Elsevier

Metabolism

Volume 63, Issue 11, November 2014, Pages 1455-1461
Metabolism

Basic Science
GPR30 mediates anorectic estrogen-induced STAT3 signaling in the hypothalamus

https://doi.org/10.1016/j.metabol.2014.07.015Get rights and content

Abstract

Objective

Estrogen plays an important role in the control of energy balance in the hypothalamus. Leptin-independent STAT3 activation (i.e., tyrosine705-phosphorylation of STAT3, pSTAT3) in the hypothalamus is hypothesized as the primary mechanism of the estrogen-induced anorexic response. However, the type of estrogen receptor that mediates this regulation is unknown. We investigated the role of the G protein-coupled receptor 30 (GPR30) in estradiol (E2)-induced STAT3 activation in the hypothalamus.

Materials/methods

Regulation of STAT3 activation by E2, G-1, a specific agonist of GPR30 and G-15, a specific antagonist of GPR30 was analyzed in vitro and in vivo. Effect of GPR30 activation on eating behavior was analyzed in vivo.

Results

E2 stimulated pSTAT3 in cells expressing GPR30, but not expressing estrogen receptor ERα and ERβ. G-1 induced pSTAT3, and G-15 inhibited E2-induced pSTAT3 in primary cultures of hypothalamic neurons. A cerebroventricular injection of G-1 increased pSTAT3 in the arcuate nucleus of mice, which was associated with a decrease in food intake and body weight gain.

Conclusions

These results suggest that GPR30 is the estrogen receptor that mediates the anorectic effect of estrogen through the STAT3 pathway in the hypothalamus.

Introduction

Estrogen reduces food intake and body weight and promotes an increase in the ratio of subcutaneous to visceral fat in both animals and humans [1]. The main targets of this CNS-mediated metabolic effect of estrogen are the hypothalamic nuclei. A lack of hypophagia and weight loss has been observed in STAT3-knockout mice following estradiol (E2) treatment [2], which suggests that the anorectic action of estrogen is mediated by STAT3 activation in the arcuate nucleus of the hypothalamus. This anorectic action of estrogen also appears to be independent of leptin, a hormone that exerts its effects on energy homeostasis via the JAK-STAT pathway in the hypothalamus [2], [3]. However, the estrogen receptor that mediates the STAT3-dependent anorexigenic effect of estrogen has not been identified.

The estrogen receptors (ERs), ERα and ERβ, are ligand-activated nuclear transcription factors that are members of the nuclear receptor superfamily. G protein-coupled receptor 30 (GPR30), which was originally identified as an orphan G protein-coupled receptor, also interacts with estrogen and is involved in the rapid effects of estrogen [4]. ERα, ERβ, and GPR30 are all reportedly expressed in several nuclei of the hypothalamus, which suggests that all three receptors play important roles in hypothalamic regulation [5]. ERα and ERβ can form homodimers as well as heterodimers [6], and GPR30 has been shown to interact and crosstalk with ERα [7]. Thus, it is plausible that the anorectic effect of estrogen is mediated by the activation of either a single type of receptor or a composite of GPR30-mediated rapid signaling and ER-mediated nuclear events. Before the discovery of GPR30-estrogen binding, the action of estrogen in the hypothalamus was assumed to be mediated by ERα and the role of ERβ in the regulation of energy balance is negligible [1], [8]. For example, ERα-knockout mice did not show E2-induced reduction of food intake and body weight compared with wild-type mice [9]. Increased body weight has also been observed in mice that lack GPR30, which indicates a potential role of GPR30 in the estrogen-mediated control of energy balance [10]. Despite these findings, it is difficult to address how ERs and GPR30 act alone or in combination to regulate feeding behavior and weight due to a lack of experimental evidence that links specific receptors to the anorectic signaling pathway.

The rapid action of the membrane-bound estrogen receptor, GPR30, has attracted considerable attention as a potential new regulatory mechanism of the estrogen metabolic network. GPR30 couples with trimeric G proteins to initiate diverse rapid signaling events. GPR30 activation has been linked to protein kinase A, protein kinase C, and the pertussis-sensitive transactivation of epidermal growth factor receptor (EGFR) [11], [12]. GPR30 plays an important role in the regulation of calcium oscillations in luteinizing hormone-releasing neurons in the hypothalamus and in the control of serotonin receptor signaling in the paraventricular nucleus [13], [14].

The development of the GPR30-specific agonist, G-1, and the GPR30-specific antagonist, G-15, has facilitated studies exploring the role of GPR30 in mediating estrogen’s actions in vivo independent from the role of ERs [15], [16]. The blockade of estrogen effects by GPR30-specific antagonists demonstrates that some of estrogen’s reproductive and non-reproductive functions are mediated through GPR30 rather than the ERs [17]. However, the contribution of GPR30 activation to the anorectic STAT3 pathway in the hypothalamus remains underexplored. In this study, we demonstrate that the activation of GPR30 by estrogen can initiate STAT3 phosphorylation, which leads to the activation of the anorectic pathway in the arcuate nucleus of hypothalamus.

Section snippets

Materials

All cell culture reagents, media, and sera were purchased from Invitrogen (Carlsbad, CA, USA) except for Dulbecco’s modified Eagle’s medium (DMEM), which was purchased from Welgene (Daegu, Korea). G-1 and G-15 were purchased from Tocris (Bristol, UK), and water-soluble β-estradiol (E2) was purchased from Sigma (St. Louis, MO, USA). All primary antibodies were purchased from Cell Signaling (Danvers, MA, USA) except the GPR30 antibody, which was purchased from Novus Biologicals (Littleton, CO,

Results

To test whether E2 can increase the phosphorylation of STAT3 in hypothalamic neurons, we cultured primary hypothalamic neurons from rat embryos and added E2 to the bathing media for 30 min. A Western blot analysis using tyrosine705-phosphorylated STAT3 (pSTAT3) antibody showed that E2 increased STAT3 phosphorylation (Fig. 1A). Because E2 can act on both ERα and GPR30, we transfected HeLa cells, which are reported to be ERα- and ERβ-negative [20], with GPR30 constructs and treated these cells

Discussion

STAT3 signaling plays critical roles in the hypothalamus-mediated regulation of food intake, energy expenditure, and body weight. Leptin, a potent anorexigenic hormone secreted by white adipocytes, binds to leptin receptor-b (LRb) and activates the JAK-STAT signaling pathway to reduce food intake and increase energy expenditure [3]. Leptin therefore plays a crucial role as an anorexigenic and catabolic hormone. Interestingly, the treatment of STAT3-ablated mice with E2 has revealed the role of

Author contributions

OK, IK, SS, MK, SK, and SRO performed experiments and statistical analyses, ESK and OK collected data, ESK, YSA and CHK designed the study and oversaw its performance. OK, SK, ESK, YSA, and CHK contributed to writing the manuscript.

Conflict of interest

The authors state that there is no duality of interest associated with this manuscript.

Acknowledgements

This work was supported by the Faculty Fund from Yonsei University College of Medicine (No. 6-2009-0079 No. 6-2009-0079 to Y. S. Ahn) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (No. 2007-0056092 to C. H. Kim and NRF-2012000891 to E. S. Kang) and a grant from Korea Health Care Technology R&D Project, Ministry of Health, Welfare and Family Affairs (No. HI09C13420200 to CHK). We thank

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