AMPK Methods and Protocols

The adenine nucleotides AMP, ADP, and ATP exert various

effects on AMPK activity [11, 12]. Moreover, the cellular energy

levels are better reflected by AMP:ATP and ADP:ATP ratios

[1, 25–27]. Since the levels of AMP are of extreme low ranges,

and ADP, as well as ATP, is rather unstable, it is challenging to

precisely measure the levels of AMP, ADP, and ATP. Here, we

describe a sensitive method for determining the cellular levels of

AMP, ADP, and ATP by using the capillary electrophoresis-mass

spectrometry (CE-MS)-based assays [28, 29].

All of the methods described here are summarized in Fig.1.Of

note, for simplicity, we only mention glucose starvation as a repre-

sentative stress in some parts of the chapter.

To define AMPK activation

via the lysosomal pathway

To determine lysosomal

localization of AXIN/LKB1 by:

To reconstitute

lysosomal AMPK activation by:

Isolation of

lysosome

Isolation

of DRM

Detection of

lysosomal association

of AXIN/LKB1

Detection of

AMPK activation

Level of p-T172 of

AMPK on light

organelles

To obtain genetic evidence by

treating AXIN-/-or LAMTOR1-/-

MEFs and mice, followed by

detection of p-T172-AMPKα

Preparation of

light organelle

Level of

phosphorylation of

ACC by AMPK

Detection of

v-ATPase-Ragulator-AXIN

interaction

Immunofluorescent

staining

Fig. 1Diagram of workflow for studying the lysosomal AMPK activation. To study the lysosomal AMPK
activation under a desired metabolic stress or pharmacological stimulus, p-Thr-172-AMPKαand p-Ser-79-
ACC are analyzed inAXIN
/
andLAMTOR1
/
mouse embryonic fibroblasts (MEFs) or mouse individuals.
The dynamic association of the AXIN/LKB1 with the lysosomal DRM can be determined in purified lysosomes/
DRMs, as well as in intact cells. The complex formation of v-ATPase-Ragulator-AXIN/LKB1-AMPK can be
determined by co-immunoprecipitation. In addition, the light organelle-based assay system provides a way to
determine whether a particular protein or drug of interest activates AMPK directly through triggering the
lysosomal pathway in vitro

Analysis of Lysosomal AMPK Activation 395