HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 49, 46046-46053, December 7, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/49/46046    most recent M108378200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kashima, Y. Articles by Seino, S. Search for Related Content PubMed PubMed Citation Articles by Kashima, Y. Articles by Seino, S. Social Bookmarking                      What's this?

Critical Role of cAMP-GEFII·Rim2 Complex in Incretin-potentiated Insulin Secretion^*

Yasushige Kashima, Takashi Miki, Tadao Shibasaki, Nobuaki Ozaki, Masaru Miyazaki^§, Hideki Yano, and Susumu Seino^¶

From the Departments of  Cellular and Molecular Medicine and ^§ General Surgery, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan

Received for publication, August 30, 2001, and in revised form, October 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic -cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII·Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic -cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretin-potentiated insulin secretion ~50%, while a combination of antisense ODNs and H-89 inhibited the secretion ~80-90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII·Rim2 pathway is critical in the potentiation of insulin secretion by incretins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Blood glucose levels are precisely controlled by insulin release from pancreatic -cells. Insulin secretion from the -cells is regulated positively and negatively by many intracellular signals generated by various factors, including nutrients, hormones, and neurotransmitters (1-4). cAMP is thought to be a most critical intracellular signal in the mechanism of potentiation of insulin secretion (5-8). In fact, insulin secretion varies significantly with treatment by adenylyl cyclase activators and inhibitors and phosphodiesterase inhibitors that alter cAMP levels in pancreatic islets (5, 6, 9). In addition, phosphorylation of many regulatory proteins by cAMP-dependent protein kinase (protein kinase A; PKA)¹ in the -cells has been suggested in the regulation of insulin secretion (10). However, despite its importance as an intracellular signal in the -cells, cAMP is not considered a primary signal in the insulin secretion process (5, 9). Rather, cAMP is thought to be a potentiating signal that modulates nutrient-induced insulin secretion, especially the potentiation of glucose-induced insulin secretion (5, 9).

It is known that glucose administered via the gastrointestinal tract induces a greater stimulation of insulin secretion than a comparable glucose challenge intravenously (11, 12). Gastrointestinal hormones, incretins including glucagon-like peptide-1 (GLP-1) (13-15), and glucose-dependent insulinotropic polypeptide (originally called gastric inhibitory polypeptide (GIP)) (13, 14, 16) mediate this effect. Incretins increase cAMP production by activating adenylyl cyclase by binding to their specific guanine nucleotide-binding protein (G-protein)-coupled receptors in the pancreatic -cell membrane (17-20). It is thought generally that the potentiating effects of incretins on insulin secretion are mediated mainly by the cAMP/PKA signaling pathway in the -cells (3, 21, 22). However, it has been suggested that incretins also exert their effects in the -cells in a cAMP-independent manner (23-25). For example, GLP-1 has been suggested to mobilize intracellular calcium by activation of inositol 1,4,5-trisphosphate receptor channels (23). GLP-1 has also been shown to elevate [Ca²⁺]_i (24) as well as to stimulate insulin gene promoter activity (25) by a PKA-independent mechanism in a -cell-derived cell line.

Until recently, PKA was the only molecule identified as a direct target of cAMP in pancreatic -cells (21, 26). Activation of PKA upon stimulation is assumed to phosphorylate regulatory proteins associated with the process of insulin secretion (21, 27). However, the substrates for PKA activated by cAMP or incretins and the roles of PKA-phosphorylated proteins in cAMP-potentiated insulin secretion are not clear. Although GLUT2 (a low K_m glucose transporter), Kir6.2, and SUR1 (subunits of the pancreatic -cell ATP-sensitive potassium channel) and -SNAP (a vesicle-associated protein), all of which are known to be expressed in pancreatic -cells, have been shown to be phosphorylated by PKA (28-31), the roles of these phosphorylations in insulin secretion are unknown. Furthermore, using electrophysiological measurements, cAMP has been found to promote exocytosis in the pancreatic -cells by a PKA-independent mechanism as well as by a PKA-dependent mechanism (32). We reported recently that the cAMP-binding protein cAMP-GEFII (33), also referred to as Epac2 (34, 35), is a direct target of cAMP in regulated exocytosis and that cAMP-GEFII, interacting with Rim2 (26), a target of the small G-protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system (26). However, the role of cAMP-GEFII in insulin secretion in native pancreatic -cells is not known.

In the present study, we have investigated the role of cAMP-GEFII·Rim2 in incretin-potentiated insulin secretion. Our data indicate that a PKA-independent mechanism involving cAMP-GEFII·Rim2 is critical in the potentiation of insulin secretion by both GLP-1 and GIP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Synthetic GLP-1 (7-36) amide and GIP were purchased from Peptide Institute, Inc. (Osaka, Japan). 8-Bromo-cyclic AMP (8-Br-cAMP), carbachol, and 3-isobutyl-1-methylxanthine were from Sigma. H-89 was from Calbiochem-Novabiochem. MDL 12330A was from RBI (Natick, MA).

Isolation of Mouse Pancreatic Islets and Batch Incubation Experiments-- All animal procedures were approved by the Chiba University Animal Care Committee. Mouse pancreatic islets were isolated by collagenase digestion method as described previously (36) and were cultured in RPMI medium 1640 (Invitrogen Corp., Carlsbad, CA) containing 10% (v/v) fetal bovine serum, 60.5 mg/liter penicillin, 100 mg/liter streptomycin, and 11.1 mM glucose under a humidified condition of 95% air and 5% CO₂. The islets then were cultured with the medium containing 4 µM of antisense phosphorothioate-substituted ODNs against mouse cAMP-GEFII (5'-CAACGGCCTTTTATCC-3') or control ODNs (5'-ACCTACGTGACTACGT-3') (BIOGNOSTIK, Göttingen, Germany) for 96 h. Batch incubation experiments were performed as described previously (36). After preincubation (30 min) of isolated islets with Hepes-Krebs buffer containing 2.8 mM glucose, five size-matched islets were collected in each tube and incubated in 500 µl of the same buffer containing glucose and test substances at the indicated concentrations for 30 min. GLP-1, GIP, H-89 as a PKA inhibitor, MDL 12330A as an adenylyl cyclase inhibitor, and 3-isobutyl-1-methylxanthine were added in the preincubation period, and 8-Br-cAMP was added in the incubation period. Insulin released into the medium was measured by radioimmunoassay (Eiken Chemical, Tokyo, Japan) (37).

Measurements of cAMP Content in the Islets-- The cAMP content of the islets was measured according to the manufacturer's instructions for the cAMP enzyme immunoassay system (Amersham Pharmacia Biotech) in the presence of 20 mM glucose. 3-Isobutyl-1-methylxanthine (250 µM) was always added to the incubation buffer. Twenty islets were used for cAMP measurements in each batch. The cAMP levels were normalized to the protein concentration.

Glutathione S-Transferase Pull-down Assay-- The isolated mouse pancreatic islets, treated with control ODNs or antisense ODNs as described above, were homogenized and incubated at 4 °C for 12 h with 2 µg of glutathione S-transferase-Rim2 (residues 1466-2453) or glutathione S-transferase alone immobilized on glutathione beads. The complexes were washed and then separated by SDS-PAGE and immunoblotted with the IgG-purified anti-cAMP-GEFII antibody (26).

Immunoblot Analysis-- Homogenates of mouse pancreatic islets, treated with control ODNs or antisense ODNs, were subjected to SDS-PAGE and immunoblotting with anti-PKA regulatory subunit II antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Rab3A antibody (Transduction Laboratories, Lexington, KY), and anti-VAMP-2 antibody (Calbiochem-Novabiochem).

Co-immunoprecipitation-- The wild-type (WT) Rim2 cDNA was subcloned into the modified pCMV containing HA epitope (pCMV-HA). The mutant cAMP-GEFII (G114E,G422D) was subcloned into the pFLAG-CMV-2 (Sigma). The WT cAMP-GEFII cDNA was subcloned into the pCMV containing GFP and Myc epitopes (pCMV-GFP, Myc). COS-1 cells were transfected with plasmid vectors for HA-tagged Rim2, FLAG-tagged mutant cAMP-GEFII (G114E,G422D), or GFP- and Myc-tagged cAMP-GEFII. For co-immunoprecipitation, cellular lysates were incubated with anti-Myc antibody (Santa Cruz Biotechnology), followed by incubation with protein G-Sepharose. The proteins were analyzed by immunoblotting, using anti-HA antibody (Roche Molecular Biochemicals), anti-GFP antibody (Living Colors A.v. Peptide Antibody; CLONTECH Laboratories, Inc., Palo Alto, CA), or anti-FLAG antibody (Sigma).

Measurement of C-peptide Secretion-- MIN6 cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose, 10% (v/v) fetal bovine serum, 60.5 mg/liter penicillin, and 100 mg/liter streptomycin under a humidified condition of 95% air and 5% CO₂ (37). MIN6 cells were transfected with human preproinsulin expression vector (pCMV-hproins) plus pCMV-luciferase, pCMV-HA-Rim2A, pFLAG-CMV-2-mutant cAMP-GEFII (G114E,G422D), pCMV-HA-Rim2, or pCMV-GFP- and Myc-cAMP-GEFII. The deletion mutant Rim2 (Rim2A) lacks the zinc finger and C₂ domains but retains the cAMP-GEFII binding region and has a dominant negative effect on interaction between WT cAMP-GEFII and WT Rim2 (26). The mutant cAMP-GEFII (G114E,G422D), in which both cAMP binding sites are disrupted, was also used (26). As control, luciferase was used. Three days after transfection, the C-peptide secretory response to 8-Br-cAMP (1 mM) in the presence of glucose (16.7 mM) for 60 min was evaluated by human C-peptide released into medium. Human C-peptide was measured by a human C-peptide radioimmunoassay kit (Linco Research Inc., St. Charles, MO).

Perifusion Experiment-- Perifusion of pancreatic islets was performed as described previously (36). Briefly, groups of 100 isolated mouse islets treated for 96 h with 4 µM control or antisense ODNs as described above were loaded onto filters in columns with Bio-Gel P-2 (Bio-Rad) and continuously perifused with Hepes-Krebs buffer at a constant flow rate of 1.0 ml/min. After a 30-min stabilization period with 2.8 mM glucose, the groups of islets were successively stimulated with 16.7 mM glucose with or without 100 µM 8-Br-cAMP. Perifusate solutions were gassed with 95% O₂ and 5% CO₂ and maintained at 37 °C.

Statistical Analysis-- Results presented are derived from at least three independent experiments performed on different days. The values are expressed as mean ± S.E. The data were compared using Student's unpaired t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GLP-1- and GIP-potentiated Insulin Secretion and cAMP Production in Pancreatic Islets-- Both GLP-1 and GIP (100 nM for each) potentiate insulin secretion in a glucose concentration-dependent manner (Fig. 1, A and B), as has been reported previously (15, 16, 38, 39). We next examined cAMP production by GLP-1 and GIP in pancreatic islets. GLP-1 (100 nM) induced about 2-fold cAMP production in the islets, compared with basal level (basal, 26.5 ± 1.8 ng/µg protein, n = 5; GLP-1 alone, 58.1 ± 7.2 ng/µg protein, n = 6, p < 0.005) (Fig. 2A). The effect of GLP-1 on cAMP production was completely blocked by treatment with MDL12330A (10 µM), an adenylyl cyclase inhibitor (39) (GLP-1 plus MDL12330A, 27.3 ± 1.2 ng/µg protein, n = 6) (Fig. 2A). MDL12330A did not affect the cAMP level at the basal state (Fig. 2A). Similarly, GIP (100 nM) induced about 2-fold cAMP production in the islets, compared with basal level (basal, 27.3 ± 1.0 ng/µg protein, n = 5; GIP alone, 56.4 ± 3.3 ng/µg protein, n = 5, p < 0.0001) (Fig. 2B). The GIP-induced cAMP production was completely abolished by treatment with MDL12330A (GIP plus MDL12330A, 27.2 ± 2.4 ng/µg protein, n = 6) (Fig. 2B).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Dose response relationships for the potentiating effects of GLP-1 and GIP on insulin secretion. A, insulin response to various concentrations of glucose in the presence or absence of GLP-1 (100 nM) was measured. B, insulin response to various concentrations of glucose in the presence or absence of GIP (100 nM) was measured. The data were obtained from three independent experiments (n = 5-6 for each point) in A and B.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of MDL 12330A on GLP-1- or GIP-induced cAMP production and GLP-1- or GIP-potentiated insulin secretion in mouse pancreatic islets. A, effect of MDL 12330A on GLP-1-induced cAMP production. cAMP levels in response to GLP-1 (100 nM) in the presence or absence of MDL 12330A (10 µM) in islets were measured (*, p < 0.005). B, effect of MDL 12330A on GIP-induced cAMP production. cAMP levels in response to GIP (100 nM) in the presence or absence of MDL 12330A (10 µM) in islets were measured (**, p < 0.0001). C, effect of MDL 12330A on GLP-1-potentiated insulin secretion. Insulin response to GLP-1 (100 nM) in the presence or absence of MDL 12330A (10 µM) in islets were measured (**, p < 0.0001). D, effect of MDL 12330A on GIP-potentiated insulin secretion. Insulin response to GIP (100 nM) in the presence or absence of MDL 12330A (10 µM) in islets were measured (**, p < 0.0001). One of three or four similar experiments is shown (n = 4-6 for each) in A-D.

Effects of MDL12330A on Incretin-potentiated Insulin Secretion-- To determine whether cAMP is an essential signal in incretin-potentiated insulin secretion, we examined the effects of MDL12330A (10 µM) on the GLP-1- and GIP-potentiated insulin secretions in the presence of 11.1 mM glucose using the batch incubation method (Fig. 2, C and D). MDL12330A did not alter the insulin secretion at the basal state (basal, 2.53 ± 0.22 ng/islet/30 min; MDL12330A alone, 2.88 ± 0.24 ng/islet/30 min, n = 4) (Fig. 2C). MDL12330A treatment remarkably inhibited GLP-1 (100 nM)-potentiated insulin secretion (GLP-1 alone, 8.50 ± 0.20 ng/islet/30 min; GLP-1 plus MDL12330A, 3.93 ± 0.14 ng/islet/30 min, n = 4, p < 0.0001) (Fig. 2C). Similarly, MDL12330A treatment inhibited GIP-potentiated insulin secretion (basal, 2.50 ± 0.18 ng/islet/30 min; MDL12330A alone, 2.76 ± 0.15 ng/islet/30 min; GIP alone, 7.72 ± 0.50 ng/islet/30 min; GIP plus MDL12330A, 3.08 ± 0.15 ng/islet/30 min, n = 5 for each) (GIP alone versus GIP plus MDL12330A, p < 0.0001) (Fig. 2D). Together, these data indicate that both the GLP-1- and GIP-potentiated insulin secretions depend critically on cAMP production in pancreatic -cells.

Effects of the Antisense ODNs against cAMP-GEFII on Incretin-potentiated Insulin Secretion-- We have shown recently that the cAMP-binding protein cAMP-GEFII is a direct target of cAMP in regulated exocytosis (26). In the present study, we investigated with the purpose of evaluating cAMP-GEFII involvement in the potentiation of insulin secretion by incretins with native pancreatic -cells. We first ascertained if treatment of the islets with antisense ODNs could suppress the level of endogenous cAMP-GEFII protein. Treatment with antisense ODNs markedly decreased the cAMP-GEFII protein level in the islets (Fig. 3A). To further confirm the specificity of the antisense ODNs, we checked the level of other proteins (i.e. the PKA regulatory subunit II, small G-protein Rab3A, and VAMP-2 (vesicle-associated membrane protein-2)). There were no differences in expression of protein levels between control ODN-treated and antisense ODN-treated pancreatic islets (Fig. 3A), indicating that the antisense ODNs are specific for cAMP-GEFII. We then examined the effect of antisense ODNs on the insulin secretory responses to GLP-1 and GIP. Glucose-induced insulin secretion was not affected by antisense ODNs treatment (Fig. 3B). The GLP-1 (100 nM)-potentiated insulin secretion in the presence of 11.1 mM glucose from pancreatic islets treated with antisense ODNs decreased significantly, compared with that treated with control ODNs (control ODN-treated, 8.51 ± 0.63 ng/islet/30 min; antisense ODN-treated, 6.26 ± 0.57 ng/islet/30 min, n = 5, p < 0.0001) (Fig. 3C). The GIP (100 nM)-potentiated insulin secretion also decreased significantly (control ODN-treated, 7.01 ± 0.79 ng/islet/30 min; antisense ODN-treated, 5.10 ± 0.44 ng/islet/30 min, n = 6, p < 0.0005) (Fig. 3D). These results indicate that cAMP-GEFII is involved in the potentiation of insulin secretion by both GLP-1 and GIP in pancreatic islets.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of antisense ODNs against cAMP-GEFII on cAMP-GEFII protein level and on incretin-potentiated insulin secretions. A, effect of antisense ODNs on the cAMP-GEFII protein level in pancreatic islets. Mouse pancreatic islets were treated with control ODNs (4 µM) or antisense ODNs (4 µM) for 96 h. Cellular lysates from the islets were incubated with glutathione S-transferase-Rim2 immobilized on glutathione beads and were then subjected to immunoblot analysis with anti-cAMP-GEFII antibody (top panel). The lysates also were subjected to SDS-PAGE and immunoblotting with antibodies against PKA regulatory subunits II, Rab3A, and VAMP-2 (bottom three panels). B, effects of antisense ODNs on glucose-induced insulin secretion. Low and high glucose concentrations used are 2.8 and 11.1 mM, respectively. C, effect of antisense ODNs on GLP-1-potentiated insulin secretion. Insulin response to GLP-1 (100 nM) in the presence of 11.1 mM glucose in the islets treated with control or antisense ODNs was measured (*, p < 0.0001). D, effect of antisense ODNs on GIP-potentiated insulin secretion. Insulin response to GIP (100 nM) in the presence of 11.1 mM glucose in the islets treated with control or antisense ODNs was measured (**, p < 0.0005). Control ODNs, pancreatic islets treated with control ODNs. Antisense ODNs, pancreatic islets treated with antisense ODNs in A-D. One of 4-6 similar experiments is shown in B-D.

Involvement of cAMP-GEFII in PKA-independent Insulin Secretion Potentiated by GLP-1, GIP, and 8-Br-cAMP-- We examined the effects of a combination of H-89 (41) and antisense ODNs on GLP-1-, GIP-, or 8-Br-cAMP-potentiated insulin secretion in mouse pancreatic islets. The insulin secretion potentiated by GLP-1 (100 nM) was measured in the presence of 11.1 mM glucose (Fig. 4A). H-89 significantly but only partially blocked GLP-1-potentiated insulin secretion (GLP-1 alone, 8.49 ± 0.23 ng/islet/30 min; GLP-1 plus H-89, 5.57 ± 0.20 ng/islet/30 min, n = 5, p < 0.0001). Combination treatment with H-89 and the antisense ODNs caused a further reduction in GLP-1-potentiated insulin secretion (4.27 ± 0.12 ng/islet/30 min, n = 5, p < 0.001). The insulin secretion potentiated by GIP (100 nM) also was measured in the presence of 11.1 mM glucose (Fig. 4B). Similarly, H-89 partially blocked GIP-potentiated insulin secretion (GIP alone, 7.27 ± 0.18 ng/islet/30 min; GIP plus H-89, 4.24 ± 0.13 ng/islet/30 min, n = 5, p < 0.0001), and combination treatment with H-89 and the antisense ODNs caused a further reduction in GIP-potentiated insulin secretion (2.99 ± 0.14 ng/islet/30 min, n = 5, p < 0.0005). To confirm the involvement of cAMP-GEFII in cAMP-dependent, PKA-independent insulin secretion, we used the cAMP analog 8-Br-cAMP in the presence of 16.7 mM glucose. Similar results were obtained with 8-Br-cAMP (Fig. 4C). These results suggest strongly that both GLP-1- and GIP-potentiated insulin secretions are mediated by PKA-independent as well as PKA-dependent mechanisms and that cAMP-GEFII participates in a PKA-independent mechanism.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of H-89 on incretin-potentiated and 8-Br-cAMP-potentiated insulin secretions in pancreatic islets treated with the antisense ODNs. A, effect of H-89 on GLP-1-potentiated insulin secretion in the islets. Pancreatic islets treated with control ODNs or antisense ODNs were preincubated for 30 min with the buffer containing 2.8 mM glucose alone or 2.8 mM glucose together with GLP-1 (100 nM), H-89 (10 µM), or GLP-1 (100 nM) plus H-89 (10 µM). The islets were then incubated for an additional 30 min with the buffer containing 11.1 mM glucose together with the same test reagents as described above. Open and filled columns indicate the insulin secretion in the absence and presence of GLP-1, respectively (*, p < 0.0001; **, p < 0.001). B, effect of H-89 on GIP-potentiated insulin secretion in the islets. The experiments were done in a similar manner as described for A, using GIP (100 nM) (*, p < 0.0001; ***, p < 0.0005). C, effect of H-89 on 8-Br-cAMP-potentiated insulin secretion in the islets. The experiment was done in a similar manner as described for A, using 8-Br-cAMP (1 mM) (***, p < 0.0005; ****, p < 0.005). 16.7 mM glucose was used to determine the potentiating effect of 8-Br-cAMP. Control ODNs, pancreatic islets treated with control ODNs. Antisense ODNs, pancreatic islets treated with antisense ODNs in A-C. The data were obtained from one of 4-6 similar experiments (n = 5-6 for each) in A-C.

cAMP-potentiated Insulin Secretion Is Mediated by the cAMP-GEFII·Rim2 Complex-- cAMP-GEFII has been shown to interact with Rim2, a target of the small G-protein Rab3 (26). To determine whether the effect of cAMP-GEFII on cAMP-potentiated insulin secretion requires its direct interaction with Rim2, we used two mutants (26) (Fig. 5A). We first examined the effect of Rim2A on 8-Br-cAMP-potentiated exocytosis from MIN6 cells in which endogenous cAMP-GEFII and Rim2 are expressed. For this purpose, we utilized MIN6 cells transfected with human preproinsulin cDNA (25, 42). Proinsulin is converted into insulin and C-peptide during the secretory process in pancreatic -cells (43). Since antibodies against human insulin cross-react with endogenous mouse insulin, we monitored secretion by measuring the human C-peptide release from MIN6 cells transfected with human preproinsulin and Rim2A (26). Overexpression of Rim2A in MIN6 cells significantly inhibited the 8-Br-cAMP-induced C-peptide secretion in the presence of 16.7 mM glucose (Fig. 5B). Coexpression of WT cAMP-GEFII with Rim2A in MIN6 cells significantly restored inhibition of the C-peptide secretion by Rim2A, suggesting that the effect of cAMP-GEFII on cAMP-potentiated insulin secretion requires interaction with Rim2. Similarly, we also assessed the effect of the mutant cAMP-GEFII (G114E,G422D). An in vivo binding experiment shows that overexpression of cAMP-GEFII (G114E,G422D) inhibits interaction between the WT cAMP-GEFII and WT Rim2 (Fig. 5C), indicating that the mutant acts as a dominant-negative inhibitor of the interaction. We reasoned that the double mutant (G114E,G422D), when overexpressed in MIN6 cells, might trap endogenous Rim2 to inhibit cAMP-potentiated C-peptide secretion. While overexpression of WT cAMP-GEFII did not alter 8-Br-cAMP-potentiated C-peptide secretion (data not shown), overexpression of the double mutant (G114E,G422D) significantly inhibited it (Fig. 5D). This inhibition of the C-peptide secretion was mostly restored by coexpression of WT Rim2, due probably to its interaction with endogenous WT cAMP-GEFII. These results indicate that cAMP-potentiated insulin secretion is mediated by the cAMP-GEFII·Rim2 complex.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of Rim2A and mutant cAMP-GEFII (G114E,G422D) on interaction of WT cAMP-GEFII and WT Rim2 and on cAMP-GEFII-mediated insulin secretion. A, schematic representation of the mutant Rim2 (Rim2A) and cAMP-GEFII (G114E,G422D) used. PDZ, PSD-95, DLG, and ZO-1; DEP, domain found in dishevelled, Egl-10, and pleckstrin. GEF, guanine nucleotide exchange factor. B, human C-peptide secretory response to 8-Br-cAMP (1 mM) in MIN6 cells transfected with human preproinsulin together with luciferase, Rim2A, or Rim2A plus WT cAMP-GEFII (*, p < 0.01). C, effects of mutant cAMP-GEFII (G114E,G422D) on interaction of WT cAMP-GEFII and WT Rim2 in COS-1 cells. Top three panels, the lysates from COS-1 cells expressed with HA-tagged WT Rim2, GFP- and Myc-tagged WT cAMP-GEFII, and FLAG-tagged cAMP-GEFII (G114E,G422D) were subjected to SDS-PAGE. Immunoblotting (IB) was performed using anti-HA antibody, anti-GFP antibody, or anti-FLAG antibody. Bottom panel, the lysates from COS-1 cells were subjected to immunoprecipitation (IP) with anti-Myc antibody, followed by immunoblotting with anti-HA antibody. D, human C-peptide secretory response to 8-Br-cAMP (1 mM) in MIN6 cells transfected with human preproinsulin together with luciferase, the mutant G114E,G422D, or the mutant G114E,G422D plus WT Rim2 (**, p < 0.0005). The C-peptide secretions are expressed as percentage increments of the secretion in the absence of 8-Br-cAMP for both B and D. The data were obtained from three independent experiments (n = 14-16) in B and D.

The Effects of cAMP-GEFII Are Dependent on Intracellular Calcium as Well as cAMP-- To determine whether the effect of cAMP-GEFII on cAMP-potentiated insulin secretion requires a rise in intracellular calcium concentrations ([Ca²⁺]_i), we examined the effects of 8-Br-cAMP (1 mM), high K⁺ (60 mM), and their combination, in the presence of 2.8 mM glucose, on insulin secretion in pancreatic islets treated with control ODNs or antisense ODNs. There were no differences in the insulin secretions stimulated by 8-Br-cAMP alone or high K⁺ alone between control ODN- and antisense ODN-treated islets. In contrast, the insulin secretion stimulated by a combination of 8-Br-cAMP plus high K⁺ in antisense ODN-treated islets was significantly lower than that in control ODN-treated islets (control ODNs, 8.81 ± 0.60 ng/islet/30 min; antisense ODNs, 6.24 ± 0.40 ng/islet/30 min, n = 10, p < 0.005) (Fig. 6A). We also examined the effect of carbachol (50 µM) on insulin secretion in the islets treated with control ODNs or antisense ODNs. While there were no differences in insulin secretion stimulated by 8-Br-cAMP alone or carbachol alone, the insulin secretion stimulated by a combination of 8-Br-cAMP plus carbachol in the antisense ODN-treated islets was significantly lower than that in the control ODN-treated islets (control ODNs, 3.48 ± 0.17 ng/islet/30 min; antisense ODNs, 2.45 ± 0.08 ng/islet/30 min, n = 16, p < 0.0001) (Fig. 6B). These results indicate that the effects of cAMP-GEFII on insulin secretion depend on intracellular Ca²⁺ as well as cAMP in pancreatic -cells.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of cAMP-GEFII on insulin secretion depend on both cAMP and intracellular calcium. A, effects of 8-Br-cAMP (1 mM, 30 min), high K+ (60 mM, 30 min), or a combination of 8-Br-cAMP plus high K+ (30 min) on insulin secretion in pancreatic islets treated with control or antisense ODNs (*, p < 0.005). 2.8 mM glucose was always added to the incubation buffer. B, effects of 8-Br-cAMP (1 mM, 30 min), carbachol (50 µM, 30 min), or a combination of 8-Br-cAMP plus carbachol (30 min) on insulin secretion in the islets treated with control or antisense ODNs (**, p < 0.0001). 2.8 mM glucose was always added to the incubation buffer. Control ODNs (open columns), pancreatic islets treated with control ODNs. Antisense ODNs (filled columns), pancreatic islets treated with antisense ODNs in A and B. The data were obtained from three or four independent experiments (n = 8-21 for each point) in A and B.

cAMP-GEFII Is Involved in Both the First and Second Phase of cAMP-potentiated Insulin Secretion-- We examined the involvement of cAMP-GEFII in the insulin secretory phase using perifused mouse pancreatic islets. No significant difference was found between control ODN-treated islets and antisense ODN-treated islets in the absence of 8-Br-cAMP (Fig. 7A). When the islets were treated with antisense ODNs, both the first phase and second phase potentiations by 8-Br-cAMP were suppressed (Fig. 7B), clearly showing that cAMP-GEFII is involved in both phases of insulin secretion.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of cAMP-GEFII on cAMP-potentiated insulin secretion in perifused islets. A, perifusion experiment in the absence of 8-Br-cAMP. The concentration of glucose was increased from 2.8 to 16.7 mM at the time indicated (between 0 and 20 min). B, potentiation of glucose-induced insulin secretion by 8-Br-cAMP (100 µM) in the islets treated with control or antisense ODNs. 8-Br-cAMP was applied 5 min prior to 16.7 mM glucose stimulation (between 5 and 15 min). Control (open circles), pancreatic islets treated with control ODNs. Antisense (filled circles), pancreatic islets treated with antisense ODNs in A and B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Incretins such as GLP-1 and GIP play an important role in the potentiation of insulin secretion (44-47). It has generally been thought that both GLP-1 and GIP potentiate glucose-induced insulin secretion primarily by cAMP/PKA signaling, which leads to phosphorylation of regulatory proteins associated with the secretory process in pancreatic -cells (15, 48, 49). A study by capacitance measurements has suggested that cAMP also promotes exocytosis in pancreatic -cells in a PKA-independent mechanism (32). In the present study, we show that MDL 12330A, an inhibitor of adenylyl cyclase, completely blocks both the GLP-1- and the GIP-stimulated cAMP production in pancreatic islets and that MDL 12330A remarkably inhibits both the GLP-1- and the GIP-induced insulin secretions. These results confirm that the effects of both GLP-1 and GIP on insulin secretion depend critically on the intracellular cAMP elevation due to activation of adenylyl cyclase. It is interesting that MDL 12330A did not completely inhibit either GLP-1- or GIP-potentiated insulin secretion under the conditions in which cAMP production was blocked. This suggests that the potentiating effects of the incretins on insulin secretion are mediated at least in part by a cAMP-independent mechanism, although the effects are small.

We recently found that the cAMP-binding protein cAMP-GEFII, by interacting with Rim2, a target of Rab3, participates in cAMP-dependent, PKA-independent exocytosis in a reconstituted system (26). In the present study, we investigated in order to find whether the cAMP-GEFII in native pancreatic -cells is involved in GLP-1- and GIP-potentiated insulin secretions and if such action is PKA-independent. Treatment of islets with antisense ODNs reduced both GLP-1- and GIP-potentiated insulin secretion, clearly indicating that the effects of the incretins are mediated in part by cAMP-GEFII. Ten µM of H-89, a widely used specific inhibitor of PKA phosphorylation in intact cells (24, 28, 41, 50), was then used to block the phosphorylation of GLUT2, a substrate of PKA in pancreatic -cells (28), to evaluate incretin-potentiated insulin secretion. Interestingly, although treatment of pancreatic islets with H-89 reduced (about 50%) both GLP-1- and GIP-potentiated insulin secretions, treatment of the islets with H-89 plus antisense ODNs further reduced the insulin secretions (80-90%), suggesting strongly that the potentiation of insulin secretion by both GLP-1 and GIP is mediated by PKA-independent as well as PKA-dependent mechanisms and that cAMP-GEFII is involved in the PKA-independent mechanism.

We then determined whether or not the potentiating effects of cAMP on insulin secretion are mediated by Rim2. Overexpression of a dominant negative mutant, Rim2 (Rim2A) or cAMP-GEFII (G114E,G422D double mutant), inhibited the potentiating effect of 8-Br-cAMP on C-peptide secretion from human preproinsulin-transfected MIN6 cells. In addition, the inhibitory effect of Rim2A or the cAMP-GEFII double mutant on C-peptide secretion was mostly restored by coexpression of WT cAMP-GEFII or WT Rim2, respectively, suggesting that the potentiating effects of the incretins are mediated by the cAMP-GEFII·Rim2 complex.

Because intracellular Ca²⁺ is essential in triggering insulin secretion, we investigated to find if the mechanism of potentiation by the cAMP-GEFII·Rim2 complex is also Ca²⁺-dependent. The effects of high K⁺ and carbachol, which triggers Ca²⁺ influx and mobilizes intracellular Ca²⁺ (51), respectively, on insulin secretion were examined in islets treated with antisense ODNs. While there were no differences in the insulin secretions stimulated by 8-Br-cAMP alone, high K⁺ alone, or carbachol alone between control ODN-treated and antisense ODN-treated islets, insulin secretion stimulated by a combination of 8-Br-cAMP plus high K⁺ or 8-Br-cAMP plus carbachol was significantly reduced in antisense ODN-treated islets. These findings indicate that the potentiation of insulin secretion through the cAMP-GEFII·Rim2 pathway depends on intracellular Ca²⁺ as well as cAMP. Since Rim2 has two C₂ domains, Ca²⁺ might modulate interaction between cAMP-GEFII and Rim2.

cAMP potentiates both phases of insulin secretion at high glucose concentrations in isolated perifused pancreas (2). Similarly, GLP-1 and GIP are both known to potentiate both the first and second phases of glucose-induced insulin secretion (15, 16). To determine the involvement of cAMP-GEFII in each phase of insulin secretion, we evaluated the potentiation of insulin secretion by 8-Br-cAMP in perifused pancreatic islets with antisense or control ODNs treatment. Both the first and second phase enhancement by 8-Br-cAMP was significantly suppressed in islets treated with antisense ODNs compared with control, showing that both the first and second phases are potentiated by cAMP in a PKA-independent mechanism.

Considering these findings together, we propose that incretins potentiate glucose-induced insulin secretion primarily by two mechanisms: the pathway involving phosphorylation of regulatory proteins by PKA activation (PKA-dependent) and the pathway involving the cAMP-GEFII·Rim2 complex (PKA-independent). The affinity for cAMP is quite different in PKA and cAMP-GEFII, with K_d of ~100 nM (52) and ~10 µM (26), respectively. cAMP at basal state in pancreatic islets has been reported in a range of micromolar concentrations (52), suggesting that many substrates for PKA already are maximally phosphorylated in pancreatic islets. This is the case with GLUT2 (28) and the sulfonylurea receptor SUR1, a subunit of the -cell K_ATP channel (29). Accordingly, PKA and cAMP-GEFII have distinct roles in cAMP-potentiated insulin secretion. The mechanism involving the cAMP-GEFII·Rim2 complex might operate upon a rise in local cAMP concentrations by stimulation. The mechanism involving PKA phosphorylation might also be controlled by PKA-anchoring protein (44, 53, 54). Since cAMP-GEFII has guanine exchange factor activity toward the small G-protein Rap1 (33), it is also tempting to speculate that Rap1, which is activated by cAMP-GEFII through incretins, might also be involved in insulin secretion.

Further elucidation of the regulation of the cAMP-GEFII·Rim2 complex by incretins should both clarify the mechanism of the potentiation of insulin secretion and suggest novel anti-diabetic drug therapy.

	FOOTNOTES

^* This work was supported by Grant-in-Aid for Creative Scientific Research 10NP0201 and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; by a Scientific Research Grant from the Ministry of Health, Labor, and Welfare, Japan; and by grants from Novo Nordisc Pharma Ltd., from Takeda Chemical Industries Ltd., and from the Yamanouchi Foundation for Research on Metabolic Disorders.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.

^¶ To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel.: 81-43-226-2187; Fax: 81-43-221-7803; E-mail: seino@med.m.chiba-u.ac.jp.

Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M108378200

	ABBREVIATIONS

The abbreviations used are: PKA, protein kinase A; GLP-1, glucagon-like peptide-1; GIP, gastric inhibitory polypeptide; G-protein, guanine nucleotide-binding protein; 8-Br-cAMP, 8-bromo-cyclic AMP; ODN, oligodeoxynucleotide; HA, hemagglutinin; WT, wild type; NS, not significant; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				B. Ponsioen, M. Gloerich, L. Ritsma, H. Rehmann, J. L. Bos, and K. Jalink Direct Spatial Control of Epac1 by Cyclic AMP Mol. Cell. Biol., May 15, 2009; 29(10): 2521 - 2531. [Abstract] [Full Text] [PDF]

				S. B. Widenmaier, A. V. Sampaio, T. M. Underhill, and C. H. S. McIntosh Noncanonical Activation of Akt/Protein Kinase B in {beta}-Cells by the Incretin Hormone Glucose-dependent Insulinotropic Polypeptide J. Biol. Chem., April 17, 2009; 284(16): 10764 - 10773. [Abstract] [Full Text] [PDF]

				O. G. Chepurny, C. A. Leech, G. G. Kelley, I. Dzhura, E. Dzhura, X. Li, M. J. Rindler, F. Schwede, H. G. Genieser, and G. G. Holz Enhanced Rap1 Activation and Insulin Secretagogue Properties of an Acetoxymethyl Ester of an Epac-selective Cyclic AMP Analog in Rat INS-1 Cells: STUDIES WITH 8-pCPT-2'-O-Me-cAMP-AM J. Biol. Chem., April 17, 2009; 284(16): 10728 - 10736. [Abstract] [Full Text] [PDF]

				K. Sugawara, T. Shibasaki, A. Mizoguchi, T. Saito, and S. Seino Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis. Genes Cells, April 1, 2009; 14(4): 445 - 456. [Abstract] [Full Text] [PDF]

				D. Islam, N. Zhang, P. Wang, H. Li, P. L. Brubaker, H. Y. Gaisano, Q. Wang, and T. Jin Epac is involved in cAMP-stimulated proglucagon expression and hormone production but not hormone secretion in pancreatic {alpha}- and intestinal L-cell lines Am J Physiol Endocrinol Metab, January 1, 2009; 296(1): E174 - E181. [Abstract] [Full Text] [PDF]

				C. Liu, M. Takahashi, Y. Li, S. Song, T. J. Dillon, U. Shinde, and P. J. S. Stork Ras Is Required for the Cyclic AMP-Dependent Activation of Rap1 via Epac2 Mol. Cell. Biol., December 1, 2008; 28(23): 7109 - 7125. [Abstract] [Full Text] [PDF]

				W. Kim and J. M. Egan The Role of Incretins in Glucose Homeostasis and Diabetes Treatment Pharmacol. Rev., December 1, 2008; 60(4): 470 - 512. [Abstract] [Full Text] [PDF]

				D. D. De Leon, C. Li, M. I. Delson, F. M. Matschinsky, C. A. Stanley, and D. A. Stoffers Exendin-(9-39) Corrects Fasting Hypoglycemia in SUR-1-/- Mice by Lowering cAMP in Pancreatic {beta}-Cells and Inhibiting Insulin Secretion J. Biol. Chem., September 19, 2008; 283(38): 25786 - 25793. [Abstract] [Full Text] [PDF]

				M. E. Sabbatini, X. Chen, S. A. Ernst, and J. A. Williams Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion J. Biol. Chem., August 29, 2008; 283(35): 23884 - 23894. [Abstract] [Full Text] [PDF]

				J. L. Jewell, E. Oh, S. M. Bennett, S. O. Meroueh, and D. C. Thurmond The Tyrosine Phosphorylation of Munc18c Induces a Switch in Binding Specificity from Syntaxin 4 to Doc2{beta} J. Biol. Chem., August 1, 2008; 283(31): 21734 - 21746. [Abstract] [Full Text] [PDF]

				N. Sonoda, T. Imamura, T. Yoshizaki, J. L. Babendure, J.-C. Lu, and J. M. Olefsky {beta}-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic {beta} cells PNAS, May 6, 2008; 105(18): 6614 - 6619. [Abstract] [Full Text] [PDF]

				M. Salehi, B. A. Aulinger, and D. A. D'Alessio Targeting {beta}-Cell Mass in Type 2 Diabetes: Promise and Limitations of New Drugs Based on Incretins Endocr. Rev., May 1, 2008; 29(3): 367 - 379. [Abstract] [Full Text] [PDF]

				W. J. Lu, Q. Yang, W. Sun, S. C. Woods, D. D'Alessio, and P. Tso Using the lymph fistula rat model to study the potentiation of GIP secretion by the ingestion of fat and glucose Am J Physiol Gastrointest Liver Physiol, May 1, 2008; 294(5): G1130 - G1138. [Abstract] [Full Text] [PDF]

				J. L. Jewell, W. Luo, E. Oh, Z. Wang, and D. C. Thurmond Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4 J. Biol. Chem., April 18, 2008; 283(16): 10716 - 10726. [Abstract] [Full Text] [PDF]

				G. Kang, C. A. Leech, O. G. Chepurny, W. A. Coetzee, and G. G. Holz Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic {beta}-cells and rat INS-1 cells J. Physiol., March 1, 2008; 586(5): 1307 - 1319. [Abstract] [Full Text] [PDF]

				A. Dov, E. Abramovitch, N. Warwar, and R. Nesher Diminished Phosphodiesterase-8B Potentiates Biphasic Insulin Response to Glucose Endocrinology, February 1, 2008; 149(2): 741 - 748. [Abstract] [Full Text] [PDF]

				T. Shibasaki, H. Takahashi, T. Miki, Y. Sunaga, K. Matsumura, M. Yamanaka, C. Zhang, A. Tamamoto, T. Satoh, J.-i. Miyazaki, et al. Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP PNAS, December 4, 2007; 104(49): 19333 - 19338. [Abstract] [Full Text] [PDF]

				E. Oh, C. J. Heise, J. M. English, M. H. Cobb, and D. C. Thurmond WNK1 Is a Novel Regulator of Munc18c-Syntaxin 4 Complex Formation in Soluble NSF Attachment Protein Receptor (SNARE)-mediated Vesicle Exocytosis J. Biol. Chem., November 9, 2007; 282(45): 32613 - 32622. [Abstract] [Full Text] [PDF]

				Y. M. Leung, E. P. Kwan, B. Ng, Y. Kang, and H. Y. Gaisano SNAREing Voltage-Gated K+ and ATP-Sensitive K+ Channels: Tuning {beta}-Cell Excitability with Syntaxin-1A and Other Exocytotic Proteins Endocr. Rev., October 1, 2007; 28(6): 653 - 663. [Abstract] [Full Text] [PDF]

				B. Ke, E. Oh, and D. C. Thurmond Doc2beta Is a Novel Munc18c-interacting Partner and Positive Effector of Syntaxin 4-mediated Exocytosis J. Biol. Chem., July 27, 2007; 282(30): 21786 - 21797. [Abstract] [Full Text] [PDF]

				K. Yamazaki, N. Yasuda, T. Inoue, E. Yamamoto, Y. Sugaya, T. Nagakura, M. Shinoda, R. Clark, T. Saeki, and I. Tanaka Effects of the Combination of a Dipeptidyl Peptidase IV Inhibitor and an Insulin Secretagogue on Glucose and Insulin Levels in Mice and Rats J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 738 - 746. [Abstract] [Full Text] [PDF]

				J. Li, K. L. O'Connor, X. Cheng, F. C. Mei, T. Uchida, C. M. Townsend Jr, and B. M. Evers Cyclic Adenosine 5'-Monophosphate-Stimulated Neurotensin Secretion Is Mediated through Rap1 Downstream of both Epac and Protein Kinase A Signaling Pathways Mol. Endocrinol., January 1, 2007; 21(1): 159 - 171. [Abstract] [Full Text] [PDF]

				G. E. Lim and P. L. Brubaker Glucagon-Like Peptide 1 Secretion by the L-Cell: The View From Within Diabetes, December 1, 2006; 55(Supplement_2): S70 - S77. [Abstract] [Full Text] [PDF]

				G. G. Holz, G. Kang, M. Harbeck, M. W. Roe, and O. G. Chepurny Cell physiology of cAMP sensor Epac J. Physiol., November 15, 2006; 577(1): 5 - 15. [Abstract] [Full Text] [PDF]

				K.-P. Yip Epac-mediated Ca2+ mobilization and exocytosis in inner medullary collecting duct Am J Physiol Renal Physiol, October 1, 2006; 291(4): F882 - F890. [Abstract] [Full Text] [PDF]

				T S McQuaid, M C Saleh, J W Joseph, A Gyulkhandanyan, J E Manning-Fox, J D MacLellan, M B Wheeler, and C B Chan cAMP-mediated signaling normalizes glucose-stimulated insulin secretion in uncoupling protein-2 overexpressing {beta}-cells. J. Endocrinol., September 1, 2006; 190(3): 669 - 680. [Abstract] [Full Text] [PDF]

				F. F. Dai, Y. Zhang, Y. Kang, Q. Wang, H. Y. Gaisano, K.-H. Braunewell, C. B. Chan, and M. B. Wheeler The Neuronal Ca2+ Sensor Protein Visinin-like Protein-1 Is Expressed in Pancreatic Islets and Regulates Insulin Secretion J. Biol. Chem., August 4, 2006; 281(31): 21942 - 21953. [Abstract] [Full Text] [PDF]

				S. Lotfi, Z. Li, J. Sun, Y. Zuo, P. P. L. Lam, Y. Kang, M. Rahimi, D. Islam, P. Wang, H. Y. Gaisano, et al. Role of the Exchange Protein Directly Activated by Cyclic Adenosine 5'-Monophosphate (Epac) Pathway in Regulating Proglucagon Gene Expression in Intestinal Endocrine L Cells Endocrinology, August 1, 2006; 147(8): 3727 - 3736. [Abstract] [Full Text] [PDF]

				A. K. Nevins and D. C. Thurmond Caveolin-1 Functions as a Novel Cdc42 Guanine Nucleotide Dissociation Inhibitor in Pancreatic beta-Cells J. Biol. Chem., July 14, 2006; 281(28): 18961 - 18972. [Abstract] [Full Text] [PDF]

				G. Liu, S. M. P. Jacobo, N. Hilliard, and G. H. Hockerman Differential Modulation of Cav1.2 and Cav1.3-Mediated Glucose-Stimulated Insulin Secretion by cAMP in INS-1 Cells: Distinct Roles for Exchange Protein Directly Activated by cAMP 2 (Epac2) and Protein Kinase A J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 152 - 160. [Abstract] [Full Text] [PDF]

				E. Oh and D. C. Thurmond The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis J. Biol. Chem., June 30, 2006; 281(26): 17624 - 17634. [Abstract] [Full Text] [PDF]

				T. Bryn, M. Mahic, J. M. Enserink, F. Schwede, E. M. Aandahl, and K. Tasken The Cyclic AMP-Epac1-Rap1 Pathway Is Dissociated from Regulation of Effector Functions in Monocytes but Acquires Immunoregulatory Function in Mature Macrophages. J. Immunol., June 15, 2006; 176(12): 7361 - 7370. [Abstract] [Full Text] [PDF]

				G. Kang, O. G. Chepurny, B. Malester, M. J. Rindler, H. Rehmann, J. L. Bos, F. Schwede, W. A. Coetzee, and G. G. Holz cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic {beta} cells and rat INS-1 cells J. Physiol., June 15, 2006; 573(3): 595 - 609. [Abstract] [Full Text] [PDF]

				T. B. Gibson, M. C. Lawrence, C. J. Gibson, C. A. Vanderbilt, K. McGlynn, D. Arnette, W. Chen, J. Collins, B. Naziruddin, M. F. Levy, et al. Inhibition of Glucose-Stimulated Activation of Extracellular Signal-Regulated Protein Kinases 1 and 2 by Epinephrine in Pancreatic {beta}-Cells. Diabetes, April 1, 2006; 55(4): 1066 - 1073. [Abstract] [Full Text] [PDF]

				M. T. Branham, L. S. Mayorga, and C. N. Tomes Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway J. Biol. Chem., March 31, 2006; 281(13): 8656 - 8666. [Abstract] [Full Text] [PDF]

				C.-C. Huang and K.-S. Hsu Presynaptic Mechanism Underlying cAMP-Induced Synaptic Potentiation in Medial Prefrontal Cortex Pyramidal Neurons Mol. Pharmacol., March 1, 2006; 69(3): 846 - 856. [Abstract] [Full Text] [PDF]

				Y. Li, S. Asuri, J. F. Rebhun, A. F. Castro, N. C. Paranavitana, and L. A. Quilliam The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane J. Biol. Chem., February 3, 2006; 281(5): 2506 - 2514. [Abstract] [Full Text] [PDF]

				H. Hatakeyama, T. Kishimoto, T. Nemoto, H. Kasai, and N. Takahashi Rapid glucose sensing by protein kinase A for insulin exocytosis in mouse pancreatic islets J. Physiol., January 15, 2006; 570(2): 271 - 282. [Abstract] [Full Text] [PDF]

				B. A. Spurlin and D. C. Thurmond Syntaxin 4 Facilitates Biphasic Glucose-Stimulated Insulin Secretion from Pancreatic {beta}-Cells Mol. Endocrinol., January 1, 2006; 20(1): 183 - 193. [Abstract] [Full Text] [PDF]

				M. Dolz, D. Bailbe, M.-H. Giroix, S. Calderari, M.-N. Gangnerau, P. Serradas, K. Rickenbach, J.-C. Irminger, and B. Portha Restitution of Defective Glucose-Stimulated Insulin Secretion in Diabetic GK Rat by Acetylcholine Uncovers Paradoxical Stimulatory Effect of {beta}-Cell Muscarinic Receptor Activation on cAMP Production Diabetes, November 1, 2005; 54(11): 3229 - 3237. [Abstract] [Full Text] [PDF]

				K. Minami, M. Okuno, K. Miyawaki, A. Okumachi, K. Ishizaki, K. Oyama, M. Kawaguchi, N. Ishizuka, T. Iwanaga, and S. Seino Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells PNAS, October 18, 2005; 102(42): 15116 - 15121. [Abstract] [Full Text] [PDF]

				E. M Sinclair and D. J. Drucker Proglucagon-Derived Peptides: Mechanisms of Action and Therapeutic Potential Physiology, October 1, 2005; 20(5): 357 - 365. [Abstract] [Full Text] [PDF]

				S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]

				G. Kang, O. G Chepurny, M. J Rindler, L. Collis, Z. Chepurny, W.-h. Li, M. Harbeck, M. W Roe, and G. G Holz A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic {beta} cells J. Physiol., July 1, 2005; 566(1): 173 - 188. [Abstract] [Full Text] [PDF]

				P. Thams, M. R Anwar, and K. Capito Glucose triggers protein kinase A-dependent insulin secretion in mouse pancreatic islets through activation of the K+ATP channel-dependent pathway Eur. J. Endocrinol., April 1, 2005; 152(4): 671 - 677. [Abstract] [Full Text] [PDF]

				T. Miki, K. Minami, H. Shinozaki, K. Matsumura, A. Saraya, H. Ikeda, Y. Yamada, J. J. Holst, and S. Seino Distinct Effects of Glucose-Dependent Insulinotropic Polypeptide and Glucagon-Like Peptide-1 on Insulin Secretion and Gut Motility Diabetes, April 1, 2005; 54(4): 1056 - 1063. [Abstract] [Full Text] [PDF]

				J. R. Wood, V. L. Nelson-Degrave, E. Jansen, J. M. McAllister, S. Mosselman, and J. F. Strauss III Valproate-induced alterations in human theca cell gene expression: clues to the association between valproate use and metabolic side effects Physiol Genomics, February 10, 2005; 20(3): 233 - 243. [Abstract] [Full Text] [PDF]

				A. K. Nevins and D. C. Thurmond A Direct Interaction between Cdc42 and Vesicle-associated Membrane Protein 2 Regulates SNARE-dependent Insulin Exocytosis J. Biol. Chem., January 21, 2005; 280(3): 1944 - 1952. [Abstract] [Full Text] [PDF]

				N. Zhong and R. S. Zucker cAMP Acts on Exchange Protein Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Protein to Regulate Transmitter Release at the Crayfish Neuromuscular Junction J. Neurosci., January 5, 2005; 25(1): 208 - 214. [Abstract] [Full Text] [PDF]

				X. Ma, Y. Zhang, J. Gromada, S. Sewing, P.-O. Berggren, K. Buschard, A. Salehi, J. Vikman, P. Rorsman, and L. Eliasson Glucagon Stimulates Exocytosis in Mouse and Rat Pancreatic {alpha}-Cells by Binding to Glucagon Receptors Mol. Endocrinol., January 1, 2005; 19(1): 198 - 212. [Abstract] [Full Text] [PDF]

				T. Shibasaki, Y. Sunaga, and S. Seino Integration of ATP, cAMP, and Ca2+ Signals in Insulin Granule Exocytosis Diabetes, December 1, 2004; 53(suppl_3): S59 - S62. [Abstract] [Full Text] [PDF]

				G. Kwon, C. A. Marshall, K. L. Pappan, M. S. Remedi, and M. L. McDaniel Signaling Elements Involved in the Metabolic Regulation of mTOR by Nutrients, Incretins, and Growth Factors in Islets Diabetes, December 1, 2004; 53(suppl_3): S225 - S232. [Abstract] [Full Text] [PDF]

				S. Yang, U. Fransson, L. Fagerhus, L. Stenson Holst, H. E. Hohmeier, E. Renstrom, and H. Mulder Enhanced cAMP Protein Kinase A Signaling Determines Improved Insulin Secretion in a Clonal Insulin-Producing {beta}-Cell Line (INS-1 832/13) Mol. Endocrinol., September 1, 2004; 18(9): 2312 - 2320. [Abstract] [Full Text] [PDF]

				S. G. Straub and G. W. G. Sharp Hypothesis: one rate-limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion Am J Physiol Cell Physiol, September 1, 2004; 287(3): C565 - C571. [Abstract] [Full Text] [PDF]

				S. A. Hinke, K. Hellemans, and F. C. Schuit Plasticity of the {beta} cell insulin secretory competence: preparing the pancreatic {beta} cell for the next meal J. Physiol., July 15, 2004; 558(2): 369 - 380. [Abstract] [Full Text] [PDF]

				M. Kaneko and T. Takahashi Presynaptic Mechanism Underlying cAMP-Dependent Synaptic Potentiation J. Neurosci., June 2, 2004; 24(22): 5202 - 5208. [Abstract] [Full Text] [PDF]

				D. A. D'Alessio and T. P. Vahl Glucagon-like peptide 1: evolution of an incretin into a treatment for diabetes Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E882 - E890. [Abstract] [Full Text] [PDF]

				M. Matsumoto, T. Miki, T. Shibasaki, M. Kawaguchi, H. Shinozaki, J. Nio, A. Saraya, H. Koseki, M. Miyazaki, T. Iwanaga, et al. Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells PNAS, June 1, 2004; 101(22): 8313 - 8318. [Abstract] [Full Text] [PDF]

				N. M. Doliba, W. Qin, M. Z. Vatamaniuk, C. Li, D. Zelent, H. Najafi, C. W. Buettger, H. W. Collins, R. D. Carr, M. A. Magnuson, et al. Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E834 - E843. [Abstract] [Full Text] [PDF]

				N. Zhong and R. S. Zucker Roles of Ca2+, Hyperpolarization and Cyclic Nucleotide-Activated Channel Activation, and Actin in Temporal Synaptic Tagging J. Neurosci., April 28, 2004; 24(17): 4205 - 4212. [Abstract] [Full Text] [PDF]

				G. Kwon, K. L. Pappan, C. A. Marshall, J. E. Schaffer, and M. L. McDaniel cAMP Dose-dependently Prevents Palmitate-induced Apoptosis by Both Protein Kinase A- and cAMP-Guanine Nucleotide Exchange Factor-dependent Pathways in {beta}-Cells J. Biol. Chem., March 5, 2004; 279(10): 8938 - 8945. [Abstract] [Full Text] [PDF]

				T. Shibasaki, Y. Sunaga, K. Fujimoto, Y. Kashima, and S. Seino Interaction of ATP Sensor, cAMP Sensor, Ca2+ Sensor, and Voltage-dependent Ca2+ Channel in Insulin Granule Exocytosis J. Biol. Chem., February 27, 2004; 279(9): 7956 - 7961. [Abstract] [Full Text] [PDF]

				G. G. Holz Epac: A New cAMP-Binding Protein in Support of Glucagon-Like Peptide-1 Receptor-Mediated Signal Transduction in the Pancreatic {beta}-Cell Diabetes, January 1, 2004; 53(1): 5 - 13. [Abstract] [Full Text] [PDF]

				M. J. DUNNE, K. E. COSGROVE, R. M. SHEPHERD, A. AYNSLEY-GREEN, and K. J. LINDLEY Hyperinsulinism in Infancy: From Basic Science to Clinical Disease Physiol Rev, January 1, 2004; 84(1): 239 - 275. [Abstract] [Full Text] [PDF]

				P. E. MacDonald, X. Wang, F. Xia, W. El-kholy, E. D. Targonsky, R. G. Tsushima, and M. B. Wheeler Antagonism of Rat {beta}-Cell Voltage-dependent K+ Currents by Exendin 4 Requires Dual Activation of the cAMP/Protein Kinase A and Phosphatidylinositol 3-Kinase Signaling Pathways J. Biol. Chem., December 26, 2003; 278(52): 52446 - 52453. [Abstract] [Full Text] [PDF]

				J. A. Ehses, V. R. Casilla, T. Doty, J. A. Pospisilik, K. D. Winter, H.-U. Demuth, R. A. Pederson, and C. H. S. McIntosh Glucose-Dependent Insulinotropic Polypeptide Promotes {beta}-(INS-1) Cell Survival via Cyclic Adenosine Monophosphate-Mediated Caspase-3 Inhibition and Regulation of p38 Mitogen-Activated Protein Kinase Endocrinology, October 1, 2003; 144(10): 4433 - 4445. [Abstract] [Full Text] [PDF]

				L. Sheu, E. A. Pasyk, J. Ji, X. Huang, X. Gao, F. Varoqueaux, N. Brose, and H. Y. Gaisano Regulation of Insulin Exocytosis by Munc13-1 J. Biol. Chem., July 18, 2003; 278(30): 27556 - 27563. [Abstract] [Full Text] [PDF]

				R. D. Burgoyne and A. Morgan Secretory Granule Exocytosis Physiol Rev, April 1, 2003; 83(2): 581 - 632. [Abstract] [Full Text] [PDF]

				K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]

				G. Kang, J. W. Joseph, O. G. Chepurny, M. Monaco, M. B. Wheeler, J. L. Bos, F. Schwede, H.-G. Genieser, and G. G. Holz Epac-selective cAMP Analog 8-pCPT-2'-O-Me-cAMP as a Stimulus for Ca2+-induced Ca2+ Release and Exocytosis in Pancreatic beta -Cells J. Biol. Chem., February 28, 2003; 278(10): 8279 - 8285. [Abstract] [Full Text] [PDF]

				L. Eliasson, X. Ma, E. Renstrom, S. Barg, P.-O. Berggren, J. Galvanovskis, J. Gromada, X. Jing, I. Lundquist, A. Salehi, et al. SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells J. Gen. Physiol., February 24, 2003; 121(3): 181 - 197. [Abstract] [Full Text] [PDF]

				D. J. Drucker Glucagon-Like Peptides: Regulators of Cell Proliferation, Differentiation, and Apoptosis Mol. Endocrinol., February 1, 2003; 17(2): 161 - 171. [Abstract] [Full Text] [PDF]

				G. Kang and G. G Holz Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic {beta} cells J. Physiol., January 1, 2003; 546(1): 175 - 189. [Abstract] [Full Text] [PDF]

				K. Fujimoto, T. Shibasaki, N. Yokoi, Y. Kashima, M. Matsumoto, T. Sasaki, N. Tajima, T. Iwanaga, and S. Seino Piccolo, a Ca2+ Sensor in Pancreatic beta -Cells. INVOLVEMENT OF cAMP-GEFII{middle dot}Rim2{middle dot}PICCOLO COMPLEX IN cAMP-DEPENDENT EXOCYTOSIS J. Biol. Chem., December 20, 2002; 277(52): 50497 - 50502. [Abstract] [Full Text] [PDF]

				M. Nakazaki, A. Crane, M. Hu, V. Seghers, S. Ullrich, L. Aguilar-Bryan, and J. Bryan cAMP-Activated Protein Kinase-Independent Potentiation of Insulin Secretion by cAMP Is Impaired in SUR1 Null Islets Diabetes, December 1, 2002; 51(12): 3440 - 3449. [Abstract] [Full Text] [PDF]

				P. E. MacDonald, W. El-kholy, M. J. Riedel, A. M. F. Salapatek, P. E. Light, and M. B. Wheeler The Multiple Actions of GLP-1 on the Process of Glucose-Stimulated Insulin Secretion Diabetes, December 1, 2002; 51(90003): S434 - 442. [Abstract] [Full Text] [PDF]

				P. E. MacDonald, A. M. F. Salapatek, and M. B. Wheeler Glucagon-Like Peptide-1 Receptor Activation Antagonizes Voltage-Dependent Repolarizing K+ Currents in {beta}-Cells: A Possible Glucose-Dependent Insulinotropic Mechanism Diabetes, December 1, 2002; 51(90003): S443 - 447. [Abstract] [Full Text] [PDF]

				S. Yamada, M. Komatsu, Y. Sato, K. Yamauchi, I. Kojima, T. Aizawa, and K. Hashizume Time-Dependent Stimulation of Insulin Exocytosis by 3',5'-Cyclic Adenosine Monophosphate in the Rat Islet {beta}-Cell Endocrinology, November 1, 2002; 143(11): 4203 - 4209. [Abstract] [Full Text] [PDF]

				C. Shiota, O. Larsson, K. D. Shelton, M. Shiota, A. M. Efanov, M. Hoy, J. Lindner, S. Kooptiwut, L. Juntti-Berggren, J. Gromada, et al. Sulfonylurea Receptor Type 1 Knock-out Mice Have Intact Feeding-stimulated Insulin Secretion despite Marked Impairment in Their Response to Glucose J. Biol. Chem., September 27, 2002; 277(40): 37176 - 37183. [Abstract] [Full Text] [PDF]

				J. Qiao, F. C. Mei, V. L. Popov, L. A. Vergara, and X. Cheng Cell Cycle-dependent Subcellular Localization of Exchange Factor Directly Activated by cAMP J. Biol. Chem., July 12, 2002; 277(29): 26581 - 26586. [Abstract] [Full Text] [PDF]

				B. Yusta, J. Estall, and D. J. Drucker Glucagon-like Peptide-2 Receptor Activation Engages Bad and Glycogen Synthase Kinase-3 in a Protein Kinase A-dependent Manner and Prevents Apoptosis following Inhibition of Phosphatidylinositol 3-Kinase J. Biol. Chem., July 5, 2002; 277(28): 24896 - 24906. [Abstract] [Full Text] [PDF]

				O. G. Chepurny, M. A. Hussain, and G. G. Holz Exendin-4 as a Stimulator of Rat Insulin I Gene Promoter Activity via bZIP/CRE Interactions Sensitive to Serine/Threonine Protein Kinase Inhibitor Ro 31-8220 Endocrinology, June 1, 2002; 143(6): 2303 - 2313. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/49/46046    most recent M108378200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kashima, Y. Articles by Seino, S. Search for Related Content PubMed PubMed Citation Articles by Kashima, Y. Articles by Seino, S. Social Bookmarking                      What's this?