Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur

Abstract

Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and in vivo, acquired with genetically-encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K⁺ coincides with a Warburg effect mediated by NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4⁺. The compounded outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na⁺ pump. We conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.

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Neurochemical Research
https://doi.org/10.1007/s11064-020-02964-w
ORIGINAL PAPER
Aerobic Glycolysis intheBrain: Warburg andCrabtree Contra Pasteur
L.FelipeBarros1 · IvánRuminot1· AlejandroSanMartín1· RodrigoLerchundi1· IgnacioFernández‑Moncada1·
FelipeBaeza‑Lehnert1
Received: 10 September 2019 / Revised: 10 January 2020 / Accepted: 16 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local
surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when
energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by
the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light
of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and invivo, acquired with genetically-
encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular
compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute
aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K+ coincides with a Warburg effect mediated by
NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4+. The compounded
outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume
both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na+ pump. We
conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not
reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains
to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.
Keywords Glucose· Lactate· Oxygen· Astrocytes· Neuron· Potassium· Nitric oxide· Glutamate· Ammonium
Introduction
The full oxidation of glucose to CO2 and H2O renders 30–32
ATPs [1]. The alternative to oxidation is the production of
lactate, which consumes no oxygen and produces only 2
ATPs. It was therefore surprising to see reports by Marcus
Raichle and colleagues showing that evoked neural activity
in human subjects is accompanied by glucose consumption
in excess of oxygen consumption [2]. This finding was later
confirmed by lactate measurements in humans and rodents
[3, 4]. As the excess glycolysis occurred in the presence
of oxygen, the phenomenon was a puzzling physiological
counterpart to the aerobic glycolysis originally described in
tumors [5]. Why would the brain shun oxygen and engage in
inefficient energy production at the time of its greatest need?
We address this question in this article. Sustained aerobic
glycolysis, as reported in the developing brain [6] and in
proliferating cells [7], will not be discussed here.
Whilst experimenting with yeast in the 1860s, Louis Pas-
teur observed that glycolysis, then known as fermentation, is
acutely suppressed by oxygen [8]. In the 1920s, Otto War-
burg christened the phenomenon Pasteursche Reaktion [9],
while reporting that it vanishes in tumor slices [5, 10]. The
Warburg effect, a term introduced by Efraim Racker in the
1970s [11], has become a prospective therapeutic target in
cancer and inflammation [7, 12]. Warburg knew that glucose
(but not amino acids and fatty acids) inhibits the respiration
of tumors [5]. However, the inhibitory effect of glucose on
respiration came to be named after Herbert Crabtree, who
published his results (obtained with a Warburg manometer)
several years later [13]. We could not find out when was the
term Crabtree effect first introduced, but it appears in the
literature as early as 1940 [14]. In contrast with the current
Special Issue: In Honor of Professor Juan Bolaños.
* L. Felipe Barros
fbarros@cecs.cl
1 Centro de Estudios Científicos—CECs, 5110466Valdivia,
Chile

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hype about the Warburg effect, the Crabtree effect remains in
relative darkness, except for brewers [15], who take advan-
tage of the ancient evolutionary invention of ethanol as a
tactical weapon [16, 17].
The Pasteur effect is the suppression of glycolysis by
mitochondrial oxidative metabolism. The Warburg effect
is the inhibition/failure of the Pasteur effect. The Crabtree
effect is the suppression of mitochondrial oxidative metabo-
lism by glycolysis, i.e. the mirror image of the Pasteur effect
(Table1). There are other oxygen sinks in mammalian cells
but for the sake of brevity, in this review we will refer to
mitochondrial oxidative metabolism as respiration. The
relationship between these three interdependent effects is
depicted in Fig.1. In a typical mammalian cell, blockage of
respiration by anoxia or mitochondrial poisons stimulates
glycolysis by a factor of 3 to 10, meaning that the Pasteur
effect inhibits glycolysis by 60–90%. The Pasteur effect is
thus a major contributor to the balance between glycolysis
and respiration (Fig.1a). The Warburg effect may be under-
stood as a deficit in the capacity of mitochondria to keep
glycolysis at bay (Fig.1b). The Crabtree effect involves
a primary increase in glycolytic flux that leads to inhibi-
tion of respiration (Fig.1c). In its original descriptions the
increase in glycolysis was secondary to glucose addition [5,
13]. Our group recently reported a variant of the Crabtree
effect in astrocytes, in which the inhibition of respiration
results not from increased glucose availability but from a
primary stimulation of the glycolytic machinery mediated
by an extracellular signal [18]. While conceptually differ-
ent, the Warburg and Crabtree effects look similar as they
both involve enhanced relative glucose to oxygen consump-
tion and augmented lactate production. So which of them is
responsible for activity-dependent aerobic glycolysis in brain
tissue? The answer to that question lies in the metabolic
behavior of neurons and astrocytes.
Neurons
Neurons are the main energy consumers of brain tissue,
accounting for > 90% of the ATP turnover triggered by
activity [19]. Substantial evidence invitro and invivo indi-
cates that neurons consume both glucose and lactate, the
latter produced by neighboring astrocytes upon neuronal
prompting [20–24]. The transfer of lactate from astrocytes
to neurons is termed Astrocyte-to-Neuron Lactate Shuttle
(ANLS), a phenomenon first proposed by Luc Pellerin and
Pierre Magistretti in the 1990s [25] and since characterized
by multiple experimental approaches [20–24]. ANLS is an
evolutionary conserved phenomenon, an extreme version of
which occurs in Drosophila melanogaster [26, 27]. How-
ever, its existence is not universally accepted [28–30].
Attempts to investigate the impact of synaptic activity on
the metabolism of cultured neurons using prolonged bath
application of glutamate or glutamate receptor agonists have
produced contrasting results. While glutamate was found to
inhibit glucose transport [31] and glycolysis [32], engaging
NMDA receptors led to glycolysis stimulation [33]. Thanks
to the availability of genetically-encoded fluorescent sen-
sors for metabolites, it has recently been possible to look
into these phenomena with improved temporal resolution.
Even short exposures of neurons to glutamate and NMDA
provoke metabolic stress, with glutamate having the most
dramatic effect [34–36]. As a more physiological activation
by electrical stimulation, which did not perturb ATP or ATP/
ADP, resulted in robust glycolytic stimulation [36], it seems
likely to us that inhibition of neuronal glucose transport and
glycolysis by glutamate represent pathological events, akin
to the generalized shutdown of metabolism observed in mul-
tiple systems under metabolic stress [37]. Two other studies
based on genetically-encoded sensors gave more direct infor-
mation on the balance between glycolysis and respiration.
In the first study, hippocampal granule cells in acute tissue
slices responded to afferent stimulation (60 pulses distrib-
uted over 3s) with transient increases in cytosolic NADH/
NAD+ ratio and lactate [38], pointing to stronger activation
of glycolysis relative to respiration. However, the neuronal
lactate surge was insensitive to blockage of the lactate trans-
porters [38], suggesting that there was no influx or efflux of
lactate and that therefore the activity-dependent extracellular
lactate surge observed invivo [4, 39] originates in another
cell type, i.e. astrocytes. In the second study, fluxes were
measured with transport-stop protocols. Exposure of cul-
tured hippocampal neurons to a short theta burst (40 pulses
distributed over 11s) elicited a strong stimulation of both
glucose consumption (200%) and mitochondrial pyruvate
consumption (300%), but did not change cytosolic pyruvate
or lactate [36]. This shows that the balance between glyco-
lysis and respiration withstood the change in flux regime.
Table 1 Interdependence of
glycolysis and mitochondrial
respiration
Effect Effector Cellular response Reference Christening
Pasteur Respiration Inhibition of glycolysis Pasteur [8] Warburg [9]
Warburg Respiration deficit Stimulation of glycolysis Warburg etal. [10]
Warburg [5]
Racker [11]
Crabtree Increase in glycolysis Inhibition of respiration Crabtree [13]
Warburg [5]
Unknown, At or before
Rosenthal etal. [14]

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Glycolysis and mitochondria were proposed to be synchro-
nized by a mechanism involving the Na+/K+ ATPasepump
independently of adenine nucleotides and Ca2+ [36]. In this
same study, tetanic stimulation (600 pulses over 30s) caused
ATP depletion and inhibition of mitochondrial pyruvate con-
sumption, indicative of mitochondrial collapse. This failure
coincided with a large increase in intramitochondrial Ca2+,
which is also observed in neurons exposed to toxic glutamate
levels. Measurements in different types of neurons at varying
levels of activation will be needed to ascertain the conditions
under which the balance between glycolysis and respiration
breaks down. The nature of the stimulation protocol is rel-
evant, as hinted by the sensitivity of long term potentia-
tion (LTP) and long term depression (LTD) to the specific
arrangement of pulse stimulation [40]. Our working model at
this stage is that at rest and at moderate levels of activation,
neurons consume glucose and also lactate from astrocytes
(more below), whereas at supraphysiological stimulation
(e.g. excitotoxicity), mitochondria fail and neurons start to
produce lactate. A fine balance between glycolysis and res-
piration in these cells is ensured by shared control of both
pathways by the Na+/K+ pump [36]. It is not known how
could the Na+ pump, which is a surface protein, exert control
over the metabolism of mitochondria, most of which lie hun-
dreds of nanometers away. On top of this, there is a robust
Pasteur effect evidenced by the strong response of neuronal
glycolysis to metabolic and oxidative stress [41]. It remains
to be seen whether neurons contribute to aerobic glycolysis
Fig. 1 Pasteur, Warburg and Crabtree. a The Pasteur effect is the
tonic inhibition of glycolysis by mitochondrial respiration that is
abrogated by anoxia. b The Warburg effect is the weakening of the
Pasteur effect, leading to lactate production despite the presence of
oxygen. c The Crabtree effect is the inhibition of mitochondrial res-
piration by augmented glycolysis, also leading to lactate production.
Note that the Warburg and Crabtree effects may not be distinguished
without detailed knowledge of biochemical events involved

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in brain tissue under physiological conditions and how much
of the incremental glucose consumption of active neurons is
diverted to the pentose-phosphate pathway, which does not
generate ATP but antioxidant power [42].
Astrocytes
Astrocytes are net lactate producers as shown by animal
experiments in culture, in slices and invivo [20, 21, 23,
24]. The robust glycolytic phenotype of these cells is partly
explained by stabilization of the master regulator of glyco-
lysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase
3 (PFKFB3); [42, 43]. PFKFB3 is the enzyme that generates
fructose-2,6-biphosphate, a potent allosteric activator of the
glycolytic enzyme 6-phosphofructokinase-1 (PFK1). Such
is the strength of glycolysis in astrocytes that they are still
able to export lactate when bathed in 10mM lactate (Valde-
benito R. and Barros L.F., unpublished data). Contributing
to this vectoriality is the expression in astrocytes of a lactate
channel gated by extracellular lactate that can export even
against a concentration gradient, using membrane potential
as the driving force [44]. Pannexin hemichannels may also
contribute to vectorial lactate export from astrocytes [45].
Astrocytic glycolysis is sensitive to several neuronal
signals, acting through different mechanisms over differ-
ent spatial and temporal domains. Stimulation by glutamate
is mediated by the Na+/glutamate cotransporter and the
Na+ pump [25], peaks at 10–20min and leaves the cell in
a stimulated state long after removal of glutamate [46]. It
is accompanied by stimulation of the glucose transporter
GLUT1, also via the Na+/glutamate cotransporter and the
Na+ pump [46–48]. Glutamate is oxidized by astrocytes
[49], but in the short term its effect on respiration [50, 51]
is smaller than its effect on glycolysis, as evidenced by a
strong lactate production [25, 46]. Throughout the brain,
postsynaptic activity is kept low by tonic GABA-mediated
inhibition despite ongoing glutamate release. This means
that astrocytes are exposed to some glutamate even if neu-
ronal energy demand, chiefly postsynaptic, is low. Consid-
ering the sluggish time course of glycolytic modulation by
glutamate, its permanency upon glutamate removal and the
fact that astrocytic glutamate uptake does not necessarily
correlate with neuronal energy demand, it seems plausible
that astrocytes integrate phasic glutamate signals into a sus-
tained metabolic signal, which primes them to receive pha-
sic information of postsynaptic energy demand, for example
via extracellular K+. This tonic function may also extend
to oligodendroglia, where glutamate facilitates glycolysis
and lactate release through slow GLUT1 translocation to the
cell surface mediated by NMDA receptors [52]. Exposure
of astrocytes to glutamate results in rapid ATP depletion
[18, 51, 53] but the stimulation of glycolysis develops much
later so there does not seem to be a fast mechanistic link
between the two phenomena. One point to be considered
is the manner of glutamate application. We have discussed
how bath application of glutamate or glutamate agonists to
neurons results in inordinate Ca2+ increases and metabolic
stress. This raises the issue of whether the astrocytic ATP
depletion observed upon bath application of glutamate is, or
is not, a physiological phenomenon. At any rate, the aerobic
glycolysis induced by glutamate in astrocytes may well be
regarded as a Warburg effect (Fig.1b).
A more faithful second-to-second reporter of neuronal
energy demand is extracellular K+. Active dendrites release
K+ equimolarly with their uptake of Na+, which is in turn
directly proportional to the ATP demand of the Na+ pump.
Using microelectrodes and microdialysis, average extra-
cellular K+ in the central nervous system was measured
at 2.5–3mM under sleep and anesthesia, rising to 4mM
in the awake state [54] and up to 6mM under physiologi-
cal stimulation [55]. The tiny size of the brain interstice
(20nm) implies that the μm electrodes create a third space
that dampens fluctuations and that local extracellular K+
variations are even larger than recorded [56]. Early inves-
tigation of the metabolic effects of K+ on astrocytes using
radioactive 2-deoxyglucose found small or no effects on gly-
colysis even at 50mM [57–59]. However, with the advent
of genetically-encoded sensors and their improved temporal
resolution, it was possible to observe a strong, immediate
stimulation of glucose consumption, even at 4mM extracel-
lular K+ [46]. The stimulation of glucose consumption by
K+ requires a functional Na+ pump [46] and is driven by the
Na+/bicarbonate cotransporter NBCe1, leading to intracel-
lular alkalinization [60–62]. In addition, the NBCe1, acting
through the bicarbonate-sensitive adenylyl cyclase, mediates
the mobilization of glycogen in response to extracellular K+
[63] and, according to cytosolic NADH measurements, is
also involved in the metabolic effects of glutamate and ATP
[62]. Extracellular K+ contributes further to aerobic glyco-
lysis in its activation of the astrocytic lactate channel [44],
leading to cytosolic lactate depletion and release of product
inhibition of glycolysis [64]. Exposure of astrocytes to K+
resulted in elevated ATP levels and inhibition of respiration
[18, 51], showing that aerobic glycolysis induced by K+ in
astrocytes resembles the Crabtree effect, where a primary
stimulation of glycolysis leads to a secondary inhibition of
respiration (Fig.1c). Whereas the effects of K+ and glu-
tamate on astrocytic metabolism do not interact linearly
[50], afferent stimulation in hippocampal slices provoked
an increase in astrocytic ATP [18], showing that the Crab-
tree effect dominates over the Warburg effect, at least in the
short term.
Additional intercellular signals involved in the control
of astrocytic glycolysis by neuronal activity are nitric oxide
(NO) and ammonium (NH4+). Astrocytes are devoid of NO
synthase but are surrounded by the highest NO synthase

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activity of the body, located in endothelial cells and in neu-
rons [65]. The initial observation that NO stimulates glyco-
lysis and lactate production in astrocytes but not in neurons
through inhibition of mitochondrial cytochrome oxidase
[66] was recently followed by the demonstration that the
modulation can be detected within seconds at nanomolar
NO, levels that are deemed to be within the physiological
range [67]. Neurons may not produce enough NO to reach
astrocytes [68] but endothelium is a stronger NO source,
activated by shear stress during local reactive hyperemia or
by neuronal signals [69–71]. NH4+ is another candidate for
the acute regulation of astrocytic metabolism. Most of the
glutamate released during excitatory neurotransmission is
returned to neurons in the form of glutamine. Within neu-
rons, glutamine is reconverted to glutamate with the gen-
eration of one NH4+ [72]. It is not clear how much of this
NH4+ is returned to astrocytes as such, or as amino acids
[73], but activity-dependent local NH4+ surges have been
recorded in several animal models [74–77]. NH4+ is effi-
ciently captured by astrocytes via channels and transporters
[78, 79]. Physiological ammonium levels in brain tissue have
been estimated at 0.2–0.45mM [79]. Intravenous infusion
of NH4+ leading to an increase of 0.7mM, caused a rapid
reversible rise in brain tissue lactate and cerebral blood flow
[80]. At 0.2mM, NH4+ provoked an acute inhibition of mito-
chondrial pyruvate consumption in astrocytes resulting in
deviation of the glycolytic flux towards lactate production
and release, but glycolysis was not stimulated [81]. This lack
of response is another example of the relative autonomy of
glycolysis in these specialized cells. Given these metabolic
effects of NH4+ it is unfortunate that so little is known about
the speed and mechanism of its release by neurons. If stored
in synaptic vesicles to be co-released with glutamate [82], its
metabolic effects would be fast. The primary target of both
NO and NH4+ (at low physiological levels) is the mitochon-
dria, so both signals can be said to induce aerobic glycolysis
of the Warburg type.
Mechanisms ofthePasteur, Warburg andCrabtree
Eects
According to classic biochemistry, the second-to-second
conversation between glycolysis and respiration is conducted
via adenine nucleotides. Glycolysis responds to ATP and
AMP (which amplifies ADP changes through adenylate
kinase) and respiration responds to ADP. Thus, the Pasteur
effect is mediated by the mitochondria sustaining high cyto-
solic ATP and low cytosolic AMP, resulting in glycolysis
inhibition at PFK1. The Warburg effect is therefore seen
as a suppression/failure of these inhibitory mechanisms,
either because not enough ATP is produced or because the
glycolytic machinery becomes insensitive to ATP or AMP.
The Crabtree effect develops when a primary stimulation
of glycolysis (e.g. by glucose or by K+) increases ATP and
decreases ADP, leading to inhibition of mitochondrial respi-
ration. All this sounds quite logical according to the test-tube
properties of isolated enzymes and organelles, but there is
no evidence that adenine nucleotides mediate these effects
in intact cells under physiological conditions. For exam-
ple, the strong NBCe1-dependent activation of glycolysis
in astrocytes that occurs despite increased cytosolic ATP
[18] demonstrates that alternative mechanisms may override
the influence of adenine nucleotides. Conversely, in astro-
cytes exposed to glutamate, glycolysis remained unstimu-
lated during several minutes despite severe ATP depletion
[18, 46, 51], so there must be another, stronger influence
interfering with the stimulatory effect of the nucleotides. In
neurons, adenine nucleotides do not seem to be paramount
either, because these cells are capable of increasing their
rates of glycolysis and mitochondrial pyruvate consump-
tion by several-fold in the absence of detectable changes in
cytosolic ATP and ADP [36]. Adenine nucleotide-mediated
control may well dominate under pathological conditions
like ischemia. For normal workloads however, it is perhaps
time to consider alternatives, for example glycolytic inter-
mediates [15] or the conspicuous mitochondrial attachment
of hexokinase to mitochondria [83, 84].
In summary, based on experiments in animals, invivo,
exvivo and in cultured cells, the main locus of acute activ-
ity-dependent aerobic glycolysis in brain tissue appears to
be the astrocyte. A fast Crabtree effect triggered by K+ coin-
cides with a fast Warburg effect mediated by NO, superim-
posed on a tonic, glutamate-dependent Warburg effect. The
time course of the Warburg effect induced by NH4+ remains
to be determined. The combined result of these modulations
is that lactate and oxygen are made available to neurons,
on demand. In the meantime neurons maintain a balance
between glycolysis and respiration mediated by parallel
upstream control of both pathways by the Na+ pump (Fig.2).
Technical developments are eagerly awaited to confirm these
observations in humans.

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Acknowledgements We thank all members of the Barros Lab for
their contributions and discussions. We also thank Karen Everett for
critical reading of the manuscript. This work was partially funded by
CONICYT-BMBF Grant 180045. The Centro de Estudios Científicos
(CECs) is funded by the Chilean Government through the Centers of
Excellence Basal Financing Program of CONICYT.
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