Download Glucose and forskolin regulate IAPP gene expression through

Am J Physiol Endocrinol Metab
281: E938–E945, 2001.
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
Received 7 December 2000; accepted in final form 26 June 2001
islet amyloid polypeptide; cyclic adenosine 3,5-monophosphate; protein kinase A
(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
ISLET AMYLOID POLYPEPTIDE
Address for reprint requests and other correspondence: L. J.
Miller, Mayo Clinic and Foundation, Guggenheim 17, Rochester, MN
55905 (E-mail: [email protected]).
E938
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 demonThe costs of publication of this article were defrayed in part by the
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0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
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Ding, Wei-Qun, Eileen Holicky, and Laurence J.
Miller. Glucose and forskolin regulate IAPP gene expression
through different signal transduction pathways. Am J Physiol
Endocrinol Metab 281: E938–E945, 2001.—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.
IAPP GENE EXPRESSION IN INS-1 CELLS
strated 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.
MATERIALS AND METHODS
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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 ␮l 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 ⱖ2 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 PCRbased, 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 PGL3APCREm.
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 ␮l of lipofectin with 5 ␮g
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 ␮l 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 ␮l of luciferase assay
reagent mixed with 50 ␮l 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
␮M forskolin or 20 nM GLP-1 at 37°C for 30 min in KrebsRinger-HEPES (KRH) medium containing (in mM) 25
HEPES, pH 7.4, 104 NaCl, 5 KCl, 1 KH2PO4, 1.2 MgSO4, 2
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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 ␮g/ml streptomycin, 1 mM sodium pyruvate,
and 50 ␮M 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 ␮g/ml streptomycin at 37°C in an environment
containing 5% CO2.
RT-PCR. First-strand cDNA was synthesized using total
RNA (2.5 ␮g) isolated from INS-1 cells with 5 ␮M 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 ␮l. 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 ␮l of the
resulting cDNA were subjected to PCR containing 0.2 ␮M 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 ␮l of
TRIzol per well. Two hundred microliters of chloroform were
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IAPP GENE EXPRESSION IN INS-1 CELLS
EDTA, 0.5 mM dithioerythritol, 50 mM NaCl, 10 mM
Tris 䡠 HCl, pH 7.5, and 5 ␮g of nuclear protein. After incubation for 5 min at room temperature, 8,000–10,000 cpm of
32
P-labeled oligonucleotides were added. The reaction was
terminated after a 20-min incubation at room temperature
by addition of 1 ␮l of loading buffer (250 mM Tris 䡠 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.
RESULTS
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 ␮g/ml aprotinin, 10 ␮g/ml
leupeptin, 2 ␮g/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 ␮l 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 ␮g/ml aprotinin, 10 ␮g/ml
leupeptin, 2 ␮g/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 ␮l, contained 50 ␮g/ml
poly(dI-dC) 䡠 poly(dI-dC), 4% glycerol, 1 mM MgCl2, 0.5 mM
AJP-Endocrinol Metab • VOL
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 ␮M forskolin, 1 ␮M PMA, and 100 ␮M 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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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
32
P-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.
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).
IAPP GENE EXPRESSION IN INS-1 CELLS
To understand potential signaling pathways regulating IAPP gene expression, INS-1 cells were stimulated
with 10 ␮M forskolin, an activator of adenylate cyclase,
1 ␮M phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC), and 100 ␮M 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.
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 ␮M forskolin. The effect
of forskolin on IAPP promoter activity was detected as
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.
281 • NOVEMBER 2001 • www.ajpendo.org
Fig. 4. Effects of glucose and forskolin on IAPP gene promoter
activity in INS-1 cells. INS-1 cells were transfected with PGL3AP1.0 and grown in RPMI-1640 medium with different glucose
concentrations (A) for 12 h and stimulated with 10 ␮M 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.
AJP-Endocrinol Metab • VOL
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 ␮M 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. 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 RPMI1640 medium with supplements. The cells were stimulated with 10 ␮M
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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IAPP GENE EXPRESSION IN INS-1 CELLS
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
Fig. 8. cAMP level and PKA activity in INS-1 cells. A:
INS-1 cells were stimulated with 10 ␮M 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 ␮M 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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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 ␮M 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.
had no effect on IAPP gene promoter activity, whereas
mannose mimicked the stimulatory effect of glucose.
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).
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 ␮M H-89 blocked 1 ␮M forskolin-stimulated
IAPP promoter activity but had no effect on glucosedriven IAPP gene transcription (Fig. 9A). The GLP-1stimulated IAPP promoter activity was also completely
inhibited by H-89 (Fig. 9B). Given that the magnitude
of 1 ␮M 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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IAPP GENE EXPRESSION IN INS-1 CELLS
Table 1. Effects of mutation of the CRE-like element
flanking the IAPP gene on forskolin- and glucosestimulated IAPP gene promoter activity
Relative Luciferase Activity
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,
PGL3-AP1.0
PGL3-APCREm
Control
10 mM Glucose
10 ␮M Forskolin
1 ⫾ 0.1
2.9 ⫾ 0.3
15.5 ⫾ 0.5
0.9 ⫾ 0.1
3.1 ⫾ 0.2
4.6 ⫾ 0.4
Data (3 independent transfections, n ⫽ 9) are expressed as
means ⫾ SE. CRE, cAMP-regulatory element; IAPP, islet amyloid
polypeptide. INS-1 cells were transfected with PGL3-AP1.0 or PGL3APCREm and treated with 10 mM glucose or 10 ␮M forskolin in the
presence of glucose for 12 h in RPMI-1640 medium with supplements. Luciferase activity was assayed and normalized for the protein amounts in each sample. Luciferase activity in cells transfected
with PGL3-AP1.0 and grown in 0 mM glucose medium was set
arbitrarily as 1.0.
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 glucoseenhanced 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.
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.
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 ␮M 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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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 ␮M) was added to the medium
20 min before 10 mM glucose or 1 ␮M 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.
Treatment
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IAPP GENE EXPRESSION IN INS-1 CELLS
AJP-Endocrinol Metab • VOL
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 PKAmediated 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
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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 glucoseindependent effect of forskolin could also be achieved
by a physiological stimulation with GLP-1, an insulinstimulatory 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
IAPP GENE EXPRESSION IN INS-1 CELLS
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.
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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.
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