Because of the critical roles played by AMPK in

Because of the critical roles played by AMPK in energy sensing and cancer cell survival, a huge number of drugs have been proposed to exert their pharmacological effects by means of AMPK activation (Kim and He, 2013). For example, metformin has been shown to activate AMPK in muscle (Sajan et al., 2010; Kristensen et al., 2014), liver (Shaw et al., 2005; Sajan et al., 2010; Tajima et al., 2013), KH 7 (Chen et al., 2009; Duan et al., 2013; Cho et al., 2015), and pancreatic cancer cells (Hinke et al., 2007; Kisfalvi et al., 2009; Sinnett-Smith et al., 2013); however, the difficulty of performing direct comparisons among experimental setups renders the effect of metformin obscure, even if we admit the pleiotropic effects of this agent. Intravital imaging of AMPK activity by using AMPKAR-EV-expressing transgenic mice has enabled us to visualize the influence of AMPK-activating reagents to different organs on the same scale. We found that effect of metformin on AMPK activity differs substantially between liver and skeletal muscle (Figure 4). The reason for this tissue-specific action of metformin is probably because a metformin transporter, organic cation transporter 1 (OCT1), is expressed preferentially in the liver (Wang et al., 2002). Similarly, the expression level of OCT1 in each cell line could affect the responsiveness to metformin in vitro. In the six cell lines analyzed in this study, the expression of LKB1 was perfectly correlated with the reactivity to metformin; however, this observation does not rule out that low OCT1 expression abolishes the reactivity to metformin in vitro. AICAR-induced AMPK activation was observed in previous studies using isolated hepatocytes from mice (Foretz et al., 2010) and rats (Corton et al., 1995) and, in the present study, using hepatic cancer-derived HepG2 cells (Figure 2B). However, only a few reports described similar results in vivo. After two weeks of administration of AICAR, AMPK activity in the liver is increased approximately two-fold in mice (Liu et al., 2015). To our knowledge, only two studies reported in vivo AMPK activation after acute administration of AICAR (Buhl et al., 2002; Sajan et al., 2010). By using obese Zucker (fa/fa) rats and Sprague-Dawley rats, the authors reported a two- to three-fold increase of AMPK activity and pAMPK (Thr172) by AICAR and a three-fold increase by metformin, indicating that metformin more potently activates AMPK in the liver than does AICAR. We also found AMPK activation by metformin in the liver but failed to detect the effect of AICAR (Figure 4). The discrepancy may be ascribable to the difference between mice and rats. Because AICAR must be transported into the liver and phosphorylated to yield ZMP for its action, the kinetics of AMPK activation by AICAR may be influenced by the transporters and kinases, of which activity may vary among species and organs. Although the mechanism of muscle-specific action of AICAR in mice is elusive, these findings give us a clue to understanding the effectiveness of metformin for type 2 diabetes mellitus and AICAR for sports doping, respectively (Hardie et al., 2012). Because many pro-AMPK reagents exert their effect through decreasing intracellular ATP concentration, use of transgenic mice expressing FRET biosensors for ATP (Imamura et al., 2009) will also be informative in understanding the regulation of AMPK in vivo.
Experimental Procedures For detailed methods, see also Supplemental Experimental Procedures.
Author Contributions
Acknowledgments We are grateful to the members of the Matsuda laboratory for their helpful input; Y. Inaoka, K. Hirano, S. Kobayashi, N. Koizumi, and A. Kawagishi for their technical assistance; and the Medical Research Support Center of Kyoto University for in vivo imaging. K. Terai was funded by JSPS KAKENHI16K19391. M.M. was funded by JSPS KAKENHI15H02397, 15H05949 “Resonance Bio,” and 16H06280 “ABiS”; CRESTJPMJCR1654; and the Nakatani Foundation.