Endocrine Abstracts

Introduction: Metformin is widely used as oral anti-hyperglycaemic agent to treat type 2 diabetes, with increasing reports of an additional, potential bone protective role.

Objective: We investigated the role of AMPK in mediating the effects of metformin on the differentiation of mesenchymal stem cells (MSCs) to either osteoblasts or adipocytes.

Methods: Confluent murine MSCs (C3H10T1/2) were treated with metformin (500 μM), a known AMPK activator (A769662; 100 μM), or the p70S6K inhibitor (rapamycin; 10 μM), in both control and adipogenic-inducing environments (using pioglitazone; 10 μM) for 5 days. Nuclear extracts were separated by SDS–PAGE and immunoblotted with primary antibodies to peroxisome proliferator-activated receptor gamma (PPARγ; marker for adipogenesis), Runt-related transcription factor 2 (Runx2; marker for osteogenesis), phosphorylated-ACC (P-ACC (Ser79); marker for AMPK activity) and phosphorylated-p70s6k (P-p70s6k (Thr389); upstream regulator of mTOR signalling. Immunoblots were scanned using a Licor fluorescent reader. PPARγ and Runx2 activities were determined using Luciferase reporter assays and adipogenesis was quantified histochemically by staining neutral lipids with Oil red O.

Results: MSCs treated with pioglitazone demonstrated marked adipogenic phenotype staining positively with Oil red O. In contrast, treatment with both metformin and A769662 impaired adipogenesis. Pioglitazone induced an (P<0.01) increase in PPARγ expression, whilst metformin and A796662 suppressed PPARγ expression to basal levels, P<0.05 and P<0.01 respectively. Runx2 activity was significantly increased by metformin (P<0.001) and A769662 (P<0.001) but not Runx2 protein levels. As expected, A769662 promotes phosphorylation of ACC, but not so with metformin. Instead, metformin suppressed (P<0.05) the phosphorylation of p70s6k, as did A769662 (P<0.05) and rapamycin (P<0.001). Luciferase reporter assays confirmed the reciprocal action of metformin on adipogenesis and osteogenesis, namely suppression of PPARγ activity (P<0.001) and induction of Runx2 activity (P<0.001).

Conclusions: Metformin suppresses adipogenesis of C3H10T1/2 cells through the reciprocal regulation of PPARγ and Runx2. These results present novel mechanisms of action for metformin on MSC differentiation which is largely AMPK-independent, involving the suppression of p70S6K activity.