Methods to Determine Lysosomal AMPK Activation

Recent studies have revealed that AMP-activated protein kinase (AMPK) can be activated in a non-canonical, AMP- and ADP-independent manner on the lysosome surface in response to low glucose. This novel mode of activation requires the participation of the glycolytic enzyme aldolase, which acts as a sensor of falling levels of the glucose metabolite fructose-1,6-bisphosphate (FBP). The FBP-unoccupied aldolase blocks the V subfamily of transient receptor potential (TRPV) cation channel to generate a local low calcium environment, under which conditions the inhibited TRPVs contact the lysosome-localized vacuolar ATPase (v-ATPase). This in turn triggers the translocation of AXIN and the associated liver kinase B1 (LKB1) to the lysosomal surface to activate AMPK thereon. Interestingly, such a lysosomal AMPK activating pathway has now been demonstrated to be shared by both the anti-diabetic drug metformin and the anti-tumor/inflammation drug mannose. Here, we describe the experimental conditions and procedures for distinguishing if a certain activation of AMPK is mediated by the lysosomal pathway. We will update the previously described methods for determining AXIN lysosomal translocation and the AMP:ATP and ADP:ATP ratios that are used for differentiating the lysosomal pathway from the canonical, AMP-dependent pathway [1, 2]. We also describe how to determine the lysosomal pH and the inhibition of TRPV activity that are prerequisite for triggering the lysosomal pathway during sensing of low glucose.