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J Biol Chem, Vol. 273, Issue 45, 29530-29539, November 6, 1998
From the Division of Cardiology, Department of Internal Medicine,
University of Texas-Houston Medical School, Houston, Texas 77030
We determined the contribution of all major
energy substrates (glucose, glycogen, lactate, oleate, and
triglycerides) during an acute increase in heart work (1 µM epinephrine, afterload increased by 40%) and
the involvement of key regulatory enzymes, using isolated working rat
hearts exhibiting physiologic values for contractile performance and
oxygen consumption. We accounted for oxygen consumption quantitatively
from the rates of substrate oxidation, measured on a minute-to-minute
basis. Total Increased heart work, usually elicited by catecholamines,
increases carbohydrate oxidation because of activation of the pyruvate dehydrogenase complex (PDC)1
by dephosphorylation (1); PDC phosphatase is stimulated by increased
mitochondrial Ca2+ (2). Activation of PDC under conditions
of high workload could be advantageous, tending to increase
carbohydrate oxidation selectively. Under the assumption that the ATP
yield per O2 consumed is higher for oxidation of
carbohydrate versus lipid, a selective increase in
carbohydrate oxidation could provide increased ATP synthesis despite
maximum oxygen extraction.
Whether increased substrate oxidation with workload is selective
for carbohydrate remains unresolved. Studies by us (3) and by
Collins-Nakai et al. (4), using isolated working rat hearts,
indicated that increased substrate oxidation is selective for
carbohydrate, with little or no increase in exogenous fatty acid
oxidation. In contrast, early studies by Neely et al. (5, 6)
and Crass et al. (7) suggested that fatty acid and
carbohydrate oxidation increase in parallel. Hall et al. (8)
showed that swine heart exhibit increased fatty acid uptake in
vivo in response to dobutamine, presumably from stimulated
The problem with existing studies is the failure to consider all
relevant exogenous and endogenous substrates. The question of whether
carbohydrate oxidation is increased selectively with the workload
cannot be answered based on measures of glucose and exogenous fatty
acid oxidation alone. Regulation of total Lactate and pyruvate are released or consumed by tissues at different
rates, since their systemic concentrations differ. We hypothesize that
heart, which, at times, displays large fluxes for lactate or pyruvate
across the plasma membrane, uses differential efflux of lactate
versus pyruvate (a redox pair) as a supplemental mechanism
for regulation of the cytosolic redox.
We previously found small amounts of simultaneous glycogen synthesis
and degradation in heart, especially during glycogen depletion (10).
Compared with total glycolytic flux, the contribution of flux through
glycogen in the absence of cyclical changes in glycogen content
("glycogen cycling" (11)) was minor. In the present study, we
examined activities for synthase and phosphorylase and their allosteric
effectors in relation to fluxes for glycogen synthesis and
glycogenolysis. We postulate that the well described reciprocal
regulation of glycogen synthase and phosphorylase, apparently designed
to prevent futile cycling of glycogen, is robust, preventing more than
minor glycogen cycling in heart.
Materials--
Isotopes were from ICN (Costa Mesa, CA). Enzymes
were from Boehringer Mannheim. Other chemicals were from Sigma. Fatty
acid synthase was purified from rat liver after inducing the enzyme; rats were fasted for 2 days and then fed bread (8% protein, no fat)
for 3 days. Enzyme was purified from the cytosol of four rat livers (53 g) as described by Awan and Saggerson (9) except that chromatography
was on DEAE-cellulose, as described by Linn (12). We obtained 68 units
(1.1 unit/mg of protein), assayed by the method of Carey and Dils
(13).
Heart Perfusions--
Hearts from chow-fed male Harlan
Sprague-Dawley rats (349 ± 8 g, n = 25) were
perfused using the working heart apparatus (14) in a gas-tight
configuration described previously (10). The filling pressure was 15 cm
H2O, and the afterload/perfusion pressure was initially 100 cm H2O, increased to 140 cm H2O at the time of
adrenergic stimulation. The initial perfusate was Krebs-Henseleit buffer containing 1.4 mM free Ca2+ (1.5 mM CaCl2 plus 0.1 mM EDTA),
equilibrated with 95% O2, 5% CO2. Hearts were
perfused in the working mode using 200 ml of recirculated perfusate,
lacking carbon substrate initially. The initial part of this protocol
(Fig. 1) was designed to deplete and then resynthesize glycogen, as we
previously described (3). After 20 min, the perfusate was supplemented
to 5 mM glucose, 40 microunits/ml regular insulin (Lilly),
5 mM sodium D-
We studied three treatment groups according to the time at which hearts
were freeze-clamped: "unstimulated" (n = 5, clamped at 55 min of the protocol), "acute stimulation" (n = 5, clamped at 58 min at the peak of glycogen oxidation, Fig. 3), and
"prolonged stimulation" (n = 15, clamped at 75 min). Hearts subjected to prolonged stimulation were randomly assigned
to one of three treatment groups according to which
14C-labeled isotope was traced: exogenous
[U-14C]glucose (n = 5, 20 µCi/liter, 4 µCi/mmol), exogenous [U-14C]lactate (n = 5, 3 µCi/liter, 6 µCi/mmol), or 14C-glycogen
(n = 5, 100 µCi/liter, 20 µCi/mmol
[U-14C]glucose included between 20 and 45 min of the
protocol). In some perfusions, we included
[9,10-3H]oleate (30 µCi/liter, 75 µCi/mmol) to
measure fatty acid oxidation and de novo triglyceride synthesis.
Oxygen Consumption (MVO2) and Contractile
Performance--
A portion of the coronary flow (10 ml/min) was pumped
through a stirred, thermostated 3-ml chamber fitted with a Clark
electrode (Yellow Springs Instrument Co.) and then returned to the
heart chamber. A second electrode was fitted to the bottom of the
oxygenator, and the two electrode currents were displayed continuously.
MVO2 was calculated from the A Analytical Methods--
14CO2 and
3H2O were determined in fresh perfusate as
described previously (3). We verified that
14CO2 is recovered quantitatively by injecting
2 µCi of [14C]NaHCO3 into the apparatus.
The sum of 14C-lactate plus 14C-pyruvate was
determined in deproteinized perfusate by paper chromatography (16).
Lactate and pyruvate migrate together in this system. The recovery of
lactate from perfusate spiked with authentic
[U-14C]lactate was 88 ± 2% (n = 5)
and was completely separated from [U-14C]glucose. Other
metabolites (lactate, pyruvate, glucose, glycerol) were measured in
deproteinized samples by established spectrophotometric enzymatic
assays (17). Metabolic rates were calculated as described previously
(3). Apparent rates from glycogen were calculated based on the specific
activity of glucose used to prelabel glycogen and then converted to
true rates by assuming uniform isotopic enrichment of 54.7%
(unstimulated treatment group in Table I).
Tissue Analysis--
Frozen hearts were weighed and ground to a
fine powder under liquid N2, and a portion was taken for
dry weight determination. High energy phosphates (ATP, ADP, AMP,
glucose 6-phosphate, phosphocreatine), Pi, pyruvate, and
malonyl-CoA were measured in freshly prepared 6% perchloric acid
extracts, adjusted to pH 5 with buffered KOH. High energy phosphates
and pyruvate were measured using established enzymatic assays (17).
Inorganic phosphate was measured with the acid molybdate reaction (18).
Malonyl-CoA was measured radiochemically with purified fatty acid
synthase (19). For analysis of triglycerides, a total lipid extract was
prepared (20), and the triglyceride fraction was isolated by thin layer
chromatography on silica gel plates (Whatman No. 4860-320) using
CHCl3 as the mobile phase. Lipid in the triglyceride band
(RF = 0.5) was extracted from the silica (20),
hydrolyzed with alcoholic KOH, neutralized with perchloric acid, and
analyzed for glycerol (17). Values were referred to acyl content
(glycerol/3). This procedure had a recovery of 80%, using tripalmitin
as an internal standard. To determine de novo synthesis, a
portion of the neutralized hydrolysate was taken for scintillation
counting, and the radioactivity referred to new acyl group
incorporation based on the specific activity of extracellular
[9,10-3H]oleate.
Glycogen was measured as described previously (3). The isolated
glycogen was free from contamination by glucose and extracellular isotopes. To measure the tissue distribution of 14C in
glycogen, heart powder (50 mg) was dispersed into 1 ml of glacial
acetic acid and then evaporated to dryness to remove
14CO2. The residue was dissolved by heating
with 1 ml of SolvableTM (Packard Instrument Co.) and then
counted after adding 10 ml of scintillation mixture (Ultima Gold;
Packard). In hearts subjected to 14C-pulse labeling
(i.e. 14C-glycogen and unstimulated treatment
groups), 85 ± 3 and 90 ± 3% of 14C was found
in glycogen, respectively. This indicates that glycogen recovery from
the isolation procedure is at least 90% (otherwise there would be more
14C-glycogen than total radioactivity) and that glycogen
was virtually the sole source of radioactivity during the chase.
Enzyme Assays--
Glycogen synthase was measured as described
by Skurat et al. (21) using the filter binding assay of
Thomas et al. (22). The glycogen phosphorylase assay was
based on the method of Gilboe et al. (23). We determined the
enzyme in the reverse direction by release of Pi measured
with the acid molybdate reaction (18). Phosphorylase a was
measured in the presence of 0.5 mM caffeine to prevent
activation by endogenous AMP (24), and total phosphorylase was measured
in the presence of 3 mM AMP. Pyruvate dehydrogenase was
measured by 14CO2 production from 2 mM [1-14C]pyruvate, as described by Harris
et al. (25). The active form was measured directly, and the
total activity was measured following incubation for 30 min at 30 °C
with 1 mM CaCl2 plus 5 mM
MgCl2, to allow dephosphorylation by endogenous PDC
phosphatase. This procedure produced stable, maximal values for the
total activity of PDC. We established that all of the enzyme assays
were linear with respect to time and amount of tissue extract used in
the assays.
Data are expressed as mean ± S.E. Statistical comparison was by
analysis of variance with post hoc comparison by Newman-Keuls multisample test. p < 0.05 was considered significant.
Physiological Performance (Fig. 2 and Appendix Table IA)--
We
performed five sets of matched perfusions, three of which were used to
measure oxidation of three different 14C-labeled substrates
(14CO2 from glucose, glycogen, or lactate).
Physiologic performance of these three groups is shown in Fig. 2. Fig.
2A shows contractile activity (hydraulic power, watts), and
the bottom panel shows oxygen consumption (MVO2). Two other
treatment groups (unstimulated, and acute stimulation) were omitted
from the figure for clarity, because the data points overlapped the
values presented. These two groups were freeze-clamped at earlier times
of the protocol, for tissue analysis. Various measures of performance
for all the groups are given in the Appendix. The five groups were well
matched for all values of performance measured. Performance is
comparable with values measured in vivo for resting and
exercising rats (26).
During the first 20 min of the protocol (Fig.
1), the absence of exogenous substrates
resulted in diminishing contractile function. The protocol was designed
to deplete endogenous substrates, evidenced by continued oxygen
consumption. The addition of substrates at 20 min (glucose plus
insulin, lactate, and D- Rates for Substrate Oxidation--
Fig.
3 shows rates of substrate oxidation
during the chase period for glycogen. Rates were measured by the Fick
principle (V Glycogen Content and Enrichment--
Table
I gives values for the content of total
glycogen, 14C-glycogen, and enrichment. In hearts clamped
at 55 min (unstimulated), the glycogen enrichment achieved by the
labeling protocol (pulse portion of the pulse-chase) was 55 ± 10%. With acute adrenergic stimulation, the decrease in glycogen
content was not large, since the duration of stimulation was short (3 min). Following prolonged stimulation, the total glycogen content
decreased by half, as did the content for 14C-glycogen. The
enrichment of residual glycogen remained the same (49 ± 9%,
14C-glycogen group in Table I). A similar value for
enrichment (49%) was calculated for the glycogen that was broken down,
correcting for the small amount of de novo synthesis
([14C]glucose group in Table I). These findings indicate
that glycogen was degraded randomly, without discretion for new
[14C]glycosyl residues and preexisting
[12C]glycogen. As expected, there was no glycogen
synthesis from lactate (Table I, last line).
We next considered the possibility that the initial glycogen degraded
was enriched to a greater extent than the rest (i.e. a
hybrid of random and ordered degradation). The distinction (purely random versus hybrid model) is important for interpretation
of the present data, as well as data from 13C NMR
spectroscopy studies. To do this, we examined the time course of
glycogen enrichment by taking advantage of oxygen consumption measurements, but it was first necessary to account for oxygen consumption from all the substrates.
Triglyceride Turnover and Pyruvate Release--
Total triglyceride
content of the hearts increased slightly (not significantly) during the
20 min of adrenergic stimulation (from 56.9 ± 6.2 (n = 5) to 59.3 ± 6.3 (n = 15)
µmol of acyl/g, dry weight for unstimulated and prolonged stimulation
treatment groups, respectively). During the same interval, de
novo synthesis, based on incorporation of
[9,10-3H]oleate into triglycerides, increased
(p < 0.05) from 1.38 ± 0.34 (n = 3) to 6.38 ± 1.40 (n = 15) µmol of acyl/g, dry
weight. Therefore, the total triglyceride pool size expanded by 2.4 µmol acyl/g, dry weight, and de novo synthesis was 5.0 µmol of acyl/g, dry weight. Using the relation degradation = synthesis
Release of pyruvate contributes slightly to oxygen consumption (0.5 mol
of O2/mol of pyruvate release) because pyruvate is more
oxidized than its precursors (glucose, glycogen, or lactate). Rates of
pyruvate release are given in Appendix Table IIA.
Oxygen Consumption from Total Substrate Oxidation and the Time
Course of Glycogen Enrichment (Figs. 4
and 5)--
Fig. 4 shows predicted and
measured rates of oxygen consumption. Predicted rates are based on the
sum for the measured rates of oxidation of every major substrate
(glucose, glycogen, lactate, oleate, triglycerides, and release of
pyruvate). To calculate rates of oxygen consumption resulting from
glycogen oxidation, we assumed that there is uniform isotopic dilution
(i.e. that a purely random pattern of synthesis and
degradation applies) and used the values given in Fig. 3. Two
conclusions can be drawn from the close agreement between measured and
predicted MVO2. First, we accounted for every major
oxidizable substrate for the heart quantitatively. Second, the
assumption of uniform isotopic dilution is valid. The second conclusion
is shown more clearly in Fig. 5, which depicts oxygen consumption
resulting from glycogen oxidation over time. The upper
curve is oxygen consumption from oxidation of all glycogen,
based on the difference between MVO2 and oxygen consumption
from every substrate except glycogen. The lower
curve is oxygen consumption from oxidation of that portion of glycogen (55% of the total) that was labeled with 14C
(i.e. the apparent rate of glycogen
oxidation × 6). The relationship between the two curves is
precisely the pattern expected based on purely random synthesis and/or
degradation. It is distinguished from the pattern expected based on the
last on-first off model or from a hybrid model.
Glycogen Synthase Activity in Relation to Glycogen Turnover (Table
II)--
There was a complete absence of
incorporation of [5-3H]glucose into glycogen
(n = 3) when the isotope was included between 45 and 55 min of perfusions in the unstimulated treatment group. Therefore,
neither synthesis nor degradation occurred during the 10 min prior to
adrenergic stimulation. There was turnover following prolonged
stimulation, since, as described above, there was robust glycogen
oxidation (Fig. 3), and de novo synthesis occurred during the same period (Table I, [14C]glucose treatment group).
Table II shows activity states for glycogen synthase at different times
of the protocol, along with the content of glucose 6-phosphate
(activator of the glucose 6-phosphate-independent form), which account
for the observed changes in de novo synthesis. The activity
state of synthase was increased by 65% following prolonged
stimulation. However, the enzyme was not activated acutely. The content
of glucose 6-phosphate was not significantly changed during the
protocol. These data indicate that a small amount of glycogen synthesis
occurred late in the protocol, since synthase did not become activated
until after prolonged stimulation. The potential for futile cycling is
therefore limited, since most of glycogenolysis occurred early (Fig.
3).
Lactate/Pyruvate Ratios, High Energy Phosphates, and Regulation of
the Redox (Appendix Tables IIA and IIIA)--
We measured high energy
phosphates and the intracellular ratio of lactate/pyruvate to determine
if hearts experienced demand ischemia upon adrenergic stimulation,
which would exaggerate glycogenolysis. The ratio of lactate/pyruvate,
which rises dramatically during ischemia, reflects the cytosolic redox
potential (NADH/NAD+), assuming lactate dehydrogenase is
near equilibrium. We did not observe an acute increase in this ratio
(Appendix Table IIA). Therefore, hearts were not ischemic during acute
stimulation, when glycogenolysis occurred. The largest change in high
energy phosphates was a 21% decrease in phosphocreatine during acute stimulation, in exchange with Pi for the most part
(Appendix Table IIIA). The ATP content was not decreased during acute
stimulation. That acute changes in high energy phosphates were small or
absent further supports the conclusion of adequate oxygen supply.
Prior to adrenergic stimulation, there was small net lactate extraction
resulting from a small lactate gradient across the plasma membrane (0.5 mM extracellular versus 0.45 mM in
the cytosol). With adrenergic stimulation, extraction switched to net
release, and there was a large transient burst of release (Appendix
Table IIA). Subsequently, lactate release quickly decreased to a new steady state, which was maintained for the remainder of the protocol. There was simultaneous oxidation of exogenous lactate during net release (Fig. 3). This phenomenon has been observed previously (27),
and could result from mixing of intracellular and extracellular lactate
pools or from cellular heterogeneity. Not unexpectedly, a pyruvate
gradient (roughly equal to the intracellular pyruvate concentration,
Appendix Table IIA) produced pyruvate efflux. Although pyruvate release
followed its concentration gradient throughout the protocol, the burst
of lactate release immediately after adrenergic stimulation (derived
primarily from glycogen) was out of proportion to the lactate gradient
(0.5 mM extracellular versus 0.87 mM
in the cytosol). The resulting large increase in the ratio for lactate efflux relative to pyruvate efflux (from Coupling of Glycolysis to Pyruvate Oxidation--
Fig.
6 shows cumulative values for metabolism
of glycogen (A) or glucose (B) by glycolysis,
oxidation, and the percentage of glycolysis that was subsequently
oxidized. We expressed the results as cumulative values (the slope is
equal to the rate), because different metabolites
(14CO2, [14C]lactate,
etc.) produced at the same time may not appear in the coronary effluent at the same rate. Differences in diffusion are averaged out over a cumulative plot. As we reported previously (16),
there is tighter coupling of glycolysis to oxidation from glycogen
compared with glucose. By the end of the protocol, 31.4 ± 2.1%
of cumulative glucose utilization was subjected to oxidation, while the
corresponding value from glycogen was 45.3 ± 5.3% (44% higher,
p < 0.05).
Temporal Dissociation of Activation of Glycogen Phosphorylase
from PDC and Regulation of Phosphorylase (Table
III)--
The burst of release of
glycogen-derived lactate plus pyruvate immediately after adrenergic
stimulation arose, first, because PDC was slow to become activated
relative to phosphorylase (Table III) and, second, because of
competition between extracellular lactate with glycogen as sources of
pyruvate for PDC (Fig. 3).
Adrenergic stimulation predictably converted a larger portion of
phosphorylase to the a form (Table III), since phosphorylase kinase is stimulated by cAMP and Ca2+. However, because
intracellular glucose was acutely increased, augmented phosphorylase
flux must have resulted largely from elevated AMP, a potent allosteric
activator of phosphorylase b. Increased Pi
(derived mostly from phosphocreatine; see Appendix Table IIIA), being a
substrate for phosphorylase, probably also contributed to increased
phosphorylase flux. Because of accumulation of intracellular glucose,
which inhibits phosphorylase a allosterically,
phosphorylation of phosphorylase to the a form may have
paradoxically inhibited flux through the enzyme. Indeed, the mechanism
for cessation of phosphorylase flux, despite 50% residual glycogen,
appears to be the return of AMP to control levels, since phosphorylase
a and intracellular glucose remained elevated. We conclude
that the transient burst of phosphorylase flux reflects the status of
high energy phosphates and not the activation state of phosphorylase kinase.
Coordination of Glycolysis from Glucose Versus
Glycogen--
During the first minutes of adrenergic stimulation,
there was a gradual switch from glycogen to glucose as the glycolytic substrate (Fig. 3). The coordination between glucose and glycogen during the switch resulted from inhibition of glucose transport by
accumulation of intracellular glucose during rapid glycogen breakdown
(Table III), partially derived from glycogen itself (i.e. the 8% or so released as glucose instead of glucose 1-phosphate). The
majority of glycolytic flux from glycogen bypasses hexokinase. Therefore, one would expect that if a bottleneck were at
phosphofructo-1-kinase, then glucose 6-phosphate would increase during
a burst of glycogenolysis, but this was not the case (Table II). We
conclude that coordination between glucose and glycogen occurs at
glucose transport.
Rates of Fatty Acid Oxidation and Regulation by
Malonyl-CoA--
Table IV gives values
for exogenous oleate oxidation and total The present study provides a comprehensive analysis of fuel
selection during acute transition from low to high workload of isolated
working rat heart. We confirm preliminary studies (3) by showing that
glycogen and, to a lesser extent, lactate are important energy
substrates for aerobic heart when the workload is acutely increased
(Fig. 3). Although exogenous fatty acid oxidation was not significantly
changed, total Triglyceride oxidation resulted from turnover of the triglyceride pool.
Our result agrees with the previous estimate that triglyceride turnover
contributes about 10% to total To answer the question posed in the Introduction, when all relevant
exogenous and endogenous substrates are examined, the increase in
carbohydrate oxidation upon adrenergic stimulation is, in fact,
selective. Carbohydrate oxidation is increased selectively because
total In very broad terms, we confirmed early studies of Neely et
al. (5, 6) and Crass et al. (7) by showing that
stimulation of heart work increases both carbohydrate and fatty acid
oxidation. However, these early studies examined only
exogenous substrate oxidation. Presently, we did not find an
increase in exogenous fatty acid oxidation (there was a 20% increase,
but this was not statistically significant). This result agrees with
our previous report (3) and a similar study by Collins-Nakai et
al. (4). Further, the small increase in total The Strategy for Substrate Selection Based on ATP
Yield--
We can now consider why the heart adopted the strategy of
preferential increase in carbohydrate oxidation to deal with increased energy demand, at least under idealized conditions of fixed substrate availability. Using a revised estimate for the stoichiometry of oxidative phosphorylation (33), the ATP/O2 ratio calculated for complete oxidation of glucose (5.2) is only 4% higher than for
oleate (5.0), and the value for complete oxidation of lactate (4.8) is
actually less than oleate. Based on these calculations, the advantage
of selective increase in carbohydrate oxidation during high oxygen
extraction seems marginal, or it is a slight disadvantage in the case
of lactate. The analysis is flawed, because carbohydrates are not
completely oxidized. The actual value for ATP/O2
is higher for glucose or glycogen relative to oleate, partially because
carbohydrates carry more of their own oxygen but also because a portion
of carbohydrates are metabolized anaerobically. Like skeletal muscle,
during high workload states, a portion of glucose taken up by heart is
diverted to the release of lactate plus pyruvate. This occurs
irrespective of whether there is net release or extraction of lactate
(Ref. 27; and compare Fig. 3 with net lactate fluxes given in Appendix
Table IIA). Using values for the distribution between oxidation and
release of lactate or pyruvate that were obtained during adrenergic
stimulation in this study, we calculate that the effective
value of ATP/O2 is 5.4 for glucose and 5.7 for glycogen.
Therefore, the advantage of using glycogen during periods of high
energy demand is pronounced.
Coordination of Glucose and Glycogen Utilization--
Upon
adrenergic stimulation, the increase in glucose oxidation was delayed
relative to the burst of glycogenolysis (Fig. 3). We explain the delay
by interaction between glucose and glycogen at the level of glucose
transport. Therefore, phosphorylase effectively regulates glucose
uptake in this setting. We also conclude that acute regulation of
phosphorylase mostly reflects the status of high energy phosphates
(particularly AMP) and not the activity state of phosphorylase kinase.
It follows that glucose uptake, in the setting of adrenergic
stimulation, is regulated indirectly by the status of high energy
phosphates. Blocked glucose uptake upon acute reduction in energy
charge, and the associated burst of glycogenolysis, obviates the
investment of ATP to prime glucose for glycolysis until after glycogen
has been exploited.
Redox Regulation during Glycogenolysis and the Coupling to
Oxidation--
We confirmed our earlier observation (16) that
glycogen-derived pyruvate is more tightly coupled to oxidation than is
pyruvate derived from extracellular glucose (Fig. 6). We hypothesized
that tighter coupling would reflect coordination between phosphorylase activation and PDC activation. Contrary to our hypothesis, PDC activation was delayed relative to the burst of glycogenolysis. We
would have predicted pronounced activation of PDC resulting from the
combined effect of Ca2+ stimulation of PDC phosphatase and
feed-forward activation resulting from inhibition of PDC kinase by high
levels of glycogen-derived pyruvate. Paradoxically, we found that
glycogen-derived pyruvate was preferentially oxidized despite the fact
that PDC activation was slow to develop. Further, the portion not
oxidized was preferentially reduced to lactate and was exported as
such. Since the relative efflux of lactate and pyruvate varied during
the protocol (Appendix Table IIA), the process contributed to
regulation of the cytosolic redox potential. During the burst of
glycolytic flux from glycogen, differential efflux of lactate
versus pyruvate prevented the occurrence of cytosolic
reduction that results when glycolytic flux is stimulated in excess of
the capacity for pyruvate oxidation. In so doing, inhibition of
glyceraldehyde-3P dehydrogenase and phosphofructo-1-kinase from
cytosolic reduction and H+ accumulation, which would
otherwise have self-limited pathway flux, was obviated.
Activation of Synthase and Phosphorylase Were Timed to
Minimize Glycogen Cycling--
We postulated that there is robust
reciprocal regulation of glycogen synthase and phosphorylase,
preventing more than minor occurrence of glycogen futile cycling in
heart. We did observe activation (dephosphorylation) of synthase late
in the protocol (Table II), accounting for a small amount of de
novo glycogen synthesis (Table I) during net glycogenolysis (Fig.
3). Synthase activation could serve to prime the system for
glycogen repletion during recovery but was timed to minimize futile
cycling, since most of glycogenolysis occurred early on. The lag phase
for synthase activation following stimulation of glycogenolysis, as
suggested by Stalmans et al. (34) in liver, could have
resulted from the progressive release of phosphorylase phosphatase,
which then becomes available to dephosphorylate/activate glycogen synthase.
Is Glycogenolysis a Physiologic Response to
Epinephrine?--
Myocardial glycogenolysis is a physiological
response to exercise of the whole animal (35), mediated in large part
by increased adrenergic tone and circulating catecholamines. However,
the glycogenolytic response to epinephrine has not been observed
consistently. There are at least three explanations for the
inconsistency. First, hearts perfused with saline in vitro
are very sensitive to epinephrine induced glycogenolysis (3, 36). This
could be an artifact resulting from low reserve for O2
delivery and from the induction of demand ischemia when no provision is
made to increase O2 delivery (37). In the present study, we
did make provision for increased O2 delivery at the onset
of adrenergic stimulation (we increased the perfusion pressure at the
same time), and we verified by various methods (intracellular ratio for
lactate to pyruvate, content of high energy phosphates, oxygen
consumption relative to contractile performance) that hearts did not
experience demand ischemia. The increased AMP and small decrease in
phosphocreatine that we observed during acute stimulation are similar
to changes in vivo in response to increased pressure
development and/or dobutamine infusion (37, 38) and therefore represent
a physiologic response. Second, we find that myocardial glycogenolysis
is blunted under conditions of high lactate and nonesterified fatty
acids2 (39). High systemic lactate and nonesterified fat
arise during exercise or infusion of epinephrine (40). This may explain
the results reported by Williams and Mayer (Ref. 41 and references therein). Third, studies of glycogenolysis detected by 13C
NMR based on pulse-chase labeling of glycogen with
[1-13C]glucose, which suggest that glycogenolysis is
insensitive to epinephrine, may be flawed because of the assumption of
the last on-first off pattern of glycogen synthesis and degradation
(42-44).
The Molecular Order of Glycogen Synthesis and Degradation Is Purely
Random in Heart--
In the present study, we examined this issue in
greater detail than previously (3, 10) by taking advantage of oxygen consumption measurements, using the fact that total substrate oxidation
should be equal to oxygen consumption if the correct model for glycogen
dynamics is employed. Specifically, we could distinguish between purely
random versus a mixed pattern for glycogen dynamics. We have
now shown by several independent techniques based on analysis of both
metabolites derived from glycogen and by analysis of glycogen itself
that there is no molecular order in the synthesis and subsequent
degradation of glycogen (Refs. 3 and 10 and the present study). The
pattern is not even a hybrid but appears to be purely random (Fig. 5),
and it is therefore different from the situation in the liver (45). By
adopting the last on-first off model proposed by Brainard et
al. (42), apparent glycogenolysis based on disappearance of the
13C NMR signal from glycogen will underestimate true
glycogenolysis by an amount determined by the degree of isotopic
dilution of [13C] glycosyl residues by preexisting
[12C]glycogen. The degree of 13C isotopic
enrichment has not generally been specified, but it is probably
considerably less than the value of 55% 14C enrichment
achieved in the present study. For example, Laurent et al.
(44) recently concluded that epinephrine does not stimulate glycogenolysis in skeletal muscle. If the degree of isotopic enrichment of glycogen in that study were as high as the value for extracellular glucose (13.9%), then glycogenolysis was underestimated by a factor of 7.
In conclusion, we characterized total substrate utilization during an
acute low to high work transition of the heart, described the mechanism
responsible for high respiratory rates from glycogen, and explained the
inconsistency among reports of epinephrine-induced glycogenolysis. High
glycogen respiration was one component of the overall pattern of
selective increase in carbohydrate oxidation. The advantage of
increased carbohydrate oxidation may relate to a higher
effective value for the ratio of ATP
synthesized/O2 consumed for carbohydrate versus
lipid, not to be confused with the ATP/O2 ratio calculated
for complete oxidation.
We thank Qiuying Han and Patrick H. Guthrie
for technical assistance.
*
This work was supported by NHLBI, National Institutes of
Health (NIH), Grant RO1-43133 and American Heart Association, Texas Affiliate, Grant-in-aid 97G-329.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.
§
Supported by NIDDK, NIH, Grant T35 DK07676.
The abbreviation used is:
PDC, pyruvate
dehydrogenase complex.
2
G. W. Goodwin and H. Taegtmeyer, manuscript
in preparation.
Tables
IA-IIIA
show physiological performance and lactate, pyruvate, and high energy
phosphate content in the treatment groups.
Regulation of Energy Metabolism of the Heart during Acute
Increase in Heart Work*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-oxidation (but not exogenous oleate oxidation) was
increased by the work jump, consistent with a decrease in the level of
malonyl-CoA. Glycogen and lactate were important buffers for carbon
substrate when heart work was acutely increased. Three mechanisms
contributed to high respiration from glycogen: 1) carbohydrate
oxidation was increased selectively; 2) stimulation of glucose
oxidation was delayed at glucose uptake; and 3) glycogen-derived
pyruvate behaved differently from pyruvate derived from extracellular
glucose. Despite delayed activation of pyruvate dehydrogenase relative
to phosphorylase, glycogen-derived pyruvate was more tightly coupled to
oxidation. Also, glycogen-derived lactate plus pyruvate contributed to
an increase in the relative efflux of lactate versus
pyruvate, thereby regulating the redox. Glycogen synthesis resulted
from activation of glycogen synthase late in the protocol but was timed
to minimize futile cycling, since phosphorylase a became
inhibited by high intracellular glucose.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-oxidation, since levels of malonyl-CoA were reduced. Increased
systemic nonesterified fatty acids resulting from peripheral lipolysis
could also have promoted fatty acid uptake in that study. Awan and
Saggerson (9) found reduced malonyl-CoA in isolated hearts and
stimulated palmitate oxidation by isolated (nonworking) heart myocytes
upon adrenergic stimulation. Therefore, the potential exists for direct
adrenergic stimulation of total -oxidation in the heart resulting
from lowered malonyl-CoA, which is a potent inhibitor of a limiting
enzyme for -oxidation, carnitine palmitoyltransferase I.
-oxidation should,
ideally, consider both exogenous and endogenous lipids. Therefore, we
extended existing studies in the following important ways. We examined
oxidation of all carbohydrates (glucose, glycogen, and
lactate) that together contribute virtually all PDC flux. We also
considered total -oxidation of exogenous and endogenous lipids. We
used a pulse-chase technique to measure glycogen turnover continuously.
Using this method, we find that the degree of coupling of glycogen
utilization to oxidation varies depending on the relation between
glycogenolysis and the capacity for carbohydrate oxidation (i.e. phosphorylase activation versus PDC
activation). To explain the tight coupling of glycogen to subsequent
oxidation, we examined the hypothesis that the burst of glycogenolysis
is coordinated with activation of PDC. Surprisingly, it was not.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-hydroxybutyrate, and 0.5 mM sodium L-lactate, and perfusion was
continued for 25 min to allow glycogen resynthesis while contractile
function returned to base line. Hearts were then switched to a
nonrecirculating mode as described previously (3). The perfusate during
this period was Krebs-Henseleit buffer containing 5 mM
glucose, 40 microunits/ml insulin, 0.5 mM sodium
L-lactate, 0.4 mM sodium oleate prebound to 3%
(w/v) bovine serum albumin (fraction V, fatty acid free, Intergen,
Purchase, NY), with 1.4 mM free Ca2+ (the
preparation was dialyzed against a large volume of albumin-free perfusate containing 1.5 mM CaCl2 and 0.1 mM EDTA). At 55 min, epinephrine bitartrate was added to 1 µM, and the height of the aortic overflow was raised to
140 cm above the heart. Hearts were freeze-clamped on their cannulae
with aluminum tongs cooled in liquid N2.
V difference times the coronary flow, using 1.06 mM for the concentration of dissolved O2 at
100% saturation (15). Electrodes were calibrated with air-saturated
water (19.6% O2 saturation after correction for water
vapor, 47 mm Hg at 37 °C). Hydraulic power (watts) is the product of
cardiac output (coronary plus aortic flow, m3/s) times the
afterload (pascals). Aortic pressure was recorded continuously with a
Millar pressure transducer (Millar Instruments, Houston, TX) at the
side arm of the aortic cannula, interfaced to a Gould physiologic
recorder (Gould model 2400S; Cleveland, OH).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-hydroxybutyrate) restored
performance (Fig. 2). We used
D--hydroxybutyrate as a co-substrate to maintain
incorporation of glucose into glycogen. Perfusions were switched to a
nonrecirculating mode starting at 45 min (beginning of the "chase"
for glycogen labeling), and the only change in substrate availability
at that time was the replacement of -hydroxybutyrate for a
physiologic long-chain fatty acid, oleate prebound to albumin.
Following a 10-min equilibration period, hearts were stimulated with
epinephrine (1 µM), and at the same time, we raised the
afterload by 40%. There was an immediate 95% increase in contractile
performance and 103% increase in MVO2. As occurs in
vivo, oxygen extraction was slightly increased by this
intervention, and O2 was almost completely extracted, but most of the increase in MVO2 resulted from an increase in
coronary flow (see Appendix). Immediately after the work jump, there
was no change in the supply/demand ratio for oxygen
(MVO2/power), suggesting that hearts did not experience
acute demand ischemia (this issue is examined further below). There was
a 14% reduction in the ratio MVO2/power after prolonged
stimulation, consistent with the well known reduction in cardiac
efficiency during adrenergic stimulation.
View larger version (14K):
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Fig. 1.
Perfusion protocol.
View larger version (25K):
[in a new window]
Fig. 2.
Physiological performance. A
shows contractile performance (milliwatts), and B shows
oxygen consumption (MVO2) throughout the protocol depicted
in Fig. 1 for three sets of matched perfusions that were used to trace
oxidation of three different 14C-labeled substrates. The
treatment groups are as follows: [14C]glucose group
( 
); [14C]glycogen group (
);
[14C]lactate group (
). Values are the mean ± S.E. for five perfusions in each treatment group.
A difference times coronary
flow, but the A side was fresh perfusate or endogenous
substrate), based on 14CO2 production for
carbohydrate oxidation, or 3H2O production from
exogenous [9,10-3H]oleate. The results are similar to
those we reported previously (3), determined in the absence of insulin
and lactate and without increasing the perfusion pressure. The new
finding is that, like glycogen, lactate oxidation is rapidly increased,
but unlike glycogen, increased lactate oxidation is sustained.
View larger version (25K):
[in a new window]
Fig. 3.
Rates of substrate oxidation. Rates
during the chase period of the protocol are depicted. ,
14CO2 from exogenous
[U-14C]glucose; , 14CO2 from
[14C]glycogen; , 14CO2 from
exogenous [U-14C]lactate; 
,
3H2O from exogenous
[9,10-3H]oleate. Values for glycogen were corrected for
incomplete labeling assuming uniform isotopic dilution at an enrichment
of 54.7% (Table I). Values are the mean ± S.E. for five
perfusions in each treatment group.
Glycogen content and enrichment
change in pool size, the value for degradation is
5.0-2.4 = 2.6 µmol/g, dry weight, and the corresponding rate
over 20 min is 0.13 µmol/min/g, dry weight (8% of exogenous oleate
oxidation). To calculate oxygen consumption, we assumed that
triglycerides were oxidized using a value of 25.5 mol of
O2/mol for the average acyl group (i.e. like oleate).
View larger version (22K):
[in a new window]
Fig. 4.
Measured and predicted values for oxygen
consumption. Oxygen consumption during the chase period of the
protocol is depicted. Open symbols, values
measured directly from the A V difference
for oxygen (MVO2, mean ± S.E., n = 15). Closed symbols, predicted values calculated
from the sum for the measured rates of oxidation of glucose, glycogen,
lactate, oleate, triglycerides, and release of pyruvate, using values
depicted in Fig. 3 and Appendix Table IIA.
View larger version (19K):
[in a new window]
Fig. 5.
Oxygen consumption resulting from total
glycogen oxidation compared with [14C]glycogen
oxidation. Oxygen consumption from glycogen oxidation during the
chase is depicted. Open symbols, oxygen
consumption resulting from oxidation of all glycogen, calculated by
taking the difference between MVO2 (shown in Fig. 4) and
oxygen consumption resulting from oxidation of every substrate except
glycogen. Closed symbols, oxygen consumption
resulting from oxidation of that portion of glycogen (55% of the
total) that was labeled with 14C (mean ± S.E.,
n = 5).
Glycogen synthase activity and the content of glucose 6-phosphate
the dependent form × 100%. Glycogen synthase
D did not differ between the different groups and was
6.9 ± 0.2 µmol/min/g, dry weight (n = 25).
0.49 ± 0.62 to
+4.42 ± 1.14, p < 0.05) prevented an acute
increase in the lactate/pyruvate ratio (Appendix Table IIA). The ratio
of lactate/pyruvate did eventually increase (after glycogenolysis had
ceased) despite persistent increase in the ratio for lactate efflux
relative to pyruvate efflux (3.85 ± 1.53, p < 0.05 versus the unstimulated state). We explain the result
by a switch from glycogen to glucose as the glycolytic substrate and
tighter coupling of glycogen to oxidation (see below), since 70% of
released lactate plus pyruvate was derived from glycogen during acute
stimulation, but it was entirely from exogenous glucose later in the protocol.
View larger version (30K):
[in a new window]
Fig. 6.
Cumulative metabolism of glucose and glycogen
by different pathways. Values during the chase period of the
protocol are depicted. A, glycogen; B, exogenous
glucose. The symbols for the A are as follows:
glycogen oxidation (14CO2 from
[14C]glycogen) ( ); glycolytic flux of glycogen
(14CO2 + [14C]lactate + [14C]pyruvate from [14C]glycogen) ();
percentage of glycolytic flux from glycogen that was oxidized (
).
The same symbols are used for exogenous glucose in
B. Values are the mean ± S.E. (n = 5 perfusions each for glucose and glycogen). *, p < 0.05 versus glucose.
Activity state of PDC and of phosphorylase and the content of effectors
of phosphorylase, AMP, and glucose
-oxidation (exogenous plus
endogenous lipids). We measured exogenous fatty acid oxidation directly
(3H2O from [9, 10-3H]oleate).
Total -oxidation was measured indirectly by a completely independent
method based on oxygen consumption not resulting from carbohydrate
oxidation. To check the validity of this approach, we calculated total
-oxidation based on the sum for oleate oxidation plus the value for
triglyceride oxidation measured during adrenergic stimulation (0.13 µmol/min/g, dry weight) that was described above (see "Triglyceride
Turnover and Pyruvate Release"). Values for total -oxidation
calculated from oleate plus triglycerides during acute stimulation
(1.71 µmol/min/g, dry weight) and prolonged stimulation (1.74 µmol/min/g, dry weight) are slightly less (not significant) than
values for total -oxidation based on oxygen consumption measurements
(Table IV). This is the expected result, since most of endogenous lipid
oxidation is from oxidation of triglycerides. Prior to adrenergic
stimulation, total -oxidation was in excellent agreement with oleate
oxidation (Table IV). This result is not surprising, because we expect
to find diminished rates of triglyceride oxidation in the unstimulated
state. We used the values for total -oxidation based on oxygen
consumption measurements as the best estimate of total -oxidation.
With adrenergic stimulation, the increase in oleate oxidation (20%)
was small and did not reach statistical significance. The increase in
total -oxidation (40%) was larger and was significant. The level of malonyl-CoA was decreased by 33% following prolonged stimulation (Table IV), which is consistent with the increase in total
-oxidation.
Rates of exogenous oleate oxidation and total -oxidation and the
level of malonyl-CoA
-oxidation was calculated from oxygen consumption not resulting from
carbohydrate oxidation.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-oxidation was increased by 40%, and the increase
was associated with a decrease in the level of malonyl-CoA. This is
expected based on the regulation of total -oxidation by malonyl-CoA
inhibition of carnitine palmitoyltransferase I (28). The increase in
total -oxidation was not in proportion to energy demand, however, so
increased carbohydrate oxidation, facilitated by activation of PDC,
became relatively more important. The pattern of substrate oxidation
that developed with more prolonged adrenergic stimulation (Fig. 3) is
in keeping with the observation in vivo that nonesterified
fatty acids and lactate are major respiratory substrates for the heart
in the exercising state (29, 30).
-oxidation in the steady state (31).
The capacity for mobilization of triglycerides, reflecting maximum
triglyceride lipase activity, is unknown. However, lipolytic capacity
is probably less than the tremendous capacity for glycogen mobilization
in heart, since triglycerides were not an important endogenous energy
substrate compared with glycogen in the short term. Glycogen may serve
to prevent transient supply-demand mismatch for carbon substrate during
acute increase in heart work, which would impair contractile function
in the short term.
-oxidation, regulated mostly by malonyl-CoA levels in the
cytosol, is independent of the activity state of PDC in the
mitochondria (compare PDC activities in Table III to malonyl-CoA levels
in Table IV). Our data do not support the suggestion that PDC
activation, by providing substrate for the synthesis of malonyl-CoA in
the cytosol (by acetyl-CoA carboxylase), is a mechanism for reciprocal
regulation of carbohydrate and fatty acid oxidation (32), at least in
the setting of adrenergic induced activation of PDC.
-oxidation that
we did find (40%) was not in step with carbohydrate oxidation or the
workloads, contrary to Neely et al. (6). We also confirmed
the report by Awan and Saggerson that epinephrine decreases the level
of malonyl-CoA in isolated hearts (9). We extend the results to include
the relation between malonyl-CoA and total -oxidation, which is
consistent with stimulation of -oxidation at carnitine palmitoyltransferase I and inhibition of carnitine palmitoyltransferase I by malonyl-CoA (28). Since the concentration of nonesterified fatty
acids was fixed in our perfusions, increased -oxidation must have
resulted directly from adrenergic stimulation and/or increased heart
work and is not secondary to changes in delivery of nonesterified fatty
acids. Although Hall et al. (8) found increased
exogenous fatty acid uptake by dobutamine in pig heart in vivo, the result does not contradict our findings, since,
as we will show elsewhere,2
the sensitivity to workload of -oxidation and triglyceride turnover becomes enhanced under metabolic conditions (high lactate and nonesterified fat) that develop in vivo during exercise or
adrenergic stimulation.
Physiologic performance of the treatment groups
Lactate and pyruvate content and fluxes
High energy phosphates, adenine nucleotides, and inorganic phosphate
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Division of
Cardiology, Dept. of Internal Medicine, University of Texas-Houston Medical School, 6431 Fannin, Houston TX 77030. Tel.: 713-500-6568; Fax:
713-500-6556.
APPENDIX
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ska, M.
(1985)
Am. J. Physiol.
249,
H799-H806
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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 M. E. Young, P. H. Guthrie, P. Razeghi, B. Leighton, S. Abbasi, S. Patil, K. A. Youker, and H. Taegtmeyer Impaired Long-Chain Fatty Acid Oxidation and Contractile Dysfunction in the Obese Zucker Rat Heart Diabetes, August 1, 2002; 51(8): 2587 - 2595. [Abstract] [Full Text] [PDF]
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L. A. Gustafson and J. H. G. M. Van Beek Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2259 - H2264. [Abstract] [Full Text] [PDF]
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M. E. Young, P. McNulty, and H. Taegtmeyer Adaptation and Maladaptation of the Heart in Diabetes: Part II: Potential Mechanisms Circulation, April 16, 2002; 105(15): 1861 - 1870. [Full Text] [PDF]
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H. Taegtmeyer, P. McNulty, and M. E. Young Adaptation and Maladaptation of the Heart in Diabetes: Part I: General Concepts Circulation, April 9, 2002; 105(14): 1727 - 1733. [Full Text] [PDF]
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 F. M. Campbell, R. Kozak, A. Wagner, J. Y. Altarejos, J. R. B. Dyck, D. D. Belke, D. L. Severson, D. P. Kelly, and G. D. Lopaschuk A Role for Peroxisome Proliferator-activated Receptor alpha (PPARalpha ) in the Control of Cardiac Malonyl-CoA Levels. REDUCED FATTY ACID OXIDATION RATES AND INCREASED GLUCOSE OXIDATION RATES IN THE HEARTS OF MICE LACKING PPARalpha ARE ASSOCIATED WITH HIGHER CONCENTRATIONS OF MALONYL-CoA AND REDUCED EXPRESSION OF MALONYL-CoA DECARBOXYLASE J. Biol. Chem., February 1, 2002; 277(6): 4098 - 4103. [Abstract] [Full Text] [PDF]
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J. J. Krueger, X.-H. Ning, B. M. Argo, O. Hyyti, and M. A. Portman Triidothyronine and epinephrine rapidly modify myocardial substrate selection: a 13C isotopomer analysis Am J Physiol Endocrinol Metab, November 1, 2001; 281(5): E983 - E990. [Abstract] [Full Text] [PDF]
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J. C. Chatham, C. Des Rosiers, and J. R. Forder Evidence of separate pathways for lactate uptake and release by the perfused rat heart Am J Physiol Endocrinol Metab, October 1, 2001; 281(4): E794 - E802. [Abstract] [Full Text] [PDF]
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M. E. Young, G. W. Goodwin, J. Ying, P. Guthrie, C. R. Wilson, F. A. Laws, and H. Taegtmeyer Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids Am J Physiol Endocrinol Metab, March 1, 2001; 280(3): E471 - E479. [Abstract] [Full Text] [PDF]
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G. W. Goodwin, D. M. Cohen, and H. Taegtmeyer [5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway Am J Physiol Endocrinol Metab, March 1, 2001; 280(3): E502 - E508. [Abstract] [Full Text] [PDF]
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H. Taegtmeyer Metabolism--the lost child of cardiology J. Am. Coll. Cardiol., October 1, 2000; 36(4): 1386 - 1388. [Full Text] [PDF]
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G. W. Goodwin and H. Taegtmeyer Improved energy homeostasis of the heart in the metabolic state of exercise Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1490 - H1501. [Abstract] [Full Text] [PDF]
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C. Depre and H. Taegtmeyer Metabolic aspects of programmed cell survival and cell death in the heart Cardiovasc Res, February 1, 2000; 45(3): 538 - 548. [Abstract] [Full Text] [PDF]
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G. W. Goodwin and H. Taegtmeyer Regulation of fatty acid oxidation of the heart by MCD and ACC during contractile stimulation Am J Physiol Endocrinol Metab, October 1, 1999; 277(4): E772 - E777. [Abstract] [Full Text] [PDF]
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J. C. Chatham, Z.-P. Gao, and J. R. Forder Impact of 1 wk of diabetes on the regulation of myocardial carbohydrate and fatty acid oxidation Am J Physiol Endocrinol Metab, August 1, 1999; 277(2): E342 - E351. [Abstract] [Full Text] [PDF]
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M. E. Young, F. A. Laws, G. W. Goodwin, and H. Taegtmeyer Reactivation of Peroxisome Proliferator-activated Receptor alpha Is Associated with Contractile Dysfunction in Hypertrophied Rat Heart J. Biol. Chem., November 21, 2001; 276(48): 44390 - 44395. [Abstract] [Full Text] [PDF]
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L. A. Gustafson and J. H. G. M. Van Beek Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2259 - H2264. [Abstract] [Full Text] [PDF]
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M. P. Chandler, H. Huang, T. A. McElfresh, and W. C. Stanley Increased nonoxidative glycolysis despite continued fatty acid uptake during demand-induced myocardial ischemia Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1871 - H1878. [Abstract] [Full Text] [PDF]
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 M. E. Young, P. Razeghi, and H. Taegtmeyer Clock Genes in the Heart : Characterization and Attenuation With Hypertrophy Circ. Res., June 8, 2001; 88(11): 1142 - 1150. [Abstract] [Full Text] [PDF]
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M. E. Young, P. Razeghi, A. M. Cedars, P. H. Guthrie, and H. Taegtmeyer Intrinsic Diurnal Variations in Cardiac Metabolism and Contractile Function Circ. Res., December 7, 2001; 89(12): 1199 - 1208. [Abstract] [Full Text] [PDF]
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