Activation of G protein-coupled receptor 30 modulates hormone secretion and counteracts cytokine-induced apoptosis in pancreatic islets of female mice

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Abstract

The role of the newly discovered estrogen receptor GPR30 in islet physiology and pathophysiology is unclear. We examined GPR30 expression in relation to hormone secretion and possible anti-apoptotic effects in isolated mouse islets using the synthetic GPR30 ligand G-1. The mRNA and protein expression of GPR30 was analyzed by qPCR, Western blot and confocal microscopy. Hormone secretion and cAMP content were determined with RIA and apoptosis in islet cells with the Annexin-V method.

GPR30 mRNA and protein expression was markedly higher in islets from females compared to male. This gender difference was not found for the genomic estrogen receptors ERα and ERβ, the ERα expression being 10-fold higher than ERβ in both genders. Confocal microscopy revealed abounden GPR30 expression in insulin, glucagon and somatostatin cells. Dose–response studies of G-1 vs 17β-estradiol in isolated islets at 1 or 12 mM glucose showed an almost identical pattern in that both compounds increased insulin and inhibited glucagon and somatostatin secretion. ICI-182,780 and EM-652, potent antagonists of the 17β-estradiol receptors (ERα and ERβ) did not influence the amplifying effect of G-1 or 17β-estradiol on cAMP content or insulin secretion from isolated islets. Cytokine-induced (IL-1β + TNFα + INFγ) apoptosis in islets, cultured for 24 h at 5 mM glucose, was almost abolished by G-1 or 17β-estradiol treatment. Addition of ICI-182,780 or EM-652 did not affect this beneficial effect of G-1 or 17β-estradiol.

Taken together, our findings show that GPR30 is expressed in most islet endocrine cells. The synthetic GPR30 ligand G-1 mimics the non-genomic effects of 17β-estradiol on islet hormone secretion, cAMP content in islets and its anti-apoptotic effects. G-1 or analogs thereof might be new potential candidates in the therapeutic strategy for type 2 diabetes in women.

Introduction

Previous studies have shown that women display not only increased insulin sensitivity, and a better glucose tolerance but also a better insulin secretory response to nutrients compared to men (Boyns et al., 1969, Yki-Järvinen, 1984) and are more resistant to nutrition-induced insulin resistance (Frias et al., 2001, Hevener et al., 2002, Soeters et al., 2007). Likewise, female mice are less prone to diet-induced insulin resistance (Corsetti et al., 2000, Zierath et al., 1997) and many genetically induced forms of insulin resistance have milder phenotypes in female compared with male mice (Corsetti et al., 2000, Li et al., 2000).

Genomic effects of estrogens through the nuclear estrogen receptors (ER) ERα and ERβ have been recognized for a long time in several tissues including the islets of Langerhans (Prossnitz et al., 2007).

Recently, however, several reports have described rapid effects by 17β-estradiol elicited through a non-classical membrane receptor, which has been shown to share the characteristics of a G protein-coupled receptor (GPCR). This receptor has been labeled GPR30 (Filardo et al., 2007, Martensson et al., 2009, Prossnitz et al., 2007). Very recently it was shown that deletion of GPR30 in female mice resulted in impaired glucose tolerance, reduced bone growth and increased vascular resistance (Martensson et al., 2009). Moreover, in that study we could show that glucose-stimulated insulin release was greatly impaired in GPR30(−/−) mice both in vitro and in vivo (Martensson et al., 2009). In addition, the estrogen-induced suppression of glucagon secretion was almost abolished in GPR30(−/−) mice (Martensson et al., 2009). Recent results from different studies suggest that 17β-estradiol protects insulin secretory processes in various diabetic states and that the prevalence of diabetes is known to be lower in females than in males (Wild et al., 2004). Hence, we found it of interest to study whether GPR30 was expressed in the different pancreatic endocrine cells and if GPR30 activation could influence insulin, glucagon and somatostatin secretion as well as islet cell survival. To this end we used a non-steroidal, high affinity, selective agonist of GPR30 referred to as G-1 (GPR30-specific compound 1; a substituted dihydroquinoline) (Prossnitz et al., 2008a, Prossnitz et al., 2008b, Prossnitz et al., 2008c). For comparative purposes, we also studied the influence of 17β-estradiol.

Section snippets

Materials

Female and male mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 25–30 g were used for the experiments. They were given a standard pellet diet (B&K) and tap water ad libitum. All animals were housed in metabolic cages with constant temperature (22 °C) and 12-h light/dark cycles. The local animal welfare committee (Lund, Sweden) approved the experimental protocols and all procedures using animals.

Collagenase (CLS 4) was from Sigma, St. Louis, MO, USA. Fatty acid free bovine serum albumin

mRNA and protein expression of GPR30 in female and male mice islets

Control experiments showed no differences between female and male mice islets with regard to mRNA expression for ERα and ERβ (Fig. 1A). Notably ERα expression was almost 10-fold higher than ERβ (p < 0.01) in both female and male mice islets (Fig. 1A). However, the expression of GPR30 mRNA relative to house keeping gene GAPDH was found to be higher in female [(9.5 ± 1.0) × 10−3] compared to male mice islets [(5.5 ± 0.7) × 10−3]; (p < 0.01; n = 10 in each group) (Fig. 1A). The mRNA expression of GPR30 in the

Discussion

It has been described recently that the putative membrane-associated estrogen receptors either are intimately linked to or distinct from the classical nuclear receptors (Filardo et al., 2007, Filardo et al., 2002, Filippo Acconcia, 2003, Govind and Thampan, 2003). Although still controversial, current studies point at the involvement of a G protein-coupled receptor for non-genomic, rapid effects of estrogen in different tissues including excitable cells such as neurons and endocrine cells (

Acknowledgements

The technical assistance of Britt-Marie Nilsson is gratefully acknowledged and F. Labrie is also acknowledged for kind supply of EM-652.

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    This study was supported by the Swedish Science Council; Medical Faculty, Lund University, Novo Nordic Foundation, Øresund Diabetes Academy and Albert Påhlsson foundation.

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