1 Division of Cardiology, University of Texas-Houston Medical School; and 2 Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 µU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 ± 0.19 for 14CO2, and 1.00 ± 0.01 for [14C]lactate + [14C]pyruvate, mean ± SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 ± 0.01 and 0.43 ± 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.
transaldolase; glycogen; tracer kinetics
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INTRODUCTION |
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WE SET OUT TO STUDY the pentose phosphate pathway (PPP) in isolated working rat hearts and examined the hypothesis that 3H2O release from [5-3H]glucose overestimates rates of glycolysis. The conventional view is that 3H2O production can be used to measure glycolytic flux from [5-3H]glucose with loss of 3H into water at the triose phosphate isomerase reaction. Should this reaction be incomplete, the remainder would be lost at the enolase reaction (22). This line of reasoning assumes that 3H2O release from the nonoxidative portion of the pentose phosphate pathway can be disregarded.
The PPP functions mainly to synthesize ribose 5-phosphate (ribose 5-P) and NADPH. Ribose is used for synthesis of nucleic acids and nucleotide cofactors. The capacity for endogenous ribose synthesis by the heart is probably small, because provision of exogenous ribose is an intervention that stimulates resynthesis of the adenine nucleotide pool following conditions (e.g., ischemia) that deplete the pool (25, 33). Ribose may also be recycled. Compared with ribose, heart has a larger requirement for NADPH to counteract oxidative stress resulting especially from mitochondrial production of reactive oxygen species. The major requirement for NADPH in heart is to maintain reduced glutathione. Based on the distribution of enzyme activities, NADP+-dependent isocitrate dehydrogenase is a major source for NADPH synthesis in normal heart (1). In hypertrophied heart, the regulatory enzymes of the oxidative PPP [glucose 6-phosphate (G-6-P) dehydrogenase and 6-phosphogluconate dehydrogenase] are upregulated and provide a further source for NADPH (34).
The PPP consists of two branches: an irreversible oxidative branch that produces NADPH and ribulose 5-P, and a reversible, nonoxidative branch that permits the interconversion of glycolytic intermediates with pentose phosphates, notably, ribulose 5-P, ribose 5-P, and xylulose 5-P. This last compound is of recent interest as a signaling molecule for transcriptional activation through the glucose response element (6) and stimulation of a type 2A protein phosphatase (23).
Horecker and Mehler (12) first noted the cyclical nature of the pathway created when G-6-P is oxidized to ribulose 5-P and then converted back to glycolytic intermediates (triose and hexose phosphates) by nonoxidative PPP ("pentose phosphate cycle" or "PPP with carbon recycling"). The nonoxidative branch of the PPP is required for synthesis of ribose 5-P from either ribulose 5-P or, in the absence of oxidative PPP, from glycolytic intermediates. Therefore, in the absence of an exogenous source of ribose, the heart has an absolute, albeit modest, requirement for the nonoxidative PPP. As a whole, the nonoxidative PPP adjusts the immediate requirement for ribose synthesis to that for NADPH.
In the present study, we used an existing method to assess the
oxidative portion of the pathway on the basis of selective decarboxylation of glucose C1 by 6-phosphogluconate dehydrogenase (the
C1/C6 ratio method, which is the relative rate of product formation
from C1 C6 of glucose). Unlike previous studies, we measured
the C1/C6 ratio for both 14CO2 and released
[14C]lactate + [14C]pyruvate,
because 14CO2 production alone is insufficient
for rigorous interpretation of the oxidative pathway in the presence of
carbon recycling. In addition, we provide new qualitative evidence
regarding the operation of the nonoxidative pathway in the heart. The
evidence for nonoxidative PPP stems from observations that we interpret as nonglycolytic detritiation of [5-3H]glucose, reduced
3H/14C ratio of glycogen (and intracellular
sugar phosphates, representing the glycogen precursor pool of hexose
phosphates), and 3H2O production from
[5-3H]glucose (a measure of unidirectional glycolytic
flux) in excess of glycolytic flux measured by other methods (net
release of lactate + pyruvate, and efflux of
[14C]lactate + [14C]pyruvate).
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MATERIALS AND METHODS |
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Sources of materials.
D-[5-3H]glucose (product TRK.290, batch 42)
was from Amersham (Arlington Heights, IL). The manufacturer's stated
radiochemical and positional isotopic purities were 97% and
"essentially 100%" (by tritium NMR), respectively. Owing to
radiolysis, the product accumulates 3H2O on
storage (~5%) at the time of the heart perfusions. For this reason,
the purity of the compound was ascertained by column chromatography
before onset of each experiment, and a small correction was applied to
the calculated specific activity.
D-[1-14C]glucose (product G5645, lot
068H9446, 98% purity) and D-[6-14C]glucose
(product G9899, lot 019H9616,
98% purity) were from Sigma (St.
Louis, MO). Sources of other materials were given previously (9).
Heart perfusions. Hearts from chow-fed male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN; 361 ± 2 g, n = 8) were perfused using a working heart apparatus (28). After pentobarbital sodium anesthesia (100 mg/kg ip) and heparin injection (100 U iv), hearts were removed and arrested in ice-cold perfusate. The aorta was cannulated, and retrograde perfusion was begun to wash out blood and cannulate the left atrium (~5 min). We then switched to a working mode (anterograde aortic flow) using recirculated perfusate (200 ml) for 5 min to obtain a stable preparation. The perfusion pressure (aortic afterload) was set at 73.5 mmHg (100 cmH2O), and the atrial filling pressure in the working mode was 11.0 mmHg (15 cmH2O). After 5 min, hearts were switched to a nonrecirculating mode supplied by fresh perfusate (600 ml for each 30-min interval) containing the indicated isotopes. The entire coronary flow was collected directly from the pulmonary artery into preweighed vials over 5-min intervals, and the vials were capped immediately. After 2 min to exchange the extracellular space, we began recirculating the aortic flow by redirecting the aortic overflow to the new reservoir. The aortic circuit was sampled at 10-min intervals to measure blank values, and the content of metabolites calculated for the coronary flow was corrected for average blanks measured in the recirculated aortic circuit. The perfusate consisted of warm (37°C) Krebs-Henseleit buffer with 5 mM glucose, 40 µU/ml regular insulin (Humulin, Eli Lilly, Indianapolis, IN), 0.5 mM sodium L-lactate, 0.05 mM sodium pyruvate, 1.5 mM CaCl2, 0.1 mM EDTA (~1.4 mM free Ca2+) and was equilibrated with 95% O2-5% CO2. The perfusate during the nonrecirculating perfusion also contained 0.4 mM sodium oleate prebound to 3% bovine serum albumin (fatty acid free, Intergen, Purchase, NY), 1.4 mM free Ca2+, 0.025 µCi/ml [5-3H]glucose, and either 0.01 µCi/ml of [1-14C]glucose or 0.01 µCi/ml of [6-14C]glucose. The free calcium was adjusted by overnight dialysis against 10 volumes of albumin-free perfusate containing 1.5 mM CaCl2 and 0.1 mM EDTA. Perfusions were continued for 30 min in the presence of one of the two [14C]glucose isotopomers and then switched to a second reservoir containing the alternate 14C isotopomer, and the perfusions were continued for another 30 min before freeze-clamping the hearts on the cannula with aluminum tongs cooled in liquid N2. There were eight perfusions total (4 for each order of addition of [1-14C]- or [6-14C]glucose). [5-3H]glucose was present at constant specific activity throughout.
Analytical procedures.
All metabolites measured in the coronary flow are duplicates.
3H2O was separated from
[5-3H]glucose in samples of fresh perfusate (0.5 ml) by
use of 1.5-ml columns of AG 1-X8 resin (hydroxide form) as described
previously (8). The assay for [14C]lactate
plus [14C]pyruvate in samples (0.3 ml) of deproteinized
perfusate, with the use of paper chromatography to separate
[14C]glucose, was described previously (8).
The recovery of [14C]lactate for the entire procedure, by
means of fresh perfusate spiked with authentic
[U-14C]lactate, is 80%, and lactate is completely
separated from [U-14C]glucose (8). Total
lactate and pyruvate were measured enzymatically in deproteinized
perfusate by means of standard enzymatic assays with lactate
dehydrogenase (2). To measure
14CO2, 10-ml portions of fresh perfusate were
transferred to 50-ml Erlenmeyer flasks, and the flasks were fitted with
a serum cap and hanging center well (Kontes, Vineland, NJ) containing a
strip of fluted filter paper and 0.5 ml of
14CO2-trapping agent (1 M hyamine hydroxide in
methanol). The perfusate was acidified by injecting 0.5 ml of 60%
perchloric acid (PCA). The vials were incubated overnight, and the
wells were measured for radioactivity in 10 ml of scintillation mixture
(Ultima Gold, Packard, Meriden, CT). Metabolic rates were calculated
from the content of radioactivity in the coronary flow (dpm/ml,
corrected for blanks determined on the recirculated aortic circuit)
divided by the specific activity measured in the aortic circuit
(dpm/µmol), multiplied by the coronary flow rate (ml/min), and
normalized to the dry weight of each heart. Fluxes for total lactate
and pyruvate, measured enzymatically, were calculated from the
concentration difference across the heart times the coronary flow and
are expressed as glycosyl units (one-half the lactate + pyruvate
flux). Flow rates were calculated from the filling time for an in-line
graduated chamber (aortic flow) or gravimetrically (coronary flow).
Contractile performance is expressed as the rate of external
pressure-volume work (hydraulic power). Average power (watts)
is the product of cardiac output (aortic plus coronary flow,
m3/s) multiplied by the pressure differential
(afterload preload) across the heart in pascal units (73.5
11.0 = 62.5 mmHg, 8,500 Pa). In this preparation, the mean
aortic pressure (calculated as one-third systolic + two-thirds
diastolic pressure) assumes a value equal to the afterload, which is
fixed by the height of the aortic overflow above the aortic valve (100 cm). Other measures of contractile performance (Table
1) were measured with a 3 French Millar
pressure transducer (Millar Instruments, Houston, TX) at the side arm
of the aortic cannula interfaced to a Gould physiological recorder
(Gould model 2400S; Cleveland, OH). Hearts were weighed and ground to a
fine powder under liquid nitrogen, and a portion of the powder was used
to measure dry weight. Glycogen was isolated from the powdered tissue
by means of a standard procedure (29). Isolated glycogen
was dissolved in acetate buffer (0.5 ml), digested to glucose with
amyloglucosidase (to avoid a 3H-quenching artifact)
following a standard procedure (2), and a portion was
mixed with 10 ml of scintillation mixture for dual isotope
scintillation counting. For total glycogen (as glucosyl units), the
digest was assayed for glucose by use of hexokinase and
glucose-6-phosphate dehydrogenase (2). In one experiment, a sample of isolated glycogen from 5 mg dry wt of heart containing 400 dpm tritium and 609 dpm 14C was added to 50 mg wet wt of
powdered heart (nonradioactive), and the glycogen isolation procedure
was repeated before the sample was subjected to digestion with
amyloglucosidase. The reisolated glycogen contained 369 dpm tritium
(93% recovery) and 518 dpm 14C (83% recovery). The result
indicated that selective detritiation during glycogen isolation was
negligible. Intracellular sugar phosphates were isolated from fresh 6%
PCA extracts of powdered heart (200 mg) following a published procedure
(10). The neutralized PCA extracts were added to 0.25 g of Dowex 2 × 8-100 (50-100 mesh) suspended in 4 ml of
0.17 M NH4OH, incubated for 1 h, and centrifuged, and
the supernatant was aspirated. The resin was washed four times with 4 ml of 0.17 M NH4OH and then treated with 1 ml of 1 M HCl. A
0.5-ml aliquot of the acid eluent containing hexose phosphates and
other phosphorylated sugars was added to 10 ml of scintillation mixture
to measure the 3H/14C ratio by dual isotope
scintillation counting. Values were corrected for blanks measured in
each heart by use of deproteinized extracts of coronary flow collected
near the end of the heart perfusions. Another portion of PCA extract of
the hearts was used for enzymatic assay of G-6-P with
glucose-6-phosphate dehydrogenase (2). For radiochemical
analysis, quench correction and simultaneous determination of
3H and 14C were performed by a routine
(spectral index analysis) supplied with the instrument (Packard 1900 TR).
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RESULTS |
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Contractile performance.
We conducted two sets of four heart perfusions, each of 1 h total
duration (30 min with one [14C]glucose isotopomer and
then 30 min with the other). The perfusions were identical in every
respect except for the order of addition of the two 14C
isotopomers of glucose. We included [1-14C]glucose first
and then [6-14C]glucose, or vice versa (crossover study
design). [5-3H]glucose was included throughout both sets.
Contractile performance, measured as hydraulic power (the product of
cardiac output and the mean pressure differential across the heart)
throughout the protocol for both sets, is shown in Fig.
1. The two sets were well matched for
performance, and all perfusions exhibited stable performance for the
duration of the protocol. Table 1 gives additional measures of
contractile activity: heart rate, coronary and aortic flow, cardiac
output, hydraulic power, maximum and minimum aortic pressure, developed
pressure, and rate × pressure products. Consistent with the crossover
design of the study, the contractile data given in Table 1 are grouped
according to which 14C isotopomer of glucose was present.
Again, the two groups were well matched for performance (Table 1). This
is necessary to obtain meaningful values for the C1/C6 ratios
calculated pairwise (i.e., rates from C1 and C6 measured
with the same heart).
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Metabolic activity for [1-14C]- and
[6-14C]glucose.
Figure 2 shows rates of oxidation
(14CO2 production), and Fig.
3 shows rates of release of
[14C]lactate plus [14C]pyruvate during the
time course of the perfusions with a given [14C]glucose
isotopomer. In each figure, values from [1-14C]glucose
and from [6-14C]glucose are shown side by side.
Consistent with the crossover design of the study, data were grouped
according to which 14C isotopomer of glucose was present,
as in Table 1. Values were calculated on the basis of the specific
activity of extracellular glucose and are reported in terms of glycosyl
(C6) units. In both cases (oxidation and 14C glycolytic
flux), hearts reached isotopic/metabolic steady state within the time
frame of perfusion with a given 14C isotopomer (30 min),
allowing us to calculate steady-state values for the C1/C6 ratios. Both
ratios were calculated from the average rate for the last three time
points (Fig. 2). The steady-state C1/C6 ratio was 1.06 ± 0.19 (n = 8) for 14CO2 and 1.00 ± 0.01 (n = 8) for [14C]lactate + [14C]pyruvate. In other words, rates of glucose oxidation
and unidirectional glycolytic 14C carbon flux from glucose
C1 and from C6 were indistinguishable.
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Glycolytic flux.
Figure 4 shows three measures of
glycolytic flux during the time course of perfusion. One of the lower
two plots shows net release of lactate + pyruvate (in C6 units)
based on concentration differences across the heart, measured
enzymatically. The separate contributions by lactate and pyruvate to
net flux averaged 2.2 and 0.4 µmol · min1 · g dry wt
1
(C6 units), respectively. The second of the two lower plots shows efflux of [14C]lactate + [14C]pyruvate
from [14C]glucose (the average of the two rates given in
Fig. 3). We did not report separate values for efflux of
[14C]lactate and [14C]pyruvate because the
radiochemical assay does not separate lactate from pyruvate.
Strictly speaking, efflux of [14C]lactate + [14C]pyruvate is a unidirectional process that may differ
from net flux of lactate + pyruvate measured enzymatically because
of exchange between intracellular (metabolically derived) lactate and
extracellular lactate (7, 8). The exchange contributes to
14C efflux but not to net flux. However, net flux and
efflux did not differ in the present study (Fig. 4). The third measure
of glycolytic flux shown in Fig. 4 is 3H2O
production from [5-3H]glucose. Like efflux of
[14C]lactate + [14C]pyruvate,
3H2O production measures a unidirectional
process. Because the 5-position is first detritiated at the level of a
triose (specifically, at triose phosphate isomerase, since this is an
equilibrium enzyme), 3H2O production from
[5-3H]glucose should measure glycolytic flux from glucose
to trioses, at least in the absence of confounding processes like
gluconeogenesis or transaldolase. The rate should include both glucose
oxidation and glycolytic flux appearing as lactate + pyruvate in
the coronary flow, although the contribution by glucose oxidation (0.1 µmol · min
1 · g dry wt
1,
Fig. 2) was negligible compared with total flux (2.6 µmol · min
1 · g dry wt
1).
Therefore, we would expect the rate for 3H2O
production to equal the other measures of glycolytic flux shown in Fig.
4 plus a very small contribution by glucose oxidation, shown in Fig. 2,
in the absence of confounding reactions. Contrary to this expectation,
the rate of 3H2O production did not exhibit
steady state. The rate increased progressively during the protocol and
reached 260% of the other measures of glycolytic flux after 30 min
(Fig. 4). We interpret this result as evidence for detritiation of
[5-3H]glucose in excess of true glycolytic flux because
of the presence of confounding reactions.
Tritium and 14C incorporation into
intracellular metabolites.
Table 2 shows tritium and 14C
radioactivity recovered in glycogen (top) and cellular
phosphorylated sugars (bottom), as well as the content of
glycogen and G-6-P in the two groups of heart perfusions.
The left column shows hearts perfused with
[1-14C]- and then [6-14C]glucose, and the
right column shows hearts perfused with
[6-14C]- and then [1-14C]glucose.
[1-14C]- and [6-14C]glucose were included
at the same specific activity, and [5-3H]glucose was
present at constant specific activity throughout the protocol. Using
values for 3H and 14C specific activity of
extracellular glucose given in the legend to Table 2, we calculated the
relative enrichment of glycogen and cellular sugar phosphates for the
[5-3H]glycosyl moiety compared with the
[14C]glycosyl moiety. We expected [14C]-
and [3H]glycosyl enrichments of glycogen to be the same.
Contrary to the expectation, the amount of new glycogen synthesis based
on incorporation of tritium was, on average, 36% of the value based on
incorporation of 14C
([3H]/[14C] glycosyl enrichment ratio of
0.33 ± 0.02 and 0.38 ± 0.01 in Table 2, P < 0.05 vs. 1). The result indicates that one or more intermediates
between glucose and glycogen were detritiated.
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DISCUSSION |
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The main finding of this study is a discrepancy between the apparent rate of glycolysis as determined by a popular isotopic tracer method and the true rate of glycolysis as determined by a second, independent method. Under present experimental conditions, the rate of detritiation continued to increase over 20 min, whereas the rate of glycolysis estimated from the rate of 14C release in lactate and pyruvate remained constant. We also observed a loss of ~60-70% of 3H relative to 14C in the glycosyl moieties of glycogen, suggesting a loss of tritium in the nonoxidative PPP.
These observations are the result of an exploration of the PPP in heart. Indeed, the nonoxidative PPP turned out to be a major source of 3H2O from [5-3H]glucose. Futile cycling between hexose and triose phosphate is unlikely to account for the loss of tritium, because the activity of fructose-1,6-bisphosphatase is negligible in the heart (24). Therefore, the discussion will focus on the PPP.
Evidence for nonoxidative PPP. Our evidence for nonoxidative PPP is qualitative and is based on the following two observations. First, we found that 3H2O production from [5-3H]glucose, unlike the other two measures of glycolytic flux, did not exhibit steady state and progressively overestimated the other measurements of glycolysis (net release of lactate + pyruvate and efflux of [14C]lactate + [14C]pyruvate from [14C]glucose). We interpret this result as detritiation in the absence of net glycolysis. Second, we found that the enrichment of glycogen (and the glycogen precursor pool of hexose phosphates, represented by cellular phosphorylated sugars) was considerably less when based on incorporation of tritium from [5-3H]glucose compared with incorporation of 14C from [1-14C]- or [6-14C]glucose. It was recognized early (13, 18) that this pattern of 3H2O production and tritium incorporation from [5-3H]glucose can occur in tissues like liver, which either exhibit the substrate cycle involving the enzyme fructose-1,6-bisphosphatase (which is undetectable in heart) and/or exhibit transaldolase activity, because both processes allow interconversion of glycolytic triose and hexose phosphates. The enzyme transaldolase (E.C. 2.2.1.2) detritiates C5 of fructose 6-phosphate. Transaldolase activity in rat heart has been reported to be 14-53% of the activity in liver (11, 14).
Because the glucose 5-position exchanges tritium with the solvent at the level of a triose, nonoxidative PPP-catalyzed isotopic exchange between glycolytic triose phosphates and hexose phosphates should be manifested as detritiation of glycolytic hexoses, which we measured in terms of the 3H/14C ratio of glycogen and intracellular glycogen precursors and in terms of 3H2O production in excess of what we believe to be true glycolytic flux. At least two nonoxidative pathways have been proposed, F-type and L-type, for fat and liver, respectively (F-type is the classical, textbook presentation). Severim and Stepanova (27) and Williams (reviewed in Ref. 29) provided evidence for an alternate (L-type) pathway that does not require transaldolase. The evidence for this pathway is controversial (20, 32). The precise reaction sequence that constitutes the (putative) L-type pathway is not completely resolved and would vary among tissues [different pathways are required to accommodate the difference in specificity of liver vs. muscle aldolase, for example (3)]. On the basis of the pattern of carbon redistribution from glucose C2 (or ribose C1) into hexose C1 and C3, it was concluded that the L-type, but not the F-type pathway, is active in the heart (15, 31). More recently, enzyme activities for both oxidative and nonoxidative pathways, including transaldolase, were found at moderate levels associated with heart sarcoplasmic reticulum (4), suggesting compartmentation of an active pathway.Appraisal of oxidative PPP in heart based on C1/C6 ratios. Interpretation of the C1/C6 ratio is a complicated issue. During glycolysis, C1 and C6 of glucose become pyruvate C3. In contrast, some of glucose C1 can be decarboxylated before reaching pyruvate, at the reaction catalyzed by 6-phosphogluconate dehydrogenase. In the presence of oxidative PPP, the overall rate of 14CO2 production from glucose C1 depends on the extent of carbon recycling. If there is extensive recycling of glucose-derived ribulose 5-P back into the glycolytic pathway (not unlikely for normal heart), then 14CO2 production from glucose C1 and C6 will be the same (14CO2 C1/C6 = 1) irrespective of the activity of oxidative PPP. Therefore, it is not possible to draw a definitive conclusion regarding the oxidative PPP from 14CO2 data alone. A different set of considerations applies to release of [14C]lactate + [14C]pyruvate. Oxidative PPP with carbon recycling decreases the specific activity of the glycolytic pathway from glucose C1 but not C6. Recycled carbon from [1-14C]glucose does not contribute to 14C in lactate or pyruvate at all. Stated differently, decreased specific activity of the glycolytic pathway from glucose C1 relative to C6 because of carbon recycling should be detectable from the C1/C6 ratio of [14C]lactate + [14C]pyruvate but not necessarily from 14CO2, because the decrease in glycolytic specific activity and the action of 6-phosphogluconate dehydrogenase oppose each other with respect to the overall rate of 14CO2 production from [1-14C]glucose. For this reason, we included measurements, lacking in previous studies of heart PPP, required to interpret the oxidative PPP rigorously based on C1/C6 ratios (i.e., we measured both 14CO2 and [14C]lactate + [14C]pyruvate).
The C1/C6 ratio of 14CO2 from heart was measured in two previous studies, with conflicting results. First, using isolated working hearts, Pfeiffer et al. (26) could not detect substrate flux through oxidative PPP. This study did not measure glucose oxidation directly; rather, it was based on a very indirect approach employing 14CO2 washout kinetics. The second study used isolated heart myocytes (5). The addition of competing substrates (pyruvate, octanoate, etc.) to myocytes increased the C1/C6 ratio of 14CO2 to a value >1. From this result, the authors concluded that there is an appreciable contribution of oxidative PPP to total glucose oxidation under conditions where mitochondrial glucose oxidation is suppressed by the other substrates (the usual situation in vivo). The authors' interpretation was criticized in a subsequent editorial reply by Katz (16), who reiterated the points, from the original work of Katz and Wood (19) and a more recent reappraisal by Larabee (21), that the C1/C6 ratio of 14CO2 is of limited quantitative value in the absence of additional measures of glucose utilization. We have provided the missing measurements in the present study. Larabee also noted potential problems associated with tissue metabolic heterogeneity, which probably apply to virtually any metabolic measurement in a heterogeneous system. This is not a moot issue in the case of the isolated working rat heart, because we previously found evidence for heterogeneity of carbohydrate metabolism for glucose and glycogen in this system (9). In any event, because we found that the C1/C6 ratio for both 14CO2 and for [14C]lactate + [14C]pyruvate is equal to 1 (the rates from [1-14C]glucose and [6-14C]glucose were indistinguishable), we suggest that flux for oxidative PPP, although not necessarily absent, is small compared with overall glucose oxidation. In summary, we have found 3H2O production from [5-3H]glucose in excess of glycolytic flux and decreased isotope incorporation into glycogen from [5-3H]glucose compared with [14C]glucose. We interpret the results as evidence for nonoxidative PPP in heart, consistent with transaldolase activity previously reported in heart sarcoplasmic reticulum. The steady-state rate of production of 14CO2 and of [14C]lactate + [14C]pyruvate was the same from [1-14C]glucose compared with [6-14C]glucose, suggesting that oxidative PPP is small compared with overall glucose oxidation. ![]() |
ACKNOWLEDGEMENTS |
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This study was supported in part by National Heart, Lung, and Blood Institute Grant RO1-43133. Dr. Cohen has been funded in part with federal funds from the US Department of Agriculture (USDA)/Agricultural Research Service under Cooperative Agreement No. 58-6250-6-001. The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement from the US government.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Taegtmeyer, Univ. of Texas-Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030 (E-mail: Heinrich.Taegtmeyer{at}uth.tmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 May 2000; accepted in final form 27 November 2000.
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