Biochemical and Biophysical Research Communications
Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues
Section snippets
Materials and methods
Materials. ATP assay kit and PKC inhibitors were from Calbiochem (La Jolla, CA). DMEM and Lipofectamine RNAiMAX were from Invitrogen (Kyoto, Japan). Antibodies for β-actin and PKCδ were from Santa Cruz Biotechnology (Santa Cruz, CA). Isoform-specific antibodies for each PKC molecule were from BD biosciences. Antibodies for the phosphorylated forms of AMPK α-subunit (Thr-172) and acetyl-CoA carboxylase (ACC; Ser-79) were from Cell Signaling Technology (Beverly, MA). Aortas from male Japanese
Rottlerin activates AMPK in vascular smooth muscle cells
The impact of rottlerin on AMPK was investigated in human VSMCs. In a dose-dependent manner, treatment with rottlerin for 2 h increased Thr-172 phosphorylation of AMPK, indicative of AMPK activation (Fig. 1A). The enzymatic activation of AMPK was also confirmed by examining Ser-79 phosphorylation of ACC, a major downstream target protein in the AMPK signaling cascade. Consistent with AMPK phosphorylation and activation, phosphorylation of ACC was elevated in rottlerin-treated HASMCs (Fig. 1A).
Discussion
Although widely thought to be a selective PKCδ inhibitor, we show in the current study that rottlerin is an AMPK activator in human and rabbit VSMCs as well as in isolated rabbit aortic strips. These findings are important because more studies using PKCδ inhibitors to characterize the cellular and physiological effects of this important signaling kinase are appearing in the literature. AMPK activation by rottlerin was also found to be associated with a reduction of cellular ATP levels. Since a
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, Japan (Nos. 16046219 to E. A. and 18590995 to T. N.) and by grant from the Japan Diabetes Foundation (to H.M.).
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