Glucagon-Like Peptide-1 Receptor Activation Modulates Pancreatitis-Associated Gene Expression But Does Not Modify the Susceptibility to Experimental Pancreatitis in Mice

Article (PDF Available)inDiabetes 58(9):2148-61 · July 2009with17 Reads
DOI: 10.2337/db09-0626 · Source: PubMed
Abstract
Clinical reports link use of the glucagon-like peptide-1 receptor (GLP-1R) agonists exenatide and liraglutide to pancreatitis. However, whether these agents act on the exocrine pancreas is poorly understood. We assessed whether the antidiabetic agents exendin (Ex)-4, liraglutide, the dipeptidyl peptidase-4 inhibitor sitagliptin, or the biguanide metformin were associated with changes in expression of genes associated with the development of experimental pancreatitis. The effects of Ex-4 when administered before or after the initiation of caerulein-induced experimental pancreatitis were determined. The importance of endogenous GLP-1R signaling for gene expression in the exocrine pancreas and the severity of pancreatitis was assessed in Glp1r(-/-) mice. Acute administration of Ex-4 increased expression of egr-1 and c-fos in the exocrine pancreas. Administration of Ex-4 or liraglutide for 1 week increased pancreas weight and induced expression of mRNA transcripts encoding the anti-inflammatory proteins pancreatitis-associated protein (PAP) (RegIIIbeta) and RegIIIalpha. Chronic Ex-4 treatment of high-fat-fed mice increased expression of PAP and reduced pancreatic expression of mRNA transcripts encoding for the proinflammatory monocyte chemotactic protein-1, tumor necrosis factor-alpha, and signal transducer and activator of transcription-3. Sitagliptin and metformin did not significantly change pancreatic gene expression profiles. Ex-4 administered before or after caerulein did not modify the severity of experimental pancreatitis, and levels of pancreatic edema and serum amylase were comparable in caerulein-treated Glp1r(-/-) versus Glp1r(+/+) mice. These findings demonstrate that GLP-1 receptor activation increases pancreatic mass and selectively modulates the expression of genes associated with pancreatitis. However, activation or genetic elimination of GLP-1R signaling does not modify the severity of experimental pancreatitis in mice.
Glucagon-Like Peptide-1 Receptor Activation Modulates
Pancreatitis-Associated Gene Expression But Does Not
Modify the Susceptibility to Experimental Pancreatitis
in Mice
Jacqueline A. Koehler, Laurie L. Baggio, Benjamin J. Lamont, Safina Ali, and Daniel J. Drucker
OBJECTIVE—Clinical reports link use of the glucagon-like
peptide-1 receptor (GLP-1R) agonists exenatide and liraglutide to
pancreatitis. However, whether these agents act on the exocrine
pancreas is poorly understood.
RESEARCH DESIGN AND METHODS—We assessed whether
the antidiabetic agents exendin (Ex)-4, liraglutide, the dipeptidyl
peptidase-4 inhibitor sitagliptin, or the biguanide metformin were
associated with changes in expression of genes associated with
the development of experimental pancreatitis. The effects of Ex-4
when administered before or after the initiation of caerulein-
induced experimental pancreatitis were determined. The impor-
tance of endogenous GLP-1R signaling for gene expression in the
exocrine pancreas and the severity of pancreatitis was assessed
in Glp1r
/
mice.
RESULTS—Acute administration of Ex-4 increased expres-
sion of egr-1 and c-fos in the exocrine pancreas. Administra-
tion of Ex-4 or liraglutide for 1 week increased pancreas
weight and induced expression of mRNA transcripts encoding
the anti-inflammatory proteins pancreatitis-associated protein
(PAP) (RegIII) and RegIII. Chronic Ex-4 treatment of high-
fat–fed mice increased expression of PAP and reduced pan-
creatic expression of mRNA transcripts encoding for the
proinflammatory monocyte chemotactic protein-1, tumor ne-
crosis factor-, and signal transducer and activator of tran-
scription-3. Sitagliptin and metformin did not significantly
change pancreatic gene expression profiles. Ex-4 administered
before or after caerulein did not modify the severity of
experimental pancreatitis, and levels of pancreatic edema and
serum amylase were comparable in caerulein-treated
Glp1r
/
versus Glp1r
/
mice.
CONCLUSIONS—These findings demonstrate that GLP-1 re-
ceptor activation increases pancreatic mass and selectively mod-
ulates the expression of genes associated with pancreatitis.
However, activation or genetic elimination of GLP-1R signaling
does not modify the severity of experimental pancreatitis in
mice. Diabetes 58:2148–2161, 2009
G
lucagon-like peptide (GLP)-1, a peptide hor-
mone secreted by enteroendocrine cells in the
distal small bowel and colon, exerts a diverse
set of complementary actions on islet -cells,
resulting in glucose-dependent augmentation of insulin
biosynthesis and secretion (1). GLP-1 also restores glu-
cose sensitivity to diabetic -cells and promotes expan-
sion of -cell mass via stimulation of -cell proliferation
and enhancement of -cell survival (2). Moreover, exoge-
nous GLP-1 administration inhibits glucagon secretion and
gastric emptying and induces satiety, leading to weight
loss after prolonged GLP-1 receptor (GLP-1R) activation
(3). Taken together, these actions of GLP-1 lead to signif-
icant improvement in glucose homeostasis and have fos-
tered the development of GLP-1R agonists, exemplified by
synthetic exendin (Ex)-4, for the treatment of type 2
diabetes (3,4).
The majority of studies examining GLP-1 biology in the
pancreas has focused on - and -cells within the endo-
crine pancreas; however, GLP-1 exerts a number of ac-
tions in the exocrine pancreas. GLP-1R agonists induce
transdifferentiation of pancreatic exocrine cells to an
endocrine cell phenotype in vitro (5), and GLP-1 inhibits
hypoglycemia-induced pancreatic bicarbonate and protein
secretion in the isolated perfused pig pancreas (6). More-
over, exogenous GLP-1 induces neural transmission con-
verging on the pancreas via depolarization of neurons
within the dorsal motor nucleus of the vagus that project
to the exocrine pancreas (7).
Although the biology of GLP-1 action in the exocrine
pancreas remains poorly understood, the clinical use of
the first approved GLP-1R agonist, exenatide, has been
associated with case reports of pancreatitis, and some
have speculated this may be related to the venomous
origin of the Ex-4 peptide from the lizard (8). Although
there is limited scientific information linking GLP-1 recep-
tor activation to the pathogenesis of pancreatic inflamma-
tion, pancreatitis has also been reported in clinical trials of
the human GLP-1R agonist liraglutide (9). In contrast,
analysis of a health care claims database did not reveal an
increased incidence of pancreatitis in hospitalized patients
previously treated with exenatide versus other antidia-
betic agents (10).
As GLP-1 inhibits pancreatic exocrine secretion (6), a
putative mechanism associated with the development of
pancreatitis (11), it seems possible that sustained GLP-1
receptor activation will increase the susceptibility for
development of pancreatic inflammation. Ex-4 regulates
the pancreatic expression of the Reg gene family (12), and
From the Department of Medicine, Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.
Corresponding author: Daniel J. Drucker, d.drucker@utoronto.ca.
Received 28 April 2009 and accepted 22 May 2009.
Published ahead of print at http://diabetes.diabetesjournals.org on 9 June
2009. DOI: 10.2337/db09-0626.
© 2009 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
2148 DIABETES, VOL. 58, SEPTEMBER 2009
changes in expression of RegIII (also known as pancre-
atitis-associated protein [PAP]) have been associated with
divergent effects on pancreatitis susceptibility and pancre-
atic necrosis in vivo (13,14).
Accordingly, we examined whether GLP-1 receptor ac-
tivation modulates expression of genes known to be
associated with the development of inflammation or acute
pancreatitis in mice. We employed two structurally dis-
tinct GLP-1R agonists, Ex-4 and liraglutide, as well as a
dipeptidyl peptidase (DPP)-4 inhibitor, sitagliptin, and the
biguanide metformin to assess pancreatitis-associated
gene expression in mice. The effect of Ex-4 administra-
tion before or after the administration of low-dose
caerulein, a chemical cholecystokinin (CCK) mimic that
produces secreatogue-induced pancreatitis (15), was
assessed in wild-type mice, and changes in pancreatic
gene expression and the susceptibility to pancreatitis
were analyzed in Glp1r
/
mice.
Experimental procedures. Experiments were carried
out according to protocols approved by the animal care
committees of the University Health Network and Mount
Sinai Hospital. Mice were housed under a 12-h light/dark
cycle with access to standard or high-fat rodent diet (45%
kcal from fat; Research Diets, New Brunswick, NJ). Male
C57Bl/6J mice (8 –10 weeks old) were obtained from
Taconic Laboratories (Hudson, NY) and were allowed to
acclimatize to the animal facility for 7 days before each
experiment. Glp1r
/
mice in the C57Bl/6J background
were generated as described (16).
Reagents. Ex-4 was from Chi Scientific (Maynard, MA)
and dissolved in PBS. Forskolin and caerulein were from
Sigma Chemical (St. Louis, MO). Caerulein was dissolved
in saline (0.9% NaCl) and forskolin in DMSO. Liraglutide
was from Novo Nordisk (Bagsværd, Denmark). Sitagliptin
was from Merck (Rahway, NJ).
Induction of experimental pancreatitis. Pancreatitis
was induced in 9- to 11-week-old male C57Bl/6J mice or
3-month-old male Glp1r
/
and littermate control
Glp1r
/
mice using the CCK receptor agonist caerulein
(17). Caerulein dose-response experiments were per-
formed in male 9- to 11-week-old C57Bl/6J mice to identify
a submaximal dose of caerulein that would produce a low
level of pancreatic inflammation. Secretagogue-induced
pancreatitis was elicited by administration of five sequen-
tial hourly intraperitoneal injections of caerulein, at doses
of 6, 12, 24, or 50 g/kg body wt as described (11,17,18);
control animals received an equal volume of saline. Mice
were killed by CO
2
inhalation 1 h after the final caerulein
injection.
Pretreatment with Ex-4. To assess whether prior
GLP-1R activation exacerbates the subsequent develop-
ment of pancreatitis, male C57Bl/6J mice were injected
(intraperitoneally) twice daily with either 10 nmol/kg Ex-4
or PBS for 7 days. Sixteen hours after the last Ex-4
injection, mice were administered 5 hourly intraperitoneal
injections of caerulein at 3 or 6 g/kg body wt, as
indicated, and killed by CO
2
inhalation 1 h after the final
caerulein injection.
Posttreatment with Ex-4. To assess whether a low level
of pancreatitis was worsened by subsequent GLP-1R acti-
vation, male C57Bl/6J mice were administered 5 hourly
intraperitoneal injections of caerulein (6 g/kg) followed
by twice daily intraperitoneal injections with either 10
nmol/kg Ex-4 or PBS for up to 6 days. Mice were killed 1,
24, 72, or 144 h (day 0, 1, 3, and 6, respectively) after the
final caerulein injection such that mice killed on day 1
received two injections of PBS or Ex-4, one in the evening
(3 h after the last caerulein injection) and one in the
morning (6 h before being killed). Similarly, the final
injection with Ex-4 or PBS was 6 h before being killed for
A
egr-1
PBS Ex-4
PBS
Ex-4
relative mRNA levels
c-fos
PBS Ex-4
0
5
10
15
20
25
30
35
PBS
Ex-4
***
relative mRNA levels
PBS
Ex-4
200x 400x
B
Egr-1
i
i
i
i
C
PBS
Ex-4
200x 400x
c-Fos
i
i
i
0
10
20
30
40
50
60
70
80
***
FIG. 1. Acute induction of egr-1 and c-fos transcripts by Ex-4. Male
C57Bl/6J mice were subcutaneously injected with a single dose of Ex-4
(1 g) or vehicle alone (PBS). A: Total RNA was isolated from pancreas
45 min after injections and reverse transcribed, and the levels of the
indicated transcripts were determined by real-time PCR, normalized to
18S rRNA content, and shown relative to the control (PBS)-treated
group. Results are expressed as means SE. ***P < 0.001 PBS- versus
Ex-4 –treated mice, n 4 in each group. Immunohistochemical local-
ization of egr-1 (B) and c-fos (C) in the pancreas of mice treated for 45
min with vehicle (PBS) or Ex-4. Photomicrographs are representative
of four mice per group. Islets are represented with an i. Magnification
200 or 400. (A high-quality digital representation of this figure is
available in the online issue.)
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2149
Pancreas weight (% BW)
PBS Ex 0.1 Ex 1 Ex 10
0.9
1.0
1.1
1.2
1.3
1.4
*
Pancreas weight
(% of body weight)
PAP (Reg IIIβ)
PBS Ex 0.1 Ex 1 Ex 10
0
5
10
15
20
25
30
***
relative mRNA levels
egr-1
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
2.5
relative mRNA levels
MCP-1
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
2.5
relative mRNA levels
Anti-inflammatory:
Pro-inflammatory:
Transcription factors:
mist1 (Bh1hb8)
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
AT F3
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
c-fos
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
2.5
relative mRNA levels
c-myc
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
ICAM-1
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
A
B
SOCS3
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
realtive mRNA levels
STAT3
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
2.5
relative mRNA levels
junB
PBS Ex 0.1 Ex 1 Ex 10
0.0
1.0
2.0
3.0
realtive mRNA levels
ndrg1
PBS Ex 0.1 Ex 1 Ex 10
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
Reg IIIα
PBS Ex 0.1 Ex 1 Ex 10
0
5
10
15
20
25
**
relative mRNA levels
ifitm3
PBS Ex 0.1 Ex 1 Ex 10
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
relative mRNA levels
absolute pancreas weight
PBS Ex 0.1 Ex 1 Ex 10
0.20
0.25
0.30
0.35
*
pancreas weight (g)
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2150 DIABETES, VOL. 58, SEPTEMBER 2009
all remaining mice (day 3 and 6). To determine whether
loss of GLP-1R signaling protected mice from development
of experimental pancreatic inflammation, pancreatitis was
induced in 3-month-old male Glp1r
/
and Glp1r
/
mice
by administration of 5 hourly intraperitoneal injections of
caerulein (6 g/kg). Mice were killed by CO
2
inhalation 1 h
after the final caerulein injection.
Acute peptide administration and high-fat feeding
studies. To assess whether GLP-1 receptor activation
regulates gene expression in the exocrine pancreas of
C
D
pancreas weight (% BW)
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
0.8
0.9
1.0
1.1
1.2
1.3
1.4
PBS
Lira
*
pancreas weight
(% of body weight)
SOCS3
0.00
0.25
0.50
0.75
1.00
1.25
#
relative mRNA levels
STAT3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
**
##
realtive mRNA levels
egr-1
0.0
0.5
1.0
1.5
2.0
2.5
realtive mRNA levels
junB
0.0
0.2
0.4
0.6
0.8
1.0
1.2
realtive mRNA levels
c-myc
0.0
0.4
0.8
1.2
1.6
relaitve mRNA levels
c-fos
0.0
0.2
0.4
0.6
0.8
1.0
1.2
relaitve mRNA levels
ATF3
0.0
0.5
1.0
1.5
2.0
2.5
realtive mRNA levels
PAP
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
PBS
Lira
*
relative mRNA levels
Anti-inflammatory:
Transcription factors:
RegIIIα
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*
relative mRNA levels
absolute pancreas weight
WT Glp-1r
-/-
0.15
0.20
0.25
0.30
0.35
PBS
Lira
*
pancreas weight (g)
FIG. 2. GLP-1R agonists increase pancreatic weight and regulate gene expression in a GLP-1R– dependent manner. A: Pancreas weight of mice
treated twice daily with the indicated doses of Ex-4 for 1 week is shown as a percentage of the final body weight (left panel) or absolute pancreas
weight (right panel). Results are expressed as means SE. *P < 0.05 PBS- versus Ex-4 –treated mice, n 5 in each group. B: Expression profiles
of genes associated with pancreatitis in the pancreas of mice treated twice daily for 1 week with the indicated doses of Ex-4 (0.1–10 nmol/kg).
Total RNA was isolated from pancreas samples and reverse transcribed, and the levels of the indicated transcripts were determined by real-time
quantitative PCR, normalized to levels of 18S or cyclophillin mRNA transcripts (for SOCS3, STAT3, junB, ndrg1, and ifitm3), and shown relative
to the control (PBS)-treated group. Results are expressed as means SE. **P < 0.01, ***P < 0.001 PBS- versus Ex-4 –treated mice, n 5 in each
group. Pancreas weight shown as a percentage of the final body weight (left panel) or absolute pancreas weight (right panel)(C), and expression
profiles of genes associated with pancreatitis (D) in the pancreas of wild-type Glp1r
/
and Glp1r
/
mice treated twice daily for 1 week with 75
g/kg liraglutide. Levels of mRNA transcripts were determined by real-time quantitative PCR, normalized to the 18S rRNA content, and shown
relative to control (PBS)-treated wild-type mice. Results are expressed as means SE. *P < 0.05, **P < 0.01 PBS- versus liraglutide-treated mice,
#P < 0.05, ##P < 0.01 wild-type versus Glp1r
/
mice, n 3 in each group.
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2151
normal mice, male C57Bl/6J mice were injected subcuta-
neously with a single dose of Ex-4 (1 g), Ex-4 15%
glucose (G X), or PBS, and mice were killed 30, 45, 60,
or 90 min later by CO
2
inhalation. To determine whether
high-fat feeding and induction of insulin resistance (19)
modifies the effects of Ex-4 on the exocrine pancreas,
4-week-old wild-type male mice were fed either standard
rodent diet or high-fat diet (45% kcal from fat) for 8 weeks;
during the last 4 weeks mice on high-fat diet were given
twice daily intraperitoneal injections of PBS or Ex-4 (24
nmol/kg). Separately, to assess the impact of diabetes on
the GLP-1R– dependent regulation of exocrine pancreatic
gene expression, 13-week-old male C57BL/6 mice were
maintained on a high-fat diet for 8 weeks, given one dose
of streptozotocin (STZ, 100 mg/kg), and after 4 more
weeks were allocated to four treatment groups for an
additional 8 weeks of 1) high-fat diet and twice daily
intraperitoneal injections of PBS, 2) high-fat diet and
metformin (500 mg kg
1
day
1
) provided in the drinking
water, 3) high-fat diet and twice daily intraperitoneal
injections of Ex-4 (3 nmol/kg), or 4) high-fat diet and
sitagliptin (370 mg kg
1
day
1
) provided in the diet.
Preparation of serum and tissue samples. Animals
were killed by CO
2
inhalation 1, 24, 72, or 144 h after the
final caerulein injection. Blood samples were collected by
cardiac puncture, placed on ice for 30 min, and centrifuged
at 4°C for 10 min at 14,000 rpm, and serum was stored at
80°C. The pancreas was removed, weighed, placed on
ice, and cut into sections for RNA, protein, histology, and
edema analyses. For RNA isolation, a section of pancreas
was homogenized in TRIzol reagent (Invitrogen Life Tech-
nologies, San Diego, CA), frozen on dry ice, and stored at
80°C until further analysis. Tissue samples for histology
were fixed in 10% neutral-buffered formalin for 24 h and
embedded in paraffin. Remaining tissue samples were snap
frozen in liquid nitrogen and stored at 80°C until further
analysis.
Evaluation of the severity of pancreatitis. Quantifica-
tion of serum amylase was performed using the Phad-
ebas amylase test (Magle Life Sciences, Cambridge, MA)
using 10 l serum (20). Pancreatic edema (21) was
quantified by measuring tissue water content after des-
iccation to a constant weight at room temperature for
72 h (dry weight). The results were calculated and
pancreas weight (% BW)
PBS Ex-4
0.8
0.9
1.0
1.1
1.2
1.3
1.4
0
3 µg/kg
6 µg/kg
##
#
pancrease weight
(% of body weight)
serum amylase
PBS Ex-4
0
5000
10000
15000
20000
25000
30000
0
3 µg/kg
6 µg/kg
*
*
amylase (U/L)
normalized for total
protein
serum amylase
P0 P3 P6 E0 E3 E6
0
10000
20000
30000
40000
amylase (U/L)
normalized for total
protein
Edema
PBS Ex-4
60
65
70
75
80
0
3 µg/kg caerulein
6 µg/kg caerulein
% water content
(wet-dry/wet*100)
C
A
B
D
1234 657
9 am
6 pm
8 sac
6 pm
Ex-4 twice daily, 1 week 5 hourly
injections
caerulein
Day
absolute pancreas weight
PBS Ex-4
0.15
0.20
0.25
0.30
0.35
#
pancreas weight (g)
FIG. 3. Effect of prior exposure to Ex-4 on the severity of caerulein-induced acute pancreatitis. Mice were treated twice daily with PBS or 10
nmol/kg Ex-4 for 1 week followed by 5 hourly injections with the indicated concentrations of caerulein. A: Schematic representation of
experimental design. B: Pancreas weight shown as a percentage of the final body weight (left panel) or absolute pancreas weight (right panel),
and pancreatic water content (edema) (C) and serum amylase (D) were assessed in mice 6 h after the initial caerulein injection. Shown are
means SE, (C and D) means SE (left panel), and individual serum amylase levels (right panel). #P < 0.05, ##P < 0.01 PBS versus Ex-4
treatment, *P < 0.05 saline versus caerulein treatment, n 4 in each group. E: Expression profiles of pancreatitis-associated genes in the
pancreas of mice treated with PBS or 10 nmol/kg Ex-4 for 1 week before caerulein administration as indicated above. Levels of the indicated
transcripts are plotted relative to levels from pancreata of control (PBS 1 week saline treatment) group as determined by real-time
quantitative PCR normalized to the 18S rRNA content or cyclophillin mRNA levels (for STAT3, SOCS3, ndrg1, and ifitm3). Results are expressed
as means SE. #P < 0.05 PBS versus Ex-4 treatment, *P < 0.05, **P < 0.01, ***P < 0.001, saline versus caerulein treatment, n 4 mice in each
group.
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2152 DIABETES, VOL. 58, SEPTEMBER 2009
PAP
PBS Ex-4
0
10
20
30
40
50
0 (saline)
3
µ
g/kg caerulein
6 µg/kg caerulein
*
#
relative mRNA levels
MCP-1
PBS Ex-4
0
20
40
60
80
100
120
140
***
***
relative mRNA levels
IL-6
PBS Ex-4
0
50
100
150
200
250
**
relative mRNA levels
TNF
α
PBS Ex-4
0
5
10
15
20
25
**
**
relative mRNA levels
egr-1
PBS Ex-4
0
250
500
750
1000
1250
*
***
***
relative mRNA levels
AT F 3
PBS Ex-4
0
100
200
300
400
**
***
relative mRNA levels
mist1
PBS Ex-4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
***
***
***
***
relative mRNA levels
c-fos
PBS Ex-4
0
25
50
75
**
**
200
300
400
relative mRNA levels
c-myc
PBS Ex-4
0.0
1.0
2.0
3.0
4.0
*
*
relative mRNA levels
Anti-inflammatory:
Pro-inflammatory:
E
Transcription factors:
STAT3
PBS Ex-4
0
1
2
3
***
***
***
***
relative mRNA levels
SOCS3
PBS Ex-4
0
5
10
15
20
*
***
***
***
#
relative mRNA levels
ifitm3
PBS Ex-4
0.0
0.5
1.0
1.5
2.0
*
*
***
#
relative mRNA levels
ndrg1
PBS Ex-4
0.0
2.0
4.0
6.0
8.0
10.0
12.0
***
***
relative mRNAlevels
junB
PBS Ex-4
0
10
20
30
40
*
***
***
***
realtive mRNA values
Reg IIIα
PBS Ex-4
0
5
10
15
20
25
#
relative mRNA values
FIG. 3. Continued.
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2153
expressed as a percentage of wet weight (wet
weightdry weight/wet weight 100).
RNA isolation and quantitative real-time PCR. RNA
from pancreatic tissue was extracted using TRIzol reagent;
after first-strand cDNA synthesis, real-time quantitative
PCR was carried out as described (22). Quantification of
transcript levels was performed with the ABI PRISM SDS
2.1 software. Cyclophilin mRNA or 18S rRNA was used for
normalization as expression of both remained unaltered
regardless of treatment.
Histology. Paraffin-embedded tissues were sectioned (5
m) and stained with hematoxylin-eosin. Immunohisto-
chemistry was carried out using indirect immunoperoxi-
dase detection with NovaRED substrate (Vector
Laboratories, Burlington, ON, Canada) followed by hema-
toxylin counterstaining. Primary rabbit polyclonal anti-
bodies were used at dilutions recommended by the
manufacturer and included: rabbit anti–c-fos (Sigma-Al-
drich), rabbit anti– egr-1 (Santa Cruz Biotechnology, Santa
Cruz, CA), and rat anti-mouse neutrophils (MCA771GA;
AbD Serotec, Kidlington, Oxford, U.K.).
Ex vivo pancreas preparation and cAMP assay. Pan-
creatic fragments were prepared from C57Bl/J6 mice.
Briefly, the pancreas was rapidly cut into small pieces,
cultured in DMEM 1% BSA 10% trasylol, washed three
times with DMEM 1% BSA 5% trasylol, resuspended in
DMEM 1% BSA 10% trasylol, and aliquoted such that
each tube contained 4% of the entire pancreas. Prepara-
tions were then stimulated with 100 nmol/l Ex-4 or 20
mol/l forskolin for 15 or 30 min at 37°C and frozen on dry
ice. Samples were thawed, sonicated with ice-cold ethanol
(65% final concentration), and cellular debris removed by
centrifugation at 13,000 rpm. cAMP was measured from
dried aliquots of ethanol extracts using a cAMP radioim-
munoassay kit (Biomedical Technologies, Stoughton, MA).
Statistical analysis. Statistical significance was assessed
by one-way or two-way ANOVA using Bonferroni’s multi-
ple comparison post hoc test and, where appropriate, by
unpaired Student’s t test using GraphPad Prism 4 (Graph-
Pad Software, San Diego, CA). A P value of 0.05 was
considered to be statistically significant.
RESULTS
Ex-4 acutely increases egr-1 and c-fos expression in
the exocrine pancreas. Mice treated with GLP-1R ago-
nists exhibit a significant increase in pancreas weight (23)
that cannot be attributed solely to increased -cell mass. To
determine whether GLP-1R activation induces a program of
gene expression in the exocrine pancreas associated with
development of pancreatic growth or inflammation, we ex-
amined whether Ex-4 enhanced the expression of immediate
early genes known to play important roles in regulating cell
proliferation. Ex-4 rapidly and robustly increased pancreatic
levels of egr-1 and c-fos (Fig. 1A), c-myc, and junB mRNA
transcripts (supplementary Fig. 1, available in an online
appendix at http://diabetes.diabetesjournals.org/cgi/content/
full/db09-0626/DC1). Moreover, the egr-1 protein was not
upregulated in islets but was localized exclusively to nuclei
of cells within the exocrine pancreas (Fig. 1B), whereas
nuclear c-fos expression was observed in both islets and
exocrine tissue (Fig. 1C). Hence, GLP-1R activation induces a
robust induction of gene and protein expression in the
exocrine pancreas.
Treatment with Ex-4 induces expression of PAP.
Egr-1 is an early response gene that encodes a transcrip-
tion factor that regulates cell proliferation, growth, and
pancreas weight (% BW)
WT Glp1r
-/-
WT Glp1r
-/-
WT W0 W6 K0 K6Glp1r
-/-
WT Glp-1r
-/-
0.6
0.8
1.0
1.2
1.4
1.6
0
6 µg/kg caerulein
pancreas weight
(% of body weight)
Edema
60
65
70
75
80
0
6 µg/kg caerulein
% water content
(wet-dry/wet*100)
B
D
A
serum amylase
0
2000
4000
6000
8000
10000
12000
0
6 µg/kg caerulein
***
***
amylase (U/L)
normalized for total
protein
serum amylase
0
2000
4000
6000
8000
10000
12000
amylase (U/L)
normalized for total
protein
absolute pancreas weight
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0
6 µg/kg caerulein
pancreas weight (g)
sac
5 hourly
injections
caerulein
C
0
FIG. 4. Susceptibility of Glp1r
/
mice to caerulein-induced pancreatitis. Glp1r
/
(wild-type) and Glp1r
/
mice were administered 5 hourly
injections of 6 g/kg caerulein. A: Schematic representation of experimental design. Pancreas weight (B) shown as a percentage of the final body
weight (left panel) or absolute pancreas weight (right panel), and pancreatic water content (edema) (C) and serum amylase (D) were assessed
in mice 6 h after the initial caerulein injection. Shown are means SE (D)(left panel) and individual serum amylase levels (right panel). ***P <
0.001 saline versus caerulein treatment, n 5 (saline) or n 4 (caerulein) mice for each genotype. E: Expression profiles of pancreatitis-
associated genes in the pancreas of wild-type and Glp1r
/
mice after caerulein administration. Levels of mRNA transcripts relative to the
wild-type control (saline)-treated group are shown normalized to 18S rRNA content. Results are expressed as means SE. *P < 0.05, **P < 0.01,
***P < 0.001 saline versus caerulein treatment. ##P < 0.01 Glp1r
/
versus Glp1r
/
mice, n 5 (saline) or n 4 (caerulein) mice for each
genotype.
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2154 DIABETES, VOL. 58, SEPTEMBER 2009
apoptosis (24). Induction of egr-1 expression occurs
early in the course of caerulein-induced pancreatitis,
and inflammation-related gene expression is attenuated
in egr-1
/
mice with experimental pancreatitis (25).
Accordingly, we examined whether agents that activate
the GLP-1Rs upregulate expression of genes associated
with pancreatitis. Mice were treated with Ex-4 for 1 week
(supplementary Fig. 2), and the expression of pancreatitis-
associated genes was assessed by real-time quantitative
PCR. Ex-4 (1 or 10 nmol/kg twice daily for 1 week)
increased pancreas weight (Fig. 2A) but had no effect on
levels of mRNA transcripts encoding the proinflammatory
mediators (monocyte chemotactic protein [Mcp]-1, intracel-
lular adhesion molecule-1, signal transducer and activator of
transcription [STAT]-3, ndrg1, or ifitm3) or transcription
factors (egr-1, activating transcription factor (ATF)-3, mist1,
c-fos, c-myc, and junB) associated with acute pancreatitis
(Fig. 2B and supplementary Table 1). In contrast, Ex-4
markedly increased pancreatic expression of the anti-inflam-
matory gene PAP (Reg III) and Reg III (Fig. 2B). The
increase in pancreatic weight and induction of PAP and
RegIII gene expression was not specific to Ex-4 but was
also observed in mice treated with the human GLP-1R
agonist liraglutide (Fig. 2C and D). These actions required a
functional GLP-1R as liraglutide had no effect on pancreatic
weight or induction of gene expression in Glp1r
/
mice
(Fig. 2C and D). Moreover, liraglutide reduced the expression
of the proinflammatory transcription factor, STAT3, in wild-
AT F 3
0
50
100
150
200
250
300
350
**
***
relative mRNA levels
IL-6
0
10
20
30
40
50
60
70
relative mRNA levels
mist1
0.0
0.5
1.0
1.5
2.0
2.5
*
relative mRNA levels
c-fos
0
50
100
150
200
**
***
relative mRNA levels
MCP-1
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
*
**
relative mRNA levels
Anti-inflammatory:
E
Pro-inflammatory:
Transcription factors:
junB
0
10
20
30
40
50
***
***
##
relative mRNA levels
SOCS3
0
5
10
15
20
25
***
***
relative mRNA levels
egr-1
0
100
200
300
400
500
**
**
relative mRNA levels
c-myc
0.0
0.5
1.0
1.5
2.0
2.5
3.0
*
relative mRNA levels
PAP
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp1r
-/-
WT Glp-1r
-/-
0.0
1.0
2.0
3.0
4.0
**
0
6 µg/kg caerulein
relative mRNA levels
RegIIIα
0.0
1.0
2.0
3.0
4.0
5.0
*
relative mRNA levels
STAT3
1.0
2.0
3.0
4.0
5.0
***
***
relative mRNA levels
FIG. 4. Continued.
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2155
type mice but not in Glp1r
/
mice (Fig. 2D). The expression
levels of most pancreatitis-associated mRNA transcripts
were comparable in Glp1r
/
versus Glp1r
/
pancreas;
however, basal levels of suppressor of cytokine signaling
(SOCS)-3 and STAT3 mRNA transcripts were lower in
Glp1r
/
mice (Fig. 2D). Taken together, these findings
demonstrate that structurally distinct GLP-1R agonists pro-
duce changes in pancreatic mass and gene expression,
actions requiring a functional GLP-1 receptor.
Effect of Ex-4 pretreatment on susceptibility to
caerulein-induced pancreatitis. To assess whether an-
tecedent activation of the GLP-1R increases the severity of
pancreatitis, mice were treated twice daily with Ex-4 for 1
week before exposure to caerulein (Fig. 3). A low dose of
caerulein was employed for these studies (3 or 6 g/kg)
based on preliminary experiments designed to induce a
detectable yet submaximal inflammatory response in the
pancreas (supplementary Fig. 3). Ex-4 significantly in-
creased pancreas weight (Fig. 3B) whereas caerulein
alone produced a small increase in pancreas weight (Fig.
3B) and a modest increase in tissue edema (Fig. 3C).
Caerulein consistently produced a low but detectable level
of inflammation, as evidenced by increased pancreatic
edema, elevated serum amylase, neutrophil infiltration,
and upregulated egr-1 and c-fos expression in the exocrine
pancreas (supplementary Figs. 3 and 4, and data not
shown). Intriguingly, serum amylase levels were lower in
mice pretreated with Ex-4 and then treated with 3 g/kg
caerulein (Fig. 3D). Ex-4 did not significantly modulate
levels of most pancreatitis-associated mRNA transcripts
regulated by caerulein administration with the exception
of Reg III and SOCS3 that were further induced in
Ex-4 –treated mice exposed to caerulein (Fig. 3E). Consis-
tent with previous findings (Fig. 2A), treatment with Ex-4
for 1 week significantly increased the transcript levels of
the anti-inflammatory protein PAP (Fig. 3E), in the pres-
ence or absence of caerulein.
Loss of GLP-1R signaling does not modify the sever-
ity of caerulein-induced pancreatitis. To determine
whether endogenous GLP-1R signaling influences suscep-
tibility to caerulein-induced pancreatitis, we assessed the
effects of a submaximal dose of caerulein on gene expres-
sion and severity of pancreatitis in Glp1r
/
versus
Glp1
/
mice. Pancreas weight, edema, and serum amy
-
lase were not significantly different in caerulein-treated
Glp1r
/
versus Glp1r
/
mice (Fig. 4
B–D). Furthermore,
GLP-1R genotype had no effect on the expression of
pancreatic genes known to be induced by caerulein,
including SOCS3, Mcp-1, egr-1, ATF-3, and c-fos (Fig. 4E).
Intriguingly, caerulein-induced levels of PAP, RegIII,c-
myc, and junB mRNA transcripts were significantly greater
in Glp1r
/
mice (Fig. 4E).
Ex-4 does not affect recovery from or severity of
caerulein-induced pancreatitis. To determine whether
GLP-1R agonists exacerbate the severity or prolong the
recovery after initiation of acute pancreatitis, mice were
treated with caerulein followed by 10 nmol/kg of Ex-4
twice daily for up to 6 days (Fig. 5A). Mice were killed
for analysis at 1, 24, 72 h, and 6 days after the final
injection of caerulein. Caerulein transiently but signifi-
Serum amylase
0
5000
10000
15000
20000
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
- - -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
- - -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
- - -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
- - -+ -+ -+
Day 0 Day 1 Day 3 Day 6
***
amylase (U/L)
normalized total
protein
B
Edema
50
60
70
80
**
% water content
(wet-dry/wet*100)
C
sac
6 pm
Ex-4 twice daily
5 hourly
injections
caerulein
Day
0
3 h
1
9 am 6 pm
sac
9 am 6 pm
3
9 am 6 pm
sac
9 am 6 pm 9 am 6 pm
6
9 am
sac
A
3 pm
Pancreas weight (% BW)
0.8
0.9
1.0
1.1
1.2
1.3
1.4
##
**
*
pancreas weight
(% of body weight)
D
Absolute pancreas weight
0.15
0.20
0.25
0.30
0.35
#
pancreas weight (g)
FIG. 5. Ex-4 does not modify severity of or recovery from caerulein-induced pancreatitis. Mice were administered 5 hourly injections with 6 g/kg
caerulein followed by twice daily injections with PBS or 10 nmol/kg Ex-4. A: Schematic representation of experimental design. Serum amylase (B),
pancreatic water content (edema) (C), and pancreas weight (D), shown as a percentage of the final body weight (left panel) or absolute pancreas
weight (right panel), were assessed in mice 6 h after the initial caerulein injection (day 0) or 24 h (day 1), 72 h (day 3), or 144 h (day 6) after
the final caerulein injection. Day 1 mice received two treatments with either PBS or Ex-4. Shown are means SE. #P < 0.05, ##P < 0.01 PBS
versus Ex-4 treatment, *P < 0.05, **P < 0.01, ***P < 0.001 saline versus caerulein treatment, n 4 mice in each treatment group. E: Expression
profiles of genes associated with pancreatitis in the pancreas of mice treated twice daily with PBS or 10 nmol/kg Ex-4 after caerulein
administration as indicated above. Shown are the levels of the indicated transcripts relative to the control (saline)-treated group (day 0) as
determined by real-time quantitative PCR normalized to the 18S rRNA content or cyclophillin mRNA levels (for STAT3, SOCS3, ndrg1, and
ifitm3). Results are expressed as means SE. #P < 0.05, ##P < 0.01, ###P < 0.001 PBS versus Ex-4 treatment, *P < 0.05, **P < 0.01, ***P < 0.001
saline (day 0) versus caerulein treatment, n 4 in each treatment group.
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2156 DIABETES, VOL. 58, SEPTEMBER 2009
c-myc
0.0
0.5
1.0
1.5
2.0
2.5
**
relative mRNA levels
mist1
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
**
#
relative mRNA levels
MCP-1
0
2
4
6
8
50
75
100
***
egr-1
0
5
10
15
600
800
1000
1200
***
#
relative mRNA levels
c-fos
0
1
2
3
4
200
300
400
***
relative mRNA levels
PAP
0
400
800
1200
2000
2500
3000
3500
***
***
*
*
**
##
relative mRNA levels
relative mRNA levels
junB
0
2
4
35
40
45
50
***
relative mRNA levels
IL-6
0
1
2
10
15
20
ND ND ND
ND: not detectable
*
relative mRNA levels
Anti-inflammatory:
Pro-inflammatory:
Transcription factors:
E
SOCS3
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
***
#
relative mRNA levels
ndrg1
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
***
relative mRNA levels
ifitm3
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
###
***
***
**
realtive mRNA levels
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
μg/kg Caerulein
Ex-4 (10 nmol/kg)
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
STAT3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
***
###
##
relative mRNA levels
-6 66 66 66
-- -+ -+ -+
Day 0 Day 1 Day 3 Day 6
FIG. 5. Continued.
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2157
cantly increased serum amylase (Fig. 5B, day 0), pan-
creatic edema (Fig. 5C, day 0), and relative pancreas
weight (Fig. 5D, day 1). However, serum amylase and
pancreatic edema returned to basal levels after 24 h,
independent of Ex-4 treatment (Fig. 5B and C). Simi-
larly, levels of mRNA transcripts encoding the proin-
flammatory proteins Mcp-1, interleukin (IL)-6, and ndrg1
were rapidly induced by caerulein but returned to basal
levels within 24 h independent of Ex-4 treatment (Fig.
5E). Levels of STAT3 and ifitm3 transcripts were ele-
vated in Ex-4 –treated mice (Fig. 5E), and mRNA tran-
scripts for the anti-inflammatory proteins PAP and
SOCS3 remained elevated in Ex-4 –treated mice. In
contrast, mRNA transcripts for c-fos, c-myc, and junB
were transiently increased in the pancreas of caerulein-
treated mice but were not affected by concomitant Ex-4
treatment (Fig. 5E), whereas levels of pancreatic mRNA
transcripts for mist1 and egr-1 were modestly but sig-
nificantly higher in Ex-4 –treated mice.
Ex-4 modulates pancreatic gene expression in mice
with metabolic stress. To determine whether GLP-1R
activation modified the expression of pancreatitis-associated
serum amylase
PBS NC PBS HF Ex-4 HF
0
2000
4000
6000
8000
amylase (U/L) normalized
for total protein
serum amylase
PBS NC PBS HF Ex-4 HF
0
2000
4000
6000
8000
amylase (U/L) normalized
for total protein
D
B
C
body weight
PBS NC PBS HF Ex-4 HF
0
5
10
15
20
25
30
35
***
###
body weight
absolute pancreas weight
PBS NC PBS HF Ex-4 HF
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
##
pancreas weight (g)
40
sac
week
HFD
age
(weeks)
84
-Ex-4
(24 nmol/kg)
twice daily
12
8
A
FIG. 6. Ex-4 modulates pancreatic gene expression in high-fat–fed mice. A: Schematic representation of experimental design. B: Pancreas weight, (C)
body weight, (D) serum amylase, and (E) pancreas gene expression profiles of mice treated twice daily with PBS or 24 nmol/kg Ex-4 for 1 month on
normal chow or high-fat diet as described (23). Results are expressed as means SE (D)(left panel) and individual serum amylase levels (right panel).
E: Shown are the levels of the indicated transcripts relative to control (PBS-treated mice on normal chow) as determined by real-time quantitative PCR
normalized to cyclophillin mRNA levels. Results are expressed as means SE. *P < 0.05, **P < 0.01, ***P < 0.001 PBS-treated mice on normal chow
versus high-fat diet. #P < 0.05, ##P < 0.01, ###P < 0.001 PBS- versus Ex-4 –treated mice on a high-fat diet, n 7 in each group.
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2158 DIABETES, VOL. 58, SEPTEMBER 2009
genes in the setting of a mild metabolic stress associated with
the development of insulin resistance (19), mice were placed
on a high-fat diet and treated twice daily with saline or Ex-4
(Fig. 6A). Consistent with previous observations, Ex-4 pre-
vented weight gain (Fig. 6C) and significantly increased
pancreas weight (Fig. 6B) but did not affect levels of serum
amylase (Fig. 6D). Moreover, high-fat diet mice treated with
saline, but not Ex-4, exhibited a significant increase in the
transcript levels of pancreatitis-associated genes including
Mcp-1, STAT3, egr-1, and ATF-3 as well as the exocrine-
specific transcription factor mist1 (Fig. 6E). In contrast, Ex-4
(but not saline) administration significantly increased levels
of mRNA transcripts for PAP, c-fos, c-myc, and junB (Fig.
6E).
mist1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PBS PBS Ex-4
HFNC
*
#
relative mRNA levels
c-fos
0
5
10
15
20
25
30
35
40
PBS PBS Ex-4
HFNC
###
relative mRNA levels
MCP-1
0
2
4
6
8
10
12
PBS PBS Ex-4
HFNC
*
#
relative mRNA levels
c-myc
0.0
1.0
2.0
3.0
4.0
5.0
PBS PBS Ex-4
HFNC
#
relative mRNA levels
AT F 3
0.0
1.0
2.0
3.0
4.0
5.0
6.0
PBS PBS Ex-4
HFNC
**
relative mRNA levels
TNFα
0.0
1.0
2.0
3.0
4.0
5.0
6.0
PBS PBS Ex-4
HFNC
relative mRNA levels
egr-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PBS PBS Ex-4
HFNC
*
#
relative mRNA levels
PAP
0
1
2
3
4
PBS PBS Ex-4
HFNC
30
40
50
###
relative mRNA levels
junB
0.0
2.0
4.0
6.0
8.0
PBS PBS Ex-4
HFNC
###
relative mRNA levels
Anti-inflammatory:
Pro-inflammatory:
Transcription factors:
E
STAT3
PBS PBS Ex-4
0.0
1.0
2.0
3.0
4.0
*
HFNC
relative mRNA levels
SOCS3
PBS PBS Ex-4
0.0
1.0
2.0
3.0
4.0
HFNC
relative mRNA levels
FIG. 6. Continued.
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2159
We next determined whether metformin, sitagliptin, or
Ex-4, antidiabetic agents associated with enhanced GLP-1
receptor activation through various mechanisms (3,4,26),
modulated pancreatitis-associated gene expression profiles
in high-fat–fed diabetic mice. Ex-4, and to a lesser extent the
DPP-4 inhibitor sitagliptin, but not metformin increased PAP
mRNA transcript levels in diabetic mice (Fig. 7). Neither
Ex-4, metformin, nor sitagliptin treatment modulated the
expression of other genes associated with pancreatitis in-
cluding SOCS3, egr-1, STAT3, c-myc, or junB (Fig. 7B).
Finally, as Ex-4 was reported to exhibit high-affinity binding
to guinea pig pancreatic acinar membranes and stimulate
cyclic AMP formation through a distinct receptor that pref-
erentially recognized Ex-4 relative to GLP-1 (27), we assessed
cyclic AMP formation in slices from wild-type mouse pan-
creas. Forskolin but not Ex-4 rapidly stimulated cyclic AMP
accumulation in pancreatic slices ex vivo (Fig. 8).
DISCUSSION
There are currently limited data regarding putative effects of
GLPs on the function of the normal or inflamed exocrine
pancreas. Intriguingly, coadministration of glucagon and
careulein attenuated the increase in pancreatic weight and
amylase expression seen in rats treated with careulein alone
(28). Similarly, oxyntomodulin, a peptide structurally related
to both glucagon and GLP-1, was shown to be 10-fold more
potent than glucagon in the suppression of rat pancreatic
exocrine secretion in the basal state or after administration
of caerulein (29). Our results extend these findings by dem-
onstrating that although acute and chronic GLP-1R activation
modulates a gene expression program in the exocrine murine
pancreas, GLP-1 receptor activation does not predispose to
or exacerbate experimental pancreatitis in mice.
Several lines of evidence imply that exocrine cells express
a functional GLP-1 and/or exendin receptor. Guinea pig
pancreatic acini contain high-affinity binding sites for Ex-4,
and Ex-4 binding was displaced from acinar cells by coincu-
bation with native GLP-1 (27). Furthermore, both Ex-4 and
GLP-1–stimulated cAMP formation but not amylase release
in dispersed guinea pig pancreatic acini (27,30), and these
stimulatory actions on cAMP were blocked by the GLP-1R
antagonist exendin (9 –39) (30,31). Similarly, Ex-4 stimulated
cyclic AMP formation in rat pancreatic slices, and potenti-
ated calcium ionophore-, neurotransmitter-, or CCK-induced
amylase release in vitro (32). In contrast, we found that
forskolin, but not Ex-4, rapidly increased cAMP formation in
murine pancreatic fragments. Whether these findings reflect
species-specific differences in the expression or signaling of
functional pancreatic Ex-4/GLP-1 receptors requires further
investigation.
We used the CCK agonist caerulein for studies of
experimental pancreatitis as previous data demonstrated a
functional interaction of CCK and GLP-1 signaling path-
ways in the endocrine and exocrine pancreas (32,33). Al-
though the expression of most pancreatitis-associated genes
was not further modified by administration of GLP-1R ago-
nists, we consistently detected upregulation of PAP after
chronic administration of Ex-4 to mice, in the presence or
absence of caerulein administration. Similarly, liraglutide
junB
PBS PBS Met Ex-4 Sit
0.0
0.5
1.0
1.5
2.0
2.5
HF + STZNC
relative mRNA levels
egr-1
PBS PBS Met Ex-4 Sit
0.0
1.0
2.0
3.0
4.0
HF + STZNC
relative mRNA levels
c-myc
PBS PBS Met Ex-4 Sit
0.0
0.4
0.8
1.2
1.6
HF + STZNC
relative mRNA levels
PAP
PBS PBS Met Ex-4 Sit
0
2
4
6
8
10
12
14
HF + STZNC
*
relative mRNA levels
SOCS3
PBS PBS Met Ex-4 Sit
0.0
0.5
1.0
1.5
2.0
HF + STZNC
relative mRNA levels
STAT3
PBS PBS Met Ex-4 Sit
0.0
0.5
1.0
1.5
2.0
2.5
HF + STZNC
relative mRNA levels
Transcription factors
Anti-inflammatory
80 12
sac
week
HFD
STZ
age
(weeks)
13 21 25
20
33
- Metformin
- Ex-4
- Sitagliptin
A
B
FIG. 7. Ex-4 increases PAP gene expression in diabetic mice. Mice on a
high-fat diet for 8 weeks were treated with a single dose of STZ (100
mg/kg). After 4 weeks of hyperglycemia on a high-fat diet, mice were
then treated with metformin (500 mg kg
1
day
1
), Ex-4 (3 nmol/kg),
or sitagliptin (370 mg kg
1
day
1
) for an additional 8 weeks on a
high-fat diet. Mean glucose levels at end of study ranged from 10 to 14
to 16 mm for Ex-4 versus metformin- versus sitagliptin-treated mice.
A: Schematic representation of experimental design. B: Expression
profiles of pancreatitis-associated genes in the pancreas of mice
treated as indicated above. Shown are the levels of mRNA transcripts
relative to control (PBS-treated mice on normal chow) as determined
by real-time quantitative PCR normalized to cyclophillin mRNA levels.
Results are expressed as means SE. *P < 0.05 PBS- versus Ex-4
treated mice on a high-fat diet STZ treatment, n 4 6 in each group.
PBS Ex-4 Fsk PBS Ex-4 Fsk
0
10
20
30
40
50
60
70
80
15 min
30 min
***
***
cAMP (pmoles)
normalized for total
protein
FIG. 8. Ex-4 does not increase cAMP levels in pancreatic fragments ex
vivo. The entire pancreas was rapidly digested and treated with 100
nmol/l Ex-4 or 20 mol/l forskolin for 15 or 30 min. Cell extracts were
analyzed for cAMP content. cAMP levels were normalized to total
protein in pancreatic extracts. Results are expressed as means SE.
***P < 0.001 PBS- versus forskolin-treated pancreas preparations, n
3 in each group.
GLP-1 AND EXPERIMENTAL MURINE PANCREATITIS
2160 DIABETES, VOL. 58, SEPTEMBER 2009
also induced PAP expression, in a GLP-1R– dependent man-
ner, and basal levels of pancreatic PAP mRNA transcripts
were modestly reduced in Glp1r
/
mice.
PAP has been shown to be mitogenic, antiapoptotic, and
anti-inflammatory and is strongly induced early in the
course of inflammatory diseases such as pancreatitis,
Crohn’s disease, and ulcerative colitis (14). Moreover,
inhibition of PAP gene expression in rats augments the
severity of acute pancreatitis (34), and PAP knockout mice
exhibit more extensive inflammation after induction of
caerulein-induced pancreatitis (13), suggesting a protec-
tive role for PAP in the inflammatory response to cellular
injury. Hence, the induction of PAP after GLP-1R activa-
tion may represent a compensatory mechanism that serves
to limit damage to the exocrine pancreas.
Our studies of GLP-1R activation on gene expression and
the development of and/or recovery from pancreatitis were
motivated, in part, by clinical reports of acute pancreatitis in
diabetic patients treated with GLP-1R agonists (8,10). Our
findings do not support the hypothesis that GLP-1R activa-
tion sensitizes the murine pancreas to the development of
pancreatic inflammation. Similarly, we did not detect any
difference in the extent of pancreatic inflammation in
Glp1r
/
mice. Taken together, the available data demon
-
strate that GLP-1R activation leads to increases in mass and
changes in expression of pancreatitis-associated genes but
does not modify pancreatitis susceptibility or severity in the
murine pancreas. Whether GLP-1R activation modifies gene
expression, enzyme secretion, or pancreatic inflammation in
the human pancreas requires further analysis.
ACKNOWLEDGMENTS
These studies were supported in part by a grant from the
Canadian Institutes of Health Research and Hoffman La-
Roche. D.J.D. is supported by the Canada Research Chairs
Program.
The Drucker Lab has received operating grant support
from Novo Nordisk in conjunction with the studies of
liraglutide. No other potential conflicts of interest relevant
to this article were reported.
We thank B. Yusta, A. Maida, and D. Holland for
experimental assistance and helpful discussions.
REFERENCES
1. Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3:153–165
2. Brubaker PL, Drucker DJ. Glucagon-like peptides regulate cell prolifera-
tion and apoptosis in the pancreas, gut and central nervous system.
Endocrinology 2004;145:2653–2659
3. Deacon CF. Therapeutic strategies based on glucagon-like peptide 1.
Diabetes 2004;53:2181–2189
4. Drucker DJ, Nauck MA. The incretin system: glucagon-like peptide-1
receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes.
Lancet 2006;368:1696–1705
5. Zhou J, Wang X, Pineyro MA, Egan JM. Glucagon-like peptide 1 and
exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-
producing cells. Diabetes 1999;48:2358–2366
6. Wettergren A, Wojdemann M, Holst JJ. Glucagon-like peptide-1 inhibits
gastropancreatic function by inhibiting central parasympathetic outflow.
Am J Physiol 1998;275:G984–G992
7. Wan S, Coleman FH, Travagli RA. Glucagon-like peptide-1 excites pancre-
as-projecting preganglionic vagal motoneurons. Am J Physiol Gastrointest
Liver Physiol 2007;292:G1474–G1482
8. Ahmad SR, Swann J. Exenatide and rare adverse events. N Engl J Med
2008;358:1970 –1971; discussion 1971–1972
9. Garber A, Henry R, Ratner R, Garcia-Hernandez PA, Rodriguez-Pattzi H,
Olvera-Alvarez I, Hale PM, Zdravkovic M, Bode B. Liraglutide versus
glimepiride monotherapy for type 2 diabetes (LEAD-3 Mono): a random-
ised, 52-week, phase III, double-blind, parallel-treatment trial. Lancet
2009;373:473– 481
10. Dore DD, Seeger JD, Arnold Chan K. Use of a claims-based active drug
safety surveillance system to assess the risk of acute pancreatitis with
exenatide or sitagliptin compared to metformin or glyburide. Curr Med Res
Opin 2009;25:1019–1027
11. Gaisano HY, Gorelick FS. New insights into the mechanisms of pancreati-
tis. Gastroenterology, 2009 [Epub ahead of print]
12. De Leon DD, Farzad C, Crutchlow MF, Brestelli J, Tobias J, Kaestner KH,
Stoffers DA. Identification of transcriptional targets during pancreatic
growth after partial pancreatectomy and exendin-4 treatment. Physiol
Genomics 2006;24:133–143
13. Gironella M, Folch-Puy E, LeGoffic A, Garcia S, Christa L, Smith A, Tebar L,
Hunt SP, Bayne R, Smith AJ, Dagorn JC, Closa D, Iovanna JL. Experimental
acute pancreatitis in PAP/HIP knock-out mice. Gut 2007;56:1091–1097
14. Graf R, Schiesser M, Reding T, Appenzeller P, Sun LK, Fortunato F, Perren A,
Bimmler D. Exocrine meets endocrine: pancreatic stone protein and regen-
erating protein–two sides of the same coin. J Surg Res 2006;133:113–120
15. Saluja AK, Lerch MM, Phillips PA, Dudeja V. Why does pancreatic
overstimulation cause pancreatitis? Annu Rev Physiol 2007;69:249 –269
16. Hansotia T, Maida A, Flock G, Yamada Y, Tsukiyama K, Seino Y, Drucker
DJ. Extrapancreatic incretin receptors modulate glucose homeostasis,
body weight, and energy expenditure. J Clin Invest 2007;117:143–152
17. Pandol SJ, Saluja AK, Imrie CW, Banks PA. Acute pancreatitis: bench to the
bedside. Gastroenterology 2007;132:1127–1151
18. Willemer S, Elsasser HP, Adler G. Hormone-induced pancreatitis. Eur Surg
Res 1992;1 (Suppl.):29–39
19. Winzell MS, Ahren B. The high-fat diet-fed mouse: a model for studying
mechanisms and treatment of impaired glucose tolerance and type 2
diabetes. Diabetes 2004;3 (Suppl.):S215–S219
20. Kowalik AS, Johnson CL, Chadi SA, Weston JY, Fazio EN, Pin CL. Mice
lacking the transcription factor Mist1 exhibit an altered stress response
and increased sensitivity to caerulein-induced pancreatitis. Am J Physiol
Gastrointest Liver Physiol 2007;292:G1123–G1132
21. Singh VP, Bhagat L, Navina S, Sharif R, Dawra RK, Saluja AK. Protease-
activated receptor-2 protects against pancreatitis by stimulating exocrine
secretion. Gut 2007;56:958–964
22. Flock G, Baggio LL, Longuet C, Drucker DJ. Incretin receptors for
glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide
are essential for the sustained metabolic actions of vildagliptin in mice.
Diabetes 2007;56:3006–3013
23. Baggio LL, Huang Q, Cao X, Drucker DJ. The long-acting albumin-exendin-4
GLP-1R agonist CJC-1134 engages central and peripheral mechanisms regu-
lating glucose homeostasis. Gastroenterology 2008;134:1137–1147
24. Thiel G, Cibelli G. Regulation of life and death by the zinc finger
transcription factor Egr-1. J Cell Physiol 2002;193:287–292
25. Ji B, Chen XQ, Misek DE, Kuick R, Hanash S, Ernst S, Najarian R, Logsdon
CD. Pancreatic gene expression during the initiation of acute pancreatitis:
identification of EGR-1 as a key regulator. Physiol Genomics 2003;14:59 –72
26. Yasuda N, Inoue T, Nagakura T, Yamazaki K, Kira K, Saeki T, Tanaka I.
Enhanced secretion of glucagon-like peptide 1 by biguanide compounds.
Biochem Biophys Res Commun 2002;298:779 –784
27. Singh G, Eng J, Raufman JP. Use of 125I-[Y39]exendin-4 to characterize
exendin receptors on dispersed pancreatic acini and gastric chief cells
from guinea pig. Regul Pept 1994;53:47–59
28. Kash FF, Wood JG, Solomon T. Glucagon inhibition of cerulein-induced
hypertrophy of the exocrine pancreas. Pancreas 1988;3:11–17
29. Biedzinski TM, Bataille D, Devaux MA, Sarles H. The effect of oxyntomodu-
lin (glucagon-37) and glucagon on exocrine pancreatic secretion in the
conscious rat. Peptides 1987;8:967–972
30. Raufman J-P, Singh L, Singh G, Eng J. Truncated glucagon-like peptide-1
interacts with exendin receptors on disperced acini from guinea pig
pancreas. Identification of a mammalian homolgue of the reptilian peptide
exendin-4. J Biol Chem 1992;267:21432–21437
31. Eng J, Kleinman WA, Singh L, Singh G, Raufman JP. Isolation and
characterization of exendin-4, an exendin-3 analogue, from Heloderma
suspectum venom. Further evidence for an exendin receptor on dispersed
acini from guinea pig pancreas. J Biol Chem 1992;267:7402–7405
32. Malhotra R, Singh L, Eng J, Raufman JP. Exendin-4, a new peptide from
Heloderma suspectum venom, potentiates cholecystokinin-induced amy-
lase release from rat pancreatic acini. Regul Pept 1992;41:149–156
33. Fehmann HC, Goke B, Weber V, Goke R, Trautmann ME, Richter G, Arnold
R. Interaction of glucagon-like peptide-1 (7–36)amide and cholecystoki-
nin-8 in the endocrine and exocrine rat pancreas. Pancreas 1990;5:361–365
34. Zhang H, Kandil E, Lin YY, Levi G, Zenilman ME. Targeted inhibition of
gene expression of pancreatitis-associated proteins exacerbates the sever-
ity of acute pancreatitis in rats. Scand J Gastroenterol 2004;39:870881
J.A. KOEHLER AND ASSOCIATES
DIABETES, VOL. 58, SEPTEMBER 2009 2161
    • "On the other hand, previous studies have shown that genetic and pharmacological interference with GLP-1R does not affect the severity of pancreatitis in cerulein model of this disease. This last observation suggests that therapeutic effect of obestatin in ischemia/reperfusion-induced AP is probably independent to GLP-1R signaling [49]. In 2005, Zhang et al. [50] isolated a new 23-amino-acid peptide derived from prepro-ghrelin and named it obestatin a contraction of obese, from the Latin "obedere," meaning to devour, and "statin," denoting suppression. "
    [Show abstract] [Hide abstract] ABSTRACT: OBJECTIVE: Several previous studies have shown that obestatin exhibits protective and regenerative effects in some organs including the stomach, kidney, and the brain. In the pancreas, pretreatment with obestatin inhibits the development of cerulein-induced acute pancreatitis, and promotes survival of pancreatic beta cells and human islets. However, no studies investigated the effect of obestatin administration following the onset of experimental acute pancreatitis. AIM: The aim of this study was to evaluate the impact of obestatin therapy in the course of ischemia/reperfusion-induced pancreatitis. Moreover, we tested the influence of ischemia/reperfusion-induced acute pancreatitis and administration of obestatin on daily food intake and pancreatic exocrine secretion. METHODS: Acute pancreatitis was induced by pancreatic ischemia followed by reperfusion of the pancreas. Obestatin (8nmol/kg/dose) was administered intraperitoneally twice a day, starting 24 hours after the beginning of reperfusion. The effect of obestatin in the course of necrotizing pancreatitis was assessed between 2 and 14 days, and included histological, functional, and biochemical analyses. Secretory studies were performed on the third day after sham-operation or induction of acute pancreatitis in conscious rats equipped with chronic pancreatic fistula. RESULTS: Treatment with obestatin ameliorated morphological signs of pancreatic damage including edema, vacuolization of acinar cells, hemorrhages, acinar necrosis, and leukocyte infiltration of the gland, and led to earlier pancreatic regeneration. Structural changes were accompanied by biochemical and functional improvements manifested by accelerated normalization of interleukin-1β level and activity of myeloperoxidase and lipase, attenuation of the decrease in pancreatic DNA synthesis, and by an improvement of pancreatic blood flow. Induction of acute pancreatitis by pancreatic ischemia followed by reperfusion significantly decreased daily food intake and pancreatic exocrine secretion. Administration of obestatin at doses used was without significant effect with regard to daily food intake or pancreatic exocrine secretion in sham-operated rats, as well as in rats with acute pancreatitis. On the other hand, obestatin abolished a statistical significance of difference in food intake between animals with AP and control animals without pancreatic fistula and induction of AP. CONCLUSION: Treatment with the exogenous obestatin reduces severity of ischemia/reperfusion-induced acute pancreatitis and accelerates recovery in this disease. The involved mechanisms are likely to be multifactorial, and are mediated, at least in part, by anti-inflammatory properties of obestatin.
    Full-text · Article · Jul 2015
    • "Experimentally, 12 weeks of exenatide treatment up-regulated genes of lipolytic proteins [31]. Exenatide has enhanced pancreatic lipid metabolism in a high fat environment in vitro and in vivo [12,20,31]. Stem cell survival is consistent with findings in b-cells where pro-survival and proliferative gene expression changes have been attributed to EXE treatment through control of Sirt-1 (also differentially expressed in the present study) and other genes that control epigenetic modifications necessary for transcription [13,14,32]. "
    [Show abstract] [Hide abstract] ABSTRACT: This study expanded upon a previous study in mice reporting a link between exenatide treatment and exocrine pancreatic injury by demonstrating temporal and dose responses and providing an initial mechanistic hypothesis. The design of the present study included varying lengths of exenatide exposure (3, 6 weeks to 12 weeks) at multiple concentrations (3, 10, or 30 µg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice on a high fat diet treated with exenatide. The morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (proliferation/regeneration) accompanied by varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles indicated increased signaling for cell survival and altered lipid metabolism in exenatide treated mice. Immunohistochemistry supported gene expression findings that exenatide caused and/or exacerbated pancreatic injury in a high fat diet environment potentially by further increasing high fat diet exacerbated lipid metabolism and resulting oxidative stress. Further investigation is required to confirm these findings and determine their relevance to human disease.
    Full-text · Article · Oct 2014
    • "Incretin-based therapies, because of their low hypoglycemic risk and apparently acceptable tolerability profile [1– 3], are gaining wider use in current clinical practice for treating type 2 diabetes. However, incongruent data from experimental studies on rodents456, recent case reports in humans789, and pharmacovigilance analyses [10, 11] have been interpreted as providing evidence for the hypothesis that exposure to incretins may be associated with an increased risk of acute pancreatitis (AP), arousing concern about the overall safety of this drug class. Interpretations of the pharmacovigilance data in particular, based on self-reported, uncontrolled cases, supported the hypothesis for an almost 25-fold excess risk of developing AP [11, 12]. "
    [Show abstract] [Hide abstract] ABSTRACT: Concerns raised by several animal studies, case reports, and pharmacovigilance warnings over incretin-based therapy potentially exposing type two diabetes patients to an elevated risk of pancreatitis have cast a shadow on the overall safety of this class of drugs. This systematic review evaluates the data from observational studies that compared treatment with or without incretins and the risk of pancreatitis. We searched PubMed for publications with the key terms incretins or GLP-1 receptor agonists or DPP-4 inhibitors or sitagliptin or vildagliptin or saxagliptin or linagliptin or alogliptin or exenatide or liraglutide AND pancreatitis in the title or abstract. Studies were evaluated against the following criteria: design (either cohort or case-control); outcome definition (incidence of pancreatitis); exposure definition (new or current or past incretins users); and comparison between patients receiving incretins or not for type 2 diabetes. Two authors independently selected the studies and extracted the data. Six studies meeting the inclusion criteria were reviewed. No difference was found in the overall risk of pancreatitis between incretin users and non-users (odds ratio 1.08; 95 % CI [0.84-1.40]). A risk increase lower than 35 % cannot be excluded according to the power calculation. This systematic review and meta-analysis suggests that type 2 diabetes patients receiving incretin-based therapy are not exposed to an elevated risk of pancreatitis. Limitations of this analysis are the low prevalence of incretin users and the lack of a clear distinction by the studies between therapy with DPP-4 inhibitors or with GLP-1 receptor agonists.
    Full-text · Article · Aug 2014
Show more