Am J Physiol Endocrinol Metab
282: E67–E73, 2002.
Control of glycolysis in contracting skeletal muscle.
I. Turning it on
GREGORY J. CROWTHER,1 MICHAEL F. CAREY,2
WILLIAM F. KEMPER,1 AND KEVIN E. CONLEY1–3
Departments of 2Radiology, 1Physiology and Biophysics, and 3Bioengineering,
University of Washington Medical Center, Seattle, Washington 98195-7115
Received 7 March 2001; accepted in final form 24 August 2001
Crowther, Gregory J., Michael F. Carey, William F.
Kemper, and Kevin E. Conley. Control of glycolysis in
contracting skeletal muscle. I. Turning it on. Am J Physiol
Endocrinol Metab 282: E67–E73, 2002.—Why does the onset
of glycolytic flux in muscle lag the start of exercise? We tested
the hypothesis that both elevated metabolite levels and mus-
cle activity are required for flux to begin. Glycolytic flux was
determined from changes in muscle pH, phosphocreatine
concentration, and Pi concentration ([Pi]) as measured by 31P
magnetic resonance spectroscopy. Eight subjects performed
rapid ankle dorsiflexions to ⬃45% of maximal voluntary
contraction force under ischemia at a rate of 1 contraction/s.
Subjects completed two bouts of exercise separated by 1 min
of ischemic rest. Glycolytic flux was activated by 27 s in the
first bout, ceased during the ischemic rest period, and was
activated more quickly in the second bout. Because the onset
in both bouts occurred at approximately the same [Pi], ADP
concentration, and AMP concentration, the activation of gly-
colysis appears to be related to the elevation of these metab-
olite concentrations. However, because no glycolytic flux oc-
curred at rest, even when metabolite levels were high, both
muscle activity and elevated metabolites are needed to turn
on this pathway. We conclude that the delayed onset of
glycolytic flux during exercise reflects the time needed to
raise metabolites to flux-activating levels.
metabolic flux control; high-energy phosphates; human tibi-
alis anterior
GLYCOLYTIC FLUX
1
is extremely low in resting skeletal
muscle (23). The onset of much higher glycolytic fluxes
in muscle is often delayed by seconds or minutes after
the start of exercise (1, 6). This delay is evident from a
lag in significant glycolytic H⫹ (1, 6) and lactate (24)
production. In fact, intracellular pH rises after the
start of exercise because phosphocreatine (PCr) break-
down consumes H⫹ in the creatine kinase reaction (1,
6, 26). After ⬃25–50 contractions, pH begins to drop,
indicating that a suprabasal glycolytic flux has begun
(6). What accounts for this delay in the activation of
glycolysis in contracting muscle?
1
We define glycogenolysis as the conversion of glycogen to glucose
6-phosphate and glycolysis as the conversion of glucose 6-phosphate
to lactic acid.
Address for reprint requests and other correspondence: K. E.
Conley, Dept. of Radiology, Box 357115, Univ. of Washing-
ton Medical Center, Seattle, WA 98195-7115 (E-mail: kconley@
u.washington.edu).
http://www.ajpendo.org 0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society
A delayed onset of glycolytic flux is, on the surface,
consistent with the conventional feedback control
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model of the pathway. This model holds that the me-
tabolites generated by muscle contraction, such as Pi,
ADP, and AMP, increase flux by acting as substrates
and allosteric activators of glycogenolytic and glyco-
lytic enzymes (10). Evidence for the role of metabolites
in turning on glycolysis is the finding that glycolytic
flux does not begin until Pi concentration ([Pi]) is ele-
vated in both resting and active isolated frog muscle
(39, 40). This finding is in accord with the role of Pi as
a substrate for glycogen phosphorylase (GP) and an
allosteric activator of phosphofructokinase (PFK; see
Ref. 10). Not consistent with the feedback control
model is the finding that elevated metabolite levels are
not sufficient to maintain glycolytic flux. Both human
and animal studies have shown that the flux subsides
at the end of exercise, even if metabolites are kept high
(12, 13, 32, 37). Thus elevated metabolite levels may be
necessary for the activation of glycolysis, but they
alone are insufficient to maintain flux.
A control scheme that could reconcile these results is
a “dual-control” model in which both metabolites and
muscle contraction (specifically intracellular Ca2⫹) af-
fect flux. Such dual control of flux occurs at the GP step
of glycogenolysis. GP is converted to its more active a
form through the action of Ca2⫹ (10). In addition, GP
requires Pi as a substrate and therefore is activated by
increases in [Pi] (33). Thus glycogenolysis is subject to
dual control by intracellular Ca2⫹ and a metabolite.
Can this same dual-control system account for the
onset and maintenance of glycolytic flux?
This study tested the hypothesis that elevated me-
tabolite concentrations, in addition to muscle activity,
are needed for glycolytic flux to begin. Our experimen-
tal design varied the metabolite levels at the start of
exercise to determine whether they affect the onset of
glycolytic flux. Specifically, subjects performed two
bouts of ischemic exercise, each consisting of voluntary
1-Hz contractions, separated by 1 min of ischemic rest.
We predicted that the onset of glycolytic flux would be
more rapid in the second exercise bout, when initial
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E67
E68 ACTIVATING GLYCOLYSIS IN SKELETAL MUSCLE
metabolite concentrations are high. A second purpose
of the study was to determine whether glycogenolysis
and glycolysis are turned on at different metabolite
levels. We used 0.5-Hz exercise to slow the activation of
these pathways, allowing us to collect data with lower
time resolution and thus a better signal-to-noise ratio.
For all experiments, we employed a voluntary ballistic
exercise protocol that recruits all muscle fibers at low
force levels (14, 15) and measured key intracellular
metabolites and pH noninvasively using 31P magnetic
resonance spectroscopy (MRS).
METHODS
Subjects
Eight adult men aged 25–61 yr and recruited from a
population of normal volunteers were studied. The experi-
mental protocols were approved by the Human Subjects
Division of the University of Washington, and voluntary,
written informed consent was obtained from each subject.
Experimental Setup and Data Acquisition
Each subject lay supine in the bore of a General Electric
Signa 1.5 Tesla spectrometer. The right leg and foot were
held in place with a plastic holder to which a strain gauge
was attached. The strain gauge measured the force exerted
by the ankle dorsiflexor muscles and was linked to a com-
puter running LabView data acquisition software (National
Instruments, Austin, TX). A 4.5 ⫻ 9.0-cm surface coil tuned
to 25.9 MHz (the resonating frequency of phosphorus at 1.5
Tesla) was placed over the anterior compartment of the right
leg. 31P magnetic resonance spectra of the ankle dorsiflexors
were then acquired with the surface coil as previously re-
ported (9). Briefly, the magnetic field homogeneity was opti-
mized by off-resonance shimming on the proton peak of
muscle water. The unfiltered PCr line width (full width at
half-maximal height) was typically 3–5 Hz. A high-resolution
control spectrum of the resting muscle was acquired under
conditions of fully relaxed nuclear spins (interpulse delay:
16 s). Sequential spectra were then obtained under partially
saturating conditions (interpulse delay: 1.5 s) throughout the
experimental protocols described below. The spectrum for
each time point consisted of four summed acquisitions taken
over 6 s or eight summed acquisitions taken over 12 s.
Rapidly acquired 6-s spectra typically had a signal-to-noise
ratio of 90:1 for the PCr peak after line broadening.
Analysis of Spectra
Free-induction decays (FIDs) were summed, baseline cor-
rected, zero filled, apodized with an exponential filter
matched to the line width of the unfiltered PCr peak (3–5
Hz), and Fourier transformed into spectra. Fully relaxed
spectra were manually phased, baseline fixed, and analyzed
with the program “MacFID” (Tecmag, Houston, TX) to deter-
mine the area of each phosphorus peak. PCr-to-ATP and
Pi-to-ATP ratios were determined from the relative areas of
the appropriate peaks; the ATP peak area was taken to be
the average of the areas of the ␣-, ␤-, and ␥-ATP peaks. PCr
peaks of partially saturated spectra were analyzed with the
“Fit-to-Standard” program (21), whereas the Pi and phospho-
monoester (PME) peaks were analyzed with “MRUI” (38)
because their line shapes changed during exercise. Absolute
PCr concentration ([PCr]) and [Pi] were then calculated using
AJP-Endocrinol Metab • VOL 282 • JANUARY 2002 •
the PCr-to-ATP and Pi-to-ATP ratios of the fully relaxed
spectra and assuming the muscle ATP concentration to be 8.2
mM (20). Quantification of the PME peak was done similarly,
except that the resting PME concentration ([PME]) was too
small to be measured accurately by our methods and was
assumed to be 1 mM on the basis of muscle biopsy data (5, 22,
36). Most PME generated during exercise are hexose phos-
phates (HP), i.e., glycolytic intermediates such as glucose
6-phosphate (G-6-P) and fructose 6-phosphate (5, 19, 22);
therefore, changes in [PME] were considered equivalent to
changes in [HP]. Free ADP concentration ([ADP]) and AMP
concentration ([AMP]) were calculated assuming the creatine
kinase and adenylate kinase reactions to be at equilibrium
(28), with adjustments made for pH and Mg2⫹ concentration
(18). The chemical shift of the Pi peak relative to PCr was
used to calculate muscle pH (37).
Voluntary Exercise
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Subjects exercised by performing voluntary isometric bal-
listic dorsiflexions against the resistance of the plastic foot
holder. Subjects used a metronome to maintain the desired
contraction frequency of 0.5 or 1 Hz (see below) and used
visual feedback from a light-emitting diode display to achieve
the desired peak force. For each contraction, subjects reached
the target force as quickly as possible and then relaxed
immediately. Rapid contractions of this type, termed “ballis-
tic contractions” by Desmedt and Godaux (14, 15), have been
found to recruit all the muscle fibers of the tibialis anterior
when ⬎25% of maximal voluntary contraction (MVC) force is
generated (14). To ensure complete recruitment, our subjects
reached ⬃45% of MVC force with each contraction.
Experimental Protocol: 1-Hz Exercise
The 1-Hz exercise trials were designed to test the hypoth-
esis that elevated metabolite concentrations are necessary
for the activation of glycolysis. The details are summarized in
Table 1 and below.
Step A: Aerobic rest (60 s), establishing baseline conditions.
Baseline data were obtained at rest to determine initial
spectral peak areas under partially saturating conditions.
Step B: Ischemic rest (300 s), depleting intracellular O2.
Circulation to the ankle dorsiflexors was stopped by inflating
a pressure cuff around the thigh to a pressure 40 mmHg
above the subject’s systolic blood pressure. (Magnetic reso-
nance imaging of blood flow to the leg was used to confirm
that this pressure is sufficient to block flow through the
popliteal artery.) The ischemic rest period after cuff inflation
allowed basal metabolism to deplete O2 stores (2), rendering
the muscles anoxic and preventing mitochondrial respiration.
Step C: Bout 1 of ischemic exercise (12, 24, 42, or 60 s),
determining the normal onset of glycolytic flux. Changes in
pH, [PCr], and [Pi] were used to calculate glycolytic flux (see
below) to determine the time to the onset of flux in a “normal”
exercise bout, i.e., a bout in which initial metabolite concen-
trations are low.
Table 1. Experimental protocol for 1-Hz exercise trials
Step Description Duration, s
A Aerobic rest 60
B Ischemic rest 300
C Ischemic exercise (bout 1) 12, 24
D Postexercise ischemia
{ 42, or 60
60
E Ischemic exercise (bout 2) 42
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 ACTIVATING GLYCOLYSIS IN SKELETAL MUSCLE
Step D: Postexercise ischemia (60 s), stopping glycolytic
flux. We wanted to make sure that glycolytic flux dropped to
basal levels before the start of bout 2 (step E) and used
changes in pH, [PCr], and [Pi] to quantify this flux.
Step E: Bout 2 of ischemic exercise (42 s), determining the
onset of glycolytic flux when metabolites are high. Because of
muscle activity in the first exercise bout, initial metabolite
concentrations were elevated in this second bout. We there-
fore quantified glycolytic flux during bout 2 to test the hy-
pothesis that the onset of flux is more rapid when metabolite
concentrations are high (i.e., during bout 2) than when they
are low (i.e., during bout 1).
Each subject completed steps A-E at least four times. The
following two parameters varied among trials: 1) the dura-
tion of step C (the first exercise bout) ranged from 12 to 60 s,
and 2) step E was omitted in trials where step C lasted 60 s to
avoid fatigue in subsequent bouts. Thus each subject com-
pleted four different trials, each consisting of alternating
periods of ischemic rest and ischemic exercise. The order of
the trials was randomized. Subjects recovered aerobically for
⬎500 s between trials, which was sufficient for PCr, Pi, and
pH to return to baseline levels.
Experimental Protocol: 0.5-Hz Exercise
A lower contraction frequency (0.5 Hz) was used to com-
pare the onset of glycolytic flux with the onset of glycogeno-
lytic flux. The 0.5-Hz trial consisted of 60 s of aerobic rest,
300 s of ischemic rest, and 84 s of ischemic voluntary con-
tractions. We quantified glycolytic flux and glycogenolytic
flux (see below) to determine whether these fluxes are turned
on at different metabolite levels.
Calculation of Glycolytic Flux
Glycolytic flux under ischemic conditions was quantified as
glycolytic H⫹ production, since the lactic acid produced by
glycolysis cannot be removed from the muscle by the circu-
latory system or be oxidized to CO2. Glycolytic H⫹ production
was calculated from changes in muscle pH, [PCr], and [Pi] as
previously described (see Fig. 3 in Ref. 6). In brief, H⫹
⫹
generation by the glycolytic pathway (Hglycol ) equals the
observed change in H concentration plus the H⫹ consumed
⫹
in the breakdown of PCr
⫹
⌬H glycol ⫽ ⌬pH ⫻ ␤tot ⫹ 共⫺␥兲 ⫻ ⌬关PCr兴 (1)
where ⌬pH is the change in muscle pH, ␤tot is the total
muscle buffer capacity (which includes buffering due to Pi;
see Ref. 6), ␥ is the proton stoichiometric coefficient of PCr
hydrolysis (27), and ⌬[PCr] is the change in [PCr]. Glycolytic
H⫹ production is reported in Figs. 3 and 4 as “glycolytic H⫹
accumulation,” i.e., total (cumulative) H⫹ glycolytic produc-
tion from the beginning of exercise to the time of each data
point.
Glycolytic flux in the 0.5-Hz trials was calculated as
glycolytic G-6-P use (⌬G-6-Pglycol) to enable a direct com-
parison of glycolytic flux and glycogenolytic flux. This calcu-
lation reflects the stoichiometry that two protons (and 2
lactate molecules) are produced for each molecule of G-6-P
catabolized
⫹
⌬G-6-Pglycol ⫽ ⌬Hglycol/2 (2)
Calculation of Glycogenolytic Flux
Glycolysis is fueled almost exclusively by glycogenolysis in
active ischemic muscle, as evident in the stoichiometry that
1.5 ATP are produced for every H⫹ generated (whereas the
breakdown of glucose taken up from the blood results in a 1:1
AJP-Endocrinol Metab • VOL 282 • JANUARY 2002 •
E69
ratio; see Ref 6 and erratum therein). Thus, for our experi-
ments, the total glycogenolytic flux equals the glycolytic flux
plus any additional glycogenolytic flux that has not passed
through the glycolytic pathway. This latter component of the
flux is represented by the buildup of HP, primarily as G-6-P
(5). Hence, glycogenolytic G-6-P production (G-6-Pglycogenol)
may be calculated as
⌬G-6-Pglycogenol ⫽ ⌬G-6-Pglycol ⫹ ⌬关HP兴 (3)
where ⌬[HP] represents the change in [HP].
Statistics
Reported values are means ⫾ SE. Glycolytic fluxes were
compared with zero by use of one-tailed t-tests, with ␣-levels
adjusted for multiple comparisons by means of a sequential
Bonferroni correction (35). The first data point in each exer-
cise bout to be significantly greater than zero (i.e., baseline)
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was considered to represent the onset of flux.
RESULTS
Ballistic Exercise
In studying voluntary contractions, we wanted to
ensure that our measurements would not be con-
founded by incomplete muscle fiber recruitment. Des-
medt and Godaux (14, 15) found that recruitment of all
fibers is achieved in submaximal voluntary contrac-
tions of the tibialis anterior and two other muscles
when the peak force (1) exceeds 25–35% of MVC force
and (2) is reached as rapidly as possible (within 100–
150 ms of the start of the contraction). To make sure
that all dorsiflexor muscle fibers were active during
exercise, we trained our subjects to exert ⬃45% of MVC
force with typical rise times of 100–200 ms (Fig. 1),
thus satisfying both criteria.
31
P MRS Measurements Before, During,
and After Exercise
Resting PCr-to-ATP and Pi-to-ATP ratios were
4.14 ⫾ 0.13 and 0.45 ⫾ 0.02, respectively, consistent
with previously reported values (8).
Figure 2 shows 31P MRS summary data for one of the
four 1-Hz exercise trials, in which ischemic rest periods
alternated with ischemic exercise. During exercise bout
Fig. 1. Time course of a representative ballistic voluntary contrac-
tion. This subject’s maximal voluntary contraction force was 270
newtons (N).
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E70 ACTIVATING GLYCOLYSIS IN SKELETAL MUSCLE

and/or AMP) is responsible for the onset of glycolytic

flux. Our test consisted of determining whether this
onset is accelerated when previous exercise has driven
up metabolite levels. A period of ischemic rest was
interposed between exercise bouts to allow short-lived
contraction-related signals such as Ca2⫹ to dissipate
while maintaining metabolite levels. A more rapid on-
set of glycolytic flux in exercise bout 2 would indicate
that this onset is governed by a signal persisting be-
tween bouts (e.g., metabolite concentrations) rather
than a short-lived contraction-linked signal (e.g., Ca2⫹).
Figure 3 shows glycolytic H⫹ production in each of
the four 1-Hz exercise trials. Glycolytic H⫹ production
began by ⬃27 s (5 spectra) in a single bout of continu-
ous exercise lasting 60 s (Fig. 3A). This onset occurred
after three spectra in a bout preceded by a 12-s bout

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(Fig. 3B) and after one or two spectra in bouts preceded
by 24- or 42-s bouts (Fig. 3, C and D). Because the
duration of prior exercise increased (from A to D in Fig.
3), the onset of glycolytic H⫹ production occurred ear-
lier. This finding that the onset time was reduced by
prior exercise means that a short-lived signal related to
contraction per se (e.g., Ca2⫹) is not solely responsible
for the onset of glycolytic flux. However, the impor-
tance of contractile activity is seen in the fact that
there was essentially no flux during the ischemic rest
periods between bouts.

Fig. 2. Concentrations of muscle metabolites and pH during steps

B-E (see Table 1) of a 2-bout exercise trial in which the first exercise
bout (step C) lasted 24 s and the second bout (step E) lasted 42 s.
Muscles remained ischemic throughout steps B-E. PCr, phosphocre-
atine. Data shown are mean values (n ⫽ 8); error bars are omitted for
clarity.

1, there was a drop in [PCr] and a rise in [Pi], [ADP],

[AMP], and pH. The rise in pH was the result of H⫹
consumption by PCr breakdown in the creatine kinase
reaction. During ischemic rest, little change was ap-
parent in metabolite levels or pH once exercise
stopped. During exercise bout 2, there was a further
drop in [PCr] and a rise in [Pi], [ADP], and [AMP], and
a decline in pH occurred when exercise was resumed.
Changes in [PCr], [Pi], and [HP] (data not shown) were
stoichiometric throughout this trial; the sum of these
compounds did not change with exercise (40.2 ⫾ 0.8
mM at rest vs. 38.6 ⫾ 1.8 mM at the end of bout 2; P ⬎
Fig. 3. Cumulative glycolytic H⫹ accumulation vs. time for each of
0.05 in a paired 2-tailed t-test). Total phosphate also the following 4 different exercise trials: a single 60-s bout (A) or a
remained constant during 0.5-Hz exercise. sequence of 2 bouts lasting 12 and 42 s (B), 24 and 42 s (C), or 42 and
42 s (D). Muscles remained ischemic throughout all exercise bouts
Activating Glycolysis and intervening rest periods; horizontal lines denote the duration of
the exercise bouts. Values plotted are means ⫾ SE (n ⫽ 8 experi-
The purpose of our 1-Hz experiments was to test ments). *First data point in each bout that is significantly greater
whether the accumulation of metabolites (Pi, ADP, than the preexercise baseline.

AJP-Endocrinol Metab • VOL 282 • JANUARY 2002 • www.ajpendo.org

 ACTIVATING GLYCOLYSIS IN SKELETAL MUSCLE
To test the hypothesis that metabolite accumulation
governs the activation of glycolysis, glycolytic H⫹ pro-
duction is replotted as a function of contraction number
(and therefore total exercise time) in Fig. 4. Glycolytic
activation occurred by ⬃27 contractions (or ⬃27 s),
regardless of whether these contractions were per-
formed within the first exercise bout or were divided
between the first and second bouts. Therefore, each
contraction in the first exercise bout reduced the num-
ber of contractions needed to turn on glycolysis in the
second bout. Table 2 lists the [Pi], [ADP], and [AMP] at
rest and after 27 contractions for the four exercise
trials, each of which drove the metabolite concentra-
tions to similar levels. Taken together, Fig. 4 and Table
2 indicate that the onset of glycolytic flux coincides
with elevated metabolite concentrations, suggesting
that one or more of these metabolites may be involved
in the activation of glycolysis.
Onset of Glycolysis vs. Glycogenolysis
We determined whether the onset time of glycolytic
flux differed from that of glycogenolytic flux using 0.5-
Hz exercise. The lower contraction frequency of 0.5 Hz
permitted the summing of signals over 12-s periods,
thus facilitating quantification of the low signal-to-
noise HP peak. Figure 5 shows that HP levels in-
creased 18 s into exercise, whereas significant glyco-
lytic G-6-P use did not occur until 54 s. Thus HP levels
were increased three spectra and 36 s before the ap-
parent onset of glycolytic flux. In addition, we found
that glycogenolytic flux and glycolytic flux began at
distinct metabolite levels: [Pi] was 10.4 ⫾ 0.8 mM at
the onset of glycogenolytic flux, whereas significant
glycolytic flux did not occur until [Pi] reached 18.0 ⫾
1.9 mM.
DISCUSSION
This study shows that elevated metabolite concen-
trations are critical to the activation of glycolytic flux
Fig. 4. Condensed version of Fig. 3 showing cumulative glycolytic H⫹
production vs. contraction number. For each of the four exercise
protocols, the flux is minimal during the first 27 contractions or so; at
a contraction frequency of 1 Hz, this corresponds to a delay of ⬃27 s.
Error bars are omitted for clarity. Symbols are as in Fig. 3.
AJP-Endocrinol Metab • VOL 282 • JANUARY 2002 •
E71
Table 2. Metabolite levels at rest and at the onset
of glycolytic flux
Aerobic Rest After 27 Contractions
[Pi], mM 3.8 ⫾ 0.2 (3.4–4.4) 18.7 ⫾ 1.0 (16.2–20.6)
[ADP], ␮M 14 ⫾ 0.3 (13–14) 94 ⫾ 5 (80–101)
[AMP], ␮M 0.024 ⫾ 0.001 (0.022–0.027) 1.06 ⫾ 0.010 (0.76–1.21)
Values are given as ⫾SE for each of the four exercise protocols,
followed by range in parentheses. Brackets denote concentrations.
Shown are concentrations of Pi, ADP, and AMP at rest and after 27
contractions during each of the four exercise protocols.
in contracting muscle. The key role of metabolites is
evident in the finding that elevated metabolite concen-
trations reduce the delay in the onset of flux. Further-
more, glycolytic flux and glycogenolytic flux turn on at
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distinct metabolite levels, suggesting that these path-
ways may be controlled separately.
Activation of Glycolysis
GP activity is responsive to both muscle activity (i.e.,
Ca2⫹) and metabolites (i.e., Pi and AMP; see Ref. 10).
Similarly, both contraction- and metabolite-related sig-
nals appear to govern the activation of glycolysis. The
importance of muscle contraction is apparent in the
result that glycolytic H⫹ production ceases once exer-
cise ends, even though metabolite levels remain high
(Fig. 2). This finding in earlier studies (32, 37) has led
to the conclusion that muscle activity (perhaps Ca2⫹ in
specific) is required to sustain high glycolytic fluxes (6).
The more rapid onset of glycolytic flux at high metab-
olite levels in this study (Fig. 3) demonstrates that
metabolites are also important in the control of this
flux. Expressing glycolytic H⫹ production as a function
of contraction number reveals that the onset of flux
occurred after ⬃27 contractions in each trial (Fig. 4),
by which time Pi, ADP, and AMP had risen far above
their basal levels (Table 2). The relatively wide ranges
listed in Table 2 reflect the fact that metabolite con-
Fig. 5. Time course of hexose phosphate (HP) levels and cumula-
tive glycolytic glucose 6-phosphate use during 0.5 Hz exercise [rest-
ing HP concentration ([HP]) was 1 mM]. Error bars show means ⫾
SE. *First data point that is significantly greater than 0 (glycolysis)
or 1 mM ([HP]).
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E72 ACTIVATING GLYCOLYSIS IN SKELETAL MUSCLE
centrations change rapidly from spectrum to spectrum
during 1-Hz exercise; nevertheless, it is clear that the
onset of glycolytic flux is associated with elevated me-
tabolite levels. These results suggest that accumula-
tion of one or more metabolites, in addition to muscle
activity, is necessary for the onset of glycolytic flux.
Glycogenolysis vs. Glycolysis
Glycolytic and glycogenolytic fluxes begin at differ-
ent times (Fig. 5) and at different metabolite levels
during 0.5-Hz exercise. We compared the two fluxes to
determine whether the onset of glycolytic flux might be
a mass-action response to the onset of glycogenolytic
flux. In theory, this is plausible because glycogenolysis
supplies glycolysis with most of its substrate (G-6-P)
during the first few minutes of exercise (16, 23). How-
ever, a disparity between the onset of glycogenolytic
flux and the onset of glycolytic flux is evident in Fig. 5.
[HP] were elevated within 18 s of the start of 0.5-Hz
exercise, whereas glycolytic flux was slower to rise and
did not reach statistical significance until 54 s into
exercise. Thus the glycogenolytic production of G-6-P
does not appear sufficient to cause the onset of glyco-
lytic flux, although it is possible that a certain “thresh-
old” level of G-6-P is required for glycolytic flux to
begin. In any case, our results coupled with the fact
that glycogenolytic flux often exceeds glycolytic flux (6,
31) suggest that the two pathways differ in their re-
spective sensitivities to the signals that control flux.
Sites and Mechanisms of Activation
Two key enzymes that affect glycolytic and glycogen-
olytic flux and that are stimulated by metabolites in
vitro are GP and PFK. Can our current knowledge of
the control of these enzymes explain our findings?
A mechanism by which elevated metabolites may
promote the onset of glycogenolytic flux has already
been established. Pi is a substrate for GP, and at AMP
levels ⱕ0.01 mM (as seen in Table 2), the in vitro
Michaelis-Menten constant (Km) of GP for Pi is 20–27
mM (3, 4, 34). If the in vivo Km is similar to that
measured in vitro, increases in [Pi] to ⬃10 mM (such as
those reported here) should increase GP activity and
glycogenolytic flux. The importance of Pi as a determi-
nant of glycogenolytic flux in resting muscle has been
shown by Ren et al. (33), who found that hypoxia-
mediated increases in [Pi] increased glycogenolysis in
isolated rat epitrochlearis muscle in the absence of a
conversion of GPb to GPa.
Our data demonstrating the role of metabolites in
turning on glycolysis are consistent with the classical
feedback control model of this pathway in which the
metabolites generated by muscle contraction increase
glycolytic flux by acting as allosteric activators of PFK
(10). However, given that PFK may not be the sole
rate-determining enzyme in glycolysis (17, 25), Pi,
ADP, and/or AMP may also modulate flux at other
enzymatic sites along the pathway. For example, ADP
is a substrate for phosphoglycerate kinase and pyru-
vate kinase, whereas Pi is a substrate for glyceralde-
AJP-Endocrinol Metab • VOL
hyde-3-phosphate dehydrogenase and has been re-
ported to relieve ATP inhibition of this enzyme in vitro
(29, 30). Thus it is possible that a metabolite-induced
activation of multiple glycolytic steps occurs via the
elevation of substrates and allosteric activators of gly-
colytic enzymes. Moreover, it is now clear that a con-
traction-related signal such as Ca2⫹ is also important
in the control of glycolytic flux (6, 32), yet the exact
mechanistic basis of this control likewise remains un-
certain (12).
In summary, we have examined the mechanisms
controlling the activation of glycolysis in contracting
human muscle. Our results show that the onset of
glycolytic flux does not simply correspond to the onset
of glycogenolytic flux but does require both muscle
activation and elevated metabolite concentrations. It is
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not yet clear which metabolites are most important,
how they promote flux, or which enzymes are involved.
Nonetheless, our results emphasize the importance of
both muscle contraction and metabolite levels in the
initiation and maintenance of glycolytic flux.
We thank R. K. Gronka, S. A. Jubrias, M. J. Kushmerick, and
E. G. Shankland for contributions to this study.
This research was supported by National Institute of Arthritis
and Musculoskeletal and Skin Diseases Grants AR-41928 and AR-
45184. G. J. Crowther and W. F. Kemper were recipients of National
Science Foundation predoctoral fellowships.
Portions of this work have been previously published in abstract
form (11).
Current address for M. F. Carey: Exercise Metabolism Unit,
Victoria University, Footscray 3011, Australia.
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