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Am J Physiol Endocrinol Metab 291: E587-E595, 2006. First published April 18, 2006; doi:10.1152/ajpendo.00612.2005
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Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression

David A. Jacobson,1 Julie Cho,2 Luis R. Landa, Jr.,1 Natalia A. Tamarina,1 Michael W. Roe,1 Joseph D. Buxbaum,2 and Louis H. Philipson1

1Department of Medicine, University of Chicago, Chicago, Illinois; and 2Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Submitted 5 December 2005 ; accepted in final form 8 April 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and {alpha}-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM–/– islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM–/– islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (~200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate {kappa}-opioid receptors expressed predominantly in {alpha}-cells of the islet, we determined the role of dynorphin A-(1–17) in glucagon secretion from the {alpha}-cell. Stimulation with dynorphin A-(1–17) caused {alpha}-cell calcium fluctuations and a significant increase in glucagon release. DREAM–/– islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of {alpha}-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

calsenilin; potassium channel interacting protein; neuroendocrine; glucagon; dynorphin

CALCIUM FLUCTUATIONS PLAY A FUNDAMENTAL ROLE in pancreatic islet hormone secretion (4, 5). When glucose levels rise, the pancreatic beta-cells respond through inhibition of ATP-sensitive potassium channels, leading to depolarization and resulting calcium influx through L-type calcium channels, triggering insulin secretion (2). Although less is known about {alpha}-cell function, when glucose levels drop the electrical activity of pancreatic {alpha}-cells increases, resulting in calcium flux through T- and L-type calcium channels stimulating glucagon secretion (41). Insulin, zinc, and {gamma}-aminobutyric acid from beta-cells also regulate {alpha}-cell function (6, 43, 51). In both situations, repolarization of the membrane potential, in part through activation of voltage-gated potassium channels, terminates hormone secretion (17). Calcium levels can dictate cellular excitability through changes in membrane polarization and regulation of a multitude of calcium-binding proteins. Although calcium is essential for regulating islet hormone secretion, many of the calcium-sensitive mechanisms affecting this pathway are unresolved.

Glucose-induced calcium entry affects not only islet hormone secretion but also islet cell transcription. Insulin transcription is stimulated through elevations of intracellular calcium, which is required for islets to replenish released insulin and keep up with the demands of increased glucose consumption (34). One pathway by which insulin transcription is increased is through calcium-mediated activation of the nuclear factor of activated T cells (NFAT); NFAT is dephosphorylated by calcineurin, allowing its translocation into the nucleus (26). Glucagon transcription, on the other hand, is stimulated through membrane depolarization and calcium influx via a cAMP-response element, which is activated by calcium through calmodulin-dependent kinase activation of cAMP response element-binding protein (44). Changes in islet cell calcium levels have been linked to alterations in the expression of over 200 genes (36). The calcium-dependent mechanisms affecting this magnitude of gene expression are diverse and require further investigation.

Most cellular processes within the islet that are affected through calcium signaling, such as transcription, involve calcium-binding proteins. Although many calcium-binding proteins, such as calmodulin, have been well characterized in islets, the importance of more recently described calcium-binding proteins has not been addressed in islet cells (33). Herein we report expression of the calcium-binding protein downstream regulatory element antagonistic modulator (DREAM), which is also termed calsenilin (CSEN) or KChIP3 (potassium channel interacting protein), in islet cells and its role as a calcium regulated transcription factor.

CSEN was first identified for its ability to interact with presenilin, an interaction that affects apoptoic processes including calcium homeostasis and Abeta-peptide formation (7, 28, 29). It was rediscovered in 1999 for its ability to bind the downstream regulatory element (DRE) in the first intron of the prodynorphin gene, where it antagonizes transcription in a calcium-dependent manner (9). It was therefore termed DREAM for downstream regulatory element antagonistic modulator. It was subsequently identified in a two-hybrid screen for Kv4 delayed rectifier interacting proteins, and termed potassium channel interacting protein (KChIP3) (1). KChIP3 is a beta-subunit of Kv4 channels that has chaperone activity, promoting surface expression of Kv4 channels (1, 3, 45). Thus calsenilin/DREAM/KChIP3, simply termed DREAM in these studies, serves multiple roles in many tissues. This study investigates the expression of DREAM and its functions in the {alpha}- and beta-cells of the endocrine pancreas.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and Culturing of Mouse Islets of Langerhans and Cell Lines

Islets were isolated from the pancreata of 1- to 5-mo-old C57BL/J6 mice (Jackson Laboratories) and DREAM–/– animals (29), using collagenase digestion and Ficoll gradients as previously described (25). Islets were used acutely or cultured in RPMI 1640 medium supplemented with 10% FCS, indicated concentrations of glucose, 100 IU/ml penicillin, and 100 µg/ml streptomycin for 16 h. Min6 and {alpha}-TC1-6 cells (generous gifts from D. F. Steiner, University of Chicago) were cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% FCS, 50 IU/ml penicillin, and 50 IU/ml streptomycin. Cells and islets were maintained in a humidified incubator at 37°C with 5% CO2. Human cadoveric islets were a generous gift from Dr. M. Garfinkel (University of Chicago).

RT-PCR and Quantitative RT-PCR

Tri Reagent (Sigma) was used to prepare total RNA from islets and cells according to the manufacturer's protocol. Total RNA (2.5 µg) was reverse transcribed with 200 units of SuperScript III RT (Invitrogen) in a 20-µl reaction with 50 ng of random hexamers, following the manufacturer's protocol. The PCR was then used to amplify various cDNA transcripts as described (37). The primer sets for beta-actin were (5'-CCT GGA GAA GAG CTA TGA GCT-3' and 5'-GGATGC CAG AGG ATT CCA TAC-3') spanning an exon that was not amplified from any of the RT preparations used in these assays. The primer sets for KChIP1 were 5'-ATG GGG GCC GTC ATG GGC ACT-3' and 5'-GCA GTT CTC TCT TGG TGA AGT-3' and for KChIP2 were 5'-ATG CGG GGC CAA GGC CGA AAG-3' and 5'-ACG CTG TTT TCA CTG ACT CAG-3'; for DREAM were 5'-AGA GAT GCA GAG GAC CAA GGA-3' and 5'-ACT GGA CAG GAT CCA CTT GAT-3'; and for KChIP4 were 5'-GCT CAT GAA GCT CTT GCC CTG CT-3' and 5'-AAT CCT CTG TAA AGA ATC TGA-3'. The PCR product was measured via a fluorescent signal generated by the binding of the fluorophore SYBR Green (SG, Molecular Probes) to double-stranded DNA. Amplification cycles were 95° for 10 min followed by 40 cycles of 95° for 30 s, 60° for 30 s, and 72° for 20 s, when the fluorescence was read followed by a melting-curve ramp up to 95°, when all points were read on a continuous basis. Aliquots (10 µl) of these reactions were analyzed with 1.5% agarose gel electrophoresis, which was visualized by ethidium bromide fluorescence. All real-time PCR reactions were performed in triplicate, and PCR mixtures lacking RT template were included to ensure specificity without contamination. Each data set was normalized to beta-actin transcript content, and relative transcript content was determined by comparative CT (cycle threshold) analysis (39) between the relative groups.

Western Blot Analysis

Protein extracts were prepared from {alpha}- and TC1-6, Min6, mouse islet, human islet, HEK cells transfected with an islet-specific CMV-DREAM expression construct and control HEK cells that show no DREAM immunoreactivity (presently not shown) by extraction with SDS loading buffer (1% SDS, 30 mmol/l Tris·HCl, pH 6.8, 5% beta-mercaptoethanol, 5% glycerol, and 0.1% bromophenol blue) and heating at 70°C for 10 min. Proteins were prepared as a Western blot on a polyvinylidene difluoride membrane (Whatman). After electrophoresis through a 14% denaturing polyacrylamide gel, DREAM antibody (Zymed) was used to probe the membrane at 1:250 dilution in PBS, 0.1% Tween, and 3% powdered dried milk, followed by goat anti-rabbit horseradish peroxidase (HRP)-coupled secondary antibody (Santa Cruz Biotechnology) at 1:5,000 in the same solution. The membranes were washed in PBS containing 0.1% Tween between and after antibody incubations; HRP was illuminated using Pico Signal (Pierce) and exposed on Kodak X-omat Blue film (Kodak). These procedures were replicated with a second DREAM antibody kindly provided by Dr. J. Buxbaum (11).

Islet beta-Galactosidase Staining and Immunofluorescence

Mouse pancreata were isolated from DREAM–/– and control animals, cut into 2.5-mm cubes, and fixed for 1 h in 4% paraformaldehyde. The tissue was washed in PBS and stained for 1 h in a solution containing 5 mM potassium ferrocyanide, 5 mM ferricyanide, 2 mM MgCl2, and 1.2 mg/ml X-galactosidase. Stained tissue was washed in PBS, frozen in OCT compound, sectioned into 80-µm sections, placed on slides, and imaged at x20 on an inverted Olympus microscope with white light for beta-galactosidase (beta-gal). Islet beta- and {alpha}-cells were stained as described (55), using insulin and glucagon antibodies at 1:300 and anti-DREAM at 1:100 in combination with fluorescently conjugated secondary antibodies, fluorescein isothiocyanate and Cy5, respectively. These procedures were also replicated with a second DREAM antibody kindly provided by Dr. J. Buxbaum (Mount Sinai School of Medicine, New York, NY) (11).

Hormone Secretion Measurements

Mouse islets. Islets were allowed to recover following isolation for 4 h. For insulin measurements, 20 islets/animal (n = 5 animals/group) were perifused with Krebs-Ringer buffer (KRB) solution containing 2 mM glucose for 20 min followed by KRB with 16 mM glucose for 10 min and return to 2 mM glucose for 10 min. Collected perfusates (1 ml/min) were analyzed for insulin content by means of an ELISA-based detection kit (Linco Research). For mouse glucagon measurements, 20 islets were equilibrated in 1 ml of HEPES (25 mM)-buffered solution (pH 7.35) containing 20 mM glucose for 30 min, switched to 1 ml of HEPES-buffered solution containing 1 mM glucose for 1 h, and switched to 1 ml of HEPES-buffered solution containing 20 mM glucose for 1 h. Aliquots of the medium (100 µl) were directly analyzed for glucagon levels with a radioimmunoassay kit (Linco Research). Glucagon measurements made in low glucose were then normalized to glucagon levels taken following the switch back to high glucose (n = 3 assays/animal, n = 10 animals/group) and presented as means ± SE. Significant differences (P < 0.05) between the data sets were determined using an unpaired t-test.

Human islets and {alpha}-TC1-6 cells. For glucagon measurements from human islets, 100 islets were incubated in HEPES (25 mM)-buffered solution (pH 7.35) containing 20 mM glucose for 30 min, switched to 1 ml of HEPES-buffered solution containing 2 mM glucose for 30 min, and switched to 1 ml of HEPES-buffered solution containing 2 mM glucose for 30 min with or without U-50488. U-50488 was used at a concentration of 5 µM, based on previous studies demonstrating increased intracellular calcium levels in response to low micromolar levels of U-50488, which are blocked by {kappa}-opioid receptor-specific antagonists (48, 52, 53). {alpha}-TC1-6 cells were plated in six-well tissue culture plates and treated as the human islets except with or without 50 mM dynorphin A-(1–17). Dynorphin A-(1–17) was used at a concentration of 50 nM, based on previous studies demonstrating an EC50 of 30 nM for inward potassium currents activated by cloned {kappa}-opioid receptors (32). Glucagon measurements made in 2 mM glucose were then normalized by glucagon levels taken following the switch back to 2 mM glucose with or without {kappa}-opioid receptor agonist (n = 6 {alpha}-TC1-6 cells; n = 2 human islet sets per treatment condition) and presented as ± SEM. Significant differences (P < 0.05) between the data sets were determined using an unpaired t-test.

Electrophoretic Mobility and Competition Assays

Min6 nuclear extracts were prepared as described (13) and dialyzed twice for 3 h in 20 mM HEPES (pH 7.6), 20% glycerol, 100 mM KCl, 1.5 mM MgCl2, and 0.2 mM EDTA with a protease inhibitor cocktail (Sigma). Electrophoretic mobility shift assays were performed as described (38). Double-stranded oligonucleotides (5'-GAAGCCGGAGTCAAGGAGGCCCCTG-3') were radiolabeled at the 5' termini using [{gamma}-32P]ATP and T4 polynucleotide kinase and purified using Qiagen DNA columns. Unlabeled nonspecific competitor DNA (5'-catcaattattcacaacaccagcaca-3') was used to test the specificity of the shifted bands at 100-fold excess of the probe concentration. When indicated, DREAM antibody was preincubated with Min6 nuclear extracts for 5 min before addition of the probe. The mobility shift mixtures were electrophoresed through nondenaturing 3% polyacrylamide gels at 100 V for ≥4 h. The gels were then vacuum dried, and signals were exposed on X-omat Blue film (Kodak).

Reporter Assays

Min6 cells were transfected using lipofection (Polyfect; Qiagen) with a 9:1 mixture of luciferase reporter and pCMV-LacZ plasmid DNAs. The cells were switched to low (2 mM) or high glucose (20 mM) conditions 12 h after transfection, and in all cases luciferase assays were conducted 36 h after transfection. Where indicated, a CMV-based DREAM expression plasmid, designed from mouse islet cDNA, was transfected in combination with the prodynorphin luciferase reporter. Cells were then harvested in ATP buffer (0.1 M NaPO4, 4 mM ATP, 1 mM PPi, and 6 mM MgCl2) with 0.2% Triton X-100; 5–15 liters of lysate was used as described (27), on an AutoLumat LB 953 luminometer (EG&G, Berthold Analytical Instruments). The data were normalized to beta-galactosidase activity (18), as measured on the same luminometer, grouped for each reporter, and presented as means SE. Significant differences (P < 0.05) between the data sets were determined using an unpaired t-test.

Intranuclear Calcium Measurements

Nuclear calcium changes of Min6 cells in response to glucose (20 mM) stimulation were measured using fluorescent changes recorded with the nuclear localized ratiometric pericam, (RPC-Nu) as described (35). Islet nuclear calcium was determined with the calcium indicator fura red, as described (49), with nuclear regions identified through confocal sections and plotted as a change in fluorescent signal.

Measurement of Cytoplasmic Calcium

Mouse {alpha}-TC1-6 cells were loaded with fura 2 for 20 min at 37°C in KRB containing (in mmol/l) 119 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 25 HEPES supplemented with 5 µmol/l acetoxymethyl ester of fura 2 (Molecular Probes). Fluorescence imaging was performed using a Nikon Eclipse TE2000-U microscope equipped with an epifluorescence illuminator and an approximately x40 oil immersion fluorescence objective (Fryer) and MetaFluor software (Universal Imaging). Cells were perifused, at a flow of 1 ml/min, with appropriate KRB-based solutions at 37°C containing 3 mM glucose. Addition of U-50488 and norbinaltorphimine was made directly to the perfusion chamber with a pipette while perfusion flow was interrupted. The ratio of fluorescence intensity at excitation wavelengths 340 and 380 nm (F340/F380) was taken every 5 s with background subtraction. Calibration of intracellular Ca2+ concentration was estimated as previously described (41).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of DREAM Expression in Pancreatic Islets

Calcium signaling plays an essential role in the islet regulating both insulin and glucagon secretion. Thus we investigated the presence of the calcium-binding protein DREAM and related family members within the islet. To determine whether DREAM is expressed in the pancreatic islet cells, total RNA from Min6 mouse beta-cells, {alpha}-TC1-6 mouse {alpha}-cells, and mouse islets were examined for DREAM (KChIP3) gene expression together with other similar KChIP family members via RT-PCR. RT-PCR of Min6 cell and islet RNA with KChIP1-, KChIP2-, DREAM-, and KChIP4-specific primers resulted in amplification of the expected size products (Fig. 1A). In contrast, RT-PCR of {alpha}-TC1-6 cell RNA produced the predicted product only for DREAM (Fig. 1A). DREAM RNA is the most abundantly expressed message in mouse islets and beta- and {alpha}-cells, as illustrated by the intensity of the RT-PCR bands for DREAM (Fig. 1A) and using quantitative real-time PCR (Fig. 1B). In addition, protein homgenates from Min6 and {alpha}-TC1-6 cells show strong expression of DREAM protein (Fig. 1C). To study the role of DREAM in the mouse islet, a transgenic animal with a disrupted DREAM gene was utilized (29). In the recombinant mice, exon 2 of the DREAM gene was replaced with an internal ribosomal entry site (IRES) and a beta-gal gene (29). Pancreatic sections from the DREAM animals assayed for beta-gal activity show prominent activity in islet-like structures (Fig. 1D). Insulin and glucagon staining of primary mouse (wild-type) islet cells in combination with staining for DREAM confirmed expression of DREAM in both {alpha}- and beta-cells of the mouse islet (Fig. 2, A and B, respectively) with abundant levels in nuclear-like structures.


Figure 1
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  		Fig. 1. Potassium channel interacting protein (KChIP) expression in pancreatic islet cells. A: identification of downstream regulatory element antagonistic modulator (DREAM) and family members KChIP1, KChIP2, and KChIP4 expression in mouse islets (3), Min6 beta-cells (2), and {alpha}-TC1-6 {alpha}-cells (1) using RT-PCR analysis with (+) or without (–) reverse transcriptase (RT). B: relative transcript abundance of DREAM and family members KChIP1, KChIP2, and KChIP4 in mouse islets and Min6 and {alpha}-TC1-6 cells quantified by real-time RT-PCR. The transcript number is presented as comparative CT calculations with normalization to beta-actin levels; n = 6. C: Western blot analysis of human islet (lane 1), mouse islet (lane 2), Min6 cell (lane 3), {alpha}-TC1-6 cell (lane 4), and overexpressed mouse islet DREAM (lane 5) protein, using a DREAM-specific antibody. D: DREAM –/– internal ribosomal entry site (IRES)-beta-galactosidase (beta-gal) pancreatic section (90 µM) stained with X-gal.

 

Figure 2
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  		Fig. 2. Islet {alpha}- and beta-cell-specific DREAM expression: A: islet {alpha}-cells stained with glucagon (left, green) and DREAM (middle, red) antibodies with overlay of both glucagon and DREAM staining (right, green and red, respectively). B: islet beta-cells stained with insulin (left, green) and DREAM (middle, red) with overlay of both insulin and DREAM (right, green and red, respectively).

 
Transcriptional Effects of DREAM in Neuroendocrine Cells

Because DREAM immunoreactivity was identified in nuclear regions of {alpha}- and beta-cells (Fig. 2, A and B), its role as a calcium-regulated transcriptional factor was investigated. Electrophoretic mobility shift assays were performed using a radiolabeled probe containing a DREAM-interacting motif from the mouse prodynorphin gene, termed the downstream regulatory element (DRE), using nuclear extracts prepared from Min6 and {alpha}-TC1-6 cells. Nuclear extracts from both sources induced gel shifts of two bands (Fig. 3, lanes 1 and 5) that were competitively inhibited by nonradiolabeled DNA containing the DRE sequence. However, neither of the shifted complexes was inhibited by nonradiolabeled DNA containing mutations in the DRE sequence (Fig. 3, lanes 3 and 4 for Min6 and lanes 7 and 8 for {alpha}-TC1-6). To distinguish whether DREAM bound to the DRE motif, nuclear extracts were preincubated with DREAM antibody followed by addition of the labeled prodynorphin probe. Incubation of this antibody with the DRE-containing probe and both Min6 and {alpha}-TC1-6 nuclear extracts competed with the top band, indicating that this complex contains DREAM (Fig. 3, lanes 2 and 6).


Figure 3
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  		Fig. 3. DREAM from {alpha}- and beta-cells binds to mouse prodynorphin (PDN) downstream regulatory element (DRE). Electrophoretic mobility shift assay performed with nuclear extracts from Min6 and {alpha}-TC1-6 cells, using a mouse PDN probe containing a DRE motif. Two bands are detected when Min6 and {alpha}-TC1-6 extracts are incubated with the probe (lanes 1 and 5): the top band (arrow) is lost with addition of a DREAM antibody (lanes 2 and 6); neither band is affected by addition of 100-fold excess unlabeled nonspecific DNA (lanes 3 and 7); and both bands are competitively inhibited by addition of 100-fold excess unlabeled probe (lanes 4 and 8).

 
To determine whether DREAM regulates transcription in a calcium-dependent manner, early-passage Min6 cells were employed, which respond to elevated glucose with increased intranuclear calcium levels (Fig. 4B), in combination with a DRE-containing luciferase construct (Fig. 4A). The DRE construct contains a functional DRE motif in a fragment from the mouse prodynorphin gene fused to the luciferase gene (Fig. 4A). Extracts from Min6 cells transfected with the DRE construct showed a doubling of luciferase activity when the cells were stimulated with 20 mM glucose compared with 2 mM glucose treatment for 24 h (Fig. 4C). In contrast, with a DRE construct where the DRE site is mutated (Fig. 4A, 2), there is no significant difference between cells stimulated with low (2 mM) or high (20 mM) glucose (Fig. 4C). In addition, when Min6 cells were transfected with both the DRE construct and a DREAM expression construct, there was a 1.7-fold decrease in luciferase expression compared with the DRE construct alone (Fig. 4C). These results demonstrate that DREAM can regulate transcription from a DRE motif in Min6 beta-cells.


Figure 4
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  		Fig. 4. DREAM regulates PDN transcription from a DRE motif in Min6 cells. A: reporter constructs with the mouse PDN 5'-flanking sequence with the endogenous DRE motif (1) or a mutant DRE motif (2) fused to the luciferase coding sequence. An expression construct containing a CMV promoter and the coding sequence of DREAM cloned from mouse islet cDNA (3) was used in some reporter assays. B: representative change in intranuclear calcium levels of Min6 cells in response to low (2 mM) and high (20 mM) glucose stimulation, measured using a nuclear localized ratiometric pericam. C: luciferase expression levels from Min6 cells transfected with indicated PDN reporter constructs in the absence or presence of the KChIP3 expression construct, with low (2 mM, open bars) or high (20 mM, gray bars) glucose stimulation. *P < 0.05.

 
Because the mouse prodynorphin gene is upregulated with glucose stimulation in Min6 cells (21), the regulation of the endogenous prodynorphin gene was investigated in freshly dissociated islets. Prodynorphin levels were determined in mouse islets treated in vitro with low (2 mM) or high (16 mM) glucose for 16 h, which induces a pronounced rise in islet intranuclear calcium (Fig. 5D). RNA harvested from the islets was assayed using quantitative real-time PCR for differences in prodynorphin transcript levels, normalizing expression to beta-gal expression. Islets treated with high (16 mM) glucose show a 10-fold increase in the level of prodynorphin message observed when islets were treated with low (2 mM) glucose (Fig. 5, A and B). To determine whether DREAM affects the glucose regulation of prodynorphin, islets harvested from animals deficient in DREAM were assayed for their prodynorphin levels under low- and high-glucose conditions. Islets from control and DREAM–/– animals both show glucose stimulation of prodynorphin message, however, the level of prodynorphin RNA was elevated nearly 80% in the knockout islets under low-glucose stimulation (Fig. 5, A and B). This implicates a role for DREAM in the calcium regulation of prodynorphin transcription.


Figure 5
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  		Fig. 5. Glucose and DREAM regulate mouse islet PDN transcription and glucagon release. A: representative RT-PCR of PDN message from wild-type (WT) or DREAM–/– (–/–) islets cultured with low (2 mM) or high (16 mM) glucose with (+) or without (–) RT. B: relative transcript abundance of PDN in control (open bars) and DREAM–/– (gray bars) islets cultured with low (2 mM) or high (16 mM) glucose quantified by real-time PCR. The transcript number is presented as comparative CT calculations with normalization to beta-actin levels, using 3 assays/animal (n = 4 for DREAM–/– and n = 6 for WT). C: glucagon secretion from isolated WT (gray bar) and DREAM–/– (filled bar) islets was measured in the presence of 1 mM glucose (1 h) and normalized to glucagon secretion measured after switch to 20 mM glucose (1 h) and is presented as the average of 3 experiments/animal for 8 animals. *P < 0.05. D: representative changes in mouse islet intranuclear calcium changed to 16 mM glucose (black arrow) from 2 mM glucose, presented as change in fluorescent signal obtained from islet nuclear regions loaded with fura red. Intensity of light decreases when fura red binds calcium; thus a drop in fluorescence is equal to a rise in calcium. *P < 0.05.

 
Dynorphin Regulates Glucagon Release

DREAM affects prodynorphin expression and because dynorphin peptides have been shown to affect islet hormone secretion the islet hormone release from DREAM–/– and control animals was assessed (23). DREAM–/– and control islets have comparable peak insulin secretion levels (29.6 ± 4.1 vs. 25.1 ± 2.2 µU/ml from 20 equal-sized islets) following glucose stimulation (16 mM, 6 min poststimulation). However, in the presence of low glucose (1 mM glucose), DREAM-deficient islets show significantly higher glucagon secretion compared with control islets, and when switched from low to high glucose (20 mM glucose) both groups of islets return to similar baseline levels of glucagon secretion (Fig. 5C). Thus increased dynorphin peptides in the DREAM islets may increase glucagon secretion from these islets.

Prodynorphin is cleaved into dynorphin peptides, which stimulate opioid receptors that have been identified in {alpha}-cells (24, 54). Therefore, the effects of dynorphin and {kappa}-opioid receptor stimulation of {alpha}-cells and islets were assessed. Treatment of {alpha}-TC1-6 cells with dynorphin A-(1–17) peptide significantly increased glucagon secretion compared with controls (Fig. 6A). These cells express the {kappa}-opioid receptor (Fig. 7); furthermore, a {kappa}-opioid-specific agonist, U-50488, significantly stimulates glucagon secretion from {alpha}-TC1-6 cells (Table 1). Similarly, human islets treated with U-50488 show increased glucagon secretion compared with unstimulated islets (Fig. 6A).


Figure 6
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  		Fig. 6. Dynorphin stimulates glucagon release. A: relative glucagon release of {alpha}-TC1-6 cells and human islets in 2 mM glucose with (+) or without (–) dynorphin A-(1–17) (50 nM) or U-50488, as indicated. Levels are calculated from total glucagon released for 30 min with or without treatment divided by total glucagon release from the same islets or cells during 30-min incubation in 2 mM glucose before treatment (see Table 1 for raw numbers). Two separate sets of human islet groups are pooled for each U-50488 treatment. B: representative cytoplasmic calcium changes of {alpha}-TC1-6 cells loaded with fura 2 and treated with U-50488 (5 µM, black horizontal bar) in 3 mM glucose. C: representative cytoplasmic calcium levels from {alpha}-TC1-6 cells treated with U-50488 (5 µM, black bar) with or without norbinaltromorhine (0.1 µM, gray bar) and 3 mM glucose. *P < 0.05.

 

Figure 7
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  		Fig. 7. {alpha}-Cell {kappa}-opioid receptor expression. {alpha}-TC1-6 cells stained with {kappa}-opioid receptor antibody (green).

 

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  		Table 1. Average glucagon levels measured from {alpha}-TC1-6 cells and human islets

 
Calcium fluctuations play an important role in G protein-coupled glucagon release from {alpha}-cells (31). This was also observed in {alpha}-T1-6 cells that show substantial fluctuations in calcium with U-50488 stimulation (Fig. 6B). Application of the {kappa}-opioid-specific antagonist norbinaltorphimine (0.1 µM) prior to U-50488 stimulation inhibited the calcium fluctuations (Fig. 6C). Thus stimulation of {kappa}-opioid receptors on {alpha}-cells causes an increase in calcium fluctuations and glucagon secretion.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Glucose-dependent regulation of transcription has an important influence on pancreatic islet cell function (34, 36). The results presented here demonstrate the expression of the calcium-binding protein DREAM and its importance as a calcium-dependent transcriptional regulator within the pancreatic islet. RT-PCR analysis and immunoflourescence performed on mouse pancreatic sections show that DREAM is expressed in pancreatic {alpha}- and beta-cells. We found DREAM staining within the nuclear regions of islet cells and defined its role as a calcium-dependent transcription factor that regulates prodynorphin transcription. Interestingly, {kappa}-opioid stimulation by dynorphin increases {alpha}-cell glucagon release, suggesting that the regulation of prodynorphin transcription may play an important role in glucagon secretion. DREAM provides yet another key regulatory mechanism employed by neuroendocrine cells adapting to changes in the glucose concentration.

DREAM was identified in nuclear regions of {alpha}- and beta-cells where it may regulate transcription in a calcium-dependent fashion, as it does in other tissues (Fig. 2)(8–10, 30). Calcium-dependent transcriptional regulation through a DRE motif was first demonstrated by elegant studies on the prodynorphin gene in neuroblastoma cells (8). The DRE motif is found in the 5'-regulatory region of many genes, and with low intranuclear calcium DREAM can interact with this motif, inhibiting transcription (8, 10, and 30). Interestingly, prodynorphin RNA expression is regulated in a glucose-dependent manner in the pancreatic beta-cell line Min6, so that when glucose is high prodynorphin levels rise (20). Similarly, glucose-dependent regulation of a luciferase reporter containing a small portion of the prodynorphin promoter, including the DRE motif, was also observed (Fig. 4C). When the DRE sequence within this reporter is mutated, not allowing a functional DREAM interaction, the glucose regulation is lost (Fig. 4C). Elevated glucose leads to increases in intracellular and nuclear calcium levels in beta-cells that would, in turn, cause decreased interaction of DREAM with the DRE motif and greater expression of prodynorphin. Prodynorphin is also regulated in the islet, where there is a 10-fold elevation of prodynorphin RNA in the presence of high glucose (16 mM) compared with low glucose (2 mM). Although glucose-dependent increases of prodynorphin are maintained in DREAM-deficient islets, there is a significant increase in prodynorphin RNA expression at low glucose concentrations (Fig. 5B). This indicates that DREAM plays a part in the glucose-induced transcriptional regulation of prodynorphin [presumably together with the other KChIP family members found in the pancreatic beta-cell, which can also bind to the DRE motif (30)]. These results suggest that DREAM is responsible only for subtle variations in prodynorphin expression at basal intracellular calcium and that other calcium-dependent pathways are also very important for regulation of prodynorphin expression. Once expressed, prodynorphin is cleaved into dynorphin peptides, which mediate diverse cellular responses as {kappa}-opioid receptor agonists.

Glucagon secretion is significantly affected by dynorphin peptides and {kappa}-opioid stimulation (16, 19, 23). Dynorphin A-(1–13) and the {kappa}-opioid agonist U-50488 individually stimulated increases in mouse insulin as well as blood glucose levels in obese (ob/ob) mice, whereas they stimulate increases in blood glucose levels only in lean control mice (23). Injection of morphine into rats also leads to hyperglycemia, due to significantly elevated glucagon release observed within 5 min of injection (14, 20). Similarly, morphine can cause hyperglycemia in both dogs and humans (16, 39). Stimulation of human islets and the {alpha}-TC1-6 cell line with U-50488 induced a significant elevation in glucagon secretion (Fig. 6A), indicating that stimulation of {kappa}-opioid receptors expressed in {alpha}-cells could potentiate glucagon secretion. Indeed, {kappa}-opioid receptors have been identified in rat and rabbit {alpha}-cells as well as in the {alpha}-TC1-6 mouse {alpha}-cell line (Refs. 24 and 54 and Fig. 7). Stimulation of G protein cascades in pancreatic {alpha}-cells, such as with epinephrine, can lead to glucagon release through increases in intracellular calcium under low-glucose conditions (31). Interestingly, stimulation of {alpha}-TC1-6 cells with U-50488 caused a significant increase in intracellular calcium levels that was blocked by the {kappa}-opioid-specific antagonist norbinaltorphimine (Fig. 6, B and C). Thus dynorphin stimulation of {alpha}-cell {kappa}-opioid receptors increases calcium fluctuation and glucagon release. Accordingly, transcriptional regulation of prodynorphin plays an important role in glucagon release, as DREAM–/– islets show significantly elevated glucagon release in response to low glucose. Elevated glucose levels and increased glucagon secretion are common in adult-onset diabetes (15). On the basis of the results presented, elevations in glucose could lead to increased prodynorphin expression. Thus it is interesting to speculate that elevated glucose levels may lead to increased dynorphin expression resulting in the increased glucagon level observed during adult onset diabetes.

Although DREAM affects islet transcription, it may also play important roles in neuroendocrine apoptosis and/or regulation of Kv4 voltage-dependent potassium channels. First described for its interactions with presenilins and linked to apoptotic processes, DREAM alters endoplasmic reticulum calcium stores and is cleaved by caspase-3 following apoptotic signaling (7, 12, 28, 29). Therefore, DREAM might influence beta-cell apoptosis related to diabetes. DREAM also interacts with Kv4 potassium channels, which are expressed in {alpha}-cells, allowing proper membrane localization of these channels (1, 46). Thus DREAM may affect repolarization of {alpha}-cells through Kv4 membrane trafficking. Further studies are required to address the exact role of DREAM in beta-cell apoptosis and {alpha}-cell plasma membrane repolarization.

In conclusion, the results show that DREAM is expressed in both {alpha}- and beta-cells, where it can affect calcium-dependent transcription of prodynorphin. Dynorphin in turn can activate glucagon secretion via stimulation of {alpha}-cell {kappa}-opioid receptors, explaining the hyperglycemic effect of many opiates that can stimulate {kappa}-opioid receptors. These results suggest that the DREAM-dynorphin pathway may provide intraislet cell communication that can affect glucagon secretion. Further studies are necessary to confirm this hypothesis.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work has been partially supported by the National Institute of Diabetes and Digestive and Kidney Diseases (DK-44840, DK-48494, DK-63493, and DK-20595), the Diabetes Research and Training Center at the University of Chicago, and the Blum-Kovler Foundation. D. A. Jacobson was supported in part by an American Diabetes Association Mentor-Based Fellowship and the Cardiovascular Pathophysiology and Biochemistry Training Program from the National Heart, Lung, and Blood Institute (5T32-HL-07237).


   ACKNOWLEDGMENTS
 
We thank Andrey Kuznetsov for expert assistance with confocal imaging. We thank Molly Dodge and James Lopez for skilled assistance with mouse islet isolation and culture.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. H. Philipson, Univ. of Chicago, MC1027/5841 S. Maryland Ave., Chicago, IL 60637 (e-mail: l-philipson{at}uchicago.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.


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 ABSTRACT
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 DISCUSSION
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