AMPK Methods and Protocols

Chapter 19

Study of AMPK-Regulated Metabolic Fluxes in Neurons

Using the Seahorse XFe Analyzer

Claudia Marinangeli, Je ́rome Kluza, Philippe Marchetti, Luc Bue ́e,

and Vale ́rie Vingtdeux

Abstract

AMP-activated protein kinase (AMPK) is the intracellular master energy sensor and metabolic regulator.
AMPK is involved in cell energy homeostasis through the regulation of glycolytic flux and mitochondrial
biogenesis. Interestingly, metabolic dysfunctions and AMPK deregulations are observed in many neurode-
generative diseases, including Alzheimer’s. While these deregulations could play a key role in the develop-
ment of these diseases, the study of metabolic fluxes has remained quite challenging and time-consuming.
In this chapter, we describe the Seahorse XFe respirometry assay as a fundamental experimental tool to
investigate the role of AMPK in controlling and modulating cell metabolic fluxes in living and intact
differentiated primary neurons. The Seahorse XFe respirometry assay allows the real-time monitoring of
glycolytic flux and mitochondrial respiration from different kind of cells, tissues, and isolated mitochondria.
Here, we specify a protocol optimized for primary neuronal cells using several energy substrates such as
glucose, pyruvate, lactate, glutamine, and ketone bodies. Nevertheless, this protocol can easily be adapted
to monitor metabolic fluxes from other types of cells, tissues, or isolated mitochondria by taking into
account the notes proposed for each key step of this assay.

Key wordsAMPK, Energy metabolism, Glycolysis, Mitochondrial respiration, Oxidative phosphory-

lation, Seahorse Analyzer, Glucose, Lactate, Ketone bodies

1 Introduction

As master energy sensor and metabolic regulator, the

AMP-activated protein kinase (AMPK) plays a central role in the

maintenance of cell energy homeostasis. AMPK is activated when

intracellular reduction of ATP/AMP ratio occurs in response to

energetic stress conditions. Once activated, AMPK modulates sev-

eral anabolic and catabolic pathways, such as fatty acid oxidation,

glucose metabolism, and mitochondrial biogenesis to reestablish

the ATP reservoir [1–3]. For instance, AMPK can increase the

intracellular availability and processing of glucose by inducing the

translocation of glucose transporters on cell membrane and by

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_19,©Springer Science+Business Media, LLC 2018

289