Glutamic acid ameliorates estrogen deficiency–induced menopausal-like symptoms in ovariectomized mice  Na-Ra Han, Hee-Yun Kim, Woong Mo Yang, Hyun-Ja Jeong, Hyung-Min Kim  Nutrition Research  Volume 35, Issue 9, Pages 774-783 (September 2015) DOI: 10.1016/j.nutres.2015.06.006 Copyright © 2015 Elsevier Inc. Terms and Conditions

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. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

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. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

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 cross-sectional μCT scanning. n = 5. E2, 17β-estradiol. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

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. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

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. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

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. Nutrition Research 2015 35, 774-783DOI: (10.1016/j.nutres.2015.06.006) Copyright © 2015 Elsevier Inc. Terms and Conditions