DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL

Mechanisms Underlying Metformin-Induced Secretion

of Glucagon-Like Peptide-1 from the Intestinal L Cell

Andrew J. Mulherin, Amy H. Oh, Helena Kim, Anthony Grieco, Lina M. Lauffer,
and Patricia L. Brubaker
Departments of Physiology (A.J.M., A.H.O., H.K., A.G., L.M.L., P.L.B.) and Medicine (P.L.B.), University of
Toronto, Toronto, Ontario, Canada M5S 1A8

Glucagon-like peptide-17-36NH2 (GLP-1) is secreted by the intestinal L cell in response to both nu-
trient and neural stimulation, resulting in enhanced glucose-dependent insulin secretion. GLP-1 is
therefore an attractive therapeutic for the treatment of type 2 diabetes. The antidiabetic drug,
metformin, is known to increase circulating GLP-1 levels, although its mechanism of action is
unknown. Direct effects of metformin (5–2000 ␮M) or another AMP kinase activator, aminoimi-
dazole carboxamide ribonucleotide (100 –1000 ␮M) on GLP-1 secretion were assessed in murine
human NCI-H716, and rat FRIC L cells. Neither agent stimulated GLP-1 secretion in any model,
despite increasing AMP kinase phosphorylation (P ⬍ 0.05– 0.01). Treatment of rats with metformin
(300 mg/kg, per os) or aminoimidazole carboxamide ribonucleotide (250 mg/kg, sc) increased
plasma total GLP-1 over 2 h, reaching 37 ⫾ 9 and 29 ⫾ 9 pg/ml (P ⬍ 0.001), respectively, compared
with basal (7 ⫾ 1 pg/ml). Plasma activity of the GLP-1-degrading enzyme, dipeptidylpeptidase-IV,
was not affected by metformin treatment. Pretreatment with the nonspecific muscarinic antag-
onist, atropine (1 mg/kg, iv), decreased metformin-induced GLP-1 secretion by 55 ⫾ 11% (P ⬍ 0.05).
Pretreatment with the muscarinic (M) 3 receptor antagonist, 1-1-dimethyl-4-diphenylacetoxypip-
eridinium iodide (500 ␮g/kg, iv), also decreased the GLP-1 area under curve, by 48 ⫾ 8% (P ⬍ 0.05),
whereas the antagonists pirenzepine (M1) and gallamine (M2) had no effect. Furthermore, chronic
bilateral subdiaphragmatic vagotomy decreased basal secretion compared with sham-operated
animals (7 ⫾ 1 vs. 13 ⫾ 1 pg/ml, P ⬍ 0.001) but did not alter the GLP-1 response to metformin. In
contrast, pretreatment with the gastrin-releasing peptide antagonist, RC-3095 (100 ␮g/kg, sc),
reduced the GLP-1 response to metformin, by 55 ⫾ 6% (P ⬍ 0.01) at 30 min. These studies elucidate
the mechanism underlying metformin-induced GLP-1 secretion and highlight the benefits of using
metformin with dipeptidylpeptidase-IV inhibitors in patients with type 2 diabetes. (Endocrinology
152: 4610 – 4619, 2011)

lucagon-like peptide-17-36NH2 (GLP-1) is an incretin apeutic for patients with type 2 diabetes mellitus (T2DM).
G hormone secreted by the distal intestinal L cell. GLP-1 However, GLP-1 is rapidly degraded upon secretion by the
exerts numerous beneficial effects in vivo, including enhance- enzyme, dipeptidylpeptidase-IV (DPP-IV), resulting in a
ment of glucose-dependent insulin secretion and inhibition short half-life of less than 2 min (11). Hence, both long-acting
of glucagon secretion, gastric emptying, and food intake (1– GLP-1 receptor agonists and DPP-IV inhibitors have been
7). Moreover, chronic treatment of rodents with GLP-1 has developed for clinical use, resulting in reductions in glycated
also been shown to stimulate ␤-cell proliferation and de- hemoglobin levels of up to 0.9% (2, 12).
crease apoptosis (8 –10). As a consequence of these biological With the introduction of DPP-IV inhibition into the
actions, GLP-1 has gained considerable attention as a ther- clinic, interest in the factors that regulate the release of

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: 4-DAMP, 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide; AICAR,
Printed in U.S.A. aminoimidazole carboxamide ribonucleotide; AMPK, AMP kinase; AUC, area under curve;
Copyright © 2011 by The Endocrine Society DPP-IV, dipeptidylpeptidase-IV; FBS, fetal bovine serum; FRIC, fetal rat intestinal cell; GIP,
doi: 10.1210/en.2011-1485 Received July 11, 2011. Accepted September 9, 2011. glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-17-36NH2; GRP,
First Published Online October 4, 2011 gastrin-releasing peptide; HBSS, Hanks’ balanced salt solution; IBMX, 3-isobutyl-1-meth-
ylxanthine; M, muscarinic; T2DM, type 2 diabetes mellitus.

4610 endo.endojournals.org Endocrinology, December 2011, 152(12):4610 – 4619

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Endocrinology, December 2011, 152(12):4610 – 4619 endo.endojournals.org 4611

endogenous GLP-1 has been heightened (13–15). GLP-1 DPP-IV-deficient rats markedly enhances plasma bioac-
secretion occurs primarily in response to nutrient inges- tive GLP-1 levels. Consistent with these findings, Migoya
tion (16), through a complex array of direct and indirect et al. (44) recently reported that a 2-d pretreatment of
mechanisms. Postprandial GLP-1 levels peak within healthy subjects with metformin enhances levels of total
15–30 min of nutrient consumption (17, 18). However, (active plus inactive forms) but not active GLP-1 after a
this response cannot be explained by direct nutrient stim- meal, whereas coadministration of metformin and sita-
ulation of the L cell, because the majority of the L cells in gliptin increases both active and total levels. Moreover,
the human and rodent intestine are localized in the distal metformin has no effect on levels of GIP, which is also
gut (19), and nutrients do not normally transit to this inactivated by DPP-IV, in humans or mice (36, 44), further
region within this timeframe (20). Consistent with these suggesting that metformin does not increase GLP-1 levels
observations, we have demonstrated the presence of an through effects on DPP-IV. Although these studies provide
indirect loop that mediates this early phase of secretion, a compelling argument for a role of metformin as a GLP-1
whereby nutrients entering the proximal gut stimulate the secretagogue, the mechanism by which metformin en-
release of GLP-1 from L cells in the distal gut. In rodents, hances GLP-1 release remains unknown. The purpose of
this pathway is mediated through activation of the vagus the current study was therefore to determine whether met-
nerve (21) by the other incretin hormone, glucose-depen- formin exerts direct effects on the intestinal L cell as a
dent insulinotropic peptide (GIP) (21, 22). This indirect GLP-1 secretagogue and, if not, to assess the indirect
pathway is further mediated by the enteric neuropeptide, mechanism(s) underlying the acute effects of metformin to
gastrin-releasing peptide (GRP) (23, 24), and the musca- increase plasma levels of GLP-1 in vivo.
rinic (M) 1 receptor (25). Similarly, administration of
either a cholecystokinin receptor antagonist or of the gen-
eral muscarinic antagonist, atropine, reduces nutrient-in- Materials and Methods
duced GLP-1 secretion in healthy subjects, indicating the
relevance of this proximal-distal loop in humans (26, 27). In vitro studies
Finally, passage of nutrients and, particularly, fats to the Cell cultures
distal ileum enhances GLP-1 release through direct inter- Fetal rat intestinal cell (FRIC) cultures were prepared as pre-
actions with the intestinal L cell (28 –31). viously described (46 – 48). In brief, intestinal cells from 19- to
The biguanide, metformin, has been used as a treatment 21-d fetal Wistar rats were enzymatically dispersed, plated at a
for T2DM for over 50 yr and, combined with lifestyle and density of 0.6 intestines per 60-mm dish in culture medium
diet change, has become the first-line therapy for the dis- [DMEM; 5% fetal bovine serum (FBS), 4.5 g/liter glucose, 40
U/ml penicillin, and 40 ␮g/ml streptomycin], and incubated over-
ease (32). Metformin reduces hyperglycemia through de- night at 37 C and 5% CO2 with constant humidity. Animal
creased hepatic glucose output and enhanced glucose up- procedures were approved by the Animal Care Committee of the
take by skeletal muscle, at least in part through an AMP University of Toronto.
kinase (AMPK)-dependent mechanism (33–35). How- Murine GLUTag and human NCI-H716 cells were propa-
ever, metformin also exerts AMPK-independent effects, gated, as previously described (49 –51), in culture media
[DMEM (GLUTag) or RPMI (NCI-H716; American Type Cul-
including sensitization of the ␤-cell to GLP-1 and GIP
ture Collection, Manassas, VA), with 5% FBS, and 4.5 g/liter
through peroxisome proliferator-activated receptor ␣ glucose] on 10-cm dishes. Two days before experimental use,
(36). Excitingly, in addition to acting as an incretin sen- cells were seeded in 24- or six-well plates coated with poly-D
sitizer, metformin has also been shown to enhance plasma lysine (GLUTag) or Matrigel (NCI-H716) for secretion or West-
levels of GLP-1. Although Molloy et al. (37) first demon- ern blot studies, respectively.
strated a link between metformin therapy and enhanced
plasma glucagon-like immunoreactivity in normal indi- Secretion experiments
On the day of the experiment, cells were washed twice with
viduals, Mannucci et al. (38, 39) reported that metformin
Hanks’ balanced salt solution (HBSS) and incubated for 2 h
increases plasma concentrations of active GLP-1 in obese, with DMEM containing 0.5% FBS, 1 g/liter glucose, and 10
nondiabetic, as well as in obese, diabetic subjects. It was ␮U/ml insulin (FRIC), DMEM plus 0.5% FBS (GLUTag), or
initially proposed that inhibition of DPP-IV could account RPMI plus 0.5% FBS (NCI-H716) alone (negative control), or
for the increase in circulating levels of the active peptide media plus forskolin (adenylyl cyclase activator) and 3-isobu-
(38, 40 – 42), although these findings are controversial tyl-1-methylxanthine (IBMX) (phosphodiesterase inhibitor; 1
or 10 ␮M each; positive control; Sigma Chemical Co., St. Louis,
(36, 43, 44). Furthermore, an in vitro study by Hinke et al.
MO), metformin (5–2000 ␮M; Sigma Chemical Co.), or amino-
(45) revealed that metformin does not directly inhibit imidazole carboxamide ribonucleotide (AICAR) (AMPK acti-
DPP-IV activity, and Yasuda et al. (43) demonstrated that vator; 100-1000 ␮M; Toronto Research Chemicals, Toronto,
acute administration of metformin or other biguanides to Ontario, Canada). For metformin-sensitization experiments,

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4612 Mulherin et al. Metformin and GLP-1 Secretion
GLUTag cells were washed with HBSS, preincubated with or
without metformin (2000 ␮M) in DMEM plus 0.5% FBS for 1 h,
washed with HBSS, and incubated for an additional 2 h with
forskolin and IBMX (10 ␮M each), bethanechol (muscarinic ag-
onist, 500-1000 ␮M; Sigma Chemical Co.), bethanechol (500 –
1000 ␮M) plus metformin (2000 ␮M), or metformin alone (2000
␮M). The timing of the metformin pretreatment was selected
based on previous studies demonstrating that metformin in-
creases plasma GLP-1 levels in rats within 1 h of administration
(43). After the incubation period for all experiments, cells were
checked by microscopy to ensure there were no observable mor-
phological differences between treatment groups. Media and cell
peptides were purified by reversed-phase adsorption (C18 Sep-
Pak; Waters, Milford, MA), as previously described (25, 52), and
stored at ⫺20 C until RIA for total GLP-1 using an anti-GLP-
1(x-36NH2) antiserum (Enzo Life Sciences, Plymouth Meeting,
PA). Percent GLP-1 secretion was calculated as the amount of
GLP-1 in the media normalized to the total GLP-1 in the dish or
well (i.e. medium plus cell content). The total content of GLP-1
in each dish or well was not affected by any of the treatments
(data not shown).
Western blot analysis
GLUTag and NCI-H716 cells were washed with HBSS and
incubated for 1 h at 37 C in DMEM plus 0.5% FBS (GLUTag)
or RPMI plus 0.5% FBS (NCI-H716) alone (negative control), or
supplemented with metformin (2000 ␮M) or AICAR (1000 ␮M),
followed by Western blot analysis for AMPK phosphorylation.
FRIC cultures were not analyzed by immunoblot, because the
heterogeneous nature of the cells precludes identification of L
cell-specific responses. Cells were then washed in HBSS, col-
lected into cold RIPA buffer containing EDTA-free protease in-
hibitors (Roche Diagnostics, Mannheim, Germany), sonicated,
and stored at ⫺20 C until immunoblot. Total protein content
was determined by Bradford assay (Bio-Rad Laboratories, Her-
cules, CA). Equal amounts of protein were separated on 7.5%
SDS-PAGE, transferred onto a polyvinyldifluoride membrane
(Bio-Rad Laboratories), and incubated overnight with a rabbit
antiphosphorylated-AMPK antiserum (1:1000; Cell Signaling,
Beverly, MA), followed by visualization using a horseradish
peroxidase-linked goat-antirabbit secondary antibody (1:
2000; Cell Signaling) and an electrochemical luminescence
detection system (Amersham Pharmacia Biotech, Baie D’Urfe,
Quebec, Canada). Membranes were then stripped and re-
probed using a rabbit anti-AMPK antiserum (1:1000; Cell
Signaling) and a rabbit antiactin antiserum (1:1000; Sigma
Chemical Co.). Membranes were imaged using a Kodak Image
Station 4000MM PRO system (Carestream Molecular Imag-
ing, New Haven, CT).
In vivo studies
Animals
Male Wistar rats (250 –350 g; Charles River Laboratories, St.
Constant, Quebec, Canada) were housed with a standard 12-h
light, 12-h dark cycle for at least 2 wk before the experiment and
were handled daily for 3–5 d before the experiment to reduce
stress. Animals were fed regular chow and fasted overnight (16
h) before each experiment. All animal procedures were approved
by the Animal Care Committee of the University of Toronto.
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Endocrinology, December 2011, 152(12):4610 – 4619
Secretion experiments
Conscious rats were treated with metformin (300 mg/kg, per os),
AICAR (250 mg/kg, sc), or an equal volume of vehicle (sterile saline,
per os and sc). For the inhibitor studies, rats were administered
atropine sulfate (general muscarinic antagonist; 1 mg/kg, iv; Si-
gma Chemical Co.), pirenzepine (M1 antagonist; 0.5 mg/kg, iv;
Sigma Chemical Co.), gallamine (M2 antagonist; 0.5 mg/kg, iv;
Sigma Chemical Co.), 1,1-Dimethyl-4-diphenylacetoxypiperi-
dinium iodide (4-DAMP) (M3 antagonist; 0.5 mg/kg, iv; Tocris
Bioscience, Ellisville, MO), RC-3095 (GRP receptor antagonist; 0.1
mg/kg, sc; Sigma Chemical Co.), or an equal volume of vehicle
(sterile saline, iv or sc, as appropriate), 30 min before treatment
with metformin or vehicle, as previously. Blood samples (200 ␮l)
were collected from the lateral saphenous vein into heparinized
glass capillary tubes and immediately placed on ice. An antico-
agulant and protease inhibitor solution [5000 KIU/ml aprotinin
(Sigma Chemical Co.), 1 mM diprotin A (DPP-IV inhibitor;
Sigma Chemical Co.), and 4.1 mM EDTA] was added to all sam-
ples, to a final concentration of 10% (vol/vol), except for those
used for the DPP-IV activity assay, in which diprotin A was
excluded. As a positive control for successful inhibition with
atropine, pirenzepine or 4-DAMP, salivary secretion was col-
lected for 1 min from the oral cavity, using a Q-tip, before and
after administration of the drug.
Plasma total and active GLP-1 were assayed using a sandwich
immunoassay kit from Meso Scale Discovery (Gaithersburg,
MD). Changes in the levels of active GLP-1 were found to closely
reflect those of total GLP-1 in response to metformin treatment
(data not shown). However, because active GLP-1 levels were
not detectable unless the animals were also pretreated with sita-
gliptin (DPP-IV inhibitor; 5 mg/kg, iv; Merck Canada, Kirkland,
Quebec, Canada), all further analyses were conducted using the
total GLP-1 kit.
Plasma DPP-IV activity was measured using a colorimetric
assay (Sigma Chemical Co.). One unit is defined as the produc-
tion of 1.0 ␮mole of 4-nitroaniline from Gly-Pro p-nitroanilide
per minute.
Bilateral subdiaphragmatic vagotomy
After a laparoscopic incision in anesthetized rats, the stomach
was retracted caudally to expose the esophagus and the anterior
and posterior branches of the vagus nerve. A small section (0.5
cm) of each branch was resected to vagotomize animals, whereas
the nerves were visualized but not cut in sham-operated animals.
The muscular and cutaneous incisions were closed with absorb-
able and silk suture, respectively, and animals were allowed to
recover for at least 1 wk before being subjected to secretion
experiments, as previously. Body weight was monitored daily; no
abnormal (i.e. ⬎20%) decreases in weight were observed.
Data Analysis
All data are expressed as the mean ⫾ SEM. In vitro GLP-1
secretion is expressed as a percentage of control secretion.
AMPK phosphorylation is expressed as the fold of control. In
vivo GLP-1 concentrations are expressed either as absolute val-
ues or as the change (i.e. ␦) from basal levels. ␦ Area under curve
(AUC) analysis was calculated using the trapezoidal rule. In some
experiments, data were converted to log10 to normalize variance
for statistical analysis. Statistical analyses were performed using
Statistical Analysis System software (SAS Institute, Cary, NC).
Endocrinology, December 2011, 152(12):4610 – 4619 endo.endojournals.org 4613

FIG. 1. Metformin increases plasma concentrations of total GLP-1 but not DPP-IV activity in rats. A, Rats were treated with metformin (300
mg/kg, per os; diamonds), AICAR (250 mg/kg, sc; squares), or vehicle (per os and sc; circles) at time ⫽ 0 min, and total GLP-1
concentrations in plasma were determined over 120 min by sandwich immunoassay (n ⫽ 6). *, P ⬍ 0.05; ***, P ⬍ 0.001 vs. vehicle-treated
rats. B, Rats were treated with metformin (diamonds and white bars) or vehicle (black bars), and plasma DPP-IV activity was measured by
colorimetric assay for 120 min (n ⫽ 5– 6).

Differences between experimental groups were determined by well as with forskolin plus IBMX as a cell function control.
Student’s t test or one- or two-way ANOVA, as appropriate, Neither metformin nor AICAR stimulated GLP-1 secre-
followed by n-1 post hoc comparisons, as appropriate. Signifi-
tion, compared with vehicle, in any of the three L cell
cance was assumed at P ⬍ 0.05.
models (Fig. 2, A–C). In contrast, treatment with forskolin
and IBMX increased GLP-1 secretion, to 4.4 ⫾ 0.7-, 4.2 ⫾
1.7-, and 2.5 ⫾ 0.3-fold of control, in FRIC, GLUTag, and
Results
NCI-H716 cells, respectively (P ⬍ 0.001). Furthermore,
Metformin increases plasma GLP-1 levels in vivo incubation of the cells with very high concentrations of
To confirm the actions of metformin in vivo, rats were metformin (2000 ␮M) or AICAR (1000 ␮M) increased
treated with metformin, AICAR, or vehicle, and plasma AMPK phosphorylation in GLUTag cells (to 1.7 ⫾ 0.2-
levels of total GLP-1 were determined over 2 h (Fig. 1A). fold, P ⬍ 0.01 and 1.2 ⫾ 0.2-fold, P ⬍ 0.05 of control,
Orally administered metformin significantly increased to- respectively), as well as in NCI-H716 cells (to 2.1 ⫾ 0.3-
tal GLP-1 concentrations at 60 and 120 min (to 212 ⫾ fold, P ⬍ 0.01 and 1.5 ⫾ 0.2-fold, P ⬍ 0.05 of control,
73%, P ⬍ 0.05; and 771 ⫾ 168%, P ⬍ 0.001, of vehicle respectively).
controls, respectively). Subcutaneous administration of To determine whether metformin increases L cell sensiti-
the AMPK agonist, AICAR, similarly increased total zation to a known L cell secretagogue, bethanechol (25, 55),
GLP-1 levels at 60 and 120 min (to 519 ⫾ 75%, P ⬍ 0.001; GLUTag cells were pretreated with or without metformin,
and 354 ⫾ 96%, P ⬍ 0.05, of vehicle controls, respec- followed by treatment with secretagogues in the absence or
tively). Because metformin treatment did not affect plasma continued presence of metformin (Fig. 2D). As previously,
DPP-IV activity (Fig. 1B), these findings suggested that the forskolin plus IBMX induced a robust increase in GLP-1
effect of metformin on plasma GLP-1 concentrations secretion, compared with control cells preincubated with
in vivo is mediated through increased peptide secretion vehicle, and this response was not affected by pre- or
rather than via decreased degradation. coincubation with metformin. Bethanechol treatment
(1000 ␮M) also increased GLP-1 secretion, to 2.6 ⫾
Metformin does not increase GLP-1 secretion 0.4-fold of control (P ⬍ 0.001) in cells preincubated with
in vitro vehicle. However, again, pre- or coincubation with met-
To establish whether metformin acts directly on the formin did not alter the L cell response to bethanechol. Fur-
intestinal L cell to stimulate GLP-1 secretion, rat FRIC, thermore, pre- or cotreatment with metformin did not alter
murine GLUTag, and human NCI-H716 cultures were GLP-1 secretion in the presence of a suboptimal concentra-
incubated with concentrations of metformin that ranged tion of bethanechol (500 ␮M). Collectively, therefore, the
from clinically relevant (5–15 ␮M) (53, 54) to supraphar- results of the in vitro studies indicated that metformin does
macologic (45–2000 ␮M). Cells were also incubated with not stimulate GLP-1 secretion through direct effects on the
AICAR as a positive control for AMPK stimulation, as intestinal L cell.

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4614 Mulherin et al. Metformin and GLP-1 Secretion Endocrinology, December 2011, 152(12):4610 – 4619

FIG. 2. Metformin does not increase GLP-1 secretion in L cell cultures. A–C, FRIC, murine GLUTag, and human NCI-H716 L cell cultures were
incubated for 2 h with vehicle (C, Negative control), forskolin and IBMX (FI) (positive control; 1 or 10 ␮M, as indicated), metformin (5, 15, 45, 150,
and 2000 ␮M), and AICAR (AMPK positive control; 100 and 1000 ␮M). Total (t) GLP-1 was measured by RIA and phosphorylated phosphorylated
(p) AMPK by immunoblot. A, FRIC cultures (n ⫽ 8 –12); B, GLUTag cells (n ⫽ 8 –23 for secretion, n ⫽ 8 for Western blot analysis); and C, NCI-
H716 cells (n ⫽ 6 –25 for secretion, n ⫽ 8 for Western blot analysis). Basal (control) GLP-1 secretion was 9.5 ⫾ 1.3% (FRIC), 4.2 ⫾ 0.6%
(GLUTag), and 3.6 ⫾ 0.6% (NCI-H716) of total cell content. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 compared with control. D, GLUTag cells
were preincubated for 1 h with vehicle (black bars) or metformin (2000 ␮M; white bars), followed by a 2-h incubation with metformin (Met) (2000
␮M), forskolin and IBMX (10 ␮M each), or bethanechol (Beth) (500 and 1000 ␮M) with or without metformin (2000 ␮M), or vehicle (control). Basal
(control) secretion was 6.6 ⫾ 1.1 and 7.0 ⫾ 1.2% of total cell content in vehicle- and metformin-pretreated cells, respectively. Total GLP-1 was
measured by RIA (n ⫽ 6 –10). *, P ⬍ 0.05; ***, P ⬍ 0.001 vs. respective controls.

Metformin-induced GLP-1 secretion in vivo is M3 induced GLP-1 secretion at 30, 60, and 90 min (by 81 ⫾
muscarinic receptor but not vagus dependent 11%, P ⬍ 0.01; 68 ⫾ 18%, P ⬍ 0.05; and 42 ⫾ 14%,
To elucidate whether the stimulatory effects of metformin P ⬍ 0.05, respectively). The ␦ AUC was thus decreased
on GLP-1 secretion in vivo involved the parasympathetic by 55 ⫾ 11% (P ⬍ 0.05) in these animals compared with
nervous system, a known regulator of the intestinal L cell (21, those treated with vehicle plus metformin alone (Fig.
25, 26, 55), rats were pretreated with the general muscarinic 3E). Pretreatment with pirenzepine or gallamine had no
receptor antagonist, atropine, or with antagonists for each of effect on either GLP-1 secretion or the ␦ AUC (Fig, 3, B,
the three muscarinic receptors previously demonstrated to be C, and E). In contrast, pretreatment with 4-DAMP re-
expressed by the rat L cell; pirenzepine, gallamine, and sulted in a significant reduction in GLP-1 secretion at 30
4-DAMP, for the M1, M2, and M3 muscarinic receptors, and 60 min (to 60 ⫾ 12%, P ⬍ 0.05; and 62 ⫾ 3%, P ⬍
respectively (25, 55). None of the antagonists alone 0.05, respectively), and the ␦ AUC was correspondingly
caused changes in circulating levels of GLP-1 (Fig. 3). In reduced, by 48 ⫾ 8%, (P ⬍ 0.05) (Fig. 3, D and E).
contrast, atropine significantly decreased metformin- Successful blockade of the muscarinic receptors was

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Endocrinology, December 2011, 152(12):4610 – 4619 endo.endojournals.org 4615

To investigate the involvement of the

vagus nerve in mediating metformin-in-
duced GLP-1 secretion, rats were sub-
jected to a bilateral subdiaphragmatic
vagotomy. Body weights decreased by
the same extent in both groups of ani-
mals for up to 1 wk after surgery, and
there was no difference between vagot-
omized and sham-operated animals on
the day of the experiment (427 ⫾ 9 and
428 ⫾ 11 g, respectively). Vagotomized
animals demonstrated a significant de-
crease in basal levels of total GLP-1 (by
38 ⫾ 6%, P ⬍ 0.05), consistent with the
known role of the vagus in stimulating
GLP-1 secretion (21). However, GLP-1
release in response to metformin treat-
ment was not different between vagot-
omized rats and sham-operated ani-
mals (Fig. 3F). The results of these
studies therefore implicate nonvagal
M3 muscarinic pathways in the regu-
lation of metformin-induced GLP-1
secretion.

Metformin-induced GLP-1
secretion is partially dependent
upon the GRP receptor
Finally, a role for the enteric neuro-
peptide, GRP, was evaluated, because
GRP has been demonstrated to be a po-
tent GLP-1 secretagogue in vitro and in
vivo (23, 50, 56, 57). Pretreatment with
the GRP receptor antagonist, RC-
3095, produced a brief, but significant,
FIG. 3. Metformin-induced GLP-1 secretion in rats requires M3 muscarinic receptors but not the decrease in metformin-induced GLP-1
vagus nerve. A–D, Total (t) GLP-1 plasma levels were determined in rats pretreated at time ⫽ ⫺30 secretion (to 52 ⫾ 6%, P ⬍ 0.05, com-
min with the muscarinic antagonists atropine (A; 1000 ␮g/kg, iv), pirenzepine (B; 500 ␮g/kg, iv),
gallamine (C; 500 ␮g/kg, iv), 4-DAMP (D; 500 ␮g/kg, iv), or vehicle (iv), before treatment at t ⫽ 0
pared with vehicle plus metformin) at
min with metformin (300 mg/kg, per os) or vehicle (per os). Diamonds, Vehicle plus metformin; 30 min, resulting in a 66 ⫾ 15% (P ⬍
circles, inhibitor plus vehicle; squares, inhibitor plus metformin. Animals treated with vehicle plus 0.05) reduction in the ␦ AUC at this
metformin (diamonds) were randomized throughout the experiments and are shown in black
in A and are replicated in B–D in gray to facilitate comparison. Basal GLP-1 secretion for time point (Fig. 4).
metformin-treated animals was 6.2 ⫾ 0.8 pg/ml and was not changed by antagonist treatment.
Data are expressed as ␦ total GLP-1. (E) ␦ AUC analysis for muscarinic inhibition curves shown in
A–D. Veh, Vehicle; Atr, atropine; Pir, pirenzepine; Gal, gallamine; 4-D, 4-DAMP; black bars,
vehicle; white bars, metformin. n ⫽ 6 –9; *, P ⬍ 0.05; **, P ⬍ 0.01 compared with vehicle. F, Discussion
Total GLP-1 plasma levels in vagotomized or sham-operated rats treated with metformin. Animals
underwent a bilateral subdiaphragmatic vagotomy and, after a 1-wk recovery, were treated with Administration of metformin increases
metformin (300 mg/kg, per os). Data are expressed as ␦ total GLP-1, and basal total GLP-1 is
shown in the inset. Circles and black bars, Sham operation plus metformin; diamonds and white
plasma concentrations of GLP-1 in nor-
bars, vagotomy plus metformin. n ⫽ 8 –10; *, P ⬍ 0.05 compared with sham. mal, diabetic, and DPP-IV-deficient ro-
dents (36, 42– 44, 58), as well as in hu-
verified by significant reductions in salivary secretion mans with and without T2DM (38, 39,
after pretreatment of rats with atropine, pirenzepine, 41, 44). These findings have led to the suggestion that
and 4-DAMP (data not shown). metformin acts as a GLP-1 secretagogue. However, the

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4616 Mulherin et al. Metformin and GLP-1 Secretion Endocrinology, December 2011, 152(12):4610 – 4619

in vitro only at concentrations that are 10,000 times

greater than those achieved by in vivo treatment. These
findings therefore suggest that although activation of
AMPK in the L cell can enhance GLP-1 secretion in vitro
under some conditions, this pathway is unlikely to have
any relevance in the physiologic or clinical setting.
Metformin has well-established actions as an insulin
and leptin sensitizer, in addition to its more recent char-
acterization as an incretin sensitizer in the ␤-cell (36, 59,
60). Therefore, we also investigated the effects of met-
formin as an L cell sensitizer in vitro, using the same time-
frame (i.e. 1 h) for pretreatment as was found to be re-
FIG. 4. ␦ Total GLP-1 plasma levels in rats pretreated with the GRP
receptor antagonist, RC-3095 (100 ␮g/kg, sc), or vehicle (sc), 30 min quired for metformin to enhance plasma GLP-1
before treatment with metformin (300 mg/kg, per os) or vehicle (per concentrations in vivo. However, preincubation of
os). A second injection of the inhibitor, or vehicle, was given 30 min GLUTag cells with metformin did not enhance GLP-1 re-
after oral gavage. Basal secretion for the vehicle plus metformin group
was 1.1 ⫾ 0.4 pg/ml and was not significantly different from basal lease in response to the general muscarinic agonist and
secretion in animals pretreated with antagonist. Diamonds, Vehicle known L cell secretagogue, bethanechol (25, 55), nor did
plus metformin; squares, inhibitor plus metformin; circles, inhibitor it enhance the effects of subthreshold concentrations of
plus vehicle. n ⫽ 6; *, P ⬍ 0.05 compared with vehicle plus
metformin.
bethanechol. Furthermore, metformin pretreatment also
had no effect on the response to the cAMP-dependent
pathway of GLP-1 secretion. Collectively, therefore, our
exact mechanism by which these effects are exerted has in vitro studies suggest that metformin does not act di-
remained unexplored. The results of the present study in- rectly on the L cell to induce GLP-1 secretion or enhance
dicate that metformin stimulates GLP-1 release through a L cell sensitivity to several known secretagogues.
mechanism that involves both muscarinic (M3) and GRP Previous studies have shown that metformin inhibits
receptor-dependent pathways but that is independent of DPP-IV activity in vivo, in humans and rodents, leading to
both DPP-IV and direct effects on the intestinal L cell. the suggestion that metformin enhances active GLP-1 con-
Three in vitro models of the intestinal L cell were em- centrations through a DPP-IV-dependent mechanism (38,
ployed to determine whether metformin directly stimu- 40 – 42). However, our data indicated that metformin in-
lates GLP-1 secretion, at concentrations ranging from clin- creases plasma total GLP-1, which is a better indicator of
ically relevant to suprapharmacologic (53, 54). The FRIC GLP-1 secretion than levels of active GLP-1, because it is
cultures are a heterogeneous population of primary FRIC independent of changes in DPP-IV activity. Of note, we
(13). In contrast, the murine GLUTag cells are a homo- also observed that metformin increases circulating levels
geneous immortalized L cell line derived from a mouse of active GLP-1, although only in rats that were pretreated
expressing a proglucagon promoter, simian virus 40 large with sitagliptin to inhibit DPP-IV activity before met-
T-antigen transgene (14), whereas the human NCI-H716 formin administration, further suggesting that the actions
cells originated from a spontaneous human cecal adeno- of metformin are DPP-IV independent. Importantly, these
carcinoma (15). These models, representing both primary findings are consistent with the results of Yasuda et al.
and immortal L cells, as well as three different species, (43), who demonstrated that metformin increases plasma
have been demonstrated to secrete GLP-1 in a regulated active GLP-1 levels in DPP-IV-deficient rats. Although we
manner in response to a wide variety of known nutritional, cannot dismiss the possibility that metformin affects the
hormonal, and neural secretagogues (16 –18). However, activity of the anchored form of DPP-IV, localized on the
treatment of these L cell models with either metformin or surface of epithelial tissues, including the capillaries in
AICAR had no direct effect on GLP-1 secretion, although close proximity to the L cell (61), our findings with total
very high concentrations of both agents increased AMPK GLP-1 argue against any actions of metformin to enhance
phosphorylation in the GLUTag and NCI-H716 cells. GLP-1 levels through effects on DPP-IV.
Notwithstanding, a recent study has suggested a role for To determine which indirect pathways might mediate
AMPK in mediating berberine-induced GLP-1 secretion in metformin-induced GLP-1 secretion, we examined several
the NCI-H716 cells (19). Berberine is a plant-derived iso- mediators previously reported to regulate the indirect ef-
quinoline alkaloid that has been demonstrated to have fects of nutrients on the intestinal L cell, specifically the
hypoglycemic effects in association with enhanced GLP-1 muscarinic-, vagal-, and GRP-dependent pathways. Inter-
release in vivo but that is effective as an L cell secretagogue estingly, our data demonstrated that both atropine and the

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Endocrinology, December 2011, 152(12):4610 – 4619 endo.endojournals.org 4617

M3 receptor antagonist, 4-DAMP, but not the M1 and present study (500 vs. 300 mg/kg). Furthermore, the total
M2 receptor antagonists, reduced metformin-induced content of GLP-1 in our in vitro studies was not altered by
GLP-1 secretion in vivo, albeit only a single dose each of 2–3 h of incubation with metformin, suggesting that trans-
metformin and the inhibitors was tested. However, the lation to active peptide does not occur within this time-
comparable inhibition profiles for atropine and 4-DAMP frame, at least in our models. Finally, as recently reported
suggest that the M3 receptor may be the primary musca- by others for mice (36), we did find that acute adminis-
rinic receptor responsible for mediating the effect of met- tration of AICAR to rats increased plasma levels of GLP-1.
formin on GLP-1. Anini et al. (25) previously reported These findings suggest a role for AMPK in mediating met-
expression of the muscarinic receptor subtypes M1-M3 by formin-induced effects on GLP-1 release. How these find-
the rat intestinal L cell in vivo. However, the results of that ings with AICAR relate to the roles of the M3 and GRP
study demonstrated a role for only the M1 receptor in receptors in the actions of metformin remains to be
nutrient-induced stimulation of GLP-1 secretion. Further- determined.
more, the M3 receptor was shown to be inactive in both Although the present study has elucidated the mech-
the rat and human L cell in vitro (25, 55). Notably, despite anism of action of metformin to increase GLP-1 secre-
this role for the M3 receptor, the effects of metformin were tion in the fasted rat under acute conditions, it is rec-
found to be independent of the vagus nerve, although ognized that this does not truly reflect the clinical
basal GLP-1 levels were reduced in vagotomized animals, setting, in which patients are normally exposed to met-
consistent with known role for the vagus in regulating formin for prolonged periods of time with regular food
GLP-1 secretion (21). Hence, these findings suggest that intake. Notwithstanding these differences, chronic
the M3-dependent effects of metformin are mediated in- treatment of humans with metformin enhances both basal
directly, rather than directly through a receptor that is and nutrient-induced GLP-1 secretion (38, 39, 44), al-
expressed by the L cell. Additionally, as the enteric neu- though acute administration has not been demonstrated to
ropeptide, GRP, has been demonstrated to play a role in increase circulating levels of GLP-1 (39 – 41). These dis-
nutrient-induced GLP-1 secretion (23, 24), this pathway crepancies are most likely due to differences in the dose of
was also evaluated as a potential mediator of metformin- metformin used in human vs. rodent studies. Future stud-
induced GLP-1 secretion. Administration of the GRP re- ies should therefore investigate the mechanisms involved
ceptor antagonist, RC-3095, reduced the effects of met- in the enhancement of GLP-1 secretion that are induced by
formin briefly but significantly. RC-3095 is known to chronic metformin treatment.
have a very short half-life in vivo (62), which may account Collectively, the results of the present study indicate
for the transient nature of the effects. Finally, although that a dose of 300 mg/kg metformin enhances circulating
atropine sulfate is known to cross the blood-brain barrier levels of GLP-1 in rats through a peripheral M3 and GRP
(63), neither 4-DAMP nor RC-3095 do so (64, 65). To- receptor-dependent mechanism. These findings reinforce
gether, these findings suggest that at least part of the ac- clinical reports of improved glycemic control in subjects
tions of metformin on GLP-1 secretion must be exerted treated with metformin plus DPP-IV inhibitors, which ap-
through a peripheral M3 receptor and GRPergic pathway. pear to be consequent to the additive effects of increased
It is recognized that the metformin-induced increases in GLP-1 release and decreased GLP-1 degradation (44, 67).
GLP-1 may have occurred through decreased renal clear- Further elucidation of the mechanisms underlying met-
ance of the peptide. However, because the rat kidney does formin-induced GLP-1 secretion will not only provide a
not appear to express M3 muscarinic receptors (64), this more comprehensive understanding of the benefits of co-
possibility seems unlikely given the ability of 4-DAMP to administration of metformin and incretin therapy but also
block the actions of metformin on GLP-1. Similarly, given a more thorough understanding of the pathways regulat-
that bile acids are potent GLP-1 secretagogues (30), it has ing GLP-1 secretion.
recently been suggested that metformin increases GLP-1
secretion by inhibiting carrier-mediated uptake of bile ac-
ids in the intestine (66). However, as the present study was Acknowledgments
conducted in fasted rats, the effects of metformin in these
animals was presumably independent of bile acids. One We thank Dr. Kate Banks, Rainer de Guzman, Frank Giuliano,
Sara Johnson, and Tracy McCook for assistance with develop-
final possibility is that metformin may increase mRNA
ment of the animal techniques, as well as Angelo Izzo for labo-
levels for the GLP-1 prohormone, proglucagon, as re-
ratory training.
cently reported by others for murine large intestine, 1 h
after administration of metformin (44). However, the dose Address all correspondence and requests for reprints to:
of metformin used in that study was higher than in the Patricia Brubaker, Ph.D., Room 3366 Medical Sciences Build-

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4618 Mulherin et al. Metformin and GLP-1 Secretion
ing, University of Toronto, 1 King’s College Circle, Toronto,
Ontario, Canada M5S 1A8. E-mail: p.brubaker@utoronto.ca.
Present address for L.M.L.: Medizinische Poliklinik Campus
Innerstadt, Klinikum der LMU, Pettenkoferstr. 8A, 80336 Mu-
nich, Germany.
This work was supported by operating grants from the Ca-
nadian Diabetes Association (#2674) and the Merck Invetigator-
initiated Study Program. A.M was supported by graduate schol-
arships from the Banting and Best Diabetes Centre (BBDC),
University of Toronto, Toronto; A.O. and A.G. by Summer
Student’ships from the BBDC; L.M.L. by postdoctoral fellowships
from the BBDC, and the European Foundation for the Study of
Diabetes (EFSD; Albert Renold and EFSD/Lilly Research Fellow-
ships); and P.L.B. by the Canada Research Chairs program.
Disclosure Summary: A.J.M., A.H.O., H.K., A.G., and
L.M.L. have nothing to disclose. P.L.B. has received previous
consulting fees from Merck.
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