Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice

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Abstract

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic β-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 μM 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 μM 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.

Research highlights

► AS1535907 represents a novel small-molecule GPR119 agonist that enhances insulin secretion at 16.8 mM glucose in rat islets. ► AS1535907 enhances the first phase of insulin secretion at 16.8 mM glucose in isolated perfused rat pancreas. ► AS1535907 reduces blood glucose levels due to the rapid secretion on insulin secretion following oral glucose loading in diabetic db/db mice.

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 and preserve pancreatic β-cells, these analogs can cause gastrointestinal side effects, pancreatitis, and require parenteral administration [5], [6], [7], [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 β-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], [10], [11], [12], [13], [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 AS1535907 (2-(4-bromophenyl)-6-methyl-N-[2-(1-oxidopyridin-3-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.

Section snippets

Animals and materials

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

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 μM 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 β-adrenergic and GLP-1 receptors (data not shown). To

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

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.

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