Several studies in rodent osteoblastic cell lines and bone marrow progenitor cells demonstrated that pharmacological Dapagliflozin AMPK activators metformin and AICAR (acadesine) induce differentiation and mineralization of osteoblasts

by upregulating the expression of Runx2 [25], [26], [27] and [28]. Moreover, the in vivo studies confirmed that metformin stimulates bone lesion regeneration in rats [29], while AMPK gene knockdown reduces bone mass in mice [30] and [31]. Recently, Kim et al. [15], using an RNA interference approach, provided the first evidence for the involvement of AMPK in osteogenic differentiation of human adipose tissue-derived MSC. The results of the present study confirm and expand these findings by demonstrating the induction of autophagy and activation

of Akt as the major early and late downstream events, respectively, in AMPK-controlled MSC osteogenic differentiation. While it has been reported that Akt is required for BMP2-stimulated osteogenesis Anti-infection Compound Library nmr in mice [14] and [32], our data for the first time demonstrate the involvement of autophagy in osteoblast differentiation. The role of AMPK in autophagy induction or Akt activation in osteoblasts has not been assessed thus far, but the present results are consistent with the ability of AMPK to induce autophagy in various cell types [33], as well as to activate Akt in leukemic cells, endothelial cells and renal tubular cells [34], [35] and [36]. The latter effect, however, seems to be cell type- and/or context-dependent, as we have previously failed to observe any influence of AMPK on Akt phosphorylation in U251 human glioma cells exposed to simvastatin or compound C [37] and [38], or in metformin-treated B16 mouse melanoma cell line [39]. While our data with AMPK shRNA clearly support the role of AMPK in Akt activation during osteogenic differentiation of hDP-MSC, it should be noted that the AMPK inhibitor compound C [40] has recently been reported to directly interfere with Akt phosphorylation in an AMPK-independent manner [38]. Therefore, although we used compound C at

quite a low dose (1 μM) as a precaution against non-specific effects, the possibility Roflumilast that its actions in the present study were partly mediated independently of AMPK inhibition could not be completely excluded. However, compound C, unlike Akt inhibitor DEBC, failed to reduce osteogenic differentiation of hDP-MSC if added 3 days after its initiation, which argues against the ability of compound C to directly inhibit Akt in our experimental setting. In addition, it has been shown that AMPK can modulate differentiation of rodent osteoblast cell lines through interference with Wnt/β-catenin and Smad1/5/8-Dlx5 signaling pathways [26] and [41]. We are currently investigating possible connections between these signaling pathways and AMPK-triggered activation of autophagy and Akt during osteoblast differentiation of human MSC.