Methods to Determine Lysosomal AMPK Activation

Authors:
Publisher website
Google Scholar
Abstract
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.

This publication has 48 references indexed in Scilit: