(Circulation. 1997;96:676-682.)
© 1997 American Heart Association, Inc.
Articles |
Glycogen Metabolism in the Aerobic Hypertrophied Rat Heart
From the Cardiovascular Research Laboratory, University of British Columbia, Vancouver (M.F.A., S.L.H., R.B.W., S.R.G., D.R.E.), and the Cardiovascular Disease Research Group, University of Alberta, Edmonton (G.D.L.), Canada.
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Abstract |
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Background Rates of glycolysis from exogenous glucose are accelerated in hypertrophied hearts. In this study, we determined whether alterations in the metabolism of glycogen, an endogenous storage form of glucose, also occur in hypertrophied hearts.
Methods and Results Rates of glycolysis ([3H]H2O production) and oxidation ([14C]CO2 production) from exogenous glucose and glycogen were measured in isolated working hearts from control and aortic-banded rats. Hearts in which glycogen was prelabeled with [5-3H]- or [U-14C]glucose were perfused with buffer containing 11 mmol/L [5-3H]- or [U-14C]glucose (different from the isotope used to prelabel glycogen), 0.4 mmol/L palmitate, 0.5 mmol/L lactate, and 100 µU/mL insulin. Rates of glycolysis from exogenous glucose were greater (3471±114 versus 2665±194 nmol glucose·min-1·g dry wt-1, P<.05, n=4 to 6, mean±SEM) and rates of exogenous glucose oxidation (445±36 versus 619±16 nmol glucose·min-1·g dry wt-1, P<.05, n=4 to 6) were lower in hypertrophied hearts than in control hearts. Rates of glycolysis and oxidation from glycogen were not different between hypertrophied and control hearts. A greater proportion of glycogen was oxidized (80% to 100%) than the proportion of exogenous glucose oxidized (13% to 24%) in both groups. Additionally, 10.5±1.4 and 12.3±1.0 µmol/g dry wt of glycogen was synthesized in hypertrophied and control hearts, respectively, indicating that simultaneous synthesis and degradation (ie, glycogen turnover) occurred in both groups.
Conclusions Thus, aerobic myocardial glycogen metabolism in the hypertrophied heart is similar to that observed in the normal heart even though exogenous glucose metabolism is altered in the hypertrophied heart.
Key Words: myocardium • hypertrophy • glycogen • metabolism
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Introduction |
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Cardiac hypertrophy is very common in our society, affecting 15% to 20% of adults in the general population1 and nearly 90% of hospitalized, adult cardiac patients2 in North America. Pressure-overload cardiac hypertrophy is a well-recognized risk factor for the development of sudden death, myocardial infarction, and congestive heart failure.3 4 Hypertrophied hearts are also more susceptible to injury during ischemia and reperfusion than normal hearts.5 6 7 8 Although the exact mechanisms responsible are not yet known, alterations in myocardial glucose metabolism have been implicated as an important factor contributing to the pathophysiology of cardiac hypertrophy.7 9 10 11 12 13 14 15
Although it is well accepted that alterations in glucose metabolism occur in the hypertrophied heart, the nature of the alterations in myocardial glucose metabolism is controversial. Some investigators suggest that hypertrophied hearts have an impaired ability to recruit glycolysis.10 11 In contrast, we have found that rates of glycolysis are accelerated in hypertrophied hearts compared with normal hearts.12 13 14 In addition, lactate production by hypertrophied hearts has also been shown to be increased during hypoxia7 and ischemia15 compared with normal hearts, suggesting that hypertrophied hearts, in fact, have an enhanced glycolytic capacity. In keeping with these observations are findings that the activity of a number of glycolytic enzymes measured in vitro is greater in hypertrophied hearts than in normal hearts16 and that isoenzymes of lactate dehydrogenase9 and creatine kinase17 shift toward more anaerobic, fetal forms. The controversy as to whether glucose metabolism is impaired or accelerated in the hypertrophied heart will most likely remain unresolved until overall glucose metabolism in the hypertrophied heart is directly measured. In this regard, determination of overall glucose metabolism in the hypertrophied heart must include assessment of the contribution of glycogen, the intracellular storage form of glucose in the myocardium.18
Glycogen had formerly been believed to make insignificant contributions to energy metabolism of the normal myocardium under nonstressful, aerobic conditions18 and was thought to contribute to energy production only if hearts were exposed to increased workloads,19 were perfused without insulin and/or fatty acid,19 20 or were subjected to hypoxic/ischemic conditions.18 Contrary to this generally held view concerning glycogen metabolism, we recently demonstrated that glycogen contributes significantly to energy production in aerobic, fatty acid–perfused normal hearts, accounting for up to 41% of ATP produced from glucose metabolism.21 Furthermore, we21 and others20 22 have found that glycogen is preferentially oxidized compared with exogenous glucose and undergoes significant turnover (ie, simultaneous synthesis and degradation) in normal, aerobic hearts. However, despite the importance of glycogen to glucose metabolism, it remains to be determined whether alterations in glycogen metabolism occur in hypertrophied hearts.
In the present study, we directly measured glycogen turnover and determined its contribution to aerobic glucose metabolism in isolated fatty acid–perfused working hypertrophied rat hearts by a technique recently developed in our laboratory.21
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Methods |
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Animal Model
A mild pressure-overload left ventricular hypertrophy was produced as previously described.12 Briefly, male Sprague-Dawley rats (3 weeks old; 50 to 70 g) were anesthetized with halothane (1.5% to 2%). The abdominal aorta was then exposed through an incision in the lateral abdominal wall. The abdominal aorta was isolated, and a minimally occlusive (0.4-mm diameter) metallic clip was placed on the suprarenal abdominal aorta. In control rats, the abdominal aorta was isolated but not clipped. The incision was then closed, and the animals were allowed to recover. Food and water were administered ad libitum. Experiments were performed on hearts excised from animals 8 weeks after surgery.
Experimental Protocol
Parallel series of pulse-chase perfusions were used to quantify
myocardial glucose and glycogen metabolism in isolated
working hearts, as previously described.21 Hearts from
anesthetized (halothane, 2% to 3%) control and aortic-banded
rats were initially perfused via the aorta in the Langendorff mode with
Krebs-Henseleit buffer, pH 7.4, containing 11 mmol/L glucose and
2.5 mmol/L calcium at a constant pressure of 60 mm Hg for 10
minutes. Excess tissue was removed and the left atrium cannulated
during this time. Myocardial glycogen was then depleted by a 30-minute
Langendorff perfusion with substrate-free, insulin-free Krebs-Henseleit
buffer.
After glycogen depletion, hearts were switched to the working mode and perfused with recirculating Krebs-Henseleit buffer at a preload of 11.5 mm Hg and an afterload of 80 mm Hg. Glycogen was resynthesized during a 60-minute pulse glycogen-labeling period with either [U-14C]glucose (series A, 29 104±2063 dpm/µmol glucose) or [5-3H]glucose (series B, 32 740±299 dpm/µmol glucose). During this period, buffer contained 1.2 mmol/L palmitate prebound to 3% albumin, 11 mmol/L [5-3H]- or [U-14C]glucose, 0.5 mmol/L lactate, and 100 µU/mL insulin. Insulin and high fatty acid were included to stimulate glycogen synthesis. After 60 minutes, hearts were switched to a recirculating perfusion buffer circuit that allowed determination of both exogenous and endogenous glucose utilization during the subsequent 40-minute chase perfusion. During this perfusion period, hearts were perfused with buffer containing 0.4 mmol/L palmitate prebound to 3% albumin, 11 mmol/L [5-3H]- or [U-14C]glucose, 0.5 mmol/L lactate, and 100 µU/mL insulin. The [5-3H]glucose (series A, 34 821±1951 dpm/µmol glucose) or [U-14C]glucose (series B, 23 944±1910 dpm/µmol glucose) used during the chase period was the opposite of the radiolabel used during glycogen labeling. During the working perfusion, heart rate and peak systolic pressure were recorded with a DIREC physiological recording system (Fine Science Tools Inc) with a pressure transducer (Viggo-Spectramed) in the aortic afterload line. All buffers were oxygenated by exposure to 95% O2/5% CO2 and maintained at 37°C.
Rates of glucose oxidation and glycolysis from [14C]- or [3H]-labeled glycogen and [U-14C]- or [5-3H]glucose were measured during the chase perfusion by quantitative measurement of rates of [14C]CO2 production (oxidation) and [3H]H2O production (glycolysis), respectively.21 In series A, we determined the rate of glucose oxidation ([14C]CO2 production) from endogenous glycogen during the chase period at the same time as rates of glycolysis ([3H]H2O production) from exogenous glucose were measured. In series B, we measured the rate of glycolysis from endogenous glycogen during the chase period while simultaneously measuring rates of glucose oxidation from exogenous glucose. In this manner, the contribution of endogenous glycogen and exogenous glucose to both oxidation and glycolysis was determined. Approximately 5% to 10% of labeled glucose used during the preceding glycogen labeling period was carried over into the chase perfusate. The degree of contamination was determined by measurement of the specific activity of the buffer at the beginning of the chase perfusion. Correction for the contribution of this exogenous source of glucose to rates of glycolysis or oxidation measured from glycogen was made with the appropriate mean rate of glycolysis or glucose oxidation from exogenous glucose during the chase period of the opposite series.21
Hearts were quickly frozen at various time points throughout the perfusion protocol by clamping with aluminum tongs cooled to the temperature of liquid nitrogen, including hearts frozen (1) immediately after removal from anesthetized rats, (2) at the end of the glycogen depletion period, (3) at the end of the glycogen labeling period, and (4) at the end of the chase perfusion.
Measurement of Glucose Oxidation and Glycolysis
Glucose oxidation and glycolysis were measured
simultaneously, as previously
described.12 21 23 24 25 Glucose oxidation rates were
measured by quantitative collection of
[14C]CO2 (liberated at the pyruvate
dehydrogenase reaction and in the citric acid cycle) released as a gas
and dissolved in the perfusate as
[14C]bicarbonate. Hearts were perfused in a closed system
in which the amount of gas entering the system was regulated. Gas
exiting the system was bubbled through a 1-mol/L hyamine hydroxide
solution that trapped the [14C]CO2 liberated
in the gaseous state. Perfusate samples containing
[14C]bicarbonate were collected throughout the perfusion
and stored under mineral oil to prevent the liberation of
[14C]CO2 until
analysis.12 23 25
Rates of glycolysis were determined by measurement of the rate of [3H]H2O production from [5-3H]glucose. As previously described,12 21 24 [3H]H2O was separated from [3H/14C]glucose in the perfusate samples by passing the samples of perfusate through columns containing Dowex 1-X4 anion exchange resin (200 to 400 mesh). Perfusate and hyamine samples were taken every 20 minutes of the pulse-labeling period and at 2, 5, 10, 15, 20, 30, and 40 minutes during the chase perfusion. Samples were ultimately placed in vials containing a scintillation cocktail and counted by standard double-isotope counting procedures.
Myocardial Metabolites
The frozen ventricular tissue was weighed and
powdered in a mortar and pestle cooled to the temperature of liquid
nitrogen. A portion of the tissue was then used to determine the ratio
of dry to wet weight. Myocardial glycogen was determined by measurement
of glucose obtained after digestion of the powdered
ventricular tissue with 30% KOH, ethanol precipitation,
and acid hydrolysis of glycogen.26 A portion of the
glycogen extract was also used for scintillation counting to determine
enrichment and specific activity of the glycogen pool by 3H
and 14C. The myocardial content of 14C-labeled
nonglycogen metabolites, such as glutamate, was determined in
neutralized acid extracts of myocardium from hearts frozen
at the end of the pulse and chase periods essentially as described by
Tempero and Kien.27
Statistical Analysis
Data are expressed as mean±SEM. Data were examined by
one-way, two-way, or three-way ANOVA with the following factors: (1)
control/hypertrophy hearts, (2)
baseline/depleted/pulse/pulse-chase series, and (3)
oxidation/glycolysis. For the pulse-chase series, the additional
repeating factor of pulse/chase was included. Because of the nature of
the experiment, the first two series, baseline and depleted, did not
contain the oxidation/glycolysis factor, and these variables were
examined in the final two series to determine whether an
oxidation/glycolysis effect existed. Interaction terms were examined
first in all models. When complex interactions were detected, the
design was reduced to simpler ANOVA models, and multiple comparisons
were applied. For comparisons of preferential oxidation, H+
production, and delta and percentage calculations, estimates
were calculated according to Mood et al.28 From these
estimates, 98.75% to 99.8% CIs (ranges accounting for multiple
comparisons allowing a .05 significance level) were determined, and
comparisons were made by examination of any overlap between groups.
Multiple variables and multiple comparisons were corrected for by
the sequential rejective Bonferroni test.29 A corrected
value of P<.05 was considered significant.
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Results |
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Heart Weight and Body Weight Data
Heart weight and body weight data are summarized in Table 1
. Heart weight and ratio of heart weight to body weight
of aortic-banded rats were 14% to 19% larger than those of
sham-operated control rats. Body weights of aortic-banded rats were not
significantly different from those of sham-operated control rats.
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Heart Function During Glycogen Labeling and Chase
Perfusions
Table 2 summarizes the heart rate, peak
systolic pressure, and heart ratexpeak systolic
pressure product data in working control and hypertrophied hearts
during both the pulse and chase perfusions. Although all
parameters tended to be lower in hypertrophied hearts than
in control hearts, no significant differences were observed between the
two groups at the end of either the pulse-labeling or chase periods.
Left ventricular function, quantified as either the heart
ratexpeak systolic pressure product or peak
systolic pressure, showed a small but significant decline
during the chase period in both groups. Heart rate at the end of the
pulse period was not different from that at the end of the chase
perfusion in either group.
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Rates of Glycolysis and Glucose Oxidation
During the chase period, accumulation of
[3H]H2O from glycolysis and
[14C]CO2 from oxidation of glucose was linear
between 10 and 40 minutes (data not shown). Mean rates of glycolysis
and oxidation from exogenous glucose and endogenous
glycogen, calculated from rates between 20 and 40 minutes of perfusion,
are summarized in Figs 1 and 2. Rates of glycolysis and oxidation from
exogenous glucose were significantly greater than those from glycogen
in both control and hypertrophied hearts. Rates of glycolysis from
exogenous glucose were significantly increased in hypertrophied hearts
(3471±114 nmol glucose·min-1·g dry
wt-1) compared with those in control hearts
(2665±194 nmol glucose·min-1·g dry
wt-1, P<.05), whereas rates of
glycolysis from glycogen were not significantly different between the
two groups (hypertrophy, 436±36 versus control, 410±14
nmol glucose·min-1·g dry
wt-1, P=NS) (Fig 1).
Oxidation of exogenous glucose (control, 619±16 versus
hypertrophy, 445±36 nmol
glucose·min-1·g dry
wt-1, P<.05) was significantly
greater in control hearts than in hypertrophied hearts, whereas
oxidation of endogenous glycogen (control, 481±35 versus
hypertrophy, 355±31 nmol
glucose·min-1·g dry
wt-1, P>.08) was not significantly
different between the two groups (Fig 2).
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Only a fraction of the exogenous glucose that passed through glycolysis was oxidized in both control and hypertrophied hearts. A significantly smaller fraction of the exogenous glucose that passed through glycolysis was oxidized in hypertrophied hearts (0.13±0.03) compared with control hearts (0.24±0.04, P<.05). The fraction of glucose from glycogen passing through glycolysis that was subsequently oxidized was significantly greater than the fraction of exogenous glucose passing through glycolysis that was oxidized in both control (1.18±0.19 versus 0.24±0.04, P<.05) and hypertrophied (0.85±0.24 versus 0.13±0.03, P<.05) hearts. Although the fraction of glucose from glycogen oxidized by hypertrophied hearts was less than that in control hearts (0.85±0.24 versus 1.18±0.19, respectively, P=NS), this difference was not significant. In other words, 80% to 100% of glucose from glycogen was oxidized, whereas only 13% to 24% of exogenous glucose was oxidized in both control and hypertrophied hearts.
Myocardial ATP Production and Tricarboxylic Acid Cycle
Activity
Rates of ATP production from myocardial glucose and
glycogen metabolism were calculated from mean rates of
glycolysis and oxidation assuming that 2 mol ATP was produced for each
mole of exogenous glucose passing through glycolysis, 3 mol ATP was
produced for each mole of glucose from glycogen passing through
glycolysis, and 36 mol ATP was produced for each mole of exogenous and
endogenous glucose oxidized to CO2. These
calculations assume that 100% of the NADH and FADH2
produced from the tricarboxylic acid (TCA) cycle is used to produce ATP
when oxidized in the electron transport chain. TCA cycle activity of
glucose metabolism was determined by calculating the rate
of acetyl coenzyme A (CoA) originating from glucose entering the TCA
cycle from oxidation of exogenous glucose and glycogen. A value of 2
mol acetyl CoA derived from each mole of glucose equivalents was used
to determine rates of acetyl CoA production.
The data summarized in Fig 3, left, show that
metabolism of glycogen contributed significantly to ATP
production from myocardial glucose use in both control and
hypertrophied hearts. In fact, 38% to 40% of ATP produced from
glycolysis and glucose oxidation by these hearts under these
experimental conditions was derived from glucose from glycogen (Fig 3, left). The vast majority of ATP from both exogenous and
endogenous sources was derived from mitochondrial oxidation
(Fig 3, left). Overall TCA-cycle acetyl CoA production from
glucose and glycogen metabolism was significantly lower in
hypertrophied hearts than control hearts (Fig 3, right).
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Myocardial Glycogen
The profile of changes occurring in the glycogen storage pool
during the course of the experiments is illustrated in Fig 4. Each group represents a series of hearts
frozen at selected time points throughout the protocol. Baseline
glycogen content tended to be greater in freshly frozen, unperfused
myocardium from hypertrophied hearts compared with control
hearts, but this difference did not reach statistical significance. As
expected, myocardial glycogen was significantly depleted by the
30-minute period of aerobic, substrate-free perfusion. Glycogen
decreased to 50.4±11.5% and 60.4±7.3% of baseline values in
hypertrophied and control hearts, respectively. During the subsequent
glycogen-labeling period, substantial resynthesis of glycogen occurred,
with levels of myocardial glycogen returning to baseline values in both
groups. There was no significant difference in myocardial glycogen
content between hearts frozen at the end of the chase-perfusion period
and hearts frozen at the end of the pulse-labeling period. Specific
activity of glycogen at the end of the chase period, corrected for
newly synthesized glycogen, (control, 10 721±1700 dpm/µmol;
hypertrophy, 11 258±2690 dpm/µmol) was also not
different from that at the end of the pulse perfusion (control,
10 421±2063 dpm/µmol, P=NS; hypertrophy,
11 258 dpm/µmol, P=NS) in either group, suggesting that
the degradation of glycogen was substantially random.
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That myocardial glycogen content at the end of the chase
perfusion was not different from that at the end of the pulse-labeling
period (Fig 4
), even though glycogenolysis was observed in both groups
during the chase period (Fig 1), indicates that glycogen synthesis also
occurred in control and hypertrophied hearts during this time.
Synthesis of glycogen was confirmed by the incorporation of labeled
glucose into glycogen during the chase period in both control and
hypertrophied hearts. On the basis of glycogen specific activities at
the end of the chase period, 12.3±1.0 and 10.5±1.4 µmol/g dry
wt glycogen were synthesized during the chase perfusion in control and
hypertrophied hearts, respectively. Differences in the amount of
glycogen synthesized from [3H]glucose and
[14C]glucose were not observed. During the chase period,
17.4±1.4 and 16.4±0.5 µmol/g dry wt glycogen were degraded in
control and hypertrophied hearts, respectively, as determined from
rates of glycolysis from glycogen. Simultaneous glycogen
degradation and synthesis during the chase period indicates that
glycogen turnover was occurring in control and hypertrophied hearts, a
finding not previously described in hypertrophied hearts.
In frozen myocardium from control (n=5) and hypertrophied (n=5) hearts exposed to 60 minutes of [14C]glucose during the pulse perfusion, the amount of 14C within the nonglycogen metabolite pool was 2.75±0.29 µmol glucose equivalents/g dry wt in hypertrophied hearts and 1.30±0.19 µmol glucose equivalents/g dry wt in control hearts (P<.05). The amount of 14C in this pool in control hearts frozen at the end of the chase perfusion (0.97±0.27 µmol glucose equivalents/g dry wt, n=3, P=NS) was not significantly different from that in control hearts frozen at the end of the pulse perfusion. However, 14C in this metabolite pool in hypertrophied hearts frozen at the end of the chase period (1.06±0.24 µmol glucose equivalents/g dry wt, n=3) was lower than that in hypertrophied hearts frozen at the end of the pulse-labeling period (P<.05).
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Discussion |
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In the present study, we found that glycogen metabolism in the hypertrophied heart was similar to that in the normal heart, even though glycolysis of exogenous glucose was increased and exogenous glucose oxidation was decreased in the hypertrophied heart compared with the normal heart. Despite the acceleration of glycolysis of exogenous glucose, absolute rates of aerobic glycogenolysis, as determined from rates of glycolysis from glycogen, were not statistically different between the two groups (Fig 1
). Taken together, these findings indicate that glycolysis is not
impaired in the aerobic hypertrophied heart. Rather, they are
consistent with the hypothesis that the hypertrophied heart has
an enhanced glycolytic capacity7 9 26 and provide support
for the concept that exogenous glucose and glycogen are metabolized by
separate pathways in the heart.21 22 In addition, the fact
that rates of glycogenolysis were not accelerated in hypertrophied
hearts suggests that the hypertrophied hearts were adequately perfused
and well-oxygenated under these perfusion conditions.
The observation that rates of aerobic glycolysis from exogenous
glucose were accelerated in hypertrophied hearts compared with control
hearts without an accompanying acceleration of glucose oxidation rates
(Figs 1 and 2) confirms our previous findings12 13 14 but is
inconsistent with the finding of net chemical extraction of
lactate by aerobic myocardium in vivo.30 This
discrepancy may be a consequence of fundamental differences between in
vitro and in vivo settings. However, studies in normal
humans30 and anesthetized dogs31
using isotopic analysis have shown that only a fraction
(
20%) of glucose taken up by the heart is immediately
oxidized30 and that extraction and release of lactate by
aerobic myocardium occur
simultaneously.31 These findings support and
are consistent with the concept that the uncoupling of
glycolysis and glucose oxidation observed in vitro also occurs in vivo.
These findings also indicate that although direct extrapolation of
metabolic data obtained from isolated heart preparations to
the in vivo setting may not be possible, the in vitro results do appear
to provide a realistic representation of the pattern of
substrate utilization that exists in vivo. The present studies were
not designed to characterize the mechanism(s) responsible for the
acceleration of aerobic glycolysis from exogenous glucose in
hypertrophied hearts. A number of possible mechanisms exist that may
explain this phenomenon. These include the upregulation of key enzymes
of glycolysis9 16 with alterations in their isoenzyme
composition,9 17 alterations in glucose
transport,32 33 and/or an attempt to normalize ATP
production.12
Our data also indicate that metabolism of glucose from
glycogen contributes significantly to ATP production in
aerobically perfused hypertrophied hearts. Nearly 40% of ATP produced
from glycolysis and glucose oxidation was found to be derived from
glycogen in these hearts (Fig 3A). The vast majority of ATP generated
from the overall metabolism of glucose was produced by
mitochondrial oxidation in both the control and hypertrophied hearts
(Fig 3A). Although the fate of glucose in the hypertrophied hearts was
comparable to that observed in the control hearts, the fraction of
exogenous glucose passing through glycolysis that was subsequently
oxidized was less in hypertrophied hearts than in control hearts (Figs 1 and 2). As previously observed in normal hearts,21 22
the metabolic fate of exogenous glucose differed
significantly from that of glucose from glycogen in hypertrophied
hearts, with glycogen being preferentially oxidized (Figs 1 and 2).
Less than 15% of exogenous glucose passing through glycolysis was
oxidized, whereas 80% of glucose from glycogen was oxidized in the
hypertrophied heart (Figs 1 and 2). It is important to note that a
number of endogenous nonglycogen metabolites, including
glutamate, would have been labeled with 14C during the
pulse-labeling period, the oxidation of which may have contributed to
the [14C]CO2 produced during the chase
perfusion. Glutamate and other nonglycogen substrates have relatively
rapid turnover rates, achieving a steady state within 20 to 30 minutes
after exposure to radiolabeled substrates.34 35 Since our
measurements of substrate utilization rely on steady-state conditions
and occurred between 20 and 40 minutes of the chase perfusion, it is
likely that the majority of 14C in this metabolite pool
that originated from 14C-labeled exogenous glucose during
the pulse-perfusion period had disappeared by this time. Furthermore,
determination of 14C activity in the nonglucose fraction of
neutralized acid extracts of myocardium from hearts at the
end of the pulse and chase perfusions showed that a maximum of 19% of
the endogenously produced
[14C]CO2 could have potentially been derived
from this pool of metabolites. When the
[14C]CO2 from nonglycogen sources is taken
into account, the proportion of glucose from glycogen that was oxidized
is still fourfold to fivefold greater than the proportion of exogenous
glucose oxidized. Thus, the oxidation of endogenous
nonglycogen metabolites cannot account for the preferential oxidation
of glycogen observed in this study. The fact that slightly more glucose
from glycogen was oxidized in control hearts than passed through
glycolysis may have resulted from a combination of preferential
oxidation of glycogen and the small pool of 14C-labeled
metabolites that were also oxidized. Identification and
characterization of the cellular and subcellular mechanisms responsible
for the dramatic differences in the fate of exogenous glucose compared
with endogenous glycogen are beyond the scope of the
present study and remain to be elucidated.
Glycolysis, when uncoupled from glucose oxidation, is a major source of H+ production in the myocardium,36 37 because each molecule of glucose that passes through glycolysis that is not oxidized yields 2 molecules of H+ by way of ATP hydrolysis.36 37 Conversely, a molecule passing through glycolysis that is subsequently oxidized produces no H+.36 37 As in our previous studies,38 we found that rates of H+ production from exogenous glucose were significantly greater in hypertrophied hearts (6055±584 nmol glucose·min-1·g dry wt-1) than those in control hearts (4092±780 nmol glucose·min-1·g dry wt-1, P<.05). Since the majority of glucose derived from myocardial glycogen was metabolized through the oxidative pathway, H+ production from glycogen metabolism was minimal in both groups. As a result, the vast majority of H+ production from myocardial glucose utilization in both control and hypertrophied hearts arose from the metabolism of exogenous glucose, with glycogen metabolism contributing <4% to 5% of the total H+ production from overall glucose metabolism. Thus, the preferential oxidation of glucose from glycogen renders glycogen a particularly good energy substrate because not only is ATP production maximized, but H+ production is also minimized for each glucose molecule metabolized. Relatively speaking, we calculate that the ATP yield from glycogen is threefold to fourfold higher for each glucose molecule metabolized from glycogen than exogenous glucose. In addition, the relative lack of H+ production from aerobic metabolism of glycogen potentially has great pathophysiological significance, because production and/or accumulation of H+ in myocardium may contribute to contractile dysfunction in normal36 39 and pathological hearts.13 14
An interesting observation from this study is that myocardial
TCA-cycle acetyl CoA production from overall glucose oxidation
was significantly lower in hypertrophied hearts than control hearts
(1.6±0.9 versus 2.2±0.8
µmol·min-1·g dry
wt-1, P<.05, respectively) (Fig 3B). The decreased TCA-cycle activity in the hypertrophied hearts was
due to a combination of a significant reduction in TCA-cycle acetyl CoA
production from metabolism of exogenous glucose and
a small but insignificant reduction in TCA-cycle acetyl CoA
production from metabolism of
endogenous glycogen in hypertrophied hearts compared with
control hearts. A number of mechanism(s) may be responsible for the
reduction in TCA-cycle acetyl CoA production from glucose and
glycogen metabolism observed in the hypertrophied hearts.
First, an impairment in the production of TCA-cycle acetyl CoA
from the metabolism of exogenous and endogenous
glucose sources per se is one possibility. Second, reduction in
TCA-cycle activity in the hypertrophied hearts may occur because these
hearts perform less cardiac "work" than the control hearts. For
example, although left ventricular function was not
significantly different between the two groups (Table 2), TCA-cycle
activity from metabolism of either glucose or glycogen was
not different between the two groups when heart function was taken into
account (data not shown). This finding suggests that the reduction in
TCA-cycle activity may be appropriate for the cardiac work performed.
Clearly, further studies are required to determine the mechanism(s)
responsible for the reduced TCA-cycle acetyl CoA production
from glucose and glycogen metabolism observed in
hypertrophied hearts in this study.
Previous investigations by ourselves21 and
others20 have shown that substantial glycogen turnover
(ie, simultaneous synthesis and degradation) occurs in the
aerobic normal heart in the presence of net glycogen loss. Conversely,
Goodwin and colleagues20 suggest that glycogen turnover is
minimal when glycogen synthesis predominates in isolated working rat
hearts exposed to supraphysiological levels of
insulin and glucose. In the present study, we made two novel
observations regarding myocardial glycogen turnover in normal and
hypertrophied hearts. First, we demonstrate that substantial glycogen
turnover occurs in the aerobic hypertrophied heart, similar to that
seen in the normal heart. The extent of glycogen turnover observed in
aerobic myocardium may seem paradoxical, given the fact
that glycogen synthesis is energetically costly (one ATP is used for
each glucose moiety incorporated into glycogen18 ).
However, the substantial turnover observed is not surprising, because
the cost of glycogen synthesis becomes insignificant relative to the
energy produced from glycogen utilization because of the preferential
oxidation of glycogen. Second, we observed that substantial glycogen
turnover occurred in both normal and hypertrophied hearts exposed to
conditions in which myocardial glycogen content did not appear to
change over time (Fig 4). The lack of a difference in glycogen content
between hearts frozen at the end of the chase perfusion and those
frozen at the end of the pulse-labeling period (Fig 4) suggests that
net glycogenolysis did not occur under these experimental conditions.
It is important to note that the methodology used in these experiments
makes it impossible to know the actual glycogen content at the end of
the pulse perfusion of the hearts studied during the chase perfusion.
Comparison of glucose incorporation into glycogen (control, 12.3±1.0
and hypertrophy, 10.5±1.4 µmol/g dry wt) and
glycogen degradation determined from steady-state rates of glycolysis
(control, 17.4±1.4 and hypertrophy, 16.4±0.5
µmol/g dry wt) during the chase perfusion supports the view that
little if any net glycogenolysis occurred, particularly when one
considers that the extent of glycogen incorporation during the chase
perfusion was underestimated because of simultaneous
degradation. However, if glycogen was degraded in a random as opposed
to an ordered manner, as suggested by data in this and previous
studies,20 21 the extent of glycogen degradation was
substantially greater than that calculated above (control, 26.9±3.2
and hypertrophy, 29.1±2.1 µmol/g dry wt). Thus,
although some of our observations are consistent with the
concept that minimal to no net glycogen degradation occurred in these
experiments, other data suggest that net glycogenolysis did, in fact,
occur during the chase perfusion. Further studies are therefore
required to determine the extent of glycogen turnover when rates of
glycogenolysis and glycogen synthesis are unquestionably
equivalent.
In summary, glycogen turnover (ie, simultaneous synthesis and degradation) occurs and glycogen metabolism contributes significantly to myocardial ATP production in the hypertrophied heart, as observed in the normal heart. Glucose from glycogen is preferentially oxidized in both normal and hypertrophied hearts, which maximizes ATP production and minimizes H+ production and renders it a particularly good energy substrate. Our results also demonstrate that glycolysis is not impaired in the hypertrophied heart, although overall glucose oxidation may be impaired. Thus, aerobic myocardial glycogen metabolism in the hypertrophied heart is similar to that observed in the normal heart despite the significant differences that exist in the metabolism of exogenous glucose between hypertrophied and normal hearts.
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Acknowledgments |
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This study was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of British Columbia and Yukon. Dr Allard is a Research Scholar of the Heart and Stroke Foundation of Canada. Sarah Henning is a graduate student trainee of the Heart and Stroke Foundation of Canada. Dr Lopaschuk is a Medical Research Council of Canada Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. The authors thank Lorraine Verburgt for help with statistical analysis and Dr Gregory Bondy for assistance with analysis of the radiolabeled nonglycogen pool in the myocardium.
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Footnotes |
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Reprint requests to Michael F. Allard, BSc, MD, Department of Pathology and Laboratory Medicine, University of British Columbia, Cardiovascular Research Laboratory, 1081 Burrard St, Vancouver, BC, Canada V6Z 1Y6.
Received May 20, 1996; revision received December 18, 1996; accepted January 15, 1997.
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