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    • Another class of AMPK regulator

    2023-02-25

    Another class of AMPK regulator is peptidyl-prolyl cis/trans isomerase (PPIase) NIMA-interacting 1 (Pin1), which binds to a number of proteins and regulates oncogenesis and metabolic diseases (Khanal et al., 2013; Zhou and Lu, 2016). Pin1 has been shown to bind to and inhibit AMPK; therefore, at least some effects of Pin1 on metabolism appear to be mediated by the Pin1-AMPK association. The AMPK activity has been studied extensively by in vitro kinase assay and immunoblotting with anti-phospho-AMPK (pAMPK) or anti-phospho-ACC (pACC), which reflect mean AMPK activity in the cell population. To examine the heterogeneity of AMPK activity, Tsou et al. (2011) developed AMPKAR, a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) for AMPK activity, and revealed a very high cell-to-cell heterogeneity in the amplitude and time course using tissue culture cells. An improved version of AMPKAR has been developed and used to examine the AMPK activity in neurons (Sample et al., 2015). A drawback of many FRET biosensors, including AMPKAR, may be low signal-to-noise ratio. We have reported that a long, flexible EV linker could significantly improve the dynamic range of many FRET biosensors by reducing the basal FRET signal (Komatsu et al., 2011) and that the resulting highly sensitive FRET biosensors enable us to visualize protein kinase activities in living mice, collectively called FRET mice (Kamioka et al., 2012). In this study, we have applied the EV linker technology to AMPKAR. The resulting AMPKAR-EV FRET biosensor exhibits three-fold higher dynamic range than AMPKAR and monitored AMPK activation in HeLa Bupivacaine HCl stimulated by 2-deoxyglucose (2-DG). Moreover, intravital imaging of transgenic mice expressing AMPKAR-EV has revealed that AMPK is predominantly activated in fast-twitch muscle fibers and that metformin activates AMPK in hepatocytes, but not in muscles. Thus, the in vivo imaging of AMPK activity will open a window to understanding the heterogeneous responses of AMPK among cell types in vivo.
    Results
    Discussion By the use of a flexible EV linker, the basal level of the FRET/CFP ratio was markedly decreased in comparison to that for the prototype, AMPKAR (Figures 1B and 1C). This decreased basal signal of AMPKAR-EV allowed us to classify cells easily into two groups based on the expression of LKB1 (Figure 2). In agreement with previous reports (Hawley et al., 2003; Woods et al., 2003; Shaw et al., 2004; Gowans et al., 2013), this significant difference in the basal AMPK activity indicates that LKB1 phosphorylates and activates AMPK even in nutrient-rich culture medium. It should be recalled that AMPK-dependent phosphorylation inactivates ACC and HMG-CoA reductase. Thus, the basal activities of LKB1 and AMPK may play a role in reserving inert ACC and HMG-CoA reductase. In this context, we may need to pay more attention to signals that reduce LKB1 activity under nutrient-rich conditions. Pin1 has been shown to bind to and inactivate AMPK (Khanal et al., 2013; Nakatsu et al., 2015). The binding of Pin1 to the CBS3 domain of the AMPK γ subunit exposes phospho-Thr172 of the α subunit for the dephosphorylation by PP2C and thereby suppresses AMPK activity (Nakatsu et al., 2015). By using cell lines expressing AMPKAR-EV, we found that Pin1 inhibits AMPK activation by LKB1, but not by CaMKK2 (Figures 3 and S2), suggesting an LKB1-specific mechanism of inhibition. LKB1 phosphorylates AMPK on a scaffold protein, Axin (Zhang et al., 2013). It remains unknown which subunit of AMPK binds to Axin; however, we could speculate that Pin1 binding to AMPK inhibits the association of AMPK with Axin and thereby prevents AMPK from LKB1-dependent phosphorylation. This scenario can also explain why Pin1 did not inhibit CaMKK2-dependent AMPK activation. The difference in the AMPK activity between slow- and fast-twitch fibers has been controversial. Narkar et al. (2011) found that AMPK was more active in the soleus (predominantly slow-twitch myofibers) than the quadriceps (predominantly fast-twitch myofibers). Meanwhile, other research groups failed to find significant difference in AMPK activity between the soleus and the extensor digitorum longus (predominantly fast-twitch myofibers) (Dzamko et al., 2008; Jensen et al., 2007; Jørgensen et al., 2004). These studies were based mostly on immunoblotting with anti-pAMPK; therefore, they do not necessarily show the difference between fast- and slow-twitch fibers. The use of AMPKAR-EV enabled us to examine the AMPK activity directly in each fiber type before and after stimulation (Figure 5). Our data strongly suggested that only the fast-twitch myofibers exhibited an increase in AMPK activity upon tetanic contraction and exercise. We may speculate that the tetanic contraction and the exercise causes ATP consumption primarily in fast-twitch myofibers, resulting in strong AMPK activation. Although we cannot rule out the possibility that AMPK activation in the slow-twitch myofibers was transient, and therefore could not be detected in our experimental protocol, it is unlikely that such transient AMPK activation alters the metabolic states of the slow-twitch myofibers. It would be interesting to test whether low-intensity, long-time exercise may activate AMPK preferentially in the slow-twitch myofibers.