Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

Abstract

The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect.
We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis.
We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis.
An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Full text

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Glucagon-Like Peptide-1 Protects ?-Cells Against
Apoptosis by Increasing the Activity of an Igf-2/Igf-1
Receptor Autocrine Loop
Marion Cornu,1Jiang-Yan Yang,2Evrim Jaccard,2Carine Poussin,1Christian Widmann,2and
Bernard Thorens1
OBJECTIVE—The gluco-incretin hormones glucagon-like pep-
tide (GLP)-1 and gastric inhibitory peptide (GIP) protect ?-cells
against cytokine-induced apoptosis. Their action is initiated by
binding to specific receptors that activate the cAMP signaling
pathway, but the downstream events are not fully elucidated.
Here we searched for mechanisms that may underlie this protec-
tive effect.
RESEARCH DESIGN AND METHODS—We performed com-
parative transcriptomic analysis of islets from control and
GipR?/?;Glp-1-R?/?mice, which have increased sensitivity to
cytokine-induced apoptosis. We found that IGF-1 receptor ex-
pression was markedly reduced in the mutant islets. Because the
IGF-1 receptor signaling pathway is known for its antiapoptotic
effect, we explored the relationship between gluco-incretin ac-
tion, IGF-1 receptor expression and signaling, and apoptosis.
RESULTS—We found that GLP-1 robustly stimulated IGF-1
receptor expression and Akt phosphorylation and that increased
Akt phosphorylation was dependent on IGF-1 but not insulin
receptor expression. We demonstrated that GLP-1–induced Akt
phosphorylation required active secretion, indicating the pres-
ence of an autocrine activation mechanism; we showed that
activation of IGF-1 receptor signaling was dependent on the
secretion of IGF-2. We demonstrated, both in MIN6 cell line and
primary ?-cells, that reducing IGF-1 receptor or IGF-2 expression
or neutralizing secreted IGF-2 suppressed GLP-1–induced pro-
tection against apoptosis.
CONCLUSIONS—An IGF-2/IGF-1 receptor autocrine loop oper-
ates in ?-cells. GLP-1 increases its activity by augmenting IGF-1
receptor expression and by stimulating secretion; this mecha-
nism is required for GLP-1–induced protection against apoptosis.
These findings may lead to novel ways of preventing ?-cell loss in
the pathogenesis of diabetes. Diabetes 58:1816–1825, 2009
T
failure of the endocrine pancreas to maintain an adequate
insulin secretory capacity due to reduced ?-cell number
and function underlies the pathogenesis of both type 1 and
type 2 diabetes. In type 1 diabetes, ?-cells are destroyed by
an autoimmune mechanism in which cytokine-induced
apoptosis is thought to play an important role (2). In the
pathogenesis of type 2 diabetes, hyperglycemia appears
when ?-cell mass and insulin secretory capacity are no
longer sufficient to compensate for insulin resistance. The
reduction in ?-cell mass results from increased apoptosis,
most probably caused by the combined action of cytokines
and increased plasma glucose and free fatty acid levels
(3,4). Therefore, finding means to protect ?-cell against
apoptosis may be useful for the treatment or prevention of
diabetes.
The gluco-incretin hormones GLP-1 and GIP can protect
?-cells against apoptosis induced by cytokines or glucose
and free fatty acids (5). Both hormones bind to specific
stimulatory G-protein (Gs)–coupled receptors, which trig-
ger cAMP formation (6,7). In ?-cells, basal cAMP levels
control glucose competence (8), i.e., the magnitude of the
insulin secretion response to a given increase in extracel-
lular glucose concentration. Increases in cAMP levels, for
instance, as stimulated by GLP-1 or GIP action, potentiate
glucose-stimulated insulin secretion by both protein ki-
nase A (PKA)-dependent and -independent mechanisms
(9); they also stimulate gene transcription through PKA-
dependent phosphorylation of the transcription factor
CREB (cAMP-response element binding) (10).
In ?-cells, increased cAMP levels also activate the
mitogen-activated protein (MAP) kinase cascade, leading
to rapid phosphorylation of Erk1/2 (11). An activation of
the phosphatidylinositol (PI) 3-kinase/Akt pathway is also
observed. PI 3-kinase may be directly activated by the ??
subunit of Gs (12), be secondary to transactivation of the
EGF receptor by betacellulin (13), or follow transcrip-
tional induction of insulin receptor substrate (IRS)-2
through the PKA/CREB pathway. The IRS-2/PI 3-kinase/
Akt pathway is known to have antiapoptotic effects (14–
16); however, it is unclear why increased expression of
IRS-2 leads to activation of its signaling pathway. IRS-2
may be downstream of the insulin receptor (IR) or IGF-1
receptor (IGF-1R). Studies of mice with ?-cell–specific
inactivation of either receptor (17–19) have indicated that
the insulin receptor was important for compensatory
growth of the ?-cells in response to insulin resistance,
he number of pancreatic islet ?-cells as well as
their capacity to secrete insulin is modulated in
normal physiological conditions to respond to
the metabolic demand of the organism (1). A
From the1Department of Physiology and Center for Integrative Genomics,
University of Lausanne, Lausanne, Switzerland; and the
Physiology and Department of Cellular Biology and Morphology, Biology
and Medicine Faculty, University of Lausanne, Lausanne, Switzerland.
Corresponding author: Bernard Thorens, bernard.thorens@unil.ch.
Received 13 January 2009 and accepted 20 April 2009.
Published ahead of print at http://diabetes.diabetesjournals.org on 28 April
2009. DOI: 10.2337/db09-0063.
© 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.
2Department of
ORIGINAL ARTICLE
1816DIABETES, VOL. 58, AUGUST 2009

Page 2

whereas the IGF-1 receptor was involved in the control of
glucose competence.
Here we studied islets from mice with inactivation of
both the GLP-1 and GIP receptor genes. We showed them
to be more sensitive than control islets to cytokine-
induced apoptosis. By comparative transcriptomic analy-
sis of islets from control and mutant mice, we found that
expression of the IGF-1R was markedly decreased in the
mutant islets. In control islets, GLP-1–activated IGF-1R
expression and signaling and GLP-1–induced IGF-1R sig-
naling required autocrine secretion of IGF-2. We provided
evidence that increasing the activity of this autocrine loop
was required for GLP-1 to protect ?-cells against apopto-
sis. These data provide an integrated description of the
interaction between the GLP-1 and IGF-1 receptor signal-
ing pathways that operate to protect ?-cells against
apoptosis.
RESEARCH DESIGN AND METHODS
Mice. Male C57BL/6 and GipR?/?;Glp-1-R?/?mice backcrossed for seven
generations into the C57BL/6 background were used between 8 and 10 weeks
C
wt
wt +
GLP-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
wtwt +
GLP-1
***
IGF-1R
ß chain
ß-actin
Expression level (a.u.)
0.2
0.4
0.6
0.8
1.0
1.2
0.0
wtdKO
***
Expression level (a.u.)
qRT-PCR
BA
wt mice
dKO mice
GLP-1
Cytokines (low)
Cytokines (high)
0
5
10
15
20
25
30
35
***
***
***
-
-
-
-
+
-
-
+
Apoptosis (%)
***
***
-
-
+
+
***
Expression level (a.u.)
0.0
0.5
1.0
1.5
wtdKO
**
IGF-1R
ß chain
ß-actin
wtdKO
D
FIG. 1. Gluco-incretin signaling controls susceptibility to apoptosis and IGF-1R expression. A: Islets from control or GipR?/?;Glp-1R?/?mice were
exposed or not to cytokines (IL-1?, TNF-?, and IFN-?) at low or high concentrations (5, 12.5, and 5 ng/ml or 10, 25, and 10 ng/ml, respectively)
and in the presence or absence of GLP-1 (100 nmol/l). B: Quantitative RT-PCR analysis of IGF-1R mRNA expression in islets from control (wt)
and GipR?/?;Glp-1R?/?mice (dKO). C: Reduced expression of IGF-1R expression in GipR?/?;Glp-1R?/?mouse islets as detected by Western blot
analysis. Lower panel: Quantitation of the expression data. D: GLP-1 (100 nmol/l) treatment for 18 h increased IGF-1R expression as detected
by Western blot analysis. Lower panel: Quantitation of the expression data. For all panels, data are means ? SD, n ? 3 independent experiments.
**P < 0.01; ***P < 0.001.
M. CORNU AND ASSOCIATES
DIABETES, VOL. 58, AUGUST 20091817

Page 3

of age. All experimental procedures received approval from the Service
Ve ´te ´rinaire du Canton de Vaud.
Antibodies. Rabbit polyclonal antibody against actin was purchased from
Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20;
sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312)
were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit mono-
clonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit
polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271),
phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were
from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2
(ab10731) was purchased from Abcam (Cambridge, U.K.).
Pancreatic islet isolation. Islets were isolated according to published
procedures (20) using a Histopaque density gradient. Islets were cultured
overnight at 37°C in 5% CO2in RPMI-1640, 10% FCS, 2 mmol/l glutamax, 100
units/ml penicillin, and 100 ?g/ml streptomycin. ?-Cells were purified by cell
sorter as described (21).
Microarray and quantitative PCR analyses. Islets were isolated from 8-to
10-week-old mice and kept in culture overnight before RNA extraction. Total
islet RNA was extracted using RNeasy Mini kit (Qiagen); purity and quality
were assessed by an Agilent bioanalyzer. After linear amplification with T7
polymerase, the RNA was reverse transcribed and labeled with Cy3 or Cy5 and
hybridized to cDNA arrays containing 17,664 different probes, prepared by the
University of Lausanne DNA Array Facility (www.unil.ch/dafl). Hybridization,
data acquisition, and quality control were performed as previously described
(22). Differentially expressed genes were identified using linear models
implemented in the Limma package from Bioconductor (http://www.
bioconductor.org). All statistical analyses were performed using the R soft-
ware (http://www.r-project.org). Real-time quantitative PCR was performed
using Light Cycler Technology (Roche, France). Primers sets were chosen to
amplify products of ?200 bp. cDNAs were obtained from 2.5 ?g total RNA
with SuperScript II RNAse H- Reverse Transcriptase (Invitrogen, Carlsbad,
CA) and 50 pmol random hexamer (Applied Biosystem). cDNA amplification
was performed in a 20-?l reaction mixture including 1? QuantiTect SYBR
Green PCR Master Mix (Qiagen) and 10 pmol of each primer. Primers for
mouse IGF-1 (sense, 5?-GGCATTGTGGATGAGTGTTG-3?; antisense, 5?-AGT-
TGCCTCCGTTACCTCCT-3?), mouse IGF-2 (sense, 5?-GTCGATGTTGGTGCT-
TCTCA-3?; antisense,5?-AAGCAGCACTCTTCCACGAT-3?),
cyclophilin (sense, 5?-TCCATCGTGTCATCAAGGAC-3?; antisense, 5?-CTTGC-
CATCCAGCCAGGAG-3?) were used. All measurements were normalized to
cyclophilin.
MIN6 cell culture and secretion tests. MIN6 cells were grown in Dulbec-
co’s modified Eagle’s medium, 15% FCS, 2 mmol/l glutamine, and 50 ?mol/l
?-mercaptoethanol and used between passages 19 and 30. For pharmacolog-
ical treatments, 106MIN6 cells were plated in 6-cm-diameter tissue culture
dishes for 4 days, the medium was replaced, and cells were incubated for 2 h
at 37°C before addition of various reagents at the concentrations and for the
periods of time indicated in the figure legends.
For gene silencing, MIN6 cells were transfected 1 day after plating with 0.5
?g of a green fluorescent protein (GFP) expression plasmid and siRNAs at a
final concentration of 100 nmol/l using lipofectamine (Invitrogen, Carlsbad,
CA). After 6 h at 37°C, the growth medium was replaced, and the cells were
used 48 h later.
For secretion tests, MIN6 cells were seeded at 106cells in sixwell plates.
Three days later, cells were washed and preincubated for 2 h in Krebs-Ringer
bicarbonate HEPES buffer (120 mmol/l NaCl, 4 mmol/l KH2PO4, 20 mmol/l
HEPES, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 5 mmol/l NaHCO3, and 0.5% BSA, pH
7.4) supplemented with 2 mmol/l glucose. Then, the medium was replaced
with Krebs-Ringer bicarbonate HEPES buffer containing 2 or 20 mmol/l
glucose for an additional hour. Secretion and cellular IGF-2 (extracted in
PBS-Tween 5%) was assessed by ELISA (no. DY702, R&D; Minneapolis, MN).
Gel electrophoresis and immunoblotting. MIN6 cells were placed on ice,
washed twice with PBS, and lysed for 30 min on ice in 50 mmol/l Tris (pH 7.5)
and mouse
B
A
IGF-1R
ß chain
ß-actin
Akt-Pi
(T308)
Akt-Pi
(S473)
Akt
Bad-Pi
Bad
siRNA:
GLP-1:
Un
+
IGF1-R
--+
UnIGF1-R
******
0
0.5
1
1.5
2
2.5
3
Akt phosphorylation
(S473) level (a.u.)
0
1
2
3
4
***
***
Bad phosphorylation
level (a.u.)
siRNA:
GLP-1:
Un
+
IGF1-R
--+
Un IGF1-R
siRNA:
GLP-1:
Un
+
IGF1-R
--+
Un IGF1-R
IGF1-R
ß chain
ß-actin
Akt-Pi
(T308)
Akt
0
1
2
3
4
5
6
7
***
IGF-1R expression
level (a.u.)
***
***
***
***
0
1
2
3
4
5
6
7
8
Akt phosphorylation
level (a.u.)
***
***
***
***
***
0
GLP-1
6 1218 2424 (hrs)6 12 182424 (hrs)0
GLP-1
6 12 182424 (hrs)0
GLP-1
FIG. 2. GLP-1 increased IGF-1R expression and signaling in MIN6 cells. A: MIN6 cells were treated with GLP-1 for the indicated periods of time,
and IGF-1R expression and Akt phosphorylation were analyzed by Western blot analysis. B: MIN6 cells were transfected with a control (Un) or
an IGF-1R siRNA and treated for 18 h with GLP-1. GLP-1 induced IGF-1 receptor expression, Akt phosphorylation on both T308 and S473, and
Bad phosphorylation. Preventing IGF-1R expression suppressed GLP-1–induced Akt and Bad phosphorylation. For all panels, data are means ?
SD, n ? 3 independent experiments. ***P < 0.001.
GLP-1–INDUCED AUTOCRINE ACTIVATION OF IGF-1R
1818 DIABETES, VOL. 58, AUGUST 2009

Page 4

containing 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton, 10 mmol/l ?-glycerol-
phosphate, 1 mmol/l Na3VO4, 50 mmol/l NaF, 5 mmol/l phenylmethylsulfonyl
fluoride, 10 ?g/ml aprotinin, leupeptin, and pepstatin. The lysates were
centrifuged for 15 min at 13,000 rpm at 4°C.
Mouse islets were washed twice with PBS and lysed in 160 mmol/l Tris (pH
6.8) containing 5 mmol/l EDTA, 20% glycerol, 10% ?-mercaptoethanol, 10%
SDS, 1 mmol/l Na3VO4, 50 mmol/l NaF, 5 mmol/l phenylmethylsulfonyl
fluoride, and 10 ?g/ml aprotinin. Lysates were sonicated for 15 s before
protein determination. Western blot analysis was performed as described (21)
with antibodies diluted in Tris-buffered saline–5% nonfat dry milk (1/500
dilution for IGF-1R, IR and phosphorylated Akt [Thr308 and Ser473], and
phosphorylated Bad and 1/1,000 for actin, total Akt, total Bad, and IGF-2) and
revealed using horseradish peroxidase–conjugated donkey anti-rabbit IgG or
horseradish peroxidase–conjugated sheep anti-mouse IgG as secondary anti-
bodies (Amersham, Buckinghamshire, U.K.). Band intensities were deter-
mined using a Bio-Rad densitometer (Strasbourg, France).
Apoptosis assay. Apoptosis was induced by exposing the MIN6 cells or islets
to cytokines at the indicated concentrations for 24 h; GLP-1(7–36)amide (100
nmol/l) was added 4 h before the cytokines and was present for the entire
cytokine incubation period (24 h). Apoptosis was assessed by scoring the
number of cells expressing GFP and displaying pyknotic nuclei labeled with
Hoechst 33342 (10 ?g/ml) (23). Islets were first dissociated in 100 ?l
dissociation buffer (Gibco, product number 13151-014) for 15 min at 37°C
before apoptosis was assessed.
Adenovirus. Recombinant adenoviruses expressing enhanced GFP alone
(Ad-GFP) or together with IGF-1R (Ad-IGF-1R) were generated using the
AdEasy system (24). Islets were infected with a multiplicity of infection of 40
in 500 ?l of medium for 3 h at 37°C and then cultured for 48 h before insulin
secretion tests, as described above. Preliminary experiments showed that
?85% of islet cells were infected and that infection did not affect glucose-
induced insulin secretion.
Statistical analysis. All experiments were performed at least three times.
Results are expressed as means ? SD. Comparisons were performed using
unpaired Student’s t test or one-way or two-way ANOVA for the different
groups followed by post hoc pair-wise multiple-comparison procedures
(Tukey test or Bonferroni, respectively).
RESULTS
Increased susceptibility to apoptosis of islets from
GipR?/?;Glp-1R?/?mice. To evaluate whether absence
of both gluco-incretin hormone receptor expression led to
increased susceptibility to cytokine-induced apoptosis,
islets from GipR?/?;Glp-1-R?/?mice were exposed to low
or high concentrations of cytokines (interleukin [IL]-1?,
tumor necrosis factor [TNF]-?, and interferon [IFN]-?),
and the percent of apoptotic cells was determined. Figure
1A shows that islets from mutant mice were significantly
more sensitive than control islets to apoptosis induced by
the low dose of cytokine; at the high dose, apoptosis was
similar in both types of islets. As expected, GLP-1 pro-
tected control but not mutant islets against cytokine-
induced apoptosis.
Gluco-incretinscontrol
signaling. To search for genes differentially expressed in
control and mutant islets and that could be involved in the
increased susceptibility to cytokine-induced apoptosis, we
performed microarray analysis of transcripts expressed in
control and mutant islets. We found that the IGF-1R mRNA
was downregulated in the mutant islets (Fig. 1B) and that
IGF-1R protein expression was also markedly decreased
(Fig. 1C). This suggested that expression of the IGF-1R
IGF-1Rexpression and
IGF-1R
ß chain
IR
ß chain
ß-actin
Akt-Pi
(T308)
Akt
12345678
0
1
2
3
4
5
***
***
**
*
*
***
siRNA:
--
Un IGF-1RIR Un IGF-1R IR
GLP-1:
-+---+++
Akt phosphorylation
level (a.u.)
A
B
00.2 2 20 200
[nM]
Insulin
IGF1-R
ß chain
ß-actin
Akt-Pi
(T308)
GLP-1: -+-- - - - - - -+ + + + + + + +
0.2 2 20200
IGF-2
0 0.2 2 20 200
Insulin
0.2 2 20 200
IGF-2
Akt
0
1
2
3
4
5
6
7
8
9
0 0.2220 200
[nM]
Insulin
IGF-2
GLP-1 + Insulin
GLP-1 + IGF-2
***
*
§§
§§
§§§
§§§
###
###
###
###
C
Akt phosphorylation
level (a.u.)
FIG. 3. GLP-1–induced Akt phosphorylation depends on IGF-1R but not
IR expression. A: MIN6 cells were transfected with control (Un, lanes
3 and 6), IGF-1R (lanes 4 and 7), or IR-specific (lanes 5 and 8) siRNAs
and exposed (?) or not (?) to GLP-1 for 18 h before Western analysis.
Lanes 1 and 2 show the induction by GLP-1 of IGF-1R expression in
nontransfected cells. Lane 3 shows the basal level of IGF-1R and IR
expression in transfected cells; reducing IGF-1R expression (lane 4)
but not the IR (lane 5) reduced Akt phosphorylation. Lane 6: GLP-1–
treated cells showed higher expression of the IGF-1R but not of the IR;
reducing IGF-1R expression (lane 7) but not IR (lane 8) reduced Akt
phosphorylation. Bottom panel: Quantitation of the data. Data are
means ? SD, n ? 3 independent experiments. **P < 0.01; ***P < 0.001.
B: MIN6 cells were treated or not for 18 h with GLP-1 to increase
IGF-1R expression, then incubated with 2 mmol/l glucose for 2 h, and
exposed for 15 min to the indicated concentrations of insulin or IGF-2.
IGF-1R and total and phosphorylated Akt were determined by Western
blot analysis. C: Quantification of the data in B. *P < 0.05, ***P < 0.001
for IGF-2 vs. insulin; §§P < 0.01, §§§P < 0.001 for IGF-2 in control vs.
GLP-1–treated cells; ###P < 0.001 for IGF-2 vs. insulin in GLP-1–
treated cells. a.u., arbitrary units.
M. CORNU AND ASSOCIATES
DIABETES, VOL. 58, AUGUST 20091819

Page 5

0
1
2
3
4
Akt phosphorylation
level (a.u.)
***
***
***
Nimodipine
Diazoxide
-
-
-
-
+
-
-
+
Glucose (mM)2 2020 20
B
IGF-1R
ß chain
ß-actin
Akt-Pi
(T308)
Akt
0
1
2
3000
4000
5000
8000
9000
10000
qRT-PCR
Expression level (a.u.)
isletsβ cells
***
IGF-1
IGF-2
ND
C
A
IGF-1R
ß chain
ß-actin
Akt-Pi
(T308)
Akt
1234
1234
5678
Akt phosphorylation
level (a.u.)
**
***
***
**
***
***
GLP-1
Nimodipine
Diazoxide
-
-
-
2
-
-
-
-
+
-
20
-
-
+
20
+
-
-
2
+
-
-
20
+
+
-
20
+
-
+
20Glucose (mM)20
0
2.5
5
7.5
10
12.5
**
D
GLP-1
Nimodipine
Diazoxide
-
-
-
2
-
-
-
-
+
-
20
-
-
+
20
Glucose (mM)
20
IGF-2 secretion (% of content)
+
-
-
2
+
-
-
20
+
+
-
20
+
-
+
20
0
5
10
15
20
25
30
35
***
***
***
***
***
***
***
FIG. 4. Activation of Akt phosphorylation by GLP-1 depends on glucose-induced secretion. A: MIN6 cells were incubated for 18 h with GLP-1 to
induce IGF-1R expression, then with 2 mmol/l glucose for 2 h, and finally for 1 h with 2 or 20 mmol/l glucose with or without GLP-1 and nimodipine
(1 ?mol/l) or diazoxide (200 ?mol/l). Bottom panel: Quantitation of the data. B: Pancreatic islets were treated as the MIN6 cells in A. C: qRT-PCR
analysis of IGF-1 and IGF-2 mRNA expression in mouse islets and cell sorter–purified ?-cells. D: Secretion of IGF-2 by MIN6 cells incubated to
2 or 20 mmol/l glucose and in the presence of nimodipine, diazoxide, or GLP-1, as indicated. Total IGF-2 content was 10 ng/4 ? 106cells. A–D: Data
are means ? SD, n ? 3 independent experiments. **P < 0.01; ***P < 0.001. a.u., arbitrary units.
GLP-1–INDUCED AUTOCRINE ACTIVATION OF IGF-1R
1820 DIABETES, VOL. 58, AUGUST 2009

Page 6

could be regulated by gluco-incretin hormones. Figure 1D
shows that GLP-1 indeed induced a marked increase in
IGF-1 receptor expression in control islets.
To investigate in more detail the regulation by gluco-
incretins of IGF-1R expression and signaling, we used
MIN6 cells as a model system and concentrated our study
on the effect of GLP-1, since GLP-1 and GIP use the same
signaling pathway. We first demonstrated that, as in pri-
mary islets, GLP-1 increased IGF-1R expression with max-
imal induction reached after ?18 h of treatment (Fig. 2A).
In addition, we found that induction of IGF-1R expression
was associated with a corresponding increase in Akt
phosphorylation on Thr308 (Fig. 2A). We then showed that
GLP-1 treatment also induced Akt phosphorylation on
Ser473 and the phosphorylation of Bad, a proapoptotic
protein inactivated by phosphorylation by IGF-1R signal-
ing (25,26) (Fig. 2B). We also determined that preventing
IGF-1R expression by transfecting the cells with an IGF-
1R–specific siRNA suppressed GLP-1–induced Akt phos-
phorylation on both sites and the phosphorylation of Bad
(Fig. 2B). GLP-1 treatment of the cells induced an approx-
imately threefold increase in IRS-2 expression (see Fig. S1
in an online-only appendix, available at http://diabetes.
diabetesjournals.org/cgi/content/full/db09-0063/DC1).
Because ?-cells also express the IR and secrete insulin,
we searched for direct evidence that the IGF-1R, and not
the IR, was responsible for the induction of Akt phosphor-
ylation. First, we transfected MIN6 cells with IGF-1R– or
IR-specific siRNAs before treating the cells with GLP-1 for
18 h and analyzing Akt phosphorylation. Figure 3A (lanes
1 and 2) shows that GLP-1 induced expression of the
IGF-1R but not the IR. The receptor-specific, but not the
unrelated, siRNAs led to a reduction of either IGF-1R or IR
expression in the basal state (in the absence of GLP-1
stimulation; Fig. 2A, lanes 3–5) or after GLP-1 treatment
(Fig. 2A, lanes 6–8). Importantly, Akt phosphorylation in
the basal state or after GLP-1 treatment was reduced only
when the IGF-1R, but not the IR expression, was de-
creased (lanes 4 and 7 vs. lanes 5 and 8).
To further evaluate the respective roles of the IGF-1R
and IR in activating the Akt pathway, we measured the
efficiency of insulin and IGF-2 to activate Akt phosphory-
lation in MIN6 cells. Indeed, insulin binds to the IR with
high affinity (Kd?1 nmol/l) and to IGF-1R with an ?100-
fold lower affinity, whereas IGF-2 binds to the IGF-1R with
high affinity (?1 nmol/l) and with much lower affinity to
the IR (27). MIN6 cells were thus pretreated or not with
GLP-1 for 18 h and kept in the presence of 2 mmol/l
glucose for 2 h to prevent insulin secretion. The cells were
then exposed to different concentrations of each hormone
for 15 min, and IGF-1R expression and Akt phosphoryla-
tion were determined (Fig. 3B). The data show that in the
Glucose (mM):
Antibody:
2
-
20
IgG anti-IGF-2
20
0
0.5
1
1.5
2
2.5
Akt phosphorylation level (a.u.)
*** **
Pro IGF-2
IGF-1R
ß chain
Akt-Pi
(T308)
Akt
ß-actin
Mature
IGF-2
B
MIN6
Glucose (mM):
Antibody:
2
-
20
IgG
20
anti-IGF-2
Akt phosphorylation level (a.u.)
**
**
0
0.5
1
1.5
2
2.5
IGF-1R
ß chain
ß-actin
Akt-Pi
(T308)
Akt
Pro IGF-2
C
Islets
Akt phosphorylation level (a.u.)
0
0.5
1
1.5
2
2.5
3
***
***
shRNA:
Glucose (mM):
Un
2
Un
20
IGF-2
2
IGF-2
20
A
MIN6
IGF-1R
ß chain
Akt-Pi
(T308)
Akt
ß-actin
Pro IGF-2
Mature
IGF-2
FIG. 5. GLP-1–increased Akt phosphorylation depends on secreted IGF-2. A: MIN6 cells were transfected with an unrelated (Un) or an
IGF-2–specific shRNA. Forty-eight hours later, they were stimulated with GLP-1 for 18 h to increase IGF-1R expression and then incubated with
2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose. B: MIN6 cells were stimulated with GLP-1 for 18 h and then incubated with
2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with nonspecific IgGs or an IGF-2 blocking antibody. C: Pancreatic islets
were stimulated with GLP-1 for 18 h and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with
nonspecific IgGs or an IGF-2 blocking antibody. A–C: Data are means ? SD, n ? 3 independent experiments. **P < 0.01; ***P < 0.001. a.u.,
arbitrary units.
M. CORNU AND ASSOCIATES
DIABETES, VOL. 58, AUGUST 20091821

Page 7

0
10
20
30
40
50
60
Apoptosis (%)
***
***
***
GLP-1: -
cytokines: -
-
+
-
-
+
-
-
-
+
+
-
-
-
-
+
-
-
+
+
-
+
+
+
-
-
-
-
+
-
+
-
+
+
+
-
+
Unrelated siRNA: -
IGF-1R siRNA: -
A
MIN6
B
GLP-1: -
cytokines: -
-
+
-
-
+
-
-
-
+
+
-
-
-
-
+
-
-
+
+
-
+
+
+
-
-
-
-
+
-
+
-
+
+
+
-
+
Unrelated shRNA: -
IGF-2 shRNA: -
Apoptosis (%)
0
10
20
30
40
50
60
******
***
MIN6
C
GLP-1:
cytokines:
Antibody:
-
-
-
-
+
-
+
+
+
+
IgGanti-IGF-2
Apoptosis (%)
***
***
**
0
2
4
6
8
10
MIN6
D
wt mice
βIGF-1R KO mice
GLP-1:
cytokines:
-
-
-
+
+
+
Apoptosis (%)
***
***
0
5
10
15
20
25
30
35
***
Islets
E
0
5
10
15
20
25
GLP-1:
cytokines:
Antibody:
-
-
-
-
+
-
+
+
+
+
IgG anti-IGF-2
Apoptosis (%)
***
***
***
Islets
cytokines:
***
0
5
10
15
20
25
30
35
-+
Apoptosis (%)
F
Ad GFP
Ad IGF-1R
***
***
Islets
FIG. 6. GLP-1–induced protection against cytokine-induced apoptosis depends on activation of an IGF-2/IGF-1R autocrine loop. A: MIN6 cells
were transfected with an unrelated or an IGF-1R siRNA, and 48 h later they were incubated in the presence or absence of GLP-1 for 4 h before
addition of cytokines (IL-1?, 10 ng/ml; TNF-?, 25 ng/ml; IFN-?, 10 ng/ml); the incubations were continued for 24 h in the continuous presence of
GLP-1. B: MIN6 cells were transfected with an unrelated or an IGF-2 shRNA and then treated as described for A. C: MIN6 cells were incubated in the
presence or absence of GLP-1 and in the presence of a nonspecific IgG fraction or an IGF-2 blocking antibody for 4 h before addition of cytokines (see
above). D: Islets from ?-cell–specific Igf-1R?/?mice or from their control littermates were incubated in the presence or absence of GLP-1
GLP-1–INDUCED AUTOCRINE ACTIVATION OF IGF-1R
1822DIABETES, VOL. 58, AUGUST 2009

Page 8

absence of GLP-1 pretreatment, IGF-2 was significantly
more efficient in inducing Akt phosphorylation than insu-
lin (Fig. 3B and C). After GLP-1 treatment, IGF-1R expres-
sion was markedly augmented and IGF-2 induced Akt
phosphorylation with a greater efficacy than in nontreated
cells; insulin-induced Akt phosphorylation, however, was
not increased.
Together, the above data indicated that GLP-1–induced
Akt phosphorylation was dependent on IGF-1R upregula-
tion and independent of IR expression.
IGF-1R activation is triggered by autocrine secre-
tion of IGF-2. To determine whether activation of the
IGF-1R signaling pathway was dependent on secretion,
we evaluated the level of Akt phosphorylation in cells,
which had previously been treated for 18 h with or
without GLP-1 to increase IGF-1R expression, then
exposed for 2 h to a low glucose concentration, and then
exposed to low or high glucose concentrations in the
presence or absence of secretion inhibitors. We first
evaluated Akt phosphorylation in cells pretreated or not
pretreated with GLP-1. In the absence of pretreatment,
high glucose induced an approximately threefold in-
crease in Akt phosphorylation, whereas this increase
was ?12-fold in GLP-1–treated cells (see Fig. S2 in the
online appendix and Fig. 4A). The increased phosphor-
ylation at 20 mmol/l glucose was suppressed by the
ATP-sensitive K?(KATP) channel opener diazoxide or
the Ca2?channel inhibitor nimodipine (Fig. 4A). When
the cells were exposed to GLP-1 during the last incuba-
tion period, Akt phosphorylation was higher than in the
absence of GLP-1 but equally suppressed by the secre-
tion inhibitors. To determine if the same regulation of
Akt phosphorylation was observed in primary ?-cells,
mouse islets were similarly treated with GLP-1 for 18 h
and then exposed to 2 or 20 mmol/l glucose in the
presence or absence of nimodipine and diazoxide. As
for MIN6 cells, phosphorylation of Akt was very low at
2 mmol/l glucose, increased at 20 mmol/l glucose, and
suppressed by the secretion inhibitors (Fig. 4B). Thus,
these observations indicated that activation of Akt
phosphorylation was dependent on active secretion.
Three ligands can potentially activate the IGF-1R: insu-
lin, IGF-1, and IGF-2. Because the data of Fig. 3 indicated
that activation of Akt phosphorylation was through the
IGF-1R but not the IR, it was highly unlikely that insulin
was the ligand activating Akt phosphorylation. On the
other hand, IGF-1 was not detectable by RT-PCR in MIN6
cells (not shown) and was expressed at an extremely low
level in islets and not detectable in cell sorter–purified
?-cells (Fig. 4C); IGF-1 was therefore not a candidate for
activating the IGF-1 receptor signaling pathway in our
experiments. Previous reports described the expression of
IGF-2 in mature ?-cells (28,29), and we confirmed that
IGF-2 mRNA was expressed at high level in islets and
purified ?-cells (Fig. 4C). Furthermore, incubation of MIN6
cells at high glucose triggered IGF-2 secretion in the
culture medium; this secretion was blocked by diazoxide
and nimodipine and was increased when GLP-1 was
present (Fig. 4D).
We thus tested whether IGF-2 was involved in activa-
tion of Akt phosphorylation in MIN6 cells exposed to
GLP-1. MIN6 cells were first transfected with an IGF-2–
specific or an unrelated shRNA, then exposed to GLP-1
for 18 h to induce IGF-1R expression, and finally ex-
posed to 2 or 20 mmol/l glucose before analysis of Akt
phosphorylation. Figure 5A shows that expression of
pro–IGF-2 and mature IGF-2 was strongly decreased by
transfection of the specific shRNA, and this suppressed
the induction of Akt phosphorylation in cells exposed to
high glucose. As a second test for an autocrine role of
IGF-2 secretion in Akt phosphorylation, MIN6 cells were
treated as in the previous experiment but the final
incubation with 20 mmol/l glucose was conducted in the
presence of an IGF-2 blocking antibody or an unrelated
IgG fraction. Figure 5B shows that the IGF-2 blocking
antibody suppressed Akt phosphorylation. The same
inhibition of Akt phosphorylation by the IGF-2 neutral-
izing antibody was made using primary islets (Fig. 5C).
Thus, both in MIN6 cells and primary islets, activation of
Akt phosphorylation by GLP-1 requires secretion of
IGF-2 under these conditions. These data also indicated
that an IGF-2/IGF-1R autocrine loop operated in ?-cells,
and its activity could be increased by GLP-1 by enhanc-
ing IGF-1R expression and by stimulating secretion in
the presence of high glucose.
GLP-1 signaling protects ?-cells against apoptosis by
increasing the IGF-2/IGF-1R autocrine loop. To deter-
mine whether the protective effect of GLP-1 against cyto-
kine-induced apoptosis was dependent on activation of the
IGF-2/IGF-1R autocrine loop, we measured apoptosis in
MIN6 cells exposed to cytokines and treated or not treated
with GLP-1. Figure 6A shows that cytokine-induced apo-
ptosis was suppressed by treatment with GLP-1 and that
this protective effect was blunted when IGF-1R expression
was prevented by transfection of a specific siRNA. The
same blunting of the GLP-1 protective effect was obtained
when the cells were transfected with an shRNA that
specifically suppressed IGF-2 expression (Fig. 6B) or
when the cells were incubated in the presence of an IGF-2
blocking antibody (Fig. 6C).
To determine whether GLP-1 protected primary islets
using the same IGF-2/IGF-1R autocrine loop, we per-
formed three sets of experiments. First, we tested
whether GLP-1 protected islets from mice with a ?-cell–
specific IGF-1R gene inactivation against cytokine-in-
duced apoptosis. As shown in Fig. 6D, apoptosis was not
suppressed by GLP-1 in these islets—in contrast to the
marked protection observed in control islets. Second,
we showed that GLP-1 protection against cytokine-
induced apoptosis was also suppressed when the islets
were incubated in the presence of the neutralizing IGF-2
antibody (Fig. 6E). Third, we overexpressed the IGF-1R
in islets from GipR?/?;Glp-1R?/?mice using a recom-
binant adenovirus (see Fig. S3 in the online appendix).
Cytokine-induced apoptosis was markedly reduced in
IGF-1R–overexpressing islets compared with control-
infected cells (Fig. 6F). Thus, both in MIN6 cells and in
primary islets, GLP-1 protected ?-cells against cytokine-
induced apoptosis by modulating the activity of the
IGF-2/IGF-1R autocrine loop.
for 4 h before addition of cytokines (see above). E: Control islets were incubated for 4 h in the presence or absence of GLP-1 and with a
nonspecific or an IGF-2 blocking antibody before cytokines treatment. F: Islets from GipR?/?;Glp-1R?/?mice were infected with a control
adenovirus (Ad-GFP) or an IGF-1R–expressing adenovirus (Ad-IGF1-R) and 1 day later exposed to cytokines, as described in A. A–F: Data are
means ? SD, n ? 3 independent experiments. **P < 0.01; ***P < 0.001.
M. CORNU AND ASSOCIATES
DIABETES, VOL. 58, AUGUST 2009 1823

Page 9

DISCUSSION
GLP-1 has previously been demonstrated to protect ?-cells
against apoptosis by increasing IRS-2 expression (14,16),
PI 3-kinase activity (30), Akt phosphorylation (31) and
Foxo1 phosphorylation, and nuclear exclusion (32). Our
data are in agreement with these previous studies but
present an integrated view of the operation of the GLP-1
and IGF-1R signaling pathways by providing novel findings
on 1) the regulation of IGF-1R expression by GLP-1, 2) the
predominant role of the IGF-1R compared with the IR in
GLP-1–induced Akt phosphorylation, 3) the role of IGF-2
as an autocrine regulator of this signaling pathway, and 4)
the modulation of this autocrine loop by GLP-1 as a basis
for this hormone antiapoptotic effect.
Induction of IGF-1R expression by GLP-1 follows rela-
tively slow kinetics, with a peak expression at ?18 h. This
is in contrast with the induction of immediate early genes,
whose expression peaks between 30 and 60 min after
initiation of GLP-1 treatment (21). Thus, increased expres-
sion of the IGF-1 receptor after GLP-1 treatment may be
due to transcriptional or posttranscriptional regulations
that are secondary to the initial wave of GLP-1–mediated
transcriptional events. A detailed understanding of these
mechanisms will, however, need further investigation. The
slow induction of IGF-1R expression is thus an adaptive
process, which, in vivo, may link the amount of GLP-1
secreted daily to the ?-cell susceptibility to apoptosis.
Because there is a link between feeding and GLP-1 secre-
tion (33), reduced susceptibility to apoptosis may also
participate in the increased ?-cell mass observed in obe-
sity. This slow induction kinetics should also be consid-
ered when using GLP-1 to protect ?-cells from apoptosis
after islet transplantation (34). To be efficacious, this
treatment may need to be started before transplanting the
islets to ensure full expression of the IGF-1R and maximal
antiapoptotic protection.
A question that had not previously been addressed is the
mechanism by which GLP-1–induced IRS-2 expression
leads to activation of its downstream signaling pathway.
Our data show that GLP-1–induced Akt phosphorylation
was suppressed by knocking-down IGF-1R but not IR
expression; furthermore, the relative efficacy of insulin
and IGF-2 induction of Akt phosphorylation in cells stim-
ulated by GLP-1 supported a primary role of the IGF-1R.
Thus, our data indicate that the IGF-1R but not the IR lies
upstream of activated Akt in our experimental conditions.
Phosphorylation of Akt in GLP-1–treated cells required
secretion of IGF-2, as supported by several data. First, the
IR is not involved in GLP-1–induced Akt phosphorylation,
and insulin is therefore unlikely to be responsible for the
autocrine activation of the IGF-1R; second, IGF-1 is not
expressed in MIN6 cells nor in cell sorter–purified ?-cells
and is present at extremely low levels in isolated islets,
which is in agreement with the reported expression of
IGF-1 in non–?-cells (35); third, IGF-2 mRNA is detected at
relatively high levels in primary islets and in cell sorter–
purified ?-cells, and its secretion can be stimulated by
glucose, which is in agreement with previous data showing
the presence of IGF-2 in insulin granules (29,36).
The importance of secreted IGF-2 in activating IGF-1R
signaling was further demonstrated by showing that IGF-2
downregulation or neutralization with a specific antibody
suppressed GLP-1–induced Akt phosphorylation. Further-
more, we showed that IGF-2 was responsible for GLP-1–
induced protection against apoptosis, since this protective
effect was suppressed by IGF-2 knockdown, by antibody-
mediated blocking of secreted IGF-2, or by suppressing
IGF-1R expression. This was observed both in MIN6 cells
and in pancreatic islets.
This proposed autocrine role of IGF-2 in protecting
?-cells against apoptosis is in agreement with previous
data suggesting a role of this hormone in the physiological
regulation of ?-cells during embryonic development (36)
and with the reported link between reduced IGF-2 expres-
sion by the pancreas during the fetal life and impaired
?-cell function in the adult GK rat (37,38). Also, in the
neonatal period, when the endocrine pancreas goes
through a wave of ?-cell apoptosis, there is a decreased
expression of IGF-2 in the islets (39). This wave of
apoptosis can, however, be prevented by transgenic ex-
pression of IGF-2 in peripheral tissues, which leads to
elevated circulating plasma levels of this hormone (40).
Also, transgenic expression of IGF-2 in ?-cells leads to
increased ?-cell mass in adult mice (41). However, a role
for IGF-2 in the control of adult islet function has not been
previously established.
Therefore, several lines of evidence support an auto-
crine role for secreted IGF-2 in the control of ?-cell mass
both during embryonic development and after birth. Inter-
estingly, recent genome-wide association studies have
identified Igf2bp2 as a gene associated with increased
incidence of type 2 diabetes (42). IGF2BP2 is an IGF-2
mRNA binding protein that regulates the translation of this
mRNA (43), suggesting that the control of IGF-2 expres-
sion may also participate in regulating ?-cell physiology.
Together, our studies provide an integrated view of the
GLP-1 and IGF-1R signaling pathways. They show that an
IGF-2/IGF-1R autocrine loop operates in ?-cells and that
GLP-1 increases its activity by augmenting IGF-1R expres-
sion, a long-term effect, and also by acutely stimulating
IGF-2 secretion. These results therefore have important
pathophysiological implications, since they can lead to
alternative ways of preventing ?-cell mass decrease: by
modulating IGF-1R expression and/or expression and se-
cretion of IGF-2.
ACKNOWLEDGMENTS
This work was supported by grants to B.T. from the Swiss
National Science Foundation (3100A0-113525), the Juve-
nile Diabetes Research Foundation (Program Project
7-2005-1158), and the European Union (Integrated Project
Eurodia LSHM-CT-2006-518153, Framework Programme 6
[FP6] of the European Community).
No potential conflicts of interest relevant to this article
were reported.
We thank the University of Lausanne DNA Array Facility
for help with cDNA microarray experiments, D. Accili and
A. Efstratiadis for ?-IGF1-R knockout mice, and C.B.
Wollheim for helpful comments on the manuscript.
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