ObjectivePrevious reports have indicated that Ahnak-deficient mice were protected from high-fat diet-induced obesity. However, the molecular mechanism in which Ahnak mediates adipocyte differentiation and high-fat diet-induced obesity is unclear. MethodsAdipocytes from Ahnak knockout (Ahnak(-/-)) mice and knockdown of Ahnak in C3H10T1/2 were used to investigate the function of Ahnak in adipocyte differentiation. Ahnak-induced adipocyte differentiation was analyzed by Oil Red O staining. ResultsAdipocytes from Ahnak(-/-) mice were smaller than those from wild-type mice. Silencing of Ahnak in C3H10T1/2 and adipose tissue-derived mesenchymal stem cells (ADSCs) from Ahnak(-/-) mice showed severely impaired adipocyte differentiation. Down-regulation of Ahnak in C3H10T1/2 cells and ADSCs from Ahnak(-/-) mice attenuated the phosphorylation and nuclear localization of Smad1 in response to BMP2, whereas Ahnak overexpression in 3T3-L1 cells significantly increased Smad1 activation. Because PPAR is a well-known transcriptional factor in adipocyte differentiation, the PPAR expression in Ahnak-mediated adipocyte differentiation was investigated. Transfection of C3H10T1/2 cells with Ahnak siRNA resulted in reduced PPAR expression apparently through inhibited binding of Smad1 to the Smad1-binding site in the PPAR promoter. These results suggest that Ahnak regulates adipogenesis by regulating Smad1-dependent PPAR expression. ConclusionsA molecular mechanism was proposed in which Ahnak regulates adipocyte differentiation through Smad1 activation.