upregulating the glycolytic flux via 6-phosphofructo-2-kinase
phosphorylation [4–7]. Additionally, AMPK is responsible for sti-
mulating mitochondrial biogenesis, via increasing the transcription
and the activity of the peroxisome proliferator-activated receptor
gamma coactivator 1α(PGC-1α)[ 8]. Therefore, AMPK represents
a key player in regulating several metabolic pathways to rebalance
processes of energy expenses and energy intake. However, the study
of metabolic fluxes has remained quite challenging and time-
consuming.
A valuable and efficient experimental tool for accessing meta-
bolic fluxes is the Seahorse XFe24 respirometry assay. This tech-
nique is based on the use of specific sensors that define a
microchamber over the cells in the wells. Once the sensors reach
the right position, they monitor in real time the glycolytic flux and
mitochondrial respiration, by measuring the extracellular acidifica-
tion rate (ECAR) and oxygen consumption rate (OCR), respec-
tively. The Seahorse XFe can be run on intact cells, including stable
and primary cells [9, 10], tissues (i.e., pancreatic islet, rodent
hippocampus slices), as well as isolated mitochondria [11, 12].
In this chapter, we propose a protocol optimized for the analy-
sis of the metabolic fluxes using various energy substrates in pri-
mary neuronal cultures. Indeed, neurons consume most of the
energy intake of the brain, an organ that per se consumes approxi-
mately 20% of the oxygen and 25% of the total glucose spent by the
human body. Neuronal functionality is strongly dependent on
intracellular metabolic homeostasis and substrates availability. The
importance of neuronal dependence on energy homeostasis is
underpinned by the observation that several neurodegenerative
diseases, such as Alzheimer’s disease, are associated to an alteration
of glucose metabolism, mitochondrial dysfunctions, and AMPK
deregulation [13, 14]. Despite glucose being the obligatory brain
energy source, neurons can use alternative energy substrates, such
as lactate and ketone bodies [15, 16]. In this context, the Seahorse
XFe assay is a powerful experimental approach for studying the role
of AMPK in regulating glycolysis and mitochondrial respiration,
herein monitoring metabolic fluxes in the presence of distinct
energy substrates, ranging from glucose to pyruvate, lactate, ketone
bodies, and glutamine.2 Materials
All solutions should be prepared using ultrapure water (prepared by
purifying deionized water to reach the sensitivity of 18 MΩ/cm at
25 C) and cell culture grade reagents.290 Claudia Marinangeli et al.