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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -cell system.

islet amyloid polypeptide; cyclic adenosine 3,5-monophosphate; protein kinase A


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -cells may prove relevant to the pathogenesis of type 2 diabetes mellitus and contribute to a better understanding of pancreatic beta -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 beta -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 beta -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 beta -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 beta -cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 beta -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.

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 beta -cell specific, because when cells of a non-beta -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.

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.

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.

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 beta -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


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous work on IAPP gene regulation has identified the DNA cis-elements that are responsible for activation of this gene in pancreatic beta -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 beta 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 beta -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 beta -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 beta -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 beta 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 beta -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 beta -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 beta -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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