Figure 9. Definition of PRR as a direct target gene of PPARγ and in differentiated 3T3 cells.

(A) Schematic illustration of the mutagenesis of the 2 PPRE sites in the promoter of PRR. (B) Luciferase assay for PRR promoter activity. The 3T3 cells were transfected with empty vector or vectors carrying a 2-kb flanking region of the promoter with or without mutagenesis of either one of the PRRE sites (n = 5, repeat 2 times). (C) The effect of rosiglitazone (Rosi) on PRR protein expression (n = 4, repeat 3 times). The cells were treated for 24 hours with vehicle or Rosi, followed by immunoblotting analysis of PRR. The same samples were run on a separate gel for detecting GAPDH. The densitometry values are shown underneath the blots. (D) The effect of Rosi on sPRR production. The cells were treated for 24 hours with vehicle, PF, Rosi, or Rosi + PF, followed by ELISA measurement of medium sPRR (n = 10). (E) The role of sPRR in mediating Rosi-induced insulin sensitivity (n = 10). The cells were treated for 24 hours with vehicle, Rosi, Rosi + PF, Rosi + PF + sPRR-His, or Rosi + sPRR ab, and then each group was divided to receive vehicle or 20-minute insulin treatment, followed by the assay for glucose uptake. *P < 0.05 vs. vehicle (C). Statistical significance was determined by using ANOVA with the Bonferroni test. Data are shown as mean ± SEM. P-Luc, PRR promoter-luciferase constructor; Δ-P-Luc, PPRE mutation of PRR promoter-luciferase construction; sPRR ab, sPRR antibody.