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Endocrinology, doi:10.1210/en.2002-0087
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Endocrinology Vol. 144, No. 7 3216-3224
Copyright © 2003 by The Endocrine Society

Urocortin III Is Expressed in Pancreatic ß-Cells and Stimulates Insulin and Glucagon Secretion

Chien Li, Peilin Chen, Joan Vaughan, Amy Blount, Alon Chen, Pauline M. Jamieson, Jean Rivier, M. Susan Smith and Wylie Vale

Clayton Foundation Laboratories for Peptide Biology (C.L., J.V., A.B., A.C., P.M.J., J.R., W.V.), Salk Institute for Biological Studies, La Jolla, California 92037; Oregon National Primate Research Center, Department of Physiology and Pharmacology (P.C., M.S.S.), Oregon Health & Sciences University, Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: Wylie W. Vale, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Urocortin (Ucn) III, or stresscopin, is a high affinity ligand for the type 2 corticotropin-releasing factor (CRFR2) receptor recently identified in rodents and human. Ucn III was initially identified as a neuropeptide expressed in discrete areas in the brain. In the present study, we demonstrate that Ucn III is expressed in pancreatic ß-cells and in a mouse ß-cell line, MIN6. Ucn III secretion from the cells was measured using a highly specific RIA, and we found that high potassium, forskolin, or high glucose can stimulate Ucn III secretion from these cells. In vivo studies showed that rats receiving an iv Ucn III injection had a significant elevation of plasma glucagon followed by plasma glucose levels compared with rats receiving vehicle. Ucn III injections also result in an increase in plasma insulin levels. The observed effects of Ucn III were blocked by pretreatment with a CRFR2 antagonist, astressin2-B. Furthermore, Ucn III stimulated glucagon and insulin release from isolated rat islets, and astressin2-B abolished the effects of Ucn III, in keeping with a CRFR2-mediated mechanism. Taken together, the present studies suggest pancreatic Ucn III acting through CRFR2 is involved in the local regulation of glucagon and insulin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HORMONES PRODUCED BY pancreatic islets play key roles in maintaining blood glucose homeostasis. Glucagon and insulin, produced by {alpha}- and ß-cells, respectively, in the islet are the two major hormones responsible for this regulation. Extensive studies have shown that insulin secretion is mainly regulated by glucose, whereas autonomic input and hormones from the islet as well as the enteric system play modulatory roles (1). The secretion of glucagon has been shown to be sensitive to glucose as well (1, 2). Nevertheless, neural input from the autonomic nervous system has been suggested as the main mechanism for hypoglycemia-induced glucagon secretion (3). Hormonal factors, including insulin and somatostatin produced from adjacent cells in the islets, have been shown to negatively modulate glucagon secretion (4, 5). Accumulating evidence demonstrates that both {alpha}- and ß-cells produce other peptides in addition to glucagon and insulin (6). For example, it has been shown that ß-cells express amylin (7, 8), cholecystokinin (9), ghrelin (10), and neuropeptide Y (11) and that {alpha}-cells express glucagon-like peptide-1 (12), ghrelin (13), and corticotropin releasing factor (CRF) (14). A defined role for many of these peptides remains to be elucidated. Available evidence has suggested that many of these peptides take part in the regulation of intraislet homeostasis through autocrine/paracrine routes.

CRF is a 41-amino-acid hypothalamic peptide that plays a key role in regulating hypothalamic-pituitary-adrenal axis response to stress and also stress-associated behavior (15, 16). In the pancreas, CRF has been shown to modulate pancreatic hormone secretions (17, 18) and also to modulate Ca2+ influx in rat pancreatic ß-cells (19). Recently, the expression of receptors for CRF family peptides in endocrine pancreas was studied by immunohistochemistry, and it was shown that both type 1 and type 2 CRF receptors (CRFR1 and CRFR2) are expressed in the islets (19). CRF has been considered as an endogenous ligand for CRFR1 due to its higher affinity for CRFR1 than for CRFR2 (15). Thus, it is likely that the effect of CRF in the pancreas is mediated by CRFR1. On the other hand, the function of CRFR2 and the possible ligand for this receptor in the pancreatic islet are unknown.

In addition to CRF, several additional members of the family have been identified in mammals: urocortin (Ucn) (20), Ucn II (21), stresscopin (22), stresscopin-related peptide (22), and Ucn III (23). The predicted mature stresscopin peptide is essentially identical with human Ucn III, with two amino acids extended from the N-terminal end of human Ucn III (22, 23), whereas stresscopin-related peptide is identical with human Ucn II with a 5-amino-acid extension from the N-terminal end of human Ucn II (21, 22). Both human and mouse Ucn III have been shown to exhibit high affinity for CRFR2 (22, 23). However, unlike CRF, Ucn, and Ucn II, Ucn III displays very low affinity for CRFR1 (22, 23). The high affinity for CRFR2 has led us to postulate that Ucn III is an endogenous ligand for CRFR2. Initial characterization shows that Ucn III is a neuropeptide with discrete expression in the hypothalamus and amygdala (23, 24). Strikingly, Ucn III neuronal fibers and axonal terminals in the brain show significant overlap with the expression of CRFR2 in the forebrain (24). This anatomical feature provides further support that Ucn III is an endogenous ligand for CRFR2 in some sites of expression.

Here we report the expression of Ucn III in the pancreatic ß-cell and in a mouse clonal ß-cell line, MIN6, along with the effects of this peptide on glucagon and insulin secretion in vivo and in vitro. This study identifies Ucn III as a new regulator produced by pancreatic islets that may be involved in paracrine regulation of islet hormone secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MIN6 cell (passage, <35), a ß-cell line derived from transgenic mice expressing the simian virus 40 large T antigen under the control of the rat insulin promoter, were cultured in DMEM supplemented with 10% fetal bovine serum, antibiotics, L-glutamine, and 114 µM ß-mercaptoethanol. For Ucn III secretion, 1 x 106 cells were seeded in 2 ml medium in 6-well plates. Media were changed the day before the experiments. On the day of the experiment, the media were removed and the cells were washed twice with HEPES-balanced Krebs-Ringer bicarbonate buffer containing 5 mM glucose and 0.2% BSA. The cells were then incubated in HEPES-balanced Krebs-Ringer bicarbonate buffer with various testing reagents for 4 h at 37 C. The incubation buffer was collected and vacuum-dried for Ucn III RIA. To measure cell content of Ucn III, cells were lysed with 1 ml of lysis buffer (0.05% Triton X-100, 0.1% ß-mercaptoethanol in 50 mM HCl), centrifuged, and the supernatants were vacuum-dried overnight. The samples were then reconstituted with RIA buffer for Ucn III RIA.

Semiquantitative RT-PCR
Total RNA was extracted from mouse brain and pancreas using Trizol RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH), and total RNA from MIN6 cells was extracted with the Rneasy mini kit (QIAGEN Inc., Valencia, CA), according to the manufacturer’s recommendations. To avoid false positive results caused by DNA contamination, we performed a deoxyribonuclease treatment for 30 min at 37 C using the RQ1 ribonuclease (RNase)-free deoxyribonuclease (Promega Corp., Madison, WI). The expression of the ribosomal protein, S-16 (25), was used as an internal control. The PCR conditions were: cDNA equivalent to 0.35 µg of RNA was amplified by PCR for 32 cycles; the annealing temperatures were 67 C and 60 C for Ucn and Ucn II/Ucn III reactions, respectively. The final MgCl2 concentration was 3 mM, and each reaction contained a 2.5 U of Taq DNA polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd., London, UK). The following specific Ucn, Ucn II, Ucn III, and S-16 oligonucleotide primers were used in the PCR: Ucn, 5'-CTTCGCCCGGAGAGCAGCCAGTG-3' and 5'-GCGTTCCAGCCCCGGGGTAGGTAA-3' corresponding to nucleotides 167–189 (sense) and 522–545 (antisense), respectively (26); the predicted size of the band is 379 bp. Ucn II, 5'-GGCCGCCGCTGAGACTGAA-3' and 5'-GGCCTGTGGACCTTAGATGGACTT-3' corresponding to nucleotides 329–347 (sense) and 707–730 (antisense), respectively (21); the predicted size of the band is 402 bp. Ucn III, 5'-CTGCTGCCACTTCTGCTGCTCCTA-3' and 5'-CAAAAGCGACCGGGGCACC-3' corresponding to nucleotides 22–45 (sense) and 345–363 (antisense), respectively (23); the predicted size of the band is 342 bp. S-16, 5'-TGCGGTGTGGAGCTCGTGCTTGT-3' and 5'-GCTACCAGGCCTTTGAGATGGA-3' corresponding to nucleotides 369–391 (sense) and 1968–1990 (antisense), respectively (25); the predicted size of the band is 309 bp.

RNase protection assay
The mouse Ucn II and Ucn III DNA templates used for the synthesis of an antisense riboprobe contain approximately 600 and 436 bp of the full-length cDNA in pBluescript IISK (Stratagene, La Jolla, CA), respectively. Ucn II cDNA containing plasmid was linearized with BamHI, and an antisense riboprobe [630 nucleotides (nt)] was synthesized using T3 RNA polymerase. Ucn III cDNA containing plasmid was linearized with XbaI, and T7 RNA polymerase was used to synthesize the probe (567 nt). The protected fragment sizes of Ucn II and Ucn III RNA are 592 nt and 414 nt, respectively. A mouse glyceraldehyde-3-phosphate dehydrogenase antisense riboprobe was synthesized using T3 RNA polymerase to yield a full-length probe of 376 nt and a protected fragment of 316 nt and was used as an internal control. RNase protection assays were performed as previously described (27) using 60 µg of total RNA extracted from various mouse tissues using TRI Reagent (Molecular Research Center, Inc.). The samples were resolved on a 4% polyacrylamide/8 M urea gel.

Immunohistochemistry
Mice were perfused transcardially with saline followed by 4% paraformaldehyde. Pancreas was removed and postfixed overnight in the same fixative at 4 C. The pancreata were sectioned at 15 µm using a cryostat, collected onto glass slides, and stored at -20 C until use. For immunohistochemistry, the pancreatic section-containing slides were rinsed in 0.05 M potassium PBS (KPBS) followed by treatment with 1% NaBH4-KPBS solution. Sections were incubated in rabbit antihuman TyrGly-Ucn III antiserum (PBL no. 6570; 1:19,000 final dilution) (24) in KPBS with 0.4% Triton X-100 at room temperature for 1 h, and then at 4 C for 48 h. The tissues were then rinsed in KPBS and incubated in biotinylated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:700) in KPBS with 0.4% Triton X-100 for 1 h at room temperature. This was followed by another 1-h incubation at room temperature in avidin-biotin complex solution (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA). The antibody-peroxidase complex was visualized with a mixture of nickel sulfate (25 mg/ml), 3,3-diaminobenzidine (0.2 mg/ml), and 3% H2O2 (0.83 µl/ml) in 175 mM sodium acetate solution. When the staining had reached the appropriate intensity, the tissue was rinsed in KPBS, dehydrated through graded alcohols, cleared in xylenes, and coverslipped with DPX mountant (Electronic Microscope Science, Fort Washington, PA). For Ucn III and insulin or glucagon double-label immuofluorescent staining, sections were incubated in rabbit antihuman Ucn III serum (1: 1000) and either guinea pig antiinsulin serum or guinea pig antiglucagon serum (1: 7000 for both antisera; Linco, St. Louis, MO). The sections were then incubated in a mixture of biotinylated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:600) and Cy3-conjugated donkey antiguinea pig IgG (1:500) followed by Cy2-conjugated streptavidin (1:1000) to visualize Ucn III immunoreactivity (ir). The slides were coverslipped with buffered glycerol.

In situ hybridization
Animals were quickly euthanized, and the pancreas was removed and frozen in dry ice. Fresh-frozen pancreas was sectioned (20 µm) on a cryostat, thaw-mounted onto glass slides, and stored at -80 C until use. An antisense cRNA probe was transcribed from a linearized 567-bp mouse Ucn III cDNA and labeled with 35S-uridine triphosphate (PerkinElmer Life Science, Boston, MA). The specific activity of the probe was approximately 1–3 x 108 dpm/µg of cRNA. The saturating concentration for the probe used in the assay was 0.3 µg/ml·kb. The procedure for in situ hybridization has been described previously (28). Briefly, fresh mouse pancreatic sections were fixed in 4% paraformaldehyde and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2x sodium chloride/sodium citrate buffer (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air-dried. The slides were exposed to the cRNA probe overnight in humidified chambers at 55 C. After incubation, the slides were washed in SSC of increasing stringency, in RNase, and then in 0.1x SSC at 63 C, dehydrated through a graded series of alcohols, and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 14 d at 4 C, and developed. After development, the slides were counterstained with cresyl violet.

Tissue extraction and RIA
Pancreas or isolated rat islets were acid-extracted and partially purified using octadecyl silica cartridges as described previously (29). The procedure for Ucn III RIA was similar to that previously described for melanin-concentrating hormone (29). Briefly, rabbit antimouse TyrGly-Ucn III serum (PBL no. 6598) was used at 1:75,000 final dilution, [125I]Tyr0 Nle12,35 mUcn III was used as tracer, and mUcn III was used as standard. Samples were tested at three to seven doses level. Free tracer was separated from tracer bound to antibody with the addition of sheep antirabbit {gamma}-globulins and 10% (wt/vol) polyethylene glycol. The EC50 and minimum detectable dose for mUcn III are 25–35 pg and 2 pg/tube, respectively.

Animal studies
Male Sprague-Dawley rats (250–300 g) were used for the in vivo studies. Intravenous cannulae were implanted through the right jugular vein into the right atrium 2 d before treatment. On the day of the experiment, rats were connected with blood sampling tubing and left in a sampling cage 3 h before the experiment. Varying doses of Ucn III or vehicle were administered iv, and blood samples (300 µl) were drawn at specified times. In experiments in which the CRFR2 antagonist astressin2-B (Ast2-B) was used, the antagonist or vehicle was administered intravenously 15 min before Ucn III treatment. Blood samples were immediately centrifuged to separate plasma from blood cells and stored at -20 C until assay. Plasma glucose levels were determined using a commercial kit (Sigma, St. Louis, MO). Glucagon and insulin levels were determined by RIA using a commercially available RIA kit (Linco). All animal procedures were approved by the Salk Institute Institutional Animal Care and Use Committee.

Islet culture studies
Islets from male Sprague Dawley rats (300–350 g) were isolated according to the method described by Lacy and Kostianovsky (30) and incubated overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum before treatment. On the day of experiment, islets were washed twice with Krebs-Ringer-buffered saline (KRB) and preincubated in KRB with 2.8 mM glucose for 60 min, followed by incubation in testing agents diluted in KRB for another 60 min. KRB with 2.8 mM glucose was used as the control. Testing buffer was then removed and stored at -20 C until assay for glucagon and insulin by RIA. The glucagon antagonist, des-His,Glu-glucagon1–29 (des-glucagon), was obtained from Bachem (Torrance, CA).

Imaging
Results from immunofluorescent staining were imaged with a Leica TCSNT confocal system (Leica Lasertechnik, GmBH, Heidlelberg, Germany). Cy2 was imaged using the 488-nm excitation line of an argon laser and an emission band pass filter of 530 ± 30 nm. Cy3 was excited with a 568-nm Kr gas laser line and imaged through a 590-nm long-pass filter. In all cases, z-sections were imaged throughout the thickness of the tissue sections. The typical thickness of a single optical section was approximately 0.5 µm. In some cases, 0.2-µm-thick optical sections were collected for detailed analysis. Images were processed and analyzed with the use of MetaMorph (Universal Imaging, West Chester, PA) and Volocity (Improvision Inc., Lexington, MA). For results obtained from in situ hybridization, the slides were examined using a Lecia light microscope, and images were captured onto Kodak Ektachrome 64T (tungsten) films. The films were scanned into a computer using a Nikon (Melville, NY) LS1000 35-mm film scanner. The images were cropped and adjusted to balance brightness and contrast in Adobe Photoshop (version 6.0) before import into Canvas (version 8.0) for assembly into figures.

Data analysis
Data are presented as means ± SEM. ANOVA is used for comparison followed by a post hoc test for between-group comparison. Differences were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ucn III is expressed in the pancreatic ß-cells and MIN6 cells
Examination of transcripts of the three Ucns by RT-PCR showed that Ucn II and Ucn III, but not Ucn, are found in the mouse pancreas (Fig. 1AGo). RNase protection assay was then employed to further examine the expression of Ucn II and III. As shown in Fig. 1BGo, RNase protection analyses revealed that the levels of Ucn III mRNA appear to be higher than Ucn II mRNA (Fig. 1BGo), suggesting that Ucn III is the more abundant CRFR2 ligand in the pancreas. The expression of Ucns was also examined in a mouse pancreatic ß-cell line, MIN6, by RT-PCR. As shown in Fig. 1Go, all three Ucn transcripts were found in the total RNA prepared from these cells, with Ucn III mRNA much more abundant than the other two Ucns. We further localized Ucn III mRNA in the mouse pancreas by in situ hybridization and found that Ucn III mRNA is almost exclusively expressed in the core of the islet in the pancreas (Fig. 1Go, C and D), suggesting that Ucn III mRNA is expressed in ß-cells. The localization of Ucn III in the ß-cells was confirmed by immunohistochemistry on mouse pancreas using a specific antiserum (24) against synthetic Ucn III peptide. Strong Ucn III-immunoreactive cells were only seen in the islet (Fig. 1EGo). Preincubation of the antiserum with synthetic Ucn III peptide completely abolished the staining (Fig. 1FGo). Double-label immunofluorescent staining for Ucn III and insulin or glucagon shows that the majority of Ucn III-immunopositive cells colocalized with insulin-positive cells (Fig. 2Go, A–C) but not glucagon (Fig. 2Go, G–I). Higher magnification (Fig. 2Go, D–F) revealed that Ucn III staining within the ß-cell resembled that of secretory granules. When Ucn III and insulin staining within ß-cells were visualized simultaneously, we found that Ucn III and insulin immunoreactivities appear to occupy different regions of the cell, and little colocalization was observed between the two substances (Fig. 2Go).



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  		Figure 1. Expression and localization of Ucn III in mouse pancreatic islets and MIN6 cells. A, Representative electrophoretic analysis of the RT-PCR products of Ucn, Ucn II, and Ucn III in mouse pancreas and MIN6 cells. S16 RNA was used as positive control. -RT, RNA samples were used for PCR without RT; -cDNA, no RT product added to the PCR. B, Representative autoradiogram showing the detection of Ucn II and Ucn III mRNAs by RNase protection assay. Total RNA isolated from mouse brain stem (for Ucn II), hypothalamus (for Ucn III), or whole pancreas was hybridized with antisense probes specific to mouse Ucn II or Ucn III and mouse glyceraldehyde-3-phosphate dehydrogenase. Ucn II probe protects a 592-nt band in the brain stem and the pancreas and Ucn III probe protects a 414-nt band in the hypothalamus and the pancreas. C, Representative dark-field photomicrograph showing a mouse pancreas section probed with mouse Ucn III riboprobe. Ucn III mRNA-positive signals (silver grain clusters) were found in the pancreatic islets. D, Bright-field photomicrograph of panel C with nissl staining showing the islets. *, Blood vessel. E and F, Representative photomicrographs showing Ucn III immunostaining in the mouse pancreatic sections without (E) or with (F) preabsorption of the anti-Ucn III serum with 30 µM of mouse Ucn III. Ucn III-positive cells (dark purple staining) were observed only in the islets. Scale bar, 50 µm.

 


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  		Figure 2. Colocalization of Ucn III and insulin in mouse pancreatic islets. Stacked serial confocal images of insulin (A) and Ucn III (B) cells in the islet. C, Visualization of both substances simultaneously showing the majority of insulin and Ucn III colocalize in the same cells. High magnification of confocal images showing insulin (D) and Ucn III (E) in a single ß-cell. F, Combined image of panels D and E to visualize insulin and Ucn III simultaneously. Note that little colocalization is observed. G–I, Stacked serial confocal images of glucagon (G), Ucn III (H), and combined image (I) of panels G and H showing no colocalization of the two substances. Scale bar, 25 µm for A–C and G–I and 1 µm for D–F.

 
Ucn III-like ir in murine pancreas and secretion from MIN6 cells
Using antiserum we raised in rabbit against mouse TyrGly-Ucn III, we developed an RIA that displays the requisite sensitivity for the measurement of Ucn III-like ir in biologic fluids and tissues. As shown in Fig. 3Go, the RIA is highly specific for mouse Ucn III (mUcn III), with less sensitivity for human Ucn III (~10%). Closely related peptide family members including rat CRF, Ucn, and Ucn II fail to displace the tracer, with cross-reactivities less than 0.01%. The mUcn III RIA has a detection limit of 2–3 pg/tube and ED50 values in the range of 25–35 pg/tube. Ucn III-like ir was measured in acid-extracted and partially purified tissue isolated from mouse or rat pancreas, rat pancreatic islets, and MIN6 cells. As shown in Table 1Go, Ucn III-like ir was approximately 440 and 1212 pg in rat and mouse pancreas, respectively, about 0.66 pg/rat islet and 1047 pg/106 MIN6 cells. Furthermore, Ucn III-like ir was detected in secreted media from MIN6 cells. The levels of the ir in the media were significantly elevated when MIN6 cells were treated with either 25 mM glucose, 30 mM KCl, or 30 µM forskolin (Fig. 4Go).



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  		Figure 3. Displacement of 125I-labeled mUcn III binding to rabbit anti-mUcn III by mUcn III ({square}) and partially purified rat pancreatic ({oplus}) or isolated rat islet () extract. Rat pancreas or isolated rat islets were acid-extracted and partially purified using octadecyl silica cartridges. Human Unc III (h Ucn III, {boxplus}) shows approximately 10% cross-reactivity. Closely related family peptides including rat Ucn (r Ucn, {circ}), rat CRF (r CRF, {diamond}), or mouse Ucn II (m Ucn II, {triangleup}) showed little or no cross-reactivity. B/B0, Bound to maximum-bound ratio.

 

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  		Table 1. Ucn III-like ir in murine pancreas, rat islets, or MIN6 cells

 


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  		Figure 4. Ucn III-like ir in secreted media from MIN6 cells. MIN6 cells (106 cells per well) were incubated in media containing various concentrations of glucose, 30 µM forskolin, or 30 mM KCl and supplemented with BSA (0.2% wt/vol) for 4 h. Cultured supernatants were collected and assayed for Ucn III. Cells were treated in triplicate, and the average of duplicate experiments is shown. *, P < 0.05 vs. 5 mM glucose; **, P < 0.01 vs. 5 mM glucose.

 
Ucn III treatments stimulate glucagon release and elevate blood glucose levels
The presence of Ucn III in the ß-cells raises the possibility that the peptide may be involved in the regulation of blood glucose levels. To investigate this issue, various doses of murine synthetic Ucn III (0.09–90 nmol/kg) or vehicle were injected iv into male rats through a jugular vein cannula. Exogenous Ucn III (9 or 90 nmol/kg) significantly increased blood glucose levels at 15 min and reached peak levels at 30 min after injection (Fig. 5AGo). After 30 min, the glucose levels gradually returned to baseline. We also measured plasma insulin levels to determine whether the elevation of glucose was due to a suppression of insulin secretion by Ucn III treatment. As shown in Fig. 5BGo, plasma insulin was also elevated and mirrored the pattern of blood glucose levels, suggesting that the elevation of blood glucose was not due to a suppression of insulin release. The elevation of both blood insulin (Fig. 5CGo) and glucose (data not shown) levels by Ucn III could be blocked by pretreating the animals with a CRFR2 selective antagonist, Ast2-B (31), further reinforcing the notion that the effects of Ucn III are mediated by CRFR2.



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  		Figure 5. The effects of either vehicle or Ucn III (0.09–9 nmol/kg, iv) on plasma glucose (A) and insulin (B) levels in male rats. C, Insulin levels at 30 min after iv vehicle or Ucn III (90 nmol/kg) with or without Ast2-B (90 nmol/kg) pretreatment. Basal glucose level, 4.25 ± 0.18 mmol/liter. B, For 9 nmol/kg group: +, P < 0.05 vs. vehicle; for 90 nmol/kg group: *, P < 0.05; **, P < 0.01 vs. vehicle. C, *, P < 0.05 vs. the rest of groups.

 
The elevation of blood glucose by Ucn III in the face of rising insulin levels suggested that Ucn III may affect glucagon secretion. Single iv Ucn III injections (90 nmol/kg) indeed induced a rapid increase of plasma glucagon at 5 min after injection (Fig. 6BGo). Glucose responses to Ucn III (Fig. 6AGo) were similar to those shown in Fig. 5Go. Moreover, the effects of Ucn III on glucagon (Fig. 6CGo), and subsequently blood glucose (data not shown), are completely abolished by pretreating the animals with Ast2-B.



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  		Figure 6. Plasma glucose (A) and glucagon (B) levels in male rats treated with vehicle or Ucn III (9 or 90 nmol/kg). Basal glucose and glucagon concentrations are 5.1 ± 0.2 mmol/liter and 52.3 ± 3.2 pg/ml, respectively. C, Glucagon levels at 5 min after vehicle or Ucn III (90 nmol/kg) injection with or without Ast2-B (90 nmol/kg) pretreatment. B, *, P < 0.05 vs. vehicle. C, *, P < 0.05 vs. the rest of groups.

 
Stimulation of glucagon and insulin release from islets by Ucn III
In vitro islet cultures were employed to directly examine the effects of Ucn III on glucagon as well as insulin secretion. As shown in Table 2Go, Ucn III stimulated both glucagon and insulin release from isolated rat islets in a dose-dependent manner. Rat/human CRF at relatively high concentrations also stimulated the secretion of the two hormones (Table 2Go). Pretreatment with 1 µM Ast2-B completely abolished the stimulatory effects of 1 nM Ucn III on both glucagon and insulin release (Fig. 7Go). Interestingly, Ucn III-induced glucagon release was also abolished when high glucose (8.4 mM) was present in the incubation medium (Fig. 7Go).


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  		Table 2. Effects of Ucn III and rat/human CRF on glucagon and insulin secretion from isolated rat islets

 


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  		Figure 7. Effects of Ast2-B on Ucn III-induced glucagon and insulin release from isolated rat islets. Ucn III (1 nM) stimulates both glucagon (A) and insulin (B) secretion in the presence of 2.8 mM glucose. Pretreatment with 1 µM Ast2-B abolishes the effects of Ucn III on secretion of both hormones. Ucn III failed to stimulate glucagon release in the presence of 8.4 mM glucose. *, P < 0.05 vs. the rest of group; #, P < 0.05 vs. all groups except 8.4 mM glucose; +, P < 0.05 vs. all groups except Ucn III plus 2.8 mM glucose.

 
As expected, 8.4 mM glucose alone stimulated insulin release (Fig. 7Go). A small enhancement of insulin release was observed in islets treated with a combination of 1 nM Ucn III and 8.4 mM glucose compared with either of treatment alone. Because glucagon has been shown to directly stimulate insulin secretion, it is possible that the insulin-releasing effect of Ucn III may have been mediated by Ucn III-induced glucagon secretion. To investigate this issue, islets were incubated with 1 nM Ucn III in the presence of a glucagon antagonist, des-glucagon. As shown in Fig. 8Go, the antagonist failed to block the insulin-releasing effect of Ucn III, but it abolished insulin release induced by 10 nM glucagon.



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  		Figure 8. Effects of a glucagon antagonist on Ucn III-induced insulin secretion from isolated rat islets. Ucn III (1 nM) and 10 nM glucagon stimulate insulin secretion. Pretreatment with 1 µM des-glucagon abolishes the effect of glucagon but not Ucn III on insulin secretion. Values from islets incubated in medium with 2.8 mM glucose serve as controls. *, P < 0.05 compared with control. +, P < 0.05 vs. control and glucagon plus des-glucagon.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ucn III was identified initially as a neuropeptide expressed in the central nervous system (23, 24). In the present study, we demonstrate that Ucn III mRNA is also expressed in the pancreas. Anatomical investigation using in situ hybridization and immunohistochemistry showed that Ucn III is expressed in the ß-cells in the pancreatic islet. Ucn III was also found in a mouse clonal ß-cell line, MIN6 (32), further supporting the expression of Ucn III in the ß-cell. The secretory granule-like appearance of Ucn III in the ß-cell suggests that Ucn III is a secretory product. Indeed, using MIN6 cells as a model, we showed that Ucn III can be secreted from these cells as measured using a highly specific RIA and that high potassium or forskolin can significantly stimulate Ucn III secretion from these cells. Taken together, these data suggest that Ucn III can be secreted from pancreatic ß-cells and may have possible paracrine and/or autocrine effects in the islet. The present study also showed that high glucose significantly stimulated Ucn III secretion from MIN6 cells, suggesting that ß-cell Ucn III is sensitive to extracellular glucose concentrations. It will be interesting to determine the dynamics of insulin and Ucn III secretion in response to glucose. Currently, the processing sites of Ucn III have not been defined, and isolation and sequencing will be required to establish its native sequence in mammalian tissues. Further studies are underway to address the size of Ucn III secreted by MIN6 cells. The amount of Ucn III ir detected in the murine pancreas suggests that that pancreas likely makes a negligible contribution to Ucn III in the general circulation, and pancreatic Ucn III is likely to serve as a local regulator in pancreatic islets.

The expression of receptors for CRF family peptides has been examined in the pancreas (19, 33). Both CRFR1 and CRFR2 were detected in pancreatic islets (19), albeit the cell types that express the receptors remain to be determined. Currently, the physiological function of the two receptors in the pancreas is not known. In the present study, Ucn III, a highly CRFR2 selective ligand, was found to elevate blood glucose levels and stimulate glucagon and insulin secretion. These effects were also observed in vitro using isolated rat islets. Most importantly, the effects of Ucn III can be abolished by pretreatment with Ast2-B, a selective CRFR2 antagonist (31). Thus, these results suggest that pancreatic CRFR2 receptor is involved in modulating islet hormone secretion.

It is well known that the secretion of insulin and glucagon is under complex paracrine regulation. For example, glucagon stimulates insulin secretion, and insulin in turn inhibits glucagon secretion. In addition, somatostatin, produced by the {delta}-cell in the islet, inhibits both insulin and glucagon secretion (4, 5). These intraislet feedback mechanisms play an important role to ensure proper levels of glucagon and insulin under a given glycemic condition. The present study suggests that Ucn III acting through CRFR2 in the islet may represent a new intraislet regulatory mechanism that adds additional levels of modulation of insulin and glucagon secretion. It is interesting to note that Ucn III exhibits an opposite effect to that of somatostatin, raising the possibility that Ucn III may act to counterbalance somatostatin in modulating insulin and glucagon secretion. The observation in the present study that, similar to somatostatin (34, 35, 36), high glucose can stimulate Ucn III secretion from ß-cells supports this possibility. Results from the present studies show that under high glucose conditions exogenous Ucn III was not effective in stimulating glucagon secretion. High glucose has been suggested to directly inhibit glucagon secretion (37). In addition, hormones released from the islet under high glucose conditions, including insulin and somatostatin, may potentially mask the effects of Ucn III on glucagon secretion. It remains to be determined whether endogenous Ucn III from the ß-cell is involved in glucagon secretion. However, results from the present study showed that Ucn III secretion from static cultured MIN6 cells was stimulated by high glucose. This result seems to question the physiological role of Ucn III on glucagon secretion, as Ucn III is released from ß-cells by high glucose concentration, and, coincidentally, exogenous Ucn III has no effect on glucagon secretion under high glucose conditions. More studies are needed to resolve this issue. For example, the time course and dynamics of endogenous Ucn III secretion in response to glucose stimulation need to be determined to further elucidate the effect of Ucn III on glucagon secretion. Interestingly, the distinct separation of Ucn III and insulin ir within the ß-cell observed in the present study suggests that Ucn III secretion from the ß-cell may occur independently of insulin secretion.

Exogenous Ucn III stimulates insulin release both in vivo and in vitro. This is in contrast to amylin, which has been shown to inhibit insulin secretion in some studies (38, 39). The effect of Ucn III on insulin release is not mediated by glucagon, as demonstrated in the islet culture study showing that pretreatment with a glucagon antagonist failed to abolish the effects of Ucn III on insulin release. Furthermore, high glucose abolished the effect of Ucn III on glucagon while having no effect on insulin secretion. This result supports the notion that Ucn III can act on insulin secretion independently of glucagon. Ucn III partially potentiated the effects of 8.4 mM glucose on insulin release, suggesting that the mechanism mediating Ucn III and glucose effects on insulin release may somewhat overlap. cAMP has been shown to be the predominant messenger mediating the effects of CRFR2 (15, 40). It is reasonable, therefore, to assume that the effect of Ucn III on insulin release is mediated by cAMP. However, it has also been suggested that cAMP alone is not sufficient to initiate insulin release (41, 42, 43). It has been shown that CRFR1 and CRFR2 can couple to additional messenger systems including MAPK (44, 45, 46), protein kinase B (47), protein kinase C (45, 48), and phospholipase C pathways (49). Thus, CRFR2 in the ß-cells may couple to additional systems or machinery that are associated with insulin release.

The present study showed that rat/human CRF appears less effective than Ucn III in stimulating both glucagon and insulin secretion from isolated rat islets. This observation raises two important points. First, it supports the notion that the effect of CRF ligands on glucagon and insulin secretion is mediated by CRFR2. Second, it suggests that CRFR1 in the islet may not be involved in hormone secretion. It has been shown that low concentrations of CRF can induce Ca2+ influx into rat pancreatic ß-cells (19), suggesting that CRF may stimulate insulin secretion. However, in agreement with an earlier report (17), the present study showed that CRF is not a potent insulin-releasing agent under the conditions tested. Thus, the exact physiological function of CRFR1 and its ligand(s) in the islet remains to be determined.

In summary, Ucn III was localized in pancreatic ß-cells. In vivo and in vitro studies show that Ucn III can stimulate glucagon secretion and, as a consequence, elevate blood glucose levels. In addition, Ucn III can also stimulate insulin release. These results suggest that Ucn III may be a new paracrine regulator in pancreatic islets to regulate glucagon as well as insulin secretion and subsequently to modulate blood glucose levels.


   Acknowledgments
 
We thank J. Gulyas, D. Kirby, and W. Low for synthesis and characterization of the peptides used in this study. We also thank Katy Metz and Jamie Tomie for technical assistance.


   Footnotes
 
This work was supported by NIH Grants DK-26741 (to W.V. and J.R.) and HD-14643 and RR-00163 (to M.S.S.), the Robert J. and Helen C. Kleberg Foundation, the Adler Foundation, and the Foundation for Research (W.V.). W.V. is an FFR Senior Investigator. J.R. is the Frederik Paulsen Chair in Neurosciences Professor. C.L. is supported by a National Research Service Award (MH-12654). P.J. is supported by the Alder Foundation.

Abbreviations: Ast2-B, Aastressin2-B; CRF, corticotropin releasing factor; CRFR1, type 1 CRF receptor; CRFR2, type 2 CRF receptor; des-glucagon, des-His,Glu-glucagon1–29; ir, immunoreactivity; KPBS, potassium PBS; KRB, Krebs-Ringer-buffered saline; mUcn III, mouse urocortin III; nt, nucleotide; RNase, ribonuclease; SSC, sodium chloride/sodium citrate buffer; Ucn, urocortin.

Received November 27, 2002.

Accepted for publication April 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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