Metformin (N, N-dimethylbiguanide) is the most widely used first-line drug for treatment of type 2 diabetes (T2D). This antihyperglycemic drug offers clinical superiority over other glucose-lowering drugs, with little induction of hypoglycemia or weight gain and with the effects to reverse fatty liver, improve insulin sensitivity, and ameliorate cardiovascular dysfunctions associated with T2D (extensively reviewed by Foretz et al., 2014). Administration of metformin to nematodes (C. elegans) and mice gave rise to extended lifespan and health span (Barzilai et al., 2016). Moreover, cancer incidence was found to be decreased in patients treated with metformin (Foretz et al., 2014). Based on the promising research results, a clinical trial named TAME (Targeting Aging with Metformin; http://www. afar. org/natgeo) has been proposed to test whether metformin can delay the onset of age-related diseases and conditions, including cancer, cardiovascular disease, and Alzheimer’s disease. The AMP-activated protein kinase (AMPK) is a master controller of various metabolic pathways (Hardie, 2014). The beneficial effects of metformin can be attributed, at least in part, to its cause of AMPK activation (Foretz et al., 2014), which depends on LKB1, the liver kinase B1 (Shaw et al., 2005). For example, activated AMPK is indispensable for the attenuation of hepatic steatosis and atherosclerosis after metformin treatment (Li et al., 2011). One recent study further revealed that a single mutation on the AMPK-mediated phosphorylation sites of ACC1/2 strongly blocks the metformin-improved insulin action and glucose tolerance in diabetic mice (Fullerton et al., 2013). Similarly, metformin retards aging in C. elegans in an AMPK-dependent manner (reviewed by Burkewitz et al., 2014). However, the mechanism for metformin to activate AMPK remains unclear and even controversial. It was shown that metformin treatment increases cellular levels of AMP through inhibiting complex I of the electron transport chain, by which ATP synthesis is uncoupled, leading to a drop in cellular ATP concentration and hence an increase of AMP levels (Foretz et al., 2014). However, some studies failed to detect the alteration of AMP levels in cultured cell lines treated with metformin (He and Wondisford, 2015). In addition, recent studies showed that treatment of primary hepatocytes with clinically relevant concentrations of metformin, $70 μM (detected in portal vein after a therapeutic dose), efficiently activates AMPK without disrupting energy state (He and Wondisford, 2015). Similar observations were also obtained in the liver of mice after chronic administration of 50 mg/kg of metformin, within the clinical dose range (He and Wondisford, 2015). Furthermore, some studies were unable to detect direct inhibition of metformin on complex I in isolated mitochondria, unless at high concentrations ($5 mM)(He and Wondisford, 2015). It is therefore evident that a clear understanding on how metformin activates AMPK awaits further studies.

The scaffold protein AXIN plays an essential role in glucose starvationinduced AMPK activation by co-translocating LKB1 to the surface of lysosome in that AXIN also docks onto the lysosomal v-ATPase-Ragulator complex (Zhang et al., 2014, 2013). It must be noted that the v-ATPase-Ragulator complex must be primed by glucose starvation or treatment of the v-ATPase inhibitor concanamycin A (conA), likely through conformational change upon glucose starvation, for AXIN to anchor onto (Zhang et al., 2014). Here, we first