Chapter 24
Studying the Role of AMPK in Autophagy
Sarah Krieg, Bernhard L€uscher, Jo ̈rg Vervoorts, and Marc Dohmen
Abstract
AMPK is an energy-sensing kinase and is required for the induction and progression of the autophagy
process. In this chapter, we describe experimental approaches to study the steady state and flux of autophagy
in response to AMPK activation. For this purpose, we provide detailed protocols for the measurement of
general as well as AMPK-specific autophagy markers by immunoblot and immunofluorescence analysis.
Key wordsAMP-activated protein kinase, Autophagy, Flux analysis, LC3, Immunofluorescence,
Immunoblot, Bafilomycin A1, AICAR, A-769662, Glucose deprivation1 Introduction
The adenosine monophosphate (AMP)-activated protein kinase
(AMPK) is generally considered the master energy sensor because
it promotes switching from anabolism to catabolism when activated
by an increased ratio of AMP to ATP under energy-deprived con-
ditions. AMPK can be activated selectively by the combination of
aminoimidazole-4-carboxamide ribonucleotide (AICAR) and
A-769662 or by glucose deprivation [1–4]. AMPK regulates a
multitude of metabolic processes by activating or repressing phos-
phorylation of key enzymes [5, 6]. The most prominent is the
phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79 that
inhibits fatty acid and isoprenoid synthesis [7, 8]. One of the
catabolic processes activated by AMPK is macroautophagy (hereaf-
ter autophagy), which is characterized by the engulfing of cytoplas-
mic components into a double lipid membrane resulting in an
isolation membrane and later the autophagosome [9]. Eventually
the autophagosome fuses with a lysosome resulting in the autolyso-
some where the cargo is degraded by the acidic lysosomal hydro-
lases and recycled to the cytoplasm where it serves as new building
blocks and an energy source to maintain homeostasis and prevent
apoptosis [5, 9].Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_24,©Springer Science+Business Media, LLC 2018
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