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Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts
Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street–WEL320, Boston, Massachusetts 02114. E-mail: jhabener{at}partners.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-cell line. In the presence of stimulatory
concentrations of GLP-1 (0.1–100 nM) on rINS1
activity, inhibition of p38 mitogen-activated protein kinase (p38 MAPK)
using SB 203580 resulted in a marked increase in promoter activity
(maximum 3-fold) over GLP-1 alone, as determined by rINS1
promoter-luciferase reporter gene expression. This effect was revealed
to be mediated via the cAMP response element (CRE) of rINS1, because
site directed mutagenesis of the CRE motif in rINS1 abolished the
increased response to SB 203580. Furthermore, inhibition of p38 MAPK
uncovered a similar, more pronounced, response in the expression of a
generic CRE promoter driven reporter gene. Time course dose-response
studies indicate that the p38 MAPK induced inhibitory response may
involve expression of immediate early genes (IEGs); maximum repression
of rINS1 activity occurred after 4 h of treatment, comparable with
regulatory responses by IEGs. In conclusion, these results demonstrate
a novel signaling mechanism whereby p38 MAPK represses rINS1 promoter
activity in response to GLP-1, suggesting the involvement
of a robust regulatory control by p38 MAPK in insulin gene expression.
The relevance of this mechanism may be most apparent during periods of
cellular stress in which p38 MAPK activity is stimulated. In this
regard, reduced insulin expression levels caused by chronic
hyperglycemia (glucotoxicity) and/or hyperlipidemia (lipotoxicity) may
be a direct consequence of this mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cells to augment glucose-induced insulin secretion
(1, 2). Apart from its role as a potent insulin
secretagogue, GLP-1 also induces multiple intracellular
mechanisms intrinsic to -cell function. Up-regulation of insulin
gene expression is central to its function in these cells and may be
achieved by a variety of mechanisms that influence transcriptional
activity through a number of different cis-acting promoter
sequence elements (3, 4). The -cell-specific
transcription factor IDX-1 (which binds A-box elements in the insulin
promoter) is up-regulated by GLP-1 coincident with
increased insulin expression (5, 6, 7). GLP-1
also stimulates insulin promoter activity through its cAMP response
element(s) (CREs) by multiple, incompletely understood signaling
pathways (8, 9). Other transcription factors (either
stimulators, inhibitors, or synergists) that regulate the activity of
the insulin gene promoter include the helix-loop-helix proteins
E47/Pan1, E12, and BETA2/NeuroD1, the bZIP proteins C/EBP, CREB,
ATF-2, and the winged helix-like protein NF-Y (10, 11, 12, 13, 14).
The complexity of the transcriptional machinery acting on the insulin
gene promoter creates difficulty in dissecting specific
GLP-1-induced effects on the promoter. Furthermore, other,
as yet unidentified, transcriptional regulators may be involved in
GLP-1-induced insulin gene expression. In addition to its
effects on insulin gene expression, GLP-1 induces other
distinct responses in -cells, which include up-regulation of several
immediate early genes such as c-Jun, c-Fos, and JunB, proposed to be
important for -cell adaptation to hyperglycemia
(15).
Further challenges in characterizing GLP-1-mediated
responses in -cells arise from the ability of the GLP-1
receptor to activate multiple signaling pathways. Early studies on the
GLP-1 receptor uncovered a robust stimulation of cAMP
levels, mediated by the activation of the G protein
Gs and adenylyl cyclase (9, 16).
Subsequent studies determined activation of several other second
messengers including mitogen-activated protein kinase (17, 18), phospholipase C (19),
Ca2+ mobilization (20, 21, 22) and
phosphatidylinositol 3-kinase (6). The ability of the
GLP-1 receptor to regulate such a diverse response
suggests a prominent role of the receptor in intracellular cross-talk.
Promiscuous G protein coupling of the GLP-1 receptor has
recently been demonstrated (17), and this phenomenon has
long been ascribed as the reason for the induction of multiple pathways
by this receptor and other receptor family members including the
calcitonin and PACAP receptors (19, 23, 24). Thus, the
reported pleiotropic effects of GLP-1 receptor signaling
prompts further investigations to characterize the functional responses
of this receptor. This need for further investigation is further
emphasized by the recent findings that secreted insulin feedback on
-cells is strongly augmented by GLP-1 by its
secretogogic activity (25, 26, 27, 28).
Recently, GLP-1-mediated activation of p38 MAP kinase (p38 MAPK) was identified in CHO cells and also in RIN-1046–38 insulinoma cells (17). p38 MAPK is characteristically induced by activation of cellular stress-mediated signaling pathways that modify inflammation, cell growth, cell differentiation, the cell cycle, and apoptosis (reviewed in Ref. 29). Substrates for p38 MAPK include several transcription factors including ATF-2 (30) and CHOP (31, 32), both of which regulate gene expression mediated by CREs in the promoters of target genes. Furthermore MAPKAP-K2 (a substrate for p38 MAPK), also activated by p38 MAPK, activates CREB, an important transcriptional activator of CRE-regulated gene expression (33, 34). These observations suggest a role for p38 MAPK in the regulation of genes under the transcriptional control of CREs, and moreover, the potential involvement of p38 MAPK in the regulation of insulin gene transcription by GLP-1. Notably, the expression of the insulin gene was recently shown to be regulated by p38 MAPK activity in response to glucose and insulin in human islets and MIN6 cells (27, 35, 36), further supporting the hypothesis that p38 MAPK is an important mediator of insulin gene transcription.
Here we describe the identification of a novel signaling mechanism stimulated by GLP-1 that involves the activation of p38 MAPK leading to a potent repression of rat insulin 1 promoter (rINS1) activity via a specific targeting of the CRE sequence in the promoter of the insulin gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
INS-1 cells (passage numbers 99–110) were cultured in RPMI 1640
containing 10 mmol/liter HEPES, 11.1 mmol/liter glucose, 10% FBS, 100
µU/ml penicillin G, 100 µg/ml streptomycin, 1 mmol/liter sodium
pyruvate, and 50 µmol/l 2-mercaptoethanol (37). Cells
were maintained at 37 C in a humidified incubator gassed with 5%
CO2. Cell cultures were passaged by
trypsinization and subcultured every 5 days.
Plasmid DNA constructs
The rat insulin promoter sequence, rINS1, was provided by Dr. M.
German (University of California at San Francisco Medical School, San
Francisco, CA). It was fused to the coding sequence of luciferase in
the pxp2-basic vector to generate -410rINS1-LUC. The construct
containing deletions (8) of the rINS1 CRE were prepared by
site-directed mutagenesis (8). The pCRE-LUC expression
plasmid was obtained from Stratagene (La Jolla, CA).
Transfection of INS-1 cells
Adherent INS-1 cells grown to 80–90% confluence in Falcon
100-mm tissue culture dishes were transfected using Lipofectamine 2000
(Life Technologies, Inc.). Cells were rinsed twice in
serum-free culture medium before the addition of 1.0 ml transfection
cocktail containing 1.0 µg plasmid DNA. Cells were incubated in this
mixture for 4 h. Cells were then trypsinized and transferred in
culture medium to 24-well plates (Costar Corning, Inc.,
Acton, MA) at 500 µl cell suspension per well and incubated for
48 h.
Luciferase assays
Test substances were dissolved in RPMI culture medium and added
to 24-well plates at a final volume of 300 µl/well. Cells were
exposed to test substances for 4 h unless stated otherwise at 37 C
in a humidified incubator. Inhibitory test substances were added 30 min
before addition of GLP-1. After 4 h, cells were
lysed, and measurements of luciferase activity were performed using a
luciferase assay kit (Promega Corp., Madison, WI) in
conjunction with a dual injection port luminometer (Wallac, Inc., Gaithersburg, MD) with automated application of ATP
and luciferin assay substrates. All experiments were carried out in
triplicate.
Immunoblotting
Immunoblotting for p38 MAPK and dual phospho-p38 MAPK was
carried out in accordance with the manufacturer’s instructions with
modifications (Cell Signaling Technology, Beverly, MA). After
treatment, cells were washed with PBS, and lysed in 70 µl lysis
buffer (25 mM Tris-phosphate (pH 7.8), 2 mM
DTT, 1% Triton X-100, 1 mM EGTA, 1 mM sodium
orthovanadate, 50 mM -glycerophosphate, 10% glycerol
and 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid). Cell lysates were then collected into Eppendorf
tubes, vortexed and frozen over night. After centrifugation at
14,000 x g for 5 min., the resultant supernatant was
collected, followed by addition of SDS-PAGE sample buffer containing 50
mM Tris-Cl, 2% SDS, 10% glycerol, 5%
-mercaptoethanol, and bromophenol blue (pH 6.8). Samples were boiled
for 2 min. and centrifuged at 14,000 x g for 2 min,
then analyzed on 10% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes. The membranes were blocked with 10% nonfat
dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS/T) for 1
h at 22 C. The blots were then treated with the primary antibody in
TBS/T containing 5% BSA at 4 C overnight. After washing,
immunolabeling was detected by LumiGLO chemiluminescent reagent
according to the manufacturer’s instructions. Membranes were then
stripped by incubation for 45 min at 65 C in a solution containing 10
mM Tris (pH 6.7), 100 mM
-mercaptoethanol, and 2% SDS. After washing, efficacy of stripping
was determined by reexposure of the membranes to LumiGLO reagent.
Thereafter, blots were reblocked and immunolabeled as described
above.
Statistical analysis
Concentration-response curves were analyzed by nonlinear
regression with variable slope, using GraphPad Software, Inc. Prism. Other graphs were generated using Microsoft Corp. Excel. Data represents mean ± SEM.
Statistical significance was determined using the Student’s
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cell line INS-1. Treatment of the INS-1 cells with
increasing concentrations of GLP-1 resulted in a
dose-dependent increase in luciferase gene expression with an
EC50 value of 0.43 nM (Fig. 1A
) corresponding
with reported observations of cAMP accumulation in INS-1 cells in
response to GLP-1 (16). The maximal response
was 7-fold above basal level, which corresponded to only 26% of that
attributed to forskolin (10 µM) treatment in the same
experiment (Fig. 1B). To determine whether CRE-directed luciferase
expression was mediated by cAMP/PKA, cells were treated with the
specific cAMP pathway inhibitors, Rp-cAMPS (50 µM), and
H-89 (10 µM). Both treatments blocked GLP-1
stimulated luciferase expression whereas the PI3-kinase specific
inhibitor wortmannin (50 nM) had no effect on CRE-directed
luciferase expression (Fig. 1C), suggesting that GLP-1
induction of the CRE luciferase reporter gene is mediated through cAMP
and PKA signal transduction pathways.
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). However, insulin promoter activity
was not inhibited by H-89 suggesting that the activity was mediated by
an alternate signaling pathway to that of the CRE promoter. Treatment
with wortmannin appeared to partially attenuate the insulin promoter
response to GLP-1, but was not statistically significant
(Fig. 2B), suggesting that PI3-K may mediate GLP-1
stimulated rINS1 activity but that this signaling pathway is not
sufficient for elicitation of the full response.
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, A and B). In the presence of the p38
MAPK-specific inhibitor SB 203580, the response was attenuated.
Treatment with 100 nM GLP-1 increased p38 MAPK
phosphorylation to a lesser extent than 10 nM
GLP-1. The immunoblot was stripped of antibody and
reprobed using a p38 antibody to confirm equal protein levels in each
lane. Anisomycin treated INS-1 cells (30 min) served as a positive
control for p38 MAPK activity.
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). This finding indicated that p38 MAPK
activity regulates rINS1 activity by a potent inhibitory mechanism in
response to GLP-1. Notably, in the absence of
GLP-1, there was no significant effect of SB 203580
treatment on basal activity of the promoter, suggesting that the p38
MAPK-mediated repression of rINS1 activity was a direct result of
GLP-1 signaling. Thus, in INS-1 cells, GLP-1
appears to induce a bifurcating signal that both activates and
represses rINS1 promoter activity.
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). In the experiments using either the pCRE-LUC or rINS1-LUC
reporter gene constructs, luciferase expression was down regulated by
GLP-1 at concentrations above 10 nM. We
contribute this effect to a desensitization of the GLP-1
receptor by prolonged elevated concentrations of agonist, which has
been demonstrated in earlier studies of -cell lines (38, 39). Furthermore, similar effects of GLP-1 occur at
the level of cAMP accumulation in RIN-1046–38 cells, also suggesting
that this observed down-regulation occurs at the receptor level
(9).
Repression of rat insulin I promoter activity by p38 MAPK is
mediated through the CRE motif
Because the inhibitory response by p38 MAPK was particularly
strong in the expression of pCRE-LUC, we investigated whether
repression of rINS1 activity was mediated through the CRE motif in the
rINS1 promoter. Mutation of the rINS1 sequence by a base deletion
within the CRE sequence generated a construct that displayed a
nonfunctional CRE sequence (rINS1
CRE-LUC) (8).
GLP-1 treatment in cells transiently expressing the
rINS1CRE-LUC plasmid induced a 40% increase in luciferase
expression above basal level compared with a 114% increase with the
similarly transiently transfected intact promoter (rINS1-LUC) (Fig. 5), presumably as a result of the
inability of GLP-1 to induce CRE-mediated promoter
activity. However, in the presence of SB 203580, luciferase expression
of rINS1CRE-LUC was not augmented, suggesting that the p38 MAPK
induced effect in rINS1-LUC activity is mediated through the CRE of the
rINS1 insulin promoter.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cells. We used luciferase reporter
constructs to assess the functional activity of the insulin gene by
employing various promoter sequences to direct luciferase expression.
Using the pCRE-LUC reporter plasmid, the ability of GLP-1
to induce CRE-mediated gene transcription to only 26% of that observed
with forskolin corresponds with observations at the level of cAMP
accumulation in INS-1 cells (16). This proportionality
suggests a direct relationship between cAMP levels and CRE-mediated
gene expression. The relatively low response to GLP-1
maybe explained by several possibilities including the low
GLP-1 receptor density in INS-1 cells (16),
receptor desensitization (38, 39), or by the potent
stimulatory effect of forskolin on adenylyl cyclase. Treatment with two
specific inhibitors of PKA activity (H-89 and Rp-cAMPS) abolished the
response to GLP-1, further evidence of the direct
signaling cascade. Conversely, GLP-1 induced activation of
the rat insulin 1 promoter (-410rINS1-LUC) was insensitive to H-89,
suggesting that the insulin gene is regulated by a different signaling
pathway in response to GLP-1. However, involvement of a
CRE-mediated response in rINS1 activity was evident because mutation of
the CRE in the rINS1 promoter construct led to a decreased
fold-induction in reporter gene expression by GLP-1 (Fig. 4; Ref. 8). Therefore, these results suggest that although
GLP-1 can induce CRE-mediated gene expression via orthodox
signaling through cAMP, PKA, and presumably CREB, induction of the rat
insulin 1 gene appears to be mediated by a distinct pathway, but which
also displays partial dependence on a functional CRE. These
observations are in agreement with the recent studies of Skoglund
et al. (8).
Following treatment of INS-1 cells with the p38 MAPK specific inhibitor
SB 203580, GLP-1 induced augmented responses in both CRE
and rINS1 promoter activity. This effect was dose dependent, suggesting
that p38 MAPK activity may be induced by GLP-1 (Fig. 4).
Indeed this was shown by immunoblot analysis of phospho-p38 MAK
activity in response to GLP-1 treatment (Fig. 3). The fact
that reporter gene expression was enhanced by SB 203580 treatment
indicated that p38 MAPK activity provokes a marked inhibition of
GLP-1 mediated promoter activity. Also, the correlating
response between the two promoter-reporter constructs implied that the
p38 MAPK-induced inhibition of rINS1 promoter activity was directed by
its CRE sequence. This conjecture was further evidenced by the finding
that a mutation of the CRE in the rINS1 insulin promoter construct
abrogated the CRE responsiveness and resulted in a complete loss of
response to SB 203580 treatment. The reduced fold expression level in
response to GLP-1 compared with the insulin promoter
construct with an intact CRE was presumably due to the abolition of
CRE-mediated activity (8). In conclusion, it appears that
the CRE motif in the insulin promoter is the direct target of
repression by p38 MAPK.
Interestingly, p38 MAPK phosphorylation was maximal at 10
nM GLP-1 and was reduced at 100
nM. This observation correlates well with the extent by
which SB 203580 treatment augmented the transcriptional response of the
CRE promoter in response to GLP-1 (Fig. 4B). This supports
the evidence that the reporter activity in response to
GLP-1 and SB 203580 directly reflects the activity of p38
MAPK.
The inhibitory effect of p38 MAPK peaked at 4 h and was reduced at
6 h, as shown in a time course experiment comparing reporter gene
expression in response to GLP-1 in the presence and
absence of SB 203580 (Fig. 6). This temporal pattern of promoter
activity may indicate the involvement of immediate early
gene (IEG) regulation as opposed to direct
phospho-activation of transcription factors by p38 MAPK or MAP/KAP-K2,
MAP/KAP-K3, or MSK1/2 (substrates for p38). p38 MAPK has been
implicated in the regulation of several IEGs including
c-jun, c-fos, and nur-77 (29).
c-jun is a particularly attractive candidate target for further investigation as previous studies provide strong evidence for the involvement of this protein in regulatory effects of p38 MAPK (29, 40, 41). Furthermore, up-regulation of c-jun expression was recently demonstrated in response to GLP-1 treatment in INS-1 cells (15) along with several other IEGs. The PACAP receptor, a member of the GLP-1 receptor family was also shown to stimulate c-jun transcription (42), which may signify a common feature of these receptors. Even more evidence can be derived from observations that activating protein-1 (AP-1) binding activity in INS-1 cells is significantly increased in response to GLP-1 (6), consistent with increased expression of the AP-1 subunits, Fos, Jun, and ATF proteins. Increased AP-1 activity was also observed in rat pancreatic carcinoma cells in response to PACAP (42). A recent study describes a complete signal transduction pathway that involves direct regulation of the c-jun promoter by GPCRs (e.g. the m1 muscarinic receptor) via p38 MAPK (41). Perhaps the most convincing existing evidence in support of the possible involvement of c-jun in the repression of promoter activity in our studies is the observation that c-jun represses human insulin promoter activity, and that the mechanism of repression may involve the inhibition of CRE-binding proteins (43). Interestingly, not only c-jun but also c-fos represses human insulin promoter activity and thus c-fos may be an equally attractive target for p38 MAPK.
The rat insulin 1 promoter CRE sequence (TGACGTCC) is positioned
between -178 and -185 relative to the transcriptional start site and
closely matches but is not identical with the CRE octamer consensus
motif (TGACGTCA) (44). Studies of transcriptional
activities of 5' deleted and point mutated promoter-reporter plasmids
demonstrated that this CRE-like sequence is necessary for cAMP
induction of rat insulin 1 gene transcription in HIT cells and INS-1
cells (8, 44). However, cellular CREB binds only weakly to
this CRE and was not detected by elecrophoretic mobility shift assays
using the insulin CRE sequence, although three protein complexes from
HIT cell nuclear extracts were identified, none of them containing
proteins with CREB like immunoreactivity (45). This
circumstance contrasts with the binding profiles of the somatostatin
and glucagon gene CRE motifs, which display characteristic CREB binding
patterns. Notably, this situation may help explain the relatively weak
stimulation of rINS1 activity through cAMP/PKA-mediated signaling in
response to GLP-1 compared with that seen with the generic
CRE promoter used in these studies. A possible reason for the unusual
behavior of the insulin gene CRE could be the overlapping CCAAT
sequence beginning two base pairs from the 3' end of the CRE motif.
Cross-reactivity with CCAAT/enhancer binding proteins (C/EBPs) and/or
NF-Y family member proteins at this site may cause steric-hindrance
resulting in a competitive binding relationship between CRE and CCAAT
binding proteins. Disruption of the 3' CCAAT motif overlapping the CRE
of the rINS1 promoter (TGACGTCCAAT) markedly enhanced the
cAMP-inducible activity of the promoter (14). Evidence was
presented that NF-Y conferred basal activity to the CRE and decreases
the ability of CREB to mediate cAMP-stimulated transcription
(14). Another possible factor in the p38 MAPK regulated
repression of insulin gene promoter activity may be the transcriptional
repressors C/EBP or CHOP, both members of the C/EBP transcription
factor family. C/EBPs have been shown to bind to and activate
transcription of CREs in the promoters of genes (46). CHOP
is activated by phosphorylation by p38 MAPK (47), although
there is as yet no evidence of up-regulation of CHOP gene expression by
p38 MAPK.
In a recent study to identify possible substrates for MAPKAP-K2 (a downstream substrate for p38 MAPK), the E2A gene product E47 was isolated (48). This transcription factor binds two distinct enhancer elements (E boxes) in the rat insulin 1 promoter and is required for transcriptional activation (49, 50). Interestingly, it was shown that MAPK-K2 repressed transcriptional activity by interaction with E47 linking the E47 function to the MAPK signaling network. However, the involvement of this mechanism in our studies is doubtful because repression of promoter activity was dependent on the CRE rather than the E boxes.
What is the mechanism by which GLP-1 activates p38 MAPK?
An increasing amount of information suggests that in contrast to the
initial lock and key concept of receptor signaling, G protein-coupled
receptors can regulate multiple signaling pathways by binding and
functionally activating several trimeric G proteins (reviewed in Ref.
51). This concept has lead to the identification of
receptor cross-talk, in which supposedly distinct GPCRs can
interregulate the functional response of each other’s signal (reviewed
in Ref. 52). The GLP-1 receptor couples to
multiple G proteins including Gs,
Gq/11, and Gi1,2
(17). Although G protein promiscuity has not been directly
demonstrated in -cells, the potential for such promiscuous signaling
of various pathways and signaling cross-talk is evident. In accordance
with this notion, activation of the GLP-1 receptor
increases p38 MAPK activity by 2-fold in RIN-1046–38 insulinoma cells
and transfected CHO cells. This response is dependent on
Gs protein activation, although not by
-subunit signaling, suggested by the failure of 8-bromo-cAMP to
activate p38 MAPK activity (17). The authors proposed that
G
subunits associated with Gs protein could
be responsible for GLP-1 induced p38 MAPK activity, and
this hypothesis is supported by studies in which p38 MAPK activation by
several GPCRs was identified, with the implication of G subunits
in the response (53).
What is the functional purpose of the repression by p38 MAPK in insulin promoter activity? In accord with our findings, p38 MAPK activity prevents a much greater expression potential of the insulin gene than is seen in response to GLP-1 treatment. Interestingly, although the time course study demonstrated a transient increase in the p38 MAPK-mediated repression in insulin promoter activity, the insulin- promoter luciferase reporter expression level at all time points was consistently similar, suggesting that the repression is a regulatory mechanism to retain insulin transcription levels at a precisely controlled level. If this is the case, it could be hypothesized that diverse p38 MAPK activating stimuli (e.g. ER stress, cytokines, growth factors, and UV irradiation) could lead to detrimental repression of insulin gene expression. This regulatory response may function as a conservation mechanism in response to a stressful environment or as an early response in the process of programmed cell death.
Glucose toxicity and lipotoxicity results in decreased levels of
insulin biosynthesis along with other important functional components
of -cells (54, 55), followed by the commitment to
programmed cell death (56, 57). Although many reports
detail a decrement of the -cell specific transcription factor IDX-1
levels in association with insulin gene transcription in response to
sustained supraphysiological levels of glucose or lipid treatment
(58, 59, 60), Kajimoto et al. (61)
demonstrated that suppression of IDX-1 causes no decrease in insulin
messenger RNA in MIN6 cells, thus it could be inferred that contrary to
general opinion, other inhibitory factors cause the suppression of
insulin gene expression, independently of IDX-1 activity, which may
simply be down regulated coincident with the down-regulation of the
insulin gene. Therefore, the mechanism described in our studies may
play an important role in the stress related decrease in insulin gene
expression.
Our findings also suggest that agents that inhibit p38 MAPK activity in vivo could lead to increased insulin transcription levels, signifying a potentially new target for the treatment of type 2 diabetes mellitus. In conclusion, our results highlight a novel mechanism whereby GLP-1 induces a bifurcating signal that imparts opposing transcriptional responses by affecting both stimulatory and inhibitory signals on the same insulin gene promoter.
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Acknowledgments |
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CRE-Luc plasmid
was constructed by M. Hussain. |
Footnotes |
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2 Investigator with the Howard Hughes Medical Institute.
Received September 6, 2000.
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