Glucose-Dependent Insulinotropic Polypeptide Is a Growth Factor for ß (INS-1) Cells by Pleiotropic Signaling
Andrea Trümper,
Katja Trümper,
Heidi Trusheim,
Rudolf Arnold,
Burkhard Göke and
Dieter Hörsch
Department of Internal Medicine (A.T., K.T., H.T., R.A.,
D.H.), Division of Gastroenterology and Metabolism,
Philipps-University, Marburg, Germany D-35033; and Department of
Medicine II (B.G.), Ludwig Maximilians University, Munich, Germany
D-81377
Address all correspondence and requests for reprints to: Dieter Hörsch, M.D., Department of Internal Medicine, Division of Gastroenterology and Metabolism, Philipps-University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail:
hoerschd{at}post.med.uni-marburg.de
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ABSTRACT
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Activation of the G-protein-coupled receptor for glucose-dependent
insulinotropic polypeptide facilitates insulin-release from pancreatic
ß-cells. In the present study, we examined whether glucose-dependent
insulinotropic polypeptide also acts as a growth factor for the
ß-cell line INS-1. Here, we show that glucose-dependent
insulinotropic polypeptide induced cellular proliferation
synergistically with glucose between 2.5 mM and 15
mM by pleiotropic activation of signaling pathways.
Glucose-dependent insulinotropic polypeptide stimulated the
signaling modules of PKA/cAMP regulatory element binder, MAPK, and
PI3K/protein kinase B in a glucose- and dose-dependent manner. Janus
kinase 2 and signal transducer and activators of transcription 5/6
pathways were not stimulated by glucose-dependent insulinotropic
polypeptide. Activation of PI3K by glucose-dependent insulinotropic
polypeptide and glucose was associated with insulin receptor substrate
isoforms insulin receptor substrate-2 and growth factor bound-2
associated binder-1 and PI3K isoforms p85
, p110
, p110ß, and
p110
. Downstream of PI3K, glucose-dependent insulinotropic
polypeptide-stimulated protein kinase B
and protein kinase Bß
isoforms and phosphorylated glycogen synthase kinase-3, forkhead
transcription factor FKHR, and p70S6K. These data indicate
that glucose-dependent insulinotropic polypeptide functions
synergistically with glucose as a pleiotropic growth factor for
insulin-producing ß-cells, which may play a role for metabolic
adaptations of insulin-producing cells during type II diabetes.
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INTRODUCTION
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THE GLUCO-INCRETIN EFFECT results in higher
insulin response and consecutive smaller increase in blood sugar after
an oral glucose load compared with intravenous administration. Two
major insulinotropic incretin hormones have been characterized:
glucagon-like peptide-1 (GLP-1) and
glucose-dependent insulinotropic polypeptide (GIP) (1, 2).
GLP-1 is synthesized in entero-endocrine L-cells of the
ileum and colon and released into circulation as bioactive truncated
GLP-1 (736 amide). GLP-1 receptors are
expressed on pancreatic ß-cells, and activation of GLP-1
receptors at high glucose levels results in potentiation of
glucose-dependent insulin secretion and activation of insulin gene
transcription (1, 2). The incretin effect of
GLP-1 is preserved in type II diabetes mellitus, which is
currently exploited in clinical studies for the therapy of type II
diabetes mellitus (2). In addition to its insulinotropic
characteristics, GLP-1 also functions as a growth and
differentiation factor for pancreatic ß-cells by pleiotropic
activation of mitogenic signaling modules (3, 4, 5, 6, 7, 8).
GIP is synthesized in duodenal K cells as a 42-amino acid peptide and
secreted as a hormone by the stimulation of the duodenum with
nutrients, mainly by fat and glucose. The GIP receptor belongs the
family of G protein-linked seven-transmembrane receptors and is highly
expressed on ß-cells. Stimulation of GIP receptors induces a rise in
cAMP and intracellular calcium, which facilitates glucose-dependent
insulin release from pancreatic ß-cells physiologically (1, 9, 10, 11). However, the incretin effect of GIP is not preserved in
type II diabetes or in relatives of type II diabetic subjects
(1). Thus, defective insulinotropic signaling by the GIP
receptor has been implicated in the pathogenesis of type II diabetes
(1, 12). In addition to its role as an insulinotropin, GIP
is involved in metabolic regulation of insulin-sensitive tissues by
sensitizing adipose cells to insulin (12). Furthermore,
several lines of evidence indicate a function of GIP as a growth and
metabolic factor for ß-cells. In Chinese hamster ovary (CHO) cells
stably expressing the GIP receptor, GIP activates MAPK, which could be
partially blocked by wortmannin, an inhibitor of PI3K
(13). Wortmannin also partially inhibits GIP-mediated
insulin secretion in ß-cells providing indirect evidence that GIP
signaling in ß cells may involve activation of mitogenic lipid kinase
PI3K (14). A knockout of the GIP receptor demonstrated not
only a defect in entero-insular axis but also the failure of ß cells
to adapt metabolically to insulin resistance (15).
Although it was not reported whether islet size is decreased in GIP
receptor knockout mice, a recent study showed that dominant negative
overexpression of the GIP receptor in pancreatic ß-cells leads to
diminished islet size (Göke, B., and A. Volz, personal
communication).
In the light of these studies, we examined whether GIP acts as a
ß-cell growth factor using the differentiated ß-cell line INS-1
(16). In addition, we elucidated patterns of signaling by
GIP on major mitogenic signaling modules in ß- cells, namely the
pathways of PKA/cAMP regulatory element binder (CREB), MAPK,
PI3K/protein kinase B (PKB), and janus kinase 2 (JAK2)/signal
transducers and activators of transcription 5/6 (STATs5/6).
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RESULTS
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Mitogenic Effects of GIP and Glucose
To determine whether GIP and glucose act as growth factors for
ß-cells, proliferation of INS-1 cells was determined by rate of DNA
synthesis by enzyme-linked detection of 5-bromo-deoxyuridine (BrdU)
incorporation (17). BrdU incorporation at 2.5
mM glucose without GIP served as control and was set at 1.
The addition of glucose at rising concentrations in the absence of GIP
induced a dose-dependent increase in proliferation, which was maximal
at 15 mM glucose (2.8 ± 0.14 mean ±
SD of control; n = 12; Fig. 1A
) and stagnated at higher
concentrations of glucose up to 25 mM (data not shown).
Addition of 10-7 M GIP instigated a
further 1.5- to 1.9-fold rise in INS-1 cell proliferation at glucose
concentrations between 2.5 and 15 mM (Fig. 1A
). GIP-induced
INS-1 cell proliferation was absent at 0 mM glucose and
stagnated at glucose concentrations higher than 15 mM (data
not shown). Statistical analysis by ANOVA revealed that the increase of
proliferation at basal levels and after stimulation with GIP were
always highly significant when compared with control levels at 2.5
mM glucose. Furthermore, the GIP-induced rise in
proliferation was highly significant when compared with respective
basal levels (Fig. 1A
).

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Figure 1. Mitogenic Effects of GIP and Glucose
A, Proliferation of INS-1 cells stimulated with a glucose gradient and
GIP. INS-1 cells were starved overnight and stimulated for 24 h
with 10-7 M GIP at 2.5 mM, 7.5
mM, and 15 mM glucose. DNA synthesis was
measured by adding BrdU for the last 6 h of the stimulation period
and subsequent detection by ELISA. Each bar represents
the mean ± SD of 12 independent experiments. They are
expressed as relative to control assigning a value of 1 to cells
stimulated with 2.5 mM glucose in the absence of GIP. B,
Dose-response of INS-1 cell proliferation by GIP at 15 mM
glucose. INS-1 cells were stimulated with 15 mM glucose and
a GIP gradient between 10-10 and 10-6
M at 15 mM glucose for 24 h. Cellular
proliferation was measured by BrdU incorporation. Each
bar represents the mean ± SD of six
independent experiments. They are expressed as relative to control
assigning a value of 1 to cells stimulated with 15 mM
glucose. Statistical analysis was performed by ANOVA. n.s.,
Nonsignificant, P = 0.08 for 10-10 and
10-9 M GIP; *, P <
0.05; **, P < 0.005.
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To elucidate whether mitogenic effects by GIP were dose-dependent,
INS-1 cells were stimulated by a rising concentration of GIP between
10-10 M and 10-6
M at 15 mM glucose. At low concentrations of
10-10 M and
10-9 M, GIP induced only a minor
rise in BrdU-incorporation, which was not statistically significant in
the ANOVA analysis. However at higher concentrations of GIP between
10-8 M and
10-6 M, INS-1 cell proliferation
increased 1.4- to 1.5 fold, which was highly statistically significant
(Fig. 1B
).
Activation of PKA/CREB by GIP and Glucose
Activation of the PKA/CREB signaling module was measured by
phosphorylation of CREB in a transactivation assay using luciferase as
a reporter gene (Fig. 2A
) and by
immunoblotting with an antibody specific for phosphorylated CREB at
serine133 (Fig. 2
, BD). Dose-response of
GIP-induced CREB phosphorylation was examined at 2.5 mM and
15 mM glucose (Fig. 2A
). CREB phosphorylation at 2.5
mM glucose served as control and was set at 1. Elevation of
glucose to 15 mM initiated a 4.1-fold rise in CREB
phosphorylation (Fig. 2A
). At 2.5 mM glucose, GIP
instigated a dose-dependent increase in CREB phosphorylation, which was
maximal at 10-7 M by 21-fold with an
EC50 of approximately 10-8
M. At 15 mM glucose, GIP stimulated CREB
phosphorylation in a dose-dependent manner in a similar pattern as at
2.5 mM, although at a much higher level of activation (Fig. 2A
). Here, maximal activation was 38-fold at
10-6 M with an
EC50 between 10- 9
M and 10-8 M GIP. At
both glucose concentrations, a sharp rise in CREB phosphorylation was
observed between 10-9 M and
10-8 M and a slower elevation at
higher concentrations of GIP (Fig. 2A
). Additional elevation of glucose
concentrations above 15 mM did not further increase basal
and GIP-stimulated CREB phosphorylation (data not shown). ANOVA
analysis revealed that glucose-induced rise of basal CREB
phosphorylation was highly statistically significant. Furthermore,
GIP-induced elevation of CREB phosphorylation was significant
(P < 0.05) or highly significant (P <
0.005) at all GIP and glucose concentrations compared with basal levels
and also when GIP-induced CREB phosphorylation was compared between low
(2.5 mM) and high (15 mM)
glucose concentrations (Fig. 2A
).

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Figure 2. Activation of the PKA/CREB Signaling Module by GIP
and Glucose
A, Dose response of GIP-induced CREB phosphorylation at low (2.5
mM) and high (15 mM) glucose concentrations.
INS-1 cells were transfected with CREB transactivator plasmid as
described in Materials and Methods. INS-1 cells were
stimulated for 16 h with different concentrations of GIP and
glucose. CREB phosphorylation was determined by the luciferase activity
of a cotransfected reporter plasmid. The luciferase activity of INS-1
cells stimulated with 2.5 mM glucose without GIP served as
control and was set at 1. Each bar represents the
mean ± SD of four to five independent experiments.
Statistical analysis was performed by ANOVA. *, P
< 0.05; **, P < 0.005. B, Time course of CREB
phosphorylation by GIP. INS-1 cells were starved overnight and
stimulated with 10-7 M GIP at indicated times.
Cells were lysed and 100 µg of proteins were separated by SDS-PAGE
and immunoblotted by Western analysis. The degree of CREB
phosphorylation was determined using activation-specific antibody for
pCREB Ser133. Proteins were detected using enhanced
chemiluminescence, and band densities were quantified by densiometry.
Data represent a typical blot of n = 3. They are expressed as
relative to control assigning a value of 1 to nonstimulated cells. C,
Dose response of CREB phosphorylation by GIP. Experiment was performed
as in panel B except that cells were stimulated with indicated
concentrations of GIP for 60 min. D, Glucose dependency of CREB
phosphorylation by GIP. INS-1 cells were stimulated with
10-7 M GIP at 2.5 mM, 7.5
mM, and 15 mM glucose for 60 min. CREB
phosphorylation of INS-1 cells stimulated with 2.5 mM
glucose only was set at 1.
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The kinetics of GIP-induced CREB phosphorylation was examined by
immunoblotting. CREB phosphorylation was rapid with an almost 4-fold
rise in phosphorylation at 10 min. Maximal phosphorylation was
increased 7-fold at 60 min. After 60 min, CREB phosphorylation
decreased down to almost control levels at 120 min (Fig. 2B
). The dose
response of CREB phosphorylation demonstrated by immunoblotting (Fig. 2C
) was similar to that shown by transactivating luciferase assays
(Fig. 2A
). In the immunoblot, maximal phosphorylation of CREB was
observed at 10-7 M GIP with an
EC50 at about 10-8 M GIP. Again,
there was a sharp elevation in CREB phosphorylation between
10-9 M and
10-8 M GIP (Fig. 2C
) as in the
transactivating luciferase assay (Fig. 2A
). Glucose dependency of
GIP-stimulated CREB phosphorylation was examined by immunoblotting
using a glucose gradient of 2.5 mM, 7.5 mM, and
15 mM glucose and by stimulation with
10-7 M GIP (Fig. 2D
). Elevation of
glucose from 2.5 mM to 15 mM instigated a rise
in CREB phosphorylation to 3.2-fold compared with the control, which
was further stimulated 2- to 3-fold above basal levels by the addition
of GIP (Fig. 2D
). Since elevation of glucose without GIP also
stimulated CREB phosphorylation, we examined the kinetic
glucose-stimulated CREB phosphorylation. When 7.5 or 15 mM
glucose was added after glucose starvation of INS-1 cells of 24 h,
increased phosphorylation was only noted after 6 h and was maximal
by 12 h (data not shown), implicating a much slower kinetics of
glucose-induced CREB phosphorylation compared with the CREB activation
by GIP. Therefore, to determine the glucose dependency of CREB
phosphorylation by GIP in the immunoblot analysis, we equilibrated
INS-1 cells in 7.5 and 15 mM glucose concentration
overnight. Equal loading of protein lysates was verified by
immunoblotting for nonphosphorylated CREB (data not shown).
Activation of MAPK by GIP and Glucose
Activation of the MAPK pathway signaling module was examined by
phosphorylation of transcription factor Elk-1 in a transactivation
assay using luciferase as a reporter gene (Fig. 3A
) and by immunoblotting with an
antibody specific for phosphorylated MAPK [extracellular
signal-regulated kinases 1 and 2 (ERK 1/2)] (Fig. 3
, BD). The
experimental design was as described for CREB phosphorylation. Glucose
at 15 mM stimulated Elk-1 phosphorylation 4.6-fold. At low
and high glucose, GIP induced a maximal Elk-1 phosphorylation at
10-6 M (10-fold at 2.5
mM and 23-fold at 15 mM). However, the
EC50 was slightly shifted to the left at 15
mM glucose (2.5 mM glucose:
EC50 at 10-8 M
GIP; 15 mM glucose: EC50 between
10-9 M and
10-8 M GIP; Fig. 3A
). As in the case
of CREB phosphorylation, there was a sudden approximately 2-fold rise
in Elk-1 phosphorylation between 10-9
M and 10-8 M GIP
(compare Figs. 2
and 3
). ANOVA analysis revealed that glucose-induced
Elk-1 phosphorylation was highly statistically significant. Elk-1
phosphorylation by GIP became significant at
10-10 at 15 mM glucose and at
10-9 at 2.5 mM glucose and was
highly statistically significant at higher concentrations of GIP. The
difference between GIP-induced CREB activation at low (2.5
mM) and high (15 mM) glucose concentrations was
always highly significant (Fig. 3A
).

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Figure 3. Activation of the MAPK Signaling Module by GIP and
Glucose at the Level of Transcription Factor Elk-1 (A) and the MAPK
ERK-1 and ERK-2 (BD)
A, Dose response of GIP-induced Elk-1 phosphorylation at low (2.5
mM) and high (15 mM) glucose concentrations.
INS-1 cells were transfected with Elk-1 transactivator plasmid as
described in Materials and Methods. INS-1 cells were
stimulated for 16 h with different concentrations of GIP and
glucose. Elk-1 phosphorylation was determined by the luciferase
activity of a cotransfected reporter plasmid. The luciferase activity
of INS-1 cells stimulated with 2.5 mM glucose without GIP
was set at 1. Each bar represents the mean ±
SD of four to five independent experiments. Statistical
analysis was performed by ANOVA. *, P < 0.05; **,
P < 0.005. B, Time course of ERK phosphorylation
by GIP. INS-1 cells were starved overnight and stimulated with
10-7 M GIP at indicated time points. Cells
were lysed and 100 µg of proteins were separated by SDS-PAGE and
immunoblotted by Western analysis. The degree of ERK phosphorylation
was determined using activation-specific antibody for pTyr
(204) of ERK-1 and ERK-2. Proteins were detected using
enhanced chemiluminescence, and band densities were quantified by
densiometry. Data represent a typical blot of n = 3. They are
expressed as relative to control, assigning a value of 1 to
nonstimulated cells. C, Dose-response of ERK phosphorylation by GIP.
Experiment was performed as in panel B except that cells were
stimulated with indicated concentrations of GIP for 60 min. D, Glucose
dependency of ERK phosphorylation by GIP. INS-1 cells were stimulated
with 10-7 M GIP at 2.5 mM, 7.5
mM, and 15 mM glucose for 60 min. ERK
phosphorylation of INS-1 cells stimulated with 2.5 mM
glucose only was set at 1.
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The kinetics, dose response, and glucose response of MAPK activation
were examined by immunoblotting for activated MAPK kinases ERK 1/2
(Fig. 3
, BD). GIP at 10-7 M
instigated a fast rise in ERK phosphorylation at 10 min, which was
maximal between 30 and 90 min and decreased at 120 min (Fig. 3B
). A
dose response similar to that for Elk-1 phosphorylation was
demonstrated for ERK with a maximal phosphorylation at
10-7 M and
10-6 M GIP with an
EC50 between 10-9
M and 10-8 M (Fig. 3C
).
Elevation of glucose to 7.5 mM and 15 mM
stimulated basal ERK phosphorylation 2.4-fold, which was further
increased by the addition of GIP (Fig. 3D
). Kinetics and dose responses
of glucose-induced Elk-1 and ERK phosphorylation were similar to that
described for CREB phosphorylation. Equal loading of protein lysates
was verified by immunoblotting for nonphosphorylated ERK (data not
shown).
Activation of PI3K/PKB by GIP and Glucose
Stimulation of PI 3K by GIP and glucose was examined at the level
of signaling molecules known to activate PI3K using antibodies for
phosphorylated tyrosine (pY), insulin receptor substrate (IRS) isoforms
IRS-1, IRS-2, and growth factor bound-2 associated binder-1 (Gab-1)
(18, 19). In addition, PI3K activation was elucidated at
the level of PI3K applying antibodies for PI3K regulatory subunit
p85
and catalytic subunits p110
, p110ß, and p110
(18, 19, 20, 21). INS-1 cells were stimulated at 2.5
mM, 7.5 mM, and 15 mM glucose
overnight and subsequently with 10-7
M GIP for 60 min, since we could show that GIP-induced PI3K
activation was maximal at this time point (data not shown). Cell
lysates were subjected to immunoprecipitation, and PI3K activity was
determined as described in Materials and Methods. PI3K
activation by GIP was difficult to reproduce at 2.5
mM glucose. Thus, only stimulation of PI3K by GIP
at 7.5 mM and 15 mM glucose
was included in the analysis. Basal levels of PI3K were elevated by
increasing glucose concentration to 15 mM, which
was most pronounced for Gab-1- and p110ß-associated PI3K activity
(Fig. 4
). The addition of GIP instigated
modest amplifications in PI3K activity at 7.5 mM
glucose except for p110
- associated PI3K activity, which was
stimulated 4.1-fold (Fig. 4G
). At 15 mM glucose,
a more robust amplification of PI3K activity in all immunoprecipitates
was observed, most notably in anti-Gab-1 and anti-p110
associated
PI3K activity (Fig. 4
). No stimulation of GIP-induced PI3K activity was
associated with anti-IRS-1, anti-insulin receptor ß-chain, anti-JAK2,
and p85ß immunoprecipitates (data not shown).
The serine-threonine kinase PKB is activated by PI3K. To elucidate
whether PKB is stimulated by GIP in ß-cells, PKB activation was
detected by immunoblotting INS-1 cell lysates with an
activation-specific antibody for pPKBSer473. This method
was used to examine the kinetic (Fig. 5A
), dose response (Fig. 5B
), and glucose
dependency (Fig. 5C
) of PKB stimulation by GIP. Compared with PKA/CREB
and MAPK, PKB activation by GIP exhibited slower kinetics with a
maximal 2.6- to 3.5-fold phosphorylation between 30 and 90 min and a
decline to almost control levels at 360 min (Fig. 5A
). ANOVA revealed a
highly significant elevation in PKB phosphorylation between 30 and 120
min (Fig. 5A
). The dose-response curve of PKB phosphorylation by GIP
demonstrated maximal 2.6-fold phosphorylation between
10-8 M and
10-6 M with a sudden rise of
activation between 10-9 M and
10-8 M, which also corresponded to
the EC50 (Fig. 5B
). At 10-9
M, GIP-induced PKB phosphorylation became highly
statistically significant. Using a glucose gradient of 2.5
mM, 7.5 mM, and 15 mM, we could
show that basal and GIP-stimulated PKB phosphorylation increased with
rising glucose concentrations (Fig. 5C
) similar to the glucose response
of CREB and ERK phosphorylation (Figs. 2D
, 3D
, and 5C
). Statistical
analysis by ANOVA revealed that the increase in basal levels and after
stimulation with GIP by elevation of glucose was statistically
significant as well as the difference between basal levels and
GIP-induced PKB phosphorylation at 2.5 mM, 7.5
mM, and 15 mM glucose (Fig. 5C
). Equal loading
of protein lysates was verified by immunoblotting for nonphosphorylated
PKB
(data not shown).

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Figure 5. Activation of PKB by GIP and Glucose
A, Time course of PKB phosphorylation by GIP. INS-1 cells were starved
overnight and were stimulated with 10-7 M GIP
at 11 mM glucose. Cells were lysed and 100 µg of proteins
were separated by SDS-PAGE and immunoblotted by Western analysis. The
degree of PKB phosphorylation was determined using activation-specific
antibody for pSer473 PKB as described in
Materials and Methods. Immunoblots using an antiserum
recognizing total PKB served as a control for equal loading.
Proteins were detected using enhanced chemiluminescence, and band
densities were quantified by densiometry. Data are the mean ±
SEM of 610 independent experiments. They are expressed as
relative to control, assigning a value of 1 to nonstimulated cells at
360 min. B, Dose-response of GIP-induced PKB phosphorylation.
Experiment was performed as in panel A except that cells were
stimulated with indicated concentrations of GIP for 60 min. C, Glucose
dependency of PKB phosphorylation by GIP. INS-1 cells were stimulated
with 10-7 M GIP at 2.5 mM, 7.5
mM, and 15 mM glucose for 60 min. PKB
phosphorylation of INS-1 cells stimulated with 2.5 mM
glucose only was set at 1. Statistical analysis was performed by ANOVA.
*, P < 0.05; **, P < 0.005.
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Multiple isoforms of PKB are expressed in pancreatic ß-cells
(21, 22) and may be differentially activated by glucose
and GIP. We therefore examined the isoform-specific stimulation of PKB
using specific antisera for PKB
, PKBß, and PKB
to perform
isoform-specific PKB assays in immunoprecipitates of glucose and
GIP-stimulated INS-1 cell lysates as described in Materials and
Methods. GIP induced activation of PKB serine kinase activity
glucose dependently between 7.5 mM and 15
mM glucose in PKB
- and PKBß- (Fig. 6A
and B) but not in
PKB
-immunoprecipitates (not shown). The transcription factor FKHR
and cytoplasmatic kinases, glycogen-synthase 3
/ß (GSK-3) and
p70S6K (21) are downstream targets
of PKB. To find out whether FKHR, GSK-3, and
p70S6K are stimulated by glucose and GIP, we used
phosphorylation-specific antibodies for activated FKHR, GSK, and
p70S6K. All targets of PKB were phosphorylated by
stimulation by glucose and GIP at 7.5 and 15 mM
glucose (Fig. 6
, CF). We noticed in the PKB assays that native GSK-3
was coimmunoprecipitated by PKB
and PKBß antibodies (data not
shown) in a similar time course as the phosphorylation in straight cell
lysates (Fig. 6D
), indicating that GSK is associated with stimulated
PKB and that PKB is a GSK kinase. Equal loading of protein lysates was
verified by immunoblotting for nonphosphorylated GSK-3, FKHR, and
p70S6K (data not shown).

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Figure 6. Activation of PKB Isoforms PKB and PKBß, the
Transcription Factor FKHR, and Cytoplasmatic Kinases GSK-3 and
p70S6K by GIP and Glucose
Cells were starved overnight and stimulated with 10-7
M GIP for 60 min at 7.5 and 15 mM glucose. A
and B, Equal amounts of cell lysates were subjected to
immunoprecipitation with antibodies to PKB (A) and PKBß (B). PKB
assays were performed as described in Materials and
Methods. Shown are representative autoradiographs of n =
3. They are expressed as relative to control assigning a value of 1 to
nonstimulated cells at 7.5 mM glucose. Phosphorylation of
FKHR (C), GSK-3 (D), and p70S6K (E and F) was determined by
immunoblotting using phosphorylation-specific antibodies. Shown are
representative autoradiographs of n = 3.
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JAK/STAT Pathways
GH is a major mitogen of ß-cells by the activation of JAK/STAT
signaling module, especially JAK2 and STAT5 (23, 24). To
determine whether GIP shares this mitogenic pathway with GH, we
stimulated INS-1 cells with 10-8 M human
recombinant GH and 10-7 M GIP and
immunoblotted cell lysates with phosphorylation-specific antibodies for
tyrosine701 of STAT1,
serine727 of STAT3,
tyrosine705 of STAT3,
tyrosine694 of STAT5, and
tyrosine641 of STAT6. Neither human GH nor GIP
induced phosphorylation of STAT1 and STAT3. In contrast, GIP failed to
phosphorylate STAT5 and STAT6, which were phosphorylated by stimulation
with GH in INS-1 cells, indicating that GIP does not activate the
mitogenic JAK2/STATs5/6 pathway in INS-1 cells (data not shown).
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DISCUSSION
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Insulin-secreting ß-cells of the pancreas are highly specialized
cells with a low mitogenic index. The ß-cell pool may be replenished
by neogenesis of ß-cells from precursor cells in pancreatic ducts or
by islet cell replication (25, 26, 27). IGF-I, GH, and PRL
have been characterized as growth factors for ß-cells. Elevated
glucose levels also induce cellular proliferation in ß-cells
(23, 24, 28, 29, 30, 31, 32). Recently, it could be shown that the
gluco-incretin hormone GLP-1 acts as a growth,
antiapoptotic, and differentiation factor for ß-cells in animal
models, in isolated islets, and clonal ß-cells (4, 5, 6, 7, 8).
These results prompted us to examine whether and how the other
gluco-incretin hormone GIP acts as growth factor for ß-cells. Here,
we show that GIP induced ß-cell proliferation in the ß-cell line
INS-1 by pleiotropic signaling. Our results indicate that GIP may be
added to the growing list of ß-cell growth factors. Recent animals
studies corroborate the hypothesis of GIP as a ß-cell growth factor.
Selective expression of a dominant negative mutant of the human
GIP receptor in ß-cells as a transgene yielded diminished islet size
and overt diabetes in mice (Göke, B., and A. Volz, personal
communication). In addition, the failure of GIP receptor knockouts to
respond to high fat diet with hyperinsulinemia like wild-type mice
points to a role of GIP signaling in the regulation of appropriate
ß-cell mass in insulin resistance (15).
The GIP receptor belongs to the family of G protein-coupled
seven-transmembrane receptors and is expressed not only in pancreatic
ß-cells but also in brain, adipose tissue, intestine, and heart
(9, 10). Stimulation of the GIP receptor leads to rise of
cytosolic cAMP by the activation of membrane-bound adenylate cyclase
and a rise in intracellular Ca++
(9, 10, 11). We examined whether GIP activates major mitogenic
signaling modules and demonstrated pleiotropic stimulation of PKA/CREB,
p44/p42 MAPK, and PI3K/PKB signaling pathways by the GIP receptor in a
similar dose response. These effects were synergistic with glucose
between 2.5 mM and 15 mM. In contrast, there
was no evidence of JAK2/STATs5/6 pathway activation by GIP in INS-1
cells (Fig. 7
). These results indicate
that signaling of ß-cell growth factors is transduced by pleiotropic,
yet specific, pathways. Receptor tyrosine kinases such as
insulin and IGF-I receptors activate PI3K/PKB and MAPK
(32, 33, 34). Cytokine-like receptors such as the GH receptor
stimulate JAK2/STAT5 and PI3K (23, 24, 30, 35). Finally, G
protein-coupled receptors such as GIP- and GLP-1 receptors
induce activation of PKA/CREB, MAPK, and PI3K/PKB (3, 31, 36), but not JAK/STAT pathways (Fig. 7
). In this context, it is
interesting to note that mitogenic signaling pathways activated by both
receptors for GIP and GLP-1 are similar in ß-cells
(3, 31, 35, 36), indicating a mitogenic signaling
redundancy of both incretin hormones (37).
Glucose concentration in the stimulation medium was essential for
the activation of all signaling modules examined. Synergistic effects
of glucose and GIP were additive in the immunoblotting analysis of
CREB, ERK1/2, and PKB phosphorylation with a relative short stimulation
of 60 min whereas transactivating luciferase assays for CREB and Elk-1
demonstrated superadditive effects at a stimulation period of 16
h. Thus, the superadditive effects seen in the transactivating
luciferase assays may be caused by the accumulation of reporter
luciferase. On the other hand, detailed analysis of PI3K signal
transmission by glucose and GIP revealed additive and superadditive
effects, implying differential substrate specificity of glucose and GIP
induced signal transduction by PI3K. Blockage of glucokinase by alloxan
(38) and prevention of glucose phosphorylation by
2-deoxy-D-glucose (39) indicated signal
transduction by derivatives of glucose metabolism in ß-cells (Ref.
40 and Trümper, A., and D. Hörsch; unpublished
results). Clearly, ATP generated from pyruvate in mitochondria and
glutamate (40) are favorite candidates as mitogenic and
antiapoptotic second messengers of glucose-induced signaling as well as
for glucose-induced synergism in GIP signaling. However, it has to kept
in mind that activation of signal transduction pathways by glucose may
not be the result of a second messenger-like ATP but reflect the
altered ADP:ATP ratio in ß-cells at high glucose levels
(40). ATP is a necessary cofactor for kinase reactions
(41). The increased availability of ATP for kinase
reactions at high glucose levels may facilitate kinase reactions and
thus mimic a second messenger effect.
Growth factors signal to the nucleus by cytoplasmatic signaling
cascades inducing the transcription of cell-specific sets of genes by
the activation of transcription factors (23, 24, 25, 28, 29, 42, 43). Here, we showed that GIP and glucose phosphorylate
transcription factors CREB, Elk-1, and FKHR. In addition, glucose and
GIP phosphorylated p70S6K, a crucial regulator of
translation (44). These data indicate that mitogenic
effects of glucose and GIP in ß-cells may not only be caused by
activation of transcription but also by initiation of protein
translation. Recently, it has been shown that a major part of the
mitogenic response of ß-cells to glucose may be attributed to the
activation of p70S6K (45).
Furthermore, a knockout of p70S6K1 leads to
diminished ß-cell size and glucose resistance in mice
(44), indicating that stimulation of
p70S6K is essential for regulation of ß-cell
growth.
Altered ß-cell function is pivotal for the development of type 2
diabetes. As the disease develops, the ability of ß-cells to secrete
compensatory amounts of insulin decreases, thereby increasing
hyperglycemia and insulin resistance (27, 46, 47, 48). Thus,
mitogenic and antiapoptotic activation of G protein-coupled receptors,
strongly expressed on ß-cells, may prove to be a therapeutic approach
for late stages of type II diabetes. Although, short-term antidiabetic
effects of GIP are not preserved in type II diabetes and glucose
intolerance due to lack of insulinotropic action (1),
long-term application of GIP may prove to be an therapeutic approach
for type II diabetes. We have shown that GIP is a growth and
antiapoptotic factor for ß-cells by pleiotropic activation of
PKA/CREB, MAPK, and PI3K/PKB pathways. Thus, long-term application of
GIP may be beneficial in the treatment of type II diabetes as a
ß-cell growth factor by improving ß-cell function.
 |
MATERIALS AND METHODS
|
---|
Materials
Silica-gel TLC plates were obtained from Merck & Co., Inc. (Darmstadt, Germany), protein G-agarose was from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Nitrocellulose paper (Optitran BA-S85) was from Schleicher & Schuell, Inc. (Keene, NH) and
[
-32P]ATP was from Amersham Pharmacia Biotech (Arlington Heights, IL). GIP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) and
recombinant human GH were obtained from Bachem (Bubendorf,
Switzerland). 5-Bromo-deoxyuridine (BrdU) incorporation and cell death
detection ELISAs were from Roche (Mannheim, Germany).
New England Biolabs, Inc. (Beverly, MA) supplied the
PKB
antiserum, phospho-specific antibodies for
serine473 and threonine308
of PKB
, serine133 of CREB,
serine411 of p70S6K,
threonine421/serine424 of
p70S6K, tyrosine701 of
STAT1, serine727 of STAT3,
tyrosine705 of STAT3,
tyrosine694 of STAT5,
tyrosine641 of STAT6, and
serine21/9 of glycogen-synthase kinase 3
/ß
(GSK-3), and GSK-3
/ß cross-tide protein, along with respective
control antibodies for nonphosphorylated kinases as well as enhanced
chemiluminescence reagents. Antibodies for Gab-1, insulin receptor
substrate (IRS) IRS-1, IRS-2, p110
, p110ß, p85
, pY, and
PKB-isoforms
, ß, and
were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies for phosphorylated ERK-1 and
ERK-2 (pERK Tyr204), control ERK-antibodies, and
p110
antibodies were from Santa Cruz Biotechnology, Inc.. Reagents for SDS-PAGE were from Bio-Rad Laboratories, Inc. (Hercules, CA), cell culture reagents were
from Life Technologies, Inc.(Karlsruhe, Germany), and all
other chemicals were from Sigma (St. Louis, MO).
Cell Culture
INS-1 cells (passage 80120) were grown as previously described
(16) in regular RPMI-1640 medium supplemented with 10%
FBS, 10 mM HEPES, 1 mM sodium pyruvate, 50
µM ß-mercaptoethanol, 100 IU/ml penicillin, and 100
µg/ml streptomycin at 37 C in a humidified (5%
CO2, 95% air) atmosphere. Before stimulation,
INS-1 cells were starved in medium without serum, glucose, and sodium
pyruvate.
BrdU Incorporation
Cells were seeded at a density of 3 x
103 in 96-well plates, grown for 24 h in
regular medium, washed once with 10 mM PBS (pH 7.4), and
subsequently starved for 24 h. They were then incubated for
24 h in RPMI medium with different glucose concentrations and test
substances. During the last 6 h of stimulation, 20 µl of a BrdU
solution was added and ELISA (17) was performed according
to guidelines provided by the manufacturer.
Transreporting System for Elk1 and CREB Phosphorylation
INS-1 cells were grown for 48 h in normal medium in
six-well plates until they reached 6080% confluency. Cells were then
washed twice with PBS, transfected with luciferase reporter gene
(pFR-Luc) and either Elk1 (pFA-2-Elk1) or CERB (pFA2-CREB)
transactivator domains (all from Stratagene, La Jolla, CA)
by lipid-based transfection (Pfx-6; Invitrogen, Groningen,
The Netherlands) for 8 h in INS-1 medium without serum.
Subsequently, cells were grown in INS-1 medium with 5 mM
glucose and 5% FBS and then stimulated for 16 h in INS-1 medium
containing 1% FBS with GIP at different glucose concentrations.
Immunoprecipitation and Immunoblotting
INS-1 cells were starved for 12 h and were then
equilibrated for another 12 h in the indicated glucose
concentrations. One hour before stimulation, the stimulation medium was
changed. Cells were lysed after stimulation in ice-cold lysis buffer
(1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 100
mM sodium pyrophosphate, 100 mM sodium
fluoride, 10 mM EDTA, 5 mM sodium vanadate, 10
µg/ml aprotinin, 5 µg/ml leupeptin, 1.5 mg/ml benzamidine, and 34
µg/ml phenylmethylsulfonyl fluoride) and sonicated for 15 sec, and
insoluble material was removed by centrifugation at 15,000 rpm in a
microfuge for 10 min. For immunoblotting, 100 µg of protein per lane
were separated by 10% SDS-PAGE, Western-transferred on nitrocellulose
membranes, and immunoblotted as previously described (18).
For immunoprecipitation experiments, 500 µg of protein lysate were
immunoprecipitated with indicated antibodies and 60 µl of protein G-
or protein A-agarose, respectively, for 24 h at 4 C. Beads were
washed twice with protein lysis buffer and either used immediately for
SDS-PAGE or frozen at -80 C. Protein bands were visualized with
enhanced chemiluminescence. Autoradiographs were scanned, and band
density was determined using Gelscan 3D software (BioSciTec, Marburg,
Germany). Statistical analysis was performed by ANOVA.
PI3K Assays
Immune-complexed PI3K activity was determined as previously
described (18). Immune complexes were incubated in a 55
µl reaction mixture containing 200 µM ATP, 5 µCi
[
-32P]ATP, 200 mM
MgCl2, and 5 µg phosphatidylinositol for 20 min
at room temperature. Reactions were stopped by the addition of 150 µl
of CHCl3/CH3OH/11.6
N HCl (33:66:0.6) and subsequently of 120 µl of
CHCL3. The organic phase was washed once with 150
µl of CH3OH/1 N HCl (1:1), 20 µl
8 N HCl, and 160 µl CHCl3/methanol
1:1. The organic phase was removed by centrifugation and applied to
silica gel TLC plates, developed in
CHCl3/CH3OH/H2O/NH4OH
(60:47:11.3:2), dried, and visualized by autoradiography. The
band representing phosphatidylinositol 3-P was quantified as
described.
PKB Assays
Immunocomplexed PKB activity was determined by incubating washed
antibodies with 200 µmol/liter ATP and 1 µg GSK3
/ß cross-tide
fusion protein. Phosphorylation of the substrate was determined by
immunoblotting with a phosphorylation-specific antibody for
serine21/9 of GSK3
/ß at 30 kDa (New England Biolabs, Inc.).
 |
ACKNOWLEDGMENTS
|
---|
We thank A. Volz and R. Göke for critical reading of the
manuscript and H. Schmidt for valuable technical support.
 |
FOOTNOTES
|
---|
Parts of this study are contained in the medical thesis of A.
Trümper.
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft to D.H. (Ho 1762/2-1).
Abbreviations: BrdU, 5-bromo-deoxyuridine; CREB, cAMP
regulatory element binder; ERK, extracellular signal-regulated kinase;
Gab-1, growth factor bound-2 associated binder-1; GIP,
glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like
peptide 1; GSK, glycogen-synthase kinase; IRS, insulin receptor
substrate; JAK, janus kinase; PKB, protein kinase B; pY, phosphorylated
tyrosine; STAT, signal transducer and activator of transcription.
Received for publication January 29, 2001.
Accepted for publication May 17, 2001.
 |
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