We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator Ice (PGC-1 alpha) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smadl /5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5(Rgsc451) mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphotylation of Smadl /5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smadl /5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgcl a and uncoupling protein-1 expression in a manner sensitive to the PI31(/ Alct inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMPresponsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway. (c) 2014 Elsevier Inc. All rights reserved.