Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Abstract

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.

AMP-activated protein kinase pathway and bone metabolism

“…In agreement with those results, adiponectin was demonstrated to inhibit the stimulation by tumour necrosis factor a and RANKL of the master transcriptional factor of RANKL-induced osteoclastogenesis, the nuclear factor of activated T cells c1, through the AMPK signaling pathway . Metformin was very recently shown to reduce osteoclast differentiation and activity by stimulating osteoprotegerin (OPG) and inhibiting RANKL mRNA and protein expression in osteoblasts (Mai et al 2011). Conflicting results were however shown by Quinn et al (2010) who demonstrated that AICAR treatment in male mice stimulates bone loss and bone turnover with elevated rates of both bone formation and bone resorption (Quinn et al 2010).…”

“…While the authors of this study confirmed that AICAR stimulates osteoclast formation in vitro, a result in contradiction with the previously mentioned in vitro studies demonstrating that AMPK activation suppresses osteoclast activity, they however showed that AICAR's effect was independent of AMPK signaling (Quinn et al 2010). The effect of metformin on bone mass in vivo was also poorly studied, but two recent studies indicate that metformin reduces the bone loss induced by ovariectomy (OVX), in part through increasing bone formation via induction of osteoblast genes such as Runx2 and Lrp5 (Gao et al 2010) and by reducing RANKL and stimulating OPG expression in osteoblasts (Mai et al 2011).…”

“…Novel findings, described in detail in this review, have shown that AMPK plays a role in bone physiology. There are in vitro studies demonstrating that AMPK modulators regulate bone cell differentiation and function (Kanazawa et al 2007, Molinuevo et al 2010, Quinn et al 2010, Shah et al 2010, Jang et al 2011, Mai et al 2011 and two publications showing that deletions of AMPKa and b subunits in mice lead to bone loss in vivo (Quinn et al 2010, Shah et al 2010. Furthermore, AMPK activity can be regulated in bone cells by hormones, such as ghrelin and norepinephrine, that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues.…”

“…Furthermore, AMPK activity can be regulated in bone cells by hormones, such as ghrelin and norepinephrine, that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. The interest in AMPK and bone physiology comes also from the fact that metformin and TZDs have significant and opposing skeletal effects (Gao et al 2010, Lecka-Czernik 2010, Mai et al 2011 and the contribution of AMPK to these bone effects is still unclear. Although the elucidation of the importance of AMPK signaling in bone is still in its commencement, the revelation that AMPK activation may affect stimulation of bone formation and bone mass places AMPK signaling as a significant pathway in skeletal physiology.…”

Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice

“…Several studies have indeed shown that TZDs increase fracture risk (10,11). In contrast, metformin, another widely prescribed anti-diabetic drug (12), is osteogenic in vitro (13,14) and reduces the risk of fracture in DM patients (15), as well as inhibiting the bone loss induced by ovariectomy (OVX) in rats (16,17). However, our recent studies showed no beneficial effect of metformin on bone mass and fracture healing in rodents (18).…”