(Circulation. 1996;93:1549-1555.)
© 1996 American Heart Association, Inc.
Articles |
Contribution of Glycogen to Aerobic Myocardial Glucose Utilization
From the Cardiovascular Research Laboratory, University of British Columbia, St Paul's Hospital, Vancouver (S.L.H., R.B.W., M.F.A.), and the Cardiovascular Disease Research Group, University of Alberta, Edmonton (B.O.S., G.D.L.), Canada.
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Abstract |
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Background We determined glycogen turnover and the contribution of glycogen as a source of glucose for aerobic myocardial glycolysis and glucose oxidation in parallel series of isolated, working rat hearts subjected to pulse-chase perfusions.
Methods and Results Myocardial glycogen of isolated, working rat hearts was radiolabeled, after 30 minutes of substrate-free glycogen depletion, by perfusion for 60 minutes with buffer designed to stimulate resynthesis of glycogen (1.2 mmol/L palmitate, 11 mmol/L [U-14C]- or [5-3H]-glucose, 0.5 mmol/L lactate, and 100 µU/mL insulin). Rates of glucose oxidation (14CO2 production) and glycolysis (3H2O production) were then measured by perfusing the hearts for 40 minutes with buffer designed to simulate physiological conditions (0.4 mmol/L palmitate, 0.5 mmol/L lactate, 11 mmol/L [5-3H]- or [U-14C]-glucose, 100 µU/mL insulin) containing radiolabeled glucose different from that used during resynthesis. During the chase perfusion, rates of glycolysis and glucose oxidation from exogenous glucose were significantly greater than those from endogenous glycogen. Nevertheless, glycogen contributed significantly to myocardial energy production (41% of the overall ATP produced from glucose), and a significantly greater fraction of the glucose from glycogen that passed through glycolysis was oxidized (>50%) compared with the fraction oxidized from exogenous glucose (<20%, P<.05). Myocardial glycogen was simultaneously synthesized and degraded (ie, glycogen turnover) during the chase perfusion, despite net glycogenolysis. Furthermore, enrichment of labeled glucose in glycogen at the end of the chase perfusion, when corrected for newly synthesized glycogen, did not differ from that at the end of the labeling period.
Conclusions Thus, glycogen contributes significantly to aerobic myocardial glucose use under these experimental conditions, and the glucose derived from glycogen is oxidized preferentially compared with exogenous glucose. Additionally, substantial myocardial glycogen turnover occurs, and the manner in which glycogen is degraded does not fit the ordered hypothesis of "last glucose on, first glucose off."
Key Words: myocardium • glucose • metabolism • glycogen
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Introduction |
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Normal myocardium uses a variety of energy substrates to meet its large energy requirements. Fatty acids, predominantly oleic and palmitic acid, are the preferred energy source of aerobically perfused normal hearts.1 2 Oxidation of fatty acids normally supplies 60% to 70% of the heart's energy needs but may provide >90% of total ATP production under certain conditions.2 The heart also contains endogenous triacylglycerol stores that can serve as a source of fatty acids for oxidative metabolism2 and can provide from 11% to 50% of the heart's energy requirements (depending on extracellular fatty acid supply).2
Exogenous glucose and lactate are the other major energy substrates of the myocardium.1 2 Glucose is metabolized by glycolysis and mitochondrial oxidation,1 2 whereas lactate may be oxidized as well as produced by the myocardium.3 As with fatty acids, exogenous glucose can also be incorporated into an endogenous storage form, glycogen.1 Since its discovery, detailed study and characterization of glycogen metabolism have led to the development of many important concepts of enzyme regulation and mechanisms of hormone action.4 5 Despite this extensive research activity, a number of key issues regarding myocardial glycogen metabolism remain to be resolved.
One key question is what fraction of glucose taken up by the myocardium directly enters glycolysis and what fraction is converted into its storage form, glycogen. Studies are divided as to the extent to which glucose taken up by the heart is converted into glycogen. For example, studies in humans have suggested that only 20% of the glucose extracted by the myocardium is immediately oxidized and only 13% is metabolized to lactate.6 Although not measured directly, it was estimated that 60% to 70% of the glucose taken up by the heart was incorporated into glycogen.6 However, direct measurements suggest that only 4.5% to 7.5% of glucose extracted by isolated, working rat hearts is used to synthesize glycogen.7 The explanation for the discrepancy between these two studies is not immediately apparent. Although differences in species and preparation may contribute to this discrepancy, the fact that the isolated rat hearts were perfused with buffer containing carbohydrate but no fatty acid may be an additional factor.7 It is well known that fatty acids significantly affect myocardial glucose and glycogen metabolism by reducing glucose use and favoring glycogen synthesis.1 8
A second unresolved issue is whether exogenous glucose and glucose from glycogen share a similar fate in the heart. When used by the myocardium, glucose from glycogen generally has been believed to be metabolized like its exogenous counterpart. Investigations of glucose use by vascular smooth muscle suggest that significant differences exist with respect to the fates of exogenous glucose and glucose from glycogen.9 10 In studies reported by Lynch and Paul,9 it was found that glycogen use correlated with oxidative phosphorylation and contractile activity, whereas glucose uptake and aerobic glycolysis were associated with release of lactate, suggesting that exogenous and endogenous glucose were metabolized by separate pathways. Since direct measurements of the contribution of glycogen to both glycolytic and oxidative pathways have not been performed in the heart, it is not known whether myocardial utilization of glucose from glycogen differs from that of exogenous glucose.
A third unresolved issue is whether glycogen is actively "turning over" in the heart under normal circumstances. The pathways of glycogen synthesis and glycogenolysis are separate,1 involving different enzymes that are regulated in a reciprocal manner such that glycogenolysis is inhibited when synthesis is stimulated and vice versa.1 The completeness of this reciprocal regulation and the extent to which simultaneous synthesis and degradation of glycogen (ie, glycogen turnover) occurs in aerobic myocardium, however, remain to be fully characterized. As occurs with endogenous triacylglycerol stores,2 Goodwin et al7 demonstrated that significant glycogen turnover occurs in aerobic, isolated, working rat hearts perfused in the absence of insulin and fatty acids. On the other hand, no significant glycogen turnover was detectable in hearts of insulin-treated rats studied in vivo by nuclear magnetic resonance (NMR) imaging11 or in isolated, working rat hearts perfused with buffer containing pharmacological concentrations of insulin but no fatty acids.7 Whether glycogen turnover occurs in the presence of fatty acids and more physiological concentrations of insulin remains to be determined.
Finally, it is also not clear whether glycogen is synthesized and degraded in an ordered or a random manner. Glycogen is a very large molecule with a branched structure that exists in multiple tiers.12 13 Brainard et al14 used [13C]-glucose and NMR methodology to study glycogen metabolism in isolated, Langendorff-perfused guinea pig hearts. They concluded that glycogen synthesis and mobilization was "mostly" ordered (ie, first glucose molecule added to glycogen was the last to be mobilized). By this reasoning, the last glucose molecule added to glycogen should also be the first off. However, Goodwin et al7 have recently provided data from isolated, perfused rat hearts that do not support the ordered concept and suggest that glycogenolysis is substantially random.
The present study was performed to address the above controversies in myocardial glycogen metabolism. The overall objectives were (1) to determine the contribution of glycogen to aerobic glucose use; (2) to determine whether glucose from glycogen is oxidized preferentially, compared with exogenous glucose; (3) to measure the extent of glycogen turnover; and (4) to assess the degree of orderedness of glycogen degradation in the heart. To accomplish these objectives, we studied parallel series of pulse-chase perfusions in isolated, fatty acid–perfused, working rat hearts in which glycogen was labeled with either [3H]- or [14C]-glucose and that were subsequently perfused with buffer containing [3H]- or [14C]-glucose different from that present in glycogen.
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Methods |
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Materials
Bovine albumin (fraction V) was obtained from Boehringer-Mannheim Canada, beef-pork insulin was obtained from Eli Lilly, and radiochemicals were obtained from NEN-Du Pont. Dowex 1-X4 anion exchange resin (200 to 400 mesh) was obtained from Bio-Rad. All other chemicals were obtained from Sigma Chemical Co or BDH and were of analytical grade.
Isolated Heart Preparation
Hearts from fed, male Sprague-Dawley rats (weight, 350
to 450 g) anesthetized with halothane (2% to 3%) were excised
and placed in ice-cold buffer, and the aorta was quickly
cannulated.15 16 Hearts were initially perfused via the
aorta in the Langendorff mode with oxygenated
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. During the initial
10-minute retrograde aortic perfusion, hearts were trimmed of excess
tissue and the left atrium was cannulated. The hearts were switched to
the working mode after an additional 30 minutes of aerobic,
substrate-free, retrograde aortic perfusion (see below) and were
perfused with recirculating buffer at a left atrial preload of 11.5 mm
Hg and an aortic afterload of 80 mm Hg. During the working mode, heart
rate and peak systolic pressure were recorded by use of a
DIREC physiological recording system (Fine
Science Tools Inc) with a pressure transducer (Viggo-Spectramed) in the
aortic afterload line.
Perfusion Protocol
Parallel series of pulse-chase perfusions (Fig 1
) were used to measure myocardial glucose use and
glycogen metabolism in the isolated, perfused hearts.
Myocardial glycogen was initially depleted by a 30-minute Langendorff
perfusion with oxygenated substrate-free,
insulin-free buffer. Hearts were then switched to the working mode
and glycogen was resynthesized during a 60-minute pulse
glycogen-labeling period with either [U-14C]-glucose
(series A, 29 104±2063 disintegrations per minute per micromole
(dpm/µmol) of glucose, n=6) or [5-3H]-glucose (series
B, 32 740±299 dpm/µmol glucose, n=7). During this period, buffer
contained 1.2 mmol/L palmitate, 11 mmol/L [5-3H]- or
[U-14C]-glucose, 0.5 mmol/L lactate, and 100 µU/mL
insulin. Insulin and high fatty acid concentrations 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, 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, n=6) or [U-14C]-glucose (series B, 23 944±1910
dpm/µmol glucose, n=7) used during the chase period was opposite to
the radiolabel used during glycogen labeling.
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Rates of glucose oxidation and glycolysis from [14C]- or
[3H]-labeled glycogen and [U-14C]- or
[5-3H]-glucose were measured simultaneously
during the chase perfusion by quantitatively measuring rates of
14CO2 production (oxidation) and
3H2O production (glycolysis),
respectively. In one series (series A, Fig 1
), we determined the rate
of glucose oxidation (14CO2 production)
from endogenous glycogen during the chase period at the
same time as rates of glycolysis (3H2O
production) from exogenous glucose were measured. In the second
series (series B, Fig 1), 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 glucose
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 measuring 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 using the appropriate mean
rate of glycolysis or glucose oxidation from exogenous glucose during
the chase period of the opposite series.
A series of hearts were quick-frozen at various time points throughout the protocol by clamping with aluminum tongs cooled to the temperature of liquid nitrogen. This included hearts frozen immediately after removal from anesthetized rats, at the end of the glycogen depletion period, at the end of the glycogen labeling period, and at the end of the chase perfusion. Relative rates of glycogen synthesis and glycogenolysis during the chase period were determined by comparing myocardial glycogen content and specific activity in hearts that were frozen either at the end of the pulse perfusion or at the end of the chase perfusion.
All buffers were oxygenated by exposure to 95% O2/5% CO2 and maintained at 37°C. The palmitate was prebound to the 3% albumin as described previously.2 15 16
Measurement of Glucose Oxidation and Glycolysis
Glucose Oxidation
Steady state rates of oxidation of glucose were determined by
measurement of the rate of 14CO2
production, as described previously.15 16 17 During
the chase perfusion, hearts were perfused in a closed system that
allowed quantitative collection of gaseous and perfusate
14CO2 originating from either exogenous
14C-glucose (series B) or endogenous
14C-glucose (series A). Perfusate and gaseous
samples were collected at 2, 5, and 10 minutes and then at 10-minute
intervals throughout the 40-minute chase period. The
14CO2 liberated in the gaseous state was
trapped in a 1 mol/L hyamine hydroxide solution in the gas outlet line.
Samples of this solution were injected directly into scintillation
liquid for counting. Perfusate samples were immediately
injected below a 1 mL volume of mineral oil to prevent liberation of
perfusate 14CO2. The
14CO2 from the perfusate was
subsequently extracted by injection of 1 mL of perfusate into a
sealed, metabolic flask containing 9N
H2SO4 and 400 µL of 1 mol/L hyamine hydroxide
in a suspended center well. The flasks were gently shaken for 1 hour to
release the 14CO2 present as
[14C]-bicarbonate. The center wells were then removed and
counted in scintillation liquid by use of standard counting procedures.
Glucose oxidation rates were expressed as nanomoles of glucose
oxidized per minute per gram of dry heart weight (dry wt).
Glycolysis
Quantitative 3H2O production was
used to measure steady state glycolytic rates.
3H2O is liberated from
[5-3H]-glucose at the triose phosphate isomerase and
enolase steps of glycolysis. Samples for 3H2O
originating from either exogenous 3H-glucose (series A) or
endogenous 3H-glucose (series B) were collected
during the chase period at the same time intervals as the
14CO2. 3H2O was
separated from [3H]-glucose and
[14C]-glucose by use of columns containing Dowex 1-X 4
anion exchange resin (200 to 400 mesh) suspended in 0.4 mol/L potassium
tetraborate.2 15 The Dowex in the columns was extensively
washed with H2O before use. A 0.2 mL volume of
perfusate was added to the column and eluted into scintillation
vials with 0.8 mL H2O. The samples were then subjected to
standard double-isotope counting procedures. The Dowex columns were
found to retain 98% to 99.6% of the total [3H]-glucose
and [14C]-glucose present in the perfusate.
The 3H2O was corrected for the small proportion
of [3H]-glucose that passed through the column. This
could be accomplished because an equal amount of
[14C]-glucose also passed through the column and could be
used as an internal standard for the degree of
[3H]-glucose contamination in the
3H2O sample. Correction was also made for the
degree of spillover of [14C] into the
[3H] counting window by measuring this degree of
spillover with standards containing [14C]-glucose.
Glycolytic rates were expressed as nanomoles of glucose metabolized
per minute per gram of dry heart weight.
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 in the
powdered ventricular tissue as glucose after digestion with
30% KOH, ethanol precipitation, and acid hydrolysis of
glycogen.18 A portion of the glycogen extract was used for
scintillation counting to determine enrichment and specific activity of
the glycogen pool by [3H] and [14C].
Statistical Analysis
Data are expressed as mean±SEM. Heart function was
analyzed by repeated measures ANOVA with one grouping factor
and one repeating factor. Rates of ATP production and initial
and final labeled glycogen were analyzed by two-way ANOVA.
Rates of glycolysis and glucose oxidation were compared by two-way
ANOVA after log transformation. Enzymatically determined glycogen was
analyzed by one-way ANOVA, whereas glycogen enrichment was
compared by independent Student's t tests. Ratios of
glucose oxidation and glycolysis were calculated as described by Mood
et al19 and compared by examining 95% CIs. A sequential
rejective Bonferroni procedure was applied to all tests to correct for
multiple tests and/or multiple comparisons. A corrected value of
P<.05 was considered significant.
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Results |
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Heart Function During Glycogen Labeling and Chase Perfusions
A number of initial experiments were performed in an attempt to optimize conditions for glycogen depletion in the heart without compromising heart function during the chase. The protocols included the use of hypoxic perfusion, inclusion of glucagon in aerobic and hypoxic perfusate, and substrate-free perfusion of the aerobic working heart with and without electric pacing. Although many of these protocols successfully depleted myocardial glycogen stores, heart function almost always was also compromised. A protocol found to preserve heart function while substantially depleting glycogen involved a 30-minute Langendorff perfusion in the absence of substrate and insulin. Table 1
summarizes the heart rate, peak
systolic pressure, and heart rate multiplied by peak
systolic pressure product data in working hearts perfused
during pulse and chase perfusions. No significant differences were
observed between groups at the end of either the pulse-labeling or
chase periods. Left ventricular function, calculated as
either the heart rate multiplied by peak systolic pressure
product or peak systolic pressure, showed a small but
significant decline during the chase period. 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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Myocardial Glycogen
The profile of changes that occurred in the glycogen storage pool
during the course of the experiments is illustrated in Fig 2. Each group represents a series of hearts
frozen at selected time points throughout the protocol. As can be seen
from Fig 2, myocardial glycogen was significantly depleted by the
30-minute period of aerobic substrate-free perfusion to 39.5±9.1%
of baseline values. During the subsequent glycogen labeling period, a
substantial resynthesis of glycogen occurred, with levels of myocardial
glycogen returning to baseline values. During the chase-perfusion
period, when glucose utilization from exogenous glucose and
endogenous glycogen was measured, a small but significant
decrease in net glycogen levels occurred, with myocardial glycogen
dropping to 70.7±17.6% of values at the end of pulse labeling for an
overall loss of 32.4±4.3 µmol/g dry wt glycogen.
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significantly different from values at the
end of the resynthesis period, P<.05.
Rates of Glycolysis and Glucose Oxidation
During the chase period, accumulation of
3H2O from glycolysis and
14CO2 from oxidation of glucose moieties was
linear (data not shown) over time after the first 5 to 10 minutes. Mean
rates of glycolysis and glucose oxidation from exogenous glucose and
endogenous glycogen, calculated from rates at 20, 30, and
40 minutes of perfusion, are summarized in Fig 3. Rates
of glycolysis from exogenous glucose (2768±72
nmol · min-1 · g-1
dry wt) were significantly greater than those from
endogenous glycogen (792±26
nmol · min-1 · g-1
dry wt, P<.05). Rates of glucose oxidation from exogenous
sources (526±8
nmol · min-1 · g-1
dry wt) were also significantly greater than oxidation of glucose from
glycogen (416±3
nmol · min-1 · g-1
dry wt, P<.05).
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As has been well described previously,2 only a portion of
the exogenous glucose that passed through glycolysis was oxidized (Fig 3). The same general pattern held true for rates of glycolysis and
oxidation of glucose that originated from glycogen (Fig 3). However,
the fraction of glucose from glycogen that passed through glycolysis
and was subsequently oxidized (0.53±0.14) was significantly greater
than the fraction of exogenous glucose passing through glycolysis that
was oxidized (0.19±0.01, P<.05). In other words, >50% of
glucose from glycogen was oxidized compared with <20% of exogenous
glucose. The majority of 14C in myocardial extracts was
found to be present in the glycogen fraction, and calculations
based on contents of myocardial glycolytic and tricarboxylic acid cycle
intermediates reported in the literature20 indicate that
nonglycogen sources are not likely to account for the preferential
oxidation of glycogen observed (data not shown).
Rates of ATP Production From Myocardial Glucose
Utilization
Rates of ATP produced by myocardial glucose utilization were
calculated from mean rates of glycolysis and glucose oxidation assuming
2 moles ATP were produced for each mole of exogenous glucose passing
through glycolysis, 3 moles of ATP for each mole of glucose from
glycogen passing through glycolysis, and 36 moles of ATP for each mole
of exogenous and endogenous glucose oxidized to
CO2. The data, summarized in Fig 4, show
that metabolism of glycogen by glycolysis and oxidation
contributes significantly to ATP production from myocardial
glucose use. In fact, 41% of ATP produced from glucose
metabolism by these hearts under these experimental
conditions is derived from glycogen. The vast majority of ATP from both
sources is derived from oxidation of glucose.
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Myocardial Glycogen Balance
Table 2 summarizes the changes observed in the
content of [3H]-glucose and [14C]-glucose
in myocardial glycogen during the chase period. The results of series A
were not significantly different from those of series B. Substantial
quantities of labeled glucose were incorporated into glycogen during
the labeling period such that 45% to 46% of the glycogen pool was
labeled. On the basis of changes in activity in myocardial glycogen
between hearts at the end of the labeling period and those at the end
of the chase perfusion and calculations made with the specific activity
of glucose in the labeling buffer, 23.8±5.0 µmol/g dry wt of
[3H]-glycogen and 22.8±5.3 µmol/g dry wt of
[14C]-glycogen were released during the 40-minute chase
period. Interestingly, despite this substantial degree of
glycogenolysis, net glycogen synthesis occurred
simultaneously, as indicated by incorporation of
[14C]-glucose and [3H]-glucose into
glycogen during the chase period. Accordingly, one would have expected
a net loss of glycogen ranging from 3.8±6.4 µmol/g dry wt for
[3H]-glycogen to 10.3±8.2 µmol/g dry wt for
[14C]-glycogen. However, myocardial glycogen content
measured enzymatically in these same hearts was reduced by 29.5±6.0
mmol/g dry wt for [3H]-glycogen and 36.0±6.4 mmol/g dry
wt for [14C]-glycogen during the chase perfusion. This
suggests that in addition to turnover of radiolabeled glycogen, a
significant amount of unlabelled glucose has also been released from
glycogen during this period.
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If the calculations are repeated assuming that degradation of glycogen is random, then total glycogen loss may be determined as the quotient of the change in labeled glycogen (23.8±5.0 µmol/g dry wt for [3H]-glycogen and 22.8±5.3 µmol/g dry wt for [14C]-glycogen) and the enrichment of glycogen (ie, the specific activity averaged over all glycogen at the end of the pulse perfusion divided by the specific activity of labeling buffer; 0.44±0.05 for [3H]-glycogen and 0.48±0.02 for [14C]-glycogen). Accordingly, it is anticipated that a reduction of glycogen content ranging from 46.1±10.7 to 51.0±10.2 µmol/g dry wt should occur during the chase period, assuming no simultaneous synthesis. If simultaneous incorporation of labeled glucose is taken into account by calculating the amount of glycogen synthesized during the chase (determined from the activity of [3H]- or [14C]-glucose in glycogen and the corresponding specific activity of the chase buffer), the predicted change in glycogen ranges from 31.0±10.1 µmol/g dry wt for [3H]-glycogen to 34.3±14.4 µmol/g dry wt for [14C]-glycogen. These values agree well with the measured changes in glycogen content.
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Discussion |
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Glycogen is a storage form of glucose that was formerly believed to make insignificant contributions to energy metabolism of the myocardium under aerobic conditions.1 Glycogen use is thought to increase if hearts are exposed to increased workloads,21 perfused without insulin and/or fatty acid,7 21 or subjected to hypoxic/ischemic conditions.1 As shown in the present study, net glycogenolysis occurred and substantial quantities of glucose from glycogen were metabolized by glycolysis and glucose oxidation in working hearts despite the presence of insulin and physiological levels of fatty acid. Our data also demonstrate that the contribution of glycogen as a source of glucose has been underestimated in previous studies because simultaneous glycogenolysis and glycogen synthesis was not considered. Although rates of glycolysis and oxidation of glucose from glycogen were less than rates of glycolysis and oxidation of exogenous glucose (Fig 3
), glycogen metabolism contributed
significantly to overall ATP production. In fact, glucose from
glycogen accounted for 41% of the ATP produced from myocardial glucose
utilization (Fig 4).
The vast majority of myocardial ATP was produced by mitochondrial
oxidation of both exogenous and endogenous glucose (Fig 4).
By combining data from the present study with substrate utilization
data from other studies of isolated, working rat hearts perfused under
similar conditions,2 15 we estimated the contribution of
glycogen to aerobic energy metabolism. Under these
conditions, exogenous palmitate is the preferred energy substrate
(>50% of ATP produced), whereas glucose and lactate oxidation
contribute 
14% and 5% of the ATP produced, respectively.
Utilization of endogenous fatty acid
(triacylglycerol) and glucose (glycogen) produces
25% of the ATP, with glycogen accounting for about 12% to 13%.
Overall, glycolysis produces 7% of the total ATP, with 1% to 2%
coming from glycolysis of glucose from glycogen. Since the rates were
not corrected for isotopic dilution of glycogen, the rate of ATP
production from glycogen represents a minimal
estimate.
Until the present study, it was assumed but not confirmed by
experimental evidence that glucose from myocardial glycogen is
metabolized in the same manner as exogenous glucose. Our data indicate
that, in fact, glucose from glycogen is preferentially oxidized
compared with exogenous glucose (Fig 3). More than 50% of glucose from
glycogen passing through glycolysis is oxidized, whereas <20% of
exogenous glucose passing through glycolysis is oxidized, a
statistically significant difference (P<.05). This finding
is entirely in keeping with observations in vascular smooth
muscle.9 10 In the myocardium, De Tata et
al22 presented evidence for transmural gradients
of glycogen metabolism. Our data suggest that energy
metabolism is also compartmentalized within cardiac
myocytes, a view consistent with reports by
others23 24 25 26 that proposed that carbohydrate
metabolism in the myocardium is regionalized.
The significance of this preferential oxidation and whether
glycogenolysis is coordinated with oxidative
phosphorylation and mechanical activity remain to be
determined.
Despite the presence of insulin and physiological
levels of fatty acid (0.4 mmol/L palmitate) in the experiments in the
current study, net myocardial glycogen loss occurred during the chase
perfusion (Fig 2). Saddik and Lopaschuk2 found that the
myocardial triacylglycerol pool also decreased in
size in the presence of physiological levels of
fatty acid. They speculated that 0.4 mmol/L palmitate was not the
concentration of fatty acid in the interstitial compartment
seen by the heart in vivo and, therefore, endogenous pools
of triacylglycerol were reduced. The same may hold
true for glycogen metabolism. In the face of
subphysiological perfusate fatty acid
levels, glycogen was used to generate energy.
We also found that significant turnover of glycogen occurred during the
aerobic chase period in the presence of substantial glycogenolysis and
an overall net reduction of myocardial glycogen content, in keeping
with the work of Goodwin et al.7 Whether myocardial
glycogen turnover occurs when net glycogen content does not change
remains to be determined. Rates of net synthesis, calculated from the
net incorporation of glucose into glycogen during the chase period,
ranged from 0.33 to 0.51
µmol · min-1 · g-1
dry wt. Although these rates of synthesis are likely an underestimate,
since they are not corrected for simultaneous
glycogenolysis of newly synthesized glycogen, they do compare favorably
with those obtained by Goodwin et al7 (0.17 to 0.62
µmol · min-1 · g-1
dry wt). Glucose taken up by the heart is either metabolized or
synthesized into a storage form.1 27 Under the
experimental conditions used in the present study, we found that
11.6% of the glucose taken up by the heart was synthesized into
glycogen. This value compares favorably with that observed by Goodwin
et al,7 who found that 4.5% to 7.5% of glucose taken up
by the heart is synthesized into glycogen. On the other hand, our data
are inconsistent with those of Wisneski et al6
and do not support the concept that the majority of glucose taken up by
the heart cycles through glycogen, as suggested by these investigators
on the basis of indirect measurements in humans. The fact that the
value in the current study is higher than that observed by Goodwin et
al7 is presumably a consequence of fatty acid in the
buffer, a situation that would favor glycogen synthesis. The reason for
the dramatic difference between the values in the current study and
those of Wisneski et al6 is not immediately clear but may
be related to methodological differences.
Glycogen is a very large molecule with a branched structure that exists
in multiple tiers.12 13 Whether glycogen is
synthesized and degraded in an ordered or a random manner remains
controversial.7 14 When changes in myocardial glycogen
content and specific activity were considered and newly incorporated
glucose was taken into account, we found the best agreement between
measured changes in glycogen content and loss of labeled glucose
moieties occurred when random degradation was assumed. On the basis of
this assumption, estimated loss of glycogen ranged from 31.0±10.1 to
34.3±14.4 µmol/g dry wt during the chase period, values that agree
well with the overall measured loss of glycogen of 32.8±4.3 µmol/g
dry wt. Furthermore, calculation of glycogen enrichment at the end of
the chase period ([3H]-glycogen, 0.45±0.08 dpm/µmol,
n=6; [14C]-glycogen 0.47±0.14 dpm/µmol, n=6), with the
newly incorporated glucose taken into account, demonstrates that
glycogen enrichment, averaged over all the glycogen, does not change
significantly in comparison with that at the end of the labeling period
([3H]-glycogen, 0.44±0.05 dpm/µmol, n=5;
[14C]-glycogen, 0.48±0.02 dpm/µmol, n=4,
P=NS). These findings are in keeping with those of Goodwin
et al7 and are consistent with the interpretation
that glycogen is degraded in a substantially random manner. Although we
anticipated that measured rates of glycolysis from
endogenous sources would correspond to loss of labeled
glucose from myocardial glycogen, rates of glycolysis were 25% to
30% higher than expected on the basis of the loss of labeled glucose
from glycogen. The explanation for this lack of correspondence is
unclear.
On the basis of these experiments, several important and novel conclusions may be made regarding aerobic myocardial glycogen metabolism. In aerobic, isolated, working rat hearts perfused with insulin and physiological concentrations of fatty acid, net glycogenolysis occurs and glycogen contributes significantly to overall myocardial glucose utilization, accounting for 41% of the ATP produced. An additional observation arising from these studies is that glucose from myocardial glycogen is oxidized preferentially compared with exogenous glucose, consistent with the concept that myocardial carbohydrate metabolism is compartmentalized. Furthermore, substantial glycogen turnover (ie, simultaneous synthesis and degradation) occurs in the aerobic heart in the presence of net glycogenolysis. Under the conditions of study, at least 11.6% of glucose taken up by the heart is directed toward glycogen synthesis despite net glycogenolysis. Finally, our data support the concept that myocardial glycogen is degraded in a random as opposed to an ordered manner.
Note Added in Proof
Since this paper was accepted for publication, Goodwin et al have
also presented data supporting the concept that glucose from
glycogen is preferentially oxidized in the heart
(Circulation. 1995;92:I-769. Abstract.).
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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 B.C. & Yukon. Dr Allard is a Research Scholar of the Heart & Stroke Foundation of Canada. Dr Lopaschuk is a Medical Research Council of Canada Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. S.L. Henning and B.O. Schönekess are graduate student trainees of the Heart and Stroke Foundations of B.C. & Yukon (S.L.H.) and Canada (B.O.S.). B.O. Schönekess is also a graduate student trainee of the Alberta Heritage Foundation for Medical Research. The authors thank Lorraine Verburgt for help with statistical analyses and Stuart Greene for assistance with the figures.
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Footnotes |
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Reprint requests to Michael F. Allard, BSc, MD, FRCPC, Department of Pathology and Laboratory Medicine, University of British Columbia, Cardiovascular Research Laboratory, St Paul's Hospital, 1081 Burrard St, Vancouver, BC, Canada V6Z 1Y6. E-mail mallard@prl.pulmonary.ubc.ca.
Received August 1, 1995; revision received October 23, 1995; accepted November 5, 1995.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Neely JR, Morgan HE. Relationship between
carbohydrate and lipid metabolism and the energy balance of
heart muscle. Annu Rev Physiol. 1974;36:413-459.
-
Saddik M, Lopaschuk GD. Myocardial
triglyceride turnover and contribution to energy substrate
utilization in isolated working rat hearts. J
Biol Chem. 1991;236:8162-8170.
-
Gertz EW, Wisneski JA, Neese RA, Bristow JD, Searle
GL, Hanlon JT. Myocardial lactate metabolism:
evidence of lactate release during net chemical extraction in
man. Circulation. 1981;63:1273-1279.
[Abstract/Free Full Text]
-
Taegtmeyer H. Energy metabolism of
the heart: from basic concepts to clinical applications.
Curr Probl Cardiol. 1994;19:59-113. [Medline]
[Order article via Infotrieve]
-
Larner J. Insulin and stimulation of glycogen
synthesis: the road from glycogen structure to glycogen synthase to
cyclic AMP-dependent protein kinase to insulin mediators.
Adv Enzymol Relat Mol Biol. 1990;63:173-231. [Medline]
[Order article via Infotrieve]
-
Wisneski JA, Gertz EW, Neese RA, Gruenke LD, Morris
DL, Craig JC. Metabolic fate of extracted glucose in
normal human myocardium. J Clin
Invest. 1985;76:1819-1827.
-
Goodwin GW, Arteaga JR, Taegtmeyer H. Glycogen
turnover in the isolated working rat heart. J
Biol Chem. 1995;270:9234-9240.
[Abstract/Free Full Text]
-
Randle PJ, Garland PB, Hales CN, Newsholme EA.
The glucose-fatty acid cycle: its role in insulin sensitivity and
metabolic disturbances of diabetes
mellitus. Lancet. 1963;1:785-789. [Medline]
[Order article via Infotrieve]
-
Lynch RM, Paul RJ. Compartmentation of
carbohydrate metabolism in vascular smooth muscle.
Am J Physiol. 1987;252:C328-C334.
[Abstract/Free Full Text]
-
Hardin CD, Kushmerick MJ.
Simultaneous and separable flux of pathways for glucose and
glycogen utilization studied by 13C-NMR.
J Mol Cell Cardiol. 1994;26:1197-1210.[Medline]
[Order article via Infotrieve]
-
Laughlin MR, Petit WA, Dizon JM, Shulman RS, Barrett
EJ. NMR measurements of in vivo myocardial glycogen
metabolism. J Biol Chem. 1988;263:2285-2291.
[Abstract/Free Full Text]
-
Calder PC. Glycogen structure and
biogenesis. Int J Biochem. 1991;23:1335-1352. [Medline]
[Order article via Infotrieve]
-
Goldsmith E, Sprang S, Fletterick R. Structure
of maltoheptose by difference Fourier methods and a model for
glycogen. J Mol Biol. 1982;156:411-427. [Medline]
[Order article via Infotrieve]
-
Brainard JR, Hutson JY, Hockenga DE, Lenhoff R.
Ordered synthesis and mobilization of glycogen in the perfused
heart. Biochemistry. 1989;28:9766-9772. [Medline]
[Order article via Infotrieve]
-
Allard MF, Schonekess B, Henning SL, English DR,
Lopaschuk GD. Contribution of oxidative metabolism
and glycolysis to ATP production in the hypertrophied
heart. Am J Physiol. 1994;267:H742-H750.
[Abstract/Free Full Text]
-
Lopaschuk GD, Spafford MA, Davies NJ, Wall SR.
Glucose and palmitate oxidation in isolated working rat hearts
reperfused after a period of transient global ischemia.
Circ Res. 1990;66:546-553.
[Abstract/Free Full Text]
-
Lopaschuk GD, McNeill J, McVeigh J. Glucose
oxidation is stimulated in reperfused heart with the carnitine
palmitoyl-transferase I inhibitor, Etomoxir.
Mol Cell Biochem. 1989;88:175-179. [Medline]
[Order article via Infotrieve]
-
Allard MF, Emanuel PG, Russell JA, Bishop SP, Digerness
SB, Anderson PG. Preischemic glycogen reduction or
inhibition of glycolysis improve postischemic recovery of
hypertrophied rat hearts. Am J Physiol. 1994;267:H66-H74.
[Abstract/Free Full Text]
-
Mood AM, Graybill FA, Boes DC.
Introduction to the Theory of Statistics. 3rd ed. New York,
NY: McGraw-Hill Publishing Co; 1974:180.
-
Randle PJ, Tubbs PK. Carbohydrate and fatty acid
metabolism. In: Berne R, Sperelakis N, eds.
Handbook of Physiology: The Cardiovascular
System—The Heart. Bethesda, Md: American
Physiological Society; 1979:805-844.
-
Neely JR, Whitefield CF, Morgan HE. Regulation
of glycogenolysis in hearts: effects of pressure development, glucose
and FFA. Am J Physiol. 1970;219:1083-1088.
-
De Tata V, Bergamini C, Cori Z, Locci-Cudecco T,
Bergamini E. Transmural gradient of glycogen
metabolism in the normal rat left ventricle.
Pflugers Arch. 1983;396:60-65. [Medline]
[Order article via Infotrieve]
-
Xu KY, Zweier JL, Becker LC. Functional coupling
between glycolysis and sarcoplasmic reticulum Ca2+
transport. Circ Res. 1995;77:88-97.
[Abstract/Free Full Text]
-
Weiss J, Hildebrand B. Functional
compartmentation of glycolytic versus oxidative metabolism
in isolated rabbit heart. J Clin Invest. 1985;75:436-447.
-
Entman ML, Kanike K, Goldstein MA, Nelson TP, Bornet
EP, Futch TW, Schwartz A. Association of glycogenolysis with
cardiac sarcoplasmic reticulum. J Biol
Chem. 1976;251:3140-3146.
[Abstract/Free Full Text]
-
Pierce GN, Philipson KD. Binding of glycolytic
enzymes to cardiac sarcolemmal and sarcoplasmic reticular
membranes. J Biol Chem. 1985;260:6862-6870.
[Abstract/Free Full Text]
-
Neely JR, Rovetto MJ, Oram JF. Myocardial
utilization of carbohydrate and lipids. Prog Cardiovasc
Dis. 1972;15:289-329.[Medline]
[Order article via Infotrieve]
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