Glucose and forskolin regulate IAPP gene expression through
different signal transduction pathways
Wei-Qun
Ding,
Eileen
Holicky, and
Laurence J.
Miller
Departments of Medicine, Biochemistry, and Molecular Biology,
Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
Molecular
mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene
expression remain unclear. In the present study, we investigated the
effects of glucose and forskolin on IAPP gene regulation in the INS-1
islet
-cell line. Both glucose and forskolin increased the level of
expression of this gene, as measured by Northern blot analysis, and
increased IAPP gene transcription in a time- and
concentration-dependent manner, as demonstrated in a reporter gene
assay. Although inhibition of protein kinase A activity with H-89
eliminated the effect of forskolin on this gene, the glucose effect was
unaffected. This supported the predominant use of a protein kinase
A-independent signaling pathway for glucose regulation of the IAPP
gene. Electrophoretic mobility shift assay further indicated that
glucose and forskolin regulated expression of this gene by targeting
different elements of the promoter. Mutation of the cAMP regulatory
element flanking the IAPP coding region resulted in the loss of most of
the forskolin-stimulated IAPP gene promoter activity, whereas
glucose-enhanced IAPP gene transcription was unaffected. These results
demonstrate parallel and distinct regulatory pathways involved in
glucose- and forskolin-induced IAPP gene expression in this model
-cell system.
islet amyloid polypeptide; cyclic adenosine 3,5-monophosphate; protein kinase A
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INTRODUCTION |
ISLET AMYLOID
POLYPEPTIDE (IAPP or amylin) is the major component of pancreatic
amyloid deposited in excess in pancreatic islets in most patients with
type 2 diabetes mellitus (5, 7). This 37-amino acid
peptide is synthesized primarily in islet
-cells, where it is
co-localized (4, 18) and normally co-secreted with insulin
(10, 21). The physiological and pathological significance
of this hormone is not well understood, but studies have shown the
association of this peptide with diabetes mellitus and its involvement
in the control of glucose homeostasis (6), such as
inhibiting glucose uptake in skeletal muscle and causing insulin
resistance in both in vitro and in vivo systems (17, 30).
The cis-acting elements of the IAPP gene that are required
for islet
-cell expression have recently been identified (3, 14). Similar elements in the promoter regions of the insulin and
IAPP genes suggest that at least some of the transcriptional regulatory
mechanisms of these two genes are shared. However, recent studies have
convincingly demonstrated dissociation of expression of IAPP and
insulin genes. For instance, overexpression of IAPP relative to insulin
has been observed in human islets cultured at high glucose
concentration (27) and in rat models of type 2 diabetes
mellitus (23). These observations indicate that the IAPP
gene is regulated independently under certain conditions and that
aberrant expression of the IAPP gene may contribute to the pathogenesis
of type 2 diabetes mellitus. Elucidation of the mechanisms controlling
IAPP gene expression in pancreatic
-cells may prove relevant to the
pathogenesis of type 2 diabetes mellitus and contribute to a better
understanding of pancreatic
-cell-specific gene expression.
It is generally accepted that glucose is an important regulator of the
IAPP gene (1, 27). Glucose treatment causes an increase in
IAPP gene expression in primary cultures of human and rat islets
(12, 13), in
-cell lines (9, 22, 25, 32),
and in an animal model (24). The intracellular signaling pathways coupling glucose to IAPP gene expression are not clear. In the
primary culture of human islets, glucose stimulates IAPP gene
expression through signals derived from glucose metabolism (12). The involvement of intracellular second messengers
such as calcium and cAMP in the glucose regulation of IAPP gene
expression has been suggested (12, 13). These
intracellular second messengers are usually elevated in response to
various extracellular stimuli and activate corresponding protein
kinases, thereby leading to alterations of intracellular events
including gene expression.
In an attempt to examine whether glucose regulates IAPP gene expression
through protein kinase A (PKA) signaling in pancreatic
-cells, the
present study investigated the effects of glucose and forskolin on IAPP
gene regulation in INS-1 cells. This cell line was derived from a rat
insulinoma and has been extensively used as a model for the
-cell
due to the advantages of its being glucose responsive and possessing
high insulin content (2). The results from this study
demonstrated that the IAPP gene is expressed in INS-1 cells, with
expression regulated by glucose and forskolin through different
signaling pathways. The dissociation of the effects of glucose and
forskolin on IAPP gene transcription indicates that glucose regulates
IAPP gene expression primarily through PKA-independent mechanisms in
pancreatic
-cells.
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MATERIALS AND METHODS |
Materials.
TRIzol reagent, Superscript II kit, and H-89 were from GIBCO-BRL.
[32P]dATP and [32P]ATP were from Amersham
Pharmacia. Luciferase assay reagents and the PGL3 plasmid were from
Promega. All other reagents were of analytical grade.
Cell culture.
INS-1 cells were the kind gift of Dr. Christopher J. Rhodes
(Southwestern Medical Center, Dallas, TX). They were cultivated in
172-mm flasks in RPMI-1640 medium supplemented with 10% fetal calf
serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium
pyruvate, and 50 µM mercaptoethanol at 37°C in an environment containing 5% CO2, as previously described
(2). Glucose concentration in the medium was 11.2 mM
unless otherwise indicated. Cells were detached with EDTA-trypsin after
reaching 80% confluence and were plated into 12-well plates (300,000 cells/well) for Northern blot analysis or into 100-mm dishes (1.5 × 106/dish) for transfection and electrophoretic mobility
shift assay (EMSA). Cells of passages 65 through 85 were used in this study.
For studying the effects of glucose treatment, INS-1 cells were washed
twice with phosphate-buffered saline (PBS), and RPMI-1640 medium
containing varied glucose concentrations was added. The cells were then
incubated for indicated time periods. The experiments in which glucose
was absent from the medium were conducted within 12 h, because
cell viability and cell number were significantly decreased in cells
cultured in glucose-free medium for 24 h (data not shown).
Panc-1 cells (human pancreatic ductular carcinoma cell line) and COS-1
cells were cultivated in DMEM medium supplemented with 10% fetal calf
serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in
an environment containing 5% CO2.
RT-PCR.
First-strand cDNA was synthesized using total RNA (2.5 µg) isolated
from INS-1 cells with 5 µM random hexamer primers, 10 mM
dithioerythritol, 1 mM dNTP mix, 1× first-strand buffer, and 20 U of
superscript II in a total volume of 20 µl. RNA was denatured at
70°C for 10 min before addition of reverse transcriptase. The reaction was allowed to proceed for 40 min at 42°C and was stopped by
being heated to 70°C for 15 min. Aliquots of 2 µl of the resulting cDNA were subjected to PCR containing 0.2 µM of both sense and antisense oligonucleotide primers for IAPP cDNA (+73/+282, sense 5'-CCT
GTC GGA AGT GGT ACC AAC-3', antisense 5'-TTA CAG GAG TAA GAA ATC CAG
GGA-3'). The cDNA was first denatured at 95°C for 10 min. Incubation
and thermal cycling conditions were 94°C for 1 min, 52°C for 2 min,
and 72°C for 3 min, over 35 cycles. Elongation in the final cycle was
at 72°C for 12 min. The PCR products were separated on 1% agarose
gel containing ethidium bromide and visualized under ultraviolet light.
The detected IAPP fragment was then purified from the gel and used as
the probe for Northern hybridization.
RNA isolation and Northern blot analysis.
Total RNA from cells in a 12-well plate was isolated using TRIzol
reagent. Briefly, after treatment, cells were lysed using 800 µl of
TRIzol per well. Two hundred microliters of chloroform were added to
the cell lysate, and the aqueous phase was separated and collected
after centrifugation. The total cellular RNA from the aqueous phase was
precipitated with 600 µl of isopropanol at
20°C for 1 h.
After centrifugation, the RNA pellet was washed with 75% ethanol,
dried, and dissolved in diethylpyrocarbonate-treated water. The RNA
from each well was separated on 1.2% agarose gel containing
formaldehyde (16%, vol/vol) and transferred to Hybond N+ (Amersham)
nylon membrane with capillary blot technique. Membranes were
prehybridized at 65°C for
2 h in 0.2× standard saline citrate (SSC), 50 mM NaH2PO4, 0.5% SDS, 2.5 mg/ml of
Ficoll 400, polyvinylpyrolidone, and bovine serum albumin.
PCR-generated IAPP DNA fragments were labeled with
[32P]dATP by use of the random priming method and added
to the hybridization buffer (1×106 cpm/ml), and
hybridization was continued overnight. Membranes were washed three
times for 15 min at 65°C in 2×, 1×, and 0.1× SSC with 1, 0.1, and
0.1% SDS, respectively, wrapped with plastic wrap, and exposed to
X-ray film at
70°C. Levels of mRNA were quantified by densitometry
and normalized to 28S rRNA that had been visualized under ultraviolet
light after RNA separation.
Plasmid construction, transfection and luciferase activity assay.
An upstream fragment of the human IAPP gene (993 bp) was derived from
pFOXCAT-AP1.0, which has been described elsewhere (28). This fragment was subcloned into PGL3-basic plasmid at
Nhe1/Kpn1 sites of the multiple cloning site
upstream of the luciferase gene. The constructed plasmid was identified
as PGL3-AP1.0. Mutagenesis in the cAMP-regulatory element (CRE)-like
sequence located at the IAPP gene promoter from
60 to
53 bp
relative to the transcriptional start site was performed by a circular
PCR-based, site-directed mutation kit (QuikChange, Stratagene) with the
following sense and corresponding antisense primers: sense (
76/
40),
5'-GGCTCTCTGA GCTGCCGCCT GTCAGAGCTG AGAAAGG-3'; antisense
(
40/
76), 5'-CCTTTCTCAG CTCTGACAGG
CGGCAGCTCA GAGAGCC-3'. The underlined nucleotides were substitutes of TGA (sense) and TCA (antisense). Mutation was verified by DNA sequencing, and the mutated construct was referred to as PGL3-APCREm.
For transfection and transient expression, 1.5 × 106
INS-1 cells were initially seeded in 100-mm dishes in 10 ml of
RPMI-1640 medium with supplements. Twenty-four hours after plating,
cells were washed with PBS and incubated with lipofectin-DNA complexes for 6 h. Lipofectin-DNA complexes were made by incubating 25 µl of lipofectin with 5 µg of plasmid DNA in 0.2 ml of Opti-MEM medium for 12 min at 22°C. The medium was exchanged with RPMI-1640 with supplements after 6 h of transfection, and cells were incubated overnight. The cells then were plated into 12-well plates at a density
of 300,000 per well.
After 48 h of transfection, cells were washed once with PBS in the
absence of Mg2+ and Ca2+ and lysed with 200 µl of reporter lysis buffer (Promega). The cell particulate was
removed by brief centrifugation, and the protein concentration was
measured. Luciferase assays were performed using a Turner TD/20E
luminometer with 80 µl of luciferase assay reagent mixed with 50 µl
of protein extract. The relative light units were normalized for the
amount of protein in each extract, and the results were reported as
relative changes in luciferase activity.
cAMP and PKA activity assay.
cAMP assay was carried out as previously described (11).
Cells were stimulated with 1 µM forskolin or 20 nM GLP-1 at 37°C for 30 min in Krebs-Ringer-HEPES (KRH) medium containing (in mM) 25 HEPES, pH 7.4, 104 NaCl, 5 KCl, 1 KH2PO4, 1.2 MgSO4, 2 CaCl2, 1 phenylmethylsulfonyl
fluoride, and 0.01% soybean trypsin inhibitor, 0.2% bovine serum
albumin, and 1 mM 3-isobutyl-1-methylxanthine. The reaction was stopped
by adding ice-cold perchloric acid. Cell lysates were cleared by
centrifugation at 3,000 rpm for 10 min, and the supernatants were
counted in a Beckman LS6000 counter. PKA activity was assayed using the
SignaTECT PKA assay system (Promega) under the conditions recommended
by the company.
Electrophoretic mobility shift assay.
The oligonucleotides containing the IAPP-CRE element (
68/
44,
5'-GAGCTGCCTG ATGTCAGAGC TGAGGA-3'), the
IAPP-A1 element (
95/
75, 5'-GATGGAAATT
AATGACAGAG G-3'), and the consensus CRE (Promega; 5'-AGAGATTGCC TGACGTCAGA GAGCTAG-3') were used for
electrophoretic mobility shift assay (EMSA). Incubations were
terminated by washing INS-1 cells with buffer containing (in mM) 10 HEPES, pH 7.9, 1.5 MgCl2, 10 KCl, 0.5 dithioerythritol, and
1 phenylmethylsulfonyl fluoride and 2 µg/ml aprotinin, 10 µg/ml
leupeptin, 2 µg/ml pepstatin, and 0.1% NP-40. Cells were
mechanically detached using the washing buffer. Cell suspensions were
placed on ice for 5 min and centrifuged at 4,000 g, 4°C
for 2 min. Pelleted cells from one 100-mm dish were suspended in 50 µl of buffer containing 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithioerythritol, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 1 mM
phenylmethylsulfonyl fluoride and then incubated on ice for 30 min.
Insoluble material was removed by centrifugation at 12,000 g
for 20 min at 4°C, and nuclear protein content was assayed.
Binding reaction, final volume 10 µl, contained 50 µg/ml
poly(dI-dC) · poly(dI-dC), 4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM dithioerythritol, 50 mM NaCl, 10 mM Tris · HCl, pH 7.5, and 5 µg of nuclear protein. After
incubation for 5 min at room temperature, 8,000-10,000 cpm of
32P-labeled oligonucleotides were added. The reaction was
terminated after a 20-min incubation at room temperature by addition of
1 µl of loading buffer (250 mM Tris · HCl, pH 7.5, 0.2%
bromphenol blue, and 40% glycerol), and samples were loaded on a
Tris-borate-EDTA-buffered 6% polyacrylamide gel
(acrylamide-bisacrylamide, 80:1) that had been prerun for 20 min at 100 V. After separation, the gel was dried and exposed to X-ray film for
6-12 h at
70°C.
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RESULTS |
The IAPP gene was expressed in INS-1 cells, supported by RT-PCR
and Northern blot analysis. PCR amplification of the cDNA made from
INS-1 cells revealed a prominent and unique band having the expected
size on an agarose gel (Fig.
1A) (26). IAPP
gene expression was confirmed by Northern blot analysis (Fig.
1B). Although IAPP mRNA could not be detected in a
pancreatic ductular carcinoma cell line (Panc-1 cells), a transcript of
0.9 kb was clearly evident in INS-1 cells. This size of the IAPP mRNA
is consistent with previous observations with the use of primary culture of rat islets (13, 26). When INS-1 cells were
treated with an increased concentration of glucose for 12 h, IAPP
mRNA level was increased in a concentration-dependent manner (Fig. 1C).

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Fig. 1.
Expression of the islet amyloid polypeptide (IAPP) gene
in INS-1 cells detected by RT-PCR (A) and Northern blot
analysis (B and C). INS-1 cells were cultured in
RPMI-1640 medium with supplements. RT-PCR was performed using RNA
purified from INS-1 cells and primers specific for IAPP cDNA.
A: left lane, DNA marker; middle lane,
negative control; right lane, a 209-bp fragment of IAPP cDNA
was detected. Northern blot analysis was performed using
32P-labeled IAPP probe to hybridize with total RNA isolated
from INS-1 and a pancreatic duct cell line, Panc-1 cells. B:
IAPP mRNA was detected in INS-1 cells but not in Panc-1 cells;
C: IAPP mRNA level was elevated by increasing glucose
concentrations for 12 h. Ethidium bromide-stained 18S and 28S rRNA
was included, indicating similar amounts of RNA being loaded. Data are
representative of 3 experiments.
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To understand potential signaling pathways regulating IAPP gene
expression, INS-1 cells were stimulated with 10 µM forskolin, an
activator of adenylate cyclase, 1 µM phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC), and 100 µM
carbamylcholine (carbachol), a muscarinic receptor agonist, for 1 or
24 h, respectively. Stimulation with these reagents for 1 h
did not cause any change in IAPP mRNA level (data not shown), but after
24 h of stimulation, forskolin increased IAPP gene expression by
163 ± 11% compared with control cells, whereas PMA and carbachol
had no effect (Fig. 2). This pattern of
IAPP gene expression resembles that of islet cells from healthy animals
(13), indicating INS-1 cells represent a suitable
-cell
model to analyze IAPP gene regulation.

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Fig. 2.
Effects of forskolin, phorbol-12-myristate-13-acetate
(PMA), and carbachol on IAPP mRNA level in INS-1 cells. INS-1 cells
were cultured in RPMI-1640 medium with supplements and stimulated with
10 µM forskolin, 1 µM PMA, and 100 µM carbachol for 24 h.
Total RNA was isolated and hybridized to 32P-labeled IAPP
probe. Ethidium bromide-stained 18S and 28S were included, indicating
similar amounts of RNA being loaded. The mRNA level was quantified by
densitometry and normalized to 28S rRNA. Data (3 independent
experiments, n = 9) are expressed as means ± SE.
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The elevated level of IAPP mRNA elicited by glucose and forskolin could
be due to changes in stability of IAPP mRNA or increased transcription
of the gene. To study whether transcription of the IAPP gene was
affected by glucose and forskolin, a luciferase reporter gene construct
was prepared by fusing a 993-bp fragment of the human IAPP gene
promoter to the luciferase gene of the reporter plasmid. The IAPP
promoter of this length has been shown to be fully responsive (9,
28). Transfection of INS-1 cells with control plasmid lacking
the IAPP promoter showed no promoter activity (Fig.
3), whereas in cells that had been
transfected with the PGL3-AP1.0 construct, basal promoter activity was
detectable and was increased nearly 15-fold after 24 h stimulation
with 10 µM forskolin. The effect of forskolin on IAPP promoter
activity was detected as early as 1 h after initiation of the
stimulation, reaching a peak at 12 h and remaining elevated for at
least another 12 h (Fig.
4B). Forskolin-stimulated IAPP
gene promoter activity seems to be
-cell specific, because when
cells of a non-
-cell line, COS-1 cells, were transfected with
PGL3-AP1.0 construct, forskolin failed to stimulate the transcription
of the gene, although the basal promoter activity was detectable (Fig.
5). The IAPP promoter activity was also
increased by glucose in a concentration-dependent manner (Fig.
4A). Compared with control cells, treatment with 10 mM
glucose increased IAPP promoter activity three- to fourfold. To study
the possible involvement of signals derived from glucose metabolism in
this increase, we examined effects of nonmetabolizable glucose analogs,
2-deoxyglucose (20 mM) and 3-O-methylglucose (20 mM), on
IAPP gene transcription. The effects of mannose (10 mM), mannitol (10 mM), and glucosamine (5 mM) were also studied. As shown in Fig.
6, mannitol, glucosamine,
3-O-methylglucose, and 2-deoxyglucose had no effect on IAPP
gene promoter activity, whereas mannose mimicked the stimulatory effect
of glucose.

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Fig. 3.
IAPP gene promoter activity in INS-1 cells
transfected with PGL3-AP1.0 constructs. INS-1 cells were transfected
with control plasmids (PGL3-Basic) and PGL3-AP1.0 constructs and grown
in RPMI-1640 medium with supplements. The cells were stimulated with 10 µM forskolin for 24 h. Luciferase activity was assayed and
normalized for the protein amounts in each sample. Luciferase activity
measured in cells transfected with PGL3-AP1.0 without forskolin
stimulation was assigned a value of 1. Data (n = 4 for
control tranfection, n = 9 for cells transfected with
PGL3-AP1.0) are expressed as means ± SE.
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Fig. 4.
Effects of glucose and forskolin on IAPP gene promoter
activity in INS-1 cells. INS-1 cells were transfected with PGL3-AP1.0
and grown in RPMI-1640 medium with different glucose concentrations
(A) for 12 h and stimulated with 10 µM forskolin for
varied times at 10 mM glucose concentration (B). Luciferase
activity was assayed and normalized for the protein amounts in each
sample. Luciferase activity of control cells was assigned a value of 1. Data (3 independent transfections, n = 9) are expressed
as means ± SE.
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Fig. 5.
Effect of forskolin on IAPP gene promoter activity
in COS-1 cells. COS-1 and INS-1 cells were transfected with PGL3-AP1.0
and stimulated with 10 µM forskolin for 24 h in RPMI-1640 medium
containing 10 mM glucose. Luciferase activity was assayed and
normalized for the protein amounts in each sample. Luciferase activity
in cells without forkolin stimulation was assigned a value of 1. Data
(2 independent transfections, n = 6) are expressed as
means ± SE.
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Fig. 6.
Involvement of glucose metabolism in glucose regulation
of IAPP gene promoter activity in INS-1 cells. INS-1 cells were
transfected with PGL3-AP1.0 and treated with 10 mM mannose, 5 mM
glucosamine, 20 mM 2-deoxyglucose, 20 mM 3-O-methylglucose,
or 10 mM mannitol for 12 h in RPMI-1640 medium. Glucose
stimulation (10 mM) was also included as positive control. Luciferase
activity was assayed and normalized for the protein amounts in each
sample. Luciferase activity of control cells was assigned a value of 1. Data (2 independent transfections, n = 6) are expressed
as means ± SE.
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These experiments demonstrated that both glucose and forskolin
stimulate IAPP gene transcription in INS-1 cells. However, the question
remains whether they regulate IAPP gene expression via the same
mechanisms. To address this issue, we further investigated forskolin-stimulated IAPP promoter activity in cells cultured in the
absence of glucose and examined the involvement of PKA signaling in
glucose-activated IAPP gene transcription.
Figure 7 shows a glucose-independent
effect of forskolin on IAPP promoter activity. In the absence of
glucose, forskolin increased the promoter activity by nearly 15 times
that of control cells. At 10 mM glucose, the effect of forskolin in
stimulating IAPP promoter activity was proportionally the same (Fig.
7A). This glucose-independent effect of forskolin on IAPP
gene transcription could be mimicked by stimulation with glucagon-like
peptide-1 (GLP-1), a known stimulant of PKA signaling in this cell line (31). The effect was relatively moderate (Fig.
7B), reflecting the capability of forskolin and GLP-1 to
stimulate cAMP accumulation in these cells (Fig.
8A).

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Fig. 7.
Forskolin-stimulated IAPP gene promoter activity in the
presence and absence of glucose. INS-1 cells were transfected with
PGL3-AP1.0 and stimulated with 10 µM forskolin for 12 h in the
absence or presence of 10 mM glucose (A) or stimulated with
20 nM glucagon-like peptide (GLP)-1 for 6 h in the absence of
glucose (B) in RPMI-1640 medium. Luciferase activity was
assayed and normalized for the protein amounts in each sample.
Luciferase activity in cells grown in 0 mM glucose was assigned a value
of 1. Data (3 independent transfections, n = 9) are
expressed as means ± SE.
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Fig. 8.
cAMP level and PKA activity in INS-1 cells. A: INS-1
cells were stimulated with 10 µM forskolin or 20 nM GLP-1 for 30 min,
and cellular cAMP level was determined (n = 6).
B: cellular extracts from INS-1 cells were subjected to an
in vitro protein kinase A (PKA) activity assay. In the presence of 1 µM cAMP, PKA activity was dramatically increased, which was blocked
dose dependently by inclusion of H-89 in the reactions
(n = 3). Data are expressed as means ± SE.
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In an attempt to block the effects of glucose and forskolin on IAPP
gene transcription, an inhibitor of PKA, H-89, was used. The
concentration of H-89 used has been previously reported to selectively
block PKA signaling (8, 16). We also determined the
ability of H-89 to block cAMP-stimulated PKA activity by use of
cellular extracts from INS-1 cells (Fig. 8B). Results from this set of experiments showed that treatment with 5 µM H-89 blocked 1 µM forskolin-stimulated IAPP promoter activity but had no effect on
glucose-driven IAPP gene transcription (Fig.
9A). The GLP-1-stimulated IAPP
promoter activity was also completely inhibited by H-89 (Fig. 9B). Given that the magnitude of 1 µM forskolin-stimulated
IAPP promoter activity was about threefold higher than that of 10 mM glucose, the selective inhibitory effect of H-89 on forskolin, but not
on glucose stimulation, indicates that cAMP-PKA signaling is not the
predominant pathway utilized by glucose to activate IAPP gene
transcription.

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Fig. 9.
Effect of the PKA inhibitor H-89 on glucose-, forskolin-,
and GLP-1-stimulated IAPP gene promoter activity. INS-1 cells were
transfected with PGL3-AP1.0 and grown in RPMI-1640 medium with
supplements for 24 h. Medium was then changed to fresh RPMI-1640
without glucose. H-89 (5 µM) was added to the medium 20 min before 10 mM glucose or 1 µM foskolin stimulation for 12 h (A)
or 20 nM GLP-1 stimulation for 6 h (B). Luciferase
activity was assayed and normalized for the protein amounts in each
sample. Luciferase activity in control cells was assigned a value of 1. Data (3 independent transfections, n = 9) are expressed
as means ± SE.
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Eukaryotic gene expression is regulated at the transcriptional level
through transcription factors that act on DNA regulatory elements
flanking the gene. The possibility that forskolin and glucose stimulate
IAPP gene transcription by targeting different DNA regulatory elements
of the gene was tested using EMSA (Fig. 10). The IAPP-A1 element has been
suggested to be important for the activation of IAPP gene transcription
in islet
-cells, which is a potential target element of glucose
signaling (3). The IAPP-CRE element of the promoter
proximal region was assumed to be a target element of this gene to PKA
signaling. The consensus CRE was also included as a positive control,
because increased binding of this element represents a typical cellular
response to PKA stimulation. Two major bands were detected for the
IAPP-A1 element with the use of nuclear proteins from
non-glucose-treated cells, although the upper band was less stable.
This is similar to previous observations (3, 32), and
these bands were enhanced after 10 mM glucose treatment but were
unchanged after forskolin stimulation. Conversely, when the consensus
CRE and IAPP-CRE were analyzed, the two major bands that were shifted
on the gel were both enhanced by forskolin but not by glucose
stimulation. The results from EMSA thus indicated that glucose and
forskolin regulate IAPP gene transcription through different DNA
regulatory elements of the promoter and that forskolin, but not
glucose, stimulates PKA signaling in this cell line. To further
dissociate the signaling pathways coupling glucose and forskolin to
IAPP gene transcription, the CRE-like element flanking the IAPP gene
was mutated as described in MATERIALS AND METHODS. This
mutation resulted in the loss of ~70% of forskolin-induced IAPP gene
transcription, whereas the basal and glucose-enhanced IAPP promoter
activity remained unchanged (Table 1).
These findings strongly support the view that glucose and forskolin
regulate IAPP gene expression through different signal transduction
pathways.

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Fig. 10.
Electrophoretic mobility shift assay of the binding of consensus
cAMP-regulatory element (CRE), the IAPP-CRE, and the IAPP-A1 elements.
Nuclear extracts were isolated from INS-1 cells that had been treated
with 10 mM glucose or 10 µM forskolin without glucose for 12 h.
Five micrograms of nuclear proteins were subjected to a binding
reaction with 32P-labeled oligonucleotide probes, and the
DNA-protein complex was resolved in 6% acrylamide gel. For competition
experiment, 50× excess of cold oligonucleotide was included in the
binding reaction.
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Table 1.
Effects of mutation of the CRE-like element flanking the IAPP gene on
forskolin- and glucose-stimulated IAPP gene promoter activity
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DISCUSSION |
Previous work on IAPP gene regulation has identified the DNA
cis-elements that are responsible for activation of this
gene in pancreatic
-cells (3, 14), whereas the
intracellular signaling events that link glucose to IAPP gene
activation are not well defined. In the present work, we have examined
the expression of the IAPP gene in INS-1 cells and demonstrated that
glucose and forskolin regulate IAPP gene expression through different cellular mechanisms.
The involvement of the cAMP-PKA signaling pathway in the regulation of
IAPP gene expression has been a matter of controversy. An early study
using a reporter gene assay reported that forskolin was unable to
stimulate IAPP promoter activity in the
TC3 cell line
(22), whereas recent studies from Gasa et al.
(13) demonstrated that forskolin increased IAPP gene
expression in primary culture of rat islets, analyzed mainly by the
Northern blot technique. These apparently contradictory observations
might be attributed to the different cell models used and to the
parameters assayed. The results from the present study demonstrate that
forskolin increases IAPP gene expression in INS-1 cells, measured by
Northern blot analysis, reporter gene assay, and EMSA, thus supporting the finding from Gasa et al.. Because forskolin is well known to
activate adenylate cyclase, leading to an increase in intracellular cAMP level, these observations indicate the participation of cAMP-PKA signaling in the control of IAPP gene expression in islet
-cells. Moreover, we found that the relative increase in IAPP promoter activity
after forskolin stimulation was proportionately the same in the
presence and absence of glucose, demonstrating a glucose-independent effect of forskolin on IAPP gene transcription. This indicates that
activation of the cAMP-PKA signaling pathway leads to upregulated IAPP
gene transcription in islet
-cells in a glucose-independent manner.
The glucose-independent effect of forskolin could also be achieved by a
physiological stimulation with GLP-1, an insulin-stimulatory hormone
that increases cAMP levels in INS-1 cells (31), although the effect of GLP-1 was only moderate.
Glucose-stimulated IAPP gene expression has previously been reported in
several cell culture models (9, 13, 25, 27) and in rat
pancreas (24). The intracellular signal pathways linking
glucose to IAPP gene expression remain poorly understood. The most
interesting finding from the present study is that glucose regulates
IAPP gene expression through a PKA-independent pathway. This was first
supported by the observation that H-89, a potent PKA inhibitor, had no
inhibitory effect on the glucose-driven IAPP gene promoter activity,
whereas it blocked the forskolin effect. Second, EMSA showed that
forskolin stimulated an increase in the binding of both consensus CRE
and IAPP-CRE, indicating an enhanced PKA signaling that is involved in
IAPP gene regulation. On the other hand, glucose increased IAPP-A1
binding but had no effect on the binding of either consensus CRE or
IAPP-CRE, suggesting that PKA signaling is less likely to be involved
in glucose-stimulated IAPP gene expression. Third, mutagenesis in the
CRE-like element of the IAPP gene promoter indicated that this element
was primarily responsible for PKA-stimulated IAPP gene transcription,
whereas it was irrelevant to the glucose effect. Thus the intracellular signal transduction pathways whereby glucose and forskolin regulate IAPP gene expression can be dissociated. It is noteworthy, however, that, relative to control cells, a proportionate increase in IAPP gene
promoter activity was seen when the cells were stimulated with
forskolin in the presence of glucose, indicating a synergistic effect
of these two pathways to stimulate IAPP gene transcription. The
dissociation of glucose-initiated signaling and PKA-mediated pathway
and their synergistic effect in stimulating IAPP gene expression
provide new information in the understanding of this gene regulation in
pancreatic
-cells. This may have clinical therapeutic value, since
inhibiting IAPP gene transcription has been proposed as one of the
possible approaches to treat type 2 diabetes mellitus
(19).
The involvement of glucose metabolism on regulation of IAPP gene
expression has been previously documented (12, 13). The results from the present study support the role of metabolically derived signals in the effect of glucose on IAPP gene expression. As
control for osmolarity, the metabolically inert monosaccharide mannitol
did not affect IAPP gene transcription. Moreover, 2-deoxyglucose, which
is phosphorylated by hexokinases but not further metabolized through
the glycolytic pathway, or 3-O-methylglucose, which is transported into the cell but cannot be metabolized, failed to mimic
the effect of glucose on IAPP gene promoter activity. These data
suggest that the effect of glucose is related to its intracellular metabolism. This was further supported by the fact that the hexose mannose increased IAPP gene transcription (2-fold). However,
glucosamine, which has been previously shown to regulate gene
transcription in rat glomerular mesangial cells (15), had
no effect on IAPP gene promoter activity in INS-1 cells after 12 h
of stimulation. This may indicate that the hexosamine biosynthesis
pathway, through which ~1-3% of glucose is normally diverted
after entering the cell (20), does not account for the
glucose effect on IAPP gene transcription. Alternatively, this may
suggest that glucosamine regulates gene transcription in a cell-type or
gene-specific manner.
Multiple elements flanking the IAPP gene, including the A1 element used
in this study, have been shown to participate in the regulation of this
gene (3, 14). The enhanced DNA binding activity of the A1
element by glucose treatment indicates the contribution of this element
in the regulation of IAPP gene transcription in INS-1 cells. Recent
studies have described the binding activity of the A1 element of the
IAPP promoter by the pancreatic and duodenal homeobox gene-1
transcription factor in
TC 3 cells (3) and MIN6 cells
(32), which is the likely explanation for the A1-binding activity detected in the present study. On the other hand, the binding
activity of the CRE-like element of the IAPP promoter has not been
previously reported in islet
-cells despite the observations of
increased IAPP mRNA level after stimulation of PKA (13).
We showed here that transcription factors bound to the CRE-like element
of the IAPP promoter could be mediated by forskolin, indicating
involvement of this element in the regulation of IAPP gene expression
on PKA activation. The signaling cascade from cAMP-PKA and CRE-binding
protein to the CRE of many target genes has been well characterized
(29). It is, therefore, not surprising that this signaling
machinery also functions for the IAPP gene in pancreatic
-cells.
However, the fact that forskolin did not stimulate IAPP gene
transcription in PGL3-AP1.0-transfected COS-1 cells suggests the
requirement of a
-cell-specific signaling machinery in initiating
this gene expression on PKA activation.
In conclusion, the present study has characterized expression of
the IAPP gene in INS-1 cells and demonstrated that glucose regulates
transcription of this gene through PKA-independent mechanisms. Further
studies are required to define the more specific signaling pathways whereby glucose stimulates IAPP gene transcription.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Michael S. German, of the Hormone Research Institute
and Department of Medicine, University of California at San Francisco,
for kindly providing the pFOXCAT-AP1.0 constructs, and Sara Erickson
for excellent secretarial assistance.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: L. J. Miller, Mayo Clinic and Foundation, Guggenheim 17, Rochester, MN
55905 (E-mail: miller{at}mayo.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 December 2000; accepted in final form 26 June 2001.
 |
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