelin and
SDS Tris tricine gels and transferred to polyvinylidene
anti-AMPK antibodies was completed. As shown in figure 2D,
difluoride membranes. Primary blotting antibodies for
ghrelin cells co-expressed AMPK. To further show AMPK
phosphorylated Thr172-AMPK and AMPK were obtained from
activation, phosphorylated AMPK was examined after 4 h
Millipore (Billerica, MA, USA). Immunocytochemistry was
of Metformin treatment. Metformin (10 mM) significantly
completed on 4% paraformaldehyde-fixed cells on coverslips
increased levels of phosphorylated AMPK (2.20 ± 0.232 fold of
with the same AMPK antibody.
control, p > 0.01, figure 2E).
Statistics
Experiments were analysed using either Student’s t-test or Conclusions
one factor analysis of variance with Dunnett’s post hoc test to
untreated control (p values <0.05 were considered statistically Our findings indicate that Metformin inhibits stomach
significant). proghrelin mRNA production and ghrelin secretion, ghrelin
cells co-express AMPK and Metformin treatment leads to
increased AMPK phosphorylation. To our knowledge, our
Results study is the first to show the direct effect of Metformin on
To determine the direct effect of Metformin on stomach ghrelin ghrelin secretion and proghrelin mRNA expression. We found
secretion, cells were incubated with Metformin (0.1–100 mM) the 1–100 mM dose range to be effective at reducing ghrelin
for 4 h. Metformin caused a significant reduction in ghrelin secretion. The 10 mM dose used in subsequent experiments

Volume 15 No. 3 March 2013 doi:10.1111/dom.12021 277


research letter DIABETES, OBESITY AND METABOLISM

A B C

percentage secrretion relative to control


percentage secrretion relative to control

ontrol
1.5 1.5 1.5
5

proghrelin/rpl13a relative to co
1.0 1.0 1.0

*
** 05
0.5 05
0.5
05
0.5

***
0.0 0.0 0.0

et

C
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sitometry
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MPK relative dens


2

1
pAMPK/AM

0
ol

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tr
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C

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M
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m
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pAMPK

AMPK

Figure 2. Metformin acts through adenosine monophosphate-activated protein kinase (AMPK) to inhibit ghrelin secretion. Acyl ghrelin secretion
was determined after 4-h incubation with 0.5 mM of the AMPK agonist AICAR (A). Acyl ghrelin secretion was determined in cells treated with
10 mM Metformin alone or in the presence of 10 μM of the AMPK inhibitor, compound C (cpdC) (B). Quantitative RT-PCR was completed for
proghrelin/Rpl13a after 24 h treatment with media alone (control), Metformin (10 mM) and Metformin (10 mM) in the presence of cpdC (10 μM) (C).
Double immunofluorescence for ghrelin (green) and AMPK (red) was completed in rat primary culture (D). Western blotting of primary stomach culture
treated with Metformin (10 mM) for 4 h was completed with phospho-specific AMPK and total AMPK antibodies (representative blot of three repeats,
E). Results are expressed as mean ± s.e.m. (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.

provided a strong effect without causing any cell death during and secretion supports its direct action on ghrelin cells.
our experiments and has been used by other groups studying One of the possible pathways by which Metformin inhibits
the effects of Metformin in vitro [10]. This is also the first ghrelin secretion is through AKT phosphorylation, as AMPK
report linking AMPK activation and reduced ghrelin secretion. activation has been shown to increase AKT activity [11]. This is
Although it is possible that the AMPK phosphorylation in supported by our previous study showing that increases in AKT
Western blots was from non-ghrelin stomach cells in the phosphorylation by insulin led to reduced ghrelin secretion [8].
preparation, our findings that the AMPK agonist AICAR The effects on mRNA production may be occurring through
strongly reduced ghrelin secretion and that the AMPK inhibitor AMPK activation of the mammalian target of Rapamycin [2]
blocked Metformin’s effect on ghrelin mRNA production pathway, however this requires further investigation.

278 Gagnon et al. Volume 15 No. 3 March 2013


DIABETES, OBESITY AND METABOLISM research letter
Most interesting is the possible link between Metformin’s References
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foundation for innovation and the IWK Research Foundation. diabetic patients: similar levels observed after treatment with metformin,
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Conflict of Interest 10. Mueller WM, Stanhope KL, Gregoire F, Evans JL, Havel PJ. Effects of
J. G. and Y. A. designed the study and planned the experiments. metformin and vanadium on leptin secretion from cultured rat adipocytes.
J. G. performed all the experiments and collected the data Obes Res 2000; 8: 530–539.

and arranged the figures. E. S. helped with the western blot 11. Leclerc GM, Leclerc GJ, Fu G, Barredo JC. AMPK-induced activation of Akt
experiments and qRT-PCR experiments. J. G. and Y. A. by AICAR is mediated by IGF-1R dependent and independent mechanisms
in acute lymphoblastic leukemia. J Mol Signal 2010; 5: 15.
analyzed the data obtained. J. G. prepared the first draft of
the manuscript and prepared the response to the reviewers. 12. Makimattila S, Nikkila K, Yki-Jarvinen H. Causes of weight gain during
insulin therapy with and without metformin in patients with Type II
E. S. and Y. A. revised the manuscript. Y. A. approved and
diabetes mellitus. Diabetologia 1999; 42: 406–412.
submitted the manuscript for publication. The authors have
no other competing interest to declare.

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