1 Division of Magnetic Resonance Research, Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; 2 Department of Nutrition, University of Montreal, Montreal, H2L 4M1 Quebec, Canada; and 3 Center for Nuclear Magnetic Resonance Research and Development, Departments of Medicine and 4 Biomedical Engineering, The University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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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.
nuclear magnetic resonance spectroscopy; gas chromatography-mass spectrometry; diabetes; palmitate
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INTRODUCTION |
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THE ARTERIAL CONCENTRATION 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 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 tissue-level 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.
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MATERIAL AND METHODS |
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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 [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-µm 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 · minMeasurement 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
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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 13C 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.
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-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.
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RESULTS |
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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).
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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 µmol · min1 · 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).
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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].
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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).
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DISCUSSION |
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We have shown for the first time that, in the isolated perfused
heart with both lactate and glucose as substrates, 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 significance 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).
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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 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 6- to 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 lactate-derived 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 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 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 glucose-derived 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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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This work was supported in part by grants HL-48789 and HL-67464 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).
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: jchatham{at}mri.uab.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 2 November 2000; accepted in final form 1 June 2001.
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