FIG. 5.

A: Tlr2 and Tlr4 mRNA expression following adenoviral infection and differentiation induction. Confluent 3T3-L1 cells were infected with human RAGE- (Ad-RAGE) or control LacZ–expressing adenovirus (Ad-LacZ) (−24 h). At 0 h, differentiation was initiated, and total RNA was isolated at indicated hours of differentiation. Tlr2 and Tlr4 mRNA expression was determined by quantitative real-time RT-PCR. mRNA expression levels of each mouse gene were determined by a comparative Ct method using 18S ribosomal RNA as endogenous reference, and mRNA level of Ad-LacZ–infected (Ad Infect) cells at −24 h was expressed as 100%. B: Knockdown of Tlr2, but not Tlr4, ameliorates adipocyte hypertrophy induced by RAGE overexpression. 3T3-L1 preadipocytes were cultured in DMEM with 10% FBS for 2 days. The cells were harvested, suspended, and transfected with predesigned siRNA for mouse Tlr2 (siTlr2-1–3; corresponding to three distinct regions of Tlr2) or Tlr4 (siTlr4-1–3; corresponding to three distinct regions of Tlr4) using electroporation system. Twenty-four hours after siRNA transfection, the cells were infected with Ad-RAGE or Ad-LacZ. One day after adenoviral infection, differentiation was induced. Hypertrophied adipocyte was determined by Oil Red O staining 6 days after differentiation. *P < 0.05 vs. Ad-LacZ, **P < 0.05 vs. control (Cont), Student t test. Right panel: Tlr2 and Tlr4 mRNA levels 24 h after respective siRNA transfection. C: Effects of palmitate, a Tlr2 ligand, on adipogenesis in 3T3-L1 preadipocytes. As shown in the left panel, addition of 0.5 or 1.0 mmol/L palmitate increased lipid droplet in adipocytes, but did not increase adipocytes with ring-like lipid staining. Right panel represents the effects of 1.0 mmol/L palmitate on adipocyte hypertrophy in the presence or absence of RAGE overexpression. HPF, high-power field. *P < 0.05 vs. Ad-LacZ.