from multiple acetyl-CoA dehydrogenases and catalyses the transfer
of electrons from flavoproteins to ubiquinone, linking the oxida-
tion of fatty acids to mitochondrial OXPHOS. Altogether, the
redox energy generated by electron flow is used to transport pro-
tons from mitochondrial matrix to the intermembrane space, gen-
erating an electrochemical gradient (or protonmotive force) that is
ultimately used for ATP synthesis through ATP synthase or
dissipated through passive reentry into the matrix (proton leak) [1].
Specific inhibition of ETC complexes and ATP synthase or
uncoupling of OXPHOS using chemical protonophores both lead
to cellular ATP depletion and activation of the energy sensor
AMP-activated protein kinase (AMPK). AMPK is a heterotrimeric
protein complex, consisting of a catalyticαsubunit and two regu-
latoryβandγsubunits, that functions as an adenylate charge-
regulated kinase which constantly senses the cellular energy status
by monitoring intracellular AMP, ADP, and ATP levels [3]. Once
activated, AMPK inhibits ATP-consuming anabolic processes and
promotes ATP-generating catabolic pathways via direct phosphor-
ylation of multiple downstream effectors, leading to restoration of
cellular energy homeostasis [4]. In this context, investigating the
impact of AMPK-activating drugs on mitochondrial OXPHOS by
measuring oxygen consumption rates in intact cells might provide
relevant information in order to elucidate the mechanism underly-
ing AMPK activation in various conditions [5]. Interestingly, the
study of mitochondrial bioenergetics has recently faced a renewed
interest, notably in the emerging field of immunometabolism, in
part due to the development of a user-friendly integrated system for
assessing cellular oxygen consumption in a semi-throughout way
using oxygen-sensing fluorophores incorporated on a cell plate
(Seahorse™). Before this technological breakthrough, the mito-
chondrial bioenergetics was reliably assessed in isolated organelles
from whole tissue or cells using Clark-type polarographic oxygen
electrode devices. A Clark electrode is composed of a polarized
platinum cathode and a silver anode in contact with a solution of
semi-saturated potassium chloride maintained by a thin Teflon
membrane permeable to oxygen and is usually connected to a
thermostated and stirred chamber containing the mitochondria/
cell suspension [6].
In this chapter, using liver and primary hepatocytes as examples
of starting materials, we describe several step-by-step protocols for
measuring mitochondrial oxygen consumption rates in intact cells,
permeabilized cells (in situ mitochondria), and isolated organelles
using both Clark-type polarographic oxygen electrode devices and
the newly developed Seahorse technology (Fig.1). Of note, these
approaches can easily be translated to other tissues and cell types
than liver and hepatocytes.274 Guillaume Vial and Bruno Guigas