Figure 5. Prdm16 is required for expression of a thermogenic gene program in subcutaneous adipocytes.

(A–G) Subcutaneous preadipocytes in the SV fraction of inguinal fat from WT mice were transduced with adenovirus expressing a shRNA targeted to Prdm16 or a scrambled control shRNA (ctl). These cultures were then induced to differentiate in vitro into adipocytes. (A) Prdm16 mRNA levels (with and without isoproterenol stimulation, as indicated). (B) GFP was expressed from adenoviral shRNA vectors, and its expression was used to reveal control shRNA– and sh-Prdm16–transduced adipocytes. (C) Oil-Red-O staining (red) for lipid accumulation. mRNA levels of general adipocyte markers (Fabp4 and AdipoQ) were also determined. (D) mRNA levels of brown fat–selective genes (Ucp1, Cidea, Cox8b, and Ppargc1a). (E) Western blot analysis for Prdm16 and Ucp1 protein. (F) Oxygen consumption, assayed using a Clark-type electrode. Oligomycin (ATPase inhibitor) and CCCP (chemical uncoupler) were added to cells to measure the rates of uncoupled and maximal respiration, respectively. (G and H) WT Prdm16^+/+ and heterozygous Prdm16^+/– littermates were treated with CL316,243 for 3 days. (G) H&E staining of inguinal adipose tissue. (H) mRNA levels of Prdm16, brown fat–selective genes Ucp1 and Cidea, and Retn in epidWAT, ingWAT, and iBAT. Original magnification, ×20 (B, C, and G). Values are mean ± SD (n = 3–5). *P < 0.05; **P < 0.01.