The following is the abstract of the article discussed in the subsequent letter:
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ABSTRACT |
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Leong, Hon Sing, Mark M. Grist, Hannah Parsons, Richard B. Wambolt, Gary D. Lopaschuk, Roger Brownsey, and Michael F. Allard. Accelerated rates of glycolysis in the hypertrophied heart: are they a methodological artifact? Am J Physiol
Endocrinol Metab 283: E1039-E1045, 2002. January 29, 2002; 10.1152/ajpendo.00507.2001.Glycolysis, measured by 3H2O production from
[5-3H]glucose, is accelerated in isolated working
hypertrophied rat hearts. However, nonglycolytic detritiation of
[5-3H]glucose via the nonoxidative pentose phosphate
pathway (PPP) could potentially lead to an overestimation of true
glycolytic rates, especially in hypertrophied hearts where the PPP may
be upregulated. To address this concern, we measured glycolysis
using [5-3H]glucose and a second, independent method in
isolated working hearts from halothane-anesthetized,
sham-operated and aortic-constricted rats. Glycolysis was accelerated
in hypertrophied hearts compared with control hearts regardless of the
method used. There was also excellent concordance in glycolytic rates
between the different methods. Moreover, activity of
glucose-6-phosphate dehydrogenase and expression of transaldolase,
enzymes controlling key steps in the oxidative and nonoxidative PPP,
respectively, were not different between control and hypertrophied
hearts. Thus nonglycolytic detritiation of [5-3H]glucose
in the PPP is insignificant, and 3H2O
production from [5-3H]glucose is an accurate means to
measure glycolysis in isolated working normal and hypertrophied rat
hearts. Furthermore, the PPP does not appear to be increased in cardiac
hypertrophy induced by abdominal aortic constriction.
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LETTER |
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To the Editor: When considering the paper by Leong et al. (4), the reader may be left with the impression that an earlier paper on a similar subject (3) is irrelevant, if not erroneous. Here we offer an explanation for the discrepancy of the results.
The validity of isotopic methods for the measurement of metabolic
fluxes rests on the assumption that the isotope is metabolized in the
same manner as unlabeled glucose. Errors can be introduced at different
levels, e.g., by inadequate models, substrate cycling, recycling of
isotopes, or lack of specific methods to measure glucose radioactivity
(2). When we noted inconsistencies in our results from
experiments using two different methods to measure myocardial glucose
metabolism, we considered the pentose phosphate pathway (PPP) by
measuring 3H2O release from
[5-3H]glucose and by a second independent method. We
found that apparent glycolytic flux obtained by
3H2O production from
[5-3H]glucose overestimated true glycolytic flux
(3). Our conclusions were based on rate determinations
obtained at 5-min intervals over a 30-min period of stable cardiac
performance and a lack of constancy of the rate of detritiation. Using
the same preparation, but imposing a three times greater workload on
the heart (which accounts for the higher rates of glucose oxidation),
Leong et al. (4) now report that significant production of
3H2O from [5-3H]glucose by
detritiation in the PPP is unlikely. The apparent discrepancy with our
results is explained by the small number of observations (n
= 3), by the small number of time points (n = 3), and
by the way the results are analyzed and displayed (cumulative counts
and not rates). In graphs of cumulative flux, any later data points
carry over the variability of earlier time points. By selecting three
of our experiments and plotting means of cumulative glycolytic flux at
three time points, we also observe an apparent linearity (Fig.
1A). The same data plotted as individual
experiments show a nonlinear increase in cumulative flux (Fig.
1B). In Fig. 1C, we have plotted rates of
glycolytic flux for the same three experiments at 5-min intervals, and
there is a significant increase over time (P < 0.05). In
short, we are not certain whether the analysis of Leong et al.
(4) had sufficient power and was performed in a manner to
detect nonlinearity. A second point is that any error is a systematic
error that should not affect the final interpretation of the results,
unless nonoxidative PPP is different in the two groups. We made a
similar systematic error in an earlier study (1), where we
were also not aware of a difference between the different tracers. This
earlier study cannot be directly compared with our later study
(3), because it addressed an entirely different issue:
ischemia and reperfusion, and the partitioning of glucose
between different pathways (glycolysis and oxidation), with glucose as
the only exogenous substrate supplied to the heart. Finally, we would
like to suggest that there is no a priori reason to suggest that flux
through the nonoxidative PPP should be affected by hypertrophy. The
authors have shown this very clearly in their study.
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REFERENCES |
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1.
Bolukoglu, H,
Goodwin GW,
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3.
Goodwin, GW,
Cohen DM,
and
Taegtmeyer H.
[5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway.
Am J Physiol Endocrinol Metab
280:
E502-E508,
2001
4.
Leong, SH,
Grist M,
Parsons H,
Wamboldt RB,
Lopaschuk GD,
Brownsey B,
and
Allard MF.
Accelerated rates of glycolysis in the hypertrophied heart: are they a methodological artifact?
Am J Physiol Endocrinol Metab
282:
E1039-E1045,
2002
Heinrich Taegtmeyer Division of Cardiology The University of Texas Houston Medical School Houston, TX 77030 | ||||||||||||
David M. Cohen Children's Nutrition Research Center Department of Pediatrics Baylor College of Medicine Houston, TX 77030 |
To the Editor: In attempting to explain
discrepant findings between their earlier paper (3) and a
more recent one from our laboratory (5), Drs. Taegtmeyer
and Cohen raise issues that warrant further discussion.
We remain puzzled by the nonlinear glycolytic flux from
[5-3H]glucose reported by the authors (3),
because linearity of 3H20 production from
[5-3H]glucose has been consistently observed by us and
others (6). In response to their concerns about the power
and manner of analysis in our recent study (5), we
reviewed our previous work. The results of one extensive study
(1) are summarized here, expressed as cumulative
glycolysis (Fig 1A) or rates
of glycolysis (Fig 1B). This representative study
unequivocally demonstrates linearity of glycolysis. Apart from the one
study cited above (3), we are not aware of any other
studies in which nonlinearity of 3H2O
production from [5-3H]glucose has been reported.
REPLY
View larger version (13K):
[in a new window]
Fig. 1.
Cumulative glycolysis (A) and rates of
glycolysis (B) in isolated rat hearts (n = 8 per group). * P < 0.05 vs. Control.
In our view, Drs. Taegtmeyer and Cohen fail to adequately address
a second and crucial discrepancy between our two studies. Rates of
glucose oxidation reported in their study are very low, despite the
fact that the hearts were perfused with lower concentrations of fatty
acid than in our study (0.4 vs. 1.2 mM). In the presence of low fatty
acid concentrations, glucose oxidation rates 10- to 20-fold higher
than those reported by the authors are commonly observed by use of
similar (4) or different methodologies (2). This issue is of critical importance, because glucose oxidation rates
are used to calculate "true" rates of glycolysis (3). Significantly, discrepancies between "true" rates of glycolysis (estimated from the sum of rates of lactate-pyruvate production and
glucose oxidation) and "apparent" glycolysis (from
[5-3H]glucose) disappear if the higher glucose oxidation
rates are used to calculate "true" glycolysis. With respect to
workload as an explanation, careful review indicates that the relevant (i.e., pressure-related) workload was at most 20% higher in our study
and not threefold higher as suggested.
We agree that there was no a priori reason to suspect upregulation of the nonoxidative pentose phosphate pathway (PPP) in cardiac hypertrophy. However, we addressed this issue because Drs. Taegtmeyer and Cohen themselves had previously noted that the oxidative PPP is upregulated in hypertrophied hearts (3), a situation that would likely raise doubts about the utility of using [5-3H]glucose methodology in hypertrophied hearts if not specifically addressed.
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FOOTNOTES |
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10.1152/ajpendo.00201.2002
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REFERENCES |
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Hypertrophied rat hearts are less responsive to the metabolic and functional effects of insulin.
Am J Physiol Endocrinol Metab
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3.
Goodwin, GW,
Cohen DM,
and
Taegtmeyer H.
[5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway.
Am J Physiol Endocrinol Metab
280:
E502-E508,
2001
4.
Kantor, PF,
Lucien A,
Kozak R,
and
Lopaschuk GD.
The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase.
Circ Res
86:
580-588,
2000
5.
Leong, HS,
Grist M,
Parsons H,
Wambolt RB,
Lopaschuk GD,
Brownsey R,
and
Allard MF.
Accelerated rates of glycolysis in the hypertrophied heart: are they a methodological artifact?
Am J Physiol Endocrinol Metab
282:
E1039-E1045,
2002
6.
Rovetto, MJ,
Whitmer JT,
and
Neely JR.
Comparison of the effects of anoxia and whole heart ischemia on carbohydrate utilization in isolated working rat hearts.
Circ Res
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Michael Allard, Roger Brownsey Departments of 1Pathology and Laboratory Medicine and 2Biochemistry and Molecular Biology University of British Columbia Vancouver, BC, Canada V6T 1Z1 | ||||||||||||
Gary Lopaschuk Departments of Pharmacology and Pediatrics University of Alberta Edmonton AB, Canada T6G 2E5 |
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