Bone Abstracts

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.

Objective:: We investigated the role of AMPK pathway in mediating the effects of metformin on the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes.

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 5 days. Cells were harvested and nuclear extracts prepared. Nuclear extracts were separated by SDS-PAGE 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.

Disclosure: The authors declared no competing interests.