FIG. 6.

Overexpression of BMP7 induced spontaneous differentiation of wild-type brown preadipocytes and rescued brown adipocyte differentiation of IRS-1KO preadipocytes. WT and IRS-1KO brown preadipocytes were stably transfected with an hBMP7 construct driven by the aP2 promoter or with empty vector (as a control). Pools of transfected clones were grown to confluence (defined as day 0) and proceeded to continuous culture in DMEM plus 10% FBS without any inducers (for WT cells) or with Dex-IBMX-insulin-indomethacinn-T3 (for IRS-1KO cells) (see Materials and Methods). On day 10, the analyses were performed. (A) hBMP7 levels in the medium supernatants from WT and IRS-1KO cells were determined by ELISA. (B and C) Oil Red O staining and microscopic views of WT cells (B) and IRS-1KO cells (C). (D and E) Expression of adipogenic markers and brown-fat-selective genes in WT cells (D) and IRS-1KO cells (E) was analyzed by Q-RT-PCR. The data are presented as the mean plus SEM from a representative of 3 independent experiments, with each performed in duplicate or triplicate. The values for vector are set as 100 arbitrarily. Statistically significant differences between vector- and hBMP7-transfected cells are indicated above the bars: *, P < 0.05; **, P < 0.01; ***, P < 0.001.