A novel class of antagonists for the FFAs receptor GPR40

doi:10.1016/j.bbrc.2009.10.004

Biochemical and Biophysical Research Communications

Volume 390, Issue 3, 18 December 2009, Pages 557-563

https://doi.org/10.1016/j.bbrc.2009.10.004 Get rights and content

Abstract

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca²⁺ elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC₅₀: 6.28 ± 1.14, 5.96 ± 1.12, 7.07 ± 1.42, 4.58 ± 1.14 μM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.

Introduction

Free fatty acids (FFAs) are not only essential regulators of the function of normal β-cell, but also play important roles in the pathogenesis of β-cell dysfunction in type 2 diabetes [1], [2], [3]. Under normal circumstances, FFAs amplify glucose-stimulated insulin secretion (GSIS). In contrast to their acute effect on GSIS, prolonged exposure of β-cells to elevated levels of FFAs impairs the cellular function, called “lipotoxicity”, including insulin secretion, gene expression, and β-cells survival [4].

GPR40, also called FFAR1, was identified as a free fatty acid receptor in 2003, which is activated by saturated and unsaturated long or medium chain fatty acids in pancreatic β-cells. It is expressed at high levels in pancreatic β-cell lines and primary islets in both human and rodent, which mediates both acute and chronic effects of the FFAs on insulin secretion, and has also been recognized as a possible link between obesity and diabetes [5], [6], [7]. As reported previously, GPR40 deficient mice are resistant to the development of hepatic steatosis, hypertriglyceridemia, hyperglycaemia and glucose intolerance on a high-fat diet. Conversely, GPR40 overexpression in islet β-cells mice show impairment of β-cell function and diabetes [8]. However, some reports found that deletion of GPR40 dose dot protect pancreatic islets against fatty acid-induced islet dysfunction [9], [10] and overexpression of GPR40 in pancreatic β-cells augmented insulin secretion and improved oral glucose tolerance in vivo [11]. Though, there is no doubt that GPR40 is directly responsible for the acute stimulatory effects of fatty acids on GSIS, the debate about the relationship between GPR40 and “lipotoxicity” is still on. Thus, more biological studies are definitely necessary to adequately understand the exact role and function of GPR40. In addition to different genetic animal models that are powerful tools for the purpose, agonists and antagonists may provide chemical probes to study the physiological and pharmacological functions of GPR40. However, to the best of our knowledge, there are only three antagonists publically reported up to date. Tikhonova et al. found two antagonists through virtual screening and bioassay [12]. Briscoe et al. reported that GW1100, a small-molecule antagonist of GPR40, partially attenuates the linoleic acid-stimulated insulin secretion [13].

In this paper, we reported that sulfonamide compounds are small organic antagonists of GPR40. One of the compounds dose-dependently inhibited GPR40-mediated Ca²⁺ elevations stimulated by various fatty acids, reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells. Therefore, these compounds are possible specific chemical probes for further exploring the function of GPR40, and potential lead structures for anti-diabetes drug development.

Section snippets

Material and methods

Materials. The mouse insulinoma cell line Min6 was kindly provided by Dr. Susumu Seino. Oleic acid, linoleic acid, lauric acid, palmitoleic acid and oleic acid-BSA were purchased from Sigma. DELFIA^® GTP binding kit was from Perkin-Elmer. Dulbecco’s modified Eagle’s medium (DMEM), pcDNA3.1 and Fluo-4/AM were from Invitrogen. The human pcDNA-GPR40 plasmid was from HD Biosciences, the pEGFP-N3 plasmid was from BD Bioscience, and other plasmids were provided by Missouri S&T cDNA Resource Center

Homology model and virtual screening

Fig. 1 shows the sequence alignment result between human GPR40 and bovine rhodopsin based on multiple sequence alignment information. Ramachandran analysis for thirty models showed that most residues in these models have reasonable orientations. For the top 10 models, orientation parameters of >99.0% residues were in allowed region. Based on the estimated binding free energies of the 10 different homology models with 1 and 2 (Table S2, Supplementary content), the best model was selected (Fig. 2

Acknowledgments

We gratefully acknowledge the kind provision of Min6 cells by Professor S. Seino. This work was supported by Grants 2007AA02Z301 and 2006AA02Z336 of the National High Technology Research and Development Program of China (863 Program) and by Grant 07G603L056 of the New Drug Basic Research Program of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

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DC260126, another new class of GPR40 antagonist (Fig. 2), repressed GPR40-mediated calcium increases promoted by palmitoleic acid, oleic acid and LA in a dose-dependent manner [70,71]. In addition, this inhibitor also diminished the ERK1/2 phosphorylation stimulated by LA in GPR40 expressing CHO cells and oleic acid induced GPR40 mRNA expression in Min6 cells [71]. Most interestingly, Zhang et al. (2010) [70] reported that DC260126 by acting as GPR40 antagonist, significantly improve the insulin tolerance, liver Akt phosphorylation and reduce serum insulin levels, hence enhanced the insulin sensitivity in obese Zucker rats [72].
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¹: These authors contributed equally to the work.

View full text

A novel class of antagonists for the FFAs receptor GPR40

Abstract

Introduction

Section snippets

Material and methods

Homology model and virtual screening

Acknowledgments

Cell Metab.

Comput. Phys. Commun.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

Minireview: secondary beta-cell failure in type 2 diabetes – a convergence of glucotoxicity and lipotoxicity

Endocrinology

Fatty acids, lipotoxicity and insulin secretion

Diabetologia

Effect of individual fatty acids on glucose uptake and glycogen synthesis in soleus muscle in vitro

Am. J. Physiol. Endocrinol. Metab.

Fatty acid metabolism and insulin secretion in pancreatic beta cells

Diabetologia

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40

Nature

Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion

Cell Tissue Res.

Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms

PLoS ONE

Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease

Diabetes

GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo

Diabetes