Introduction: Insulin sensitising agents are reported to have a diverse range of effects on bone with metformin exerting positive effects and thiazolidinenediones (TZDs) exerting negative effects. 5′AMP-activated protein kinase (AMPK) plays a critical role in cellular energy homeostasis. It is widely expressed in the body and can be activated by metformin.
Aim: We investigated the role of AMPK pathway in mediating the effects of metformin on the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs).
Methods: Confluent murine MSCs (C3H10T1/2) were treated with 500 μM metformin and 100 μM A769662 (AMPK activator) respectively, in an adipogenic-inducing environment (the TZD, pioglitazone 10 μM) for five days. Cells were harvested and nuclear extracts prepared. Nuclear extracts were separated by SDSPAGE and immunoblotted with primary antibodies to peroxisome proliferator-activated receptor gamma (PPARγ; marker for adipogenesis) and runt-related transcription factor 2 (Runx2; marker for osteogenesis). Immunoblots were scanned using a Licor fluorescent reader. Adipogenesis was also quantified histochemically by fixing with 10% formalin followed by staining neutral lipids with Oil Red O.
Results: MSCs treated with pioglitazone for 5 days demonstrated marked adipogenic phenotype with accumulation of lipid-rich vacuoles that stained positively with Oil Red O. Pioglitazone induced a significant (P<0.01) increase in PPARγ1 and PPARγ2 expression compared to diluent control, as determined by western blotting. In the presence of pioglitazone, metformin suppressed PPARγ expression (P<0.001) to basal diluent levels, as did the AMPK activator, A769662 (P<0.01), which suggests that metformin acts through the AMPK pathway, at least to a degree, to suppress adipogenesis in MSCs. Runx2 expression was unaffected by treatment with either metformin or A769662, suggesting that AMPK is not involved in the induction of osteogenesis in these cells.
Conclusion: Metformin appears to exert its bone protective effects on MSCs by reducing adipogenesis, through activation of AMPK signalling, with no direct effect on osteogenesis.