Elsevier

Bone

Volume 77, August 2015, Pages 135-141
Bone

Original Full Length Article
Activation of AMP-activated protein kinase protects against homocysteine-induced apoptosis of osteocytic MLO-Y4 cells by regulating the expressions of NADPH oxidase 1 (Nox1) and Nox2

https://doi.org/10.1016/j.bone.2015.04.025Get rights and content

Highlights

  • Homocysteine (Hcy) dose-dependently increased apoptosis of osteocytic MLO-Y4 cells.

  • Treatments with an antioxidant and NADPH oxidase (Nox) inhibitors prevented Hcy-induced apoptosis.

  • Activators of AMP-activated protein kinase (AMPK) suppressed Hcy-induced apoptosis.

  • Hcy increased Nox1 and Nox2 mRNA expressions, which were decreased by AMPK activation.

  • AMPK activation decreases Hcy-induced apoptosis via suppressing upregulation of Nox1 and Nox2.

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.

Introduction

Homocysteine (Hcy) is a sulfur-containing amino acid formed by the demethylation of methionine, and high plasma Hcy levels are often caused by vitamin B12 and folate insufficiency [1] and polymorphism in the gene encoding the folate-metabolizing enzyme methylenetetrahydrofolate reductase [2], [3]. Accumulating evidence has shown that elevated plasma Hcy level is a risk factor for osteoporotic fracture [4], [5], [6], [7]. In accordance with the epidemiological studies, previous animal studies have shown that diet-induced hyperhomocysteinemia decreases bone quality [8], [9]. However, the mechanisms underlying Hcy-induced bone fragility have not been completely elucidated thus far. Previous studies have shown that Hcy increases the apoptosis of osteoblast lineage cells such as bone marrow stromal cells [10] and osteoblasts [11]. Hcy is a potent pro-oxidant [12], [13], [14], it induces the apoptosis of bone marrow stromal cells by increasing oxidative stress [8]. Furthermore, Hcy suppresses the expression of lysyl oxidase in osteoblasts, resulting in inhibiting enzymatic collagen cross-links [15]. These findings indicate that Hcy has negative impacts on cell viability and function of osteoblasts.

AMP-activated protein kinase (AMPK) plays a pivotal role as an intracellular energy sensor and is associated with the regulation of appetite and glucose and fat metabolism [16]. Metformin is frequently used for the treatment of type 2 diabetes mellitus and is known to improve insulin sensitivity by activating AMPK. We previously showed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), a pharmacological AMPK activator, and metformin stimulates the differentiation and mineralization of osteoblastic MC3T3-E1 cells [17]. In addition, AMPK-knockout mice show a significant reduction in bone volume [18]. Thus, AMPK may have positive effects on bone formation.

Oxidative stress is regulated by many oxidant and antioxidant enzymes. Oxidative stress is predominantly induced by NADPH oxidase (Nox) [19], one of the oxidant enzymes, and is prevented by antioxidant enzymes such as superoxide dismutase (SOD) [20]. Several studies have shown that AMPK activation ameliorates oxidative stress in various cells by regulating the activities of Nox and SOD [21], [22], [23], [24], [25], [26]. However, whether AMPK activation decreases Hcy-induced oxidative stress by regulating oxidant–antioxidant enzymes in osteoblast lineage cells has not been clarified thus far.

Osteocytes are the most abundant cells in the bone, and they play important roles in coordinating the functions of osteoblasts and osteoclasts [27]. Estrogen deficiency [28] as well as mechanical loading [29] and glucocorticoid administration [30] are associated with apoptosis of osteocytes, resulting in increased bone fragility. However, to our best knowledge, no study has described the effects of Hcy and AMPK on osteocytes. Thus, the purpose of our study was to examine the effects of Hcy and AMPK activators on the apoptosis of osteocytic MLO-Y4 cells and on the expressions of Nox and SOD in the cells.

Section snippets

Reagents

Cell culture medium and supplements were purchased from GIBCO-BRL (Rockville, MD). AICAR was purchased from Cell Signaling (Beverly, MA). dl-homocysteine, N-acetyl-l-cysteine (NAC), and Ara-A were purchased from Sigma–Aldrich (St. Louis, MO). The Nox inhibitors apocynin and diphenyleneiodonium chloride (DPI) were purchased from Santa Cruz Biotech (Santa Cruz, CA) and Enzo Life Sciences (New York, NY), respectively. Metformin was kindly provided by Sumitomo Dainippon Pharma (Osaka, Japan).

Hcy induced apoptosis of MLO-Y4 cells

The effects of Hcy on apoptosis of MLO-Y4 cells were examined using a DNA fragment detection ELISA kit. Incubation with Hcy at the indicated concentrations significantly increased the apoptosis in a dose-dependent manner (at least p < 0.05; Fig. 1A). Moreover, TUNEL staining confirmed that 5 mM Hcy significantly increased the apoptosis of MLO-Y4 cells (Fig. 1B and C).

An anti-oxidant (NAC) and Nox inhibitors, apocynin and DPI, ameliorated Hcy-induced apoptosis of MLO-Y4 cells

Further, to investigate whether oxidative stress is involved in Hcy-induced apoptosis, we examined the effect of NAC, an

Discussion

Several clinical studies have shown that moderate hyperhomocysteinemia increases the risk of osteoporotic fracture independent of the BMD [4], [5], [6], [7], which suggests that the deterioration of bone quality may be a dominant cause of Hcy-induced bone fragility. Previous studies have shown that Hcy increases apoptosis of bone marrow stromal cells and osteoblasts [10], [11]; however, no studies have examined the effects of Hcy on osteocytes to date. Osteocytes play pivotal roles in

Acknowledgments

This study had no funding support. Authors' roles: Study design and conduct: AT and IK. Performed the experiments and analyzed the data: AT and MN. Contributed equipment/materials: MY, IK, TY and TS. Wrote the paper: AT and IK. Approving final version: all authors. IK takes responsibility for the integrity of the data analysis. The authors thank Keiko Nagira for technical assistance.

Conflicts of interest

None.

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