Download Glutamine breakdown in rapidly dividing cells: waste or investment?

Hypothesis
Glutamine breakdown in
rapidly dividing cells:
waste or investment?
J. Carlos Aledo
Summary
Tumours, and in general rapidly dividing cells, behave as
dissipative devices that apparently waste glutamine,
since its consumption seems to exceed both energetic
and nitrogen needs. Although not conclusive, there is
compelling evidence suggesting that the consumption of
such large amounts of glutamine is essential to sustain
high rates of cellular proliferation. Herein, I first review the
experimental evidence linking proliferation with high
rates of glutamine breakdown. Then, the current knowledge on the proteins and activities involved in this high
glutamine consumption will be summarized. Finally, the
significance of the apparent waste of glutamine will be
discussed on bioenergetic grounds. The discussion
leads to the hypothesis that glutamine breakdown might
energize some endergonic processes, as well as accelerating other exergonic processes related to cellular
proliferation. BioEssays 26:778–785, 2004.
ß 2004 Wiley Periodicals, Inc.
Introduction
Growing cells need a constant and fast supply of both energy
and nitrogen substrates. Glutamine is the most abundant
amino acid in the plasma, where it functions as a non-toxic
nitrogen vehicle(1) and a respiratory fuel.(2) Thus, it is not
surprising that glutamine behaves as a key nutrient for rapidly
dividing cells. What may be surprising is the very high rate of
glutamine utilization exhibited by these cells. It is widely
accepted that the breakdown of glutamine greatly exceeds the
anabolic and energetic requirements of proliferating cells.(3,4)
In this article, I will argue that the low efficiency of glutamine
metabolism can be shown to be optimal for maximal growth
rate. Therefore, this apparent ‘‘waste’’ of glutamine can be
explained as the cost that the organism must pay to grow
rapidly.
Departamento de Biologı́a Molecular y Bioquı́mica, Facultad de
Ciencias, Universidad de Málaga, 29071 Málaga, Spain.
E-mail: [email protected]
DOI 10.1002/bies.20063
Published online in Wiley InterScience (www.interscience.wiley.com).
778
BioEssays 26.7
High rates of proliferation demand large
amounts of glutamine
The glutaminolytic pathway is initiated, after translocation of
glutamine across the plasma and inner mitochondrial membranes,(5) by the enzyme phosphate-activated glutaminase(6)
(EC 3.5.1.2), which catalyzes the hydrolysis of the amide
group of glutamine, yielding stoichiometric amounts of ammonium and glutamate. Glutaminase activity correlates well with
glutamine consumption and growth rate.(7) Although many
cells may require large amounts of glutamine for biosynthetic
purposes, only a limited amount (<5%) of the extracted
glutamine is used for such pathways.(8) However, it is
noteworthy that, in rapidly dividing cells, the flux through
glutaminase greatly exceeds the capacity of the mitochondria
to oxidize glutamate.(2) A set of detailed studies carried out in
vivo has revealed a net flux of glutamine from host tissues
towards tumour cells, while a reverse flux of glutamate and
aspartate takes place from tumour to plasma.(1,9 –11) Interestingly, a glutamine/glutamate cycle similar to that described
between tumours and their hosts appears to be operative
between fetus and placenta: the placenta supplies fetus with
very large amounts of glutamine, while concurrently removing
glutamate from fetal circulation.(12) Thus, depending on the
cell type and the metabolic requirements, a variable proportion
of the glutamine-derived glutamate will be excreted as an end
product, while the remaining can undergo partial oxidation to
aspartate.(13) Therefore, glutamine consumption by rapidly
dividing cells has been described by some authors as almost
completely dissipative.(14,15) Nevertheless, there is compelling evidence pointing to this apparent waste of glutamine as
an essential requisite to sustain high rates of cellular
proliferation. Indeed, glutamine clearance by infusion of pure
glutaminase into the bloodstream of patients suffering from
cancer stops tumour growth. Unfortunately, this kind of therapy lacks specificity and interferes with other healthy rapidly
dividing cells, provoking multiple side effects.(14,15) Experiments carried out using tumour cells from diverse origins, also
support the view that glutamine breakdown is a process
closely linked to cellular proliferation. Intracellular glutamine
concentration has been negatively correlated with growth
rate.(16) In contrast, high extracellular glutamine levels
BioEssays 26:778–785, ß 2004 Wiley Periodicals, Inc.
Hypothesis
stimulate proliferation, while a reduction of glutamine availability induces phenotypical and functional differentiation.(17,18) Taking all these observations together, it seems
that the key fact is glutamine hydrolysis itself. Recently, we
have provided more direct evidence supporting this view.
Using an antisense approach, glutaminase activity of tumour
cells was partially reduced. This reduction in glutaminolytic
capacity was followed by a longer doubling time and a
decrease in the saturation density and plating efficiency of
the transfected cells.(19) All these changes point to a moredifferentiated and less-transformed phenotype of those cells
with a reduced glutaminolytic capacity.
A closer look to the glutaminolytic pathway
At a first glance, it is difficult to explain the glutamine cycle
established between rapidly growing tumour cells and their
host. The dividing cell takes up glutamine, hydrolyzes the
amide group and gets rid of the products without any apparent
profit. However, this is an oversimplification of the glutaminolytic pathway, which we will now look at in more detail.
Glutamine uptake
In mammalian cells, glutamine can be recognized as a
substrate by several transport systems (Table 1A). Tumour
cells exhibit enhanced transport of glutamine across their
plasma membrane.(20,21) While increased amino acid trans-
port could in principle be accommodated by increased
expression of those transport proteins already present in the
non-proliferating cells, there is evidence suggesting that the
transformed phenotype involves a change in the pattern of
the transporters expressed.(22) Work carried out in different
laboratories, with cells of diverse origins, have led to the
hypothesis that the ASC and L systems may be related to
the growth and proliferation of both tumour and normal cells
during tissue development.(23–32) In fact, proteins belonging to
these transport agencies have been proposed as prognostic
markers.(25,29) It is noteworthy that these two systems do not
depend directly on the sodium electrochemical potential to
drive the glutamine uptake. The possible functional significance of this fact will be discussed later.
Glutamine metabolism
Once glutamine is inside the cell, there are two major fates for
this amino acid; either, it is excreted as glutamate plus
ammonium after suffering hydrolysis in a non-redox reaction
catalyzed by glutaminase, or it is partially oxidized to aspartate. This partial oxidation can be coupled to the formation of
9 moles of ATP. By contrast, if glutamine were completely
oxidized, up to 27 moles of ATP could be obtained. For many
years (see Box 1), the high glutamine consumption and the low
ATP yield observed in proliferating cells in our opinion
have been misinterpreted. It has been argued that energy
Table 1. Glutamine transporters
System
Protein
A: Glutamine and anionic amino acid transporters
A
ATA1
ATA2
ASC/B8
ASCT2/ATB8
Gene
Mechanism and properties
Slc38a1
Slc38a2
Slc1a5
Naþ-dependent. Ubiquitous expression. Short chained neutral amino acid
transport.
Electroneutral amino acid exchanger. Ubiquitous expression. Up-regulated in
proliferating cells.
Electrogenic, it takes 2 Naþ and 1 Cl per amino acid. Broad specificity for
neutral and cationic substrates. Restricted expression.
Naþ-independent amino acid exchanger. Broad specificity for neutral and
cationic substrates.
Naþ-independent ubiquitously expressed exchanger for large hydrophobic
amino acids. Up-regulated in proliferating cells.
Electroneutral transport of Q, N, H and in some instances, S, G and A,
coupled to the inward movement of 1 Naþ and of 1 Hþ in the opposite
direction. Restricted expression.
Electroneutral Naþ-dependent cationic-neutral amino acid exchanger.
B8,þ
ATB8,þ
Slc6a14
b8,þ
b8,þAT
Slc7a9
LAT1
LAT2
SN1
SN2
Slc7a5
Slc7a8
Slc38a3
Slc38a5
yþLAT1
yþLAT2
Slc7a7
Slc7a6
ASCT2/ATB8
EAAT1/GLAST
EAAT2/GLT
EAAT3/EAAC
EAAT4
EAAT5
xCT
Slc1a5
Slc1a3
Slc1a2
Slc1a1
Slc1a6
Slc1a7
Slc7a11
ClC-3 ?
Others ?
Clcn3
—
L
SN
YþL
B. Anionic amino acid transporters
ASC/B8
X-AG
x-c
VSOAC
Under slightly acid conditions it can recognize Glu and Asp as substrates.
1 Glu or Asp is taken with 3 Naþ and 1 Hþ in exchange for 1 Kþ. 30–40%
identity with carriers belonging to system ASC. Down-regulated in
proliferating cells.
Naþ-independent electroneutral Glu/Cystine exchanger. Broadly distributed.
Up-regulated in proliferating cells.
Volume-sensitive organic anion channels are potential source of acidic amino
acids efflux. Ubiquitous expression.
BioEssays 26.7
779
Hypothesis
Box 1. What is the role of high rates of
glutamine utilization?
Indeed, the complete oxidation of glutamine yields more
moles of ATP than its partial oxidation. However, the key
question is what pathway does supply ATP quicker? In
other words, the stoichiometry of the process must not
be confused with its kinetics, as it has often happened
Year
Sentence
1985
‘‘. . . energy generation per se may not be
the correct explanation for high rates
of glutaminolysis in these cells since
oxidation is only partial.’’
‘‘. . . if glutamine was vitally important in
energy production it would be expected
that more would be converted to
acetyl-CoA for complete oxidation via
the Krebs cycle.’’
‘‘. . . if energy formation per se was the
major reason for the high rate of
glutamine utilization, why is the
oxidation only partial?’’
‘‘. . . under conditions of extended cell
culture, glutamine can be fully
oxidized . . . and thus may become
a major oxidative fuel under these
conditions.’’
1985
1991
1999
Reference
(3)
(69)
(70)
(71)
generation may not be the correct explanation for high rates of
glutaminolysis in these cells, since oxidation is only partial.
Growing cells use large amounts of ATP, therefore why does
the cell get rid of energetically rich substrates such as
glutamate and aspartate? Here we propose just the opposite:
because glutamine oxidation is only partial, ATP can be
supplied at a fast enough rate for rapid proliferation. This
interpretation has been hampered by confusing ATP stoichiometry (moles of ATP formed by mol of glutamine consumed)
with kinetics (moles of ATP formed per unit time). In other
words, rather than the amount of ATP needed for cell division,
the key variable is the time required to produce it, at least when
the fastest growth capacity is the main goal. This argument is
well illustrated by the comparison of anaerobic glycolysis with
oxidative phosphorylation. Although the yield of ATP produced
by mol of glucose consumed is 18–19 times higher in oxidative
phosphorylation, the rate of ATP production by glycolysis can
be up to 100 times faster than that of oxidative phosphorylation.(33) Nevertheless, although a partial oxidation of glutamine
to aspartate and CO2 may offer an important kinetic advantage
with respect to the complete oxidation, we still have to explain
why a large proportion of the glutamine utilized is returned to
the medium as glutamate.
Glutamate and aspartate release
Mammalian cells express a number of different proteins able to
mediate this transport process (Table 1B). We will focus on the
780
BioEssays 26.7
activities present in rapid-dividing cell, paying special attention
to those proteins that are upregulated in cancer cells specifically. Perhaps the best understood model is the astrocytederived cancer cell. Glioma cells release large amounts of
glutamate,(34) which traces an excitotoxic pathway around the
tumour, thus allowing for rapid tumour expansion.(35) In
addition, glutamate can act as an autocrine factor on glioma
cell’s AMPA receptors, promoting migration and suppressing apoptosis.(36) Glutamate release is mainly mediated by
system xc.(37) Because, in most cells, this transporter is a 1:1
cystine–glutamate exchanger, it has been proposed that the
physiological role of this agency is to act as a cystine transporter that uses the transmembrane gradient of glutamate as a
driving force.(38) In line with that, extracellular glutamate
inhibits cystine uptake while aspartate has little(39) or no
effect.(40) The increased activity of system xc observed in
gliomas, is accompanied by a decrease in the uptake of
glutamate mediated by excitatory amino acid transporters
(EAATs).(37) Interestingly, Guo et al observed that ectopically
expressed EAAT2/GLT is toxic to U251 glioma cells as well as
to undifferentiated primary astrocytes.(41) In cancer cells other
than those derived from the brain, dicarboxylic amino acid
transport is poorly characterized. Although a defective
transport of glutamate/aspartate via EAAT seems to be a trait
of proliferative cells,(37,41,42) the expression of diverse EAAT
isoforms in some tumour cell lines has been reported.(22,43)
May EAATs operate in the efflux mode in these cells? The
transport cycle is reversible at all its stages; therefore,
gradients of substrate concentrations will determine the
direction of the transport. The [Glu]in/[Glu]out ratio found in
tumours and other tissues able to set large transmembrane
glutamate gradients, is between 102 and 104.(10) Nevertheless, EAATs are very concentrative transporters because
they thermodynamically couple the uptake of one molecule of
glutamate to the downhill movement of three Naþ and one Kþ.
The concentrative capacity of EAAT can be expressed as the
transmembrane glutamate concentration ratio that will be
found at equilibrium, ([Glu]in/[Glu]out)eq, which is a function of
the membrane potential (Cm) and the electrochemical
gradients of the ions involved in the transport process.
Considering Cm 70 mV and [Naþ]i/[Naþ]o 0.1, [Kþ]o/
[Kþ]i ¼ 5 102 and [Hþ]i/[Hþ]o ¼ 2.5, which are sound values
for these variables in mammalian cells, then the concentrative capacity of EAAT is around 106. This ratio is far above
that usually found in any cell, and it is too high to allow the
reversion of glutamate uptake. However, Rossi and coworkers have convincingly proved that glutamate release
after brain ischaemia is mainly due to reversed uptake.(44)
Under these conditions, ATP is depleted and the cell becomes
depolarized, making feasible the operation of EAATs in the
efflux mode. Interestingly, tumour cells may exhibit higher
(depolarized) membrane potential with respect to normal
tissues. For instance, the resting membrane potentials of
Hypothesis
unsynchronized MCF-7 cells during exponential growth
phase, when measured using sharp glass microelectrodes,
range from 58.6 mV to 2.7 mV.(45) Furthermore, the mean
membrane potential in breast biopsy tissue from women with
infiltrating ductal carcinoma is significantly depolarized,
compared with values measured in tissue from women with
benign breast disease.(46,47) In this context, I feel tempted to
speculate that, in certain tumours and under certain circumstances, the release of glutamate could partially contribute to
the maintenance of the electrochemical gradients of sodium
and potassium, by driving the transport mediated by EAAT
outwards. Regardless of the validity of this speculative
hypothesis, tumours overexpressing EAATs are unusual.(43)
The trend seems to be just the opposite, that is, cellular
transformation is accompanied by a downregulation of EAATs
and an increase in the activities of systems xc and ASC/
B0.(13,20,48) System ASC was originally described by Christensen as an entity serving for zwitterionic amino acid
transport.(49) Later on, this author(50) and others(51) realized
that the same agency can also serve as a transporter for
anionic amino acids under slightly acidic conditions, while the
transport of glutamine by this carrier shows little pH dependence.(51) Stimulation of anionic amino acids transport via
ASC by reduction of the ambient pH has been widely
documented in several cellular systems. The effect of the pH
has been ascribed to protonation of the anionic substrate(51)
but mostly to protonation of the transporter(50) that results in
an increased affinity for glutamate.(52) Although measurement
of pH in vivo has shown that the microenvironment in tumours
is generally more acidic than in normal tissue,(53) one might yet
question the physiological importance of ASC in mediating
glutamate transport. However, Nunck and Nunck have shown
that, because of its high capacity, system ASC can transport
glutamate at significant rates, even in the presence of serine at
pH 7.2.(52) Furthermore, ASCT2 has been proposed to mediate the observed efflux of L-aspartate across the blood–brain
barrier.(54) Thus, ASC could help to drive a glutamine–
aspartate (or glutamate) exchange, while an important part
of the glutamine-derived glutamate could be exchanged with
cystine via system xc, which has been linked to proliferation
in both tumour and embryonic tissues. In the intracellular
environment, cystine is reduced to cysteine,(55) the ratelimiting substrate for glutathione synthesis. Glutathione levels
are of critical importance to tumour cells, affecting their ability
to withstand oxidative attack.(56) Furthermore, the chemosensitivity of tumour cells has been related to intracellular
glutathione levels.(57)
Can glutamine hydrolysis energize amino
acid transport?
Transport across the plasma membrane is essential for
supplying cells with nutrients for cellular metabolism. Many
of these nutrients are available in the extracellular milieu in
lower concentrations than those required within the cell. Such
substances must be actively transported. Most animal cells
make use of the existing Naþ gradient as a driving force that
energizes transport processes. This Naþ gradient is created
and maintained with the participation of the (Naþ,-Kþ)ATPase. This protein pumps three Naþ out of the cell and
two Kþ into the cell, against their respective electrochemical
gradients at the expense of one molecule of ATP. The
(Naþ,Kþ)-ATPase is one of the single major users of cellular
energy, responsible for 5–40% of the steady-state energy
consumption.(58) Rapidly dividing cells possess an enhanced
metabolism accompanied by faster nutrient uptake. This
means that a high amount of the cellular ATP produced should
be expended in powering transport, to the detriment of other
ATP-utilizing processes such as macromolecule biosynthesis,
which is quite strongly controlled by ATP supply.(59) The idea
that I would like to put forward is simple. If the cell were able to
pay the active transport bill with a currency that did not involve
ATP (neither directly nor indirectly through the generation of
Naþ gradients across the membrane), it would give an important advantage. Furthermore, the established paradigm, that
the Naþ gradient is sufficient to explain the steady-state
entrance of amino acids in eukaryotic cells, has been questioned.(60,61) Consequently, other energy sources need to be
postulated for amino acid transport. Because growth is an
energetically expensive task, a maximal rate of proliferation
must be paralleled by an optimal ability to withdraw energy
from the environment. In order to illustrate this point, let’s think
about a simplified cell model, where an energetic substrate,
say glutamine, is oxidized with the concomitant production of
ATP. If this catabolic pathway is working at maximal capacity,
then any increase in glutamine availability will not be reflected
in a further increase in ATP supply. However, if under these
circumstances, the cell were able to use the excess of glutamine, not to make ATP but to drive the uptake of nutrients, this
would give an obvious advantage in growing cells. In this
context, I hypothesize that glutamine hydrolysis free energy
can partially contribute to the energization of amino acid uptake
in rapidly dividing cells. At this point, it may be convenient to
summarise what I have reviewed concerning the glutaminolytic pathway in a previous section. This summary is illustrated
in a schematic drawing in Fig. 1. Briefly, (1) rapidly dividing
cells show high fluxes of glutamine (inwards), glutamate
(outwards) and aspartate (outwards). (2) Transport systems
ASC, L and xc are related to proliferation. (3) System ASC
allows the interchange of neutral amino acids by aspartate or
glutamate. (4) A significant amount of the glutamine-derived
glutamate seems to cross the membrane via system xc, in
interchange with cystine. (5) Upon cell entry cystine is rapidly
reduced to cysteine.(38) (6) Tumour cells establish a large
transmembrane gradient of cysteine.(10) (7) Cysteine is a good
substrate for systems L and ASC, and therefore it can be
interchanged for other amino acids. (8) Outside the cell, under
BioEssays 26.7
781
Hypothesis
Figure 1. Systems ASC, L, xc and glutaminase
form the energy transduction machinery coupling
the glutamine breakdown to the uptake of amino
acids. A high glutaminase activity (GA) facilitates
the buildup of an intracellular glutamate pool. This
pool can be directly or indirectly used to drive the
endergonic uptake of those amino acids recognized as substrates by systems xc, L and ASC.
Glutamate can also be partially oxidized to aspartate, which not only supply ATP but also may help to
speed up the glutamine uptake in the growing cell.
Aa0 represents those amino acids related to
glutamine metabolism, such as Ala and Cys, that
are highly concentrated within the cell and they are
good substrates for system ASC.
an oxidant environment, cysteine is likely to be oxidized to
cystine and again serves as substrate for cystine–glutamate
exchange.(37)
From the above exposition, it follows that a high glutaminase activity, able to exceed the capacity of the mitochondria
to oxidize glutamate, facilitates the buildup of an intracellular
glutamate pool. This pool could be directly used to drive
the endergonic uptake of those amino acids recognized as
substrates by system ASC. Nevertheless, we must note that
system ASC as a substantial contributor to anionic amino acid
transport is a hypothesis and not a confirmed mechanism.
Indirectly, the potential energy stored in the intracellular
glutamate pool could be used to speed up the uptake of
cystine via system xc; this, in turn, would result in the
establishment of a cysteine gradient across the cell membrane, which could drive the uphill uptake of those amino acids
susceptible to being interchanged with cysteine via system L
and ASC. In this sense, plasma glutamine represents a pool of
chemical free energy that could be exploited by rapidly dividing
cells. One may question why evolution has not led to the
establishement, in every mammalian cell, of such a bioenergetic strategy. If we consider the energetic economy of the
whole organism, it becomes obvious that such strategy is very
costly. In order to keep the levels of plasmatic glutamine
constant, organs such as liver, kidney and muscle must
expend large amounts of ATP to synthesize and release
782
BioEssays 26.7
glutamine.(62) If the thermodynamic system considered now is
the organism as a whole, it can be stated that the uphill amino
acid uptake in the rapid dividing cells is energized by the
hydrolysis of ATP which has taken place far away, in those
tissues that show a net glutamine production. In this view,
glutamine can be described as an energy transducing agent.
Glutamine breakdown speeds up the
metabolism of amino acids
Taking data from the literature,(10,63,64) I have calculated the
actual Gibbs free-energy change for the interchange of amino
acids catalyzed by systems ASC, L and xc (Fig. 2). The DG0
calculated in this manner, can be regarded as an index of the
accumulative potential of the transporter under current
conditions. The interchange of sodium ions by amino acids
that are substrates of system A, has been included for
comparative purposes. It can be noted that the active uptake
of amino acids driven by sodium ions may become seriously
threatened in depolarized cells (Fig. 2, see the sign for the
uptake of amino acid through system A). It is noteworthy to
emphasize that this observation may be physiologically relevant because tumour cells exhibit higher (depolarized)
membrane potential with respect to normal tissues. In
contrast, transport processes working far from equilibrium
are less sensitive to depolarization. Fig. 2 also shows the
thermodynamic efficiency of each transport process. The
Hypothesis
Figure 2. Thermodynamic analysis of amino
acid transport in growing Ehrlich tumour cells.
Taking data from the literature,(10,63,64) the thermodynamic efficiencies and average actual Gibbs
free-energy changes for the interchange of amino
acids catalyzed by systems ASC, L and xc, have
been calculated. Aa and Aa 0 represent the
substrates for ASCT2/ATB8 and LAT2, respectively. The cotransport of sodium ions with Aa00 ,
amino acids that are substrates for system A,
has been included for comparative purposes.
All the calculations have been carried out at two
membrane potential: 58.6 and 2.7 mV. These
are reported values for tumour cells growing
exponentially.(45)
efficiencies were calculated using the equation:
h¼ ðDGdriven =DGdriver Þ 100%
ð1Þ
With the exception of system A, which is operating near
equilibrium, the calculated thermodynamic efficiencies are
well below 100%. I would like to stress that these low
efficiencies can be understood as optimal with respect to
growth rate. Indeed, low thermodynamic efficiencies are
related to high fluxes.(65) In the extreme situation of 100%
efficiency, the system is at equilibrium and thus, the rate of the
process is zero. In contrast, negative values are obtained for
the efficiencies of the interchanges mediated by systems ASC
and xc. Negative thermodynamic efficiency may need some
clarification. Usually, the driver process provides the freeenergy that is necessary to impulse the driven process against
its own free-energy. In these cases the efficiency is always a
value in the range between 0 and 100%. However, when an
exergonic process is coupled to another exergonic process,
we obtain a negative value for the efficiency of this coupling.
One may question, what is the point of coupling two exergonic
processes? The answer is to speed up the global process.
Although this conclusion can be formally derived from the
phenomenological non-equilibrium thermodynamics,(66) herein, I will provide only an intuitive explanation. To this end, I
would like to give an example previously used by Westerhoff
et al.(67) Let us think about the combustion of petrol to move a
car up a hill. Part of the free-energy released in petrol
combustion is recovered in the form of increased gravitational
potential energy of the car; this is a process with a positive
thermodynamic efficiency because an exergonic (petrol
combustion) and an endergonic (car moving uphill) process,
are coupled. Nevertheless, the car can also operate at a
negative efficiency: when going downhill, pressing the gas
pedal will couple the petrol combustion (exergonic process) to
the descending movement of the car (also an exergonic
process), yet this can be useful because the car reaches its
destination more quickly.
Conclusions
Glutamine can be removed from the plasma, hydrolyzed within
the cell, and the products released to the plasma. The
calculated value for the actual Gibbs free-energy of the global
process is 49.3 kJ/mol.1 That is, the hydrolysis of one mole of
glutamine can release almost as much free energy as the
hydrolysis of ATP (50 kJ/mol).(68) Thus, plasma glutamine
represents a pool of chemical free energy that can be released
upon hydrolysis. Nevertheless, this high potential for glutamine hydrolysis cannot affect the potential for change in other
systems unless some form of mechanical coupling exists
between them. In the case of rapidly dividing cells, it is
suggested that glutaminase and systems ASC, L and xc are
key elements of the energy converter that couples the
breakdown of glutamine to the uphill uptake of nutrients. Not
surprisingly, all these proteins have been shown to be
overexpressed in rapidly dividing cells. When the energetic
economy of the whole organism is considered, it should be
noted that such strategy is very costly, since non-proliferating
tissues must expend large amounts of ATP to provide the
glutamine consumed by the rapidly growing cells. In this
sense, glutamine can be described as an energy transducing
agent. In other words, glutamine allows the coupling of
exergonic and endergonic processes that do not necessarily
take place, either simultaneously or in the same tissue. This
versatility has a cost in terms of thermodynamic efficiency.
1
Data and Calculations can be provided on request.
BioEssays 26.7
783
Hypothesis
It could be said that some efficiency must be sacrificed to make
the process run faster. Once, François Jacob wrote ‘‘the
dream of every cell is to become two cells’’. Using Jacob’s
aphorism, it could be said that making this dream come true
costs large amounts of ATP, but making the dream come true
quickly costs large amounts of ATP and lots of glutamine.
Acknowledgments
The author is grateful to Alicia Esteban del Valle and Miguel
Angel Medina for their comments on the manuscript.
References
1. Carrascosa JM, Martı́nez P, Núñez de Castro I. 1984. Nitrogen
movement between host and tumor in mice inoculated with Ehrlich
ascetic tumor cells. Cancer Res 44:3831–3835.
2. Moreadith RW, Lehninger AL. 1984. The pathways of glutamate and
glutamine oxidation by tumor cell mitochondria. J Biol Chem 259:6215–
6221.
3. Newsholme EA, Crabtree B, Ardawi MSM. 1985. The role of high rates of
glycolysis and glutamine utilization in rapidly dividing cells. Biosci
Reports 5:393–400.
4. Medina MA, Sánchez-Jiménez F, Márquez J, Quesada AR, Núñez de
Castro I. 1992. Relevance of glutamine metabolism to tumor cell growth.
Mol Cell Biochem 113:1–15.
5. Molina M, Segura JA, Aledo JC, Medina MA, Núñez de Castro I, et al.
1995. Glutamine transport by vesicles isolated from tumour-cell
mitochondrial inner membrane. Biochem J 308:629–633.
6. Aledo JC, de Pedro E, Gómez-Fabre PM, Núñez de Castro I, Márquez J.
1997. Submitochondrial localization and membrane topography of
Ehrlich ascitic tumour cell glutaminase. Biochim Biophys Acta
1323:173–184.
7. Aledo JC, Segura JA, Medina MA, Alonso FJ, Núñez de Castro I, et al.
1994. Phosphate-activated glutaminase expression during tumor development. FEBS Lett 341:39–42.
8. Curthoys NP, Watdford M. 1995. Regulation of glutaminase activity and
glutamine metabolism. Annu Rev Nutr 15:133–159.
9. Quesada AR, Medina MA, Márquez J, Sánchez-Jiménez F, Núñez de
Castro I. 1998. Contribution by host tissues to circulating glutamine in mice
inoculated with Ehrlich ascitic tumor cells. Cancer Res 48:1551–1553.
10. Márquez J, Sánchez-Jiménez F, Medina MA, Quesada AR, Núñez de
Castro I. 1989. Nitrogen metabolism in tumor bearing mice. Arch
Biochem Biophys 268:667–675.
11. Márquez J, Núñez de Castro I. 1991. Mouse liver free amino acids during
the development of Ehrlich ascites tumour. Cancer Lett 58:221–224.
12. Battaglia FC. 2000. Glutamine and glutamate exchange between the
fetal liver and the placenta. J Nutr 130:974S–977S.
13. Collins CL, Wasa M, Souba WW, Abcouwer SF. 1998. Determinants of
glutamine dependence and utilization by normal and tumor-derived
breast cell line. J Cell Physiol 176:166–178.
14. Medina MA. 2001. Glutamine and cancer. J Nutr 131:2539S–2542S.
15. Souba WW. 1993. Glutamine and cancer. Ann Surg 218:715–728.
16. Sebol JS, Weber G. 1984. Negative correlations of L-glutamine
concentration with proliferation rate in rat hepatomas. Life Sci 34:301–
306.
17. Spittler A, Oehler R, Goetzinger P, Holzer S, Reissner CM, et al. 1997.
Low glutamine concentrations induce phenotypical and functional
differentiation of U937 myelomonocytic cells. J Nutr 127:2151–2157.
18. Turowski GA, Rashid Z, Hong F, Madri JA, Basson MD. 1994. Glutamine
modulates phenotype and stimulates proliferation in human colon cancer
cell lines. Cancer Res 54:5974–5980.
19. Lobo C, Ruı́z-Bellido MA, Aledo JC, Márquez J, Núñez de Castro I,
et al. 2000. Inhibition of glutaminase expression by antisense mRNA
decreases growth and tumourigenicity of tumour cells. Biochem J 348:
257–261.
20. Bode BP, Souba WW. 1999. Glutamine transport and human hepatocellular transformation. J Parenter Enteral Nutr 23:S33–S37.
784
BioEssays 26.7
21. Bode BP. 2001. Recent molecular advances in mammalian glutamine
transport. J Nutr 131:2475S–2485S
22. McGivan JD. 1998. Rat hepatoma cells express novel transport system
for glutamine and glutamate to those present in normal rat hepatocytes.
Biochem J 330:255–260.
23. Bode BP, Fuchs BC, Hurley BP, Conroy JL, Suetterlin JE, et al. 2002.
Molecular and functional analysis of glutamine uptake in human
hepatoma and liver-derived cells. Am J Physiol 283:G1062–G1073.
24. Dolinska M, Dybel A, Zablocka B, Albrecht J. 2003. Glutamine transport
in C6 glioma cells shows ASCT2 system characteristics. Neurochem Int
43:501–507.
25. Witte D, Ali N, Carlson N, Younes M. 2002. Overexpression of the neutral
amino acid transporter ASCT2 in human colorectal adenocarcinoma.
Anticancer Res 22:2555–2557.
26. Wasa M, Wang HS, Okada A. 2002. Characterization of L-glutamine
transport by a human neuroblastoma cell line. Am J Physiol 282:C1246–
C1253.
27. Hara K, Kudoh H, Enomoto T, Hashimoto Y, Masuko T. 1999. Malignant
transformation of NIH3T3 cells by overexpression of early lymphocyte
activation antigen CD98. Biochem Biophys Res Commun 262:720–
725.
28. Yagita H, Masuko T, Hashimoto Y. 1986. Inhibition of tumor cell growth
in vitro by murine monoclonal antibodies that recognize a proliferationassociated cell surface antigen system in rats and human. Cancer Res
46:1478–1484.
29. Nikolova M, Guenova M, Taskov H, Dimitrova E, Staneva M. 1998. Levels
of expression of CAF7 (CD98) have prognostic significance in adult
acute leukemia. Leuk Res 22:39–47.
30. Yanagida O, Kanai Y, Chairoungdua A, Kim DK, Segawa H, et al. 2001.
Human L-type amino acid transporter 1 (LAT1): characterization of
function and expression in tumor cell lines. Biochem Biophys Acta
1514:291–302.
31. Wolf DA, Wang S, Panzica MA, Bassily NH, Thompson NL. 1996.
Expression of a highly conserved oncofetal gene, TA1/E16, in human
colon carcinoma and other primary cancers: homology to Schistosoma
mansoni amino acid permease and Caenorhabditis elegans gene products. Cancer Res 56:5012–5022.
32. Sang J, Lim YP, Panzica M, Finch P, Thompson NL. 1995. TA1, a highly
conserved oncofetal complementary DNA from rat hepatoma, encodes
an integral membrane protein associated with liver development,
carcinogenesis, and cell activation. Cancer Res 55:1152–1159.
33. Voet D, Voet JG. 1995. Biochemistry, 2nd ed. New York: Wilely. p 464–
470.
34. Ye ZC, Sontheimer H. 1999. Glioma cells release excitotoxic concentrations of glutamate. Cancer Res 59:4383–4391.
35. Takano T, Lin JHC, Arcuino G, Gao Q, Yang J, et al. 2001. Glutamate
release promotes growth of malignant gliomas. Nat Med 7:1010–1015.
36. Ishiuchi S, Tsuzuki K, Yoshida Y, Yamada N, Hagimura N, et al. 2002.
Blockage of Ca2þ-permeable AMPA receptors suppresses migration
and induces apoptosis in human glioblastoma cells. Nat Med 8:971–
978.
37. Ye ZC, Rothstein JD, Sontheimer H. 1999. Compromised glutamate
transport in human glioma cells: reduction-mislocalization of sodiumdependent glutamate transporters and enhanced activity of cystineglutamate exchange. J Neurosci 19:10767–10777.
38. Bannai S, Ishii T. 1988. A novel function of glutamine in cell culture:
utilization of glutamine for the uptake of cystine in human fibroblasts.
J Cell Physiol 137:360–366.
39. Sato H, Tamba M, Ishii T, Bannai S. 1999. Cloning and expression of a
plasma membrane cystine/glutamate exchange transporter composed
of two distinct proteins. J Biol Chem 274:11455–11458.
40. Bannai S. 1986. Exchange of cystine and glutamate across plasma
membrane of human fibroblasts. J Biol Chem 261:2256–2263.
41. Guo H, Lai L, Butchbach MER, Lin CLG. 2002. Human glioma cells and
undifferentiated primary astrocytes that express aberrant EAAT2 mRNA
inhibit normal EAAT2 protein expression and prevent cell death. Mol Cell
Neurosci 21:546–560.
42. Mordrelle A, Jullian E, Costa C, Corment-Boyaka E, Benamouzig R, et al.
2000. EAAT1 is involved in transport of L-glutamate during differentiation
of the Caco-2 cell line. Am J Physiol 279:G366–G373.
Hypothesis
43. Pollard M, McGivan JD. 2000. The rat hepatoma cell line H4-II-E-C3
express high activities of the high-affinity glutamate transporter GLT-1A.
FEBS Lett 484:74–76.
44. Rossi DJ, Oshima T, Attwell D. 2000. Glutamate release in severe
ishaemia is mainly by reversed uptake. Nature 403:316–321.
45. Wonderlin WF, Woodfork KA, Strobl JS. 1995. Changes in membrane
potential during the progression of MCF-7 human mammary tumor cells
through the cell cycle. J Cell Physiol 165:177–185.
46. Marino AA, Iliev IG, Schwalke MA, Gonzalez E, Marier KC, et al. 1994.
Association between cell membrane potential and breast cancer. Tumor
Biol 15:82–89.
47. Cuzick J, Holland R, Barth V, Davies R, Faupel M, et al. 1998.
Electropotential measurements as a new diagnostic modality for breast
cancer. Lancet 352:359–363.
48. Makowske M, Christensen HN. 1982. Contrast in transport systems for
anionic amino acids in hepatocytes and hepatoma cell line HTC. J Biol
Chem 257:5663–5670.
49. Christensen HN, Liang M, Archer EG. 1967. A distinct Naþ-requiring
transport system for alanine, serine, cysteine, and similar amino acids.
J Biol Chem 242:5237–5246.
50. Vadgama JV, Christensen HN. 1984. Wide distribution of pH-dependent
service of transport system ASC for both anionic and zwitterionic amino
acids. J Biol Chem 259:3648–3652.
51. Utsunomiya-Tate N, Endou H, Kanai Y. 1996. Cloning and functional
characterization of a system ASC-like Naþ-dependent neutral amino acid
transporter. J Biol Chem 271:14883–14890.
52. Munck BG, Munck LK. 1999. Effects of pH changes on systems ASC and
B in rabbit ileum. Am J Physiol 276:G173–G184.
53. Webb SD, Sherratt JA, Fish RG. 1999. Mathematical modelling of tumour
acidity: regulation of intracellular pH. J theor Biol 196:237–250.
54. Tetsuka K, Takanaga H, Ohtsuki S, Hosoya K, Terasaki T. 2003. The
L-isomer-selective transport of aspartic acid is mediated by ASCT2 at
the blood-brain barrier. J Neurochem 87:891–901.
55. Bannai S, Ishii T. 1982. Transport of cystine and cysteine and cell growth
in culture human diploid fibroblasts: effect of glutamate and homocysteate. J Cell Physiol 112:265–272.
56. Matés JM, Pérez-Gómez C, Núñez de Castro I, Asenjo M, Márquez J.
2002. Glutamine and its relationship with intracellular redox status,
oxidative stress and cell proliferation/death. Int J Biochem Cell Biol
34:439–458.
57. Okuno S, Sato H, Kuriyama-Matsumura K, Tamba M, Wang H, et al.
2003. Role of cystine transport in intracellular glutathione level and
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
cisplatin resistance in human ovarian cancer cell lines. Brit J Cancer
88:951–956.
Ewart HE, Klip A. 1995. Hormonal regulation of the Naþ-Kþ-ATPase:
mechanisms underlying rapid and sustained changes in pump activity.
Am J Physiol 269:C295–C311.
Buttgereit F, Brand MD. 1995. A hierarchy of ATP-consuming processes
in mammalian cells. Biochem J 312:163–167.
Garcı́a-Sancho J, Sánchez A, Handlogten ME, Christensen HN. 1977.
Unexpected additional mode of energetization of amino-acid transport
into Ehrlich cells. Proc Natl Acad Sci USA 74:1488–1491.
Medina MA, Núñez de Castro I. 1990. Plasma membrane oxidoreduction
and amino acid transport. In: Crane FL, Morré DJ, Löw HE, editors.
Oxidoreduction at the plasma membrane: relation to growth and
transport. Boca Raton: CRS Press. p. 247–255.
Aledo JC, de Pedro E, Gómez-Fabre PM, Núñez de Castro I, Márquez J.
2000. Changes in mRNAs for enzymes of glutamine metabolism in the
tumor-bearing mouse. Anticancer Res 20:1463–1466.
de Graaf-Hess A, Trijbels F, Blom H. 1999. New method for determining
cystine in leukocytes and fibroblasts. Clin Chem 45:2224–2228.
Hidelbrandt W, Kinscherf R, Hauer K, Holm E, Dröge W. 2002. Plasma
cystine concentration and redox state in aging and physical exercise.
Mech Ageing Dev 123:1269–1281.
Aledo JC, Esteban del Valle A. 2002. Glycolysis in wonderland: the
importance of energy dissipation in metabolic pathways. J Chem Educ
79:1336–1339.
Aledo JC. 2001. Metabolic pathways: does the actual Gibbs freeenergy change affect the flux rate? Biochem Mol Biol Educ 29:142–
143.
Westerhoff HV, Hellingwerf KJ, van Dam K. 1982. Thermodynamic
efficiency of microbial growth is low but optimal for maximal growth rate.
Proc Natl Acad Sci USA 80:305–309.
Mathews CK, van Holde KE, Ahern KG. 2002. Biochemistry, 3rded. San
Francisco: Addison Wesley Longman. p. 463–498.
Newsholme EA, Crabtree B, Ardawi MS. 1985. Glutamine metabolism in
lymphocytes: its biochemical, physiological and clinical importance. Q J
Exp Physiol 70:473–489.
Newsholme EA, Board M. 1991. Application of metabolic-control logic to
fuel utilization and its significance in tumor cells. Adv Enzyme Regul
31:225–246.
Newsholme P, Curi R, Pithon-Curi TC, Murphy CJ, Garcia C, et al. 1999.
Glutamine metabolism by lymphocytes, macrophages, and neutrophils:
its importance in health and disease. J Nutr Biochem 10:316–324.
BioEssays 26.7
785