Background and aim: Metformin is widely used in Type 2 diabetes, with increasing reports of a potential bone protective role. We investigated the role of AMPK in mediating the effects of metformin on mesenchymal stem cell (MSC) differentiation to either osteoblasts or adipocytes.
Methods: Confluent mouse MSCs (C3H10T1/2), wild type (WT) and AMPK knockout (KO) mouse embryo fibroblasts (MEFs) were treated with metformin(500 μM), AMPK-activator A769662(100 μM), AMPK-inhibitor Compound C(1 μM/10 μM) and p70S6K-inhibitor rapamycin(10 μM), in both control and adipogenic-inducing environment (pioglitazone;10 μM). Nuclear extracts were prepared and separated by SDS-PAGE and immunoblotted with primary antibodies to the adipogenic markers; peroxisome proliferator-activated receptor gamma (PPAR?) and CCAAT-enhancer binding protein (C/EBPβ), the osteogenic marker; Runt-related transcription factor 2 (Runx2), the AMPK activity marker; phosphorylated-ACC (P-ACC(Ser79)) and the marker of mTOR signalling; phosphorylated-p70s6k (P-p70s6k(Thr389)). PPARγ and Runx2 activities were determined using Luciferase reporter assays and adipogenesis was quantified histochemically with Oil Red O.
Results: MSCs treated with pioglitazone demonstrated a marked adipogenic phenotype, whilst both metformin and A769662 impaired adipogenesis. Pioglitazone induced increase in PPARγ expression (P<0.05), whilst metformin (P<0.05) and A796662 (P<0.05) suppressed this to basal levels. Runx2 activity, but not protein levels, was increased by metformin (P<0.001) and A769662 (P<0.001). As expected, A769662 promoted phosphorylation of ACC, but not so with metformin. Instead, metformin suppressed the phosphorylation of p70s6k (P<0.01), as did A769662 (P<0.001) and rapamycin (P<0.001). Luciferase 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). Pioglitazone-treated WT MEFs, but not AMPK KO-MEFs, exhibited adipogenesis suggesting a basal AMPK requirement for adipogenesis. Both metformin and A769662 (both P<0.05) significantly induced phosphorylation of ACC, indicating an AMPK-dependent mechanism in MEFs.
Conclusions: Metformin suppresses adipogenesis of C3H10T1/2 cells through the reciprocal regulation of PPARγ and Runx2, involving a novel AMPK-independent mechanism of action on MSC differentiation, through suppression of p70S6K. However, metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context.
10 Sep 2016 - 12 Sep 2016