Journal of Cerebral Blood Flow & Metabolism
24:1235–1239 © 2004 The International Society for Cerebral Blood Flow and Metabolism
Published by Lippincott Williams & Wilkins, Baltimore
Commentary
The Role of Hypotheses in Current Research, Illustrated by
Hypotheses on the Possible Role of Astrocytes in Energy
Metabolism and Cerebral Blood Flow: From Newton to Now
Harold K. Kimelberg
Neural and Vascular Biology Theme, Ordway Research Institute, Albany, New York
Scientists currently strive to obtain reliable data on the
properties of astrocytes in situ, and pari passu to define
what the term astrocyte should encompass. As is the
current fashion, they are also required to propose in a
formal sense hypotheses for astrocytic function that
serve as guides for such research. It has become the
fashion to raise up “hypothesis-driven” science and denigrate descriptive science that lacks this formal introduction as merely cataloguing. Terms such as “fishing expedition” are often used pejoratively to describe
scientific studies that are viewed as simply collecting
data with no end in view. This seems as though it would
be rather rare and would anyway describe the efforts of
a rather inexperienced angler who does not have sufficient experience and skill to determine the types of fish
that are likely to be caught where he or she is planning
to fish.
There is no dispute as to whether the postulation of
hypotheses will lead to experiments. The real question,
however, is whether always starting with hypotheses
leads to more discoveries and a better-organized, more
reliable database. To be consistent, this would have to be
resolved scientifically; to test this hypothesis in the usual
carefully controlled fashion with defined measures of
whether so called hypothesis-driven science provides
better data or more insights than so called non–
hypothesis-driven science. It would be a simple exercise
for the research professional to restate this question in
the form of a hypothesis and develop a research plan to
address it. To put it into practice, however, will rapidly
lead to complications. For example, we would first need
to define what non–hypothesis-driven science actually is
and how it differs from the hypothesis-driven science.
The lack of a formal hypothesis does not mean there is
not some end in view. It may simply be that in the hypothesis-driven variety we take a positive view that we
can guess pretty well what the mechanism or relation is,
whereas in the latter case, we leave the question open and
will come to this later because we believe we don’t have
enough data to form a sensible hypothesis. I would hope
that an individual scientist’s opinion, as an expert in his
or her field on this issue, would be respected and not
dismissed out of hand. You obviously need a background
of reliable observations (that is, facts) to propose a hypothesis, and it may well not be clear to another observer
when you are ready to propose it. The criticism on a
case-by-case basis would be that any reasonable scientist, given the state of knowledge, should be able to come
up with a reasonable hypothesis for the area of study
under question. This is, in practice, unpredictable and
when successful is referred to as scientific intuition. I
will assume that everyone agrees that randomly obtained
data, however reliable, with no question in mind (although
by chance it may come up with important data leading to
insights) is not acceptable because it is simply too much
of a gamble, and this leaves one very uncomfortable.
The two astrocyte-related hypotheses that I will cover
to illustrate the problems of this common current practice
are as follows: 1) that glucose enters the CNS via the
astrocytic processes where it is converted by aerobic glycolysis to lactate which then serves as the principle
source of energy for neurons (Magistretti et al., 1994;
Pellerin and Magistretti, 2003; Voutsinos-Porche et al.,
2003), and 2) that the processes of astrocytes that surround blood vessels at the levels of the arterioles affect
smooth muscle contractility and therefore blood flow by
releasing vasoactive agents. (Anderson and Nedergaard,
2003; Simard et al., 2003; Zonta et al., 2002).
In a recent commentary regarding their well-known
astrocyte-neuron lactate shuttle hypothesis (ANLSH),
Received January 8, 2004; accepted June 9, 2004.
Address correspondence and reprint requests to Dr. Kimelberg,
Neural and Vascular Biology Theme, Ordway Research Institute,
150 New Scotland Ave., Albany, NY 12208; e-mail: hkimelberg@
ordwayresearch.org
1235
DOI: 10.1097/01.WCB.0000138668.10058.8C
1236
H. KIMELBERG
Pellerin and Magistretti (2003) write, “The important
point here is not so much to decide, based upon the actual
pieces of evidence, whether an hypothesis is right or
wrong but rather to point out what is heuristically valid
in it, what we have learned, what remains to be assessed,
what new hypotheses can be proposed, and which experiments are critical for this.” An alternative viewpoint
was written some 330 years ago by Isaac Newton: “For
the best and safest method of philosophizing seems to be,
first to enquire diligently into the properties of things,
and establishing those properties by experiments and
then proceed more slowly to hypotheses for explanations
of them. For hypotheses should be subservient only in
explaining the properties of things, but not answered in
determining them; unless so far as they may furnish experiments. For if the possibility of hypotheses is to be the
test of truth and reality of things, I see not how certainty
can be obtained in any science” (Christianson, 1984).
Now, Newton was responding to criticisms by Ignance
Gaston Pardies, a French Jesuit and professor of Rhetoric
at the Collège de Louis-le-Grand in Paris, of Newton’s
work with prisms that demonstrated that the simplest
explanation of the well-known visible spectrum is that
white light is a mixture of primary colors with different
refractions. This is clearly a hypothesis in the best sense
of the term that has been raised to the rank of a theory
because it has been repeatedly shown to be a valid explanation of all sorts of optical phenomena, with now, of
course, a much deeper understanding of what is going
on. This is very different from the astrocyte-neuron lactate shuttle hypothesis where things are very much up in
the air, as Pellerin and Magistretti do point out.
But the question that I am addressing is why there is a
modern insistence on the supremacy of hypotheses compared with Newton consigning them to a subsidiary role.
Has the philosophical basis of the scientific method
changed? Clearly the astrocyte-neuron lactate shuttle hypothesis arose from previous work. Pellerin and Magistretti thought these were sufficient to codify them into an
hypothesis in 1994. But why did they propose a hypothesis? Did it help their thinking? If so, then it was a
personal tool that did not need publicizing at that time
when the evidence was (and still is) inconclusive. Did it
lead to more experiments? Indeed yes, so for this aspect
of Newton’s statement it was a good thing. But the authors could simply have said we need to know more
about how glucose is handled by the brain. It has to enter
from the blood and will therefore first encounter an astrocytic process, so we will study how glucose is handled
by astrocytes. Wouldn’t the result be the same? The point
I would like to make is that hypotheses tend to lock us
into an either/or situation. This is the concern of the
quote from Pellerin and Magistretti. Namely, that their
hypothesis was never intended to be either/or. However,
readers will tend to accept the hypothesis as stated withJ Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
out the caveats. After all, if there are caveats, then it is
not a very precise hypothesis and is difficult to refute
experimentally, which, as Karl Popper has pointed out,
is logically all you can do with hypotheses anyway
(Lindh, 1993).
So why hypotheses? They can definitely suggest new
experiments, often clarify thinking, and make easy targets for grant reviews. However, Newton said that they
should not determine anything, and this is particularly so
for the merit of any proposed research whose usefulness
can, a priori, only be guessed at. Hypotheses also clarify,
as Newton fully realized: “And therefore because I have
observed the heads of some great virtuoso’s to run much
upon Hypotheses, as if my discourses wanted an Hypothesis to explain them by, & found, that some when I could
not make them take my meaning when I spake of the
nature of light & colors abstractly, have readily apprehended it when I illustrated my Discourse by an Hypothesis” (Christianson, 1984).
The underlying observation for both hypotheses 1 and
2, which have been known since the times of Golgi
(1885) and Ramón y Cajal (1913), is that the blood vessels in the central nervous system are surrounded by
astrocytic processes, and we now know that this can be
close to 100% (Virgintino et al., 1997). This fundamental
fact has given rise to many hypotheses, ranging from
development of the blood-brain barrier because of signals derived from the astrocytes as the central nervous
system develops to the ASLNH hypothesis. It seems that
all material that does not diffuse between the astrocytic
processes will have to pass through the thin astrocytic
processes or diffuse through the entire astrocyte. Now
the spaces between the astrocytic processes are of the
order of a few hundreds of angstroms wide. They could
still form a major short circuit path as the membranes
would be a major resistance pathway for polar substances unless there are sufficient carriers on the astrocytic processes, such as the Glu-1 carrier. There is also
the question of why there are a high density of high
affinity Glu-3 transporters on neuronal membranes
(Leino et al., 1997) if the sequence is as follows:
Glucose → endothelial cells → astrocytes
→ lactate → neuron
Pellerin and Magistretti (2003) point out that this sequence is not obligatory, and parallel pathways can coexist, but then it will be very difficult to test this hypothesis because there are no limits to the degree to which
deviations may be explained away.
The proximity of astrocytic processes surrounding
blood vessels also suggests a role in the control of blood
flow (Anderson and Nedergaard, 2003; Zonta et al.,
2002). For this, it is necessary for the control to be exerted at the level of the arterioles because the capillaries
COMMENTARY: THE ROLE OF HYPOTHESES IN CURRENT RESEARCH
lack the smooth muscle needed to cause contraction or
relaxation to change the blood vessel diameter (Edvinsson et al., 1993; Traystman 1997). Note here that there
must always be some basic tone to keep the arterioles at
an intermediate diameter from which they can constrict
or relax (Traystman, 1997). This hypothesis seems to be
on firmer ground than the ASNLH. Indeed, Mulligan and
MacVicar (2004) reported that Ca2+ in perivascular astrocyte processes leads to constriction of contiguous
small arterioles via release of arachidonic acid. The astrocytic ensheathment of central nervous system blood
vessels originates as blood vessels penetrate the brain
parenchyma early in development from the arachnoid to
the brain parenchyma and carry with them the glia limitans that are astrocytic processes. Interactions between
the astrocytic sheath and the vascular endothelial cells
are thought to be responsible for the formation of the
interendothelial tight junctions that form the blood-brain
barrier, which occurs at around the end of the first trimester in man (Abbott, 2002). However, if the role of the
astrocyte is purely developmental, then why does the
vascular ensheathment persist through adulthood? Presumably, they are then converted to physiologic roles or
a constant astrocytic influence is needed to maintain the
blood-brain barrier. Clearly, the former can be the
ASLNH (Magistretti et al., 1994), pH control (Tschirgi,
1958), control of ingress of compounds such as glucose
and amino acids or egress of waste metabolites (Pardridge, 1997), and control of [K+]o and blood flow (Newman, 1987; Zonta et al, 2002).
These theoretical possibilities need to devolve into
specific testable proposals that can be labeled as hypotheses, for the sine qua non of hypotheses, whether you are
not too enamored of them or you cannot work without
them, is that they have to be testable and that means in a
direct and relevant manner and system. Evidence that
there are the right lactate transporters (Allaman et al.,
2000; Pellerin et al., 1998) is clearly only circumstantial
and also needs to be shown in situ. Although cyclooxygenase-dependent products have been shown to be involved in vascular dilation in slices when astrocytes are
directly stimulated (Zonta et al., 2002), there is no evidence that either COX1 or 2 are present in astrocytes in
situ (Hoozemans et al., 2001). However, there are alternative pathways by which the astrocytic endfeet can influence the arterioles and the cyclooxygenase may be
located elsewhere with the astrocytic influence being indirect, to explain the findings of Zonta et al. (2002).
In general, evidence is very weak or perhaps even
misleading when it only derives from astrocyte cultures.
Differences in activities and protein profiles have been
shown for primary astrocyte cultures as compared with
freshly isolated GFAP(+) astrocytes (Kimelberg et al.,
1997), or for Muller cells with increasing time in culture
(Hauck et al., 2003). Thus it is a major weakness when
1237
both the original ASLNH hypothesis and the recent detailed rebuttal (Chih and Roberts, 2003) both use data for
astrocytic and neuronal cultures to support and refute.
The expression of the genome is plastic and influenced to
a major degree by changes in the cellular environments
via surface receptors (Peng et al., 1998). The latter follows logically from modern understanding of the biology
of gene expression.
Hypothetically, one reason why astrocytes may be developmentally retained to modulate arteriole diameter
and therefore blood flow to respond to the varying energy needs of changes in brain activity is illustrated in
Fig. 1. One should acknowledge, however, that this is
only one possible hypothesis among many that can be
proposed. Astrocytes are known to have a variety of
receptors in situ as well as in vitro (Kimelberg, 1988,
1995; Porter and McCarthy, 1997) that can then integrate
messages from neurons in the form of released transmitters so that the results of a wide range of different brain
activities can be integrated and relayed to the blood vessels. In terms of the restraint, I propose that this is a good
example of a reasonable hypothesis that one would have
trouble supporting meaningfully. The existence of several receptors subtypes on freshly isolated astrocytes
(Zhu and Kimelberg, 2004) is clearly consistent but not
in any meaningful way because there could be numerous
reasons for this coexistence. One possible approach that
comes to mind is to determine whether the perivascular
astrocytes are a specific subtype that can be specifically
manipulated to see if it affects blood flow response to
brain activity. This assumes the conclusion, as do all
FIG. 1. Models comparing the hypothetical control of arteriolar
diameter directly by individual neurons (A), with integration of
inputs from different neurons through the perivascular astrocytic
end feet (B). (A) The integration would be at the level of the
arteriole or more strictly the smooth muscle cell, which would
then have to integrate the different direct neuronal inputs. (B) The
astrocyte acts as the integrator that translates the neuronal inputs
for its receptors into an appropriate transmitter output for contraction or relaxation (black arrow). There is no obvious structure
additional to the smooth muscle within the blood vessel that can
serve this integrator role.
J Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
1238
H. KIMELBERG
hypotheses, that there are vascular specific types, and
this should be a doable task. After this is achieved, then
one could talk about specific manipulation. It is not clear
if all astrocytes have processes that surround blood vessels, although there is current evidence for the functional
heterogeneity of astrocytes (Nedergaard et al., 2003;
Zhou and Kimelberg, 2001). Note, though, that the demonstration of a specific perivascular astrocyte would
have important implications for the ANLSH, as well as a
host of other possible hypotheses. So, again, why can’t
we state that our objective is to look for this cell? Then,
the discussion would be only about whether proposed
methods are likely to be successful, which is less a matter
of opinion than the worth of some hypothesis.
Direct evidence in situ is lacking for the ASNLH. The
1:1 relation between glucose uptake and glutamate turnover (Sibson et al., 1998) is consistent with a number of
possible models and therefore is not definitive because
other scenarios can be proposed to explain this relationship (Chih and Roberts, 2003). However, definitive tests
of hypotheses regarding processes in organisms need to
be obtained in vivo and that is why genetic knockouts are
increasingly used. Such manipulations will need to be
astrocyte-specific and possibly astrocytic subtypespecific, such as for the putative perivascular astrocyte
just discussed. Thus the relation shown in Fig. 1 may
only apply to a subgroup of astrocytes. Knockout experiments could be designed to see if 1) the elimination of
the perivascular astrocytic glucose transporter essentially
starves neurons, and 2) if, after knockout of astrocytic
COX enzymes, the brain loses blood flow regulation. It
will needed to be assessed if the knockouts are specific
enough and then to see if the results are amenable of a
clear interpretation. As always, it is better to conduct
some pilot experiments, but, because of their complexity
and the time needed to generate the animals, knockouts
are not traditional pilot experiments.
A hypothesis is only the first step in a laborious scientific investigation. Thus, as Newton wrote, hypotheses
are a guide for experiments, but perhaps to be consistent
with the scientific method we need only the publication
of hypotheses supported by data. However, supporting
data for the ANSLH are almost exclusively experiments
with primary GFAP(+) astrocyte cultures. Because these
cells have active glutamate transporters and, of course,
the ubiquitous Na pump, the cells will naturally take up
glutamate with Na, the intracellular accumulation of
which then conventionally activates the pump. There will
then be an increased uptake of glucose and, being
adapted to a limited supply of oxygen, relative to consumption, through unstirred layers, the monolayer cultures are likely to mainly metabolize the glucose glycolytically with lactate production. The question is whether
this occurs under the conditions that normally pertain in
vivo. The ANLSH seems prima facie a reasonable hyJ Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
pothesis because it gives reasons for the persistence of
astrocyte processes around all the blood vessels—the key
finding—as well as other features. On one hand, we have
a role for astrocytes in integrating signals from a diversity of neurons via receptors to balance blood flow to
activity (Fig. 1), and, on the other hand, we set forth that
a signal of increased activity, namely glutamate, can signal the astrocyte to increase uptake of glucose from the
blood and convert this to substrate for the neurons in the
form of lactate in a simple servo-feedback type mechanism. However, the evidence for this is inconsistent, if
not in opposition, and the data fit other scenarios as has
recently been put forward in considerable detail (Chih
and Roberts, 2003).
The need for a hypothesis is that thinking about why
we are doing particular measurements in terms of how
they advance understanding is the most important aspect.
Here is where the need for a hypothesis is advanced as
necessary: to avoid indiscriminate collection of data with
no end in mind, to force the scientist to have an end, and
clarify for others what that end is. However, I hope I
have pointed out that hypotheses only illustrate what one
is driving toward. They do not determine that one is on
the right track, and insisting upon them as a prerequisite
to any proposed scientific investigation means that areas
in which there is insufficient information to propose any
sensible hypothesis will not be investigated or only investigated clandestinely. Looking at history, we see that
it is not only not how major discoveries in the biological
science have been made, but the proposal of hypotheses
depends upon data in the inductive scientific method
pioneered by Isaac Newton and which is the same
method as we use today (Feynman, 1965). What if the
existent data seems insufficient to an individual investigator to propose a sensible hypothesis? It then becomes
a matter of opinion, which will be decided first by one’s
peers and then by history. A not entirely satisfactory
system but perhaps, like democracy, better than any others one can think of.
It would be useful for the modern experimental biological sciences if this intellectual straight-jacket was
recognized for what it is: simply a useful but easily disposed of intellectual technique. Then we would not get
into these unending either/or arguments. The question of
what mechanisms are subserved by the astrocytic processes that persist around the CNS blood vessels, and
also the astrocytic processes that surround synapses
(Araque et al., 1999), are critical ones that beg for answers. After sufficient data have been obtained to suggest a plausible mechanism, which as discussed above is
a matter of opinion, the essence of the scientific method
is that we test the implications of these mechanisms to
see if they correspond with reality (Feynman, 1965).
These tests have to be specific in that no reasonable
COMMENTARY: THE ROLE OF HYPOTHESES IN CURRENT RESEARCH
alternative mechanism should lead to the same phenomena. One must test the actual hypothesis directly: if one
is talking about how astrocytes behave in the brain, the
tests must be performed in the brain. Reductionism in the
sense of using simpler and more experimentally tractable
systems can only be suggestive and are in the final analysis not compelling. Thus after Kuffler et al. (1966) usefully used the simple leech nervous system and then the
amphibian optic nerve to show that glial cells were actually high K+ cells and their membranes showed selective K+ currents, rather than high Na cells that functioned
as the extracellular space of the brain (Grossman, 1972),
a number of studies addressed themselves to whether the
same properties translated to mammalian glia and specifically astroglia (Somjen, 1975). These investigations
are still ongoing because the ion channels of astrocyte
membranes are more complex than indicated in the earlier electrophysiologic experiments (Barres et al., 1990;
Sontheimer, 1995; Verkhratsky and Steinhauser, 2000).
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1240
L. PELLERIN AND P. MAGISTRETTI
Letter to the Editor
Empiricism and Rationalism: Two Paths Toward
the Same Goal
We appreciate the scholarly contribution of Dr. Kimelberg, although we do not share his pessimistic view
about hypothesis-driven research, nor do we embrace his
position upon how research should be conducted. Of
course, we do not believe in the supremacy of one form
of scientific enquiry over the others, even when promoted by a giant such as Isaac Newton. We rather prefer
the pluralistic view that there are many different ways to
probe the mysteries of nature, all of them being equally
valuable. Some researchers like to collect data, others
prefer to formulate hypotheses and try to confirm or
infirm them. Some individuals formulate theories, leaving the task to skilled experimentalists to find out whether data support proposed models. Finally, for many of us,
several of these different strategies might be used at one
point or another during our work. It is important to recognize that all of these approaches (or stages toward a
coherent explanation of natural phenomena) play an important role in the progress of science, and even if one
were to have the stature of Newton in his or her field, it
appears presumptuous to recommend to future generations of scientists to favor one particular mode of investigation. The development of modern physics is there to
remind us that it was fortunate that some individuals
could escape the Newtonian vision of the world and formulate their own models, sometimes out of very little
data, to be proven experimentally only decades later. As
nicely summarized by Changeux (2004), the debate between empiricists and rationalists started by Aristotle and
Plato, and then pursued by Bacon, Locke, and Hume on
the empiricist side and by Descartes and Kant on the
rationalist side, to name a few, is still ongoing. According to empiricists, theory should come only after the
facts. The rationalist view calls for an anticipatory role of
theory (models), which in this way can structure and
orient experimentation. Changeux goes on to argue that
the two approaches may reflect two modes of functioning of the brain, which are influenced by the sociocultural environment.
In biology, studies performed upon in vitro preparations have demonstrated their capacity to provide tremendous insights about the cellular and molecular
mechanisms operating in various tissue, including the
central nervous system. The formulation of hypotheses
based upon in vitro data represents the best way such
insights can be rigorously expressed and disseminated in
Received March 16, 2004; final version received June 16, 2004;
accepted June 21, 2004.
Address correspondence and reprint requests to Dr. Pellerin, Institut
de Physiologie, 7 Rue de Bugnon, 1005 Lausanne, Switzerland; e-mail:
luc.pellerin@physiol.unil.ch
J Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
the scientific literature to be tested by many researchers
with different approaches. Of course, we agree with Dr.
Kimelberg that it is important to obtain at some point a
validation in vivo of hypotheses developed on the basis
of in vitro studies. This is precisely what we have done
for the lactate shuttle model that was elaborated and
refined on the basis of a series of in vitro experiments
(Bouzier-Sore et al., 2003; Brunet et al., 2004; Chatton et
al., 2003; Debernardi et al., 2003; Pellerin and Magistretti, 1994, 1996, 1997; Pellerin et al., 1998a; Pierre et
al., 2003), whereas some aspects were subsequently
tested ex vivo and in vivo (Bittar et al., 1996; Cholet et
al., 2001, 2002; Pellerin et al., 1998b; Pierre et al., 2000,
2002; Voutsinos-Porche et al., 2003). In fact, it is doubtful that in vivo experiments would have been performed
without the insight provided by in vitro studies. Considering the number of interesting findings that have been
made so far and the perspectives opened by the aforementioned hypothesis with new experiments being pursued by different laboratories, we can (still) predict a
bright future to hypothesis-driven research. In this regard, it is instructive to consider Louis Pasteur’s statement (1880): “The illusions of the experimenter form a
great part of his power. These are the preconceived ideas
which serve to guide him. Many of these vanish along
the path which he must travel, but one fine day he discovers and proves that some of them are adequate to the
truth. Then he finds himself master of facts and new
principles, the applications of which, sooner or later, bestow their benefits.”
Luc Pellerin and
Pierre J. Magistretti
Department of Physiology
Université de Lausanne
Switzerland
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THE ASTROCYTE-NEURON LACTATE SHUTTLE
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286
The Astrocyte-Neuron Lactate Shuttle:
A Challenge of a Challenge
Leif Hertz
College of Basic Medical Sciences, China Medical University, Shenyang, China; and the Department of Pharmacology,
University of Saskatchewan, Saskatoon, Canada
The Journal of Cerebral Blood Flow and Metabolism
is to be applauded for opening up a discussion on the
astrocyte-neuron lactate shuttle hypothesis (Chih and
Roberts, 2003; Pellerin and Magistretti, 2003). It is also
helpful to know that some of the prepositions made by
Pellerin et al. (1998) and Magistretti et al. (1999) apparently have been deleted from the present version of the
astrocyte-neuron lactate shuttle hypothesis (Pellerin and
Magistretti, 2003), for example, that an activity-dependent aerobic glycolysis in astrocytes, triggered by glutamate uptake and glutamine synthesis, sets the pace for
total energy metabolism in brain cortex (Magistretti et
al., 1999). Accordingly, the present definition of the
shuttle model has been modified as consisting of two,
and only two, components. These are i) enhancement of
aerobic glycolysis in astrocytes in response to neuronal
activation of glutamatergic synapses, reflecting a dependency of astrocytic glutamate uptake upon glycolytically
derived energy, and ii) oxidation in neurons of lactate
produced by astrocytes.
Address correspondence and reprint requests to Dr. Hertz, RR 2,
Box 245, Gilmour, K0L 1W0, Ontario, Canada; e-mail: lhertz@
northcom.net
Does astrocytic glutamate uptake depend upon
aerobic glycolysis?
The observation by Pellerin and Magistretti (1994)
that uptake of glutamate by astrocytes in primary cultures causes a distinct stimulation of deoxyglucose (DG)
phosphorylation and lactate production was almost immediately confirmed by Takahashi et al. (1995) in the
Sokoloff laboratory, but the magnitude of the response
was much smaller. However, other investigators have
found that glutamate either has no effect upon DG phosphorylation and glucose use in cultured astrocytes or that
it causes a decrease rather than an increase, except under
anoxic conditions, suggesting that glutamate normally
may be oxidized as an alternative fuel (Dienel and Cruz,
2004; Hertz et al., 1998; Liao and Chen, 2003; Peng et
al., 2001; Qu et al., 2001; Swanson et al., 1990). It is
known that glutamate is oxidatively degraded not only in
cultured astrocytes (Hertz and Hertz, 2003; McKenna et
al., 1996; Yu et al., 1982, 1992), but also in brain slices
and in the brain in situ (Taylor et al., 1996; Zielke et al.,
1998). Moreover, it is well established that glutamate
uptake into cultured astrocytes can be fueled by either
glycolytically or oxidatively derived energy (Huang et
al., 1993; Swanson and Benington, 1996).
J Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
1242
L. HERTZ
A possible explanation of the discrepancy between the
results obtained by the Pellerin-Magistretti-Sokoloff
groups and those reporting lack of stimulation of astrocytic glycolysis by glutamate is that the cultures used by
the former groups resort to accumulating glutamate by
the aid of glycolytically derived energy because they are
deficient in oxidative metabolism. Many types of cultured cells, including brain cells (Dittmann et al., 1973),
tend to become glycolytic and show a reduced oxidative
metabolism (Guminska et al., 1969; Langvad, 1970). The
predominance of glycolysis in cultured cells appears to a
large extent to depend upon the culturing technique
(Felder et al., 2002; Gstraunthaler et al., 1999). It can be
counteracted by improved culturing conditions, especially by facilitating oxygen diffusion to the tissue
(Booher et al., 1971; Kondo et al., 1997) and by not
using excessive glucose concentrations (such as the 25
mM glucose used by the Pellerin-Magistretti-Sokoloff
groups) in the medium (Abe et al., 2003).
The Pellerin-Magistretti group has never provided any
values for oxidative metabolism in their astrocyte preparations, but Itoh et al. (2003) have shown that the cultures used by the Sokoloff group have a low rate of
oxidative metabolism of [U-14C]glucose (nominal rate
0.12 nmol/[min/mg protein]). This rate is five times
lower than the respiratory rate observed by the same
authors in cultured neurons. A five times lower respiratory rate in astrocytes than in neurons cannot reflect the
in vivo situation because it has recently been demonstrated by three groups that astrocytes, which occupy less
than 30% of the volume in the brain cortex (Pope, 1978;
Williams et al., 1980; Wolff and Chao, 2004), account
for approximately 15% of its oxidative metabolism
(Blüml et al., 2002; Gruetter et al., 2001; Lebon et al.,
2002). Also, substantially higher respiratory rates (approximately 1.0 nmol/[min/mg protein]) have been reported by Hertz and coworkers in both astrocytes and
cerebellar granule neurons with [U-14C]glucose as the
precursor (e.g., Peng et al., 1994; Yu and Hertz, 1983)
and by Lopes-Cardozo et al. (1986), using [2-14C]glucose, and Vicario et al. (1993) found virtually identical
respiratory rates in cultures of neurons and astrocytes.
Accordingly, it cannot be concluded that cultured astrocytes depend upon glycolysis for glutamate uptake.
However, this does not negate the possibility that there
could be situations in the brain in vivo, where glutamate
uptake is fueled by glycolytically derived energy (e.g., in
the most peripheral parts of both neurons and astrocytes,
which are too minute to contain mitochondria).
It has been used as a powerful argument in favor of the
astrocyte-neuron lactate shuttle that interference with astrocytic glutamate uptake decreases glucose utilization
during in vivo activation of the brain, including its barrel
cortex, regardless whether the inhibition occurs by administration of the transport inhibitors beta-D,LJ Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
threohydroxyaspartate (THA) or pirrolidine-2-4dicarboxylate (PDC) (Demestre et al., 1997), by injection
of antisense mRNA to the transporter (Cholet et al.,
2001) or by the use of mutant mice without one or the
other of the two astrocytic glutamate transporters GLT-1
and GLAST (Voutsinos-Porche et al., 2003). However,
these observations are not conclusive. One problem is
that 10-day-old rats were used for these experiments.
Oxidative metabolism of glucose is not fully developed
in the 10-day-old rat or mouse brain. Ketone bodies,
which use the monocarboxylic acid transporter (MCT)
for uptake, are an important fuel for suckling rats (Cremer, 1982; Medina et al., 1999; Nehlig, 1999), and the
rate of glutamate oxidation in cultured mouse astrocytes
is lower in cells corresponding to postnatal day 10 than
in more mature cells (Yager et al., 1994). It would, therefore, have been preferable to use adult animals in the
study by Voutsinos-Porche et al. (2003), especially since
the authors indicate that the mutant mice may mature at
a reduced rate.
Another caveat against the conclusion that the reduced
metabolic response to brain activation must be a reflection of a diminished workload by the astrocytes is provided by a multitude of experimental observations that
many facets of glutamatergic activities are impaired
when glutamate uptake is reduced (e.g., Maki et al.,
1994; Niederberger et al., 2003; Turecek and Trussel,
2000). Inhibition of glutamate uptake might lead to reduction of presynaptic glutamate release, activation of
inhibitory metabotropic glutamate receptors, postsynaptic desensitization, or disturbance of glutamate recycling.
Excitatory postsynaptic currents in the rat barrel cortex
are inhibited by glutamate uptake inhibitors, and neurons
in the developing neocortex are particularly sensitive to
glutamate transporter function (Kidd and Isaac, 2000).
As indicated in the report by Voutsinos-Porche et al.
(2003), several aspects of glutamatergic transmission are
unaltered in the adult mutant mice used in their work.
However, others are not. In the very same GLT-1−/−
mutant as that used by Voutsinos-Porche et al. (2003),
long-term potentiation (LTP) induced by tetanic stimulation is reduced by two-thirds (Katagiri et al., 2001).
Development of LTP in brain slices is known to enhance
Na+,K+-ATPase activity (Glushchenko and Izvarina,
1997) and thus increases energy demand. It must therefore be concluded that neither studies in cultured astrocytes nor in vivo studies in animals with glutamate transporter deficiencies prove the dependence of astrocytic
glutamate transport upon aerobic glycolysis.
How much lactate is produced in activated brain in
vivo, and is there a substantial lactate flux through
extracellular fluid?
An essential component of the lactate shuttle hypothesis is that there must be a large, directed transfer of
THE ASTROCYTE-NEURON LACTATE SHUTTLE
lactate from astrocytes to neurons in vivo, but this has
never been demonstrated. It is well established that in
spite of sufficient oxygen availability, activation of brain
leads to an increase in intracellular and extracellular lactate from a “resting” value of approximately 1.0 ␮mol/g
wet wt. and 1 mM, respectively, to approximately twice
these values, and that lactate rapidly returns to its previous level after the activation is terminated (Dienel et al.,
2002; Korf; 1996; Prichard et al., 1991). However, it is
uncertain whether this lactate simply represents a larger,
static pool, that is, an increased level of tissue lactate
arising from the need of a higher concentration of pyruvate to stimulate pyruvate dehydrogenase activity sufficiently for TCA cycle flux to match an increased rate
of glycolysis. To demonstrate a large flux from astrocytes to neurons through the enlarged lactate pool
requires determination of the cells that are the main
lactate producers and the main lactate consumers in the
brain as well as quantification of lactate production
and degradation.
Which cell type is the predominant producer
of lactate in the brain in vivo?
It has not been established with certainty from which
cell type(s) lactate is released during brain activation in
vivo nor by which mechanism(s) the resting level is reestablished. There is evidence that at least some of the
released lactate originates from astrocytes. Thus microdialysis performed 1 to 2 weeks after the insertion of a
microdialysis probe, when the probe is surrounded by an
adhering scar of reactive glia, shows a stress-induced
increase in extracellular lactate, which is similar to that
seen 1 to 2 days after the insertion of the probe, when
access of neuronally released compounds to the probe is
indicated by a large K+-mediated transmitter release
(Korf, 1996). This observation does not, however, exclude the possibility that neurons also release lactate.
Because stimulation of glucose use in response to increased energy demand is fundamental to regulation of
cellular energy metabolism (see, e.g., Chih and Roberts,
2003; Hertz and Dienel, 2002), and because neurons are
well equipped with glucose transporters (Dwyer et al.,
2002), it is difficult to envision that glycolysis in neurons
should be unaffected when neuronal energy requirements
are increased. Moreover, the glycolytic enzyme hexokinase has a high activity in synaptosomes (Wilson, 1972),
glycolytic ATP formation has been demonstrated in postsynaptic densities (Wu et al., 1997), and in cultured glutamatergic neurons the rate of 14CO2 production from
labeled glucose is as high as in astrocytes and distinctly
increased during K+-mediated depolarization (Peng et
al., 1994).
In cell cultures, both neurons and astrocytes release
large amounts of lactate to the medium, although the
release from astrocytes (25–35 nmol/min/mg protein) is
1243
two to three times larger than the neuronal release (5–11
nmol/min/mg protein) (Dienel and Hertz, 2001; Schousboe et al., 1997; Waagepetersen et al., 2000; Walz and
Mukerji, 1988). Monocarboxylate transporter (MCT)mediated facilitated diffusion, which is driven by concentration gradients, is responsible for lactate transport in
and out of cells (Halestrap and Price, 1999; Juel, 2001),
and a major reason for the high rates of membrane transport in the cultured cells is the large volume of extracellular medium, which initially contains no lactate. Because of the slow rise of the extracellular lactate
concentration, a concentration gradient can be maintained between even a low intracellular lactate concentration and the extracellular lactate concentration, promoting continuous release of lactate. In the brain in vivo
lactate release would be delayed by rapid increase in
adjacent extracellular fluid. The rate of continued release
of lactate would be determined by its removal by extracellular diffusion, uptake into adjacent cells with a lower
concentration of lactate, and under a few pathologic
conditions (Dienel and Cruz, 2003) lactate release to
the circulation.
Lactate production in astrocytes occurs not only during the glycolytic part of glucose degradation, but also
during breakdown of glycogen (Dringen et al., 1993),
which is stimulated during brain activation and can be
very fast (Dienel et al., 2002; Swanson, 1992; Swanson
et al., 1992). Because glycogen and activity of its degrading enzyme, glycogen phosphorylase, within brain
parenchyma is virtually restricted to astrocytes (Ibrahim,
1975; Richter et al., 1996), its breakdown products must
be released from astrocytes or further metabolized in
astrocytes. The content of glycogen in brain, and thus
also activity-induced glycogenolysis, is larger than previously recognized (Cruz and Dienel, 2002; Kong et al.,
2002), and during brain activation the rate of lactate
equivalents produced from glycogen exceeds that of lactate production from glucose (Dienel and Cruz, 2003,
2004). Within the activated tissue formation of lactate
from glycogen, which under generally used experimental
conditions is unlabeled, should therefore be expected to
greatly dilute the specific activity of lactate generated
from [14C]labeled glucose. However, recent experiments
have shown that the specific activity of lactate produced
in the brain from the administered labeled glucose is not
diluted by any nonlabeled lactate (Dienel et al., 2002).
This remarkable observation demonstrates that the two
pools of lactate (and pyruvate), one from glycogen and
one from the [14C]glucose delivered by blood, are segregated. A segregation can be explained by cytosolic
“channeling” of metabolites from one enzyme to the next
in a pathway. Such a channeling is known in vascular
smooth muscle, where lactate formed by glycolysis is
segregated from that formed by glycogenolysis (Allen
J Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
1244
L. HERTZ
and Hardin, 2000; Lynch and Paul, 1983). The channeling may not be restricted to the individual astrocyte in
which lactate was produced. Rather, glycogen-derived
lactate appears to be transported away from the activated
area, probably by gap-junction mediated transport to
other cells in the astrocytic syncytium (Medina et al.,
1999). A segregation of lactate obtained by glycolysis
and glycogenolysis is virtually incompatible with release
of astrocytic lactate from both glucose and glycogen to a
presumably nonchanneled extracellular space (a precondition for an astrocyte-neuron lactate shuttle).
Does oxidation of lactate primarily occur
in neurons?
The concept that glucose-derived lactate, released
from astrocytes, is a preferred substrate for neurons is a
key component of the shuttle model. It has been taken as
support of this hypothesis that Itoh et al. (2003) and
Bouzier-Sore et al. (2003, 2004) found that unlabeled
lactate profoundly reduced the production of 14CO2 from
labeled glucose in hippocampal and cortical neurons, respectively. In contrast, there was little, if any, inhibition
by unlabeled glucose of production of 14CO2 from labeled lactate. The main reason for the inhibition of neuronal 14CO2 production from labeled glucose by unlabeled lactate was postulated by Itoh et al. (2003) to be
that conversion of lactate to pyruvate converts NAD+ to
NADH + H+ and therefore reduces the availability of
NAD+ for oxidation of glyceraldehyde-3-phosphate during glycolysis. However, if this mechanism were the major reason for the reduced 14CO2 production from
[14C]glucose in the presence of lactate, one would expect
that pyruvate, which does not consume NAD+ before its
oxidation, would be a less efficient inhibitor of 14CO2
production from [14C]glucose than lactate. In fact, the
opposite is the case, because unpublished experiments by
Peng and Hertz have shown that 14CO2 production from
7.5 mM [14C]glucose in the glutamatergic cerebellar
granule neurons is reduced from 1.3 ± 0.19 to 0.29 ± 0.02
nmol/min/mg protein (n ⳱ 6), that is, by 77.1%, in the
presence of 5 mM pyruvate, but only by 47.1% in the
presence of 5 mM lactate (pyruvate vs. lactate: P < 0.01).
Larger inhibition of release of labeled CO2 by addition
of unlabeled pyruvate than by addition of unlabeled lactate suggests that the dilution of the specific activity of
[14C]glucose-derived pyruvate/lactate by extracellular
lactate or pyruvate has a greater impact than NAD+ availability. The strong influence of exogenous substrates on
dilution of specific activity in endogenous pyruvate is to
be expected because brain pyruvate levels are low, that
is, 50 to 100 ␮M (Siesjö, 1978). Flooding of cultured
cells with large amounts of unlabeled lactate, facilitated
by the high MCT activities, and a rapid and reversible
interchange between lactate and pyruvate (Wolfe, 1990;
Wolfe et al., 1988), overwhelm the intracellular meJ Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
tabolite levels. Isotope dilution of glucose-derived
pyruvate/lactate will cause a large reduction of the production of labeled CO2 from glucose, regardless of
whether glucose metabolism is suppressed. That a potential impairment of glucose metabolism by lactate
caused by competition for NAD+ plays at most a minor
role is also suggested by the observation by Bliss and
Sapolsky (2001) that DG phosphorylation in hippocampal neurons is not inhibited at physiologically relevant
lactate levels and that even highly abnormal
lactate/glucose ratios (e.g., 0.5 mM glucose and 5 mM
lactate) have only a very modest inhibitory effect (approximately 15%).
The possibility of inhibition of neuronal and astrocytic
lactate oxidation by glucose has been examined experimentally by using the reverse experimental situation, that
is, measurement of production of 1 4 CO 2 from
[U-14C]lactate in the presence of different concentrations
of glucose. Because glucose use is governed by product
inhibition of key enzymes, especially phosphofructokinase (reviewed by Hertz and Dienel, 2002), little effect
of elevated glucose level on lactate metabolism would be
expected as long as the extracellular glucose concentration is maintained above the level necessary to secure
sufficient glucose transport into the cell. This extracellular glucose level could be 2.5 mM or perhaps slightly
higher because the Km for the neuronal glucose transporter is 2 to 3 mM (Maher et al., 1996). Raising the
concentration of glucose above this level would not increase delivery of carbon to downstream metabolic
pools, which is consistent with the observation that hyperglycemia does not change glucose use rates in brain in
vivo (Orzi et al., 1988). This reasoning explains a modest
effect of unlabeled glucose, regardless of its concentration, upon 14CO2 production from 2 mM [14C]lactate in
the experiments by Itoh et al. (2003). The larger inhibition of 14CO2 production from [U-14C]lactate by glucose
in astrocytes observed by these authors is to be expected
because of the higher rate of glycolysis in the astrocytes,
leading to a higher concentration of unlabeled glucosederived pyruvate/lactate, which accordingly can compete
more efficiently with exogenous lactate. In two studies
by Bouzier-Sore et al. (2003, 2004), a dilution of specific activity of glucose-derived pyruvate by addition
of nonlabeled lactate must in a similar manner invalidate an otherwise ingenious approach to quantitate metabolism of glucose, exogenous lactate and glucosederived lactate.
It is also against the notion that lactate predominantly
should be oxidized in neurons that Peng et al. (1994)
observed similar rates of 14CO2 formation from [14C]lactate (5 mM) in cultured GABAergic cerebral cortical
neurons, cerebellar granule neurons, and astrocytes.
However, using “trapping” of label from either [13C]glucose or [13C]lactate into glutamate as a determination of
THE ASTROCYTE-NEURON LACTATE SHUTTLE
tricarboxylic acid cycle activity, Waagepetersen et al.
(1998) found that incorporation of label from [13C]lactate into glutamate in cultured cortical astrocytes was
only 50% of that in cortical neurons. However, these
experiments were performed with either 0.5 mM glucose
or 1 mM lactate as the metabolic substrate, and stimulation of glycogenolysis in the aglycemic lactate medium
may have caused a decrease in the specific activity of
intracellular lactate. This type of experiment does accordingly also not provide solid evidence that lactate
should be a preferred substrate for neurons.
Publications by Bouzier et al. (2000) and by Tyson et
al. (2003) claim to show that lactate is primarily oxidized
in neurons in vivo, based upon the finding that intravenous injection of [3-13C]lactate gives rise to identical
incorporation of label into the C-2 and the C-3 position
of glutamate and glutamine, rather than to selective incorporation into the C-2 position, indicative of pyruvate
carboxylation. Based upon this observation, the authors
conclude that lactate is metabolized in a compartment
expressing no pyruvate carboxylase activity, that is, a
neuronal, not an astrocytic compartment. However, the
oxaloacetate produced by pyruvate carboxylation is
known to readily equilibrate with the symmetrical fumarate, thereby giving rise to equal labeling of C-2 and C-3
in glutamate and glutamine. For example, previous studies from the same laboratory by Merle et al. (1996) concluded, based upon a mathematical modeling of
[1-13C]glucose metabolism in cultured astrocytes that
39% of oxaloacetate synthesized by pyruvate carboxylation equilibrates with fumarate before it is condensed
with acetyl coenzyme A to form citrate, from which glutamate is produced. Sonnewald et al. (1993) found an
even more extensive, and possibly quantitative equilibration between oxaloacetate and fumarate. Moreover, pyruvate carboxylation from exogenous lactate in intact
brain has been demonstrated by other authors (Qu et al.,
2000), although it was less pronounced than when glucose was the substrate. Finally, demonstration of pyruvate carboxylation provides only a positive identification
of one part of astrocytic metabolism because dehydrogenation via acetyl coenzyme A in the astrocytic compartment is not determined. In fact, in intact brain the calculated rate of pyruvate dehydrogenation in astrocytes
exceeds that of pyruvate carboxylation by a factor of
almost 2 (Gruetter et al., 2001).
Another observation in intact nervous tissue used to
support the astrocyte-neuron lactate shuttle is that
Schwann cells in a stimulated isolated vagus nerve
preparation account for as much as 78% of the total
deoxyglucose phosphorylation in the nerve (Vega et al.,
2003). Pellerin and Magistretti (2003) compared this
value with a calculated estimate by Attwell and Laughlin
(2001) of glial expenditure of energy in cortical gray
matter as 5% of total energy consumption (a value that
1245
must be too low, considering that astrocytes account for
15% of the oxygen consumption in brain), and they concluded that the most likely explanation for this apparent
paradox is that the remaining 73% of phosphorylated
glucose were transferred to the axon in the form of lactate. However, such a comparison between a peripheral
nerve and cortical gray matter is not legitimate because
experiments measuring rates of oxygen consumption in
crayfish giant axons and in their ensheathing glial cells
have led to the estimate that in a normally functioning
axon-glial cell system the glial sheath accounts for 90%
of the oxygen consumption by the tissue (Hargittai and
Lieberman, 1991). Provided this value also applies to
mammalian peripheral nerves, the Schwann cells themselves are likely to oxidize all glucose they phosphorylate. That mammalian Schwann cells probably have a
high rate of oxidative metabolism is indicated by an extremely high mitochondrial density in these cells (Rydmark et al., 1998). Moreover, an inhibitor of the MCT
supposed to carry lactate from the Schwann cells to the
axon had no effect upon the action potential or the distribution of radioactivity after exposure to labeled deoxyglucose in the experiments by Vega et al. (2003).
Along similar lines, mobilization of the glycogen contained in a rat optic nerve sustains axonal action potential
in the absence of glucose (Wender et al., 2000). It was
concluded that this may be due to lactate transport from
glial cells to neurons (Brown et al., 2003), although approximately 80% of the released K+ is likely to be initially accumulated by the glial cells, mainly by active
transport mechanisms (Ransom et al., 2000). Because of
the narrowness of the periaxonal space and the low resting extracellular K+ concentration, the relative change in
extracellular K+ during the action potential is much
larger than the changes in intraaxonal ion concentrations.
That accumulation of extracellular K+ during energy failure leads to axonal block long before changes in intraaxonal ion concentrations affect excitability was demonstrated by Shanes (1951), who showed that axonal
conduction block in squid nerve during anoxia could be
reversed by simple wash with anoxic artificial sea water.
The distribution of different isotypes of MCT’s between astrocytes and neurons is also cited as being in
favor of directed transport of lactate from astrocyte to
neuron (Pellerin, 2003). However, because lactate transport across the cell membrane occurs by facilitated diffusion, its direction is determined by concentration gradients (not only of lactate, but also of H+, which is
cotransported), not by transporter type (Halestrap and
Price, 1999; Juel, 2001). Moreover, a Km value of the
mainly neuronal MCT-2 for lactate is approximately 0.7
mM, whereas those of the mainly astrocytic MTC-1 and
MCT-4 are 3 to 5 mM or higher (Bergersen et al., 2001;
Broer et al., 1997, 1999; Dimmer et al., 2000; Halestrap
J Cereb Blood Flow Metab, Vol. 24, No. 11, 2004
1246
L. HERTZ
and Price, 1999); this means that they are more responsive to the increase in the lactate concentration occurring
during brain activity (Hertz and Dienel, 2004).
What conclusions, if any, can be made regarding
a lactate shuttle?
The purpose of this challenge of a challenge has been
to critically evaluate the prepositions of the present version of the astrocyte-neuron lactate shuttle hypothesis:
i) pronounced aerobic glycolysis, especially in astrocytes, ii) stimulation of aerobic glycolysis in astrocytes
triggered by glutamate uptake, and iii) oxidation of lactate primarily in neurons. There is evidence that glycolysis combined with glycogenolysis may be more pronounced in astrocytes than in neurons, especially during
brain activation. Nevertheless, neurons almost certainly
carry out glycolysis and they are therefore likely to contribute substantially to aerobic glycolysis and lactate production in the brain in vivo. Glutamate uptake in astrocytes can be metabolically fueled by either glycolytically
or oxidatively derived energy, but there is no good evidence that it relies upon glycolytically derived energy.
Because there is net production of lactate in vivo, and in
most cases no release to the circulation, it is likely that
lactate is also degraded in the brain, although this process
may be temporally dissociated from its production, as
suggested by a mismatch between the increase in glucose
use and in oxygen consumption during many types of
brain activation (Fox and Raichle, 1986; Fox et al.,
1988). The site of lactate use is also likely to be spatially
separated from its site of production. There is, however,
no solid indication that lactate oxidation should primarily
occur in neurons, and there is very good evidence that it
is not exclusively a neuronal process. The magnitude and
direction(s) of any lactate flux(es) in the functioning
brain constitute two major unknowns. If the magnitude is
small, then the proposed shuttle process is of little functional significance. If it is large, then its direction(s)
would be of major interest, but it could at least equally
well occur within a glial syncytium as from astrocytes
to neurons.
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