GPR30 mediates anorectic estrogen-induced STAT3 signaling in the hypothalamus

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Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

GPR30 mediates anorectic estrogen-induced STAT3 signaling in the hypothalamus Obin Kwon a, b, 1 , Eun Seok Kang c, 1 , Insook Kim d , Sora Shin a, b , Mijung Kim a , Somin Kwon a , So Ra Oh a, e , Young Soo Ahn a,⁎, Chul Hoon Kim a, b, e,⁎⁎ a

Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea c Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea d Division of Metabolic Disease, Department of Biomedical Science, National Institutes of Health, Osong, Korea e Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea b

A R T I C LE I N FO Article history:

AB S T R A C T Objective. Estrogen plays an important role in the control of energy balance in the

Received 24 December 2013

hypothalamus. Leptin-independent STAT3 activation (i.e., tyrosine705-phosphorylation of

Accepted 29 July 2014

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

Keywords:

this regulation is unknown. We investigated the role of the G protein-coupled receptor 30

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

Estrogen STAT3

and G-15, a specific antagonist of GPR30 was analyzed in vitro and in vivo. Effect of GPR30

Food intake

activation on eating behavior was analyzed in vivo.

Hypothalamus

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. © 2014 Elsevier Inc. All rights reserved.

1.

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

Abbreviations: CNS, central nervous system; DAB, 3,3′-diaminobenzidine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; ER, estrogen receptor; E2, estradiol; GPR30, G protein-coupled receptor 30; ICV, intracerebroventricular; JAK, Janus kinase; PLC, phospholipase C; POMC, proopiomelanocortin; pSTAT3, tyrosine705-phosphorylation of signal transducer and activator of transcription 3; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; STAT3, signal transducer and activator of transcription 3. ⁎ Correspondence to: Y. S. Ahn, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Tel.: +82 2 2228 1736; fax: +82 2 313 1894. ⁎⁎ Correspondence to: C. H. Kim, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Tel.: + 82 2 2228 1738; fax: +82 2 313 1894. E-mail addresses: [email protected] (Y.S. Ahn), [email protected] (C.H. Kim). 1 These authors equally contributed to this work. http://dx.doi.org/10.1016/j.metabol.2014.07.015 0026-0495/© 2014 Elsevier Inc. All rights reserved.

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(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 ligandactivated 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 proteincoupled 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 GPR30mediated 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.

2.

Materials and methods

2.1.

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, USA).

2.2.

Cell line and primary hypothalamic neuron culture

HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum in a 5% CO2 atmosphere at 37 °C. Primary hypothalamus cultures were extracted from Sprague Dawley rat embryos on the 18th day of gestation using a technique reported in Ref. [18] with modifications. Briefly, medial portions of the hypothalamic tissue were dissected and incubated at 37 °C for 10 min in a solution containing 0.05% trypsin in Hank’s balanced salt solution (HBSS). The tissue was washed and triturated in HBSS by pipetting. Cells were centrifuged and the resulting pellet was re-suspended in Neurobasal medium supplemented with B-27 and 2 mmol/L L-glutamine. Cells were plated onto 6-well plates that were pre-coated with poly-Dlysine, and cultured in a 5% CO2 atmosphere at 37 °C. Primary cultures were used for drug treatment 2 weeks after plating.

2.3.

Transfection and Western blot

HeLa cells were transfected with either human GPR30 cDNA or a mock vector by Lipofectamine (Invitrogen, Grand Island, NY, USA). Cells were starved of serum and treated with E2 or G-1 48 h after transfection. For Western blot, HeLa cells or primary hypothalamic neurons were lysed with 1% Triton X-100 plus 0.1% SDS solution containing protease/phosphatase inhibitors. Lysates were separated by SDS-polyacrylamide gel electrophoresis and then subjected to immunoblotting analysis using antibodies for STAT3 (1:5000), phospho-STAT3 (1:1000), and GPR30 (1:1000).

2.4.

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA from MCF-7 and HeLa cells were isolated and extracted, and the cDNA was synthesized as templates. The following primer sequences for human ERα and ERβ were used: ERα forward 5′-CTACTGCATCAGATCCAAGG-3′ and reverse 5′-GTCATTGGTACTGGCCAATCT-3′, ERβ forward 5′CGATGCTTTGGTTTGGGTGAT-3′ and reverse 5′GCCCTCTTTGCTTTTACTGTC-3′. The following PCR reaction conditions were used: 95 °C (5 min), followed by 34 cycles at 95 °C (30 sec)/53 °C (60 sec) for ERα or 57 °C (60 sec) for ERβ/ 72 °C (60 sec), followed by 72 °C (5 min).

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2.5.

Measurement of intracellular calcium

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Changes in intracellular [Ca2+]i were measured by the method described by Kim et al. [19] with modifications. HeLa cells attached to coverslips were transfected with human GPR30 cDNA or a mock vector. The cells were preloaded with Fura-2 for 30 min at 37 °C and stimulated by the perfusion of E2 solution. [Ca2+]i was measured with excitation at 340 and 380 nm and emission at 510 nm.

of vehicle solution or G-1 (0.1 mg/kg over 2 min). Other groups of mice (n = 12–13) were treated with two consecutive ICV injections as follows: vehicle + vehicle (control group), vehicle + E2 (0.1 mg/kg), G-15 (0.9 mg/kg) + E2 (0.1 mg/kg). The second compound was injected 20 min after the first injection. Immediately following the injection, mice were fed with pre-weighed bait pellets. Food intake was measured 2, 4, 8, and 24 h after injection and the mice were weighed 24 h after injection.

2.6.

2.10.

Animals

Male C57BL/6 mice 8 weeks of age were obtained from Orient Bio (Seoul, Korea). Animals were housed in a controlled temperature environment under a 12:12 light:dark cycle and fed a standard chow diet ad libitum. All procedures were conducted in accordance with the Yonsei University College of Medicine Animal Care and Use Committee and the NIH Guide for the Care and Use of Laboratory Animals.

2.7.

Stereotaxic injection

Anesthetized mice were placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA). A 26-gauge cannula was implanted into the lateral ventricle (0.5 mm caudal to the bregma and 1.0 mm lateral to the midline, 1.5 mm below the surface of the skull) and sealed with a dummy cannula until drug injection. Mice were allowed to recover for 7 days prior to the injection experiment. G-1 was dissolved in dimethylformamide (DMF) and G-15 was dissolved in dimethyl sulfoxide (DMSO). Each solution was then diluted with phosphate buffered saline (PBS) or ethanol. E2 was dissolved in PBS. Mice were starved for 18 h before intracerebroventricular (ICV) injections. Each single injection included 2 μl of volume that was administered over 2 min.

2.8. ICV drug administration and immunohistochemical staining Independent groups of mice (n = 4–5) were treated with two consecutive ICV injections as follows: vehicle + vehicle (control group); vehicle + G-1 (0.1 mg/kg); G-15 (1.5 mg/kg) + G-1 (0.1 mg/kg). The second compound was injected 15 min after the first injection. Thirty minutes after the second injection, anesthetized mice were intracardially perfused with 4% paraformaldehyde. Brain coronal slices, including slices from the hypothalamus, were incubated overnight with a rabbit phospho-STAT3 antibody (1:100) at 4 °C, followed by incubation with biotinylated anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA, USA). Immunoreactive proteins were visualized by incubation with 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories, Burlingame, CA, USA). Tissue slices were mounted and viewed using a light microscope (Carl Zeiss, Jena, Germany). The number of tyrosine705-phosphorylated STAT3-positive neurons in the hypothalamus was quantified using the ImageJ analysis software (NIH, version 1.47).

2.9.

Measurement of food intake and body weight change

Independent groups of mice (n = 6–7) were weighed just before ICV injection and then treated with a single injection

Statistical analysis

All data are reported as means ± SEM. Statistical comparisons were made using a Mann–Whitney U test and a one-way analysis of variance (ANOVA) followed by Bonferroni post-tests.

3.

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 tyrosine 705 -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 with increasing concentrations of E2. E2 increased the phosphorylation of STAT3 in a dose-dependent manner in these transfected HeLa cells (Fig. 1B). To ensure that the HeLa cells used in this experiment did not express ERα or ERβ that are also able to stimulate pSTAT3 through a non-genomic action [21], we performed RT-PCRs. The expressions of both receptors were not observed in HeLa cells; in contrast, both receptors were expressed in MCF-7 cells, which we used as a positive control (Fig. 1C). To confirm that the overexpressed GPR30 was functional in the transfected HeLa cells, we measured the level of calcium transients, which is another non-genomic response of GPR30 activation. E2 elicited calcium transients in cells overexpressing GPR30, but not in non-transfected control cells (Fig. 1D). GPR30 is also reported to transactivate EGFR, which is sensitive to pertussis toxin (which indicates an association with Gi/o proteins), in cancer cells [11]. To examine whether EGFR transactivation occurs with STAT3 phosphorylation in the HeLa cells transfected with GPR30, we measured E2-induced STAT3 phosphorylation following a pretreatment with either AG1478, an EGFR inhibitor, or pertussis toxin. AG1478 and pertussis toxin both inhibited E2-induced STAT3 phosphorylation in cells expressing GRP30 (Fig. 1E). However, U73122, a phospholipase C (PLC) inhibitor, did not affect E2-induced STAT3 activation (Fig. 1E), which suggests that the selective pathway from various GPR30-initiated signals is involved in STAT3 activation. Together these results indicate that GPR30 is functional and sufficient for mediating E2-induced STAT3 activation in HeLa cells expressing GPR30. To further prove that GPR30 mediates the effects of E2 in hypothalamic neurons, we treated both HeLa cells transfected with GPR30 and primary cultures of hypothalamic neurons

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Fig. 1 – E2 induces tyrosine 705-phosphorylation of STAT3. A. Tyrosine 705-phosphorylation of STAT3 detected by a Tyr 705-phosphorylated STAT3-specific monoclonal antibody 30 min after E2 treatment. Total STAT3 was used as a loading control. B. STAT3 phosphorylation in HeLa cells transfected with pcDNA3-GPR30 30 min after E2 treatment. C. PCR analysis of ERα and ERβ mRNA expression in HeLa cells. MCF-7 cells were used as a positive control. D. HeLa cells were transfected with pcDNA3-GPR30 or a mock vector. E2-induced (1 nmol/L) calcium transients were measured over 10 min using a ratiometric calcium indicator, Fura-2. E. HeLa cells expressing GPR30 were pretreated with U73122 (10 μmol/L), pertussis toxin (PTX) (100 ng/mL), or AG1478 (50 μmol/L) 30 min prior to treatment with E2 (1 nmol/L) for 30 min. The Tyr705-phosphorylation of STAT3 was measured using a phospho-STAT3 (Tyr705) antibody.

with G-1, a GPR30-specific agonist [15]. G-1 increased STAT3 phosphorylation in both GPR30-expressing HeLa cells and primary cultures of hypothalamic neurons (Fig. 2A). A dosedependent increase in STAT3 phosphorylation by G-1 was observed in the primary cultures of hypothalamic neurons (Fig. 2B). To determine whether E2-STAT3 signaling is mediated by GPR30, the hypothalamic neurons were pretreated with G-15 (100 nmol/L), a GPR30-specific antago-

nist [16], and then stimulated with E2 (10 nmol/L). G-15 inhibited the activation of STAT3 by E2 in hypothalamic neurons, which suggests that GPR30 mediates the E2 effect on STAT3 signaling in the hypothalamic neurons (Fig. 2C). Because the anorectic action of E2 is mediated by STAT3 signaling in the hypothalamic neurons of the arcuate nucleus, we tested whether the in vivo administration of G-1 increases the number of phosphorylated STAT3-immunopositive cells.

Fig. 2 – GPR30 activation induces STAT3 activation. A. G-1 (100 nmol/L), a GPR30-specific agonist, stimulated tyrosine 705-phosphorylation in HeLa cells overexpressing GPR30 and primary cultures of hypothalamic neurons. B. A dose-dependent increase in STAT3 phosphorylation by G-1 was observed in the primary cultures of hypothalamic neurons. C. Primary cultures of hypothalamic neurons were pretreated with G-15 (100 nmol/L) for 15 min, then treated with E2 (10 nmol/L) for additional 30 min. STAT3 phosphorylation was analyzed using phospho-STAT3 (Tyr705) antibody.

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Cerebroventricular injection of G-1 (0.1 mg/kg, over 2 min) increased the number of pSTAT3-positive neurons in the arcuate nucleus, an increase which was blocked by G-15 (Fig. 3A). As shown in Fig. 3B, the percentage of pSTAT3 neurons increased 4.5-fold in G-1-treated mice compared with control mice and significantly decreased by 65% in G-15pretreated mice compared with G-1-treated mice. These results indicate that GPR30 can activate the anorectic STAT3 signaling pathway in the arcuate nucleus. A change in the number of pSTAT3-positive neurons was hypothesized to affect the feeding behavior of the G-1-treated mice. To test this, G-1 was stereotaxically injected into ventricular space of mice and then the consumption of food and the changes in body weight were measured for 24 h. Food intake and body weight gain decreased over 24 h when G-1 was administered into the ventricular space of mice after overnight fasting compared with control mice (Fig. 4A and B). Furthermore, mice injected with E2 into ventricular space also showed decreased food intake and body weight gain, which was inhibited by pretreatment with G-15 (Fig. 4C and D). These results indicate that GPR30-stimulated STAT3 activation plays a role in the regulation of feeding behavior in the hypothalamus.

4.

Discussion

STAT3 signaling plays critical roles in the hypothalamusmediated 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

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plays a crucial role as an anorexigenic and catabolic hormone. Interestingly, the treatment of STAT3-ablated mice with E2 has revealed the role of estrogen as an additional anorexigenic hormone with actions in the hypothalamus. Estrogen adopts the same pathway as leptin to exert its anorexigenic effect, and the anorectic action of estrogen occurs even in leptinreceptor deficient (db/db) and leptin-deficient (ob/ob) mice [2]. The effects of estrogen on food intake and body weight are abrogated in mice with a nestin-Cre knockout of STAT3, which indicates that STAT3 is a crucial signaling molecule that mediates estrogen as well as leptin [2]. The activation of STAT3 in the arcuate nucleus by G-1 further supports the anorexigenic effect of estrogen, and indicates that this effect can be mediated by the transmembrane estrogen receptor, GPR30. The results of our study also imply that, like leptin, the anorectic effect of estrogen can be mediated initially by stimulating cytoplasmic signaling molecules rather than binding estrogen response element as an estrogen–ER complex to regulate transcription of target genes. In the later phases, this non-genomic response is linked to the genomic response, for example, transcriptional activation of proopiomelanocortin (POMC) by pSTAT3. Estrogen appears to share the strong anorectic signaling pathway in the hypothalamus with leptin through GPR30. Several questions regarding the effect of estrogen on the STAT3 pathway remain. For example, it remains unclear whether ERα is involved in estrogen-stimulated STAT3 activation in the hypothalamus. ERα is highly expressed in the hypothalamic neurons that are involved in the regulation of food intake [5], and the metabolic phenotype of ERα-knockout mice is similar to leptin- or leptin receptor-deficient mice [9]. However, ovariectomy itself produced the difference in food intake and

Fig. 3 – G-1 activates STAT3 in mouse arcuate nucleus. A. Immunohistochemical detection of tyrosine705-phosphorylated STAT3-positive neurons in coronal sections of hypothalamus (30 μm) obtained from 12-week-old mice injected with G-1 (0.1 mg/kg) or G-15 (1.5 mg/kg) into ventricular space. Co-treated mice were pretreated with G-15 15 min prior to treatment with G-1 for 30 min. B. Quantification of the number of phospho-STAT3 (Tyr705)-positive cells (mean ± SEM) in the arcuate nucleus. ARC, arcuate nucleus; VMN, ventromedial nucleus; Veh, vehicle. The statistical analysis was carried out by ANOVA followed by Bonferroni post-tests. **P< 0.01 versus vehicle control. ††P< 0.01 versus G-1. The ARC regions were used for quantification of phosphorylated STAT3-positive cells.

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Fig. 4 – GPR30 activation reduces food intake and body weight in mice. (A and B) Effect of intracerebroventricular (ICV) administration of G-1 (0.1 mg/kg) on (A) food intake and (B) body weight in overnight-fasted 12-week-old mice (n = 6 for control, n = 7 for G-1-treated mice). The statistical analysis was carried out by Mann–Whitney U test. *P<0.05 versus vehicle control. (C and D) Effect of ICV administration of E2 (0.1 mg/kg) and G-15 (0.9 mg/kg) on (C) food intake and (D) body weight in overnightfasted 12-week-old mice (n = 12 for control and G-15-pretreated mice, n = 13 for E2-treated mice). G-15 was administered 20 min prior to treatment with E2. Food intake and body weight change were measured at 24 h after E2 treatment. The statistical analysis was carried out by ANOVA followed by Bonferroni posttests. **P<0.01, ***P<0.001 versus vehicle control. †P<0.05 versus E2-treated mice.

body weight between wild-type and ERα-knockout mice, which make it difficult to delineate the role of ERα in mediating E2 response. Although ERα has been shown to activate STAT3 signaling through non-classical pathway in non-neuronal cell types, this phenomenon has not yet been observed in the hypothalamus. Interestingly, leptin-induced STAT3 activation is impaired in ERα-knockout mice, which suggests that crosstalk exists between ERα and leptin-induced STAT3 signaling [22]. Given that E2 can activate STAT3 signaling in the arcuate nucleus independent from leptin [2], GPR30-mediated STAT3 activation may represent another self-contained pathway for the anorexigenic action of estrogen that is distinct from ERα-initiated signaling. Our results showed that the activation of GPR30 alone is able to initiate the STAT3 signaling pathway and a GPR30specific antagonist inhibited E2-induced STAT3 activation and anorectic behavior. Considering the important roles of estrogen in female metabolism, use of only male mice here is a limitation of this study, which necessitates the future research into estrogen’s

effects on food intake and weight gain in both male and female. A more detailed analysis of STAT3 signaling in ERα-knockout mice as well as a study to probe the possible interaction or crosstalk between GPR30 and ERα in STAT3 signaling will further our understanding about the pharmacology and mechanism of anorectic estrogen in the hypothalamus. Our immunohistochemical data indicate that G-1-responsive neurons (pSTAT3-positive) are confined to the arcuate nucleus, yet GPR30 is reportedly expressed in the arcuate nucleus, ventromedial nucleus, and paraventricular/periventricular nucleus [5]. We do not know the reason why GPR30-expressing cells present in the other hypothalamic nuclei do not activate the STAT3 pathway in response to G-1, but it might suggest that the GPR30-STAT3 pathway has a more dedicated role in “first-order neurons” in the anorexigenic pathway in the arcuate nucleus. The physiological role of GPR30 signaling as distinct from the role of leptin signaling also remains unclear. Leptin is an anti-obesity hormone that is secreted from adipocytes to signal energy status in the body to the CNS [23]. In contrast, estrogen does not convey energy balance information to the CNS. Therefore, it is not clear whether the functions of leptin and estrogen overlap or if there is an adaptive advantage for having two hormones that act on the same anorectic pathways in the hypothalamus. Future studies that explore whether the simultaneous or sequential STAT3 activation that is elicited by two different receptors is additive, synergistic, or occlusive in different types of hypothalamic neurons will support and further clarify the role of GPR30 in hypothalamic feeding and energy control. In this study, we used specific agonists and antagonists to show that GPR30 regulates the estrogen-induced anorexigenic signaling pathway in the arcuate nucleus. GPR30 may therefore be a novel target for anti-obesity drugs. We hypothesize that the GPR30-mediated anorectic effect of estrogen in the hypothalamus may provide a basis for future therapeutic interventions.

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.

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. 20070056092 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 Yonsei-Carl Zeiss Advanced Imaging Center, Yonsei University College of Medicine, for technical assistance. We thank Dong-Su Jang (Medical Illustrator, Medical Research

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Support Section, Yonsei University College of Medicine) for his help with the figure.

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

REFERENCES

[1] Mauvais-Jarvis F, Clegg DJ, Hevener AL. The role of estrogens in control of energy balance and glucose homeostasis. Endocr Rev 2013;34(3):309–38. [2] Gao Q, Mezei G, Nie Y, et al. Anorectic estrogen mimics leptin’s effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals. Nat Med 2007;13(1):89–94. [3] Bates SH, Stearns WH, Dundon TA, et al. Stat3 signalling is required for leptin regulation of energy balance but not reproduction. Nature 2003;421(6925):856–9. [4] Revankar CM, Cimino DF, Sklar LA, et al. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 2005;307(5715):1625–30. [5] Hazell GG, Yao ST, Roper JA, et al. Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues. J Endocrinol 2009;202(2):223–36. [6] Li X, Huang J, Yi P, et al. Single-chain estrogen receptors (ERs) reveal that the ERalpha/beta heterodimer emulates functions of the ERalpha dimer in genomic estrogen signaling pathways. Mol Cell Biol 2004;24(17):7681–94. [7] Sirianni R, Chimento A, Ruggiero C, et al. The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17beta-estradiol on mouse spermatogonial GC-1 cell line. Endocrinology 2008; 149(10):5043–51. [8] Harris HA. Estrogen receptor-beta: recent lessons from in vivo studies. Mol Endocrinol 2007;21(1):1–13. [9] Geary N, Asarian L, Korach KS, et al. Deficits in E2-dependent control of feeding, weight gain, and cholecystokinin satiation in ER-alpha null mice. Endocrinology 2001;142(11):4751–7.

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[10] Sharma G, Hu C, Brigman JL, et al. GPER deficiency in male mice results in insulin resistance, dyslipidemia, and a proinflammatory state. Endocrinology 2013;154(11):4136–45. [11] Filardo EJ, Quinn JA, Bland KI, et al. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000;14(10):1649–60. [12] Prossnitz ER, Arterburn JB, Smith HO, et al. Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annu Rev Physiol 2008;70:165–90. [13] Terasawa E, Noel SD, Keen KL. Rapid action of oestrogen in luteinising hormone-releasing hormone neurones: the role of GPR30. J Neuroendocrinol 2009;21(4):316–21. [14] McAllister CE, Creech RD, Kimball PA, et al. GPR30 is necessary for estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the rat hypothalamus. Psychoneuroendocrinology 2012;37(8):1248–60. [15] Bologa CG, Revankar CM, Young SM, et al. Virtual and biomolecular screening converge on a selective agonist for GPR30. Nat Chem Biol 2006;2(4):207–12. [16] Dennis MK, Burai R, Ramesh C, et al. In vivo effects of a GPR30 antagonist. Nat Chem Biol 2009;5(6):421–7. [17] Zucchetti AE, Barosso IR, Boaglio AC, et al. G-protein-coupled receptor 30/adenylyl cyclase/protein kinase a pathway is involved in estradiol 17β-D-glucuronide-induced cholestasis. Hepatology 2014;59(3):1016–29. [18] Vaccaro DE, Leeman SE, Messer A, et al. Primary cultures of dispersed hypothalamic cells from fetal rats: morphology, electrical activity, and peptide content. J Neurobiol 1980;11(4): 417–24. [19] Kim CH, Kim JH, Moon SJ, et al. Pyrithione, a zinc ionophore, inhibits NF-kappaB activation. Biochem Biophys Res Commun 1999;259(3):505–9. [20] Green S, Walter P, Kumar V, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 1986;320(6058):134–9. [21] Bjornstrom L, Sjoberg M. Signal transducers and activators of transcription as downstream targets of nongenomic estrogen receptor actions. Mol Endocrinol 2002;16(10):2202–14. [22] Park CJ, Zhao Z, Glidewell-Kenney C, et al. Genetic rescue of nonclassical ERalpha signaling normalizes energy balance in obese ERalpha-null mutant mice. J Clin Invest 2011;121(2):604–12. [23] Ahima RS. Central actions of adipocyte hormones. Trends Endocrinol Metab 2005;16(7):307–13.