Ayumu Takeno1, Ippei Kanazawa2, Ken-Ichiro Tanaka3, Masakazu Notsu4, Maki Yokomoto5, Toru Yamaguchi6, Toshitsugu Sugimoto7. 1. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: atakeno@med.shimane-u.ac.jp. 2. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: ippei.k@med.shimane-u.ac.jp. 3. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: ken1nai@med.shimane-u.ac.jp. 4. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: mnotsu25@med.shimane-u.ac.jp. 5. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: pinyan@med.shimane-u.ac.jp. 6. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: yamaguch@med.shimane-u.ac.jp. 7. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: sugimoto@med.shimane-u.ac.jp.
Abstract
BACKGROUND: Elevated plasma homocysteine (Hcy) level is associated with the risk of osteoporotic fracture. While Hcy increases oxidative stress, AMP-activated protein kinase (AMPK) activation ameliorates it. This study aimed to investigate whether Hcy induces apoptosis of osteocytic MLO-Y4 cells through regulating expressions of oxidant and anti-oxidant enzymes and determine the effects of AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and metformin on the Hcy-induced apoptosis of the cells. RESULTS: DNA fragment ELISA and TUNEL staining assays showed that Hcy treatments (0.1-5.0 mM) induced apoptosis of MLO-Y4 cells in a dose-dependent manner. The detrimental effect of Hcy was partly but significantly reversed by an antioxidant (N-acetylcysteine) and NADPH oxidase (Nox) inhibitors (apocynin and diphenyleneiodonium). In addition, treatment with AICAR (0.05-0.1 mM) and metformin (10-100 μM) ameliorated Hcy-induced apoptosis of the cells. The favorable effect of metformin on Hcy-induced apoptosis was completely canceled by an AMPK inhibitor Ara-A. Hcy increased the expression levels of Nox1 and Nox2, while it had no effects on the expressions of Nox4 or the anti-oxidant enzymes, superoxide dismutase 1 and 2. Hcy-induced increases in the expressions of Nox1 and Nox2 decreased significantly by treatments with AICAR. CONCLUSION: These findings suggest that Hcy induces apoptosis of osteocytes by increasing the expressions of Nox1 and Nox2, and AMPK activation by AICAR and metformin effectively prevents the detrimental reactions. Thus, AMPK activation may be a potent therapeutic candidate for preventing Hcy-induced osteocyte apoptosis and the resulting bone fragility.
BACKGROUND: Elevated plasma homocysteine (Hcy) level is associated with the risk of osteoporotic fracture. While Hcy increases oxidative stress, AMP-activated protein kinase (AMPK) activation ameliorates it. This study aimed to investigate whether Hcy induces apoptosis of osteocytic MLO-Y4 cells through regulating expressions of oxidant and anti-oxidant enzymes and determine the effects of AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and metformin on the Hcy-induced apoptosis of the cells. RESULTS: DNA fragment ELISA and TUNEL staining assays showed that Hcy treatments (0.1-5.0 mM) induced apoptosis of MLO-Y4 cells in a dose-dependent manner. The detrimental effect of Hcy was partly but significantly reversed by an antioxidant (N-acetylcysteine) and NADPH oxidase (Nox) inhibitors (apocynin and diphenyleneiodonium). In addition, treatment with AICAR (0.05-0.1 mM) and metformin (10-100 μM) ameliorated Hcy-induced apoptosis of the cells. The favorable effect of metformin on Hcy-induced apoptosis was completely canceled by an AMPK inhibitor Ara-A. Hcy increased the expression levels of Nox1 and Nox2, while it had no effects on the expressions of Nox4 or the anti-oxidant enzymes, superoxide dismutase 1 and 2. Hcy-induced increases in the expressions of Nox1 and Nox2 decreased significantly by treatments with AICAR. CONCLUSION: These findings suggest that Hcy induces apoptosis of osteocytes by increasing the expressions of Nox1 and Nox2, and AMPK activation by AICAR and metformin effectively prevents the detrimental reactions. Thus, AMPK activation may be a potent therapeutic candidate for preventing Hcy-induced osteocyte apoptosis and the resulting bone fragility.