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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
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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
A B C
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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.
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.