FIGURE 8.

CDDO-Me treatment increases AMPK Thr-172 and LKB1 phosphorylation in vivo in a murine model. Activation of AMPK is LKB1-dependent. A–D, the tissue extracts were prepared from Western diet-fed mice treated with CDDO-Me and vehicle through gavage for 2 weeks. Western blot analysis showed that CDDO-Me increases AMPK Thr-172 and LKB1 phosphorylation in liver (A and C) and in muscle (B and D) homogenate. Blot with AMPK and LKB1 served as loading controls for muscle as well as liver tissue. E–H, immunoprecipitation of liver homogenate (E and G) and muscle homogenate (F and H) was further characterized to show the association between AMPK and LKB1. The precipitate was subjected to Western analysis with antibodies to p-AMPK and p-LKB1. The levels of precipitated AMPK (E and F) were assessed with p-LKB1-specific antibody and precipitated LKB1 (G and H) with p-AMPK-specific antibody and showed higher phosphorylation of LKB1 and AMPK in CDDO-Me over vehicle-treated mice. Western blot with same antibody used for immunoprecipitation served as a loading control. The corresponding bar graphs show the quantification of the blot. I and J, ERK1/2, an upstream target of the LKB1-AMPK pathway, was activated by CDDO-Me treatment and displayed increased phosphorylation with CDDO-Me treatment in C₂C₁₂ cells (I) and isolated hepatocytes (J). K, MAPK inhibitor U0126 blocked ERK1/2-mediated AMPK activation by CDDO-Me, suggesting ERK1/2 as a possible upstream target. IP, immunoprecipitation; IB, immunoblot.