JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 JPETThis Fast article Forward. has not been Published copyedited and on formatted. April 4, The 2007 final asversion DOI:10.1124/jpet.107.120592 may differ from this version.

JPET #120592 1

Nateglinide and mitiglinide, but not , induce secretion through a mechanism mediated by calcium

release from endoplasmic reticulum Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

Makoto Shigeto, Masashi Katsura, Masafumi Matsuda, Seitaro Ohkuma, Kohei Kaku*

Division of Diabetes and Endocrinology, Department of Medicine, Kawasaki Medical School (M.S, M.M, K.K)

Department of Pharmacology, Kawasaki Medical School (M.K, S.O)

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 2

Running title: Glinides Induce Insulin Secretion via Ca2+ Released from ER

* Corresponding Author: Kohei Kaku, M.D.

Division of Diabetes and Endocrinology, Department of Medicine, Kawasaki Medical School

577 Matsushima, Kurashiki City, Okayama 701-0192, Japan Downloaded from

Tel: +81-86-462-1111

FAX:+81-86-462-1199 jpet.aspetjournals.org E:mail: [email protected]

Number of text pages: 23 at ASPET Journals on October 2, 2021 Number of tables: 0

Number of figures: 8

Number of reference: 30

Word count in the abstract: 235 words

Word count in the introduction: 477 words

Word count in the discussion: 842 words

Abbreviations: ATP, adenosine triphosphate; EGTA; ethyleneglycol-bis(β-aminoethylether)-N,N,N´,N´ –tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; ER, Endoplasmic reticulum; KATP channel, ATP sensitive ; KRBH, Krebs-Ringer bicarbonate HEPES buffer; LDH, Lactate dehydrogenase; SUR, receptor; JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 3

Recommended section assignment: Endocrine and Diabetes Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 4

ABSTRACT

Nateglinide and mitiglinide (glinides) are characterized as rapid-onset and short-acting insulinotropic agents. Although both compounds do not have a sulfonylurea structure, it has been postulated that insulin secretion is preceded by their binding to Kir6.2/SUR1 complex, KATP channels, and a mechanism of insulin secretion of glinides has been accounted for by this pathway. However, we hypothesized the involvement of additional mechanisms of insulin Downloaded from secretion enhanced by glinides, and we analyzed the pattern of time course of insulin secretion from MIN6 cells with existence of agents that have specific pharmacologic actions. Dose jpet.aspetjournals.org dependent effects of , , nateglinide, and mitiglinide were observed.

Insulin secretion induced by 3 µM tolbutamide and 1 nM glibenclamide was completely inhibited by 10 µM and 3 µM . While the latter half of insulin secretion induced by 3 at ASPET Journals on October 2, 2021 µM nateglinide or 30 nM mitiglinide remained with the existence of those agents. Glinides enhanced insulin secretion even in Ca2+ depleted medium, and its pattern of secretion was same as the pattern with existence of verapamil. The latter half was suppressed by 1 µM dantrolene, and concomitant addition of verapamil and dantrolene completely suppressed the entire pattern of insulin secretion enhanced by nateglinide. Thus, we conclude that glinide action is demonstrated through two pathways; dependently and independently from the pathway through

KATP channels. We also demonstrated that the latter pathway involves the intracellular calcium release from endoplasmic reticulum via ryanodine receptor activation. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 5

INTRODUCTION

A decrease in the initial insulin secretion observed in an early stage of induces postprandial hyperglycemia. A large-scale clinical trial has demonstrated that postprandial hyperglycemia is closely related to complications of diabetes mellitus, particularly cardiovascular disorders affecting survival, and that the correction of postprandial hyperglycemia is important for the prevention of vascular events (DECODE study group, 2001). Downloaded from

Sulfonylureas, which are widely used for the treatment of diabetes mellitus, are considered to bind to Kir6.2/SUR1 present in pancreatic β cells and promote insulin secretion by jpet.aspetjournals.org 2+ closing the KATP channel and opening the voltage-dependent Ca channel (Doyle and Egan,

2003). However, sulfonylureas as the long acting insulin secretagogues, are likely to cause hypoglycemia and body weight gain, and are insufficient to control postprandial hyperglycemia at ASPET Journals on October 2, 2021 (Shapiro et al., 1989; UKPDS Group, 1998). Also, problems including secondary failure have been suggested (Salas and Caro, 2002).

Rapid-onset and short-acting insulinotropic agents (glinides), typically nateglinide and mitiglinide, are often used in an early stage of diabetes mellitus, because they alleviate postprandial hyperglycemia by quickly increasing insulin secretion after a meal and less frequently induce hypoglycemia than sulfonylureas (Sato et al., 1991). The insulin secretion-stimulating action of glinides is considered to be derived from a series of reactions triggered by their binding with Kir6.2/SUR1, similarly to sulfonylurea (Akiyoshi et al., 1995).

Clinical effects of glinides, whose first site of action is considered to be the same as that of sulfonylureas, have been reported to appear significantly earlier and to disappear more rapidly than those of SUs (Hollander et al., 2001). These differences cannot be explained by the JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 6 absorption rate (Ikenoue et al., 1997) or binding characteristics with SUR alone (Proks et al.,

2002; Hu et al., 2003).

The presence of pathways dependent/not dependent on the KATP channel has been clarified in the mechanism of glucose-stimulated insulin secretion (GSIS), and the results of electrophysiologic studies and studies of Ca2+ influx suggest that the first phase is dependent on the KATP channel, whereas the second phase, mediated by glucose metabolites, is not (Straub and

Sharp, 2002; Rorsman and Renstrom, 2003). However, the first phase of GSIS decreases but Downloaded from does not disappear in SUR1-defective patients, SUR1-knockout mice, and cells in culture

(Seghers et al., 2000; Shiota et al., 2002; Shigeto et al., 2006), and is considered to be caused by a jpet.aspetjournals.org complicated insulin secretion mechanism mediated by metabolism.

In this study, we noted the differences in the characteristics of insulin secretion-promoting actions between sulfonylureas and glinides, and evaluated the mechanism of at ASPET Journals on October 2, 2021 insulin secretion at the cellular level using MIN6 cells, which have the same insulin secretion mechanism as isolated pancreatic β cells (Miyazaki et al., 1990; Ishihara et al., 1993). As a result, we demonstrated the presence of a pathway mediated by Ca2+ release from endoplasmic reticulum, which is not dependent on the KATP channel, in the mechanism of glinide-induced insulin secretion. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 7

MATERIALS AND METHODS

Reagents - Nateglinide and mitiglinide were generously provided from Ajinomoto Corp. (Tokyo,

Japan) and Kissei Pharmaceutical Corp. (Nagano, Japan). Tolbutamide and glibenclamide were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Mouse insulin enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shibayagi (Gunma, Japan). Lactate dehydrogenase (LDH) cytotoxicity detection kit was purchased from Takara (Otsu, Japan). Downloaded from

Dantrolene was purchased from Tocris Cookson Ltd. (Northpoint, United Kingdom). Caffeine, dibucaine and verapamil were from Nakalai Tesque Inc. (Kyoto, Japan). Diazoxide and ryanodine jpet.aspetjournals.org were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Other chemicals used were locally available and of analytic grade. at ASPET Journals on October 2, 2021 MIN6 cell culture and insulin secretion analysis - MIN6 cells were kindly gifted from Dr.

Junichi Miyazaki at Osaka University (Miyazaki et al., 1990). The cells (passages 20 – 30) were cultured with modified DMEM (containing 15% FBS, 5 µl/l 2-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin, 3.3 g/l NaHCO3, 25 mM glucose) in tissue culture flasks under humidified atmosphere at 37 °C with 5% CO2 / 95% Air. The culture medium was removed and replaced with fresh media every 24 h and the cells were used for experiments at

80% confluent.

For determination of insulin secretion, MIN6 cells were cultured in 35 mm diameter dish with

2 ml of the medium for 72 h, and then preincubated for 30 min in 1 ml of 3 mM glucose KRBH.

The cells were pretreated with diazoxide, verapamil, dantrolene, and for 60 s before glucose stimulation. Following the pretreatment, the buffer was exchanged for a medium containing 10 mM glucose KRBH with or without agents. The buffer was quickly collected by JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 8 aspiration every 30 s with addition of the fresh buffer. After the buffer collection, insulin left in the sample tube was confirmed to be below the detection sensitivity of insulin measurement. The same protocol was used for testing the effects of the sulfonylureas. A Ca2+ free KRBH (135 mM NaCl,

3.6 mM KCl, 2 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.0 mM EGTA and 10 mM

HEPES, 3.0 mM glucose; pH 7.4, 95% O2/5% CO2 saturated) was used in the experiments assessing the influence of the extracellular calcium. In this experiment, the Ca2+ free KRBH was added 60 s before glucose stimulation. Insulin content was measured by mouse insulin ELISA kit. Downloaded from

Measurement of [45Ca2+] influx into the MIN6 cells - [45Ca2+] influx into the MIN6 cells were jpet.aspetjournals.org measured by the method previously reported . MIN6 cells were preincubated for 30 min in 3 mM glucose KRBH buffer. Following the incubation with 3 mM glucose Ca2+-free KRBH buffer at

37 °C for 10 min, the buffer was exchanged with fresh and warm (37 °C) 3 mM glucose at ASPET Journals on October 2, 2021 2+ 45 2+ Ca -free KRBH buffer. The reaction was initiated by the addition of 2.7 mM [ Ca ]Cl2 (3.7

45 2+ MBq [ Ca ]Cl2 per dish) with or without 3 µM nateglinide. The cells were incubated at 37 °C for 5 min. The rate of [45Ca2+] uptake into the MIN6 cells was measured at 30 seconds interval as the following procedure. The reaction was terminated by rapid aspiration of the buffer containing

45 2+ [ Ca ]Cl2 followed by five times washings of the cells with ice-cold KRBH buffer and then the cells were scraped off with 0.5 N NaOH. The procedure to stop the reaction was carried out within 40 s. An aliquot of alkaline-digested cells was neutralized with equimolar acetic acid and was subjected to measure the radioactivity accumulated in the cells with scintillation cocktail

BioFluor (DuPont, Boston, MA, USA) by liquid scintillation spectrometry. The content of protein in the alkaline-digested MIN6 cells was measured by the method of Lowry using bovine serum albumin as standard. The rate of calcium influx per each period (30seconds) was calculated by the following equation: [calcium influx] = actual radioactivity/ mg protein – JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 9 previous radioactivity/ mg protein.

Immunohistochemical study of ryanodine receptor – Immunohistochemical study was carried out according to the method previously reported with a minor modification (Katsura et al., 2004).

The cells were fixed with 0.1 M phosphate buffer saline (PBS; pH 7.4) containing 4% paraformaldehyde, 0.2% picric acid and 0.3% glutaraldehyde (4 °C, 10 min), and then fixed again with 4% paraformaldehyde and 0.2% picric acid dissolved in 0.1 M PBS (4 °C, 30 min). Downloaded from

After washing five times with 15% sucrose-containing 0.1 M PBS, the cells were incubated with the same sucrose buffer (4 °C, 2 days), and 0.3% Triton X-100-containing 0.1 M PBS (4 °C, 4 jpet.aspetjournals.org days). Immunostaining was performed by using ABC kit (Vector, CA, USA) with 1st antibody and Anti-ryanodine receptor 1,2 and 3 antibodies (Chemicon, CA, U.S.A.) were diluted with

0.01% Tween 20 containing PBS (1/200). at ASPET Journals on October 2, 2021

Cytotoxicity study – The LDH in the perfusate was measured by LDH cytotoxicity detection kit in all experiments using reagents to assess the cell damage.

Statistical analysis - Each value represents the means ± SD. The experimental number was described in each figure legend, and each experiment was done in quaternion. The statistical analysis was determined by the method described in each figure legend following the application of the one-way ANOVA. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 10

Results

Insulin secretion by sulfonylureas and glinides

We had already reported the dose dependent manner of tolbutamide and glibenclamide on insulin secretion from MIN6 cells (Shigeto et al., 2006). Dose dependent effects of nateglinide and miglitinide on insulin secretion from MIN6 cells were also confirmed inthis study (Figure 1).

The pattern of insulin secretion induced by mitiglinide was same as the pattern with existence of Downloaded from nateglinide except the concentration of mitiglinide was only 1/100 of nateglinide (Figure 2). Our previous report demonstrated that tobutamide-induced insulin secretion was initiated between 60 jpet.aspetjournals.org s and 90 s after addition of the drug with a peak at 120 s (Shigeto et al., 2006). On the other hand, insulin secretion induced by 3 µM nateglinide appeared earlier with a steep peak at 60 s, and observed untill approximately 300 s. Both 3 µM tolbutamide- and 1 nM glibenclamide-induced at ASPET Journals on October 2, 2021 insulin secretion from MIN6 cells were completely inhibited by addition of 10 µM diazoxide, a

KATP or 3 µM verapamil, a L-type voltage-dependent blocker.

However, the effects of diazoxide and verapamil on nateglinide-indueced insulin secretion were quite different from those on the effect of sulfonylurea drug. The earlier half component of nateglinide-induced insulin secretion was inhibited by addition of diazoxide and verapamil, but the latter half component was still remained (Figure 2).

Insulin secretion by glinides under Ca2+ depleted condition

Effects of nateglinide and mitiglinide on insulin secretion from MIN6 in the extracellular Ca2+ free condition were examined. Both 3 µM nateglinide and 30 nM mitiglinide stimulated insulin secretion with a peak at 150 s even in the medium condition with Ca2+ depletion by 1 mM EGTA, although the peak level was lower than the original one (Figure 3). Interestingly, the pattern of JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 11 insulin secretion in this condition was same as the pattern with the existence of 10 µM diazoxide or 3 µM verapamil as shown in Figure 2.

Tracing of Ca2+ ion after nateglinide stimulation

Effect of nateglinde on the intracellular Ca2+ influx was shown in Figure 4. Addition of 3 µM nateglinde evoked a rapid influx of Ca2+ ion into MIN6 cells with a significant peak between 60 and 90 s after an onset of nateglnide stimulation. The duration until the induction of Downloaded from intracellular Ca2+ influx by nateglinide coincided with that to appear a rapid peak of insulin secretion. We confirmed that 30 nM mitiglinide also induced a rapid influx of Ca2+ ion into jpet.aspetjournals.org MIN6 cells as well as nateglinide (data not shown).

Ca2+ handling at the level of endoplasmic reticulum and ryanodine receptor characterzation at ASPET Journals on October 2, 2021 Addition of 10 mM caffeine, which is known to be a ryanodine receptor agonist, induced insulin secretion from MIN6 cells with a peak at 240 s after an onset of stimulation (Figure 5A).

The latter half of nateglinide-induced insulin secretion was completely blocked by 1 µM dantrolene, a nonspecific blocker of calcium efflux from endoplasmic reticulum (Figure 5B). On the other hand, an addition of 5 mM caffeine augmented the latter half of insulin secretion with a peak at 150 s (Figure 5C). Nateglinide-stimulated insulin secretion was completely blocked by concomitant addition of 1 µM dantrolene and 3 µM verapamil (Figure 6). In the same condition, the effect of 30 nM mitiglinide on insulin secretion was also completely inhibited (data not shown).

Immunohistochemical analysis revealed that MIN6 cells possessed the ryanodine receptor typeው, 2 and 3, and also demonstrated the existence of cells which did not recognize the JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 12 antibodies against ryanodine receptor type 1, 2 and 3 (Figure 7). In order to confirm the role of ryanodine receptors on insulin secretion, we examined the effect of ryanodine on insulin secretion from MIN6 cells. As shown in Figure 8A, ryanodine stimulated insulin secretion from

MIN6 cells in a dose-dependent manner. Insulin secretion induced by 1nM ryanodine showed a peak at 120 s, and was not affected by addition of 3µM verapamil. On the other hand, addition of

1µM dantrolene completely inhibited insulin secretion induced by ryanodine (Figure 8B). Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 13

DISCUSSION

There have been a number of reports suggesting that the response of MIN6 cells to glucose and various is nearly identical to that of pancreatic islets (Miyazaki et al., 1990; Ishihara et al.,

1993; Shigeto et al., 2006). Therefore, the results obtained in this study are considered to be important for the clarification of in vitro responses.

While glinides have been considered to induce insulin secretion by binding with Downloaded from sulfonylurea receptors, the present study showed that insulin secretion induced by glinides appeared significantly earlier than that by sulfonylurea, suggesting different action mechanisms jpet.aspetjournals.org between two insulin secretagogues including binding character to sulfonylurea receptors. Our study first demonstrated the presence of a pathway other than the KATP channel-dependent pathway in the mechanism of induction of insulin secretion by glinides. Direct evidence of this at ASPET Journals on October 2, 2021 finding is that neither diazoxide nor verapamil, which are inhibitors of the KATP channels and voltage-dependent Ca2+ channels, completely inhibited glinide-induced insulin secretion. There may be an argument that the suppression of glinide action was insufficient because of the low concentrations of these inhibitors, but this possibility can be rejected for the following reasons: 1)

The inhibitors used in this study had been confirmed to completely inhibit the action of sulfonylureas at the same concentrations; 2) since responses similar to those observed with the addition of verapamil were obtained even without the presence of extracellular Ca2+, verapamil is considered to sufficiently inhibit the influx of extracellular Ca2+. Therefore, the concentrations of inhibitors are considered to have been appropriate, because they were sufficient to block their target pathway, and because no sign of toxicity was noted.

It is worthy to note that the optimal concentration of the drugs used in this study for stimulation of insulin secretion were lower than those used in many previous studies. We JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 14 determined the drug concentrations on the basis of in vitro results (Shigeto et al., 2006) after confirming that the drugs do not damage MIN6 cells at any of these concentrations. Moreover, the optimal concentration obtained in this study was close to the circulating drug concentrations at clinical doses (Ikenoue et al., 1997; Doyle and Egan, 2003). In addition, the insulin secretion patterns of MIN6 cells under stimulation with nateglinide and glibenclamide were similar to clinical changes in the blood insulin concentration (Hu et al., 2001). These observations suggest that the results obtained in this study reflect in vivo pharmacological actions and are extremely Downloaded from useful for the analysis of action mechanisms of various drugs.

In SUR-defective patients or SUR1- and Kir6.2-knockout mice, sulfonylureas failed to jpet.aspetjournals.org stimulate insulin secretion (Seghers et al., 2000; Grimberg et al., 2001), indicating that sulfonylureas act via a KATP channel-dependent pathway alone also in vivo. With glinides, on the other hand, the latter half of insulin secretion (component 2), but not the early half at ASPET Journals on October 2, 2021 (component 1), persisted even in the absence of extracellular Ca2+, strongly suggesting the existence of KATP channel-independent pathway.

An increase in the intracellular Ca2+ concentration is generally considered necessary for insulin secretion from pancreatic β cells (Rorsman and Renstrom, 2003), and we evaluated the possibility that component 2 is induced by an increase in the intracellular Ca2+ concentration due to Ca2+ release from endoplasmic reticulum. As a result, component 2 was affected by addition of dantrolene and caffeine, so that we concluded that component 2 is due to the Ca2+ release from endoplasmic reticulum via ryanodine receptors. Experimental results concerning the Ca2+ influx also supported this conclusion. Dantrolene suppressed GSIS in one report (O'Mara et al., 1995) but did not affect GSIS in another (Johnson et al., 2004). However, both reports were based on an evaluation at high concentrations, and whether they reflect physiologic phenomena is JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 15 questionable.

Ryanodine was known to stimulate insulin secretion associated with an increase in intracellular Ca2+ concentration (Johnson et al., 2004). We also confirmed that ryanodine stimulated insulin secretion from MIN6 cells dose-dependently, and the effect of ryanodine was completely inhibited by addition of dantrolene, but not by verapamil. Dantrolene has been used as a ryanodine receptor blocker in several organ tissues such as skeletal muscle cells, cardiac muscle cells, and nerve cells (Brillantes et al., 1994; Parness and Palnitkar, 1995; Lee et al., 2005; Downloaded from

Zhang and Bourque, 2006). On the other hand, the existence of ryanodine receptor type 1 and 2 has been demonstrated in pancreatic β cells (Johnson et al., 2004), and we also confirmed a jpet.aspetjournals.org presence of the receptor type 1, 2 and 3 in MIN6 cells. In this study, we demonstrated that glinide-induced insulin secretion was partly inhibited by dantrolene. Thus, it is obvious that action mechanism of glinides on insulin secretion from MIN6 cells involves a pathway through at ASPET Journals on October 2, 2021 the ryanodine receptors. Okamoto et al. demonstrated that ryanodine receptors and cADP ribose play an important role in GSIS under physiological conditions (Okamoto and Takasawa, 2002), and this finding is considered to be useful for explaining GSIS, in which glucose metabolites are considered to be involved in a complicated manner. In our evaluation, also, significant suppression was observed with low-concentration dantrolene, being in agreement with this observation.

A decrease in the ryanodine receptor function has been demonstrated in multiple diabetic animal models and patients with type 2 diabetes (Islam, 2002; Patti et al., 2003). Thus glinides, which stimulate the ryanodine receptor function, may be considered to enhance insulin secretion by a more physiologic mechanism than sulfonylureas. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 16

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FOOTNOTES

This study has been approved by the Animal Use Committee of the Kawasaki Medical School

(no. 03-093), and was conducted in compliance with the Animal Use Guidelines of the Kawasaki

Medical School. This work is supported by a Grant-in-Aid for Scientific Research (principal investigator K. Kaku; no 18591008) and Research Project Grants (principal investigator K. Kaku; no 18-501). Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 22

LEGENDS FOR FIGURES

Figure 1

Dose dependency of nateglinide (A) and mitiglinide (B) on insulin secretion from MIN6 cells.

Each value represents the mean ± S.D. of four separate experiments. *P<0.05 vs. control

(Dunnett's test). Downloaded from

Figure 2

Effects of 10 µM diazoxide and 3 µM verapamil on insulin secretion from MIN6 cells stimulated jpet.aspetjournals.org by 3 µM nateglinide (A) and 1 nM mitiglinide (B). Each value represents the mean ± S.D. of four separate experiments. at ASPET Journals on October 2, 2021 Figure 3

Time course of insulin secretion from MIN6 cells stimulated by 3 µM nateglinide (A) and 30 nM mitiglinide (B) under the extracellular Ca2+-free condition. Each value represents the mean ± S.D. of four separate experiments.

Figure 4

Effect of nateglinide on the rate of [45Ca2+] influx into the MIN6 cells. Each value represents the mean ± S.D. of four separate experiments.

Figure 5

A. Time course of insulin secretion from MIN6 cells stimulated by 10 mM caffeine and the effect of 1 µM dantrolene on caffeine-stimulated insulin secretion. B. Effect of 1 µM dantrolene on JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #120592 23 insulin secretion from MIN6 cells stimulated by nateglinide. C. Effect of 5 mM caffeine on insulin secretion from MIN6 cells stimulated by nateglinide. Each value represents the mean ±

S.D. of four separate experiments.

Figure 6

Effect of concomitant addition of 3 µM verapamil and 1 µM dantrolene on insulin secretion from

MIN6 cells stimulated by nateglinide. Each value represents the mean ± S.D. of four separate Downloaded from experiments. jpet.aspetjournals.org Figure 7

Immunohistochemical study to demonstrate the presence of ryanodine receptor (RyR) type 1, 2 and 3 in MIN6 cells. A. control B. ryanodine receptor type 1 C. ryanodine receptor type 2 D. at ASPET Journals on October 2, 2021 ryanodine receptor type 3 E. ryanodine receptor type 1, 2 and 3.

Figure 8

A. Dose dependency of ryanodine on insulin secretion from MIN6 cells. Each value represents the mean ± S.D. of four separate experiments. *P<0.05 vs. control (Dunnett's test). B. Time course of insulin secretion from MIN6 cells stimulated by 1 nM ryanodine and effects of 3 µM verapamil and 1 µM dantrolene on ryanodine-stimulated insulin secretion. Each value represents the mean ± S.D. of four separate experiments. JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021 JPET Fast Forward. Published on April 4, 2007 as DOI: 10.1124/jpet.107.120592 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021