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Biochemical and Biophysical Research Communications 402 (2010) 280–285
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice Shigeru Yoshida ⇑, Takahide Ohishi, Tetsuo Matsui, Hirotsugu Tanaka, Hiroyuki Oshima, Yasuhiro Yonetoku, Masayuki Shibasaki Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
a r t i c l e
i n f o
Article history: Received 13 September 2010 Available online 16 October 2010 Keywords: GPR119 AS1535907 First-phase insulin secretion Isolated perfused pancreas Glibenclamide
a b s t r a c t G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic b-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC50 value of 4.8 lM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 lM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Depletion of glucose-stimulated insulin secretion (GSIS), particularly loss of the first phase, is a characteristic feature in the pathology of type 2 diabetes mellitus (T2DM) and results in postprandial hyperglycemia [1,2]. In the clinical management of T2DM patients, sulfonylurea derivatives (SUs) are the most widely used hypoglycemic agents [3]; however, SUs can cause severe and prolonged hypoglycemia because of their long duration and glucose-independent mode of action [4]. Recent strategies for promoting normoglycemia have focused on enhancing GSIS through the targeting of G protein-coupled receptors (GPCRs), such as the glucagon-like peptide 1 (GLP-1) receptor. Although GLP-1 analogs, such as exendin-4, have been shown to effectively stimulate GSIS
Abbreviations: GPR119, G protein-coupled receptor 119; GSIS, glucose-stimulated insulin secretion; T2DM, type 2 diabetes mellitus; SU, sulfonylurea; GPCR, G protein-coupled receptor; GLP-1, glucagon-like peptide-1; HEK, human embryonic kidney; LPC, lysophosphatidylcholine; OEA, oleoylethanol amide. ⇑ Corresponding author. Address: Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Fax: +81 29 852 2955. E-mail address: [email protected] (S. Yoshida). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.015
and preserve pancreatic b-cells, these analogs can cause gastrointestinal side effects, pancreatitis, and require parenteral administration [5–8]. The limitations of SUs and GLP-1 analags underly the need for the development of oral insulin secretagogues that are capable of inducing normoglycemia and preserving the first phase of GSIS in T2DM patients. GPR119 represents a promising anti-diabetic therapeutic target as it is predominantly expressed in pancreatic b-cells and intestinal L-cells and promotes GSIS and indirectly increases GLP-1 level. Although the downstream pathways are unclear, activation of GPR119 by endogenous ligands, such as lysophosphatidylcholine (LPC) and oleoylethanol amide (OEA), or small-molecule agonists, leads to the accumulation of intracellular cAMP and subsequent insulin and GLP-1 release [9–14]. We previously identified a novel structural class of small-molecule GPR119 agonists, consisting of 2,4,6-tri-substituted pyrimidine cores, which were orally active and displayed higher activity than existing T2DM therapeutics, such as extendin-4 [5,8]. The first identified compound, AS1269574, was capable of inducing GSIS in vitro and in vivo, and improved glucose tolerance in normal mice [15]. In an effort to identify an agonist in the identical structural class with a lower effective dosage and the ability to specifically induce the first phase of GSIS, compound
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AS1535907 (2-(4-bromophenyl)-6-methyl-N-[2-(1-oxidopyridin3-yl) ethyl] pyrimidin-4-amine), which was modified from AS1269574, was selected as a promising candidate. Here, we report the in vitro and in vivo characterization of the novel small-molecule GPR119 agonist AS1535907 in rat islets, isolated pancreata, and diabetic db/db mice. We specifically compared the effects of AS1535907 with the anti-diabetic SU glibenclamide on glucose tolerance, glucose levels, and insulin secretion in these animal systems.
lated pancreata were then perfused (flow rate, 2 ml/min) through the celiac artery with HEPES-balanced KRBB containing 2.8 or 16.8 mM glucose and 0.1% fatty acid-free BSA. The preparation was then placed in an acrylic chamber containing KRBB (37 °C) and the effluent perfusate from the portal vein cannula was collected at 1–2 min intervals in fraction tubes containing aprotinin (500 units/tube). The samples were stored at 20 °C until insulin concentrations were measured using an Insulin Radioimmunoassay Kit (Amersham Pharmacia Biotech).
2. Materials and methods
2.5. Oral glucose torelance test (OGTT) in diabetic db/db mice
2.1. Animals and materials
Six-week-old male db/db mice were fasted overnight and then orally administered 0.5% methyl-cellulose (vehicle, n = 7), or 10 mg/kg AS1535907 (n = 7), and 10 mg/kg glibenclamide (n = 7). After 30 min, glucose was given orally at a dose of 2 g/kg/10 ml, and blood samples were collected from tail veins after 0, 5, 10, 15, 30, 60, 120, and 180 min. Blood glucose levels was determined using the Glucose CII test (Wako, Osaka, Japan), while plasma insulin level was measured by radioimmunoassay (Amersham Biosciences), following the manufacturers’ instructions.
Male diabetic db/db mice and male Sprague–Dawley (SD) rats were obtained from CLEA Japan, Inc. (Kanagawa, Japan). All surgical procedures performed were approved by the Animal Ethical Committee of Astellas Pharma Inc. The compounds AS1535907 (2-(4-bromophenyl)-6-methyl-N-[2-(1-oxidopyridin-3-yl) ethyl] pyrimidin-4-amine) was synthesized in-house at Astellas Pharma Inc. (Ibaraki, Japan), and was modified from the 2,4,6-tri-substituted pyrimidine core agonist AS1269574 [15] Glibenclamide was obtained from Sigma (St. Louis, MO, USA). Prior to use in assays, AS1535907 and glibenclamide were dissolved in dimethyl sulfoxide (DMSO) to yield a stock solution of 10 mM. 2.2. Plasmid construction and generation of HEK293-hGPR119 cells The human GPR119 ORF (GenBank Accession No. BD169091) was amplified by PCR from human pancreas cDNA (Clontech, Tokyo, Japan) with the sense, 50 -AAAATCTAGAATGGAATCATCTTTCTC ATTTG-30 , and antisense, 50 -CGGCTCTAGATTAGCCATCAAACTCTG AGCTGG-30 , primers (XbaI restriction sites are underlined). The amplified product was subcloned into the mammalian expression vector plasmid pcDNA3.1 (Invitrogen, Tokyo, Japan). The resulting pcDNA-GPR119 plasmid and pCRE (cAMP responsive element)-Luc plasmid (Clontech) were cotransfected into human embryonic kidney (HEK) 293 cells using standard procedures. Candidate clones stably expressing human GPR119 and pCRE-Luc were selected by resistance to 400 lg/ml geneticin, and were confirmed by the activity of LPC, an endogenous ligand of GPR119. 2.3. Insulin secretion by islet cells Islets of Langerhans were isolated from male SD rats by collagenase digestion as described previously [16] and subsequently handpicked under a stereomicroscope. The isolated islets were then cultured overnight at 37 °C in RPMI-1640 medium (Invitrogen) supplemented with 11.1 mM glucose and 10% fetal bovine serum. After 2 days of culture, the ilets were washed with HEPES-balanced Krebs–Ringer bicarbonate buffer (KRBB) containing 2.8 mM glucose and 0.1% BSA, and preincubated for 60 min at 37 °C in the same medium. After preincuation, cells were stimulated with test compound in HEPES-balanced KRBB (pH 7.4) containing 16.8 mM glucose at 37 °C for 60 min. Insulin secreted into the supernatant was collected and measured using an Insulin Radioimmunoassay Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.4. Perfusion of isolated rat pancreata The pancreata of male SD rats (300–400 g) were isolated as described previously [9], with the introduction of a few modifications. Briefly, the animals were anaesthetized using intrapertioneal sodium pentobarbital (50 mg/kg) followed by dissection of the pancreas and associated spleen, stomach and duodenum. The iso-
2.6. Statistical analysis For the statistical analysis in the OGTT experiment, the area under the curve (AUC) was calculated. Significant differences were determined using Dunnett’s multiple comparison test. A value of P < 0.05 was considered to represent statistical significance. All data are expressed as the mean ± SE. All statistical analyses were performed using the SAS 8.2 software package (SAS Institute Japan Ltd., Tokyo, Japan). 3. Results 3.1. Human GPR119 agonist activity of AS1535907 and its effect on insulin secretion by rat islets To evaluate the human GPR119 agonist activity of AS1535907, this compound was used to treat HEK293 cells stably expressing human GPR119 and pCRE-Luc. AS1535907 significantly evoked intracellular cAMP accumulation in a dose-dependent manner and displayed an EC50 value of 4.8 lM for human GPR119 (Fig. 1A). Significantly, AS1535907 had no effect on HEK293 cells expressing control vector and was inactive towards several other GPCRs, including the b-adrenergic and GLP-1 receptors (data not shown). To confirm that AS1535907 has direct effects on insulin secretion, we next examined the ability of AS1535907 to induce GSIS in isolated rat islets. The exposure of rat islets to 10 lM AS1535907 resulted in an increase of insulin secretion at high glucose levels (16.8 mM) (Fig. 1B). 3.2. Insulin secretion in isolated perfused rat pancreata We next investigated the effect of AS1535907 on first-phase insulin secretion in vitro using isolated perfused rat pancreata. Specifically, we examined and compared the effects of AS1535907 and the T2DM therapeutic agent glibenclamide on the kinetics of insulin release in pancreata in the presence of 2.8 or 16.8 mM glucose. At a basal glucose concentration (2.8 mM), treatment of pancreata with 10 lM AS1535907 had no observable effect (Fig. 2A and C). This contrasted with the effect of glibenclamide, which markedly stimulated insulin secretion within 5 min of treatment under basal glucose levels. At 16.8 mM glucose, 10 lM AS1535907 specifically enhanced first-phase insulin secretion by pancreata, as indicated by the time–response curve and the AUC between 30–40 and
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3.3. Effect of AS1535907 on glucose tolerance and plasma insulin levels after a single treatment in diabetic db/db mice
Fig. 1. Initial characterization of the small molecule GPR119 agonist. (A) Human GPR119 agonist activity of AS1535907 in HEK293 cells stably expressing human GPR119 and CRE-luc. (B) In vitro insulin secretion by isolated rat islets exposed to 10 lM AS1535907 in the presence of 2.8 and 16.8 mM glucose. The data are presented as the mean ± SE (n = 3) of three replicate experiments. *p < 0.05 vs. control group (0 lM, vehicle), as determined using the Student’s t-test.
40–60 min (Fig. 2A and D), whereas the identical concentration of glibenclamide induced second-phase insulin secretion to a much greater extent than the first phase (Fig. 2B–D).
After demonstrating that AS1535907 altered the first phase of insulin in vitro, the role of AS1535907 in the acute regulation of insulin secretion was evaluated in vivo by monitoring blood glucose and plasma insulin levels during an OGTT using db/db mice. Compared to the vehicle control and glibenclamide, a single oral treatment with 10 mg/kg AS1535907 resulted in a clear decrease in the blood glucose of db/db mice (Fig. 3A, and B). This result was reflected in the blood glucose AUC after both 0.5 h (AUC0–0.5 h) and 3 h (AUC0–3 h) of administration, which were significantly reduced compared to the vehicle control (Fig. 3C and D). In contrast, although a single oral treatment with 10 mg/kg of glibenclamide significantly reduced the blood glucose AUC after 3 h (AUC0–3 h) of administration (Fig. 3D), no effect on the blood glucose AUC after 0.5 h (AUC0–0.5 h) of administration was observed (Fig. 3C). Compared to the vehicle control and glibenclamide, a single oral treatment with 10 mg/kg AS1535907 resulted in a clear increase in the early phase-plasma insulin of db/db mice (Fig. 4A and B). The plasma insulin levels at 5 min and the AUC0–10 min after administration were significantly higher in the mice exposed to AS1535907 compared to those treated with the vehicle control (Fig. 4C and D). However, in the db/db mice treated with glibenclamide, plasma insulin levels were not significantly increased (Fig. 4C and D).
Fig. 2. Insulin secretion in isolated perfused SD rat pancreata. The effect of 10 lM AS1535907 (A) and 10 lM glibenclamide (B) on insulin secretion at 2.8 and 16.8 mM glucose (n = 3) were evaluated. The effect of 10 lM AS1535907 (C) and 10 lM glibenclamide (D) on the area under curve (AUC) for insulin secretion at 2.8 mM glucose and first- and second-phase insulin secretion at 16.8 mM glucose (n = 3). The data are presented as the mean ± SE (n = 3) of three replicate experiments. *p < 0.05, **p < 0.01 vs. control group (0 lM, vehicle), as determined using Dunnett’s multiple-range test.
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Fig. 3. Effects of AS1535907 and glibenclamide on glucose tolerance after a single oral treatment in diabetic db/db mice. The effect of 10 mg/kg po AS1535907 (A) and glibenclamide (B) on blood glucose levels in db/db mice during an OGTT. (C) The area under the blood glucose concentration–time curve for 3 h (AUC0–0.5 h) in an OGTT. (D) The area under the blood glucose concentration–time curve for 0.5 h (AUC0–3 h) in an OGTT. Data are presented as the mean ± SE for each treatment (n = 7). Dunnett’s multiple comparison tests, *p < 0.05, **p < 0.01 vs. vehicle control.
Fig. 4. Effect of AS1535907 and glibenclamide on plasma insulin levels after a single oral treatment in diabetic db/db mice. The effect of 10 mg/kg po AS1535907 (A) and glibenclamide (B) on plasma insulin levels in db/db mice during an OGTT. (B) and (C) The area under the plasma insulin concentration–time curve for 10 min (AUC0–10 min) in an OGTT. (D) The effect on plasma insulin levels at 5 min in an OGTT. Data are presented as the mean ± SE for each treatment (n = 7). Dunnett’s multiple comparison tests, *p < 0.05, **p < 0.01 vs. vehicle control.
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4. Discussion In the present study, we characterized a novel small-molecule GPR119 agonist, AS1535907, which displayed high specificity for activating human GPR119 and induced in vitro insulin secretion from rat islets under high-glucose conditions. Although AS1535907 affected both the first and second phase of GSIS under high-glucose conditions in isolated perfused rat pancreata, the first phase was specifically induced. Significantly, the action of AS1535907 was distinct from the SU glibenclamide, which had much less effect on the first phase of GSIS and predominantly potentiated the second phase. The ability of AS1535907 to affect the acute regulation of insulin secretion was further shown in diabetic db/db mice, in which a single oral treatment with AS1535907 resulted in a clear decrease in the blood glucose levels within the first 30 min. From our characterization of AS1535907, we conclude that this novel GPR119 agonist has significant potential as an effective antihyperglycemic agent for the treatment of T2DM, through the specific induction of the first phase of GSIS. AS1535907 was modified from the 2,4,6-tri-substituted pyrimidine core GPR119 agonist AS1269574, which we described in a previous report [15], in an attempt to lower the effective dosage and target the first phase of GSIS. AS1535907 displayed an EC50 of 4.8 lM for human GPR119, which is similar to the values of 2.5 and 3.2 M determined for AS1269574 and OEA, respectively [12]. After demonstrating that the treatment of rat islets with 10 lM AS1535907 led to a rapid and significant increase in insulin release, we performed a detailed comparison of this compound with the widely used anti-diabetic SU glibenclamide using isolated perfused rat panreata. When rat panreata were infused with 2.8 mM glucose, glibenclamide induced a monophagic and long-lasting release of insulin that was not observed for AS1535907. As the stimulation of insulin secretion under low-glucose conditions often leads to hypoglycemia, our findings indicate that AS1535907 has the potential to avoid this response, which is a key factor in preventing downstream diabetic complications. In addition, although glibenclamide induced biphasic insulin secretion at high glucose concentrations (16.8 mM), the second phase of GSIS was predominantly stimulated. This contrasted with the action of AS1535907, which although resulted in significant stimulation of both the first and second phases of GSIS, displayed a clear specificity for the first phase. Our results indicate that AS1535907 induces an insulin-secretory profile that is more rapid in onset and returns to pre-stimulatory levels faster than glibenclamide, regardless of the surrounding glucose levels. Overall, AS1535907 appears to be superior to glibenclamide in the treatment of T2DM due to its improved stimulation of the first phase of insulin secretion and apparent avoidance of a hypoglycemic response. Early-phase insulin secretion is decreased not only in T2DM patients, but also individuals with impaired glucose tolerance (IGT), and is regarded as an important cause of postprandial hyperglycemia. Low insulin response is a characteristic feature of T2DM that eventually leads to high levels of glucose under fasting conditions and various complications related to glucose homeostasis [17,18]. In recent years, strategies for treating this disorder have focused agents that are capable of either directly or indirectly stimulating GSIS in pancreatic b-cells and have improved pharmacodynamic profiles over those typically obtained with SUs [19–22]. We have demonstrated that the GPR119 agonist AS1535907 is able to elevate insulin production in diabetic db/db mice within 5 min of treatment, an effect that was not observed for glibenclamide. As AS1535907 also displayed a shorter duration of action than glibenclamide, we consider that this GPR119 agonist is potential therapeutic value for T2DM patients due to its ability to closely mimic normal physiological insulin responses to food consumption.
In conclusion, AS1535907 is a novel small-molecule GPR119 agonist that specifically induces the first phase of GSIS only under high-glucose conditions. As the metabolic stimulation of pancreatic b-cell activity is blunted in T2DM patients and the first phase of insulin release is lost, the kinetics of the AS1535907-induced insulin release appear appropriate to improve the impaired insulin response to glucose and restore rapid insulin secretion. As our results show that AS1535907 induces a more physiological insulin-secretory profile than other oral insulin secretagogues, such as glibenclamide, it has the potential to improve post-meal glycemic control while reducing the risk of post-absorptive hypoglycemic events. Conflict of interest The authors have no conflict of interest to declare. Acknowledgment The authors sincerely thank everyone in the Department of Drug Discovery Research at Astellas Pharma Inc. for their support with this study. S.Y. was involved in design, conduct/data collection and writing manuscript. T.O., T.M., H.T., H.O., and Y.Y. were involved in conduct/data collection. M.S. was involved in his helpful advice in this study. References [1] R.A. DeFronzo, R.C. Bonadonna, E. Ferrannini, Pathogenesis of NIDDM. A balanced overview, Diabetes Care 15 (1999) 318–368. [2] S.I. Taylor, D. Accili, Y. Imai, Insulin resistance or insulin deficiency. Which is the primary cause of NIDDM?, Diabetes 43 (1994) 735–740 [3] J. Meece, Pancreatic islet dysfunction in type 2 diabetes: a rational target for incretin-based therapies, Curr. Med. Res. Opin. 23 (2007) 933–944. [4] Y. Seino, M.F. Rasmussen, T. Nishida, K. Kaku, Efficacy and safety of the once-daily human GLP-1 analogue, liraglutide, vs glibenclamide monotherapy in Japanese patients with type 2 diabetes, Curr. Med. Res. Opin. 26 (2010) 1013–1022. [5] M.A. Nauck, T. Vilsbøll, B. Gallwitz, A. Garber, S. Madsbad, Incretin-based therapies: viewpoints on the way to consensus, Diabetes Care 32 Suppl 2 (2009) S223–S231. [6] B. Ahrén, Emerging dipeptidyl peptidase-4 inhibitors for the treatment of diabetes, Expert. Opin. Emerg. Drugs 13 (2008) 593–607. [7] J. Doupis, A. Veves, DPP4 inhibitors: a new approach in diabetes treatment, Adv. Ther. 25 (2008) 627–643. [8] C.W. Chia, J.M. Egan, Incretin-based therapies in type 2 diabetes mellitus, J. Clin. Endocrinol. Metab. 93 (2008) 3703–3716. [9] T. Soga, T. Ohishi, T. Matsui, T. Saito, M. Matsumoto, J. Takasaki, S. Matsumoto, M. Kamohara, H. Hiyama, S. Yoshida, K. Momose, Y. Ueda, H. Matsushime, M. Kobori, K. Furuichi, Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor, Biochem. Biophys. Res. Commun. 28 (2005) 744–751. [10] Y. Sakamoto, H. Inoue, S. Kawakami, K. Miyawaki, T. Miyamoto, K. Mizuta, M. Itakura, Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells, Biochem. Biophys. Res. Commun. 351 (2006) 474–480. [11] H.A. Overton, A.J. Babbs, S.M. Doel, M.C. Fyfe, L.S. Gardner, G. Griffin, H.C. Jackson, M.J. Procter, C.M. Rasamison, M. Tang-Christensen, P.S. Widdowson, G.M. Williams, C. Reynet, Deorphanization of a G proteincoupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents, Cell. Metab. 3 (2006) 167–175. [12] Z.L. Chu, R.M. Jones, H. He, et al., A role for beta-cell-expressed G proteincoupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release, Endocrinology 148 (2007) 2601–2609. [13] Z.L. Chu, R.M. Jones, H. He, C. Carroll, V. Gutierrez, A. Lucman, M. Moloney, H. Gao, H. Mondala, D. Bagnol, D. Unett, Y. Liang, K. Demarest, G. Semple, D.P. Behan, J. Leonard, A role for intestinal endocrine cell-expressed GPR119 in glycemic control by enhancing GLP-1 and GIP release, Endocrinology 149 (2008) 2038–2047. [14] R.M. Jones, J.N. Leonard, D.J. Buzard, J. Lehmann, GPR119 agonists for the treatment of type 2 diabetes, Expert. Opin. Ther. Pat. 19 (2009) 1339–1359. [15] S. Yoshida, T. Ohishi, T. Matsui, M. Shibasaki, Identification of a novel GPR119 agonist, AS1269574, With in vitro and in vivo glucose-stimulated insulin secretion, Biochem. Biophys. Res. Commun. 400 (2010) 437–441. [16] C.B. Wollheim, P. Meda, P.A. Halban, Isolation of pancreatic islets and primary culture of the intact microorgans or of dispersed islet cells, Methods Enzymol. 192 (1990) 188–223.
S. Yoshida et al. / Biochemical and Biophysical Research Communications 402 (2010) 280–285 [17] L.A. Leiter, A. Ceriello, J.A. Davidson, M. Hanefeld, L. Monnier, D.R. Owens, N. Tajima, J. Tuomilehto, Postprandial glucose regulation: new data and new implications, Clin. Ther. 27 Suppl B (2005) S42–S56. [18] H.J. Woerle, C. Neumann, S. Zschau, S. Tenner, A. Irsigler, J. Schirra, J.E. Gerich, B. Göke, Impact of fasting and postprandial glycemia on overall glycemic control in type 2 diabetes Importance of postprandial glycemia to achieve target HbA1c levels, Diabetes Res. Clin. Pract. 77 (2007) 280–285. [19] B. Ahrén, Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes, Nat. Rev. Drug. Discov. 8 (2009) 369–385.
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[20] M.S. Winzell, B. Ahrén, G-protein-coupled receptors and islet functionimplications for treatment of type 2 diabetes, Pharmacol. Ther. 116 (2007) 437–448. [21] N.G. Morgan, S. Dhayal, G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell, Biochem. Pharmacol. 78 (2009) 1419–1427. [22] U. Shah, GPR119 agonists: a promising new approach for the treatment of type 2 diabetes and related metabolic disorders, Curr. Opin. Drug Discov. Dev. 12 (2009) 519–532.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice Shigeru Yoshida ⇑, Takahide Ohishi, Tetsuo Matsui, Hirotsugu Tanaka, Hiroyuki Oshima, Yasuhiro Yonetoku, Masayuki Shibasaki Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
a r t i c l e
i n f o
Article history: Received 13 September 2010 Available online 16 October 2010 Keywords: GPR119 AS1535907 First-phase insulin secretion Isolated perfused pancreas Glibenclamide
a b s t r a c t G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic b-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC50 value of 4.8 lM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 lM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Depletion of glucose-stimulated insulin secretion (GSIS), particularly loss of the first phase, is a characteristic feature in the pathology of type 2 diabetes mellitus (T2DM) and results in postprandial hyperglycemia [1,2]. In the clinical management of T2DM patients, sulfonylurea derivatives (SUs) are the most widely used hypoglycemic agents [3]; however, SUs can cause severe and prolonged hypoglycemia because of their long duration and glucose-independent mode of action [4]. Recent strategies for promoting normoglycemia have focused on enhancing GSIS through the targeting of G protein-coupled receptors (GPCRs), such as the glucagon-like peptide 1 (GLP-1) receptor. Although GLP-1 analogs, such as exendin-4, have been shown to effectively stimulate GSIS
Abbreviations: GPR119, G protein-coupled receptor 119; GSIS, glucose-stimulated insulin secretion; T2DM, type 2 diabetes mellitus; SU, sulfonylurea; GPCR, G protein-coupled receptor; GLP-1, glucagon-like peptide-1; HEK, human embryonic kidney; LPC, lysophosphatidylcholine; OEA, oleoylethanol amide. ⇑ Corresponding author. Address: Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Fax: +81 29 852 2955. E-mail address: [email protected] (S. Yoshida). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.015
and preserve pancreatic b-cells, these analogs can cause gastrointestinal side effects, pancreatitis, and require parenteral administration [5–8]. The limitations of SUs and GLP-1 analags underly the need for the development of oral insulin secretagogues that are capable of inducing normoglycemia and preserving the first phase of GSIS in T2DM patients. GPR119 represents a promising anti-diabetic therapeutic target as it is predominantly expressed in pancreatic b-cells and intestinal L-cells and promotes GSIS and indirectly increases GLP-1 level. Although the downstream pathways are unclear, activation of GPR119 by endogenous ligands, such as lysophosphatidylcholine (LPC) and oleoylethanol amide (OEA), or small-molecule agonists, leads to the accumulation of intracellular cAMP and subsequent insulin and GLP-1 release [9–14]. We previously identified a novel structural class of small-molecule GPR119 agonists, consisting of 2,4,6-tri-substituted pyrimidine cores, which were orally active and displayed higher activity than existing T2DM therapeutics, such as extendin-4 [5,8]. The first identified compound, AS1269574, was capable of inducing GSIS in vitro and in vivo, and improved glucose tolerance in normal mice [15]. In an effort to identify an agonist in the identical structural class with a lower effective dosage and the ability to specifically induce the first phase of GSIS, compound
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AS1535907 (2-(4-bromophenyl)-6-methyl-N-[2-(1-oxidopyridin3-yl) ethyl] pyrimidin-4-amine), which was modified from AS1269574, was selected as a promising candidate. Here, we report the in vitro and in vivo characterization of the novel small-molecule GPR119 agonist AS1535907 in rat islets, isolated pancreata, and diabetic db/db mice. We specifically compared the effects of AS1535907 with the anti-diabetic SU glibenclamide on glucose tolerance, glucose levels, and insulin secretion in these animal systems.
lated pancreata were then perfused (flow rate, 2 ml/min) through the celiac artery with HEPES-balanced KRBB containing 2.8 or 16.8 mM glucose and 0.1% fatty acid-free BSA. The preparation was then placed in an acrylic chamber containing KRBB (37 °C) and the effluent perfusate from the portal vein cannula was collected at 1–2 min intervals in fraction tubes containing aprotinin (500 units/tube). The samples were stored at 20 °C until insulin concentrations were measured using an Insulin Radioimmunoassay Kit (Amersham Pharmacia Biotech).
2. Materials and methods
2.5. Oral glucose torelance test (OGTT) in diabetic db/db mice
2.1. Animals and materials
Six-week-old male db/db mice were fasted overnight and then orally administered 0.5% methyl-cellulose (vehicle, n = 7), or 10 mg/kg AS1535907 (n = 7), and 10 mg/kg glibenclamide (n = 7). After 30 min, glucose was given orally at a dose of 2 g/kg/10 ml, and blood samples were collected from tail veins after 0, 5, 10, 15, 30, 60, 120, and 180 min. Blood glucose levels was determined using the Glucose CII test (Wako, Osaka, Japan), while plasma insulin level was measured by radioimmunoassay (Amersham Biosciences), following the manufacturers’ instructions.
Male diabetic db/db mice and male Sprague–Dawley (SD) rats were obtained from CLEA Japan, Inc. (Kanagawa, Japan). All surgical procedures performed were approved by the Animal Ethical Committee of Astellas Pharma Inc. The compounds AS1535907 (2-(4-bromophenyl)-6-methyl-N-[2-(1-oxidopyridin-3-yl) ethyl] pyrimidin-4-amine) was synthesized in-house at Astellas Pharma Inc. (Ibaraki, Japan), and was modified from the 2,4,6-tri-substituted pyrimidine core agonist AS1269574 [15] Glibenclamide was obtained from Sigma (St. Louis, MO, USA). Prior to use in assays, AS1535907 and glibenclamide were dissolved in dimethyl sulfoxide (DMSO) to yield a stock solution of 10 mM. 2.2. Plasmid construction and generation of HEK293-hGPR119 cells The human GPR119 ORF (GenBank Accession No. BD169091) was amplified by PCR from human pancreas cDNA (Clontech, Tokyo, Japan) with the sense, 50 -AAAATCTAGAATGGAATCATCTTTCTC ATTTG-30 , and antisense, 50 -CGGCTCTAGATTAGCCATCAAACTCTG AGCTGG-30 , primers (XbaI restriction sites are underlined). The amplified product was subcloned into the mammalian expression vector plasmid pcDNA3.1 (Invitrogen, Tokyo, Japan). The resulting pcDNA-GPR119 plasmid and pCRE (cAMP responsive element)-Luc plasmid (Clontech) were cotransfected into human embryonic kidney (HEK) 293 cells using standard procedures. Candidate clones stably expressing human GPR119 and pCRE-Luc were selected by resistance to 400 lg/ml geneticin, and were confirmed by the activity of LPC, an endogenous ligand of GPR119. 2.3. Insulin secretion by islet cells Islets of Langerhans were isolated from male SD rats by collagenase digestion as described previously [16] and subsequently handpicked under a stereomicroscope. The isolated islets were then cultured overnight at 37 °C in RPMI-1640 medium (Invitrogen) supplemented with 11.1 mM glucose and 10% fetal bovine serum. After 2 days of culture, the ilets were washed with HEPES-balanced Krebs–Ringer bicarbonate buffer (KRBB) containing 2.8 mM glucose and 0.1% BSA, and preincubated for 60 min at 37 °C in the same medium. After preincuation, cells were stimulated with test compound in HEPES-balanced KRBB (pH 7.4) containing 16.8 mM glucose at 37 °C for 60 min. Insulin secreted into the supernatant was collected and measured using an Insulin Radioimmunoassay Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.4. Perfusion of isolated rat pancreata The pancreata of male SD rats (300–400 g) were isolated as described previously [9], with the introduction of a few modifications. Briefly, the animals were anaesthetized using intrapertioneal sodium pentobarbital (50 mg/kg) followed by dissection of the pancreas and associated spleen, stomach and duodenum. The iso-
2.6. Statistical analysis For the statistical analysis in the OGTT experiment, the area under the curve (AUC) was calculated. Significant differences were determined using Dunnett’s multiple comparison test. A value of P < 0.05 was considered to represent statistical significance. All data are expressed as the mean ± SE. All statistical analyses were performed using the SAS 8.2 software package (SAS Institute Japan Ltd., Tokyo, Japan). 3. Results 3.1. Human GPR119 agonist activity of AS1535907 and its effect on insulin secretion by rat islets To evaluate the human GPR119 agonist activity of AS1535907, this compound was used to treat HEK293 cells stably expressing human GPR119 and pCRE-Luc. AS1535907 significantly evoked intracellular cAMP accumulation in a dose-dependent manner and displayed an EC50 value of 4.8 lM for human GPR119 (Fig. 1A). Significantly, AS1535907 had no effect on HEK293 cells expressing control vector and was inactive towards several other GPCRs, including the b-adrenergic and GLP-1 receptors (data not shown). To confirm that AS1535907 has direct effects on insulin secretion, we next examined the ability of AS1535907 to induce GSIS in isolated rat islets. The exposure of rat islets to 10 lM AS1535907 resulted in an increase of insulin secretion at high glucose levels (16.8 mM) (Fig. 1B). 3.2. Insulin secretion in isolated perfused rat pancreata We next investigated the effect of AS1535907 on first-phase insulin secretion in vitro using isolated perfused rat pancreata. Specifically, we examined and compared the effects of AS1535907 and the T2DM therapeutic agent glibenclamide on the kinetics of insulin release in pancreata in the presence of 2.8 or 16.8 mM glucose. At a basal glucose concentration (2.8 mM), treatment of pancreata with 10 lM AS1535907 had no observable effect (Fig. 2A and C). This contrasted with the effect of glibenclamide, which markedly stimulated insulin secretion within 5 min of treatment under basal glucose levels. At 16.8 mM glucose, 10 lM AS1535907 specifically enhanced first-phase insulin secretion by pancreata, as indicated by the time–response curve and the AUC between 30–40 and
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3.3. Effect of AS1535907 on glucose tolerance and plasma insulin levels after a single treatment in diabetic db/db mice
Fig. 1. Initial characterization of the small molecule GPR119 agonist. (A) Human GPR119 agonist activity of AS1535907 in HEK293 cells stably expressing human GPR119 and CRE-luc. (B) In vitro insulin secretion by isolated rat islets exposed to 10 lM AS1535907 in the presence of 2.8 and 16.8 mM glucose. The data are presented as the mean ± SE (n = 3) of three replicate experiments. *p < 0.05 vs. control group (0 lM, vehicle), as determined using the Student’s t-test.
40–60 min (Fig. 2A and D), whereas the identical concentration of glibenclamide induced second-phase insulin secretion to a much greater extent than the first phase (Fig. 2B–D).
After demonstrating that AS1535907 altered the first phase of insulin in vitro, the role of AS1535907 in the acute regulation of insulin secretion was evaluated in vivo by monitoring blood glucose and plasma insulin levels during an OGTT using db/db mice. Compared to the vehicle control and glibenclamide, a single oral treatment with 10 mg/kg AS1535907 resulted in a clear decrease in the blood glucose of db/db mice (Fig. 3A, and B). This result was reflected in the blood glucose AUC after both 0.5 h (AUC0–0.5 h) and 3 h (AUC0–3 h) of administration, which were significantly reduced compared to the vehicle control (Fig. 3C and D). In contrast, although a single oral treatment with 10 mg/kg of glibenclamide significantly reduced the blood glucose AUC after 3 h (AUC0–3 h) of administration (Fig. 3D), no effect on the blood glucose AUC after 0.5 h (AUC0–0.5 h) of administration was observed (Fig. 3C). Compared to the vehicle control and glibenclamide, a single oral treatment with 10 mg/kg AS1535907 resulted in a clear increase in the early phase-plasma insulin of db/db mice (Fig. 4A and B). The plasma insulin levels at 5 min and the AUC0–10 min after administration were significantly higher in the mice exposed to AS1535907 compared to those treated with the vehicle control (Fig. 4C and D). However, in the db/db mice treated with glibenclamide, plasma insulin levels were not significantly increased (Fig. 4C and D).
Fig. 2. Insulin secretion in isolated perfused SD rat pancreata. The effect of 10 lM AS1535907 (A) and 10 lM glibenclamide (B) on insulin secretion at 2.8 and 16.8 mM glucose (n = 3) were evaluated. The effect of 10 lM AS1535907 (C) and 10 lM glibenclamide (D) on the area under curve (AUC) for insulin secretion at 2.8 mM glucose and first- and second-phase insulin secretion at 16.8 mM glucose (n = 3). The data are presented as the mean ± SE (n = 3) of three replicate experiments. *p < 0.05, **p < 0.01 vs. control group (0 lM, vehicle), as determined using Dunnett’s multiple-range test.
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Fig. 3. Effects of AS1535907 and glibenclamide on glucose tolerance after a single oral treatment in diabetic db/db mice. The effect of 10 mg/kg po AS1535907 (A) and glibenclamide (B) on blood glucose levels in db/db mice during an OGTT. (C) The area under the blood glucose concentration–time curve for 3 h (AUC0–0.5 h) in an OGTT. (D) The area under the blood glucose concentration–time curve for 0.5 h (AUC0–3 h) in an OGTT. Data are presented as the mean ± SE for each treatment (n = 7). Dunnett’s multiple comparison tests, *p < 0.05, **p < 0.01 vs. vehicle control.
Fig. 4. Effect of AS1535907 and glibenclamide on plasma insulin levels after a single oral treatment in diabetic db/db mice. The effect of 10 mg/kg po AS1535907 (A) and glibenclamide (B) on plasma insulin levels in db/db mice during an OGTT. (B) and (C) The area under the plasma insulin concentration–time curve for 10 min (AUC0–10 min) in an OGTT. (D) The effect on plasma insulin levels at 5 min in an OGTT. Data are presented as the mean ± SE for each treatment (n = 7). Dunnett’s multiple comparison tests, *p < 0.05, **p < 0.01 vs. vehicle control.
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4. Discussion In the present study, we characterized a novel small-molecule GPR119 agonist, AS1535907, which displayed high specificity for activating human GPR119 and induced in vitro insulin secretion from rat islets under high-glucose conditions. Although AS1535907 affected both the first and second phase of GSIS under high-glucose conditions in isolated perfused rat pancreata, the first phase was specifically induced. Significantly, the action of AS1535907 was distinct from the SU glibenclamide, which had much less effect on the first phase of GSIS and predominantly potentiated the second phase. The ability of AS1535907 to affect the acute regulation of insulin secretion was further shown in diabetic db/db mice, in which a single oral treatment with AS1535907 resulted in a clear decrease in the blood glucose levels within the first 30 min. From our characterization of AS1535907, we conclude that this novel GPR119 agonist has significant potential as an effective antihyperglycemic agent for the treatment of T2DM, through the specific induction of the first phase of GSIS. AS1535907 was modified from the 2,4,6-tri-substituted pyrimidine core GPR119 agonist AS1269574, which we described in a previous report [15], in an attempt to lower the effective dosage and target the first phase of GSIS. AS1535907 displayed an EC50 of 4.8 lM for human GPR119, which is similar to the values of 2.5 and 3.2 M determined for AS1269574 and OEA, respectively [12]. After demonstrating that the treatment of rat islets with 10 lM AS1535907 led to a rapid and significant increase in insulin release, we performed a detailed comparison of this compound with the widely used anti-diabetic SU glibenclamide using isolated perfused rat panreata. When rat panreata were infused with 2.8 mM glucose, glibenclamide induced a monophagic and long-lasting release of insulin that was not observed for AS1535907. As the stimulation of insulin secretion under low-glucose conditions often leads to hypoglycemia, our findings indicate that AS1535907 has the potential to avoid this response, which is a key factor in preventing downstream diabetic complications. In addition, although glibenclamide induced biphasic insulin secretion at high glucose concentrations (16.8 mM), the second phase of GSIS was predominantly stimulated. This contrasted with the action of AS1535907, which although resulted in significant stimulation of both the first and second phases of GSIS, displayed a clear specificity for the first phase. Our results indicate that AS1535907 induces an insulin-secretory profile that is more rapid in onset and returns to pre-stimulatory levels faster than glibenclamide, regardless of the surrounding glucose levels. Overall, AS1535907 appears to be superior to glibenclamide in the treatment of T2DM due to its improved stimulation of the first phase of insulin secretion and apparent avoidance of a hypoglycemic response. Early-phase insulin secretion is decreased not only in T2DM patients, but also individuals with impaired glucose tolerance (IGT), and is regarded as an important cause of postprandial hyperglycemia. Low insulin response is a characteristic feature of T2DM that eventually leads to high levels of glucose under fasting conditions and various complications related to glucose homeostasis [17,18]. In recent years, strategies for treating this disorder have focused agents that are capable of either directly or indirectly stimulating GSIS in pancreatic b-cells and have improved pharmacodynamic profiles over those typically obtained with SUs [19–22]. We have demonstrated that the GPR119 agonist AS1535907 is able to elevate insulin production in diabetic db/db mice within 5 min of treatment, an effect that was not observed for glibenclamide. As AS1535907 also displayed a shorter duration of action than glibenclamide, we consider that this GPR119 agonist is potential therapeutic value for T2DM patients due to its ability to closely mimic normal physiological insulin responses to food consumption.
In conclusion, AS1535907 is a novel small-molecule GPR119 agonist that specifically induces the first phase of GSIS only under high-glucose conditions. As the metabolic stimulation of pancreatic b-cell activity is blunted in T2DM patients and the first phase of insulin release is lost, the kinetics of the AS1535907-induced insulin release appear appropriate to improve the impaired insulin response to glucose and restore rapid insulin secretion. As our results show that AS1535907 induces a more physiological insulin-secretory profile than other oral insulin secretagogues, such as glibenclamide, it has the potential to improve post-meal glycemic control while reducing the risk of post-absorptive hypoglycemic events. Conflict of interest The authors have no conflict of interest to declare. Acknowledgment The authors sincerely thank everyone in the Department of Drug Discovery Research at Astellas Pharma Inc. for their support with this study. S.Y. was involved in design, conduct/data collection and writing manuscript. T.O., T.M., H.T., H.O., and Y.Y. were involved in conduct/data collection. M.S. was involved in his helpful advice in this study. References [1] R.A. DeFronzo, R.C. Bonadonna, E. Ferrannini, Pathogenesis of NIDDM. A balanced overview, Diabetes Care 15 (1999) 318–368. [2] S.I. Taylor, D. Accili, Y. Imai, Insulin resistance or insulin deficiency. Which is the primary cause of NIDDM?, Diabetes 43 (1994) 735–740 [3] J. Meece, Pancreatic islet dysfunction in type 2 diabetes: a rational target for incretin-based therapies, Curr. Med. Res. Opin. 23 (2007) 933–944. [4] Y. Seino, M.F. Rasmussen, T. Nishida, K. Kaku, Efficacy and safety of the once-daily human GLP-1 analogue, liraglutide, vs glibenclamide monotherapy in Japanese patients with type 2 diabetes, Curr. Med. Res. Opin. 26 (2010) 1013–1022. [5] M.A. Nauck, T. Vilsbøll, B. Gallwitz, A. Garber, S. Madsbad, Incretin-based therapies: viewpoints on the way to consensus, Diabetes Care 32 Suppl 2 (2009) S223–S231. [6] B. Ahrén, Emerging dipeptidyl peptidase-4 inhibitors for the treatment of diabetes, Expert. Opin. Emerg. Drugs 13 (2008) 593–607. [7] J. Doupis, A. Veves, DPP4 inhibitors: a new approach in diabetes treatment, Adv. Ther. 25 (2008) 627–643. [8] C.W. Chia, J.M. Egan, Incretin-based therapies in type 2 diabetes mellitus, J. Clin. Endocrinol. Metab. 93 (2008) 3703–3716. [9] T. Soga, T. Ohishi, T. Matsui, T. Saito, M. Matsumoto, J. Takasaki, S. Matsumoto, M. Kamohara, H. Hiyama, S. Yoshida, K. Momose, Y. Ueda, H. Matsushime, M. Kobori, K. Furuichi, Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor, Biochem. Biophys. Res. Commun. 28 (2005) 744–751. [10] Y. Sakamoto, H. Inoue, S. Kawakami, K. Miyawaki, T. Miyamoto, K. Mizuta, M. Itakura, Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells, Biochem. Biophys. Res. Commun. 351 (2006) 474–480. [11] H.A. Overton, A.J. Babbs, S.M. Doel, M.C. Fyfe, L.S. Gardner, G. Griffin, H.C. Jackson, M.J. Procter, C.M. Rasamison, M. Tang-Christensen, P.S. Widdowson, G.M. Williams, C. Reynet, Deorphanization of a G proteincoupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents, Cell. Metab. 3 (2006) 167–175. [12] Z.L. Chu, R.M. Jones, H. He, et al., A role for beta-cell-expressed G proteincoupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release, Endocrinology 148 (2007) 2601–2609. [13] Z.L. Chu, R.M. Jones, H. He, C. Carroll, V. Gutierrez, A. Lucman, M. Moloney, H. Gao, H. Mondala, D. Bagnol, D. Unett, Y. Liang, K. Demarest, G. Semple, D.P. Behan, J. Leonard, A role for intestinal endocrine cell-expressed GPR119 in glycemic control by enhancing GLP-1 and GIP release, Endocrinology 149 (2008) 2038–2047. [14] R.M. Jones, J.N. Leonard, D.J. Buzard, J. Lehmann, GPR119 agonists for the treatment of type 2 diabetes, Expert. Opin. Ther. Pat. 19 (2009) 1339–1359. [15] S. Yoshida, T. Ohishi, T. Matsui, M. Shibasaki, Identification of a novel GPR119 agonist, AS1269574, With in vitro and in vivo glucose-stimulated insulin secretion, Biochem. Biophys. Res. Commun. 400 (2010) 437–441. [16] C.B. Wollheim, P. Meda, P.A. Halban, Isolation of pancreatic islets and primary culture of the intact microorgans or of dispersed islet cells, Methods Enzymol. 192 (1990) 188–223.
S. Yoshida et al. / Biochemical and Biophysical Research Communications 402 (2010) 280–285 [17] L.A. Leiter, A. Ceriello, J.A. Davidson, M. Hanefeld, L. Monnier, D.R. Owens, N. Tajima, J. Tuomilehto, Postprandial glucose regulation: new data and new implications, Clin. Ther. 27 Suppl B (2005) S42–S56. [18] H.J. Woerle, C. Neumann, S. Zschau, S. Tenner, A. Irsigler, J. Schirra, J.E. Gerich, B. Göke, Impact of fasting and postprandial glycemia on overall glycemic control in type 2 diabetes Importance of postprandial glycemia to achieve target HbA1c levels, Diabetes Res. Clin. Pract. 77 (2007) 280–285. [19] B. Ahrén, Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes, Nat. Rev. Drug. Discov. 8 (2009) 369–385.
285
[20] M.S. Winzell, B. Ahrén, G-protein-coupled receptors and islet functionimplications for treatment of type 2 diabetes, Pharmacol. Ther. 116 (2007) 437–448. [21] N.G. Morgan, S. Dhayal, G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell, Biochem. Pharmacol. 78 (2009) 1419–1427. [22] U. Shah, GPR119 agonists: a promising new approach for the treatment of type 2 diabetes and related metabolic disorders, Curr. Opin. Drug Discov. Dev. 12 (2009) 519–532.