Download Evidence of separate pathways for lactate uptake and release by the

Am J Physiol Endocrinol Metab
281: E794–E802, 2001.
Evidence of separate pathways for lactate uptake and
release by the perfused rat heart
JOHN C. CHATHAM,1,3 CHRISTINE DES ROSIERS,2 AND JOHN R. FORDER4
1
Division of Magnetic Resonance Research, Department of Radiology, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205; 2Department of Nutrition, University of Montreal,
Montreal, H2L 4M1 Quebec, Canada; and 3Center for Nuclear Magnetic Resonance Research and
Development, Departments of Medicine and 4Biomedical Engineering, The University of Alabama
at Birmingham, Birmingham, Alabama 35294
Received 2 November 2000; accepted in final form 1 June 2001
of lactate in the resting
state in humans (30, 49) and in animals such as rats
and dogs (16, 25, 26, 52) is in the range of 0.5–1 mM
and is readily increased after physiological stimuli
such as exercise, insulin, or hypoxia (29, 30, 42, 52).
Drake et al. (17) demonstrated that lactate extraction
by the heart is proportional to its circulating concentration, providing up to 90% of substrate oxidation.
Under in vivo conditions, the majority of lactate taken
up by the heart is subsequently oxidized (2, 19) and
thus represents a significant source of energy for the
heart (2, 17, 48, 52). In light of the importance of
lactate for myocardial energy production, an increasing number of investigators are now including both
glucose and lactate as substrates in ex vivo perfusion
studies of the regulation of cardiac metabolism (1, 13,
20, 40). An intriguing observation that has been reported in both in vivo and ex vivo studies is that, under
conditions where there is oxidation of exogenous lactate, there appears to be simultaneous efflux of lactate
originating from metabolism of exogenous glucose (1, 2,
19, 20, 30, 40, 49).
To date, the simultaneous uptake and efflux of lactate in the heart have not been satisfactorily explained.
A variety of explanations could account for this observation, including tissue level heterogeneity resulting
from decreased blood flow in subendocardium (49) or
inter- or intracellular compartmentation of lactate metabolism. Arguing against tissue heterogeneity is an in
vivo study in newborn lambs (2), in which simultaneous myocardial lactate uptake and release were observed despite reports that, in the lamb, blood flow is
higher in the endocardial than in the epicardial region
(18). However, this study did not address the possibility of compartmentation of lactate metabolism either
at the tissue level due to the release of lactate from
other cell types such as endothelial or smooth muscle
cells (intercellular compartmentation) or at the cellular level due to the physical separation of enzymes
involved in lactate production and uptake or interactions between the enzymes of these pathways leading
to metabolic channeling (32, 43–45).
Recent studies have provided some evidence that
compartmentation of lactate metabolism might exist in
the myocardial cell. For example, Xu et al. (51) have
shown functional association of the glycolytic pathway
with the sarcoplasmic reticulum. Furthermore, Brooks
et al. (7) have demonstrated the presence of lactate
Address for reprint requests and other correspondence: J. C.
Chatham, Center for NMR Research and Development, CNIR Bldg.,
Univ. of Alabama at Birmingham, 828 Eighth Court South, Birmingham, AL 35294–4470 (E-mail: [email protected]).
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.
nuclear magnetic resonance spectroscopy; gas chromatography-mass spectrometry; diabetes; palmitate
THE ARTERIAL CONCENTRATION
E794
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
Chatham, John C., Christine Des Rosiers, and John
R. Forder. Evidence of separate pathways for lactate uptake
and release by the perfused rat heart. Am J Physiol Endocrinol Metab 281: E794–E802, 2001.—The simultaneous release and uptake of lactate by the heart has been observed
both in vivo and ex vivo; however, the pathways underlying
these observations have not been satisfactorily explained.
Consequently, the purpose of this study was to test the
hypothesis that hearts release lactate from glycolysis while
simultaneously taking up exogenous lactate. Therefore, we
determined the effects of fatty acids and diabetes on the
regulation of lactate uptake and release. Hearts from control
and 1-wk diabetic animals were perfused with 5 mM glucose,
0.5 mM [3-13C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate.
Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which
enabled the simultaneous, but independent, measurements
of the rates of 1) uptake of exogenous [13C]lactate and 2)
efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both
similarly inhibited by the addition of palmitate, (i.e., the
ratio of lactate uptake to efflux remained constant), the ratio
of lactate uptake to efflux was significantly higher in the
controls compared with the diabetic group (1.00 ⫾ 0.14 vs.
0.50 ⫾ 0.07, P ⬍ 0.002). These data, combined with heterogeneous 13C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production
and oxidation of exogenous lactate operate as functionally
separate metabolic pathways. These results are consistent
with the concept of an intracellular lactate shuttle.
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
MATERIAL AND METHODS
Animal experimentation conforms to the Guide for the
Care and Use of Laboratory Animals (National Academy of
Sciences, 1996).
Experimental protocol. Hearts from control and 1-wk diabetic rats were isolated and perfused as described in Heart
perfusion. After ⬃30 min of equilibration, the perfusion medium was switched to buffer containing [3-13C]lactate and
[3-13C]pyruvate and perfused for an additional 60 min. The
concentrations of lactate and glucose were the same in all
experiments, whereas four different concentrations of palmitate were used: 0, 0.1, 0.32, and 1.0 mM. Each experimental
group consisted of 5–6 hearts.
Perfusate and coronary effluent were collected during the
last 5 min of perfusion with 13C-labeled substrates for subsequent determination of lactate concentration and fractional enrichment as detailed in Measurement of lactate concentration and fractional enrichment. From the combination
of the arterial and venous lactate concentrations, the lactate
fractional enrichment, and the coronary flows the rate of
AJP-Endocrinol Metab • VOL
[3-12C]lactate efflux and [3-13C]lactate uptake were calculated as described in Calculation of lactate uptake and efflux.
At the end of the experiment, hearts were freeze clamped and
acid extracted, and the tissue lactate, alanine, and pyruvate
fractional enrichments were determined as described in Estimation of intracellular alanine, pyruvate, and lactate fractional enrichment.
In previous studies with comparable workloads, we have
shown that perfusate and tissue lactate and tissue alanine
enrichment reach an isotopic steady state 10–20 min after
the start of perfusion with 13C-labeled substrates (11). Therefore, we are confident that the measurements made in this
study after 55–60 min of perfusion with 13C-labeled substrates are at isotopic steady state.
Induction of diabetes. Diabetes was induced in male
Sprague-Dawley rats (270–320 g) with streptozotocin (STZ,
60 mg/kg iv; Upjohn Laboratories, Kalamazoo, MI) as previously described (9, 12, 13); untreated age-matched male
Sprague-Dawley rats were used as controls. One week after
treatment with STZ, rats were killed, and hearts were isolated and perfused as described in Heart perfusion. This dose
of STZ resulted in a marked increase in serum glucose, with
only a moderate increase in ␤-hydroxybutyrate, as previously
reported (9, 12, 13).
Heart perfusion. Rats were anesthetized with ketamine
(100 mg/kg ip), heparinized (500 U/100 g ip), and decapitated.
Hearts were excised and perfused in a modified Langendorff
mode with Krebs-Henseleit bicarbonate buffer equilibrated
with 95% O2-5% CO2 (38°C, pH 7.4). The buffer contained
3% bovine serum albumin (BSA, essentially fatty acid free;
Sigma Chemical, St. Louis, MO) and the following components (in mM): 118 NaCl, 4.8 KCl, 1.2 Mg2SO4, 1.4 CaCl2, 1.2
KH2PO4, 25 Na2HCO3, 5 glucose, 0.5 sodium lactate, 0.05
sodium pyruvate, and 0, 0.1, 0.32, or 1 sodium palmitate and
5 mU/ml insulin.
The glucose and lactate concentrations were chosen to
represent the resting fed state in vivo (16, 25, 52). A small
amount of pyruvate was added to the perfusate to produce a
lactate-to-pyruvate ratio of 10:1, typical of that found in vivo
(27, 34), to minimize perturbations of the cytosolic redox
state that would occur with lactate alone. The high insulin
concentration was used to minimize the effects of differences
in insulin sensitivity between the control and diabetic
groups. The BSA was prepared as previously described (13).
The buffer was filtered through a 0.45-␮m low protein-binding filter (Millipore, HV type) before use.
After 30 min of equilibration, the perfusate was switched
to one where lactate and pyruvate were entirely replaced by
99% enriched [3-13C]lactate and [3-13C]pyruvate. The concentrations of all the components of the perfusate were identical to those used during the equilibration period.
Cardiac function. Contractile function was determined via
a fluid-filled balloon inserted into the left ventricle through
the mitral valve and connected to a Gould P23Db pressure
transducer on a four-channel chart recorder (Gould RS 3400,
Cleveland, OH). The balloon volume was adjusted to obtain a
left ventricular end diastolic pressure of 5 mmHg. Coronary
flow was adjusted to maintain perfusion pressure at 75
mmHg.
Cardiac function in these experiments has been previously
reported (13). We found that the addition of palmitate had no
effect on cardiac function or coronary flow in either control or
diabetic groups. Coronary flow rates were similar in control
and diabetic groups (7–8 ml 䡠 min⫺1 䡠 g wet wt⫺1); however,
cardiac function expressed as rate pressure product (i.e.,
heart rate ⫻ developed pressure) was significantly depressed
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
dehydrogenase (LDH) in liver, skeletal muscle, and
cardiac mitochondria. They have suggested that this is
part of an intracellular lactate shuttle, which could
account for the simultaneous release and uptake of
lactate in well-oxygenated tissue. However, despite
this evidence, there have been no studies specifically
designed to determine whether lactate efflux from the
myocardium that occurs simultaneously with myocardial lactate uptake could be due to intracellular compartmentation.
Therefore, the purpose of this study was to test the
hypothesis that lactate metabolism in the heart is
compartmentalized, consisting of separate pathways
for glycolytic production of lactate and uptake of exogenous lactate. Specifically, we reasoned that if lactate
efflux reflects glycolysis in the same cells that are
oxidizing lactate, the addition of another oxidizable
substrate such as fatty acids should decrease lactate
efflux consistent with an anticipated decrease in glycolysis (39). However, if lactate release is due to tissuelevel heterogeneity resulting from regions of hypoxia or
reduced flow, this should be unaffected by the presence
of an additional oxidizable substrate. Furthermore, in
light of reports that diabetes has differential effects on
glucose and lactate oxidation as well as modulated
fatty acid regulation of carbohydrate oxidation (12, 13),
we anticipated that diabetes would also impact the
effects of fatty acids on lactate efflux and uptake.
To determine the rates of lactate efflux and uptake,
hearts were perfused with unlabeled glucose and
[3-13C]lactate and [3-13C]pyruvate. Under these experimental conditions, lactate produced by glycolysis from
glucose or glycogen is unlabeled (i.e., [3-12C]lactate)
and could be distinguished from the [3-13C]lactate
added to the perfusate by 1H NMR spectroscopy,
whereas the uptake of lactate could be quantified from
the decrease in the perfusate [13C]lactate concentration. Lactate efflux and uptake were measured in
hearts from control and 1-wk diabetic rats in response
to different concentrations of the long-chain fatty acid
palmitate.
E795
E796
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
关 13 C-Lac兴FE ⫽ 关13C-Lac兴/共关13C-Lac兴 ⫹ 关12C-Lac兴兲
where [13C-Lac] is the intensity of the resonances from
[3-13C]lactate, and [12C-Lac] is the intensity of the reso-
nances from unlabeled lactate. By definition, the fraction of
the lactate in the sample that is unlabeled, [12C-Lac]FE, is
equal to 1 ⫺ [13C-Lac]FE. Therefore, the rate of unlabeled
lactate efflux in micromoles per minute per gram was calculated from the arteriovenous difference of the unlabeled lactate as
关共 ART关12C-Lac兴FE ⫻ 关Lac兴ART兲 ⫺ 共VEN关12C-Lac兴FE
⫻ 关Lac兴VEN兲兴 ⫻ CF ⫽ lactate efflux
where [Lac]ART and [Lac]VEN are the concentrations of lactate in the arterial and venous perfusate, respectively, in
micromoles per milliliter, and CF is the coronary flow in
milliliters per minute per gram. The rate of exogenous
[13C]lactate uptake in micromoles per minute per gram was
calculated in the same manner
关共 ART关13C-Lac兴FE ⫻ 关Lac兴ART兲 ⫺ 共VEN关13C-Lac兴FE
⫻ 关Lac兴VEN兲兴 ⫻ CF ⫽ lactate uptake
Measurement of tissue pyruvate enrichment. NMR spectroscopy could not be used to measure the tissue fractional
enrichment of pyruvate because the concentration of pyruvate was below the limit of detection. Therefore, we used gas
chromatography-mass spectrometry (GC-MS) to determine
the enrichment of pyruvate in heart extracts. The methods
used to determine 13C mass isotopomer distribution of pyruvate in heart have been described in detail previously (14,
15). Briefly, small samples (100 mg) of frozen heart were
extracted with an 8% solution of sulfosalicylic acid containing
10 mM hydroxylamine hydrochloride. Samples were neutralized to pH 6–7, centrifuged, and stored at ⫺20°C until
extraction with diethyl ether for analysis with GC-MS. The
13
C mass isotopomer distribution of pyruvate was analyzed
as the tert-butyldimethylsilyl derivative of its oxime on a
Hewlett-Packard 5890 Series II Plus gas chromatograph
coupled to a 5972 mass selective detector equipped with an
HP-5 fused silica capillary column. The mass spectrometer
was operated in the electron impact mode (70 eV) after
calibration. The split injection ratio was ⬃10:1, the carrier
gas was helium (0.6–0.7 ml/min), and the injection port was
at 280°C.
Fig. 1. 1H NMR spectra showing the methyl protons of
lactate from buffer before passing through the heart (A)
and same buffer collected after passing through the
heart (B). In this example, the palmitate concentration
was 0.32 mM. Note that doublet centered on 1.3 ppm is
from unlabeled lactate, i.e., [3-12C]lactate and the pair
of doublets at ⬃1.43 and 1.16 ppm are from [3-13C]lactate. Therefore, the ratio of the intensity of the pair of
doublets to the total intensity of all 3 doublets ⫽ the
fractional enrichment of lactate in the sample. It is
clear that there was an increase in [3-12C]lactate in
buffer after it passed through the heart.
AJP-Endocrinol Metab • VOL
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
in the diabetic group compared with controls (⬃19,000 vs.
⬃26,000 mmHg/min)(13).
Measurement of lactate concentration and fractional enrichment. Perfusate lactate concentration was measured
spectrophotometrically using a lactate assay kit from Sigma
Chemical. The fractional enrichment of lactate in the perfusate and venous effluent was determined using 1H NMR
spectroscopy as previously described (10, 11). Briefly, 20–30
ml of buffer were deproteinized with perchloric acid, neutralized, and freeze dried. The resulting powder was dissolved in
⬃1 ml of phosphate-buffered 2H2O, and the pH was adjusted
to ⬃7.2. 1H NMR spectra were collected using a Bruker
500-MSL NMR spectrometer equipped with an 11.75 T magnet using a commercial 5-mm probe. Spectra were obtained
under fully relaxed conditions with a sweep width of 6 kHz
and collected in 32-kb data blocks. Water suppression was
achieved with a presaturation pulse before acquisition. Spectra were processed with a 0.2-Hz line broadening.
Hearts were freeze clamped and acid extracted with 6%
perchloric acid, as previously described (13). The neutralized
extract was freeze dried, and the resulting powder was dissolved in phosphate-buffered 2H2O (pH 7.2), and 1H NMR
spectra were collected as described for determination of tissue alanine and lactate fractional enrichment.
Calculation of lactate uptake and efflux. The efflux of
unlabeled lactate and uptake of exogenous [13C]lactate were
calculated by combining measurements of lactate concentration and fractional enrichment in samples of arterial perfusate and venous effluent. The total concentration of lactate in
the samples was determined by enzymatic assay, as described in Measurement of lactate concentration and fractional enrichment. The proportion of lactate that was 13C
labeled can be determined directly from the 1H NMR spectra
of the samples. As can be seen in Fig. 1, resonances from
[3-13C]lactate are clearly distinguishable from those of unlabeled lactate. Because the resonance intensity is directly
proportional to concentration, the fraction of the total lactate
that was 13C labeled ([13C-Lac]FE) can be calculated as
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
E797
tailed Student’s t-test. Groups and repeated measurements
were compared by analysis of variance (ANOVA) in combination with Scheffé’s for post hocs. The analyses were performed with Statview statistical software (Abacus Concepts,
Berkeley, CA). Significance was established at P ⬍ 0.05.
RESULTS
Due to technical difficulties, we were unable to reliably
determine tissue pyruvate enrichment in all of the samples.
Estimation of intracellular alanine, pyruvate, and lactate
fractional enrichment. In the 1H NMR spectra of perfusate
effluent, there were no resonances from alanine (data not
shown); therefore, we assumed that the alanine resonances
seen in the tissue extracts primarily reflect intracellular
alanine. However, because [3-13C]lactate and [3-13C]pyruvate were added to the perfusate, the tissue fractional enrichment of lactate and pyruvate reflects the average of the
enrichment over both the intra- and extracellular compartments. Therefore, to estimate the intracellular lactate and
pyruvate enrichment, we assumed 1) that the intracellular
volume represented 70% of the total tissue volume (4), 2) that
the extracellular fractional enrichment was the same as that
added to the perfusate (i.e., 99%), and 3) that the concentrations of lactate and pyruvate were similar in the extracellular
and intracellular compartments. Any errors in these assumptions should not impact the differences between lactate and
pyruvate enrichment. However, a decrease in the average
extracellular fractional enrichment and/or a decrease in the
relative size of the total extracellular space would tend to
decrease the differences between alanine enrichment and the
enrichments of lactate and pyruvate.
Statistics. Data are presented as means ⫾ SE throughout.
Single comparisons of means were performed using two-
Fig. 3. A: effect of exogenous palmitate on the rate of
[3-12C]lactate efflux in hearts from control and diabetic
rats. ANOVA indicated no significant group differences,
but there was a significant effect of palmitate (P ⬍
0.0001). †P ⬍ 0.03 vs. all other palmitate concentrations; *P ⬍ 0.01 vs. control. B: effect of exogenous
palmitate on the rate of [3-13C]lactate uptake in hearts
from control and diabetic rats. ANOVA indicates significant group and treatment effects (P ⬍ 0.0002 in both
cases). †P ⬍ 0.05 vs. all other palmitate concentrations;
#P ⬍ 0.02 vs. all other palmitate concentrations; §P ⬍
0.02 vs. 1.0 mM palmitate, *P ⬍ 0.04 vs. control.
AJP-Endocrinol Metab • VOL
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
Fig. 2. Fractional enrichment of lactate in the venous effluent in
hearts from control and diabetic rats perfused with different concentrations of palmitate. Note that the fractional enrichment of lactate
in the perfusate was 99%, and the enrichment in the effluent is
significantly lower under all conditions. There was no difference
between the control and diabetic groups. *P ⬍ 0.008 vs. fractional
enrichment at 0, 0.1, and 0.32 mM palmitate.
Typical 1H NMR spectra from perfusate before and
after passing through the heart are shown in Fig. 1.
Before it passed through the heart, there is very little
unlabeled lactate in the perfusate; the fractional enrichment determined from the spectra is consistent
with the 99% enrichment specification of the [13C]lactate added to the perfusate. It is evident that, after
passing through the heart, the resonances from unlabeled lactate are increased relative to that added to the
perfusate. Using these spectra, we were able to determine the fractional enrichment of lactate in the perfusate before and after it passed through the heart as
described in MATERIAL AND METHODS. We found that,
under all conditions, the enrichment of lactate in the
effluent perfusate was significantly lower than that in
the buffer before it passed through the heart, indicating the production of unlabeled lactate (Fig. 2). There
were no differences in lactate fractional enrichment
between the control and diabetic groups at any fatty
acid concentration; however, at 1 mM palmitate, the
lactate fractional enrichment was significantly higher
in both groups compared with all other fatty acid concentrations (Fig. 2).
The rate of lactate efflux was similar in both control
and diabetic groups, except in the absence of palmitate,
where lactate efflux was almost 40% higher in the
diabetic group (0.63 ⫾ 0.03 vs. 0.88 ⫾ 0.06, P ⫽ 0.01;
Fig. 3A). An increase in palmitate concentration from 0
to 0.1 mM palmitate increased the rate of lactate efflux
in control hearts from 0.63 ⫾ 0.03 to 0.92 ⫾ 0.12
␮mol 䡠 min⫺1 䡠 g wet wt⫺1, but this did not reach statistical significance (P ⫽ 0.099). A further increase in
palmitate concentration from 0.1 to 1.0 mM significantly decreased the rate of lactate efflux by ⬃70%.
Although diabetes had no impact on the rate of lactate
efflux, the lactate uptake rate was significantly reduced in the diabetic group compared with control (P ⬍
0.0002; ANOVA, Fig. 3B). The addition of palmitate
decreased [13C]lactate uptake in both groups (Fig. 3B).
E798
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
Table 1. Fractional enrichment of lactate, pyruvate,
and alanine
Palmitate, mM
Lactate
Pyruvate
Alanine
0
0.1
0.32
1.0
10.8 ⫾ 4.2
17.3 ⫾ 6.1
17.7 ⫾ 3.8
37.6 ⫾ 3.0
24.0 ⫾ 8.4
26.7 ⫾ 2.5
31.6 ⫾ 2.3†
60.9 ⫾ 3.9†
44.5 ⫾ 1.7†
40.0 ⫾ 3.1†
48.3 ⫾ 1.2*
58.2 ⫾ 3.1†
22.7 ⫾ 3.2
34.7 ⫾ 4.6†
47.8 ⫾ 1.8*
16.6 ⫾ 5.1
27.3 ⫾ 1.1
24.6 ⫾ 4.8
39.7 ⫾ 4.7
28.3 ⫾ 7.3
39.2 ⫾ 3.2
46.4 ⫾ 15.1
48.9 ⫾ 8.0
41.1 ⫾ 1.4†
42.2 ⫾ 5.7
51.8 ⫾ 2.8†
62.8 ⫾ 2.1†
26.2 ⫾ 3.0
40.7 ⫾ 5.1†
49.8 ⫾ 2.5*
Control
Total
Diabetic
0
0.1
0.32
1.0
Total
Values are means ⫾ SE. The fractional enrichments of the intracellular pyruvate and lactate were calculated from the formula: Tfe ⫽
(Efe ⫻ ECV) ⫹ (Ife ⫻ ICV), where Tfe ⫽ total tissue enrichment, Efe ⫽
extracellular fractional enrichment, Ife ⫽ intracellular fractional
enrichment, ECV ⫽ extracellular volume, and ICV ⫽ intracellular
volume and ECV ⫹ ICV ⫽ 1.0. The value for Efe was the fractional
enrichment of pyruvate added to the perfusate (i.e., 99%), and the
ECV was taken as 0.3, as previously reported (4). The tissue enrichment of alanine was assumed to reflect intracellular enrichment,
because no alanine was added to the perfusate and none was observed in the effluent from the heart. * P ⬍ 0.05 vs. pyruvate and
lactate; † P ⬍ 0.05 vs. lactate.
strates, the simultaneous uptake and efflux of lactate
can be modulated by the addition of fatty acids as well
as by diabetes. The data are consistent with compartmentation of lactate metabolism in the heart, where
glycolytically derived lactate is released, and simultaneously, exogenous lactate is taken up for oxidation.
These data are also in agreement with reports of mitochondrial LDH (7) and are consistent with the
concept of an intracellular lactate shuttle (6, 7). Furthermore, the data indicate a potential functional sig-
DISCUSSION
We have shown for the first time that, in the isolated
perfused heart with both lactate and glucose as sub-
Fig. 4. A: effect of exogenous palmitate on the ratio of lactate uptake
to efflux in hearts from control and diabetic rats. ANOVA indicated
a significant group effect (P ⬍ 0.002) but no significant effect of
palmitate; *P ⬍ 0.04 vs. control. B: average ratio of lactate uptake to
efflux in the control group and diabetic groups; *P ⬍ 0.002.
AJP-Endocrinol Metab • VOL
Fig. 5. Fractional enrichment of intracellular alanine, pyruvate, and
lactate in combined control and diabetic groups split by palmitate
concentration. ANOVA indicated no effect of the control and diabetic
groups on fractional enrichment but a significant interaction between tissue fractional enrichment and palmitate concentration (P ⬍
0.001 for all groups). *P ⬍ 0.05 vs. pyruvate and lactate; †P ⬍ 0.05
vs. lactate; #P ⬍ 0.05 vs. 0, 0.1, and 0.32 mM palmitate; $P ⬍ 0.05 vs.
0 and 0.1 mM palmitate.
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
In contrast to lactate uptake or efflux, the addition of
palmitate had no effect on the ratio of lactate uptake to
efflux in either the control or diabetic groups (Fig. 4A).
However, the ratio of lactate uptake to efflux was lower
in the diabetic group; this is especially evident when
the data from the different palmitate groups are combined [Fig. 4B; 1.00 ⫾ 0.14 (control) vs. 0.50 ⫾ 0.07
(diabetic), P ⬍ 0.002].
To provide additional evidence for the compartmentation of lactate metabolism, we measured the fractional enrichments of lactate, pyruvate, and alanine in
heart extracts. The intracellular fractional enrichments of alanine, pyruvate, and lactate for all experimental groups are shown in Table 1, and the data for
the combined control and diabetic groups are summarized in Fig. 5. In Table 1, it can be seen that, in all but
one group, tissue alanine enrichment is higher than
the intracellular pyruvate enrichment, and in all cases,
intracellular pyruvate enrichment is higher than intracellular lactate enrichment. However, as noted earlier, we were unable to reliably determine tissue pyruvate enrichment in all of the samples due to technical
problems. Consequently, the low “n” precludes many of
the differences between the individual groups reaching
statistical significance. However, ANOVA on the complete data set clearly indicated that the fractional enrichments of alanine, pyruvate, and lactate are all
significantly different from one another (P ⬍ 0.0001).
This was also the case when the control and diabetic
groups were split but grouped via palmitate concentration (Table 1). There were no significant differences in
fractional enrichment between the control and diabetic
groups; however, there was a significant interaction
between tissue fractional enrichment and palmitate
concentration (P ⬍ 0.001). This was due to an increase
in fractional enrichment in lactate, pyruvate, and alanine with increasing palmitate concentration, which
was significant at 1.0 mM palmitate (Fig. 5).
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
Fig. 6. Metabolic scheme demonstrating compartmentation of lactate metabolism. GT, glucose transporter; G-6-P, glucose 6-phosphate; MCT, monocarboxylate transporter. The asterisk indicates
13
C-labeled substrates and intermediates. The thicker arrows represent the preferential conversion of glycolytically derived pyruvate to
lactate and exogenously derived pyruvate to acetyl-CoA. The fact
that tissue alanine appears to be more closely linked to [13C]lactatederived pyruvate than to glycolytic pyruvate is also indicated by a
thicker arrow. The main pathway for oxidation of exogenous lactate
involves mitochondrial lactate dehydrogenase and is consistent with
the recent report by Brooks et al. (7). In the intracellular lactate
shuttle (6, 7), there is also mitochondrial oxidation of glycolytic
lactate. We have no direct evidence of this in this study; however, we
have indicated this as a possibility by a dashed arrow. See DISCUSSION
for more details.
AJP-Endocrinol Metab • VOL
concentrations (13). If glucose oxidation and lactate
efflux are both decreased by similar amounts after the
addition of palmitate, the net flux through glycolysis
must also decrease to the same extent.
Therefore, taken together, these data are consistent
with there being separate pathways for lactate metabolism. However, it has been suggested that, because of
rapid equilibration between lactate and pyruvate, the
efflux of unlabeled lactate is a result of simple isotope
exchange at the level of LDH (24, 50). If isotopic equilibration were the sole mechanism leading to formation
of unlabeled lactate in the perfusate, we would anticipate that the tissue enrichment of lactate and pyruvate
should be similar; however, this is not the case (Table
1 and Fig. 5). Furthermore, the observation that, in the
diabetic group, lactate efflux exceeded lactate uptake
under all conditions also suggests that the formation of
unlabeled lactate in the perfusate cannot be accounted
for by simple isotopic exchange. Therefore, although
we cannot completely exclude a contribution from
isotopic exchange to the apparent simultaneous uptake and efflux of lactate, we believe that the data are
more consistent with compartmentation of lactate
metabolism.
Another possible explanation for simultaneous lactate uptake and efflux is metabolic heterogeneity
within the heart. Under normal conditions, regional
myocardial blood flow has been shown to vary over a 6to 10-fold range (3), and recent data suggest that there
may be a correlation between local myocardial blood
flow and local oxygen consumption (41). Therefore,
lactate efflux could reflect anaerobic metabolism in
regions of the heart where blood flow is reduced. Similarly, other cell types such as coronary endothelial
cells, which are primarily glycolytic in nature (23),
could also be a site of lactate production. However, if
the bulk of the lactate efflux was due to such heterogeneity, then our data (Fig. 3A) would suggest that the
addition of palmitate decreases this heterogeneity. It is
unclear how the presence of palmitate could decrease
lactate production originating from hypoxic regions or
from cells that preferentially use glucose for energy
production. Thus, although we cannot absolutely rule
out that lactate efflux was due to tissue heterogeneity,
we believe that our results are more consistent with
compartmentation of lactate metabolism within the
myocyte.
Separate pathways for the metabolism of glycolytically derived pyruvate to lactate and the uptake of
exogenous lactate and its subsequent metabolism are
shown schematically in Fig. 6. The preferential metabolism of glucose-derived pyruvate to lactate rather
than to acetyl-CoA is indicated by the thicker arrow.
The preferential contribution of exogenous lactatederived pyruvate to acetyl-CoA formation is distinguished in the same manner. From a thermodynamic
point of view, the different pathways for lactate production and consumption need to be in physically
separate compartments, and/or channeling must be
present. Recently, Brooks et al. (7) reported the presence of mitochondrial LDH in the heart and proposed
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
nificance of this compartmentation, because lactate
uptake was decreased by diabetes, whereas lactate
efflux was unaffected. To account for these observations, we propose that pathways of glycolytic lactate
production and oxidation of exogenous lactate are functionally separate in the heart, as illustrated in Fig. 6.
We believe that this model provides a framework for
integrating the observations of myocardial lactate uptake and release in vivo and in vitro (1, 2, 19, 20, 30, 40,
49) with earlier reports of pyruvate compartmentation
in the heart (8, 28, 31, 35, 46).
Let us consider the evidence from this study that
indicates that the decrease in the fractional enrichment of lactate in the perfusate is a combination of a
decrease in [13C]lactate concentration due to net uptake plus an increase in [12C]lactate concentration due
to net lactate production from glycolysis. Under most
conditions, the rate of [3-13C]lactate uptake measured
in this study was ⬃30% greater than the rate of lactate
oxidation determined using [13C]glutamate isotopomer
analysis under similar conditions (13). This indicates
that ⬃70% of the exogenous lactate taken up by the
heart was subsequently oxidized, which is consistent
with previous studies of lactate metabolism in vivo (2,
19). Second, the release of unlabeled lactate from the
myocardium in the control group was decreased by
⬃70% by increasing the palmitate concentration from
0.1 to 1.0 mM (Fig. 3A). This is similar to the decrease
in glucose oxidation over the same range of palmitate
E799
E800
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
AJP-Endocrinol Metab • VOL
pools. This is analogous to the observation that amino
acids taken up for protein synthesis are charged onto
tRNA without equilibrating with total intracellular
amino acid pool (5). The different intracellular fractional enrichments of pyruvate, lactate, and alanine
(Table 1 and Fig. 5) also support the scheme shown in
Fig. 6. As proposed, pyruvate formed via glycolysis
is rapidly converted to lactate; exogenous [13C]lactate
is taken up and metabolized to [13C]pyruvate. Thus it
is likely that the majority of tissue lactate originates
from glycolysis and is unlabeled, whereas the majority
of tissue pyruvate originates from exogenous [13C]lactate and is 13C enriched. As a result, the fractional
enrichment of lactate would be lower than tissue pyruvate, which is consistent with our observations. The
higher fractional enrichment of alanine compared with
lactate suggests that alanine is more closely linked to
[13C]lactate-derived pyruvate than unlabeled glucosederived pyruvate. This would suggest an association of
pyruvate kinase with LDH and MCT, resulting in the
rapid conversion of glycolytic pyruvate to lactate and
thus decreasing the possibility of exchange with alanine.
In control hearts, the ratio of lactate uptake to lactate efflux was 1.0 ⫾ 0.14 and was unaffected by
palmitate (Fig. 4). In contrast, in the diabetic group,
the rate of lactate efflux was greater than lactate
uptake under all conditions. The decrease in lactate
uptake in the diabetic group could be a result of a
decreased expression of MCT; for example Hajduch et
al. (21) reported a marked decrease in MCT expression
in adipocytes after diabetes. However, we (12) have
shown previously that diabetes did not affect MCT
expression in the heart. The modulation by diabetes of
the lactate uptake-to-efflux ratio could be related to the
increased cytosolic NADH/NAD⫹ ratio that has been
reported after diabetes (12, 13, 36–38, 47). Whether
this is a cause or a consequence of the increase in
cytosolic NADH/NAD⫹ is difficult to assess without
further clarification of the physical characteristics underlying the compartmentation of lactate metabolism
in the heart. Nevertheless, irrespective of the mechanism involved, the decreased lactate uptake-to-efflux
ratio in the diabetic group not only provides further
support for separate, independently regulated pathways for lactate uptake and efflux, but also indicates
potential functional significance.
It is impossible to completely exclude tissue heterogeneity and isotope exchange as potential contributory
factors to the simultaneous uptake and efflux of lactate
from the heart. Nevertheless, we believe that the results of this study are most consistent with the concept
of intracellular compartmentation of lactate metabolism. It should be noted that these experiments were
carried out in the isolated perfused rat heart, and there
are both physiological and metabolic differences between this and the heart in vivo. We used concentrations of substrate designed to closely mimic those seen
in vivo, whereas the insulin concentration was supraphysiological and thus could potentially influence both
lactate uptake and efflux. Furthermore, the isolated
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
that mitochondria were the main site of lactate metabolism to pyruvate in heart and skeletal muscle. This
could also be facilitated by the fact that, in these
tissues, mitochondrial density is higher close to the
sarcolemma compared with deep within the muscle
fibers (33). Thus, once exogenous lactate has entered
the myocyte, it will be readily transported into the
mitochondria and subsequently oxidized. We have
shown this schematically in Fig. 6.
In Fig. 6, we have also indicated that glycolytically
derived pyruvate is preferentially metabolized to lactate rather than to acetyl-CoA; this is consistent with
the fact that the equilibrium constant for LDH is far in
the direction of lactate and the change in free energy is
large. Given the presence of mitochondrial LDH once
cytosolic lactate is formed, in addition to being transported out of the cell it could enter the mitochondria
and be oxidized. Indeed, Brooks and colleagues (6, 7)
have suggested that metabolism of glycolytic lactate,
rather than pyruvate, by the mitochondria is the predominant pathway for the oxidation of glycolytic carbons. However, our results provide no insight into the
relative flux split between transport of glycolytic lactate out of the myocyte vs. entry into the mitochondria.
Thus, although we have indicated the possibility of
mitochondrial oxidation of glycolytic lactate, we have
no direct evidence of this pathway.
As noted above, the density of mitochondria is greatest at the subsarcolemmal surface compared with
deeper within the muscle fibers (33); therefore, one
might expect that glycolytically derived lactate would
have a fairly high probability of entering the mitochondria before transport across the sarcolemma. However,
exogenous lactate contributed more than twice as
much acetyl-CoA to the tricarboxylic acid cycle compared with glucose-derived pyruvate in hearts perfused with 11 mM glucose and 0.5 mM lactate (12).
This could be interpreted as the inhibition of glycolysis
by exogenous lactate. However, it could also indicate a
greater probability of exogenous lactate oxidation compared with oxidation of glycolytic lactate. In other
words, our data suggest that lactate produced via glycolysis is more likely to be transported out of the cell
than to enter the mitochondria and be oxidized. Indeed
an association between glycolytic lactate formation and
lactate transport could be advantageous, because increased lactate production is typically associated with
oxygen limitation and accumulation of lactate in the
cell would be detrimental. Thus our proposed scheme
would predict a functional association between LDH
and the monocarboxylate transporter (MCT). This
would also be consistent with reports that MCT is
localized to specific sites in the sarcolemma (22). Thus,
in some sites the transporters may be closely linked
with subsarcolemmal mitochondria and lactate uptake, and in other sites they may be associated with the
glycolytic machinery and lactate efflux.
The higher fractional enrichment of alanine compared with pyruvate and lactate suggests that exogenous [13C]lactate is converted to alanine without equilibrating fully with the intracellular lactate and pyruvate
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
We are grateful to Dr. A.-M. Seymour for stimulating discussions
on this work, which were facilitated by a NATO Collaborative Research Grant (CRG-940624) awarded to Dr. Seymour.
This work was supported in part by grants HL-48789 and HL67464 from National Heart, Lung, and Blood Institute, National
Institutes of Health (J. C. Chatham), and Grant MOP-9575 from the
Canadian Institutes of Health Research (C. Des Rosiers).
REFERENCES
1. Allard MF, Schönekess BO, Henning SL, English DR, and
Lopaschuk GD. The contribution of oxidative metabolism and
glycolysis to ATP production in the hypertrophied heart. Am J
Physiol Heart Circ Physiol 267: H742–H750, 1994.
2. Bartelds B, Knoester H, Beaufort-Krol GCM, Smid GB,
Takens J, Zijlstra WG, Heymans HSA, and Kuipers JRG.
Myocardial lactate metabolism in fetal and newborn lambs.
Circulation 99: 1892–1897, 1999.
3. Bassingthwaighte JB and Li Z. Heterogeneities in myocardial
flow and metabolism: exacerbation with abnormal excitation.
Am J Cardiol 83: 7H–12H, 1999.
4. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Norwell, MA: Kluwer Academic, 1991. p. 258.
5. Biolo G, Chinkes D, Zhang XJ, and Wolfe RR. A new model
to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle. J Parenter
Enteral Nutr 16: 305–315, 1992.
6. Brooks GA. Intra- and extra-cellular lactate shuttles. Med Sci
Sports Exerc 32: 790–799, 2000.
AJP-Endocrinol Metab • VOL
7. Brooks GA, Dubouchaud H, Brown M, Sicurello JP, and
Butz CE. Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle. Proc Natl Acad
Sci USA 96: 1129–1134, 1999.
8. Bünger R. Compartmented pyruvate in perfused working heart.
Am J Physiol Heart Circ Physiol 249: H439–H449, 1985.
9. Chatham JC and Forder JR. A 13C-NMR study of glucose
oxidation in the intact functioning rat heart following diabetesinduced cardiomyopathy. J Mol Cell Cardiol 25: 1203–1213,
1993.
10. Chatham JC and Forder JR. Lactic acid and protein interactions: implications for the NMR visibility of lactate in biological
systems. Biochim Biophys Acta 1426: 177–184, 1999.
11. Chatham JC and Forder JR. Metabolic Compartmentation of
lactate in the glucose-perfused rat heart. Am J Physiol Heart
Circ Physiol 270: H224–H229, 1996.
12. Chatham JC, Gao ZP, Bonen A, and Forder JR. Preferential
inhibition of lactate oxidation relative to glucose oxidation in the
rat heart following diabetes. Cardiovasc Res 43: 96–106, 1999.
13. Chatham JC, Gao ZP, and Forder JR. The impact of 1 wk of
diabetes on the regulation of myocardial carbohydrate and fatty
acid oxidation. Am J Physiol Endocrinol Metab 277: E342–E351,
1999.
14. Comte B, Jetté M, Bouchard B, Cordeau S, and Des
Rosiers C. A 13C-mass isotopomer study of anaplerotic pyruvate
carboxylation in perfused rat hearts. J Biol Chem 272: 26125–
26131, 1997.
15. Comte B, Vincent G, Bouchard B, and Des Rosiers C.
Probing the origin of acetyl-CoA and oxaloacetate entering the
citric acid cycle from the 13C-labeling of citrate released by
perfused rat hearts. J Biol Chem 272: 26117–26124, 1997.
16. Connolly CC, Stevenson RW, Neal DW, Wasserman DH,
and Cherrington AD. The effects of lactate loading on alanine
and glucose metabolism in the conscious dog. Metabolism 42:
154–161, 1993.
17. Drake AJ, Haines JR, and Noble IM. Preferential uptake of
lactate by the normal myocardium in dogs. Cardiovasc Res 14:
65–72, 1980.
18. Fisher DJ, Heymann MA, and Rudolph AM. Regional myocardial blood flow and oxygen delivery in fetal, newborn, and
adult sheep. Am J Physiol Heart Circ Physiol 243: H729–H731,
1982.
19. Gertz EW, Wisneski JA, Stanley WC, and Neese RA. Myocardial substrate utilization during exercise in humans: dual
carbon-labelled carbohydrate isotope experiments. J Clin Invest
82: 2017–2025, 1988.
20. Goodwin GW, Taylor CS, and Taegtmeyer H. Regulation of
energy metabolism of the heart during acute increase in heart
work. J Biol Chem 273: 29530–29539, 1998.
21. Hajduch E, Heyes RR, Watt PW, and Hundal HS. Lactate
transport in rat adipocytes: identification of monocarboxylate
transporter 1 (MCT1) and its modulation during streptozotocininduced diabetes. FEBS Lett 479: 89–92, 2000.
22. Johannsson E, Nagelhus EA, McCullagh KJ, Sejersted
OM, Blackstad TW, Bonen A, and Ottersen OP. Cellular and
subcellular expression of the monocarboxylate transporter
MCT1 in rat heart. A high-resolution immunogold analysis. Circ
Res 80: 400–407, 1997.
23. Krutzfeldt A, Spahr R, Mertens S, Siegmund B, and Piper
HM. Metabolism of exogenous substrates by coronary endothelial cells in culture. J Mol Cell Cardiol 22: 1393–1404, 1990.
24. Landau BR and Wahren J. Nonproductive exchanges: the use
of isotopes gone astray. Metabolism 41: 457–459, 1992.
25. Large Y, Soloviev M, Brunengraber H, and Beylot M.
Lactate and pyruvate isotopic enrichments in plasma and tissues of postabsorbative and starved rats. Am J Physiol Endocrinol Metab 268: E880–E888, 1995.
26. Larkin LM, Horwitz BA, and McDonald RB. Effect of cold on
serum substrate and glycogen concentration in young and old
Fischer 344 rats. Exp Gerontol 27: 179–190, 1992.
27. Laughlin MR, Taylor J, DeGroot M, and Balaban RS.
Pyruvate and lactate metabolism in the in vivo dog heart. Am J
Physiol Heart Circ Physiol 264: H2068–H2079, 1993.
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
perfused heart performs less mechanical work than the
heart in vivo, and energy demand could also impact
both lactate uptake and efflux. However, simultaneous
lactate uptake and efflux have been observed both in
isolated heart studies with physiological insulin levels
(1, 20, 40) and in vivo (2, 19, 30, 49). Therefore, we do
not believe that our observations are a consequence of
the high insulin levels or due to reduced workload.
In conclusion, our data are consistent with the hypothesis that the simultaneous efflux and uptake of
lactate in the heart reflect separate pathways for glycolytic lactate production and the uptake and subsequent oxidation of exogenous lactate. Consequently, we
have proposed that, in the presence of exogenous lactate, glucose-derived pyruvate formation is tightly coupled to lactate production and subsequent transport
out of the cell, whereas exogenous lactate-derived
pyruvate is preferentially oxidized in the mitochondria. This model accounts for the observations of separate pyruvate pools in the isolated perfused heart (8,
28, 31, 35) and the reports of simultaneous myocardial
lactate uptake and efflux observed in vivo and in vitro
(1, 2, 19, 20, 30, 40, 49). It is also consistent with the
intracellular lactate shuttle hypothesis (6, 7). The
model is also testable, because it predicts that pyruvate kinase, LDH, and MCT should exist as a functional unit. Furthermore, the model suggests that the
ratio of lactate uptake to efflux may be sensitive to
alterations in cytosolic redox state. Additional investigations are clearly warranted to better understand the
importance of compartmentation of lactate metabolism
and the regulation of lactate uptake and efflux. Nevertheless, future studies of cardiac carbohydrate metabolism need to consider glycolytically derived lactate
production and oxidation of exogenous lactate as functionally separate pathways.
E801
E802
COMPARTMENTATION OF CARDIAC LACTATE METABOLISM
AJP-Endocrinol Metab • VOL
40. Schönekess BO, Allard MF, and Lopaschuk GD. Recovery of
glycolysis and oxidative metabolism during postischemic reperfusion of hypertrophied rat hearts. Am J Physiol Heart Circ
Physiol 271: H798–H805, 1996.
41. Schwanke U, Deussen A, Heusch G, and Schipke JD. Heterogeneity of local myocardial flow and oxidative metabolism.
Am J Physiol Heart Circ Physiol 279: H1029–H1035, 2000.
42. Siliprandi N, Di Lisa F, Pieralsi G, Ripari P, Maccari F,
Menabo R, Giamberardino MA, and Vecchiet L. Metabolic
changes induced by maximal exercise in human subjects following L-carnitine administration. Biochim Biophys Acta 1034: 17–
21, 1990.
43. Srere PA. Macromolecular interactions: tracing the roots.
Trends Biochem Sci 25: 150–153, 2001.
44. Srere PA and Mosbach K. Metabolic compartmentation: symbiotic, organellar, multienzymic, and microenvironmental. Annu
Rev Microbiol 28: 61–83, 1974.
45. Srere PA and Ovadi J. Enzyme-enzyme interactions and their
metabolic role. FEBS Lett 268: 360–364, 1990.
46. Sumegi B, Podanyi B, Forgo P, and Kover KE. Metabolism
of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies. Biochem J
312: 75–81, 1995.
47. Trueblood N and Ramasamy R. Aldose reductase inhibition
improves altered glucose metabolism of isolated diabetic rat
hearts. Am J Physiol Heart Circ Physiol 275: H75–H83, 1998.
48. Van der Vusse GJ and de Groot MJM. Interrelationship
between lactate and cardiac fatty acid metabolism. Mol Cell
Biochem 116: 11–17, 1992.
49. Wisneski JA, Gertz EW, Neese RA, Gruenke LD, Morris
DL, and Craig JC. Metabolic fate of extracted glucose in normal human myocardium. J Clin Invest 76: 1819–1827, 1985.
50. Wolfe RR. Isotopic measurement of glucose and lactate kinetics.
Ann Med 22: 163–170, 1990.
51. Xu KY, Zweier JL, and Becker LC. Functional coupling between glycolysis and sarcoplasmic reticulum Ca2⫹ transport.
Circ Res 77: 88–97, 1995.
52. Young LH, Zaret BL, and Barrett EG. Physiologic hyperinsulinemia stimulates lactate extraction by heart muscle in the
conscious dog. Metabolism 38: 1115–1119, 1989.
281 • OCTOBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 6, 2017
28. Lewandowski ED. Metabolic heterogeneity of carbon substrate
utilization in mammalian heart: NMR determination of mitochondrial vs. cytosolic compartmentation. Biochemistry 31:
8916–8923, 1992.
29. Margaria R, Cerretelli P, Aghemo P, and Sassi G. Energy
cost of running. J Appl Physiol 18: 367–370, 1963.
30. Mazer CD, Stanley WC, Hickey RF, Neese RA, Cason BA,
Demas KA, Wisneski JA, and Gertz EW. Myocardial metabolism during hypoxia: maintained lactate oxidation during increased glycolysis. Metabolism 39: 913–918, 1990.
31. Mowbray J and Ottaway JH. The effect of insulin and growth
hormone on the flux of tracer from labelled lactate in perfused
rat heart. Eur J Biochem 36: 369–379, 1973.
32. Ovadi J and Srere PA. Macromolecular compartmentation
and channeling. Int Rev Cytol 192: 255–280, 2000.
33. Palmer JW, Tandler B, and Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated
from rat cardiac muscle. J Biol Chem 252: 8731–8739, 1977.
34. Panchal AR, Comte B, Huang H, Kerwin T, Darvish A, Des
Rosiers C, Brunengraber H, and Stanley WC. Partitioning
of pyruvate between oxidation and anaplerosis in swine hearts.
Am J Physiol Heart Circ Physiol 279: H2390–H2398, 2000.
35. Peuhkurinen KJ, Hiltunen JK, and Hassinen IE. Metabolic
compartmentation of pyruvate in isolated perfused rat heart.
Biochem J 210: 193–198, 1983.
36. Puckett SW and Reddy WJ. A decrease in the malate-aspartate shuttle and glutamate translocase activity in heart mitochondria from alloxan-diabetic rats. J Mol Cell Cardiol 11:
173–187, 1979.
37. Ramasamy R, Oates PJ, and Schaefer S. Aldose reductase
inhibition protects diabetic and nondiabetic rat hearts from
ischemic injury. Diabetes 46: 292–300, 1997.
38. Ramasamy R, Trueblood N, and Schaefer S. Metabolic effects of aldose reductase inhibition during low-flow ischemia and
reperfusion. Am J Physiol Heart Circ Physiol 275: H195–H203,
1998.
39. Randle PJ, Newsholme EA, and Garland PB. Regulation of
glucose uptake by muscle 8: effects of fatty acids, ketone bodies
and pyruvate, and of alloxan-diabetes and starvation, on the
uptake and metabolic fate of glucose in rat heart and diaphragm
muscles. Biochem J 93: 652–665, 1964.