CR activates transcription factors that induce the expression of genes associated with adipogenesis. Animals (n=5–8) maintained on either AL or CR diet for 2- and 25-mo were sacrificed in a fasted state. Total RNA was isolated from frozen epididymal fat depot, and mRNA levels were compared by semiquantitative multiplex RT-PCR. After normalization of the specific gene to the internal control gene, 18S rRNA, mRNA levels are represented as means ± SEM, expressed as relative levels of 2-mo AL group. Representative PCR products, PPARγ (A, left panel) and adiponectin (E) are also presented. Additionally, equal amounts of total proteins from each group resolved with SDS-PAGE were detected with antibodies against PPARγ, CEBPα, Cdk-4 and CEBPβ. Data are expressed as percentage of changes in PPARγ (A, right panel), CEBPα (B), Cdk-4 (C) and CEBPβ (2D) versus β-actin (served as loading control). * P<0.05, ** P<0.01 versus AL counterparts according to two-way (age by diet regimen) ANOVA with post-hoc Fisher’s tests.