Biochemical Society Transactions (2002) Volume 30, part 2

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Received 22 November 2001

Lactate shuttles in Nature

G. A. Brooks1
Exercise Physiology Laboratory, Department of Integrative Biology, 5101 Valley Life Sciences Building, University of
California, Berkeley, CA 94720-3410, U.S.A.
and intracellular lactate shuttle concepts describe the roles of lactate in the delivery of
oxidative and gluconeogenic substrates, as well as
in cell signalling. Examples of cellcell shuttles include lactate exchanges between white-glycolytic
and red-oxidative fibres within a working muscle
bed, between working skeletal muscle and heart,
and between tissues of net lactate release and
gluconeogenesis. Lactate exchange between astrocytes and neurons that is linked to glutamatergic signalling in the brain is an example of a
lactate shuttle supporting cellcell signalling. Lac-

Abstract
Once thought to be the consequence of oxygen
lack in contracting skeletal muscle, the glycolytic
product lactate is formed and utilized continuously under fully aerobic conditions. Cellcell

Key words : cellcell signalling, energy substrates, exercise, gluconeogenesis, redox.

Abbreviations used : LDH, lactate dehydrogenase ; MCT, monocarboxylate transport protein ; PO2, partial pressure of O2 ; V} O2, O2
consumption.
1
e-mail GBrooks!Socrates.Berkeley.Edu

# 2002 Biochemical Society

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Skeletal Muscle Energetics and Exercise Tolerance

tate uptake by mitochondria and pyruvatelactate

exchange in peroxisomes are examples of intracellular lactate shuttles. Lactate exchange between
sites of production and removal is facilitated by
monocarboxylate transport proteins, of which
there are several isoforms, and, probably, also by
scaffolding proteins. The mitochondrial lactate
pyruvate transporter appears to work in conjunction with mitochondrial lactate dehydrogenase,
which permits lactate to be oxidized within actively respiring cells. Hence mitochondria function to establish the concentration and proton
gradients necessary for cells with high mitochondrial densities (e.g. cardiocytes) to take up
and oxidize lactate. Arteriovenous difference measurements on working cardiac and skeletal muscle
beds as well as NMR spectral analyses of these
tissues show that lactate is formed and oxidized
within the cells of formation in vivo. Glycolysis
and lactate oxidation within cells permits high flux
rates and the maintenance of redox balance in the
cytosol and mitochondria. Other examples of
intracellular lactate shuttles include lactate uptake
and oxidation in sperm mitochondria and the
facilitation of -oxidation in peroxisomes by
pyruvatelactate exchange. An ancient origin to
the utility of lactate shuttling is implied by the
observation that mitochondria of Saccharomyces
cerevisiae contain flavocytochrome b , a lactate
#
cytochrome c oxidoreductase that couples lactate
dehydrogenation to the reduction of cytochrome c.
The presence of cellcell and intracellular lactate
shuttles gives rise to the notion that glycolytic and
oxidative pathways can be viewed as linked, as
opposed to alternative, processes, because lactate,
the product of one pathway, is the substrate for the
other.

cellular redox via exchange and conversion into its

more oxidized analogue, pyruvate, through the
actions of lactate dehydrogenase (LDH). Furthermore, when lactate is released into the systemic circulation and taken up by distal tissues and
organs, it also affects the redox state in the cells,
tissues and organs of removal. Consequently,
during exercise lactate becomes a pseudohormone, a lactormone .
Recognition that there exist both intra- and
extra-cellular effects of lactate production and removal has led to a renaming of the original Lactate Shuttle Hypothesis [1], to the CellCell
Lactate Shuttle [2]. In addition, rapid progress in
ongoing research has led to an extension of the
original hypothesis to include an intracellular
component. The Intracellular Lactate Shuttle
Hypothesis was articulated when it was realized
that mitochondria isolated from rat heart, skeletal
muscle or liver oxidize lactate directly. Thus
lactate is exchanged on a quantitative basis, both
between and within cell compartments. Although
controversial only a few years ago, the concept of
lactate shuttles within and between cells has been
confirmed by others, who have observed lactate
exchange between diverse cells and tissues, including astrocytes and neurons [3].

Historical perspective
Before advancing to consideration of contemporary concepts, long-standing ideas portraying lactate in a negative context can be traced throughout
the history of biochemistry and muscle physiology. The notion that lactic acid is formed as the
result of oxygen lack can be traced to work of
Louis Pasteur in the 18th century. According to
Keilin [4] and Leicester [5], key observations of
Pasteur [6,7] were that some micro-organisms can
live and proliferate in the absence of air and cannot
use oxygen [7]. In fact, O poisons those
#
organisms. Pasteur also found that some facultative cells are capable of living both in the
presence and in the absence of air. Moreover, he
found that, in the presence of air, such cells respire
normally and cause very little fermentation, but in
its absence they show very active fermentation [6].
Then, at the beginning of the 20th century, studies
on isolated frog muscles produced results that
caused investigators to find common threads in the
metabolism of yeast and muscles of lower vertebrates [4,5].
The first measurements on muscles studied in
situ are attributable to Fletcher and Hopkins [8],
who in 1907 demonstrated that when excised frog

Introduction
The theoretical construct is that, together with
blood glucose, glycogen reserves in diverse tissues
are mobilized to provide lactate, a glycolytic
product that is either used within the cells of
formation or transported through the interstitium
and vasculature to adjacent and anatomically
distributed cells for utilization. Hence lactate is a
quantitatively important oxidizable substrate and
gluconeogenic precursor, as well as a means by
which metabolism in diverse tissues is coordinated. This is especially the case during
physical exercise, when sympathetic stimulation
of muscle glycogenolysis and recruitment of fastglycolytic muscle fibres cause lactate flux to be
high. Moreover, lactate functions as a regulator of

259

# 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 2

stature of the investigators must be recognized as

primary. Krogh, Hill and Meyerhof became Nobel
Laureates. D. B. Dill (of Margaria et al.) became
Director of the Harvard Fatigue Laboratory and
eventually served as President of the American
Physiological Society. Professor of Physiology in
Milan, Rudolfo Margaria ascended to a position of
pre-eminence in Europe. Thus in the 1920s and
1930s the luminaries in science had adopted a
Pasteur Effect\O Debt model of interpreting
#
data on glycolytic metabolism. World events and
science moved on and the O debt model was
#
immortalized in textbooks of physiology and biochemistry. Obviously, traditional and contemporary lactate shuttle concepts are very different in
terms of how biochemical and physiological processes are organized.

muscles were stimulated to contract, lactate

accumulated. Further, they showed that when
fatigued muscles were placed in oxygen-rich
environments, lactate disappeared. In 1910, A.V.
Hill [9] showed that the immediate processes of
muscle contraction did not require the consumption of oxygen, with the heat of contraction being
the same in the presence or absence of O .
#
However, extra heat was liberated in recovery, but
only if O was present. In 1920, using frog muscle
#
preparations Otto Meyerhof [10] identified glycogen as the precursor of lactic acid. He also
provided evidence strongly linking contraction to
lactate formation and oxidative recovery to glycogen restoration.
Following the 1920 paper of Krogh and
Lindhard [11], who first reported the exponential
decline in O consumption in men after exercise,
#
Hill and associates turned their attention to studies
of humans in an attempt to unite the new knowledge of muscle biochemistry and human metabolism. In 1923 Hill and Lupton [12] defined
oxygen debt as the total amount of oxygen
used, after cessation of exercise in recovery
therefrom . Recognizing that during exercise
onset and maximal exercise conditions there was
a deficit in oxygen consumption, Hill and
associates sought to measure the O debt to obtain
#
an energy equivalent of the anaerobic lactateproducing work done during exercise.
In 1933 Margaria, Edwards and Dill [13]
adopted terminology from the study of phosphagens and used it to re-interpret the biphasic
curve describing whole-body O consumption
#
(VI ) during recovery from exercise. Using hu#
man subjects running for various durations,
Margaria et al. [13] observed that, immediately
after hard exercise, the blood lactate concentration
remained elevated while VI fell rapidly during
#
the first, fast, O debt period. Subsequently, the
#
blood lactate concentration declined during
the second, slow, O debt period. Therefore Mar#
garia et al. [13] concluded that the rapid O debt
#
phase was a result of the restoration of phosphagen in recovering muscle. This rapid O debt
#
phase was termed alactacid , i.e. not having to do
with lactic acid removal. The investigators also
termed the second, slow, O debt phase that co#
incided with the decline in blood lactate concentration as the lactacid O debt [13].
#
Of the several reasons responsible for the
longevity of the belief that exercise-induced lactic
acidosis and the post-exercise oxygen debt were
attributable to O lack in working muscle, the
#
# 2002 Biochemical Society

The cellcell lactate shuttle

The original lactate shuttle hypothesis articulated
in 1984 [1] was based on results of isotope tracer
studies and knowledge of lactate release from
metabolically active tissues. Results of subsequent
investigations, as well as more careful reading of
previously published results, supported the concept. Some of the findings are enumerated here.
Lactate release and uptake by skeletal muscle
Measurements of blood flow and of arterial and
venous lactate concentrations in the blood perfusing a muscle bed allow quantification of net
lactate release both in situ and in vivo. The
hypothesis that muscle lactate production is
caused by a limitation to mitochondrial oxygen
supply in contracting canine skeletal muscle was
initially investigated by Jo$ bsis and Stainsby [14].
Using fluorescence spectroscopy, they observed
that the mitochondrial NAD+\NADH becomes
more oxidized during contractions sufficient to
elicit significant net lactate release. Subsequently,
Connett et al. [15] used a myoglobin cryomicroscopy technique to assess oxygenation status
in contracting canine muscles producing lactate.
They found lactate production to occur in fully
oxygenated contracting muscles. These results
[15] confirmed the initial observations of Jo$ bsis
and Stainsby [14] that lactate production occurs in
fully oxygenated contracting muscles in which
mitochondrial electron transport is not restricted
by O lack.
#
The initial observations of Stainsby and
colleagues that working canine muscle both
produces and removes lactate have been replicated
several times in working human muscle [1618].

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Skeletal Muscle Energetics and Exercise Tolerance

The observation that lactate is produced and

removed under fully aerobic conditions in humans
has been demonstrated many times [16,18,2527].
Most recently, Bergman et al. [25] conducted a
longitudinal training study of lactate flux during
rest and exercise in men. Among other findings,
they demonstrated significant lactate production
and oxidation in resting and exercising men in the
post-absorptive state. During exercise most
(7580 %) lactate was removed via oxidation, with
much of the remainder converted into glucose
[28].

Most notably, in their studies on humans,

Richardson, Wagner and associates [19] utilized
progressive exercise protocols and the dual technologies of proton NMR spectroscopy, to measure
myoglobin saturation and classical arteriovenous
concentration differences, to measure O con#
sumption and the lactate balance of working
human quadriceps muscle. From the myoglobin
spectra of muscle, and knowing the shape of the
myoglobin dissociation curve, Richardson and
colleagues [19] were able to calculate the intramuscular partial pressure of O (P ) during
#
#
graded efforts up to VI max. Their results indicated
#
that intracellular P is quite high in resting
#
muscles of healthy subjects breathing atmospheric
(normal) air at sea level. However, at exercise
onset, Richardson et al. [19] observed a dramatic
decline in muscle P to approx. 4 Torr (533 Pa),
#
a value still well above the critical mitochondrial
P . Moreover, the intracellular P was main#
#
tained well above the critical mitochondrial P as
#
power output increased. Working muscle released
lactate at power output equivalent to approx. 50 %
of VI max despite no signs of muscle O lack.
#
#

Facilitated lactate exchange transport

across membranes
The facilitated transport of lactate across membranes is accomplished by a family of monocarboxylate transport proteins (MCTs) that are
differentially expressed in cells and tissues. Initial
evidence of carrier-mediated lactate transport
across muscle cell membranes, obtained using rat
sarcolemmal vesicles [29,30], was followed by the
cloning and sequencing of the first lactate (monocarboxylate) transport protein (MCT) [31]. That
discovery was soon followed by the cloning and
sequencing of several additional isoforms that are
differentially expressed in mammalian tissues [32].
In the study of Bergman et al. [25], muscle biopsies
were taken and Western blots showed training
effects on the expression of muscle sarcolemmal
and mitochondrial MCT1, but not MCT4 [33].
Training-induced changes in the expression of
sarcolemmal MCT1 and mitochondrial proteins
resulted in an increase in lactate clearance during
exercise.

Blood lactate kinetics in mammals during

exercise
Using continuous infusion of [U-"$C]lactate into
dogs at rest and during continuous steady-state
exercise, Depocas et al. [20] made several key and
fundamental findings regarding whole-body lactate metabolism. These findings included : (1)
there is active lactate turnover during the resting
post-absorptive condition ; (2) a large fraction
(approx. 50 %) of lactate formed during rest is
removed through oxidation ; (3) the rate of turnover of lactate increases during exercise as compared with that at rest, even if there is only a minor
change in the blood lactate concentration ; (4) the
fraction of lactate removed through oxidation
increases to approx. 75 % during exercise ; and (5)
a minor fraction (1025 %) of the lactate removed
is converted into glucose via the Cori Cycle during
exercise. In terms of quantification, when glucose
and lactate fluxes were measured with radiotracers and values were compared in resting and
exercising rats [21,22], it became obvious that
much of the glycolytic flux passed through lactate, especially during exercise when the metabolic
rate was high. Although the values are subject
to species and experimental variations, essentially
similar results have been reproduced in studies on
rats [22], dogs [23] and horses [24].

The intracellular lactate shuttle

Concepts similar to that of the lactate shuttle as
articulated here were evolved independently by
several investigators, but the ideas never took root
or were generalized to a theory of biochemical
organization. Histochemical localization of LDH
in the mitochondria of rat heart and skeletal muscle
is probably attributable to the efforts of Baba and
Sharma [34], who used electron microscope histochemistry and showed LDH to be associated with
the inner membrane and matrix of rat pectoralis
and cardiac muscle mitochondria. Baba and
Sharma [34] were probably the first to speculate
on the presence of a lactate shuttle , but in the

261

# 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 2

pyruvate exchange across peroxisomal membranes

must be accomplished. Our preliminary efforts
indicate that an MCT is involved in this exchange
mechanism (G. B. McClelland, S. Khanna,
G. Gonza! lez, C. E. Butz and G. A. Brooks, unpublished work).
The results of studies using proton- and "$CNMR support the contention of lactate shuttles in
vivo, but the data suggest that our knowledge of
cellcell and intracellular lactate exchange and
metabolism is in its infancy. For instance, while
results from NMR spectroscopy show preferential
lactate oxidation in skeletal muscle [46] and heart
[4749], the pathways are not necessarily as expected. Pyruvate tracer given into the circulation
is rapidly converted into lactate, probably through
uptake via the action of LDH in erythrocytes and
cell lactatepyruvate exchange mediated by
MCT1. With tracers injected directly into the
myocardial circulation, ["$C]pyruvate exchanges
with lactate and alanine in the cytosol, with all
three peaks being visualized in spectra [48]. However, when ["$C]lactate is injected, cytosolic
pyruvate is not visualized [48]. Most recently,
Chatham et al. [47] have elaborated on this
apparent compartmentation of lactate metabolism,
and the results of their studies indicate preferential
oxidation of exogenous lactate in the heart, with
glycolytically derived lactate exported from this
organ.

absence of physiological or biochemical data they

were unable to expand on the physiological significance of their discovery. In contrast, others
[35,36] made similar observations, but regarded
the appearance of LDH in mitochondrial fractions
as a contaminant.
Perhaps the first depiction of an intracellular
lactate shuttle was by Hochachka [37], who linked
the presence of a unique LDH isoform (LDH-C)
to the ability of sperm mitochondria to oxidize
lactate. Hochachka fully recognized the physiological and evolutionary significance of lactate
oxidation by sperm mitochondria, but he did not
extend his findings to other cell systems.
Using cell fractionation techniques, in the
late 1980s Brandt, Kline and colleagues [38,39]
demonstrated the presence of LDH in rat liver,
kidney and heart mitochondria. Further, they
showed that isolated liver mitochondria were
capable of oxidizing lactate at least as fast as
pyruvate [39]. They interpreted their results as indicating a lactate shuttle [38]. Subsequently, the
ability of isolated muscle mitochondria to oxidize
lactate was confirmed [40,41], as was the intramitochondrial localization of LDH [2,42]. Additionally, the mitochondrial lactate\pyruvate
transporter has been identified as MCT1 [33,40].
Because proton and concentration gradients are
necessary for lactate flux via diffusion and
facilitated transport, and because removal of lactate occurs via oxidation and gluconeogenesis
(both mitochondrial processes), actively respiring
mitochondria are essential for lactate shuttles to
operate.
Today we know that some types of facultative
cells can be cultured with lactate as a preferred fuel
because their mitochondria possess the means to
consume and oxidize lactate directly without
conversion into pyruvate in the cytosol. For
instance, the mitochondria of Saccharomyces cerevisiae contain flavocytochrome b , a lactate
#
cytochrome c oxidoreductase [43] that couples the
dehydrogenation of lactate to the reduction of
cytochrome c [44].
In addition to a cytosol-to-mitochondrial
lactate shuttle, other intercellular lactate shuttles
are likely to exist, for instance between the cytosol
and peroxisomes, where it is known that a system
for the re-oxidation of NADH is essential for
the functioning of -oxidation. In this context, the
work of Baumgart et al. [45] is noteworthy, as
these workers have identified LDH to be the only
glycolytic enzyme located in peroxisomes. To play
a role in peroxisomal redox balance, lactate

# 2002 Biochemical Society

Summary
This is a rapidly changing field, and our contemporary understanding of the role of lactate
metabolism has changed dramatically from classical views. Once thought to be the consequence of
oxygen lack in contracting skeletal muscle, we
know now that lactate is formed and utilized
continuously under fully aerobic conditions. Lactate is oxidized actively at all times, especially
during exercise, when oxidation accounts for
7075 % of removal, with gluconeogenesis accounting for most of the remainder. Working
skeletal muscle both produces and uses lactate as a
fuel, with much of the lactate formed in glycolytic
fibres being taken up and oxidized in adjacent
oxidative fibres. Because it is more reduced that its
keto-acid analogue, sequestration and oxidation of
lactate to pyruvate affects cell redox, both promoting energy flux and signalling cellular events.
Lactate diffusion and carrier-mediated lactate
exchange occur down proton and concentration
gradients. These gradients are established by
mitochondrial respiration that is responsible for

262

Skeletal Muscle Energetics and Exercise Tolerance

18

oxidation and gluconeogenesis. Facilitated lactate

transport is accomplished by a family of MCTs
that are differentially expressed in cells and tissues.
The mitochondrial lactatepyruvate transporter
appears to work in conjunction with mitochondrial
LDH, permitting lactate to be oxidized within
actively respiring cells, thereby establishing the
gradients driving lactate flux. Glycolysis accompanied by lactate oxidation within cells
permits high flux rates and the maintenance of
redox balance in the cytosolic and mitochondrial
compartments. The presence of cellcell and
intracellular lactate shuttles gives rise to the notion
that glycolytic and oxidative pathways can be
viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other.

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20
21
22
23
24

25

26

This work was supported by National Institutes of Health grants

AR 42906 and DK 19577. Thanks are due to G. B. McClelland and
B. F. Miller for reading and commenting on the manuscript.

27
28

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Received 21 November 2001

Different modes of activating phosphofructokinase, a key regulatory enzyme of

glycolysis, in working vertebrate muscle
G. Wegener*1 and U. Krause
*Institut fu$ r Zoologie, Molekulare Physiologie, Johannes Gutenberg-Universita$ t, Saarstrasse 21, D-55099 Mainz,
Germany, and Christian de Duve Institute of Cellular Pathology, Hormone and Metabolic Research Unit,
UCL-ICP 7529, Avenue Hippocrates 75, B-1200 Bruxelles, Belgium
increased ATP turnover in contracting muscle
cells. This does not apply to F2,6P , which is
#
likely to respond to extracellular signals and could
be involved in mechanisms by which muscle
metabolism is integrated into the metabolism of
the whole body. Whether this phenomenon exists
in vertebrates other than the frog, and maybe even
in humans, and how the content of F2,6P in
#
muscle is controlled are intriguing open questions.

Abstract
Glycolytic flux in white muscle can be increased
several-hundredfold by exercise. Phosphofructokinase (PFK ; EC 2.7.1.11) is a key regulatory
enzyme of glycolysis, but how its activity in muscle
is controlled is not fully understood. In order not
to neglect integrative aspects of metabolic regulation, we have studied in frogs (Rana temporaria)
a physiological form of muscle work (swimming)
that can be triggered like a reflex. We analysed
swimming to fatigue in well rested frogs, recovery
from exercise, and repeated exercise after 2 h of
recovery. At various times, gastrocnemius muscles
were tested for glycolytic intermediates and
effectors of PFK. All metabolites responded similarly to the two periods of exercise, with the
notable exception of fructose 2,6-bisphosphate
(F2,6P ), which we proved to be a most potent
#
activator of frog muscle PFK. The first bout of
exercise triggered a more than 10-fold increase in
F2,6P ; PFK activity and the content of F2,6P
#
#
in muscle were well correlated. F2,6P decreased
#
to pre-exercise levels in fatigued frogs and it virtually disappeared during recovery. Varying by a
factor of 70, F2,6P was the most dynamic of all
#
metabolites in muscle. Even more surprisingly,
F2,6P did not respond at all to a second bout of
#
exercise. Other activators of PFK, such as Pi,
AMP and ADP, are increased as a consequence of

Introduction
Locomotion is the most conspicuous physiological
activity of animals and humans. It requires skeletal
muscles to transform chemical into mechanical
energy. Working muscles are subjected to more
strain and stress than any other organ system, yet
maintain their structural and metabolic integrity
when they go from rest to heavy work. A case in
point is vertebrate white muscle, which is
specialized for a fight or flight type of activity,
i.e. relatively short periods of heavy work which
soon cause fatigue. During bouts of activity, ATP
turnover in muscle may increase several-hundredfold. The regulatory mechanisms that maintain
metabolic homoeostasis of muscle cells, despite
large variations in metabolic rate, have been
studied intensively over several decades, and much
progress has been made in this field (reviewed in
[13]). However, it is still not fully understood
how relatively small changes in the concentrations
of substrates, intermediates and metabolic
effectors bring about the massive changes in
metabolic rate required for muscle function [2,4].
We will focus on the control of glycolysis in
vertebrate white muscle, and especially address
phosphofructokinase (PFK ; EC 2.7.1.11), a key
regulatory element of this pathway. White muscle

Key words : exercise, frog gastrocnemius, fructose 2,6-bisphosphate, metabolic integration, 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase.
Abbreviations used : F6P, fructose 6-phosphate ; F1,6P2, fructose 1,6-bisphosphate ; F2,6P2, fructose 2,6-bisphosphate ; PCr,
phosphocreatine ; PFK, phosphofructokinase ; PFK-2/FBPase-2,
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
1
To whom correspondence should be addressed (e-mail
gwegener!mail.uni-mainz.de).

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