Introduction

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Type 2 diabetes is characteristically associated with impaired insulin secretion and/or insulin resistance (O'meara and Polonsky, 1994; Porte and Kahn, 1995). Sulfonylureas (SUs), such as tolbutamide, glibenclamide (glyburide), and glipizide have been widely used to treat patients with type 2 diabetes. They inhibit the -cell ATP-sensitive K⁺ (K_ATP) channel, a complex of the SU receptor and an inward rectifier K⁺ channel (Inagaki et al., 1995), which leads to stimulation of insulin release and consequent reduction of blood glucose (Gylfe et al., 1984; Panten et al., 1992). However, SUs also have disadvantages: profound hypoglycemia resulting from a long-lasting and glucose-independent action to stimulate insulin release (Henquin, 1990), and the secondary failure to treatment with SUs (Groop et al., 1986), which may be due to the -cell exhaustion resulting from prolonged stimulation (Greco et al., 1992). To minimize these disadvantages, non-SU oral hypoglycemic agents, including nateglinide (Fujitani and Yada, 1994; Hirose et al., 1994; Akiyoshi et al., 1995), KAD-1229 (Ohnota et al., 1994), and repaglinide (Gromada et al., 1995; Ladriere et al., 1997), members of the meglitinide family (Malaisse, 1995), have recently been developed. Their advantages are rapid and shorter lasting insulinotropic and hypoglycemic effects. These compounds, however, act on -cells via similar mechanisms as SUs and potentially have an ability to stimulate insulin release at hypoglycemic states (Fujitani and Yada, 1994; Hirose et al., 1994; Ohnota et al., 1994; Akiyoshi et al., 1995; Gromada et al., 1995; Malaisse, 1995; Ladriere et al., 1997). Accordingly, a novel compound that activates -cells to release insulin only at hyperglycemic states has been long awaited.

A new non-SU oral hypoglycemic agent, trans-4-(4-methylcyclohexyl)-4-oxobutyric acid (JTT-608), when administered before glucose loading, potentiates insulin release and improves glucose tolerance without causing a decrease in fasting glucose levels in normal rats, Goto-Kakizaki, and neonatally streptozotocin-treated type 2 diabetic rats (Shinkai et al., 1998; Ohta et al., 1999a,b). JTT-608 enhances insulin secretion only with glucose loading, whereas SUs enhance it irrespective of glucose levels (Ohta et al., 1999a,b). In isolated perfused pancreas from normal, neonatally streptozotocin-treated, and Goto-Kakizaki rats, JTT-608 enhances glucose-stimulated, but not basal, insulin secretion (Shinkai et al., 1998; Ohta et al., 1999a,b). In a mouse insulinoma cell line, MIN6 cells, JTT-608 potently enhances insulin secretion at elevated glucose levels without inhibiting the binding of [³H]glibenclamide to membrane fractions (Furukawa et al., 1999), suggesting that JTT-608 enhances glucose-stimulated insulin secretion via a different mechanism from SUs. These studies on experimental animals suggest a possible beneficial role of JTT-608 in achievement of an improved glycemic control in patients with type 2 diabetes.

The glucose-dependent stimulation of insulin release by JTT-608 appears to account for its ability to correct hyperglycemia without inducing hypoglycemia. However, the mechanisms underlying the glucose-dependent action of JTT-608 are yet largely unknown. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) regulates insulin release in -cells (Wollheim and Sharp, 1981; Ammala et al., 1993). The first aim of the present study was to examine whether JTT-608 increases [Ca²⁺]_i in -cells and, if so, whether this process is tightly regulated by glucose and linked to insulin release. Furthermore, possible involvement of cAMP-PKA pathway in the production of [Ca²⁺]_i signals was investigated because this pathway has well been recognized as the potentiator of glucose-induced insulin release.

Oscillations of [Ca²⁺]_i in islet -cells have been suggested to be involved in the pulsatile insulin secretion, which contributes to the physiological control of glucose metabolism (Matthews et al., 1983; O'rahilly et al., 1988; Hellman et al., 1992; Gilon et al., 1993; O'meara and Polonsky, 1994; Bergsten, 1995). Heterogeneity of pancreatic -cells, with respect to electrical, metabolic, [Ca²⁺]_i, and secretory responses to glucose, has been observed both in vitro and in vivo, and the existence of glucose-unresponsive -cells has been evidenced (Pipeleers, 1987; Giordano et al., 1991; Holz et al., 1993; Yada et al., 1997). In type 2 diabetic animals, an increased fraction of glucose-unresponsive -cells is suggested to partly account for the impaired glucose-induced insulin release (Tsuura et al., 1993; O'meara and Polonsky, 1994). Islet -cells in vivo are perfused with physiological potentiators of insulin release, which include the intestinal peptide GLP-1(7-36)amide (Yada et al., 1993; Holz et al., 1995; Wang et al., 1995; Nauck, 1998), the pancreatic peptide PACAP38 (Yada et al., 1994, 1997; Filipsson et al., 1999), and the parasympathetic neurotransmitter ACh (Bergman and Miller, 1973; Garcia et al., 1988; Yada et al., 1995). The second aim of the present study was to examine whether JTT-608 could evoke [Ca²⁺]_i oscillations, recruit glucose-unresponsive -cells into [Ca²⁺]_i increases, and cooperate with physiological neurohormones.

	Materials and Methods

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Preparation of Islets and Single -Cells. Islets and single -cells were prepared as previously reported (Yada et al., 1992, 1994, 1995). Briefly, islets of Langerhans were isolated from Wistar rats aged 10 to 16 weeks by collagenase digestion. Animals were anesthetized with an intraperitoneal injection of pentobarbitone at 80 mg/kg. The abdomen was opened, and collagenase (3 mg/ml) dissolved in 5 mM Ca²⁺-containing Krebs-Ringer-bicarbonate buffer (KRB) was injected into the common bile duct at the distal end after ligation of the duct proximal to the pancreas. The rats were killed by cervical dislocation. The pancreas was dissected out and incubated at 37°C for 17 min. Islets were hand-collected.

Measurements of Insulin Release from Islets. Measurements of insulin release were carried out as previously described (Yada et al., 1994, 1997). Briefly, groups of nine isolated islets were first incubated for 30 min in KRB containing 2.8 mM glucose for stabilization. Islets were then incubated at 37°C for 30 min in 1 ml of KRB. Insulin concentration was determined by enzyme immunoassay using a kit (Morinaga, Yokohama, Japan).

Measurements of [Ca²⁺]_i in Single -Cells. [Ca²⁺]_i was measured by dual-wavelength fura-2 microfluorometry combined with digital imaging as previously reported (Yada et al., 1992, 1994). Briefly, cells on coverslips were loaded with fura-2 by incubation with 2 µM fura-2 acetoxymethylester in KRB containing 2.8 mM glucose for 30 min at 37°C. The cells were then mounted in a chamber and superfused with KRB at a rate of 1 ml/min at 37°C. The cells were excited at 340 and 380 nm alternately every 2.5 s, emission signals at 510 nm were detected with an intensified charge-coupled device camera, and ratio images were produced by an Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan). Ratio values were converted to [Ca²⁺]_i according to calibration curves (Yada et al., 1992).

Measurements of PKA Activity in Single -Cells. PKA activity was measured using the newly developed fluorescence probe DR II according to the original report (Higashi et al., 1997) with slight modification. DR II is a fluorescence PKA substrate obtained by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II that contains a specific autophosphorylation site. Its fluorescence intensity was shown to decrease in response to a rise in intracellular cAMP concentration (Higashi et al., 1997). The single islet cells were incubated with 25 µg/ml DR II in KRB for 120 min at 23°C. The cells were superfused under the same condition as that for [Ca²⁺]_i measurements, excited at 380 nm every 5 s, the emission signal at 475 nm was detected with a cooled charge-coupled device camera, and digital images were produced by a HiSCA system (Hamamatsu Photonics).

Solutions and Chemicals. KRB was composed of 121.7 mM NaCl, 4.4 mM KCl, 1.2 mM KH₂PO₄, 2.0 mM CaCl₂, 1.2 mM MgSO₄, 5.0 mM NaHCO₃, and 10 mM HEPES at pH 7.4 with NaOH supplemented with 0.1% bovine serum albumin.

Statistical Analysis. The calculated values were expressed as the mean ± S.E.M. (n = number of cells). The statistical analysis was carried out with the Student's t test. Differences were considered statistically significant when P < 0.05.

	Results

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Concentration- and Glucose-Dependent Effects of JTT-608 to Increase [Ca²⁺]_i in Single -Cells. A rise in glucose concentrations from 2.8 to 8.3 mM increased [Ca²⁺]_i in a biphasic manner: an initial large increase followed by a moderate elevation that was occasionally superimposed with an oscillation (Fig. 1A). In the presence of 8.3 mM glucose, administration of JTT-608 at 300 µM, but not 30 µM, in the superfusion solution increased [Ca²⁺]_i in a rat pancreatic -cell (Fig. 1A), thus showing a concentration-dependent effect. JTT-608 at 300 µM increased [Ca²⁺]_i in the presence of 8.3 mM but not 2.8 mM glucose (Fig. 1B). The mean amplitude of the [Ca²⁺]_i response to 300 µM JTT-608 at 8.3 mM glucose was 390 ± 15 nM (n = 31). The drug at a suprapharmacological concentration of 1000 µM induced a small increase in [Ca²⁺]_i [amplitude 63 ± 15 nM (n = 3)] only in 10% (3 of 31) of cells at 2.8 mM glucose, whereas at 8.3 mM glucose it induced a much larger increase in [Ca²⁺]_i [467 ± 16 nM (n = 15)] in 88% (15 of 17) of cells (Fig. 1C). The fraction of -cells with [Ca²⁺]_i responses to JTT-608 is expressed as percentage (Fig. 2, A and B): at 8.3 and 16.7 mM glucose JTT-608 (30-1000 µM) increased [Ca²⁺]_i in a concentration-dependent manner, whereas at 2.8 mM glucose only 1000 µM JTT-608 exerted a significant effect. In sharp contrast, a sulfonylurea tolbutamide at 30 µM increased [Ca²⁺]_i in 30% (3 of 10) of single -cells at 2.8 mM glucose and in 55% (5 of 11) of cells in 8.3 mM glucose (Fig. 2B). Tolbutamide at 300 µM increased [Ca²⁺]_i in 94% (15 of 16) and 95% (19 of 20) of cells at 2.8 and 8.3 mM glucose, respectively (Fig. 2B). Thus, the effect of tolbutamide on [Ca²⁺]_i was only loosely related to the glucose concentration, whereas that of JTT-608 was tightly glucose-dependent.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Concentration- and glucose-dependent effects of JTT-608 (JTT) to increase [Ca2+]i in rat single -cells. A, in the presence of 8.3 mM glucose, JTT concentration dependently increased [Ca2+]i in a single -cell that also responded to 8.3 mM glucose and 300 µM tolbutamide (Tolb). B, JTT at 300 µM increased [Ca2+]i in a single -cell in the presence of 8.3 mM (G8.3), but not 2.8 mM, glucose. C, JTT at high concentration of 1000 µM increased [Ca2+]i in both 2.8 and 8.3 mM glucose, and the amplitude of the [Ca2+]i increase was much smaller in 2.8 mM glucose. Among 17 cells examined, three cells responded to 1000 µM JTT at both 2.8 and 8.3 mM glucose, whereas 14 cells responded to JTT only at 8.3 mM glucose. [Ca2+]i was measured under superfusion conditions. The results shown are representative of 10 cells from three independent experiments in A, 59 from five in B, and three from two in C.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Percentage of -cells that responded to JTT and Tolb with increases in [Ca2+]i is expressed as a function of the concentration of JTT in the presence of 2.8, 8.3, and 16.7 mM glucose (A) and as a function of glucose concentration with 30 to 1000 µM JTT (B). Numbers near each point indicate the number of cells responded over that examined.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Small changes in glucose concentration around 5 mM turn on and off the effect of JTT to increase [Ca2+]i. A, at 4.2 mM glucose JTT failed to significantly increase [Ca2+]i, whereas it increased [Ca2+]i once glucose concentration was raised to 5.0 mM, as well as 8.3 mM. The results shown are representative of five cells from two independent experiments. B, percentage of -cells that responded to 300 µM JTT with increases in [Ca2+]i is expressed as a function of glucose concentration. Numbers near each point indicate the number of cells responded over that examined.

Inhibition of JTT-608-Induced [Ca²⁺]_i Increase under a Ca²⁺-Free Condition and by a Ca²⁺ Channel Blocker. In a Ca²⁺-free condition achieved in KRB with 0.1 mM EGTA and no added Ca²⁺, 300 µM JTT-608 failed to increase [Ca²⁺]_i, and after bringing 1 mM Ca²⁺ back, the response to JTT-608 was observed (n = 7) (Fig. 4A). In the presence of 10 µM nitrendipine, a blocker of the L-type Ca²⁺ channel in -cells, the [Ca²⁺]_i response to JTT-608 was completely inhibited in a reversible manner (n = 13) (Fig. 4B).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inhibition of [Ca2+]i responses to 300 µM JTT under a Ca2+-free condition (A) and by 10 µM nitrendipine (B). The Ca2+-free condition was achieved in KRB with 0.1 mM EGTA and no added Ca2+. Recovery of response was obtained after restoring 2 mM Ca2+ and upon washing out nitrendipine. The cells subsequently responded to Tolb. Glucose concentration is 8.3 mM. The results shown are representative of seven cells from two independent experiments in A and 13 from two in B.

JTT-608 Activates PKA, a PKA Inhibitor Blocks the Effect of JTT-608, and a PKA Activator Mimics JTT-608 in Increasing [Ca²⁺]_i in Single -Cells. In the presence of 8.3 mM glucose, administration of an adenylate cyclase activator, forskolin (10 µM), decreased the DR II fluorescence in a single -cell (Fig. 5A). Administration of 300 µM JTT-608 also evoked a rapid decrease in the DR II fluorescence in a single -cell, in a manner similar to that induced by forskolin (Fig. 5B). The reduction of DR II fluorescence indicates an activation of PKA (Higashi et al., 1997).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Coupling of PKA pathway to Ca2+ signaling in response to JTT. A and B, administration of forskolin (10 µM) (A) and JTT (300 µM) (B) in the superfusion solution containing 8.3 mM glucose rapidly decreased the DR II fluorescence in single -cells. A decrease in fluorescence corresponds to an increase in PKA activity. Dotted lines indicate the extrapolated baseline. The results shown are representative of 28 cells from two independent experiments in A and 25 from two in B. C, in a -cell that had been pretreated for 120 min with H89 (60 µM), a specific PKA inhibitor, JTT, and db-cAMP failed to increase [Ca2+]i. The results shown are representative of 15 cells from two independent experiments.

JTT-608 Glucose Dependently Stimulates Insulin Release from Islets and Its Inhibition under a Ca²⁺-Free Condition. Insulin release from isolated islets under static incubation was stimulated by 8.3 mM glucose. The glucose-stimulated insulin release was further increased by 300 µM JTT-608 significantly (P < 0.01), whereas it had no effect on the basal insulin release at 2.8 mM glucose (Fig. 6). Next, to examine the role of extracellular Ca²⁺ under conditions that might not chelate intracellular Ca²⁺, we used a nominal Ca²⁺-free KRB made with no added Ca²⁺ but without EGTA. In this nominal Ca²⁺-free condition, the increase in insulin release by JTT-608 was significantly (P < 0.01) reduced (Fig. 6). Thus, JTT-608 increased insulin release in a glucose- and Ca²⁺-dependent manner.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Glucose-dependent effect of JTT to stimulate insulin release from rat islets under static incubation. In the presence of 8.3 mM, but not 2.8 mM, glucose, 300 µM JTT increased insulin release, and it was attenuated under the nominal Ca2+-free condition achieved in KRB with no added Ca2+. The results are expressed as the mean ± S.E.M. of five independent experiments.

JTT-608 Recruits Glucose-Unresponsive -Cells into [Ca²⁺]_i Responses. The effect of JTT-608 on glucose-unresponsive -cells was investigated. Stimulation with 8.3 mM glucose failed to increase [Ca²⁺] in a single -cell (Fig. 7A). This cell was subsequently confirmed to be the -cell by its positive staining with insulin antibody (Fig. 7B) and by the [Ca²⁺]_i response to 300 µM tolbutamide (Fig. 7A), thereby exhibiting the property of glucose-unresponsive -cells. Administration of JTT-608 recruited these glucose-unresponsive -cells into [Ca²⁺]_i increases in a concentration-dependent manner (Fig. 7, A and C), whereby 300 µM JTT-608 aroused [Ca²⁺]_i responses in as many as 50% of glucose-unresponsive -cells.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effects of JTT on [Ca2+]i in glucose-unresponsive -cells. A and B, JTT at 300 µM increased [Ca2+]i in a single -cell that had failed to respond to 8.3 mM glucose but responded to Tolb (A). This cell was subsequently stained positively with insulin antibody (B). The results shown are representative of 15 cells from three independent experiments. C, percentage of glucose-unresponsive -cells that responded to JTT with increases in [Ca2+]i is expressed as a function of the concentration of JTT. Glucose concentration is 8.3 mM. Numbers near each point indicate the number of the cells that responded to JTT over the glucose-unresponsive -cells examined.

JTT-608 Induces [Ca²⁺]_i Oscillations in -Cells. Whether JTT-608 could induce [Ca²⁺]_i oscillations in -cells was examined. In the present study, when [Ca²⁺]_i went up and down four times or more in a repetitive manner, it was considered as the [Ca²⁺]_i oscillation. In the presence of 8.3 and 5.6 mM glucose, administration of JTT-608 at 300 to 1000 µM induced [Ca²⁺]_i oscillations (Fig. 8, A and B). JTT-608 also provoked [Ca²⁺]_i oscillations in -cells that failed to respond to 8.3 mM glucose, the glucose-unresponsive -cells (data not shown). These [Ca²⁺]_i oscillations were characterized by a slow frequency component that was occasionally superimposed with a fast frequency component. JTT-608 induced [Ca²⁺]_i oscillations in a concentration-dependent manner (Fig. 8C).

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Induction of [Ca2+]i oscillations by JTT in -cells. A, JTT at 300 µM induced [Ca2+]i oscillations in a single -cell in the presence of 8.3 mM glucose. B, JTT at a higher concentration of 1000 µM induced [Ca2+]i oscillations in a single -cell in the presence of 5.6 mM glucose. C, percentage of -cells that exhibited induction or potentiation of [Ca2+]i oscillations in response to 300 µM JTT in the presence of 5.6 mM glucose. Numbers near each point indicate the number of -cells with induction or potentiation of [Ca2+]i oscillations over that examined. The results shown are representative of five cells from three independent experiments in A and 20 from three in B.

JTT-608 Acts in Concert with PACAP, GLP-1, and ACh to Increase [Ca²⁺]_i. In a -cell in which neither JTT-608 at relatively low concentration (100 µM) nor PACAP38 (1 nM) affected [Ca²⁺]_i, the combination of these two agents induced a large increase in [Ca²⁺]_i (Fig. 9A), and it occurred in 15 of 57 cells. Similar effects were observed for the combination of JTT-608 and GLP-1 (1 nM) in 5 of 22 cells (Fig. 9B) and for the combination of JTT-608 and ACh (100 nM) in 22 of 55 cells (Fig. 9C). The cooperative effect was observed not only in the glucose-responsive but also in the glucose-unresponsive -cells with GLP-1 (n = 4) (Fig. 9B), PACAP38 (n = 3), and ACh (n = 3) (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Cooperative effects of JTT and insulinotropic neurohormones. A, neither PACAP38 (109 M) nor JTT (100 µM) had an effect on [Ca2+]i, whereas these two agents in combination increased [Ca2+]i in a -cell that responded to 8.3 mM glucose and Tolb. B, neither GLP-1 (109 M) nor JTT (100 µM) had an effect on [Ca2+]i, but the combination of the two increased [Ca2+]i in a glucose-unresponsive -cell. C, neither ACh (107 M) nor JTT (100 µM) had an effect on [Ca2+]i, but the combination of the two increased [Ca2+]i in a -cell. The results shown are representative of 15 cells from two independent experiments in A, four from two in B, and 22 from two in C.

	Discussion

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JTT-608 increased [Ca²⁺]_i and stimulated insulin release in rat islet -cells in the presence of 8.3 mM, but not 2.8 mM, glucose. The increase in [Ca²⁺]_i and stimulation of insulin release in response to JTT-608 were inhibited under Ca²⁺-free conditions. These results strongly suggest that JTT-608-induced [Ca²⁺]_i increase is linked to insulin release. Further analysis revealed that the ability of JTT-608 to increase [Ca²⁺]_i is strictly glucose-dependent (Figs. 1-3): as judged by the fraction of -cells with [Ca²⁺]_i responses to 300 µM JTT-608, a nearly half-maximal effect of JTT-608 (29%) was obtained in the presence of 5.6 mM glucose, whereas in 4.2 mM glucose only a minor effect (5.4%) was observed (Fig. 3B). Thus, the threshold glucose concentration which turns on the ability of JTT-608 to increase [Ca²⁺]_i appears to exist around 5 mM, a value that corresponds to the normal blood glucose level. The results indicate that JTT-608 activates [Ca²⁺]_i signaling to a substantial extent only when the glucose level is above the normal, whereas the activation ceases when the glucose level is below the normal. These results indicate that the production of [Ca²⁺]_i signals is the process strictly regulated by glucose. This provides a mechanistic basis for the previous observation in rats that JTT-608 stimulates insulin release only at normoglycemic and hyperglycemic states and therefore does not induce hypoglycemia (Shinkai et al., 1998; Ohta et al., 1999a,b).

Our study demonstrates that JTT-608 activates PKA and a PKA inhibitor antagonizes the effect of JTT-608 on [Ca²⁺]_i (Fig. 5, B and C). Furthermore, an activator of cAMP-PKA pathway mimicked JTT-608: db-cAMP and JTT-608 both increased [Ca²⁺]_i in a manner that is dependent on glucose concentration and Ca²⁺ influx through L-type Ca²⁺ channels (Fig. 5, D and E). These results strongly suggest that the effect of JTT-608 on [Ca²⁺]_i is mediated, at least partly, by cAMP-PKA pathway.

The present study shows that a substantial population (5-25%) of insulin-containing -cells from normal rats is glucose-unresponsive (Fig. 7), thereby confirming previous reports of the heterogeneity of pancreatic -cells and existence of glucose-unresponsive -cells (Pipeleers, 1987; Giordano et al., 1991; Holz et al., 1993; Yada et al., 1997). We found that many (24-50%) of the glucose-unresponsive -cells are recruited into [Ca²⁺]_i increases by JTT-608. Notably, it occurred with JTT-608 at 100 to 300 µM, the concentration range that was used and shown to be effective in the perfused pancreas (200 µM) and in vivo experiments (50-500 µmol/kg) in rats (Shinkai et al., 1998; Ohta et al., 1999a,b). A larger number of glucose-unresponsive -cells observed in type 2 diabetic animals is suggested to partly account for the impaired glucose-induced insulin release characteristically associated with this disease (Tsuura et al., 1993; O'meara and Polonsky, 1994). Therefore, the ability of JTT-608 to arouse [Ca²⁺]_i responses in the glucose-unresponsive -cells may be a beneficial property for a potential therapeutic agent for type 2 diabetes.

The present study shows that JTT-608 induces [Ca²⁺]_i oscillations in a substantial fraction (26-40%) of -cells (Fig. 8C). [Ca²⁺]_i oscillations in islet -cells have been implicated in the pulsatile insulin secretion, which contributes to the physiological control of glucose metabolism (Matthews et al., 1983; O'rahilly et al., 1988; Hellman et al., 1992; Gilon et al., 1993; O'meara and Polonsky, 1994; Bergsten, 1995). It has recently been shown that glucose-induced oscillations in [Ca²⁺]_i in a -cell line, MIN6 cells, are linked to a long-lasting increase in mitochondrial Ca²⁺ (Nakazaki et al., 1998), a factor that is considered to be important in the maintenance of the -cell metabolism (Civelek et al., 1996; Kennedy et al., 1996; Nakazaki et al., 1998). Regulation of gene expression by [Ca²⁺]_i oscillations has also been demonstrated (Dolmetsch et al., 1998; Li et al., 1998). Therefore, it is likely that induction of [Ca²⁺]_i oscillations by JTT-608 is related to the enhanced metabolism of -cells, increased efficiency of insulin release, and, possibly, insulin biosynthesis.

An interesting finding of this study is that the Ca²⁺ signaling is elicited more effectively by a cooperative action of JTT-608 with physiological neurohormones GLP-1, PACAP38, and ACh, whose insulinotropic effects are also glucose-dependent (Fig. 9). The cooperative effect between JTT-608 and neurohormones is observed not only in glucose-responsive but also in glucose-unresponsive -cells. GLP-1 is an intestinal incretin hormone currently under investigation as a novel therapeutic agent in the treatment of type 2 diabetes (Nauck, 1998). PACAP38 has been demonstrated to be a pancreatic peptide that acts as an endogenous intraislet amplifier of glucose-induced insulin secretion (Yada et al., 1997; Filipsson et al., 1999). ACh is a neurotransmitter that innervates pancreatic islets. Therefore, the present finding suggests that the -cells in pancreatic islets perfused with these physiological neurohormones may be activated more effectively by JTT-608 than single -cells.

Conclusion. JTT-608 evokes protein kinase A-mediated increase in [Ca²⁺]_i in rat islet -cells in a strictly glucose-dependent manner. The [Ca²⁺]_i increase is linked to stimulation of insulin release. JTT-608 also recruits glucose-unresponsive -cells into [Ca²⁺]_i increases and induces oscillations of [Ca²⁺]_i, which may also partly contribute to the insulin release by JTT-608. JTT-608 may act in concert with physiological neurohormones GLP-1, PACAP, and ACh.