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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GLP-1 is synthesized in entero-endocrine L-cells of the ileum and colon and released into circulation as bioactive truncated GLP-1 (7–36 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) 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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).
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, B–D). 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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). 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, B–D). 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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, B–D). 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).
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), 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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, 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, C–F). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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).
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 80–120) 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 60–80% 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 2–4 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.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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to the protein kinases PKB and MAPK. Science 282:293–296This article has been cited by other articles:
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S.-J. Kim, C. Nian, and C. H. S. McIntosh Activation of Lipoprotein Lipase by Glucose-dependent Insulinotropic Polypeptide in Adipocytes: A ROLE FOR A PROTEIN KINASE B, LKB1, AND AMP-ACTIVATED PROTEIN KINASE CASCADE J. Biol. Chem., March 23, 2007; 282(12): 8557 - 8567. [Abstract] [Full Text] [PDF]
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 K. B. Pedersen, P. Zhang, C. Doumen, M. Charbonnet, D. Lu, C. B. Newgard, J. W. Haycock, A. J. Lange, and D. K. Scott The promoter for the gene encoding the catalytic subunit of rat glucose-6-phosphatase contains two distinct glucose-responsive regions Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E788 - E801. [Abstract] [Full Text] [PDF]
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R. E Pratley, A. Salsali, and G. Matfin Review: Inhibition of dipeptidyl peptidase-4 with vildagliptin: a potential new treatment for type 2 diabetes The British Journal of Diabetes & Vascular Disease, July 1, 2006; 6(4): 150 - 156. [Abstract] [PDF]
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 L.-X. Li, P. E. MacDonald, D. S. Ahn, G. Y. Oudit, P. H. Backx, and P. L. Brubaker Role of Phosphatidylinositol 3-Kinase{gamma} in the {beta}-Cell: Interactions with Glucagon-Like Peptide-1 Endocrinology, July 1, 2006; 147(7): 3318 - 3325. [Abstract] [Full Text] [PDF]
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I. Alana, J. C. Parker, V. A. Gault, P. R. Flatt, F. P. M. O'Harte, J. P. G. Malthouse, and C. M. Hewage NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water J. Biol. Chem., June 16, 2006; 281(24): 16370 - 16376. [Abstract] [Full Text] [PDF]
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H. A Walz, L. Harndahl, N. Wierup, E. Zmuda-Trzebiatowska, F. Svennelid, V. C Manganiello, T. Ploug, F. Sundler, E. Degerman, B. Ahren, et al. Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B. J. Endocrinol., June 1, 2006; 189(3): 629 - 641. [Abstract] [Full Text] [PDF]
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B. N Friedrichsen, N. Neubauer, Y. C Lee, V. K Gram, N. Blume, J. S Petersen, J. H Nielsen, and A. Moldrup Stimulation of pancreatic {beta}-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways. J. Endocrinol., March 1, 2006; 188(3): 481 - 492. [Abstract] [Full Text] [PDF]
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T. L. Mazzuco, O. Chabre, N. Sturm, J.-J. Feige, and M. Thomas Ectopic Expression of the Gastric Inhibitory Polypeptide Receptor Gene Is a Sufficient Genetic Event to Induce Benign Adrenocortical Tumor in a Xenotransplantation Model Endocrinology, February 1, 2006; 147(2): 782 - 790. [Abstract] [Full Text] [PDF]
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 N. Irwin, B. D. Green, M. H. Mooney, B. Greer, P. Harriott, C. J. Bailey, V. A. Gault, F. P. M. O'Harte, and P. R. Flatt A Novel, Long-Acting Agonist of Glucose-Dependent Insulinotropic Polypeptide Suitable for Once-Daily Administration in Type 2 Diabetes J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1187 - 1194. [Abstract] [Full Text] [PDF]
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S.-J. Kim, W. S. Choi, J. S. M. Han, G. Warnock, D. Fedida, and C. H. S. McIntosh A Novel Mechanism for the Suppression of a Voltage-gated Potassium Channel by Glucose-dependent Insulinotropic Polypeptide: PROTEIN KINASE A-DEPENDENT ENDOCYTOSIS J. Biol. Chem., August 5, 2005; 280(31): 28692 - 28700. [Abstract] [Full Text] [PDF]
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V. A. Gault, N. Irwin, B. D. Green, J. T. McCluskey, B. Greer, C. J. Bailey, P. Harriott, F. P.M. O'Harte, and P. R. Flatt Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action by Daily (Pro3)GIP Administration Improves Glucose Tolerance and Ameliorates Insulin Resistance and Abnormalities of Islet Structure in Obesity-Related Diabetes Diabetes, August 1, 2005; 54(8): 2436 - 2446. [Abstract] [Full Text] [PDF]
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S.-J. Kim, K. Winter, C. Nian, M. Tsuneoka, Y. Koda, and C. H. S. McIntosh Glucose-dependent Insulinotropic Polypeptide (GIP) Stimulation of Pancreatic {beta}-Cell Survival Is Dependent upon Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (PKB) Signaling, Inactivation of the Forkhead Transcription Factor Foxo1, and Down-regulation of bax Expression J. Biol. Chem., June 10, 2005; 280(23): 22297 - 22307. [Abstract] [Full Text] [PDF]
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 J. Nyberg, M. F. Anderson, B. Meister, A.-M. Alborn, A.-K. Strom, A. Brederlau, A.-C. Illerskog, O. Nilsson, T. J. Kieffer, M. A. Hietala, et al. Glucose-Dependent Insulinotropic Polypeptide Is Expressed in Adult Hippocampus and Induces Progenitor Cell Proliferation J. Neurosci., February 16, 2005; 25(7): 1816 - 1825. [Abstract] [Full Text] [PDF]
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 B. Ahren, R. Gomis, E. Standl, D. Mills, and A. Schweizer Twelve- and 52-Week Efficacy of the Dipeptidyl Peptidase IV Inhibitor LAF237 in Metformin-Treated Patients With Type 2 Diabetes Diabetes Care, December 1, 2004; 27(12): 2874 - 2880. [Abstract] [Full Text] [PDF]
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P. E. MacDonald, J. W. Joseph, D. Yau, J. Diao, Z. Asghar, F. Dai, G. Y. Oudit, M. M. Patel, P. H. Backx, and M. B. Wheeler Impaired Glucose-Stimulated Insulin Secretion, Enhanced Intraperitoneal Insulin Tolerance, and Increased {beta}-Cell Mass in Mice Lacking the p110{gamma} Isoform of Phosphoinositide 3-Kinase Endocrinology, September 1, 2004; 145(9): 4078 - 4083. [Abstract] [Full Text] [PDF]
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J. J. Holst and J. Gromada Role of incretin hormones in the regulation of insulin secretion in diabetic and nondiabetic humans Am J Physiol Endocrinol Metab, August 1, 2004; 287(2): E199 - E206. [Abstract] [Full Text] [PDF]
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P. L. Brubaker and D. J. Drucker Minireview: Glucagon-Like Peptides Regulate Cell Proliferation and Apoptosis in the Pancreas, Gut, and Central Nervous System Endocrinology, June 1, 2004; 145(6): 2653 - 2659. [Abstract] [Full Text] [PDF]
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D. J. Drucker Enhancing Incretin Action for the Treatment of Type 2 Diabetes Diabetes Care, October 1, 2003; 26(10): 2929 - 2940. [Abstract] [Full Text] [PDF]
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J. A. Ehses, V. R. Casilla, T. Doty, J. A. Pospisilik, K. D. Winter, H.-U. Demuth, R. A. Pederson, and C. H. S. McIntosh Glucose-Dependent Insulinotropic Polypeptide Promotes {beta}-(INS-1) Cell Survival via Cyclic Adenosine Monophosphate-Mediated Caspase-3 Inhibition and Regulation of p38 Mitogen-Activated Protein Kinase Endocrinology, October 1, 2003; 144(10): 4433 - 4445. [Abstract] [Full Text] [PDF]
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 B. N. Friedrichsen, H. E. Richter, J. A. Hansen, C. J. Rhodes, J. H. Nielsen, N. Billestrup, and A. Moldrup Signal Transducer and Activator of Transcription 5 Activation Is Sufficient to Drive Transcriptional Induction of Cyclin D2 Gene and Proliferation of Rat Pancreatic {beta}-Cells Mol. Endocrinol., May 1, 2003; 17(5): 945 - 958. [Abstract] [Full Text] [PDF]
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N. Pamir, F. C. Lynn, A. M. J. Buchan, J. Ehses, S. A. Hinke, J. A. Pospisilik, K. Miyawaki, Y. Yamada, Y. Seino, C. H. S. McIntosh, et al. Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis Am J Physiol Endocrinol Metab, May 1, 2003; 284(5): E931 - E939. [Abstract] [Full Text] [PDF]
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J. A. Pospisilik, J. Martin, T. Doty, J. A. Ehses, N. Pamir, F. C. Lynn, S. Piteau, H.-U. Demuth, C. H.S. McIntosh, and R. A. Pederson Dipeptidyl Peptidase IV Inhibitor Treatment Stimulates {beta}-Cell Survival and Islet Neogenesis in Streptozotocin-Induced Diabetic Rats Diabetes, March 1, 2003; 52(3): 741 - 750. [Abstract] [Full Text] [PDF]
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K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]
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D. Hallmann, K. Trumper, H. Trusheim, K. Ueki, C. R. Kahn, L. C. Cantley, D. A. Fruman, and D. Horsch Altered Signaling and Cell Cycle Regulation in Embryonal Stem Cells with a Disruption of the Gene for Phosphoinositide 3-Kinase Regulatory Subunit p85alpha J. Biol. Chem., February 7, 2003; 278(7): 5099 - 5108. [Abstract] [Full Text] [PDF]
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D. D. De Leon, S. Deng, R. Madani, R. S. Ahima, D. J. Drucker, and D. A. Stoffers Role of Endogenous Glucagon-Like Peptide-1 in Islet Regeneration After Partial Pancreatectomy Diabetes, February 1, 2003; 52(2): 365 - 371. [Abstract] [Full Text] [PDF]
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Y. Li, T. Hansotia, B. Yusta, F. Ris, P. A. Halban, and D. J. Drucker Glucagon-like Peptide-1 Receptor Signaling Modulates beta Cell Apoptosis J. Biol. Chem., January 3, 2003; 278(1): 471 - 478. [Abstract] [Full Text] [PDF]
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L. Farilla, H. Hui, C. Bertolotto, E. Kang, A. Bulotta, U. Di Mario, and R. Perfetti Glucagon-Like Peptide-1 Promotes Islet Cell Growth and Inhibits Apoptosis in Zucker Diabetic Rats Endocrinology, November 1, 2002; 143(11): 4397 - 4408. [Abstract] [Full Text] [PDF]
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J. A. Ehses, S. L. Pelech, R. A. Pederson, and C. H. S. McIntosh Glucose-dependent Insulinotropic Polypeptide Activates the Raf-Mek1/2-ERK1/2 Module via a Cyclic AMP/cAMP-dependent Protein Kinase/Rap1-mediated Pathway J. Biol. Chem., September 27, 2002; 277(40): 37088 - 37097. [Abstract] [Full Text] [PDF]
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A. A. Hardikar, X. Y. Wang, L. J. Williams, J. Kwok, R. Wong, M. Yao, and B. E. Tuch Functional Maturation of Fetal Porcine {beta}-Cells by Glucagon-Like Peptide 1 and Cholecystokinin Endocrinology, September 1, 2002; 143(9): 3505 - 3514. [Abstract] [Full Text] [PDF]
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J. A. Pospisilik, S. G. Stafford, H.-U. Demuth, C. H.S. McIntosh, and R. A. Pederson Long-Term Treatment With Dipeptidyl Peptidase IV Inhibitor Improves Hepatic and Peripheral Insulin Sensitivity in the VDF Zucker Rat: A Euglycemic-Hyperinsulinemic Clamp Study Diabetes, September 1, 2002; 51(9): 2677 - 2683. [Abstract] [Full Text] [PDF]
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