Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice

N U TR IT ION RE S E ARCH XX ( 2 0 15 ) X XX– X XX

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Original Research

Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice Na-Ra Han a, 1 , Hee-Yun Kim a, 1 , Woong Mo Yang b , Hyun-Ja Jeong c,⁎, Hyung-Min Kim a,⁎⁎ a

Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea College of Korean Medicine and Institute of Korean Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea c Department of Food Technology and Inflammatory Disease Research Center, Hoseo University, Asan, Chungcheongnam-do, 336-795, Republic of Korea b

ARTI CLE I NFO

A BS TRACT

Article history:

Some amino acids are considered alternative therapies for improving menopausal

Received 28 February 2015

symptoms. Glutamic acid (GA), which is abundant in meats, fish, and protein-rich plant

Revised 15 May 2015

foods, is known to be a neurotransmitter or precursor of γ-aminobutyric acid. Although it is

Accepted 9 June 2015

unclear if GA functions in menopausal symptoms, we hypothesized that GA would

Keywords:

hypothesis was to examine an estrogenic effect of GA in ovariectomized (OVX) mice,

Glutamic acid

estrogen receptor (ER)–positive human osteoblast–like MG-63 cells, and ER-positive human

Microcomputed tomography

breast cancer MCF-7 cells. The results demonstrated that administration with GA to mice

Alkaline phosphatase

suppressed body weight gain and vaginal atrophy when compared with the OVX mice. A

Estrogen receptor-β

microcomputed tomographic analysis of the trabecular bone showed increases in bone

Estrogen response element

mineral density, trabecular number, and connectivity density as well as a significant

Extracellular signal-regulated kinase

decrease in total porosity of the OVX mice treated with GA. In addition, GA increased serum

attenuate estrogen deficiency–induced menopausal symptoms. The objective to test our

phosphorylation

levels of alkaline phosphatase and estrogen compared with the OVX mice. Furthermore, GA induced proliferation and increased ER-β messenger RNA (mRNA) expression, estrogen response element (ERE) activity, extracellular signal–regulated kinase phosphorylation, and alkaline phosphatase activity in MG-63 cells. In MCF-7 cells, GA also increased proliferation, Ki-67 mRNA expression, ER-β mRNA expression, and ERE activity. Estrogen response element activity increased by GA was inhibited by an estrogen antagonist. Taken together, our data demonstrated that GA has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. © 2015 Elsevier Inc. All rights reserved.

Abbreviations: μCT, microcomputed tomography; 3D, 3-dimensional; ALP, alkaline phosphatase; BMD, bone mineral density; BrdU, bromodeoxyuridine; Conn.D, connectivity density; DMSO, dimethyl sulfoxide; E2, estrogen; ER, estrogen receptor; ERE, estrogen response element; ERK, extracellular signal–regulated kinase; FSH, follicle-stimulating hormone; GA, glutamic acid; HRT, hormone replacement therapy; mRNA, messenger RNA; OVX, ovariectomized; PCR, polymerase chain reaction; Tb.N, trabecular number. ⁎ Correspondence to: H.J. Jeong. Tel.: +82 41 540 9681; fax: +82 41 542 9681. ⁎⁎ Correspondence to: H.M. Kim. Tel.: +82 2 961 9448; fax: +82 2 967 7707. E-mail addresses: [email protected] (H.-J. Jeong), [email protected] (H.-M. Kim). 1 Na-Ra Han and Hee-Yun Kim contributed equally to this report. http://dx.doi.org/10.1016/j.nutres.2015.06.006 0271-5317/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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1.

Introduction

Although menopause is a natural biological period during the female lifecycle, many women experience metabolic syndromes that include obesity or systemic skeletal diseases, such as osteoporosis, due to a deficiency of estrogen (E2) during this period [1]. Estrogen is a key regulator of growth and function in tissues, such as the reproductive tract or skeletal system [2]. Osteoporosis is characterized by a systemic decrease in bone density and increases in the possibility of fragility fractures [3]. Hormone replacement therapy (HRT) is often conducted to alleviate these symptoms and to protect women against an E2 deficiency. However, because HRT also increases the risk of breast cancer [4], menopausal women often seek complementary and alternative therapies for the symptoms [5]. Glutamic acid (GA) clearly plays a role in the biosynthesis of arginine, which is an essential amino acid [6]. An arginine diet decreases hot flashes and endothelial dysfunction in postmenopausal women [7]. A deficiency of amino acids, including GA, in menopause contributes to metabolic and cardiovascular risks [8]. Osteocalcin functions as a regulator of bone mineral maturation via vitamin K–dependent GA carboxylation [9]. Estrogen modulates cognitive functions by raising GA-induced intracellular calcium, and HRT can maintain cognitive functions during menopause [10]. Porcine placenta, which is a reservoir of a large number of amino acids including GA, enhances neuroprotection and cognition in postmenopausal women [11,12]. The predominant biological effects of E2 are induced through intracellular E2 receptors (ERs), ER-α and ER-β[13]. The biological function of the ER is mediated through the ability of ER to regulate the expression of genes containing an E2 response element (ERE) sequence in their promoter [14]. Estrogen-ER complexes binding to an ERE regulate gene transcription and subsequent tissue responses, such as cell proliferation [2]. Cell survival and proliferation are mediated principally through extracellular signal–regulated kinase (ERK) MAPK pathways [15]. Alkaline phosphatase (ALP) activity, which is a marker of osteoblast differentiation and bone formation [16], is up-regulated via the activation of ERK signaling [3]. The analysis of bone cell–specific markers, such as ALP, is frequently used to characterize osteoblasts [17]. We hypothesized that dietary GA would lessen E2 deficiency–induced menopausal symptoms. To investigate this hypothesis, we attempted to clarify the mechanism of E2-like activity of GA on menopausal-like symptoms in a mouse model of E2 loss. The approach was to investigate the effect of GA on menopausal-like symptoms in ovariectomized (OVX) mice that present loss of ovary functions [18]. We also examined if GA would have an E2-like function such as proliferation and ALP activity using ER-positive human osteoblast–like MG-63 cells and ER-positive human breast cancer MCF-7 cells.

2.

Methods and materials

2.1.

Glutamic acid and E2 preparation

Glutamic acid (no. G1251, minimum 99% pure; Sigma Chemical Co, St Louis, MO, USA) was dissolved in distilled water and

prepared at a dose of 10 mg/kg, based on previous reports [19,20]. Estrogen (no. E8875; Sigma Chemical Co) was dissolved in 1% dimethyl sulfoxide (DMSO) and prepared at a dose of 100 nmol/L, based on a previous report [21]. Genistein (no. G6649; Sigma Chemical Co) and fulvestrant (no. I4409; Sigma Chemical Co) were dissolved in DMSO and prepared at a dose of 1 μmol/L, respectively, according to previous reports [22,23].

2.2.

Animal study design

Female mice (7-week-old Balb/c) were purchased from DaeHan Experimental Animal Center (Eumsung, Republic of Korea). The mice were acclimatized for 2 weeks to local vivarium conditions. Ovariectomy was conducted, as described previously [24]. Briefly, mice were anesthetized with a combination of Zoletil and Rompun, and their ovaries were bilaterally removed. The mice in the sham-operated group were anesthetized, laparotomized, and sutured but leaving their ovaries. After 3 weeks of recovery from their ovariectomies, the mice were divided into 4 groups (n = 5 per group): sham, OVX, OVX administered orally with GA using an oral sonde (10 mg/kg per day), and OVX administered orally with E2 using an oral sonde (100 nmol/L per day). Glutamic acid or E2 was given to OVX mice consecutively for 8 weeks, based on previous reports [25,26]. The body weights of all groups were measured once a week until the last day of administration. After the mice were euthanized by cervical dislocation, blood samples and tissue specimens were collected. All animal experiments were conducted according to guidelines approved by the institutional animal care committee of Kyung Hee University (KHUASP (SE)-14-024).

2.3.

Methylene blue staining

The dissected vaginas were fixed with formaldehyde. The vaginas were embedded in paraffin and cut into 4-μm-thick sections. The sections were stained with 1% methylene blue for 45 minutes, according to a previous procedure [27].

2.4.

Microcomputed tomography

Microcomputed tomographic (μCT) scans were performed on fixed tibia using a high-resolution μCT scanner. Trabecular bone parameters were determined at approximately 0.4 to 0.9 mm from the growth plate. Volumetric analysis was completed using the associated software applications, as described previously [28,29]. Reconstruction was carried out using Sky Scan Nrecon software (Sky Scan, Ltd, Kartuizersweg, Kontich, Belgium). The x-ray source was set at 75 kV and 100 μA. Four hundred projections were acquired over an angular range of 180°. The image slices were reconstructed using cone-beam reconstruction software, based on the Feldkamp algorithm (Dataviewer; Sky Scan, Ltd., Kartuizersweg, Kontich, Belgium). The trabecular bone was extracted by drawing ellipsoid contours with CT analyzer software. Three-dimensional (3D) parameters were analyzed from a Marching cubes-type model with a rendered surface. To analyze the 3D parameters, the entire bone was scanned, and 600 slices were placed through the former area. After this, trabecular number (Tb.N), connectivity density (Conn.D), and total porosity were determined.

Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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2.5.

Assessment of serum

The serum levels of ALP (no. ab83369; Abcam, Cambridge, UK), E2 (no. KAQ0621; Invitrogen, Carlsbad, CA, USA), and folliclestimulating hormone (FSH) (no. ab108641; Abcam) were analyzed with an enzyme-linked immunosorbent assay, according to the manufacturer's instructions.

cells and MCF-7 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 48 hours, and the cells were treated with GA or E2 for 48 hours. The luciferase activity was measured using a luminometer 1420 luminescence counter (Perkin Elmer, Inc, Waltham, MA, USA). The relative luciferase activity was determined as the ratio of firefly to Renilla.

2.10. 2.6.

Assessment of proliferation

The proliferation of both MG-63 cells and MCF-7 cells was analyzed using a colorimetric immunoassay, based on the measurement of bromodeoxyuridine (BrdU) incorporated by DNA synthesis (no. 11 647 229 001; Roche Diagnostics GmbH, Mannheim, Germany).

2.8.

Quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (PCR) was conducted according to previously described methods [30,31]. Total RNA was isolated from MG-63 cells or MCF-7 cells with an easy-BLUE RNA extraction kit, according to the manufacturer's instructions (iNtRON Biotech, Seongnam, Republic of Korea). The complementary DNA synthesis was conducted for 60 minutes at 42°C and 5 minutes at 94°C with a complementary DNA synthesis kit (Bioneer Corporation, Daejeon, Republic of Korea). A SYBR Green master mix was used for Quantitative Real-Time PCR using an ABI StepOne real-time PCR System (Applied Biosystems, Foster City, CA, USA). Table 1 shows primer sequences of ER-β, Ki-67, and GAPDH. The relative expression of the target gene was normalized to GAPDH.

2.9.

Assessment of ERE luciferase reporter assay

We analyzed ERE luciferase activity following a method previously described [32]. Estrogen response element directly linked to a TATA box was constructed with the enhanced luciferase reporter gene. We transiently transfected pERETATA-Luc and pSV40-Luc reporter gene constructs into MG-63

Table 1 – Primer sequences used in this study

ER-β Ki-67 GAPDH

Western blot analysis

Cell cultures

MG-63 cells and MCF-7 cells were purchased from the Korean Cell Line Bank (KCLB; Seoul, Republic of Korea). The cells were cultured in Dulbecco Modified Eagle Medium (Gibco BRL, Grand Island, NY, USA), with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin at 37°C in 5% CO2 with 95% humidity.

2.7.

3

Sense (5′-3′)

Antisense (5′-3′)

TTC CCA GCA ATG TCA CTA ACT T ATA AAC ACC CCA ACA CAC ACA A TCG ACA GTC AGC CGC ATC TTC TTT

TTG AGG TTC CGC ATA CAG A GCC ACT TCT TCA TCC AGT TAC ACC AAA TCC GTT GAC TCC GAC CTT

Western analysis was performed according to our previous report [33]. In brief, lysates from MG-63 cells were separated through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in phosphate-buffered saline (PBS) Tween-20 and incubated with either an anti-ERK antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or an anti–phosphorylated ERK antibody (Santa Cruz Biotechnology). Then, the membrane was incubated with secondary antibodies. Finally, the expression level was visualized using an enhanced chemiluminescent assay (Amersham Co, Newark, NJ, USA).

2.11.

Statistical analyses

A power analysis was used to determine an appropriate sample size. Using 2 independent-sample t tests, we calculated the sample sizes and G power. The sample size (5 mice per group: type I error, 0.05; power, 90%) was based on a pilot study. All data obtained from the 3 independent experiments were presented as the means ± SEM. The statistical values were followed by an independent t test or an analysis of variance with a least significant difference post hoc test using IBM SPSS version 21 statistics software (IBM, Armonk, NY, USA). Results were considered at a value of P < .05.

3.

Results

3.1. Regulatory effect of GA on body overweight in the OVX mice First, we examined whether GA would regulate body weight in OVX mice. The body weights of OVX mice were significantly increased compared with the sham-operated mice (P < .05; Fig. 1). Glutamic acid treatment significantly inhibited the body weight in OVX mice (P < .05; Fig. 1). In a previous report, an E2 treatment prevented an increase in the body weight of OVX mice [34]. Thus, we used E2 as a positive control. In this study, E2 also inhibited the body weight in OVX mice (P < .05; Fig. 1). It was reported that no differences were found between the body weights of the OVX group and the OVX group treated with DMSO [35,36]. Thus, we did not include an OVX group of mice administered orally with DMSO.

3.2.

Regulatory effect of GA on vaginal atrophy in OVX mice

Vaginal atrophy is the most common menopause symptom [37]; therefore, we examined whether GA would regulate vaginal atrophy in OVX mice. As shown in Fig. 2A, the vaginal epithelium of the OVX mice was atrophic with thin epithelial

Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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3.3.

Regulatory effect of GA on osteoporosis in OVX mice

Postmenopausal osteoporosis is a major bone disease associated with rapid bone loss and an increased risk of fragility fractures [39]. A μCT analysis of the trabecular bone (Fig. 3) revealed significant increases in bone mineral density (BMD), Tb.N, and Conn.D, in addition to a significant decrease in total porosity in the OVX mice treated with GA or E2 compared with the OVX mice (P < .05; Table 2).

3.4. Regulatory effect of GA on serum E2 and ALP in OVX mice Fig. 1 – Glutamic acid reduced the body weight gain typically found in the OVX mice. The body weights of all groups were measured once a week until the last day of GA administration. The change refers to a difference between the body weight on the first day and body weight on the last day of the study period. Each value represents the means ± SEM of 3 independent experiments. The statistical values were followed by an independent t test. #P < .05, significantly different from the sham-operated mice; ⁎P < .05, significantly different from the OVX mice. n = 5. E2, 17β-estradiol.

thickness. In addition, the vaginal cells of OVX mice were smaller than those of sham-operated mice. However, a GA or E2 treatment ameliorated the epithelial thickness and the size of vaginal cells. As Balakrishnan et al [38] reported that vaginal atrophy could result in a decrease in vaginal weight, we analyzed whether GA might affect the vaginal weight. Glutamic acid or E2 was observed to significantly increase the vaginal weight in OVX mice (P < .05; Fig. 2B).

Alkaline phosphatase plays a critical role in osteoid formation and bone mineralization and is used as a valid biomarker to diagnose postmenopausal women with low BMD [40]. Ovariectomized mice revealed a significant reduction in serum ALP levels compared with the sham-operated mice (P < .05). However, GA or E2 significantly increased the serum ALP level compared with the OVX mice (P < .05; Fig. 4A). In addition, GA or E2 significantly improved the serum E2 level in the OVX mice (P < .05; Fig. 4B). There were no significant differences between the serum E2 levels of the OVX group and OVX group treated with DMSO [36]. Based on this, we evaluated serum E2 levels by E2 compared with the untreated OVX group, not the OVX group treated with DMSO. Glutamic acid or E2 had no significant effects on serum FSH level (Fig. 4C).

3.5. Regulatory effect of GA on osteogenic activity in MG-63 cells To investigate the mechanism of the osteogenic effects of GA, MG-63 cells were treated with different concentrations of GA. Glutamic acid or E2 induced significant proliferation of MG-63

Fig. 2 – Glutamic acid regulated vaginal atrophy in the OVX mice. After ovariectomy, the mice received an oral dose of GA (10 mg/kg per day) or E2 (100 nmol/L per day) for the duration of the study period. A, Histologic analysis was performed by staining vaginas with methylene blue. Upper photomicrographs were taken to observe the vaginal epithelium (scale bar, 150 μm) and lower photomicrographs were taken to observe the size of the cells (scale bar, 75 μm). B, The vaginas were dissected and immediately weighed at the end of the study. This graph shows the relative values from mice vaginal weight to the sham-operated mice in percentages. Each value represents the means ± SEM of 3 independent experiments. The statistical values were followed by an independent t test. #P < .05, significantly different from the sham-operated mice; ⁎P < .05, significantly different from the OVX mice. n = 5. E2, 17β-estradiol. Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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Fig. 3 – Glutamic acid enhanced new bone formation in OVX mice. After ovariectomy, the mice received an oral dose of GA (10 mg/kg per day) or E2 (100 nmol/L per day) for the duration of the study period. Microcomputed tomographic scans were performed on fixed tibia using a high-resolution μCT scanner. Modeling of the tibial metaphysis is representative crosssectional μCT scanning. n = 5. E2, 17β-estradiol.

cells (P < .05; Fig. 5A). Glutamic acid or E2 significantly increased the ER-β messenger RNA (mRNA) expression and ERE activity in MG-63 cells (P < .05; Fig. 5B and C). Extracellular signal–regulated kinase phosphorylation was induced by GA or E2 in MG-63 cells (Fig. 5D). Glutamic acid or E2 significantly increased ALP activity (P < .05; Fig. 5E).

3.6. Regulatory effect of GA on estrogenic activity in MCF-7 cells Estrogen influences the growth, differentiation, and function of many target tissues, such as mammary glands, the uterus, vagina, and ovaries [41]. Because MCF-7 cells have the ability to process E2 via cytoplasmic ERs, they have been used to study the estrogenic effects of phytoestrogens [42,43]. Thus, we investigated whether GA would regulate the proliferation of ER-positive MCF-7 cells to further understand the estrogenic activity of GA. The proliferation was significantly induced by GA or E2 in MCF-7 cells (P < .05; Fig. 6A). Ki-67, a proliferation marker, can serve as a useful alternative to BrdU in observing either an increase or decrease of proliferation [44]. Similar to the results of the BrdU of GA and E2, the Ki-67

Table 2 – Analysis of the trabecular bone in mice by μCT

BMD Tb.N Conn.D Total porosity

Sham

OVX

1.75 2.21 5.08 0.89

1.00 1.00 1.00 1.00

± ± ± ±

0.01 0.14 1.34 0.00

± ± ± ±

GA 0.03 ⁎ 0.05 ⁎ 0.17 ⁎ 0.01 ⁎

1.68 5.89 4.21 0.93

E2 ± ± ± ±

0.11 ⁎⁎ 1.50 ⁎⁎ 0.00 ⁎⁎ 0.02 ⁎⁎

1.37 1.21 3.63 0.95

± ± ± ±

0.01 ⁎⁎ 0.00 ⁎⁎ 0.21 ⁎⁎ 0.00 ⁎⁎

Each volumetric value in mice for each group was determined as compared to the OVX group. Bone mineral density was measured using 2-dimensional μCT. Trabecular number, Conn.D, and total porosity of proximal tibia were measured using 3D μCT. Each datum represents the means ± SEM of 3 independent experiments. The statistical values were followed by an analysis of variance with a least significant difference post hoc test; n = 5. E2, 17β-estradiol. ⁎ P < .05, significantly different from the sham-operated mice. ⁎⁎ P < .05, significantly different from the OVX mice.

mRNA expression of E2 was significantly higher than that of GA (P < .05; Fig. 6B). In addition, GA or E2 significantly increased ER-β mRNA expression in MCF-7 cells (P < .05; Fig. 6C). We compared the E2-like activity between GA and genistein, which was reported to enhance the proliferation E2 dependently and luciferase activity ERE dependently in MCF-7 cells [22,45]. Glutamic acid significantly elevated ERE activity, and the ERE activity enhanced by GA was similar to that of genistein in MCF-7 cells (P < .05; Fig. 6D). The ERE activity enhanced by GA was completely abolished by fulvestrant, an ER antagonist (P < .05; Fig. 6D). Estrogen plus fulvestrant also inhibited ERE activity significantly (P < .05; Fig. 6D).

4.

Discussion

Amino acids play a critical role in many of the body's functions, including aging, cellular repair, and hormonal regulation. The significance of amino acids in menopause has been reported due to their ability to combat uncomfortable menopausal symptoms [46]. In the present study, we observed the estrogenic and osteogenic activities of GA in OVX mice, MG-63 cells, and MCF-7 cells. Obesity might result from visceral fat accumulation in menopause, and visceral fat secretes adverse adipokines that correlate with increased adiposity [47]. Estrogen is involved in the regulation of adipokine expression [48,49] and a vital protective factor against obesity in women. Postmenopausal women have a higher rate of obesity caused by deficient ovary functions than premenopausal women [18]. In this study, it appears that GA inhibited excess body weight by increasing the serum E2 level in OVX mice. However, further evidence is needed to better elucidate the regulatory effect of GA on adipokine secretion in the OVX mice because this study lacked a good control, such as a postmenopausal mouse that did not have an increase in body weight due to caloric restrictions. Postmenopausal women experience vaginal atrophy, which is characterized by histologic vaginal changes such as epithelial thinning and cell immaturation [50]. Vaginal atrophy involves vaginal dryness, itching, pallor, and dysuria

Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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Fig. 4 – Glutamic acid improved serum ALP and E2 in the OVX mice. After ovariectomy, the mice received an oral dose of GA (10 mg/kg per day) or E2 (100 nmol/L per day) for the duration of the study period. Blood samples were collected from the heart at the end of the study. Serum levels of ALP (A), E2 (B), and FSH (C) were analyzed, according to the manufacturer's directions. Each value represents the means ± SEM of 3 independent experiments. The statistical values were followed by an independent t test. #P < .05, significantly different from the sham-operated mice; ⁎P < .05, significantly different from the OVX mice. n = 5. E2, 17β-estradiol.

[51]. Thus, the symptoms related to vaginal atrophy with deficient E2 severely affect the quality of women's lives [52]. Tibolone, an HRT option, improves the problems of obesity and vaginal atrophy and is used as a steroid hormone drug in menopause [53], but phytoestrogen is a safer alternative to

treat vaginal atrophy in postmenopausal women [54]. In this study, GA inhibited vaginal atrophy and improved cell maturation by increasing the serum E2 level in the OVX mice. However, further evidence is needed to determine the role of GA on the status of estrus and vaginal health.

Fig. 5 – Glutamic acid improved osteogenic activity in MG-63 cells. A, MG-63 cells were treated with GA for 48 hours. Proliferation was evaluated with a BrdU incorporation assay. B, MG-63 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 10 hours. The ER-β mRNA expression was determined with quantitative real-time PCR analysis. C, MG-63 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 48 hours. The ERE luciferase activity was measured with a luciferase assay. D, MG-63 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 5 minutes. The phosphorylation of ERK was visualized by Western blotting. E, MG-63 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 48 hours. The ALP activity was measured, according to the manufacturer's directions. Each value represents the means ± SEM of 3 independent experiments conducted in triplicate. The statistical values were followed by an independent t test. ⁎P < .05, significantly different from untreated cells. E2, 17β-estradiol; pERK, phosphorylation of ERK. Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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Fig. 6 – Glutamic acid improved estrogenic activity in MCF-7 cells. A, MCF-7 cells were treated with GA or E2 (100 nmol/L) for 48 hours. Proliferation was evaluated with a BrdU incorporation assay. B, MCF-7 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 24 hours. The Ki-67 mRNA expression was determined with quantitative real-time PCR analysis. C, MCF-7 cells were treated with GA (10 μg/mL) or E2 (100 nmol/L) for 10 hours. Estrogen receptor β mRNA expression was determined with quantitative real-time PCR analysis. D, MCF-7 cells were treated with fulvestrant (1 μmol/L) an hour before the treatment with GA (10 μg/mL) or E2 (100 nmol/L) for 48 hours. Genistein (1 μmol/L) was also treated in MCF-7 cells for 48 hours. The ERE luciferase activity was measured with a luciferase assay. Each value represents the means ± SEM of 3 independent experiments conducted in triplicate. The statistical values were followed by an independent t test. ⁎P < .05, significantly different from untreated cells. E2, 17β-estradiol.

Xu et al [55] reported that the serum E2 level was approximately 900 pg/mL by oral intake of E2 154 μg/kg in the OVX mice. In this study, the serum E2 level was approximately 780 pg/mL by the oral intake of E2 27 μg/kg (100 nmol/L) in the OVX mice. The dose of oral intake by E2 was much lower than those of previous reports [55,56], and as a result, serum E2 might not be markedly raised by the oral intake of E2. However, in this study, the serum E2 level measured against the amount of intake was higher than those of previous reports [55,56] and significantly greater compared to that of OVX mice. Thus, the increase in the serum E2 level by GA might improve obesity and vaginal atrophy of the OVX mice. Estrogen is also a potent stimulator of bone formation, increasing cancellous bone volume [57]. There is a direct relationship between E2 deficiency and osteoporosis development in menopause [58]. The serum ALP level and trabecular bone parameters were decreased in the OVX mice [59]. However, E2 administration improved the serum ALP level and trabecular bone parameters in the OVX mice [59,60]. Glutamic acid also increased the serum ALP level in the OVX mice. In the tibia metaphysis, GA induced significant increases in BMD, Tb.N, and Conn.D and decreased porosity

in the OVX mice. Based on our findings that GA enhanced new bone formation, we investigated the osteogenic activity of GA using MG-63 cells. Osteoporosis is associated with osteoclast proliferation [61], and thus, bone loss was prevented by stimulating the process of osteoblast proliferation and suppressing the process of osteoclastogenesis in the OVX mice [62]. Osteoblast proliferation is regulated via binding to the ERE of the E2-ER complex and ERK activation [63,64]. In addition, Proliferation of osteoblasts shows ALP activity, which is greatly enhanced during in vitro bone formation [65]. Alkaline phosphatase activity was increased via ER activation and an ERK pathway in MG-63 cells [66]. Estriol prevented bone loss by increasing ALP activity as well as increasing the proliferation of MG-63 cells through the ER [67]. Phytoestrogens stimulated ERE activity via ER-β, having the osteogenic activity in MG-63 cells [68]. Glutamic acid also induced significant proliferation via ER activation and ERK phosphorylation and increased the ALP activity in MG-63 cells. Therefore, GA might have osteogenic effects due to increasing ALP activity via ER activation. Because E2 has biological functions via ER [13], we studied the estrogenic effect of GA in ER-positive MCF-7 cells.

Please cite this article as: Han N-R, et al, Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.06.006

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Estrogen produced a robust stimulation of proliferation of MCF-7 cells [69]. Genistein had estrogen-like effects through ER in MCF-7 cells and in the OVX mice [22]. Isoflavones triggered ER-β–mediated transcriptional pathways and low incidence of menopausal symptoms or osteoporosis [70]. Glutamic acid induced significant proliferation via ER activation, and ERE activity increased by GA was inhibited by an E2 antagonist. These observations have potential implications for the estrogen-like effect of GA. In conclusion, we found that GA was functional as an osteogenic and estrogenic supplement in a mouse model that mimicked postmenopausal women. This effect of GA resulted from ER activation and an increase in ALP activity. Therefore, we suggest that GA may be of value in the treatment of E2 deficiency–induced menopausal symptoms, such as postmenopausal weight gain, vaginal atrophy, and osteoporosis. This supplementation could provide an alternative instead of HRT during menopause. However, further research is needed to characterize the mechanisms and effects of GA before its clinical use in humans. Because there is limited evidence to clarify the regulatory effect of GA on various menopausal symptoms in this study, further studies are needed concretely to explore the mechanisms of GA regulating other E2 deficiency–induced menopausal symptoms. In addition, because GA is a nonessential amino acid and is interconverted to important metabolites, such as glutamine or glutamate in the body, further investigation is needed to determine which GA metabolites are responsible for this effect via an ER pathway in the OVX mice. Nevertheless, these results partially support our hypothesis that GA can regulate E2 deficiency–induced menopausal symptoms.

Acknowledgment This research was supported by the Ministry of Trade, Industry and Energy, Korea, through the Education Support program for Creative and Industrial Convergence.

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