Chapter 20
Investigating the Role of AMPK in Inflammation
Sarah J. Mancini and Ian P. Salt
Abstract
In addition to the well-characterized role of AMPK in the regulation of nutrient metabolism, it is
increasingly clear that AMPK activation has multiple actions on inflammatory signalling. Here we describe
methods to identify effects of AMPK activity on pro-inflammatory signalling, specifically (1) the nuclear
localization of the key inflammatory mediators nuclear factor-κB (NFκB) and phosphorylated c-Jun
N-terminal kinase (JNK), (2) preparation of conditioned medium to analyze the secretion of cytokines/
chemokines, and (3) the pro-inflammatory adhesion of leukocytes to cultured cells.
Key wordsInflammation, Nuclear localization, Cytokine, Conditioned medium, Monocyte adhesion1 Introduction
Inflammatory signalling plays a key role in the development of many
chronic pathological disorders, including type 2 diabetes, atheroscle-
rosis, and cancer [1–4]. Intriguingly, AMPK has been reported to
have anti-inflammatory actions, dependent and independent of its
effects on nutrient metabolism [5–7]. We and others have demon-
strated that AMPK activation suppresses multiple distinct
pro-inflammatory signalling cascades, including the
pro-inflammatory c-Jun N-terminal kinase (JNK), nuclear factor-κ
B(NFκB), and Janus kinase-signal transducer and activator of tran-
scription (JAK-STAT) pathways [8–13]. Here, we describe methods
to investigate the effect of AMPK activation on cultured cells sub-
jected to pro-inflammatory stimuli. In particular, we describe meth-
ods to examine translocation of NFκB to and levels of activated JNK
in the nucleus, chemokine/cytokine secretion, and adhesion of
monocytes to endothelial cells. Translocation of NFκBfromthe
cytosol to the nucleus and nuclear activation of JNK are key stages
in pro-inflammatory signalling, as this process ultimately stimulates
the transcription of an overlapping set of pro-inflammatory genes
encoding cytokines and chemokines, thereby amplifying the inflam-
matory response [7, 8, 14–16]. Immunofluorescence and confocalDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_20,©Springer Science+Business Media, LLC 2018
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