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1 Department of Physiology and Biophysics and 2 Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-4970; and 3 Department of Nutrition, University of Montreal, Montreal, Quebec H3C 3Y7, Canada
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-13C3]lactate and/or [U-13C3] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 ± 0.3 and 41.5 ± 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.
cardiac; citric acid cycle; pyruvate dehydrogenase; pyruvate carboxylation; myocardial metabolism
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE HEART IS DEPENDENT
ON a high rate of citric acid cycle (CAC) flux to support ATP
regeneration and contractile function. Although oxaloacetate (OAA) is
recycled in the CAC, there is a constant mitochondrial efflux of CAC
intermediates (cataplerosis) in normal cardiomyocytes (Fig.
1). Cataplerosis is compensated for by an
equivalent entrance of CAC intermediates (anaplerosis). Because the
pool of CAC intermediates is small compared with the throughput of the
cycle, maintenance of heart function requires exact matching between
anaplerosis and cataplerosis. Perfusion of isolated rat hearts with
anaplerotic substrates such as pyruvate (4, 29), succinate
(5), fumarate (14), or glutamate (2) results in improved postischemic recovery and
decreased reperfusion injury. This suggests that anaplerosis plays an
important role in the correction of metabolic alterations resulting
from ischemia and reperfusion.
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The well-perfused heart readily oxidizes pyruvate formed from glycolysis and the oxidation of lactate (25). The primary fate of pyruvate in the normoxic heart is decarboxylation through mitochondrial pyruvate dehydrogenase (25). In isolated, perfused rat hearts, pyruvate is also carboxylated to form OAA or malate, presumably via pyruvate carboxylase or malic enzyme (8, 9, 11, 12, 26, 27) (Fig. 1). Evidence for pyruvate carboxylation in rat hearts is based on substantial activities of pyruvate carboxylase and malic enzyme (13, 27) and the incorporation of label from [1-14C]pyruvate into malate and citrate in perfused hearts (15, 21). Furthermore, inhibition of malic enzyme by hydroxymalonate decreased the incorporation of 14C from [1-14C]pyruvate into citrate and malate and impaired the contractile power of the heart (21). However, although these reports demonstrated the existence and importance of pyruvate carboxylation in the isolated heart, they did not quantify the rate or the relative contribution of pyruvate carboxylation to the CAC flux.
Recent experiments with 13C substrates have expanded
the field through the analysis of 13C isotopomers of CAC
intermediates. One can now assess either positional or mass isotopomers
of the CAC intermediates using NMR or gas chromatography-mass
spectrometry (GC-MS), respectively. Studies with
[13C]acetate (6, 7), propionate
(11), or aspartate (12) and NMR analysis have
demonstrated substantial incorporation of carbon into the CAC through
anaplerotic pathways. In these experiments, the labeling pattern of
glutamate was measured as a surrogate of that of 
-ketoglutarate
(7).
Comte and colleagues (8, 9) recently measured the relative contributions of pyruvate carboxylation and decarboxylation to citrate formation in isolated rat hearts perfused with [U-13C3]pyruvate and [U-13C3]lactate. These relative rates were computed from the mass isotopomer distribution (MID) of effluent citrate. Under conditions of physiological concentrations of pyruvate ([pyruvate], 0.2 mM) and lactate ([lactate], 1 mM), pyruvate carboxylation accounted for 6.3% whereas pyruvate decarboxylation was responsible for 4.1% of citrate formation. When fatty acid concentrations were decreased from 0.2 to 0.02 mM, pyruvate carboxylation decreased to 3.7% yet pyruvate decarboxylation increased to 36% of citrate formation. These hearts were not perfused with long-chain fatty acids but rather with the medium-chain fatty acid octanoate. Octanoate enters the mitochondria independent of carnitine palmitoyl transferase I and inhibits pyruvate dehydrogenase flux.
Because all of the above data were gathered in isolated perfused hearts, it is not clear whether the findings reflect cardiac pyruvate metabolism in vivo, where the heart is subjected to the combined influence of multiple substrates, hormones, and neurological stimuli. Therefore the goals of the present investigation were 1) to develop a reliable method to assess simultaneously the relative and absolute rates of pyruvate carboxylation and decarboxylation in vivo, and 2) to determine whether inhibiting pyruvate dehydrogenase flux with octanoate results in stimulation of pyruvate carboxylation. Our studies were performed in anesthetized swine subjected to an intracoronary infusion of [U-13C3]pyruvate and [U-13C3]lactate. Metabolic rates were computed from the MIDs of the CAC intermediates assayed by GC-MS.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chemicals
Chemicals, enzymes, and coenzymes were purchased from Boehringer Mannheim (Indianapolis, IN) and Sigma-Aldrich Chemicals (Milwaukee, WI). [2H6]succinic acid, [U-13C3]lactate, and [U-13C3]pyruvate were obtained from Isotec (Miamisburg, OH). The derivatization agent N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide was supplied by Regis Chemical (Morton Grove, IL). Intralipid solution was obtained from Baxter Healthcare (Deerfield, IL).Experimental Model
An in vivo technique was used to deliver 13C-labeled substrates directly into the left anterior descending (LAD) coronary artery of swine (23, 24). Overnight-fasted domestic swine (27-36 kg) of either sex were sedated with Telazol (6 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg + 5 mg · kg
1 · h1 iv),
intubated via a tracheotomy, and ventilated to maintain arterial blood
gases in the normal range (PO2 > 100 mmHg, PCO2 35-45 mmHg, and pH
7.35-7.45). Rectal temperature was continuously monitored and
maintained between 35 and 37°C with heating blankets. The right
femoral artery and vein were cannulated for extracorporeal bypass and
venous infusions of heparin and Intralipid, respectively. A 7-Fr
high-fidelity pressure-transducer catheter (Millar, Houston, TX) was
positioned in the left ventricle via the carotid artery. The heart was
exposed via a midline sternotomy and suspended in a pericardial sling.
The animal was then heparinized (300 U/kg bolus + 150 U · kg1 · h1 iv) and
infused with a 20% triglyceride emulsion (Intralipid 20%, 0.3 ml · kg1 · h1 iv) to
increase plasma free fatty acids (FFA) to 0.6 mM (23). This concentration of FFA is comparable to that measured in
overnight-fasted humans (36). Coronary blood flow in the
anterior wall was controlled by an extracorporeal circuit as previously
described (23, 24). The LAD coronary artery was cannulated
and perfused at 30-40 ml/min via roller pumps with blood from the
femoral artery (23, 24). The anterior interventricular
vein was cannulated to collect venous blood samples from the perfusion
territory of the LAD. Previous studies have shown that 91% of the
interventricular venous blood is derived from the LAD coronary artery
(18). The coronary perfusion pump flow was adjusted to
give an interventricular venous Hb saturation of 35-40%
(23, 24). Left ventricular pressure was continuously recorded using an online data acquisition system (BioPac Acknowledge).
Experimental Protocols
All three protocols included an equilibration period of 40 min followed by a 60-min intracoronary infusion of labeled substrates. Stock solutions of 99% [U-13C3]lactate and/or 99% [U-13C3]pyruvate and unlabeled octanoate were infused directly into the LAD perfusion circuit from 0 to 60 min at a rate of 6.5 µl/ml of LAD blood flow. In group I (n = 7), the concentrations of [U-13C3]lactate and [U-13C3]pyruvate in the infusate were 154 and 15.4 mM, respectively, so that the [lactate] and [pyruvate] in LAD blood would be raised by 1.0 and 0.1 mM, respectively. In group II (n = 7), the concentration of [U-13C3]pyruvate in the infusate was 154 mM so that the [pyruvate] in LAD blood would be raised by 1 mM. Group III (n = 7) received [U-13C3]pyruvate as in group II and unlabeled octanoate from
40 to 60 min at a concentration of 154 mM, to yield an octanoate concentration of 1 mM in the LAD blood.
In all three groups, recordings of left ventricular pressure,
end-diastolic pressure, peak first derivative of the left ventricular pressure with time (dP/dt), heart rate, and arterial and
venous blood samples were taken at 12, 5, 5, 15, 20, 25, 35, 45, and 55 min. Plasma samples were stored at 80°C until further
analysis. At 60 min, a large punch biopsy (3 g) of the LAD bed was
quickly taken, freeze-clamped, and stored at 80°C until analysis.
The heart was excised and black ink was infused down the right and left
main coronary arteries to identify the LAD perfusion bed, which was
dissected and weighed (30.8 ± 1.5 g).
Analytical Methods
Arterial and venous pH, PCO2, and PO2 were determined on a blood-gas analyzer, and Hb concentration and saturation were measured on a hemoximeter (ABL3 and OSM3, Radiometer America, Cleveland, OH). The concentrations of plasma FFA, glucose, lactate, pyruvate, citrate, and
-hydroxybutyrate as well as tissue lactate, pyruvate, and malate
were determined using spectrophotometric enzymatic assays (1, 24,
32). Tissue concentrations were measured immediately following
homogenization in neutralized perchloric acid extracts to prevent loss
of pyruvate from freeze/thaw (34).
Isotopic enrichments of plasma lactate and pyruvate were determined from the GC-MS analysis of the tert-butyldimethylsilyl (TBDMS) derivatives (8, 9). The MID of tissue lactate, pyruvate, citrate, succinate, fumarate, malate, and the OAA moiety of citrate were assayed as the TBDMS derivatives as described previously (8, 9, 14, 31). Analyses were performed on a Hewlett-Packard 5890 series II gas chromatograph with an HP-5 capillary column (50 m length, 0.2 mm inside diameter, 0.3 µm film thickness) coupled to a model 5970 mass selective detector. Helium gas flow in the capillary column was 0.8-1.0 ml/min. Individual enrichments are averages of two or three GC-MS injections. The tissue concentrations of citrate, succinate, and fumarate were also assayed by GC-MS in samples of 250 mg of tissue spiked with 0.25 µmol of tricarballylic acid and 0.21 µmol of [1,2,2,3,3,4-2H6]succinate as internal standards (10, 14). The TBDMS derivative of tricarballylic acid was eluted at 21.1 min and was monitored at the mass-to-charge ratio (m/z) of 461. Standard curves were used to determine the tissue concentrations.
Calculations.
Myocardial oxygen consumption (M
O2) and
uptakes of glucose and FFA were calculated as the product of the
arteriovenous concentration difference and the myocardial blood flow.
The myocardial blood flow was calculated as the LAD perfusion pump flow
divided by the mass of the LAD perfusion bed.
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(1) |
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(2) |
O2 and the
stoichiometric relationships between oxygen consumption and citrate
formation from fat and carbohydrate. For groups I and
II, it was assumed that 1 µmol of consumed O2
resulted in the formation of 0.6, 0.348, and 0.353 µmol of citrate
from glucose, palmitate, and oleate, respectively. It was
further assumed that palmitate and oleate supply 40 and 60% of the
fatty acids oxidized by the heart, respectively (35). The
CAC flux was calculated as
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(3) |
Statistical Analyses
Data are presented as means ± SE. The hemodynamic variables were compared among the three protocols using repeated measure two-way ANOVA. Statistical significance for the isotopic enrichments and the rates of pyruvate carboxylation and decarboxylation were determined using one-way ANOVA followed by a nonparametric post hoc test (Dunn's test).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiovascular Parameters
There were no significant changes over the course of the experiment or between groups at any time point in heart rate, peak systolic left ventricular pressure, peak left ventricular dP/dt, MO2 (Fig.
2), or in left ventricular end-diastolic
pressure or myocardial blood flow (data not shown).
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Plasma Metabolites
Plasma [pyruvate] and [lactate] and enrichments.
Before the infusions of [U-13C3]lactate
and/or pyruvate, the arterial plasma [lactate] values were 1.37 ± 0.08, 1.38 ± 0.05, and 1.40 ± 0.10 mM; and [pyruvate]
values were 0.11 ± 0.02, 0.17 ± 0.04, and 0.15 ± 0.02 mM, for groups I, II, and III, respectively (Fig.
3). After 15 min of labeled substrate
infusion, the plasma [lactate] and [pyruvate] in the blood entering
the LAD territory stabilized at 2.16 ± 0.07 and 0.24 ± 0.02 mM in group I, 1.70 ± 0.07 and 1.13 ± 0.07 mM in
group II, and 1.51 ± 0.10 and 1.10 ± 0.07 mM in
group III, respectively (Fig. 3). The M3 enrichments of
lactate and pyruvate stabilized after 15 min at 24.6 ± 4.0 and
44.5 ± 2.6% in group I, 2.3 ± 0.3 and 70.0 ± 2.9% in group II, and 2.3 ± 0.4 and 72.7 ± 3.2% in group III, respectively (Fig. 4).
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FFA and glucose uptakes.
The LAD plasma FFA levels were stable and identical in groups
I and II (0.62 ± 0.04 and 0.57 ± 0.07 mM,
respectively). In group III, the LAD arterial FFA
concentration increased significantly from 5 to 60 min (1.76 ± 0.14 mM at 55 min) as a result of the infusion of octanoate
(P < 0.05). The net FFA uptakes from 45 to 55 min were
similar in groups I and II (0.10 ± 0.05 and
0.07 ± 0.08 µmol · g1 · min1,
respectively) and significantly higher in group III
(0.51 ± 0.19 µmol · g1 · min1)
(P < 0.05). The rates of glucose uptake at the end of
the study were similar in all groups (0.23 ± 0.06, 0.15 ± 0.04, and 0.12 ± 0.05 µmol · g1 · min1, respectively).
Citrate and -hydroxybutyrate balance.
The LAD plasma citrate concentrations were unchanged over the
time course of the study, and there was no difference among the groups
(54 ± 2, 46 ± 3, and 46 ± 2 µM for groups
I, II, and III, respectively, from 45 to 55 min). The net citrate releases were stable and similar in all groups
(12 ± 7, 13 ± 4, and 9 ± 3 nmol · g1 · min1, for
groups I, II, and III,
respectively). The LAD arterial -hydroxybutyrate concentrations were unchanged over the time course of the study and
there were no differences among groups (0.116 ± 0.009, 0.076 ± 0.005, and 0.082 ± 0.009 mM for groups I,
II, and III, respectively, at 45 to 55 min). The
net balance of -hydroxybutyrate (arteriovenous difference × flow) was variable and similar among the groups (1 ± 4, 10 ± 5, and 10 ± 5 nmol · g1 · min1 for
groups I, II, and III, respectively).
Tissue Metabolites
Tissue [lactate] and [pyruvate] and enrichments.
The tissue [pyruvate] and [lactate] and the
[lactate]-to-[pyruvate] ratio were similar in groups I
and II despite the high rate of
[U-13C3]pyruvate infusion without
[U-13C3]lactate in group II (Table
1). The octanoate-treated myocardium (group III) had more than double the [pyruvate] found in
groups I and II without increase in tissue
lactate. The lower [lactate]/[pyruvate] ratio in group
III probably resulted from a decrease in pyruvate decarboxylation
because octanoate provided an alternative source of acetyl-CoA.
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CAC intermediate tissue concentrations and enrichments.
The tissue concentrations of citrate, succinate, fumarate, and malate
were similar in groups I and II (Table
2). The infusion of octanoate
(group III) resulted in a significant increase in the tissue
content of these intermediates.
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Flux Parameters
Relative rates of pyruvate carboxylation and decarboxylation.
The relative rates of pyruvate carboxylation and decarboxylation were
calculated from the enrichments of the M2 acetyl-CoA moiety of citrate,
corrected M3 OAA moiety of citrate, and M3 tissue pyruvate (see
MATERIALS AND METHODS). The relative rates of pyruvate
decarboxylation were similar in groups I and II
(41.5 ± 2.0 and 34.3 ± 1.3% of citrate formation,
respectively) but were reduced by 93% by octanoate infusion in
group III (3.1 ± 0.4%; Fig.
5). The relative rates of pyruvate
carboxylation were similar in all groups (4.7 ± 0.3, 5.7 ± 0.3, and 3.4 ± 0.3%, respectively; Fig. 5). Note the similarity
of these fluxes despite more than a 10-fold difference in the degree of
13C recycling in the CAC, as reflected in the difference
between the uncorrected and corrected M3 OAA moiety of citrate (~60%
vs. 4%; Table 3). This demonstrates the robustness of our method for
calculating pyruvate carboxylation and shows that it is independent of
the extent of correction for 13C recycling.
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Absolute substrate fluxes.
Absolute rates of pyruvate decarboxylation and carboxylation were
calculated from the rate of CAC flux, which was calculated from the
MO2 as described in MATERIALS AND
METHODS. The rates of CAC flux were not different among the three
groups (1.6 ± 0.1, 1.2 ± 0.1, and 1.3 ± 0.2 µmol · g1 · min1 for
groups I, II, and III, respectively).
The rate of pyruvate decarboxylation was similar in group I
(658 ± 103 nmol · g1 · min1) and
group II (418 ± 80 nmol · g1 · min1) but was
markedly inhibited by octanoate infusion in group III (45 ± 20 nmol · g1 · min1). The
rates of pyruvate carboxylation were similar in group I (71 ± 11 nmol · g1 · min1),
group II (68 ± 11 nmol · g1 · min1), and
group III (43 ± 7 nmol · g1 · min1).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This is the first quantitative study on anaplerosis via pyruvate carboxylation in the heart in vivo. Using [U-13C3]pyruvate and [U-13C3]lactate, we measured the partitioning of pyruvate between decarboxylation and anaplerotic carboxylation in the swine heart. We found that pyruvate carboxylation accounts for ~3-6% of the CAC flux under normal metabolic and hemodynamic conditions when arterial [pyruvate] is between 0.2 and 1.1 mM. Furthermore, the rate of pyruvate carboxylation was not significantly modulated by inhibition of pyruvate decarboxylation in the presence of octanoate.
The role of pyruvate carboxylation in the maintenance of cardiac
function is not well understood. The heart has a high rate of CAC flux
(1.6 µmol of citrate
formed · g1 · min1 in the
present study) with low concentrations of intermediates (Table 2). This
results in relatively rapid turnover times of metabolite pools (from
56 s for citrate to 4 s for succinate). Anaplerosis
replenishes CAC intermediates lost from the cycle by cataplerosis, thus
maintaining the concentrations of the intermediates and optimal CAC
flux. In support of this concept, Russell and Taegtmeyer
(21) found that inhibition of malic enzyme in perfused rat
hearts results in 1) 75% and 93% decreases in the
incorporation of [1-14C]pyruvate into citrate and malate,
respectively; 2) depletion of CAC intermediates; and
3) reduction in the contractile power of the heart. The
present investigation further supports the concept that pyruvate
carboxylation is a constitutive process in the heart, and is necessary
for maintenance of normal CAC flux.
Infusion of 1 mM octanoate markedly decreased the contribution of pyruvate to acetyl-CoA production from 34 to 3%, but did not affect the relative (Fig. 5) or absolute rates of pyruvate carboxylation. Despite the dramatic reduction in pyruvate decarboxylation, there were no changes in the net uptakes of pyruvate and lactate (Fig. 3). Although there was a doubling of tissue [pyruvate] (Table 1) that accounted for a small portion of the lactate and pyruvate uptake in this group, it is unclear how the remaining pyruvate was metabolized by the heart. It should also be noted that octanoate increased the tissue content of CAC intermediates (Table 2) without changing the rates of CAC flux or anaplerotic pyruvate carboxylation. There is an increase in the concentration of CAC intermediates when there is an excess availability of acetyl-CoA (7, 28, 33). Our results add further support to the concept that the size of the pool of CAC intermediates is not solely related to the rate of CAC flux, but also to the availability of substrates (28).
Anaplerotic fluxes balance equivalent fluxes out of the CAC through cataplerosis. Because the rate of anaplerosis by pyruvate carboxylation is 3 to 6% of the CAC flux, the minimum rate of cataplerosis must also be ~3-6% of the CAC flux. The exact rate of in vivo cardiac cataplerosis is difficult to derive because the efflux of enriched CAC intermediates is not readily followed. Some potential sites of cataplerosis may be decarboxylation of malate (via malic enzyme) and citrate efflux. In the liver, malic enzyme can operate as either a carboxylase or a decarboxylase; a similar reversibility has been suggested for the heart in 13C NMR studies (10). On the other hand, Sundqvist and colleagues (26, 27) found that the energetics of the malic enzyme reaction in the normal rat heart would not allow for the enzyme to function as a decarboxylase. In the present investigation, cataplerotic decarboxylation of malate to pyruvate would result in substantial M1 and M2 labeling of pyruvate. We observed no M1 or M2 enrichment of pyruvate, demonstrating no detectable loss of CAC carbon from cataplerosis via malic enzyme. A similar finding was reported for the heart perfused ex vivo (9, 31).
Regarding citrate cataplerosis, there was net citrate release by the
heart (12, 13, and 9 nmol · g1 · min1 for
groups I, II, and III, respectively).
These values are comparable to those obtained in isolated rat hearts
[17-22
nmol · g1 · min1, assuming
a heart wet weight of 1 g (31)] and catheterized human hearts [~5
nmol · g1 · min1, assuming
a myocardial blood flow of 1.0 ml · g1 · min1(30)].
In the present investigation the net citrate efflux amounted to 17, 19, and 21% of the rate of pyruvate carboxylation for the respective
groups. This suggests that there are other major cataplerotic processes
(e.g., -ketoglutarate to glutamate). Alternatively, the net release
of citrate might underestimate citrate cataplerosis if some citrate is
cleaved by cytosolic ATP-citrate lyase, as proposed for skeletal muscle
(22). Therefore, the lack of correspondence between
citrate release and absolute pyruvate carboxylation cannot be taken as
evidence for the contribution of other anaplerotic pathway(s).
We did not observe significant increases in tissue [pyruvate] or MPE, or a decrease in the [lactate]-to-[pyruvate] ratio when arterial pyruvate was increased fivefold from 0.24 to 1.1 mM (Fig. 3, Table 1). This unexpected finding suggests that pyruvate is either not readily taken up by the myocardium, or it is taken up and rapidly converted to lactate by lactate dehydrogenase. Consequently, there was no difference in the rates of pyruvate decarboxylation or carboxylation between groups I and II. Additional investigations are required to assess whether higher arterial [pyruvate] are needed to increase contribution of pyruvate to citrate formation in vivo. Bunger and Mallet and co-workers (4, 29) demonstrated that adding 1-5 mM pyruvate to the perfusate of isolated guinea pig heart improved contractile function after ischemia. Furthermore, the addition of 5 mM pyruvate resulted in a 15-fold increase in tissue citrate concentration after ischemia (29), suggesting anaplerosis from pyruvate. It is possible that the heart more readily takes up pyruvate during times of stress, such as increased contractile work, myocardial ischemia, and postischemic reperfusion.
The labeling patterns of CAC intermediates could not shed definite
light on whether pyruvate carboxylation in the heart is catalyzed by
pyruvate carboxylase or malic enzyme. It is not known which of these
two pathways predominates in the heart. The activities of malic enzyme
and pyruvate carboxylase have been measured in rat hearts (0.695 and
0.116 µmol · g1 · min1,
respectively) (27). The respective roles of malic enzyme
and pyruvate carboxylase have been examined by modulating these
activities by pharmacological or nutritional perturbations. In isolated
rat hearts, inhibition of malic enzyme with hydroxymalonate results in
a decrease in the content of CAC intermediates and a decrease in the
incorporation of [1-14C]pyruvate into malate
(21). On the other hand, biotin deficiency, which results
in a 90% reduction in pyruvate carboxylase activity, causes no change
in the incorporation of [1-14C]pyruvate into malate in
isolated rat hearts (27). These findings suggest that
malic enzyme, and not pyruvate carboxylase, predominates in the
isolated rat heart. Individual fluxes through malic enzyme and pyruvate
carboxylase cannot be separately measured with current isotopic
techniques; thus a thorough understanding of the enzymatic regulation
of pyruvate carboxylation awaits new methodological developments.
The fluxes of pyruvate carboxylation measured in our in vivo study are similar to rates measured by Comte and colleagues (8, 9) in hearts perfused with 11 mM glucose, 0.5-1.0 mM lactate, 0.05-0.2 mM pyruvate, and 0.02-0.2 mM octanoate (2.5-8% of citrate synthase flux). More recently, Vincent and coworkers (31) reported a rate of pyruvate carboxylation corresponding to 2.4% of citrate synthase flux in hearts perfused with more physiological substrates (5 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.4 mM oleate, and 8 nM insulin). This value is similar to the relative fluxes measured in groups I, II, and III (4.7, 5.7, and 3.4%, respectively).
Methodological limitations encountered in assessing anaplerotic pyruvate carboxylation in vivo were also similar to those reported for perfused rat hearts (8, 9). The first constraint on the use of [U-13C3]lactate and [U-13C3]pyruvate to measure the rates of pyruvate carboxylation and decarboxylation of in vivo hearts results from the need to impose a substantial enrichment of substrate in the blood as it enters the organ. The lower limit on the amount of [U-13C3]lactate and [U-13C3]pyruvate that needed to be infused was imposed by the precision in the measurement of the enrichment of the OAA moiety of citrate. To achieve the required M3 enrichments of influent lactate and pyruvate, the baseline plasma [pyruvate] was increased from 0.11 ± 0.02 to 0.24 ± 0.02 mM, and the [lactate] was increased from 1.37 ± 0.08 to 2.16 ± 0.07 mM in group I. These high but physiological concentrations of pyruvate and lactate might result in higher rates of pyruvate carboxylation and decarboxylation than would occur in the unperturbed swine heart. Another potential limitation of our method is the use of whole-tissue pyruvate M3 enrichment, rather than intracellular enrichment, in the calculation of the rates of pyruvate carboxylation and decarboxylation. Use of whole tissue pyruvate M3 enrichment would result in a slight underestimation of the actual rates of pyruvate carboxylation and decarboxylation.
In conclusion, under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the CAC flux even when pyruvate dehydrogenase is inhibited by octanoate. Cataplerotic citrate efflux represents at least 17-21% of the rate of pyruvate carboxylation. Our data suggests that pyruvate carboxylation is a constitutive process in the heart that appears to be necessary for maintenance of normal CAC flux.
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ACKNOWLEDGEMENTS |
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The authors thank F. David, M. Chandler, and C. Paxson for assistance in conducting this study.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-58653 (to W. C. Stanley) and HL-59219 (to H. Brunengraber), and Grant-in-Aid 005031N from the American Heart Association National Center (to W. C. Stanley).
Address for reprint requests and other correspondence: W. C. Stanley, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: WCS4{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 March 2000; accepted in final form 30 May 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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