Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

https://doi.org/10.1016/j.bbrc.2008.09.007Get rights and content

Abstract

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cδ, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCδ by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK 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.).

References (33)

  • H. Morawietz et al.

    AT1 blockade and cholesterol-dependent oxidative stress: the EPAS trial

    Circulation

    (2006)
  • V.J. Dzau1 et al.

    Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies

    Nat. Med.

    (2002)
  • D.G. Hardie

    AMP-activated protein kinase as a drug target

    Annu. Rev. Pharmacol. Toxicol.

    (2007)
  • H. Motoshima et al.

    AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer

    J. Physiol.

    (2006)
  • W.J. Lee et al.

    α-Lipoic acid prevents endothelial dysfunction in obese rats via activation of AMP-activated protein kinase

    Arterioscler. Thromb. Vasc. Biol.

    (2005)
  • M. Zang et al.

    Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice

    Diabetes

    (2006)
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