Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic {beta}-cells

Yoshikazu Miura and Hisao Matsui

Department of Hygiene, Dokkyo University School of Medicine, Tochigi 321-0293, Japan

Submitted 6 January 2003 ; accepted in final form 17 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.

cytosolic Na+; cytosolic Ca2+; pancreatic islet cells; cyclic adenosine 5'-monophosphate; hamster


GLUCAGON-LIKE PEPTIDE-1-(7-36) AMIDE (GLP-1) lowers blood glucose level by stimulating insulin secretion from pancreatic {beta}-cells, and this is under intensive clinical investigation for the treatment of type 2 diabetes mellitus (44, 45). GLP-1 is derived by posttranslational processing of proglucagon and released from the intestinal L cells into blood after a meal intake (33). GLP-1 has been proposed as an important incretin candidate (16, 33, 48) and is known to stimulate insulin secretion in a glucose-dependent manner (33, 50). The augmentation of glucose-stimulated insulin secretion by GLP-1 plays a critical role in the regulation of systemic glucose homeostasis (33). This insulinotropic action is shown both in vivo in humans (22 33), pig (49), and mouse (13) and in vitro in rat islets (11, 53) and in other species. GLP-1 stimulates cAMP production (20) through plasma membrane receptors (56, 60), which increases PKA-dependent or PKA-independent intracellular Ca2+ concentration ([Ca2+]i) (3, 29) and insulin secretion (29). Potentiation of insulin secretion by GLP-1 has been shown to involve a number of mechanisms, including inhibition of ATP-sensitive K+ channels (7, 26), acceleration of Ca2+ influx through voltage-gated Ca2+ channels (58), stimulation of Ca2+ release from intracellular stores (19), and activation of nonspecific cation channels (35) in addition to direct effects on exocytosis (20). However, it remains to be examined whether other signaling mechanisms for GLP-1 exist in pancreatic islet {beta}-cells.

Previous studies have shown that GLP-1 elevates the [Ca2+]i in rat {beta}-cells (12, 58), depolarizes the rat pancreatic {beta}-cells, and stimulates insulin secretion in an extracellular Na+-dependent manner (4, 30). Na+ dependency was also shown in the efflux of 45Ca2+ induced by GLP-1 (10). These findings suggest that an increase in membrane Na+ permeability is a mechanism for the actions induced by GLP-1. Glucose also promotes both the entry and the extrusion of the Na ion in the pancreatic {beta}-cells (51).

In this study, to investigate a possible role of Na+ in the action of GLP-1 on insulin secretion from pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in overnight-cultured hamster islet cells in various concentrations of extracellular Na+.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. Male Syrian hamsters, weighing 95–125 g (aged 9–10 wk), were obtained from Experimental Animal Supply (Saitama, Japan) and housed under controlled conditions (22 ± 1°C, 50 ± 5% relative humidity, 12:12-h light-dark cycle). They received laboratory mouse, rat, and hamster chow (MF, Oriental Yeast, Tokyo, Japan) and tap water ad libitum.

This study was carried out under the control of the Animal Care and Use Committee, Dokkyo University School of Medicine, in accordance with guidelines for the care and use of laboratory animals.

Chemicals. GLP-1-(7–36) amide was obtained from Peptide Institute (Osaka, Japan), dispase from Godo-Shusei (Chiba, Japan), Ficoll 400 from Pharmacia Fine Chemicals (Uppsala, Sweden), DMEM from Nissui Pharmaceutical (Tokyo, Japan), Conray 400 (sodium iotalamate) from Daiichi Pharmaceutical (Tokyo, Japan), and IBMX from Aldrich (Milwaukee, WI). 8-pCPT-2'-O-Me-cAMP was from BIOLOG Life Sciences Institute (Bremen, Germany). Collagenase and TTX were purchased from Wako Pure Chemical Industries (Osaka, Japan), EDTA, fura 2-AM, and BAPTA-AM from Dojindo Laboratories (Kumamoto, Japan), sodium-binding benzofuran isophthalate (SBFI)-AM, and pluronic F-127 from Molecular Probes (Eugene, OR), forskolin, dibutyryl cAMP, SQ-22536, ouabain, gramicidin D, somatostatin, calphostin C, and thapsigargin from Sigma Chemical (St. Louis, MO), and H-89 and H-85 from Seikagaku (Tokyo, Japan). All other chemicals were of analytical grade.

Preparation of pancreatic islets and islet cells. Hamsters were anesthetized by intraperitoneal injection of pentobarbital sodium at 50 mg/kg, and pancreas was dissected. Pancreatic islets were isolated after collagenase digestion of the pancreas (39) from fasted hamsters. Islets were separated from the collagenase digestion by the method of Ficoll-Conray gradient centrifugation (47) and individually chosen by stereoscopic microscopy in DMEM supplemented with 2% FCS. Dissociated islet cells were prepared by further digestion with dispase (0.33 mg/ml) of the isolated islets in Ca2+- and Mg2+-free HBSS as described by Takaki and Ono (55). To test the changes of stimulus-induced both [Ca2+]i and [Na+]i in pancreatic islet cells, dissociated islet cells (5 x 104 cells) were plated onto the 0.2% poly-L-lysine-coated circular glass coverslips (13.2 mm diameter), and cultured overnight at 37°C in DMEM containing 5% FCS and 5.5 mM glucose in an atmosphere of 5% CO2 in humidified air. The proportion of pancreatic {beta}-cells from hamsters was estimated by staining cells with anti-human insulin serum. The proportion of pancreatic {beta}-cells (n {beta} 5) that showed insulin immunoreactivity was estimated at ~80% of cells. The viability of islet cells and islets in hamsters consistently exceeded 95% as determined by erythrosine B dye exclusion test (41).

Insulin release from pancreatic islet cells. The dissociated islet cells (3–4 x 104 cells/dish, 15 mm) were cultured overnight at 37°C in RPMI 1640 medium containing 11.1 mM glucose supplemented with 10% FCS. Islet cells were washed twice with bicarbonate-buffered medium (pH 7.4) containing 8 mM glucose. Islet cells were incubated for 60 min at 37°Cin bicarbonate-buffered medium containing 8 mM glucose and 10 nM GLP-1 in the presence of extracellular Na+ (0, 13.5, or 135 mM) with or without somatostatin (50 nM), gadolinium (100 µM), ouabain (100 µM), TTX (2 µM), SQ-22536 (1 mM), and H-89 (10 µM). Islet cells were also incubated for 60 min in medium containing 8 mM glucose and various concentration of 8-pCPT-2'-O-Me-cAMP (29) in the presence of extracellular Na+ (0, 13.5 or 135 mM), and with or without H-89 (10 µM). A portion of the medium was withdrawn from the incubation, centrifuged, and appropriately diluted for the insulin assay. Insulin was measured with a double-antibody RIA kit from Eiken Chemical (Tokyo, Japan) (42). The intra- and interassay coefficients of variation were <10 and 12%, respectively. The minimum detectable sensitivity was 5 µU/ml, and the ED50 was 45 µU/ml.

[Ca2+]i and [Na+]i measurements. [Ca2+]i and [Na+]i were measured by a modification of the method of Gilon and Henquin (15), and Miura and colleagues (38, 40, 41). Because of the heterogeneity of [Ca2+]i and [Na+]i responses in single cells, all experiments were carried out with clusters of 3–5 islet cells. For [Ca2+]i measurement, the overnight-cultured islet cells (4 x 104 cells) were loaded with fura 2 for 45–60 min at 37°C in a HEPES-buffered medium containing (in mM) 120 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, 24 NaHCO3, and 10 HEPES, pH 7.4, containing 1 µM fura 2-AM and 5.5 mM glucose. HEPES-buffered Na+-free solutions were prepared by substituting N-methyl-D-glucamine chloride for NaCl (15, 24). HEPES-buffered 13.5 mM Na+-containing solution was prepared by diluting with a HEPES-buffered Na+-free medium. For [Na+]i measurement, the overnight-cultured islet cells were loaded with SBFI during 1.5–2 h of incubation in DMEM containing 5% FCS with 5 µM SBFI-AM and 0.02% of the nonionic dispersing agent pluronic F-127. Thereafter, the cells were equilibrated for 20 min in a HEPES-buffered medium containing (in mM) 135 NaCl, 4.8 KOH, 2.5 CaCl2, 1.2 MgCl2, and 10 HEPES, similar to that with which the subsequent experiment started. The coverslips with the fura 2- or SBFI-loaded cells were placed in HEPES medium (37°C) containing either 3 or 8 mM glucose and fixed in a hand-made chamber (fitted with a peristaltic pump for perifusion) mounted on the stage of an inverted IX 70 microscope (Olympus, Tokyo, Japan). The loaded cells were excited at 340 and 380 nm, the fluorescence emitted at 510 nm was captured by an intensified charge-coupled device camera, and the images were analyzed by the QuantiCell 700 system (Applied Imaging, Sunderland, UK). The changes of [Ca2+]i in single islet cells were calculated from the ratio (R) of the fluorescence measured with excitation at 340 nm to that at 380 nm using the following equation (21): [Ca2+]i (nM) {beta} Kd x (R — Rmin)/(Rmax — R) x {beta}, where Kd is the dissociation constant for fura 2 (224 nM), Rmax and Rmin are the ratios for unbound and bound forms of the fura 2 · Ca2+ complex, respectively, and {beta} is the ratio of fluorescence of fura 2 at 380 nm excitation in minimum calcium and saturating calcium. Rmax and Rmin were estimated with the fluorescence intensities of fura 2 solution (1 µM) containing 10 mM CaCl2 and 5 mM EGTA, respectively. The fluorescent signal generated by [Ca2+]i binding to fura 2 is not influenced by changes in the pH of the bathing solution (5, 15) over the range of 6.70–7.05. Filipsson et al. (9) also indicate that cytosolic pH is 7.41 ± 0.04 in the presence of extracellular Na+ and 7.02 ± 0.11 when Na+ in the medium is replaced with N-methyl-D-glucamine chloride. Therefore, in this study, we used the Kd for fura 2, 224 nM, in the presence or absence of extracellular 135 mM Na+. For [Na+]i measurements, [Na+]i in single islet cells were calculated by comparing the ratio of the signals successively acquired at 340 and 380 nm to calibration curves. The calibration curve was established by exposing reference cells loaded with SBFI to 10 µg/ml gramicidin D, an ionophore that equilibrates transmembrane Na+ and K+ concentrations, and perifusing them stepwise with solutions containing different Na+ (from 5 to 100 mM) and K+ concentrations (Na+ + K+ {beta} 135 mM, 105 mM gluconate, 30 mM NaCl or KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 3 mM glucose) (15, 38, 41).

Measurement of cAMP content. cAMP content was measured by a modification of the method of Nelson et al. (46). Dissociated islet cells (3 x 104 cells/dish, 15 mm) were cultured overnight at 37°C in DMEM containing 5% FCS. Islet cells were washed twice with Krebs-Ringer solution buffered with bicarbonate (KRB; pH 7.4) containing 8 mM glucose and incubated for 30 min at 37°C in 0.4 ml of KRB containing 1 mM IBMX and stimulus in the presence of 8 mM glucose. The reactions were stopped by addition of 0.2 ml of ice-cold TCA to a final concentration of 6%. The culture plates were shaken, left at room temperature for 15 min, and centrifuged at 7,800 g for 15 min. The supernatants were thoroughly mixed with 1.5 ml of diethyl ether, and the ether phase containing TCA was discarded. This step was repeated three times to ensure complete elimination of TCA. The extracts and cAMP standards were evaporated, added to 400 µl of KRB, and assayed for cAMP with an RIA kit (Yamasa Shyoyu, Choshi, Japan) in which the samples and standards are succinylated.

Statistics. The data are expressed as means ± SE. The statistical significance of differences between means was assessed by a repeated-measures ANOVA (Figs. 2, 3, and 9) or by a one-way ANOVA, followed by Scheffé's F-test (Figs. 1 and 4, 5, 6, 7, 8). Differences were considered significant at P < 0.05. Experiments were carried out with islet cells of at least three different preparations from hamsters (n >= 3).



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Fig. 2. Effects of GLP-1 (10 nM) on the 8 mM glucose-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in islet cells in the presence of 135 (A), 13.5 (B), and 0 mM Na+ (C). Cells were preincubated with a buffer containing 135 or 0 mM Na+ and 3 mM glucose for 3 min before [Ca2+]i imaging started. Traces correspond to mean responses (±SE) in 26–52 islet cells (n {beta} 3–4). P < 0.05.

 


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Fig. 3. Effects of GLP-1 (10 nM) on the 8 mM glucose-induced increase in intracellular Na+ concentration ([Na+]i) in islet cells in the presence of 135 (A), 13.5 (B), and 0 mM Na+ (C). Cells were preincubated with a buffer containing 135, 13.5, or 0 mM Na+ and 3 mM glucose for 3 min before [Ca2+]i imaging started. Traces correspond to mean responses (±SE) in 26–52 islet cells (n {beta} 3–4). P < 0.05.

 


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Fig. 9. Effects of 8-pCPT-2'-O-Me-cAMP (50 µM) on the 8 mM glucose-induced increase in [Na+]i in islet cells in the absence (A) and presence (B) of H-89 (10 µM). Cells were preincubated with both 135 mM Na+- and 8 mM glucose-containing buffer in the absence and presence of H-89 (10 µM) for 3 min before [Na+]i imaging started. Traces correspond to mean responses (±SE) in 26–52 islet cells (n {beta} 3–4). P < 0.05.

 


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Fig. 1. Effects of glucagon-like peptide (GLP)-1 (10 nM) on the insulin release from 4 x 104 islet cells stimulated by 8 mM glucose in a medium containing 135 mM, 13.5 mM (diluted with a medium containing N-methyl-D-glucamine instead of Na+), or 0 mM Na+ (with N-methyl-D-glucamine instead of Na+). Pancreatic islet cells were cultured overnight in RPMI 1640 containing 10% FCS and 11.1 mM glucose at 37°C in 5% CO2-95% O2. After a wash, islet cells were incubated for 60 min in a medium containing 8 mM glucose or 10 nM GLP-1 plus 8 mM glucose in the presence of 135, 13.5, or 0 mM Na+. Data are expressed as means ± SE (n {beta} 8). P < 0.05.

 


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Fig. 4. Effects of somatostatin, gadolinium (Gd3+), tetrodotoxin (TTX), or ouabain on insulin release from 3 x 104 islet cells stimulated by GLP-1 in a medium containing 8 mM glucose and 135 mM Na+. Pancreatic islet cells were cultured overnight in RPMI 1640 containing 10% FCS and 11.1 mM glucose at 37°C in 5% CO2-95% O2. After a wash, islet cells were incubated with 8 mM glucose or 10 nM GLP-1 plus 8 mM glucose for 60 min at 37°C with or without 50 nM somatostatin or 100 µM Gd3+ (A) or 10 nM GLP-1 plus 8 mM glucose with 2 µM TTX or 100 µM ouabain (B). Data are expressed as means ± SE (n {beta} 4–9). *P < 0.05.

 


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Fig. 5. Inhibitory effect of SQ-22536, an inhibitor of adenylate cyclase, on cAMP content in cultured single islet cells stimulated with GLP-1 and forskolin in the presence of 8 mM glucose. Pancreatic islet cells (3 x 104 cells) were cultured overnight in DMEM containing 5% FCS and 5 mM glucose. In the presence of 8 mM glucose, they were incubated with 1 mM IBMX alone, 10 nM GLP-1 plus 1 mM IBMX, or 10 µM forskolin plus 1 mM IBMX for 30 min at 37°C with or without SQ-22536 (1 mM). Data are expressed as means ± SE (n {beta} 8–21). *P < 0.05.

 


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Fig. 6. Effects of SQ-22536 and H-89, an inhibitor of PKA, on insulin release from 4 x 104 islet cells stimulated by GLP-1 or forskolin in a medium containing 8 mM glucose and 135 mM Na+. Pancreatic islet cells were cultured overnight in RPMI 1640 containing 10% FCS and 11.1 mM glucose at 37°C in 5% CO2-95% O2. After a wash, in the presence of 8 mM glucose, islet cells were incubated with 10 nM GLP-1 or 10 µM forskolin for 60 min at 37°C with or without SQ-22536 (1 mM) or by 10 nM GLP-1 plus 8 mM glucose with 10 µM H-89. Data are expressed as means ± SE (n {beta} 4–12). *P < 0.05.

 


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Fig. 7. Effects of 8-pCPT-2'-O-Me-cAMP, a PKA-indpendent cAMP analog, on insulin release from 3 x 104 islet cells incubated in a medium containing 8 mM glucose and 135 mM Na+ with or without H-89. Pancreatic islet cells were cultured overnight in RPMI 1640 containing 10% FCS and 11.1 mM glucose at 37°C in 5% CO2-95% O2. After a wash, islet cells were incubated with 8-pCPT-2'-O-Me-cAMP (0–300 µM) in the presence of 8 mM glucose for 60 min at 37°C with or without H-89 (10 µM). Data are expressed as means ± SE (n {beta} 4–12). *P < 0.05.

 


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Fig. 8. Effects of 8-pCPT-2'-O-Me-cAMP (50 µM) on insulin release from 3 x 104 islet cells stimulated by 8 mM glucose in the presence of various concentration of extracellular Na+. Pancreatic islet cells were cultured overnight in RPMI 1640 containing 10% FCS and 11.1 mM glucose at 37°C in 5% CO2-95% O2. After a wash, islet cells were incubated for 60 min in a medium containing 8 mM glucose or 50 µM 8-pCPT-2'-O-Me-cAMP plus 8 mM glucose in the presence of 135 mM, 13.5 mM (diluted with a medium containing N-methyl-D-glucamine instead of Na+), or 0 mM Na+ (with N-methyl-D-glucamine instead of Na+). Data are expressed as means ± SE (n {beta} 4–8). *P < 0.05.

 


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GLP-1-induced insulin secretion in various concentrations of extracellular Na+. Figure 1 shows the insulin secretion from 4 x 104 islet cells stimulated by 8 mM glucose or both 10 nM GLP-1 and 8 mM glucose in various concentrations of Na+ (0, 13.5, or 135 mM). In the presence of 135 mM Na+, GLP-1 significantly increased the insulin secretion from islet cells (1,080 ± 70.4 vs. 8 mM glucose 573 ± 42.7 µU/4 x 104 cells, P < 0.05, n {beta} 8). However, a decrease in [Na+] from 135 to 13.5 mM (diluted with a medium containing N-methyl-D-glucamine instead of Na+) strongly reduced the GLP-1-induced insulin secretion from islet cells (626 ± 56.1 vs. 135 mM Na+ 1,080 ± 70.4 µU/ 4 x 104 cells, P < 0.05, n {beta} 8). In a Na+-free medium (with N-methyl-D-glucamine instead of Na+), the insulin secretion by GLP-1 was further decreased (P < 0.05). However, a decrease in [Na+] from 135 to 13.5 mM slightly reduced the 8 mM glucose-induced insulin secretion from islet cells. There was no significant difference between the insulin secretion from islet cells induced by 8 mM glucose in a medium containing 135 and 13.5 mM Na+.

Glucose and GLP-1-induced changes in [Ca2+]i in various concentrations of extracellular Na+. Elevation of glucose concentration to 8 from 3 mM increased [Ca2+]i to the peak value of 89.4 ± 4.6 nM from the basal level of 58.7 ± 0.9 nM in 52 clusters of islet cells. Application of GLP-1 (10 nM) increased the [Ca2+]i in a biphasic manner in 92.8% of the clusters of islet cells. The first phase reached the peak [Ca2+]i level (122.6 ± 8.1 nM), followed by the second phase of [Ca2+]i (96.7 ± 1.6 nM, n {beta} 52; Fig. 2A). In the presence of 8 mM glucose, changing of [Na+] to 13.5 mM from 135 mM-containing medium gradually decreased the [Ca2+]i to 103.8 ± 0.6 nM (15–20 min) from 113.6 ± 0.8 nM in 38 clusters of islet cells (P < 0.05). In this condition, addition of 10 nM GLP-1 slightly increased the [Ca2+] in 71% of clusters of islet cells. This suppression was immediately removed by introducing 135 mM Na+ (Fig. 2B). In the absent of extracellular Na+, elevation of glucose concentration to 8 from 3 mM gradually increased the [Ca2+]i to 87.6 ± 1.2 nM (15–20 min) from 71.3 ± 0.4 nM in 41 clusters of islet cells (P < 0.05). Addition of 10 nM GLP-1 further increased the [Ca2+]i to 105.1 ± 8.0 nM from 87.6 ± 1.2 nM in the Na+-free medium (n {beta} 41, P < 0.05) (Fig. 2C).

GLP-1-induced changes in [Na+]i in clusters of islet cells. To determine whether GLP-1 could affect the changes of [Na+]i in islet cells, we measured the [Na+]i induced by 10 nM GLP-1 and 8 mM glucose in a medium containing 135 or 13.5 mM Na+ and that in a Na+-free medium. The basal level of [Na+]i was 18.0 ± 0.2 mM in 19 clusters of islet cells perifused with a medium containing 3 mM glucose and 135 mM Na+. An increase in glucose concentration from 3 to 8 mM did not affect the values of [Na+]i (Fig. 3A). Addition of 10 nM GLP-1 to the medium resulted in an increase of [Na+]i in 90% of the clusters of islet cells (19 of 21) studied. GLP-1 rapidly increased the [Na+]i and maintained the elevated level (22.0 ± 0.2 mM, 30–35 min, P < 0.05). This increase was reversed by replacement with an impermeant molecule (Fig. 3A) and did not occur when GLP-1 was added to the Na+-free medium (Fig. 3C). The GLP-1-induced increase of [Na+]i was reduced to 12.2 ± 0.1 mM from 19.3 ± 0.1 mM (n {beta} 42) by changing the medium containing 135 mM Na+ to 13.5 mM Na+ (Fig. 3B). There was no statistically significant difference between the [Na+]i induced by GLP-1 in the medium containing 13.5 mM Na+ and that in the Na+-free medium in 42 clusters of islet cells (Fig. 3B).

Effects of somatostatin, Gd3+, TTX, or ouabain on GLP-1-induced insulin secretion. Figure 4 shows the GLP-1-induced insulin secretion from 3 x 104 cultured islet cells in the presence of 50 nM somatostatin (54), 100 µM gadolinium (Gd3+), a blocker of nonselective cation channels (59), 2 µM TTX, an inhibitor of the voltage-gated Na+ channel (43), or 100 µM ouabain (17), an inhibitor of Na+-K+-ATPase. GLP-1 (10 nM) significantly increased the insulin secretion from islet cells in the presence of 8 mM glucose (773 ± 34.0 vs. 8 mM glucose alone 320 ± 33.4 µU/3 x 104 cells, P < 0.05, n {beta} 8–9). Somatostatin significantly decreased the GLP-1-induced insulin secretion from islet cells (406 ± 105.9 vs. 10 nM GLP-1 773 ± 34.0 µU/3 x 104 islet cells, P < 0.05, n {beta} 6–9). Gd3+ also reduced the GLP-1-induced insulin secretion (431 ± 29.4 vs. 10 nM GLP-1 773 ± 34.0 µU/3 x 104 islet cells, P < 0.05, n {beta} 5–9; Fig. 4A). Ouabain slightly increased the glucose-induced insulin secretion from islet cells (378 ± 38.7 vs. 8 mM glucose alone 320 ± 33.4 µU/3 x 104 islet cells, n {beta} 5–8). However, ouabain and TTX did not influence the GLP-1-induced insulin secretion from islet cells (n {beta} 4–9; Fig. 4B). The GLP-1-induced rise in [Na+]i was not affected by 2 µM TTX (data not shown). In the presence of 100 µM ouabain, GLP-1 gradually increased the [Na+]i in 69 clusters of islet cells. However, in the presence of 1 mM ouabain, GLP-1 did not influence the rise of [Na+]i (data not shown). The addition of GLP-1 to the medium containing 1 µM thapsigargin (38), an inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump, gradually increased the [Na+]i to 24.8 ± 0.1 mM from 22.1 ± 0.1 mM (P < 0.05, n {beta} 38 clusters; data not shown).

Effects of SQ-22536 on cAMP contents. To determine whether SQ-22536 (57), an inhibitor of adenylate cyclase, affects the increase of cAMP, we measured the cAMP content in 3 x 104 islet cells (Fig. 5). GLP-1 (10 nM) significantly increased the cAMP content in the presence of 1 mM IBMX, a phosphodiesterase inhibitor (7.3 ± 0.4 vs. 8 mM glucose 2.0 ± 0.3 pM, P < 0.05, n {beta} 18–21). Forskolin (10 µM) (25), an activator of adenylate cyclase, also markedly increased the cAMP content in islet cells in the presence of 1 mM IBMX (51.4 ± 4.3 pM vs. 8 mM glucose 2 ± 0.3 pM, P < 0.05, n {beta} 9–18). SQ-22536 (1 mM) completely inhibited an increase of cAMP content induced by 10 nM GLP-1 (P < 0.05, n {beta} 21). SQ-22536 also significantly reduced the increase in cAMP content by forskolin to the value of cAMP content in islet cells induced by GLP-1 (P < 0.05, n {beta} 13).

Effects of SQ-22536 or H-89 on insulin secretion induced by GLP-1 and forskolin. Figure 6 shows the insulin secretion from 4 x 104 cultured islet cells. GLP-1 (10 nM) increased the insulin secretion from islet cells (1,019 ± 55.6 vs. 8 mM glucose alone 492 ± 56.1 µU/4 x 104 cells, P < 0.05, n {beta} 7–12). Forskolin (10 µM) also increased insulin secretion from islet cells (1,141 ± 173.4 vs. 8 mM glucose 492 ± 56.1 µU/4 x 104 cells, P < 0.05, n {beta} 7–8). SQ-22536 significantly decreased the both GLP-1- and forskolin-induced insulin secretion from islet cells (P < 0.05, n {beta} 4). H-89 (30), a potent inhibitor of PKA, also reduced the GLP-1-induced insulin secretion (489 ± 39.7 vs. 10 nM GLP-1 1,019 ± 55.6 µU/islet cells, P < 0.05, n {beta} 8–12). Calphostin C (100 nM), an inhibitor of PKC (18), also significantly reduced the GLP-1-induced insulin secretion from islet cells in the presence of 8 mM glucose (P < 0.05; data not shown).

SQ-22536 and H-89 incompletely inhibited the increase in [Na+]i induced by GLP-1 in clusters of islet cells (data not shown). Activation of adenylate cyclase by forskolin resulted in an increase of [Na+]i in the presence of 8 mM glucose in 82.6% of clusters of islet cells (19 of 23) studied (data not shown). Db-cAMP (2 mM) (58), a membrane-permeant cAMP analog, significantly increased the [Na+]i to 21.6 ± 0.1 mM in the presence of 8 mM glucose (P < 0.05, n {beta} 25 clusters; data not shown). Application of 50 µM BAPTA-AM (35) gradually increased the [Na+]i. Under this condition, 10 nM GLP-1 further increased the [Na+]i (data not shown).

8-pCPT-2'-O-Me-cAMP-induced insulin secretion in various concentrations of extracellular Na+ with or without H-89. Figure 7 shows the insulin secretion from 3 x 104 islet cells stimulated by 8 mM glucose, 135 mM Na+, and various concentrations of the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (0–300 µM) (29) with and without 10 µM H-89. In the presence of 8 mM glucose, the insulin secretion induced by 8-pCPT-2'-O-Me-cAMP was increased in a dose-dependent manner and strongly increased in the concentration range of 50 to 300 µM with or without H-89 (P < 0.05).

Figure 8 shows the insulin secretion from 3 x 104 islet cells stimulated with 8 mM glucose or both 50 µM 8-pCPT-2'-O-Me-cAMP and 8 mM glucose in various concentrations of Na+ (0, 13.5, or 135 mM). In the presence of 135 mM Na+, 8-pCPT-2'-O-Me-cAMP significantly increased the insulin secretion from islet cells (451.1 ± 13.5 vs. 8 mM glucose 211.2 ± 23.8 µU/ 3 x 104 cells, P < 0.05, n {beta} 8). However, a decrease of [Na+] from 135 to 13.5 mM strongly reduced the 8-pCPT-2'-O-Me-cAMP-induced insulin secretion from islet cells (247.9 ± 13.5 vs. 135 mM Na+ 451.1 ± 13.5 µU/ 3 x 104 cells, P < 0.05, n {beta} 8). In a Na+-free medium, addition of 8-pCPT-2'-O-Me-cAMP showed the same insulin secretion as that of extracellular 13.5 mM Na+.

8-pCPT-2'-O-Me-cAMP-induced changes in [Na+]i in islet cells. To determine whether 8-pCPT-2'-O-Me-cAMP could affect the changes of [Na+]i in clusters of islet cells, we measured the [Na+]i induced by 50 µM 8-pCPT-2'-O-Me-cAMP and 8 mM glucose in a 135 mM Na+-containing medium with or without 10 µM H-89 (Fig. 9). The basal level of [Na+]i was 8.7 ± 0.3 mM in 12 clusters of islet cells perifused with a medium containing 8 mM glucose and 135 mM Na+. Addition of 50 µM 8-pCPT-2'-O-Me-cAMP gradually increased the [Na+]i to 10.2 ± 0.3 mM (20–25 min) in 12 clusters of islet cells. Changing to a Na+-free medium decreased the [Na+]i to 7.8 ± 0.4 mM (Fig. 9A). In the presence of H-89, 50 µM 8-pCPT-2'-O-Me-cAMP gradually increased the [Na+]i to 24.0 ± 0.3 mM (22–25 min) from 20.5 ± 0.2 mM (0–5 min) in 32 clusters of islet cells (P < 0.05; Fig. 9B).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study showed that, in a physiological concentration of glucose and extracellular Na+, GLP-1 increased the [Na+]i via cAMP in primary cultured islet cells of the hamster. We also confirmed that GLP-1 increased the [Ca2+]i and insulin secretion in cultured islet cells in a Na+-dependent manner.

An individual pancreatic {beta}-cell is often unresponsive to glucose alone but becomes responsive when stimulated in combination with hormones such as insulinotropic hormone GLP-1 (50). A role for increased Na+ permeability in the depolarization and insulin release in response to ACh (24) and GLP-1 (10, 30) has been suggested. In this study in hamster islet cells, GLP-1 plus 8 mM glucose also stimulated the insulin release and [Ca2+]i in the presence of 135 mM Na+ but not in low-Na+ condition (Figs. 1 and 2, A and B). These would suggest that an increase in Na+ permeability affects the GLP-1-induced insulin secretion and [Ca2+]i in hamster pancreatic {beta}-cells in the presence of a physiological glucose concentration and a normal extracellular Na+ condition.

GLP-1 caused a progressive rise in [Na+]i in islet cells in the presence of 8 mM glucose and 135 mM Na+. However, the GLP-1-induced response was completely blocked in the medium containing 13.5 mM Na+ and 0 mM Na+. These results indicate that GLP-1 increases the [Na+]i by augmenting Na+ influx through plasma membrane of the islet {beta}-cells. Interestingly, there was clear rise in the [Ca2+]i by increasing glucose concentration from 3 to 8 mM (Fig. 2A), but no change was observed in the [Na+]i when the glucose concentration was changed in the same manner (Fig. 3A). Moreover, there was no significant difference between the insulin secretion from islet cells induced by 8 mM glucose alone in media containing 135 vs. 13.5 mM Na+ (Fig. 1). These results indicate that physiological glucose concentration may not affect Na+ permeability in islet cells. An elevation of glucose concentration from 3 to 15 and 20 mM gradually decreases the [Na+]i (1, 38). These inhibitory effects of high glucose on [Na+]i are consistent with the report that total Na+ content of islets (measured by integrating flame photometry) is lowered by 20 mM glucose (52). The lack of a rise in [Na+]i in the presence of high glucose can be explained by the facilitation of Na+ extrusion that the sugar produces (1). However, high glucose increases the rate of ouabain-dependent rise of [Na+]i, indicating that glucose promotes not only the entry but also the extrusion of Na+ in pancreatic {beta}-cells (51).

It has been proposed that GLP-1 and cAMP-elevating agents activate an inward Na+ current through nonselective cation channels, thereby depolarizing the cell and increasing the [Ca2+]i in rat {beta}-cells and insulinoma cells (27, 36). TTX, a blocker of the voltage-dependent Na+ channel, did not suppress the rise in the [Na+]i (data not shown) and the insulin secretion (Fig. 4) induced by GLP-1 and glucose. These results indicate that the effects of GLP-1 seem to be mediated by activation of a Na+ channel other than the voltage-dependent Na+ channel in the islet {beta}-cells.

Somatostatin inhibited the GLP-1-induced increase in [Na+]i (data not shown) and insulin secretion (Fig. 4). This action is likely to be mediated through two mechanisms. One is the blockade of the Na+ channel activated by GLP-1 as in the case where somatostatin blocks the growth hormone-releasing hormone-induced Na+ current on somatotrophs (35). The other is inhibition of adenylate cyclase and subsequent decrease in cAMP formation (28).

We examined the two additional possibilities to cause the rise in [Na+]i and found that neither is involved. Na+-K+-ATPase is responsible for the active transport of Na+ and K+ across the cell membrane. The inhibition of Na+-K+-ATPase may not be involved, because GLP-1 augmented the [Na+]i and the insulin secretion in the presence of 100 µM ouabain (Fig. 4), an inhibitor of Na+-K+-ATPase (15, 17, 41). To check the possibility of incomplete inhibition of Na+-K+-ATPase by 100 µM ouabain, the effect of 1 mM ouabain was examined, which showed a continuous increase of [Na+]i without reaching a stable level (data not shown). It was difficult to observe the effects of GLP-1 in the presence of 1 mM ouabain. Store-operated channels may not be involved, because thapsigargin (40), a blocker of SERCA, did not suppress the increase in [Na+]i induced by GLP-1, as shown in the action of ACh on mouse {beta}-cells (38).

Thus it is likely that GLP-1 augments the membrane Na+ permeability via cAMP formation. To further confirm an involvement of cAMP formation in the action of GLP-1, we examined the effects of SQ-22536, an inhibitor of adenylate cyclase (57). SQ-22536 strongly inhibited the increase in cAMP content (Fig. 5), the [Na+]i and insulin secretion induced by either GLP-1 or forskolin. The next question is how cAMP augments Na+ permeability. Involvement of PKA is likely because the response was mildly inhibited by H-89, an inhibitor of PKA. Similar results are reported in pituitary somatotrophs (32). cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac) (14). Recently, it was reported that the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP induces Ca2+-induced Ca2+ release and exocytosis in pancreatic {beta}-cells (8, 29). In the presence of glucose, 8-pCPT-2'-O-Me-cAMP increased the insulin secretion and the [Na+]i without H-89 (Figs. 7 and 8). Moreover, the insulin secretion by 8-pCPT-2'-O-Me-cAMP was reduced in low extracellular Na+ (Fig. 8), indicating that the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP increases Na+ permeability though a PKA-independent mechanism.

We conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.


    ACKNOWLEDGMENTS
 
We are obliged to Dr. M. Kato (Department of Physiology 1, Nippon Medical School, Tokyo, Japan) and Drs. S. Matsuzaki, M. Ikeda, and K. Kimura at our university for much helpful advice. We also thank T. Kawanobe for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Miura, Dept. of Hygiene, Dokkyo Univ. School of Medicine, 880 Mibu, Tochigi 321-0293, Japan (E-mail: y-miura{at}dokkyomed.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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