THE JOURNAL OF BIOLOGICAL CHEMISTRY

2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 27, Issue of July 5, pp. 2441124419, 2002

Printed in U.S.A.

Regulation of Glycolytic Flux in Ischemic Preconditioning

A STUDY EMPLOYING METABOLIC CONTROL ANALYSIS*
Received for publication, February 4, 2002, and in revised form, May 1, 2002
Published, JBC Papers in Press, May 2, 2002, DOI 10.1074/jbc.M201138200

Achim M. Vogt, Mark Poolman, Cordula Ackermann, Murat Yildiz, Wolfgang Schoels,
David A. Fell, and Wolfgang Ku
bler
From the Medizinische Universitatsklinik (Ludolf-Krehl-Klinik), Abteilung Innere Medizin III (Schwerpunkt Kardiologie,
Angiologie und Pulmologie), Bergheimer Strae 58, D-69115 Heidelberg, Germany and the School of Molecular and
Biological Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom

Myocardial survival in states of supply/demand imbalance

critically depends on cellular energy status (1). To limit energy
deficit in conditions of energy shortage, e.g. hypoxia and ischemia, myocardial energy production switches from the preferential use of fatty acids to carbohydrates, thereby allowing maintenance of adequate ATP synthesis when decreased oxygen
availability becomes the limiting factor (25). However, in zeroflow ischemia, experimental analysis has shown increased glycolysis to be a double-edged sword, as the accumulation of
glycolytic end products (6, 7) outweighs the potential benefits of
increased ATP synthesis.
In accordance with this paradigm, myocardial protection by
prior exposure to ischemic preconditioning (IP)1 employs a
limitation in myocardial energy deficit (8, 9). Because IP tremendously reduces ischemic myocardial energy demands (8),
energy deficit is largely decreased at even reduced rates of
anaerobic glycolytic ATP formation in zero-flow ischemia.
There is good evidence that this attenuation in ischemic lactate
accumulation represents an important mechanism whereby IP
myocardium better withstands the challenges of sustained ischemia. Although a decrease in anaerobic glycolytic flux is a
consistent finding in myocardium protected by ischemic preconditioning (10 12), the precise mechanism used to adjust
anaerobic glycolytic flux still remains unclear. The elucidation
of this adaptive mechanism, which is not merely of theoretical
interest, was the aim of our study.
To analyze the regulation of metabolic pathways, traditional
concepts mostly imply that control over a pathway is achieved
by action on a single pathway enzyme, which is often assumed
to be a nonequilibrium step near the beginning of the pathway
subjected to feedback inhibition (13, 14). However, because the
rise of metabolic control analysis (MCA) (15) there is growing
evidence that these comfortable, time honored concepts may
mislead more than enlighten. Hence, the tools of MCA were
used to obtain a state-of-the-art analysis of glycolytic regulation in ischemic myocardium protected by preceding ischemic
preconditioning.
MATERIALS AND METHODS

* This work was supported in part by a grant from the Deutsche

Forschungsgemeinschaft, Bonn, Germany, within the Sonderforschungsbereich 320, Herzfunktion und ihre Regulation, and the University
of Heidelberg (Teilprojekt C14), Germany (to A. V.). The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-622156-8611; Fax: 49-6221-56-5515; E-mail: Achim_Vogt@med.uniheidelberg.de.
Supported by Wellcome Trust, UK, Showcase awards 048728 and
056275.
This paper is available on line at http://www.jbc.org

The experimental protocol described in this study was approved by

the Bioethical Committee of the District of Karlsruhe, Germany. All
animals in this study were handled in accordance with the guiding
principles in care and use of animals as approved by the American

1
The abbreviations used are: IP, ischemic preconditioning; MCA,
metabolic control analysis; LAD, left anterior descending coronary artery; 2-PG, 2-phosphoglycerate; 3-PG, phosphoglycerate; DHAP, dihydroxyacetone phosphate; PGM, phosphoglyceromutase; GAPDH, gluceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance;
PFK-1, phosphofructokinase-1; LDH, lactate dehydrogenase.

24411

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Exact adjustment of the Embden-Meyerhof pathway

(EMP) is an important issue in ischemic preconditioning
(IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However,
precise mechanisms of glycolytic flux and its regulation
in IP remain to be elucidated. In open chest pigs, IP was
achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index
ischemia and 120-min reperfusion. Myocardial contents
in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial
myocardial biopsies under control conditions and IP.
Detailed time courses of metabolite contents allow an
in-depth description of EMP regulation during index
ischemia using metabolic control analysis. IP reduced
myocardial infarct size (control, 90.0 3.1 versus 5.05
2.1%; p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 4.47 versus
10.3 1.26 mol/wet weight, p < 0.0001), whereby a
decrease in anaerobic glycolytic flux by at least 70%
could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the
mechanisms of glycolytic regulation were investigated.
The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory key
enzymes, as multisite regulation was employed for flux
adjustment. In myocardium subjected to IP, an even
pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux
was because of multisite modulation without any evidence for flux limitation by substrate availability or a
key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns
could not be explained on the basis of substrate kinetics.
Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must
be considered. These altered biochemical properties of
the EMP enzymes have not yet been described.

24412

Glycolysis in Ischemic Preconditioning

calculated from the accumulation of lactate and pyruvate and glyceraldehyde 3-phosphate (GA3P) (22).
J

FIG. 1. Control animals (C), following a stabilization period of

2 h, were subjected to 45 min of index ischemia and 120 min of
reperfusion. In the IP group, a preconditioning cycle of brief LAD
occlusion (10 min) and reperfusion (30 min) was performed twice. LAD
occlusions are indexed by thick-lined boxes, reperfusion periods by
white bars. The numbers indicate duration of these periods in minutes.
The arrows mark the time points when myocardial biopsies were taken
(before as well as at 2, 5, 10, 20, and 45 min after onset of index
ischemia).

0.154

2-PG]
[3-PG]

(Eq. 1)

The equilibrium of isomerase and phosphoglyceromutase are known

to be unaffected by ischemia (13). Free myocardial ADP content was
calculated from the myocardial contents of ATP and AMP, assuming an
equilibrium state of the adenylate kinase reaction (21). Glycolytic flux
(J) (in micromole of C6-units/g wet weight/min) during ischemia was

(Eq. 2)

Our experimental animal model employs total myocardial ischemia.

As a consequence, the use of extracellular glucose, which is taken up by
the cell via glucose transporters and enters glycolysis after phosphorylation to glucose 6-phosphate (Glu-6-P) by hexokinase can be quantitatively neglected. In total myocardial ischemia, fuel for glycolysis is only
provided by the glycosyl units resulting from glycogen breakdown that
enter glycolysis via glucose 1-phosphate (Glu-1-P), and to a lesser
extent, free glucose.
As 1,3-bisphosphoglycerate could not be detected, glyceraldehyde
dehydrogenase and phosphoglycerate kinase were considered as a single glycolytic step. As 2- and 3-PG, as well as of DHAP, contents were
calculated the given values are not mathematically independent. Therefore, also GAPDH and 3-PGK as well as PGM and enolase were also put
together.
On basis of these calculations, anaerobic glycolytic regulation was
investigated using flux:metabolite co-responses as described in detail
previously (23).
Oflux,metabolite
ln flux
ln flux
flux/flux

(Eq. 3)
ln metabolite ln metabolite metabolite/metabolite
Note that it follows from the definition of a co-response OJ,S that J
cSO, where c is a constant. Where an enzyme converts metabolite S to
metabolite P the co-responses OJ,S and OJ,P involve the same flux J, so
OJ,S/OJ,P lnP/lnS, and if the enzyme had no change in its mass:
action ratio (P/S) when lnP lnS then the co-responses will be equal.
If OJ,S is greater than OJ,P then there has been a relatively larger
increase in product concentration, and the reaction has moved closer to
equilibrium, whereas if OJ,S is smaller than OJ,P, the reaction has
moved away from equilibrium. Where only a single enzyme in a pathway is altered, flux:substrate co-response coefficients are likely to be
less than 1 (or less than the Hill coefficient for cooperative enzymes)
(23). Under several known physiological conditions (e.g. hypoxia) (23),
observed co-response coefficients are larger than this; exact mechanistic
explanations of this are not known, but in theory, parallel activation of
several steps (the universal method (24), multisite modulation (25,
26), or proportional activation (27)) can account for this. Enzymes that
are extremely close to equilibrium (25) can show large co-responses
during passive adaptation to a change in metabolic flux, but this would
imply virtually no detectable change in the displacement from equilibrium, and hence ln (P/S) 0, with virtually identical substrate and
product co-responses as described above. A negative flux:substrate coresponse corresponds to a classic crossover effect, where activation of an
enzyme has induced a decrease in its substrate. Thus the co-response
coefficients provide insight into the events associated with a change in
flux.
AssumptionsOur study is based on two major assumptions. (i)
Essentially maintained tissue integrity during index ischemia and (ii)
constant levels and activities in the enzymes of anaerobic glycolysis,
which are both supported by a vast body of evidence. (i) Lethal myocardial injury is initialized and determined during ischemia, whereas the
demarcation of necrosis happens during reperfusion. In this context, it
is one of the seminal findings of myocardial pathophysiology that myocytes have suffered severe injury before reperfusion, and that the dramatic consequences of reperfusion are simply postmortem manifestations of lethal injury made possible by the sudden availability of large
volumes of plasma water, calcium, or both. In other words, reperfusion
accelerates the undertaking events associated with cell death (28). (ii)
Because protein synthesis and degradation are inhibited in myocardial
ischemia (29), and because of the short time course of index ischemia,
constant levels and unchanged activities in the enzymes of the EmbdenMeyerhof pathway can be assumed.
In accordance to these basic findings, we feel justified to perform our
analysis of anaerobic glycolytic regulation in myocardium with an unchanged enzymatic composition and an essentially maintained tissue
integrity prior to reperfusion. Thus, lactate accumulation could be used
as a measure of anaerobic glycolytic flux.
Statistical AnalysisComparisons of metabolite levels between
groups were performed using ANOVA, for comparisons within groups
repeated measures of ANOVA (Scheffe -test) was used. Analysis of vari-

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Physiological Society and the investigation conformed with the Guide

for Care and Use of Laboratory Animals, United States National Institutes of Health.
Animal Preparation12 castrated German domestic pigs with body
weights between 25.0 and 32.5 kg (27.8 0.32 kg) were used in this
study employing an established model of coronary occlusion and reperfusion as described in detail previously (16).
Experimental GroupsThe experimental protocol is shown in Fig. 1.
In all animals, myocardial ischemia causing infarction was induced by
a 45-min LAD occlusion (index ischemia). The animals of the control
group (C, n 6) were only subjected to index ischemia followed by 120
min reperfusion. In the IP group, the animals were subjected to two
ischemic episodes (10 min each, with 30 min reperfusion between)
before the onset of the 45-min index ischemia (IP long; n 6).
Myocardial drill biopsies were taken at the end of the stabilization
period, i.e. before any experimental intervention, from virgin, nonischemic myocardium, and directly before occluding the LAD at the
onset of index ischemia. During the 45-min LAD occlusion, biopsies
were taken from the center of the ischemic area after 2, 5, 10, 20, and
45 min.
Determination of the Infarcted Area and Quantification of Myocardial ProtectionDetermination of myocardial infarct size was determined according to standard methods by identifying ischemic myocardium using fluorescent microspheres and detecting infarcted
myocardium following incubation in triphenyltetrazolium chloride.
Quantification was performed by normalizing infarcted myocardial
mass (infarcted region) to left ventricular myocardium or to the mass of
ischemic myocardium (risk region). These ratios are given in percent.
Biopsies and Metabolite AnalysisLeft ventricular drill biopsies
(20 mg each) were taken at various time points (Fig. 1). The biopsies
were immediately frozen (within 5 s) and kept in liquid nitrogen until
further use. Homogenization and deproteinization were performed in
ice-cold 60% acetonitrile (v/v) using a Branson sonifier. Using two
different high performance liquid chromatography protocols, the myocardial contents in ATP, ADP, and AMP, and the intermediary metabolites and end products of glycolysis were determined (17, 18). The
injury caused by the biopsies did not interfere with the triphenyltetrazolium chloride stainings. Myocardial glycogen contents was assessed
according to Ref. 19.
Data AnalysisDihydroxyacetone phosphate (DHAP) and 1,3bisphosphoglycerate could not be detected. The DHAP content was,
therefore, calculated from the glyceraldehyde 3-phosphate content assuming the equilibrium state of the triose-phosphate isomerase reaction (20). Two- and 3-phosphoglycerate (2-PG and 3-PG, respectively)
were not always clearly separated in the chromatograms, although its
common peak could be easily detected. The individual proportions were
calculated according to the equilibrium of the phosphoglyceromutase
(PGM) reaction as shown.

C6 units metabolized 0.5lactate

t
t

Glycolysis in Ischemic Preconditioning

ance was also employed to calculate regression differences. The obtained correlations were compared using the Spearmans rank correlation coefficient. Statistical differences were assumed at p values smaller
than 0.05.
RESULTS

Myocardial ProtectionAt constant areas at risk (C, 17.2

0.12%; IP, 15.8 0.72%), IP limits infarct size (C, 90.0 3.1
versus 5.05 2.1%; p 0.001).
Anaerobic Glycolytic FluxAs ischemia in control animals
proceed, anaerobic glycolytic flux is slowed down. In preconditioned myocardium, glycolytic flux is decreased at the onset of
index ischemia, showing an even more pronounced deceleration
during the subsequent phase (Fig. 2). Glycogen content at the
onset of index ischemia did not differ between normal and IP
myocardium (control, 99.0 8.09 mol of C6-units/g wet
weight; IP, 96.2 16.7).
Flux:Metabolite Co-responsesMyocardial metabolite contents are given in Table I. Flux:metabolite co-responses are
illustrated in Figs. 3 and 4, whereby the co-responses of metabolites within glycolysis itself are depicted in Fig. 3, and the
co-responses of non-glycolytic metabolites are shown in Fig. 4.
The corresponding regression data are given in Table II, significant differences could be attributed to every alteration in
the flux:metabolite co-response between control and IP myocardium. As kinetic properties of an enzyme may depend on
both substrate as well as product levels, the following description of glycolytic regulation during acute myocardial ischemia
and its modulation by preceding ischemic preconditioning focuses on the enzymes of the Embden-Meyerhof pathway.
Glycogen Phosphorylase/SynthaseGlycogen is the source
of glucose 6-phosphate during ischemia, so the reducing glycolytic flux corresponds to a reduced net flux through this potential substrate cycle.
In control myocardium, Glu-1-P levels decrease as the net
flux through the enzymes breaking down glycogen to Glu-1-P
increases. The co-response (in the range 0.5 to 1) could
easily be consistent with a slowing of this step by product
inhibition of the glycogen phosphorylase and substrate activation of glycogen synthesizing reactions (UDP-glucose phosphatase/synthase). In IP myocardium, the decrease in flux for a
given Glu-1-P level is significantly more pronounced. Because
the graph for IP does not represent an extension of the graph
for controls the altered flux:metabolite co-responses indicate
changed biochemical properties of these enzymes.
The myocardial contents in its co-substrate Pi and allosteric
activator AMP only showed poor correlations (not significant)

with glycolytic flux under control conditions. Thus, it is unlikely that they contributed to flux regulation at this level.
Although there was a better formal correlation between Pi (p
0.05) and AMP (not significant) following IP, decreases in Pi
and AMP with increasing flux still do not seem sufficient to
account for the change in enzyme activity observed.
PhosphoglucomutaseIn control animals, the decrease in
glycolytic flux during index ischemia is associated with an
increase in the substrate (G1P) content of PGM. This occurs at
basically stable levels in the product of PGM, Glu-6-P.
These findings indicate that the observed inhibition of glycolytic flux during acute myocardial ischemia in control myocardium is not because of limited G1P availability. Substrate
accumulation at increasing net flux, corresponding to a classical crossover effect, suggests a regulatory step downstream
from the Embden-Meyerhof pathway, causing a passive increase in Glu-1-P. However, as changes in net flux were independent of Glu-6-P levels, product inhibition of PGM may not
account for this alteration. Hence, other regulatory mechanisms must be considered. Moreover, the observation of decreasing substrate contents at stable product levels indicates
that the displacement from equilibrium must be decreasing
detectably during ischemia, even though this reaction is commonly viewed as close to equilibrium.
Following IP, comparable increases in Glu-1-P content during ischemia were associated with even more inhibition of flux
through PGM. Correspondingly, the correlation between flux
inhibition and substrate accumulation shows a significantly
steeper slope in IP myocardium. Moreover, the graph for preconditioned myocardium does not represent an extension of the
correlation, which has been calculated for control myocardium.
This altered correlation between PGM and changing substrate
concentrations indicates a different kinetic behavior of this
enzyme. Moreover, as substrate content changed independently of product levels, a shift in the PGM mass:action ratio
also occurred in this case, although these shifts were in an
opposite direction as observed in control myocardium.
Also following IP, the decrease in glycolytic flux throughout
index ischemia occurred at basically stable Glu-6-P levels.
However, there was a significantly negative flux-Glu-6-P coresponse. So although there was potentially an increase in
product inhibition by Glu-6-P, it is doubtful whether it could
counteract the larger increase in Glu-1-P.
As a consequence, the kinetic response of this enzyme does
not seem accounted for by the changes in its substrate and
product according to classical substrate and product kinetics.
Together with the fact that the response following IP is clearly
different from that of controls, suggests that preconditioning
has had an effect on this step by an unknown mechanism,
reinforced by the fact that for a given Glu-1-P concentration the
flux is much lower in IP hearts than in controls.
Glucose-phosphate IsomeraseIn control myocardium, the
decrease in flux through glucose-phosphate isomerase is only
very weakly correlated to substrate content, and no significance can be attached to the co-response coefficient with glucose 6-phosphate. Hence, the behavior of this enzyme cannot be
explained by substrate kinetics. However, as Fru-6-P content
was increased at reduced flux levels, product inhibition may
contribute to enzymatic regulation. As substrate levels change
independently of product concentration, a changing mass:action ratio (closer to equilibrium) must be assumed for glucosephosphate isomerase.
In IP myocardium, the changes in substrate and product
co-response coefficients are indicative of a shift of the mass:
action ratio away from equilibrium as ischemia progresses.
However, because of the large negative co-responses, mecha-

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FIG. 2. Anaerobic glycolytic flux in control (filled) and IP myocardium (unfilled).

24413

24414

Glycolysis in Ischemic Preconditioning

TABLE I
Myocardial metabolite content, given as mean S.E. in control myocardium and myocardium subjected to ischemic preconditioning
In addition, values for anaerobic glycolytic flux are also provided (in mol/g/min). Italic numbers indicate significant differences between control
and IP myocardium.
Time
Metabolite

Glu-1-P

Group

10

20

45

Mean

S.E.

Mean

S.E.

Mean

S.E.

Mean

S.E.

Mean

S.E.

Mean

S.E.

Mean

S.E.

0.048
0.056
0.310
0.287
0.037
0.035
0.022
0.017
0.063
0.057
0.010

0.004
0.022
0.060
0.040
0.005
0.006
0.012
0.004
0.023
0.020
0.002

0.045
0.048
0.274
0.440
0.023
0.037
0.040
0.042
0.082
0.139
0.014

0.004
0.017
0.056
0.075
0.005
0.005
0.003
0.012
0.019
0.038
0.009

0.143
0.229
0.471
0.440
0.095
0.062
0.067
0.035
0.236
0.110
0.014

0.044
0.058
0.054
0.056
0.014
0.009
0.004
0.006
0.082
0.053
0.002

0.285
0.374
0.388
0.427
0.156
0.084
0.051
0.057
0.218
0.135
0.010

0.060
0.089
0.051
0.080
0.016
0.013
0.010
0.029
0.082
0.064
0.003

0.550
0.595
0.546
0.463
0.215
0.085
0.089
0.072
0.224
0.156
0.019

0.108
0.094
0.078
0.061
0.028
0.017
0.030
0.032
0.109
0.037
0.004

1.048
0.870
0.502
0.604
0.355
0.129
0.040
0.048
0.168
0.104
0.019

0.125
0.098
0.114
0.096
0.076
0.026
0.013
0.014
0.123
0.038
0.003

1.382
1.192
0.402
0.549
0.546
0.093
0.018
0.028
0.023
0.090
0.007

0.198
0.165
0.068
0.098
0.128
0.031
0.007
0.013
0.006
0.046
0.002

0.009
0.239
0.213
0.041
0.039
0.006
0.006
0.009
0.009
0.051
0.047
2.573
2.688
3.142
2.995
0.352
0.330
0.091
0.088
0.351
0.330
3.970
4.187
0.139
0.128

0.001
0.041
0.036
0.015
0.014
0.002
0.002
0.002
0.002
0.010
0.015
0.498
0.506
0.314
0.277
0.128
0.136
0.032
0.036
0.102
0.095
0.493
0.443
0.029
0.033

0.028
0.336
0.661
0.047
0.066
0.007
0.010
0.012
0.019
0.041
0.046
2.926
4.092
3.357
2.427
0.254
0.220
0.030
0.063
0.296
0.342
4.700
6.624
0.175
0.304

0.002 0.038
0.207 0.337
0.055 0.913
0.017 0.074
0.025 0.067
0.003 0.011
0.004 0.010
0.003 0.019
0.003 0.012
0.013 0.066
0.015 0.062
0.704 7.246
1.002 6.384
0.313 2.365
0.362 2.009
0.025 1.029
0.038 0.831
0.008 0.597
0.029 0.400
0.039 1.125
0.107 0.825
0.143 10.158
1.091 10.225
0.053 0.426
0.065 0.352
1.080
0.573

0.012 0.030
0.060 0.449
0.287 0.717
0.026 0.107
0.019 0.070
0.004 0.016
0.003 0.011
0.004 0.018
0.003 0.014
0.021 0.070
0.041 0.079
2.067 19.770
0.836 9.402
0.273 1.559
0.107 0.994
0.107 1.066
0.035 0.753
0.069 0.748
0.049 0.557
0.127 1.027
0.049 0.716
1.011 13.452
0.975 10.602
0.034 0.349
0.064 0.325
0.228 0.682
0.051 0.217

0.006
0.087
0.150
0.035
0.024
0.005
0.004
0.004
0.004
0.020
0.024
2.827
1.227
0.153
0.120
0.157
0.073
0.098
0.086
0.068
0.077
1.427
0.722
0.027
0.061
0.162
0.099

0.022
0.462
0.522
0.089
0.053
0.014
0.008
0.013
0.015
0.068
0.096
31.862
10.344
1.094
0.954
0.536
0.664
0.262
0.500
0.501
0.621
12.388
11.567
0.235
0.391
0.605
0.047

0.003
0.068
0.072
0.032
0.020
0.005
0.003
0.002
0.003
0.028
0.029
4.473
1.263
0.267
0.156
0.115
0.038
0.061
0.100
0.113
0.045
1.958
1.555
0.045
0.085
0.172
0.014

0.024
0.165
0.564
0.045
0.053
0.007
0.008
0.012
0.012
0.095
0.093
49.120
15.868
0.147
0.599
0.310
0.513
0.555
0.461
0.249
0.484
11.758
10.862
0.077
0.186
0.345
0.110

0.009
0.049
0.214
0.017
0.011
0.003
0.002
0.003
0.003
0.022
0.037
8.227
3.770
0.028
0.100
0.039
0.082
0.155
0.072
0.026
0.052
1.375
1.636
0.015
0.036
0.127
0.061

nisms distinct from classical substrate or product-dependent

enzyme kinetics are apparently operative in IP.
Phosphofructokinase-1In control myocardium, the decrease in glycolytic flux is accompanied with an increase in the
substrate Fru-6-P for PFK-1. Fru-1,6-P2 increases with increasing flux, showing a positive flux co-response and a change
in mass:action ratio away from the equilibrium. Strictly, the
ADP:ATP ratio should be included in this calculation, but as it
changes in the same sense as the Fur-1,6-P2:Fru-6-P ratio
(from the relative sizes of the ATP and ADP co-response coefficients), it reinforces rather than counteracts the change.
These observations imply that under control conditions an
inhibition of glycolytic flux was not because of limited Fru-6-P
availability. Moreover, Fru-6-P accumulation suggests a regulatory step in downstream glycolysis, causing a passive increase in Fru-6-P. This finding is consistent with an inhibitory
role of PFK-1 under these conditions.
AMP, ADP, citrate, and especially Fru-2,6-P2 are potent
activators of PFK-1. Hence, the finding of decreased contents in
these metabolites at decreased net fluxes through PFK-1 may
account for the behavior of this enzyme, assuming that low
concentrations of these stimulatory factors have more impact
on regulation of PFK-1 than its inhibitor ATP, for which a
decreased content was found. However, control myocardium
shows a reduced glycolytic rate with increased Fru-6-P, and

0.011 0.044
0.037 0.250
0.273 1.064
0.033 0.049
0.031 0.063
0.005 0.007
0.005 0.010
0.005 0.014
0.003 0.014
0.017 0.067
0.017 0.107
1.548 12.949
0.765 7.233
0.236 2.183
0.220 0.987
0.057 1.056
0.122 0.761
0.078 0.675
0.110 0.651
0.074 1.179
0.156 0.774
0.744 10.283
1.420 9.976
0.050 0.336
0.055 0.312
0.383 0.951
0.150 0.142

little change in allosteric effector AMP or co-substrate ATP (at

least relative to Km for ATP).
In IP myocardium, the decrease in glycolytic flux is associated with a steep increase in Fru-6-P content, whereas the
decrease in flux through PFK-1 occurs independent of Fru1,6-P2 levels. For this enzyme under IP conditions, substrate
and product contents are indicative of a change in mass:action
ratio away from equilibrium.
According to the altered flux/substrate relationship, the
mechanism involved in downstream regulation has apparently
changed following IP. Under this condition, PFK-1 regulation
may not be explained on the basis of co-response, as large
positive co-responses of PFK flux to Fru-6-P are not consistent
with an effect of substrate kinetics. Moreover, the change in
flux through PFK-1 relative to controls occurred at largely
unchanged contents in citrate, AMP, and Fru-2,6-P2. Again,
mechanisms distinct and independent from substrate levels
and contents of effector metabolites are apparently operative.
Phosphofructokinase 2/Fructose-2,6-bisphosphataseAs PFK1/
Fru-2,6-P2ase represents a dead-end side branch of the EmbdenMeyerhof pathway, flux through this enzyme differs from net glycolytic flux. However, Fru-2,6-P2 has been shown to be important
for regulating the biochemical characteristics of PFK-1.
As PFK-1/Fru-2,6-P2ase is known to be inhibited by Fru-6-P,
its decreased content as glycolytic flux increases may well

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 6, 2015

Control
IP
Glu-6-P
Control
IP
Fru-6-P
Control
IP
F1.6P2
Control
IP
F2.6P2
Control
IP
Glyceraldehyde Control
3-phosphate
IP
DHAP
Control
IP
3-PG
Control
IP
2-PG
Control
IP
PEP
Control
IP
Pyr
Control
IP
Lac
Control
IP
ATP
Control
IP
ADP
Control
IP
AMP
Control
IP
Free ADP
Control
IP
Pi
Control
IP
Citrate
Control
IP
Glycolytic flux Control
IP

80

Glycolysis in Ischemic Preconditioning

24415

account for the increased contents in Fru-2,6-P2 in control

myocardium. In addition to the pattern of regulation found for
PFK-1, this finding is in good agreement with a regulatory role
for PFK-1 under control conditions. Thus, an increased content
in the positive regulator of Fru-2,6-P2 may be involved in
adjusting glycolytic flux.
In IP myocardium, a significant correlation between Fru2,6-P2 content and glycolytic flux was not observed. This finding fits well to the altered pattern for PFK-1 (see above) with
respect to flux adjustment following IP, which appears to be
independent of its traditional regulators.
AldolaseIn control myocardium, a decrease in aldolase flux
was accompanied by a decrease in Fru-1,6-P2 content. Hence,
for aldolase, the positive co-response observed indicates that
changes in substrate content may well account for changes in
aldolase flux. At aldolase, the courses of substrate and product
content indicate a shift of the mass:action ratio toward equilibrium conditions.

In contrast to the control myocardium, no significant correlations between substrate or product content and net flux could
be observed following IP. This finding is not in favor of a
flux-regulatory role for aldolase under these conditions.
GAPDH and 3-PGKIn control myocardium, the observed
reductions in flux occurs with poor correlations with glyceraldehyde 3-phosphate and 3-PG content. To the extent that the
co-responses with respect to substrate and product are comparable, the pattern observed indicates a similar inhibition of
substrate supply and product demand, implying that these
enzymes apparently do not play major roles in flux regulation.
No relevant shift in mass:action ratio could be seen.
In IP myocardium, again no significant correlation between
substrate content and net flux was observed. For 3-PG content,
a significant correlation was calculated. However, the large
value for the co-response might indicate that in addition to
product concentration, additional modifications of the enzyme
activities themselves may be responsible for the altered behav-

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 6, 2015

FIG. 3. Flux:metabolite graphs for glycolytic metabolites (mean S.E.). Filled, control; unfilled, ischemic preconditioning.

24416

Glycolysis in Ischemic Preconditioning

TABLE II
Regression data (power, y a xb) and correlation coefficients (r2 values) for the regressions displayed in Figs. 3 and 4. p Values for statistical
significances of the observed correlations were calculated using the Spearmans rank correlation coefficients. The regressions between control
and IP myocardium were compared using ANOVA (p values given).

C
versus
IP

1.094
4.824
3.417
0.186
1.167
1.989

0.615
0.623
0.951
0.006
0.077
0.438

0.005
0.05
0.001
0.2
0.2
0.05

0.037
0.022
0.002
0.017
0.015
0.012

0.293
1.142
1.354E 10
2.963E 14
2.24E 5
4.889

1.989
5.390
5.390
6.789
3.572
1.542

0.438
0.626
0.626
0.382
0.657
0.355

0.05
0.05
0.05
0.1
0.02
0.1

0.012
0.023
0.023
0.028
0.022
0.004

0.150
0.045
0.452
1.555E 11
1.379E 7

1.522
1.833
2.685
11.690
0.235

0.516
0.136
0.386
0.535
0.005

0.05
0.2
0.05
0.05
0.2

0.007
0.019
0.009
0.007
0.010

Control
a

Glu-1-P
Glu-6-P
Fru-6-P
Fru-1.6-P2
Fru-2.6-P2
Glyceraldehyde
3-phosphate
DHAP
3-PG
2-PG
PEP
Pyr
Lac
ATP
AMP
ADPfree
Pi
Citrate

Ischemic preconditioning
2

0.502
0.937
0.270
4.126
1.522
5.158

0.444
0.410
0.627
0.589
0.401
0.466

0.858
0.018
0.923
0.620
0.800
0.202

0.01
0.2
0.001
0.05
0.01
0.1

1.174
1.593
2.889
252.901
0.001
3.718

0.466
0.318
0.318
1.409
2.650
0.571

0.202
0.071
0.071
0.424
0.814
0.913

0.1
0.2
0.2
0.05
0.01
0.001

0.668
0.808
0.849
95.469
1.616

0.374
0.274
0.622
2.021
0.617

0.911
0.065
0.884
0.299
0.887

0.001
0.2
0.02
0.1
0.001

ior of these glycolytic steps in adjusting flux in IP myocardium.

Mass:action ratio shifted away from equilibrium.
PGM/EnolaseIn control myocardium, the change in flux
was poorly correlated with the substrate and product levels. This
pattern does not suggest a regulating role for these enzymes, and
the mass:action ratios remained largely unchanged.
In IP myocardium, the biochemical properties of these enzymes have changed as different correlations between enzyme
fluxes and substrate content were seen in the co-response val-

0.181
0.005
0.000
0.088
1.912
163.041

ues. The values are large and positive for the substrate but not
significant for the product. Because changes in flux occur at
almost unchanged substrate and product contents, the behaviors of these enzymes may show that they are very close to
equilibrium.
Pyruvate KinaseIn control myocardium, the decrease in
glycolytic flux was not accompanied by statistically significant
changes in substrate content. Although for its product pyruvate
a significant co-response could be observed, the value was too

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 6, 2015

FIG. 4. Flux:metabolite graphs for non-glycolytic metabolites (mean S.E.). Filled, control; unfilled, ischemic preconditioning.

Glycolysis in Ischemic Preconditioning

24417

TABLE III
Summary of enzymatic properties of the glycolytic enzymes in control and IP myocardium
Control
Enzyme

Change in
equilibrium
during index
ischemia?

Role for flux

adjustment

Change in
equilibrium
during index
ischemia?

Active

NDa

Active
Active

GAPDH/3-PGK

Towards
equilibrium
No change

Product
inhibition
Substrate
kinetics/
allosteric
Substrate
kinetics
Other

Towards
equilibrium
Away from
equilibrium
Away from
equilibrium

PGM/enolase

No change

PK

Towards
equilibrium
Away from
equilibrium

Glucose-phosphate
isomerase
PFK-1
Aldolase

LDH

Away from
equilibrium
Towards
equilibrium
Away from
equilibrium

Active

Mechanism of
flux adjustment

Role for flux

adjustment

Product
inhibition
Other

Active

Other

Active

Other

Active

Active

Active

No change

Other

Passive

Passive

Away from
equilibrium

Active

Other

Passive

Active

Other

Active

Other

Active

Product
inhibition

Active

Towards
equilibrium
Towards
equilibrium
Away from
equilibrium

Substrate
kinetics
and other
Other

Product
inhibition
and other

Active

ND, not determined.

large to explain the behavior of this enzyme on the basis of

product inhibition, so other mechanisms must be considered.
In IP myocardium, flux through PK again changes at basically stable levels for substrate content. For its product pyruvate, again significant co-responses could be observed, although this correlation did differ with controls. For both
experimental conditions, there was little evidence for a change
in the mass:action ratio of the reaction, because the co-responses to PEP and pyruvate are comparable within experimental error. These observations imply that this enzyme takes
part in flux adjustment under both experimental conditions,
although its role could not be attributed to substrate or product
kinetics and distinct mechanisms must be considered.
Lactate dehydrogenaseAs flux through LDH decreases,
pyruvate content remains basically stable, whereas lactate content increases. These data not only indicate a shift in mass:
action ratio away from equilibrium but suggest that product
inhibition of LDH may be a contributory factor.
In IP myocardium, the substrate for LDH again remained at
basically stable levels, but the correlation between lactate content and glycolytic flux has lost its statistical significance. The
slope of the regression graph was much lower than 1.0, indicating that regulatory mechanisms distinct from classical metabolite-flux kinetics must occur.
Response of Glycolytic Flux to Changes in Non-glycolytic
MetabolitesAs changes in glycolytic flux may occur in response to alterations in contents of non-glycolytic metabolites
acting as modulators of certain glycolytic enzymes, their possible contribution to flux adjustment was also investigated.
In control myocardium, a decrease in glycolytic flux with
decreasing ATP content could be seen. Although there was a
comparable response of glycolytic flux at high ATP concentrations in IP myocardium, the deceleration in glycolytic flux with
decreasing ATP content was even more pronounced. A comparable pattern was found for myocardial- free ADP content,
although the flux:metabolite co-responses for free ADP were
not significant in IP myocardium. For AMP content, no significant correlation to glycolytic flux was observed under control
and IP conditions. In control myocardium, inorganic phosphate
content did not significantly correlate to glycolytic flux. Although this correlation formally improved in IP myocardium,

flux increased as Pi remained stable, indicating an independence of flux adjustment from Pi levels. In control myocardium,
anaerobic flux decelerated with decreasing citrate contents,
whereas for IP myocardium, no significant correlation could be
observed.
Summarizing ConsiderationsUnder both experimental
conditions, glycolytic flux slows down as index ischemia precedes. However, this inhibition is more pronounced in IP myocardium and employs distinct mechanisms of flux adjustment
(Table III).
In control myocardium, decreasing substrate concentrations
account for decreased fluxes at PFK-1 and aldolase levels. The
regulating properties of glycogen phosphorylase, glucose-phosphate isomerase, and lactate dehydrogenase could be attributed to product inhibition. For phosphoglucomutase, GAPDH/
3-PGK, PGM/enolase, and PK, the mechanisms employed for
flux adjustment were found to be independent of traditional
substrate kinetics. Of these enzymes, a regulating role could be
found for phosphoglucomutase and PK, whereas the block consisting of GAPDH/3-PGK and PGM/enolase showed a rather
passive behavior.
In IP myocardium, the pattern of flux regulation has
changed. Decreasing substrate concentrations do not account
for decreased fluxes through PFK-1 and aldolase. Here, the
characteristics of these enzymes have obviously changed, and
for both enzymes, mechanisms distinct to traditional substrate kinetics, such as covalent modification, must be considered. For adjustment of flux by glycogen phosphorylase/
synthase and LDH, product inhibition could also be shown in
IP myocardium. However, the sensitivities of these enzymes
were modulated by IP, and for LDH, an additional mechanism appeared. Aldolase has lost its importance for flux
adjustment following IP, whereas formerly insignificant enzymes (block from GAPDH to enolase) play active roles in
modulating flux in IP myocardium.
DISCUSSION

Metabolic Control AnalysisFor almost every known enzyme, numerous data describing their biochemical characteristics are readily available (30). On the basis of these kinetic
and thermodynamic properties, various concepts were devel-

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 6, 2015

Product
inhibition
Other

Glycogen
phosphorylase/synthase
Phosphoglucomutase

NDa

Mechanism of
flux adjustment

Ischemic preconditioning

24418

Glycolysis in Ischemic Preconditioning

cogen content was unchanged in control and IP myocardium)
nor to one certain flux regulating the key enzyme. Again,
multisite modulation could be observed, whereas the pattern
observed along the glycolytic chain has been altered by the
preceding proconditioning protocol.
Although the regulation at the glycogen phosphorylase/synthese level was also characterized by product inhibition, the
sensitivity of this enzyme to react to changing product concentrations has been changed, indicating a modification of this
enzyme. As it was seen in control myocardium, the properties
of PGM could not be explained on the basis of classical substrate kinetics. However, the much better correlation observed
between flux and substrate content indicates an alteration in
the biochemical property of the enzyme. Also for glucose-phosphate isomerase, a change in enzyme characteristics could be
documented, as the regulatory mechanism following IP could
no longer be explained by product inhibition. Comparable findings were obtained for PFK-1 and aldolase, whereby the latter
enzyme has lost its active role for flux regulation in IP myocardium. As their biochemical properties had changed, the
formerly rather passive enzymes downstream from aldolase
are now actively involved in flux adjustment. For LDH, an
increase in its degree of product inhibition could be shown,
whereas additional regulatory mechanisms must be considered. Unlike in control myocardium, the adjustment of glycolytic flux following IP was independent of the contents in
citrate.
As the biochemical behaviors and the regulatory properties
of most glycolytic enzymes have changed following IP, the
description of the mechanisms responsible for these partially
unexpected alterations is an important issue. In this context,
the method used for analyzing glycolytic regulation validly
allows to differentiate whether an enzyme employs substrate/
product kinetics in classical terms or uses distinct regulatory
mechanisms. Although the latter mechanisms cannot be specifically characterized using the co-response approach, a differentiation whether or not a change in the regulating mechanism
occurs can be performed.
Anaerobic glycolysis was always assumed to be a metabolic
pathway, which is exclusively regulated on the basis of substrate kinetics or allosteric mechanisms (13, 3335). Our data
indicate that in addition to these traditional mechanisms, other
principles must also be considered to explain flux adjustment
by glycolytic enzymes. For glycogen phosphorylase, its regulation by enzyme phosphorylation is firmly established. Therefore, this mechanism must also be considered by analyzing the
alterations in enzyme behavior following IP. Interestingly,
there is increasing evidence that also the kinetic behavior of
glycolytic enzymes may be influenced by enzyme modification,
which must not only be because of phosphorylation (36 44). As
it could already be seen in control myocardium, glycolytic flux
decreased as ATP levels declined, although the sensitivity of
changing glycolytic flux to alterations in ATP content was even
increased, supporting the view that decelerating lactate accumulation rather than ATP depletion indicates a useful adaptive
mechanism to severe myocardial ischemia.
Summary and Possible ImplicationsThe deceleration of
glycolytic flux in myocardium protected by ischemic preconditioning is achieved by multisite modulation, whereby the regulatory mechanism of the glycolytic enzymes could only partly
be explained by classical enzyme kinetics. Moreover, previously
unknown patterns of flux adjustment were observed. Because
the attenuation of lactate accumulation represents a major
protective mechanism of IP, the analysis of these regulatory
mechanisms might have therapeutic implications.

Downloaded from http://www.jbc.org/ at NYU School of Medicine Library on February 6, 2015

oped to explain metabolic regulation (13, 14). These traditional

concepts mostly convey that control over a pathway is achieved
by action on a single pathway enzyme, which is often assumed
to be a non-equilibrium step near the beginning of the pathway
being subjected to feedback inhibition. However, starting with
the rise of MCA, there is growing evidence that these comfortable, traditional concepts may mislead more than enlighten
(23). Therefore, MCA was first applied to obtain a state-of-theart analysis of glycolytic flux, and its regulation in myocardium
was subjected to zero flow ischemia with and without prior
ischemic preconditioning.
Regulation of Anaerobic Glycolytic Flux during Ischemia in
Control MyocardiumIn control myocardium, a continuous
decrease in anaerobic glycolytic flux was observed. As regulatory roles for most pathway enzymes could be shown, our data
strongly contradict concepts implying glycolytic flux to be adjusted by only one or two regulatory sites (key enzymes).
Rather, as the tools for altering glycolytic flux were distributed
along the entire Embden-Meyerhof pathway, the findings of
our study are in good agreement with the concept of multisite
modulation, enabling rapid and tight adjustments of glycolytic
flux to changing demands (23). This cannot be achieved if only
one regulatory site is operative. Moreover, as Glu-1-P content
did not decrease but increased with decelerating glycolytic flux,
the concept of substrate availability as regulator of flux
through glycolysis in acute zero flow myocardial ischemia (31)
is not supported by our findings.
Of the steps of the glycolytic chain, only the downstream
block from GAPDH to PK was shown not to play dominating
roles in flux adjustment. For the remaining enzymes, the fluxsubstrate (product) analyses revealed an active participation,
whereas the mechanism used for regulating enzymatic properties were different. Product inhibition accounted for the behaviors of glycogen phosphorylase/synthase, glucose-phosphate
isomerase, and LDH, whereas for PFK-1 a regulatory pattern
consistent with traditional substrate kinetics could be observed. Moreover, the in vivo properties of PFK-1 for flux
adjustment as documented by MCA show a good correlation to
the known biochemical in vitro properties of this enzyme, such
as its modulation by changing concentrations in adenine nucleotides, Fru-2,6-P2, and citrate. For phosphoglucomutase,
however, MCA suggests that this step may no longer be seen
only passively linking glycogen phosphorylase to glucose-phosphate isomerase. Moreover, the mechanism of the regulation of
this enzyme could not be explained on the basis of substrate
kinetics, so that other regulatory mechanisms must be taken
into account.
According to traditional concepts of glycolytic regulation, it
was assumed that decreasing concentrations in ATP and increasing levels in ADP, AMP, and citrate, indicating energy
shortage, will increase flux through glycolysis. In our analysis,
this concept was supported by considering the co-responses of
glycolytic flux to citrate and ADP, whereas for AMP, regression
analysis could not prove a dependence of net flux from this
metabolite. Moreover, glycolytic flux decreased as ATP levels
declined, contradicting traditional models. However, as lactate
accumulation appears to be a stronger noxious stimulus for
ischemic myocardium than ATP depletion under the conditions
of zero flow ischemia (32), this finding might indicate a useful
adaptive myocardial mechanism.
Regulation of Anaerobic Glycolytic Flux during Ischemia in
Myocardium Protected by Ischemic PreconditioningFollowing
ischemic preconditioning, the deceleration of anaerobic glycolytic flux was even more marked. As it was shown for control
myocardium, the adjustment of anaerobic glycolytic flux could
neither be attributed to substrate availability (myocardial gly-

Glycolysis in Ischemic Preconditioning

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24419

METABOLISM AND BIOENERGETICS:

Regulation of Glycolytic Flux in Ischemic
Preconditioning: A STUDY EMPLOYING
METABOLIC CONTROL ANALYSIS

J. Biol. Chem. 2002, 277:24411-24419.

doi: 10.1074/jbc.M201138200 originally published online May 2, 2002

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Achim M. Vogt, Mark Poolman, Cordula

Ackermann, Murat Yildiz, Wolfgang Schoels,
David A. Fell and Wolfgang Kbler