INTRODUCTION

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ARTERIAL LACTATE concentration is decreased at absolute (14, 20, 33) and relative (2, 22, 30) exercise intensities after endurance training. Mechanisms responsible for the attenuated blood lactate response to exercise are equivocal. By using tracers, increased whole body lactate clearance during exercise was first observed in trained rats (14). Subsequently, training was shown to decrease lactate appearance rate (R_a) in humans exercising at given relative intensities (30). In contrast, others found unchanged lactate turnover in rats (14) and men (33) exercising at given absolute workloads after endurance training. Thus, although it is possible to conclude that increased lactate clearance contributes to dampened arterial lactate concentration during exercise after training, the importance of altered lactate appearance is unclear.

Limb lactate balance has also been used to evaluate effects of endurance training on lactate metabolism. After training, decreased limb net lactate release () for the first 10-15 min (19, 22) or throughout 50 min of exercise (39) at a given absolute workload has been reported. At the same relative exercise intensity, was similar in trained and untrained subjects (45). However, limb lactate uptake and oxidation during (10-12, 42) confound as a measure of intramuscular lactate production. Therefore, interpretation of limb lactate balance is tenuous without isotope measurements to quantitate lactate uptake and total (_tot).

The purpose of the present investigation was to quantitate whole body and active-limb lactate metabolism by using both tracer and limb balance techniques at given absolute and relative exercise intensities before and after endurance training. Specifically, we evaluated the hypotheses that training decreases lactate production at absolute workloads and increases lactate clearance at given absolute and relative exercise intensities. Additionally, we evaluated whether maintenance of elevated arterial lactate concentration during exercise is due to sustained active muscle .

	METHODS

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Subjects

1

1

Experimental Design

Preliminary Testing

Dietary Protocol

Catheterizations

Tracer Protocol

Muscle Biopsies and Analyses

Blood Sampling

Hemodynamics

Metabolite Analyses and Isotope Enrichments

For GCMS analyses, the injector temperature was set at 200°C; the initial oven temperature was set at 80°C, the transfer line at 250°C, the source temperature at 286°C, and the quadrapole temperature at 126°C. The carrier gas was helium, and splitless injection was used with a 35:1 ml/min ratio. Methane was used for chemical ionization, and selective ion monitoring was used to monitor ion mass-to-charge ratios of 327.25 and 328.25 for [¹²C]- and [¹³C]lactate, respectively.

Training Protocol

Calculations

Net metabolite exchange. Net metabolite exchange differences were calculated as the product of leg blood flow and arteriovenous differences, where arterial (a) and venous hematocrit (v) values were used to correct for changes in plasma volume



Tracer-measured leg lactate fractional extraction (F_ex) was measured by using two methods

Leg lactate oxidation (R_ox) rate was also measured by using two methods

Blood CO₂ content. Blood PCO₂, PO₂, pH, and Hb were measured in both arterial and venous samples and used in the calculations by Douglas et al. (15) and Kelman (27) for determination of blood CO₂ content (CCO_{2 blood}) (2, 3).

Lactate kinetics. Lactate R_a and rate of disappearance (R_d), metabolic clearance rate (MCR), and oxidation were calculated by using equations defined by Steele and modified for use with stable isotopes (47)




where F represents isotope infusion rate, IE₁ and IE₂ are lactate isotopic enrichments at sampling time points 1 (t₁) and 2 (t₂), respectively; C₁ and C₂ are lactate concentrations at t₁ and t₂, respectively; V is the estimated volume distribution of lactate (180 ml/kg); 89.08 is the molecular weight of lactate; and k is a correction factor for retention of tracer in CO₂ pools (0.65 during rest and 0.90 during exercise). Isotopic enrichments of lactate were corrected for background enrichments from blood samples taken before isotope infusion. Calculations of steady-state lactate kinetics were performed during the last 15 min of rest (75 and 90 min) and 30 min of exercise (30, 45, and 60 min) when arterial lactate concentration and enrichments were stable, thus minimizing assumptions related to volume and arterial concentration.

Statistical Analyses

	RESULTS

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Subject Characteristics

Muscle Lactate Concentrations

Arterial Lactate and Glucose Concentrations

View larger version (95K): [in this window] [in a new window]   Fig. 1.   A: arterial lactate concentration during rest and exercise before (Pre) and after endurance training. Values are means ± SE for 8-9 subjects. ABT, absolute workload; RLT, relative intensity. B: leg venous-arterial (v-a) lactate concentration difference during rest and exercise before and after training. Values are means ± SE for 7-8 subjects. C: net lactate release during rest and exercise before and after endurance exercise training. Values are means ± SE for 7-8 subjects. D: arterial lactate isotopic enrichments during rest and exercise before and after endurance exercise training. Values are means ± SE for 8-9 subjects.

Leg Lactate Metabolism

Resting limbs released lactate under all conditions, and limb was unchanged at rest due to training (Fig. 1C). Under all exercise conditions during commencement of exercise increments in leg blood flow (3) and [lactate]_v-a caused an increase in ; however, because of changing [lactate]_v-a, waned over time despite the constancy of limb blood flow. During exercise, scaled to exercise intensity, with 210% greater release at 65% compared with 45% pretraining O_{2 peak} (P < 0.05), and 55% greater release at RLT compared with ABT (P < 0.05). Compared with before training, decreased 60% (P < 0.05) at ABT but was not significantly different at RLT.

Arterial lactate isotopic enrichment is shown in Fig. 1D. On the basis of prior experience, the tracer infusion rate was adjusted among trials. Consequently, although enrichment fell during exercise, there were no differences in enrichment among trials.

Leg lactate F_ex approximated 15% (Fig. 2), was unchanged at rest before or after training, and did not change significantly from rest during exercise at any intensity. Before training at 65% compared with 45% O_{2 peak}, fractional extraction decreased 50% (P < 0.05). After training compared with 65% pretraining O_{2 peak}, it increased 65 and 90% at ABT and RLT, respectively. Fractional extraction was not significantly different between ABT and RLT.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Tracer-measured leg fractional lactate extraction during rest and exercise before and after training. Values are means ± SE for 7-9 subjects.

Tracer-measured leg lactate uptake rate (Fig. 3) and oxidation (not shown) were unchanged at rest before and after training and increased from rest to exercise at all intensities. Leg lactate uptake and oxidation scaled to exercise intensity before training, increasing 160% from 45 to 65% O_{2 peak}, and after training, increasing 70% from ABT to RLT (P < 0.05). Compared with 65% pretraining, leg lactate uptake and oxidation after training were unchanged at ABT and were 44% greater (P < 0.05) at RLT.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Tracer-measured leg lactate uptake during rest and exercise before and after training. Values are means ± SE for 7-9 subjects.

The relationship between leg lactate oxidation and arterial lactate concentration is shown in Fig. 4. Before training, leg lactate oxidation displayed saturation, where leg lactate oxidation plateaued with increased arterial lactate concentration. After training, saturation was not apparent at the concentrations achieved. Table 3 shows the contribution of leg lactate oxidation to leg carbohydrate oxidation determined from leg respiratory quotient (3). Before and after training, resting lactate oxidation accounted for 50% of leg carbohydrate oxidation, which decreased to 15% during exercise at all intensities. There were no differences during exercise before or after training in contribution of leg lactate oxidation to leg carbohydrate oxidation.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Relationship between tracer-measured leg lactate oxidation rate and arterial lactate concentration ([lactate]a) before and after training. Values are means ± SE for 7-9 subjects.

Whole Body Lactate Kinetics

View larger version (121K): [in this window] [in a new window]   Fig. 5.   A: effects of exercise intensity and training on lactate rate of appearance (Ra). Values are means ± SE for 8-9 subjects. There is a positive linear relationship between whole body lactate Ra and total leg lactate release [tot; r = 0.99; pretraining: lactate Ra = 1.32(tot) + 1.65, posttraining: lactate Ra = 1.35(tot) + 1.03]. B: effects of exercise intensity and training on lactate rate of disappearance (Rd). Values are means ± SE for 8-9 subjects. There is a positive linear relationship between whole body lactate Rd and leg lactate uptake [r = 0.99; pretraining: lactate Rd = 2.18(leg lactate uptake) + 1.09, posttraining: lactate Rd = 1.81(leg lactate uptake) + 0.72]. C: effects of exercise intensity and training on lactate metabolic clearance rate (MCR). Values are mean ± SE for 8-9 subjects. D: effects of exercise intensity and training on whole body lactate oxidation rate. Values are means ± SE for 8-9 subjects.

Lactate MCR was similar at rest before and after training (Fig. 5C). Lactate MCR increased significantly from rest to exercise by 150% at 45% pretraining O_{2 peak}, 100% at ABT, and 130% at RLT. Before training, MCR decreased 40% at 65% compared with 45% O_{2 peak} (P < 0.05). During exercise at ABT after training, MCR tended to increase, but the rise in MCR was not significant (P = 0.06). During exercise at RLT after training, lactate MCR increased 70% compared with 65% pretraining O_{2 peak} (P < 0.05).

Whole body lactate oxidation at rest was not significantly different between training states and increased during exercise regardless of intensity (Fig. 5D). Before training, lactate oxidation increased (P < 0.05) 700 and 2,000% during exercise compared with rest at 45 and 65% O_{2 peak}, respectively. After training, lactate oxidation during exercise also increased from rest by 1,300 and 2,500% at ABT and RLT, respectively. Similar to lactate turnover (R_a and R_d), lactate oxidation increased (P < 0.05) 160% at 65 vs. 45% O_{2 peak} before training, and 90% after training at RLT compared with ABT. Compared with 65% pretraining O_{2 peak}, lactate oxidation after training was 30% lower at ABT (P < 0.05), and unchanged at RLT.

Table 3 shows that, during exercise, the percentage of lactate R_d oxidized increased from rest regardless of training state or exercise intensity. Under all exercise conditions, most (60-80%) lactate was disposed of through oxidation. The percentage of R_d oxidized increased significantly with increments in exercise intensity, both before and after training, with no differences during ABT or RLT compared with values obtained during the 65% pretraining O_{2 peak} trial. The contribution of whole body lactate oxidation to whole body carbohydrate oxidation is also shown in Table 3. The percentage of total carbohydrate-derived CO₂ accounted for by lactate oxidation increased in the transition from rest to exercise when lactate oxidation accounted for 15-25% of total carbohydrate-derived CO₂. There were no differences at rest due to training, with increased contributions compared with rest at 65% pretraining O_{2 peak} and RLT. After training, the percentage of whole body carbohydrate oxidation from whole body lactate oxidation was 47% greater (P < 0.05) at RLT compared with ABT.

Tracer Lactate Uptake and _tot

Active Limb Contribution to Total Body Lactate Kinetics

View larger version (111K): [in this window] [in a new window]   Fig. 6.   A: %whole body lactate Ra from leg tot before and after training. Values are means ± SE for 5-8 subjects. There is a positive linear relationship between %lactate Ra from leg tot and exercise intensity [r = 0.99; pretraining: %Ra from tot = 0.59[%peak O2 consumption (O2 peak)] + 21.99, posttraining: %Ra from tot = 1.05 (%O2 peak) + 14.47]. B: %whole body lactate Rd from tracer-measured leg lactate uptake before and after training. Values are means ± SE for 7-9 subjects. There is a positive linear relationship between %lactate Rd from leg lactate uptake and exercise intensity [r = 0.98 pretraining: %Rd from leg lactate uptake = 0.38(%O2 peak) + 19.58, r = 0.96 posttraining: %Rd from leg lactate uptake = 0.65(%O2 peak) + 13.80]. C: %whole body lactate oxidation from leg lactate oxidation before and after training. Values are means ± SE for 6-8 subjects.

During exercise, approximately one-half of whole body lactate R_d could be explained by active-limb lactate uptake, which was greater during exercise compared with rest (Fig. 6B). Increasing exercise intensity before and after training did not alter the percentage of lactate R_d accounted for by limb lactate uptake. After training, at ABT leg lactate uptake accounted for 30% more of lactate R_d compared with 65% pretraining O_{2 peak}. Even though active legs accounted for a majority of lactate disposal during ABT and RLT after training (Fig. 6B), active legs contributed more to lactate R_a (Fig. 6A) than to R_d (Fig. 6B). There was a positive linear relationship between the percentage of lactate R_d from leg lactate uptake and exercise intensity [r = 0.98; pretraining: %R_d from leg lactate uptake = 0.38(%O_{2 peak}) + 19.58; r = 0.96; posttraining: %R_d from leg lactate uptake = 0.65(%O_{2 peak}) + 13.80].

There was no training effect on the percentage of whole body lactate oxidation attributable to the legs at rest (Fig. 6C). Active limbs accounted for the majority (70%) of whole body lactate oxidation during exercise before and after training (Fig. 6C). The percentage of whole body lactate oxidation from limb lactate oxidation did not increase during exercise compared with rest before or after training. After training at ABT, the percentage of whole body lactate oxidation from active limbs increased 30% compared with 65% pretraining O_{2 peak}, and 20% compared with RLT.

There were strong correlations (r = 0.99) between our two measures of lactate production (R_a and _tot) during rest and exercise both before and after training (Fig. 5A). Similarly, there were strong correlations (r = 0.97) between whole body lactate R_d and leg lactate uptake before and after training (Fig. 5B).

	DISCUSSION

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This is the first longitudinal investigation of training effects on lactate metabolism by using the combination of tracer technology and limb net exchange measurements. As such, our approach provided two estimates of lactate production during rest and exercise: tracer-derived blood lactate R_a and _tot. In general, values are highly correlated (r = 0.99, Fig. 5A) and show active muscle is the predominant, but not exclusive, site of lactate turnover during exercise.

Our data indicate that mechanisms for dampened arterial lactate concentration after endurance training vary depending on exercise intensity. At the same absolute workload, active trained limbs maintained similar lactate R_ox despite attenuated whole body lactate turnover and arterial concentration, due to increased F_ex. Thus, during moderate-intensity exercise after training (i.e., ABT), decreased lactate R_a and _tot and increased muscle lactate clearance contributed to dampened arterial lactate concentration. However, at a fixed relative exercise intensity (i.e., RLT), lactate turnover was the same after training as before, but leg lactate oxidation and whole body and leg lactate clearance increased.

With regard to circulating lactate concentration during exercise, we found that active-limb lactate release cannot explain the maintained elevation of arterial lactate concentration (Fig. 1A) as limb fell to close to zero by the end of exercise under all conditions (Fig. 1C). Therefore, other tissues must release lactate on net bases as exercise duration progresses.

Finally, regarding the use of as a measure of lactate production, underestimated _tot before and after training. Because limbs simultaneously take up and release lactate, alone cannot quantitate limb lactate production. Critically, measurements of differences between arterial and venous concentrations miss important parameters of muscle lactate uptake and oxidation.

Training Adaptations

Muscle Lactate Concentration

Several investigators have reported significantly decreased muscle lactate concentrations at the beginning of exercise in trained compared with untrained subjects (16, 26). Those data are consistent with our observations of a training effect that dampens most dramatically at the start of exercise (Fig. 1, B and C). Low muscle lactate concentrations after exercise (Table 2) are attributable to high R_ox (Figs. 4 and 5D).

Limb Lactate Exchange

Muscle rates computed from arteriovenous difference and blood flow obscure lactate uptake and oxidation during (11, 12, 42). Stanley et al. (42) reported _tot ( + tracer-measured lactate uptake) to be roughly twice by using [3-¹⁴C]lactate infusion during graded-intensity leg cycling in men. Subsequently, using [3-¹³C]lactate infusion, Brooks et al. (11) reported _tot to be 400% greater than during sea-level leg cycling at 51% O_{2 peak}. We found similar results in the present study, with mean _tot 280% greater than pretraining and 320% greater posttraining (Table 4). Thus results of our study show that blood concentration and are inadequate measures of either whole body or tissue lactate metabolism.

Our data show active limb decreased to close to zero after 45 min of exercise (Fig. 1C), whereas arterial lactate concentration remained elevated (Fig. 1A); similar results have been reported before (1, 11, 19, 45). We interpret these data to suggest that muscle contributes to elevated arterial lactate concentration at the beginning of steady-rate exercise, but, as exercise duration progresses, other tissues become important for maintaining circulating lactate concentration and providing lactate as a substrate for working muscle. Several other tissues, including skin (24), adipose (23), and intestine (40), have been shown to release lactate on net bases. It is possible increased in these tissues as exercise duration progressed. Thus, our data, as well as those of others (1, 19, 45), suggest that muscle is not responsible for maintaining elevated arterial lactate concentration after as little as 45 min of steady-rate exercise.

Intramuscular Lactate Metabolism

Whole Body Lactate Kinetics

Our results diverge from the literature on effects of endurance training on lactate turnover as we found significantly decreased lactate R_a and R_d during ABT after training compared with those parameters determined during the same task before training (Fig. 5, A and B). Thus our data suggest that endurance training promotes decreased arterial lactate concentration at a given absolute workload by decreasing whole body lactate R_a (Fig. 5A), active-leg total lactate production (Table 4), and (Fig. 1C, Table 4). We note in this regard that the training-induced increase in whole body lactate MCR at ABT approached, but did not achieve, significance (P = 0.06).

There are several potential mechanisms that may have promoted decreased lactate production (R_a and _tot) during exercise at ABT after training. In working muscle, decreased glycogen degradation after training at a given absolute workload (2, 22, 39) should result in less lactate formation because of decreased glycolytic flux. Similarly, although training increases muscle GLUT-4 content, less is translocated to the sarcolemma during exercise at a given power output after training (35). Perhaps more importantly, increased mitochondrial mass enhances intramuscular pyruvate and lactate oxidation and therefore decreases after training (8). At other tissue sites, glycogenolysis leading to lactate production may have been attenuated by the decline in circulating epinephrine. Consistent with this interpretation, -adrenergic blockade decreases arterial lactate concentration, whereas muscle is minimally affected (10).

Donovan and Brooks (14) and Phillips et al. (33) reported unchanged lactate turnover and increased MCR in rats and humans, respectively, exercising at an absolute workload after endurance training. Like the aforementioned researchers, we found that whole body lactate MCR tended to increase after endurance training at ABT (P = 0.06) (Fig. 5C); however, we found decreased lactate turnover at ABT. Phillips et al. employed a short, 10-day training program that may explain why they did not find dampened whole body lactate R_a at ABT. Donovan and Brooks (14) also reported unchanged lactate turnover after endurance training in rats. Possibly, the inability to precisely control exercise intensity of rats during treadmill running may explain the lack of agreement with the present study. Thus the previous data on running rats may be more like the present results obtained during RLT than ABT.

Ours is the first study to report endurance training effects on lactate kinetics at a given relative intensity under steady-state conditions. Our data suggest endurance training does not alter whole body lactate appearance at RLT (Fig. 5A) but increases whole body and leg lactate oxidation and clearance (Figs. 4 and 5, C and D; Table 4). Additionally, (Fig. 1C) and _tot (Table 4) were unchanged at RLT compared with 65% pretraining O_{2 peak}. We attribute the lack of a training effect on whole body lactate flux and increase in leg lactate oxidation at RLT after training to the effect of muscle contraction on glycolysis. Compared with before training, the power output required to elicit 65% of O_{2 peak} increased by 22 W, or 15%, which elicited a similar or greater glycolytic flux. Thus both before and after endurance exercise training, whole body lactate kinetics closely parallel working-muscle lactate production, and relative exercise intensity dictates whole body and active-muscle lactate metabolism.

Donovan and Brooks (14) and Brooks and Gaesser (9) first reported that oxidation was the major fate of lactate during and after exercise, respectively, with 75-80% oxidation of infused tracer. Similarly, using [1-¹³C]lactate in exercising humans, Mazzeo et al. (31) reported 82% oxidation of lactate R_d at 50% maximal O₂ consumption and 78% oxidation at 75% maximal O₂ consumption. Our data are consistent with literature showing oxidation as the main fate of lactate R_d during exercise (Table 3, Fig. 5D). At rest, only 20 and 25% of lactate R_d was oxidized before and after training, respectively. However, exercise dramatically increased lactate oxidation such that, before training, oxidation accounted for 60 and 70% of lactate R_d at 45 and 65% O_{2 peak}. After training, 70 and 80% of lactate R_d was oxidized at ABT and RLT, respectively. Thus oxidation is the major fate of whole body lactate disposal during exercise; lactate oxidation scales to exercise intensity and increases at a given RLT after training.

Leg total lactate production accounted for 54% of whole body lactate R_a before training and 77% after training (Fig. 6A). Thus our data suggest that most of decreased lactate R_a after training may be attributable to decreased active muscle lactate release. Active skeletal muscle was slightly less influential in determining whole body lactate R_d, with only 41% of lactate R_d attributable to active-muscle lactate uptake before training, and 53% after training (Fig. 6B). Other tissues, such as liver and inactive skeletal muscle (34), must have contributed to lactate clearance during exercise before and after training. The majority of whole body lactate oxidation was also due to active muscle lactate oxidation. Leg oxidation accounted for 70% of whole body lactate oxidation before and after training (Fig. 6C). Thus it appears that active muscle is largely responsible for alterations in whole body lactate turnover and oxidation during exercise, both before and after training.

Assumptions and Limitations

As with the lack of specificity of venous lactate concentration measurements, a thermodilution technique is unable to determine alterations in blood flow to different muscle fiber types. We assumed that blood flow to individual muscle fibers was unchanged after endurance training, and alterations in leg lactate metabolism were due to changes in skeletal muscle cellular metabolism. It is possible that increased capillary density around type I fibers after training (29) may have decreased transit time specific to type I fibers, resulting in altered muscle fiber perfusion patterns that could alter lactate exchange.

Different equations were used to calculate lactate uptake and oxidation during rest and exercise. Ninety minutes of rest were insufficient to achieve isotopic equilibrium in CO₂ pools as we found ¹³CO₂ consumption across resting limbs. Therefore, to estimate lactate oxidation during rest we determined tracer lactate uptake from isotopic dilution of lactate in femoral venous compared with arterial blood. Thus we may have overestimated resting-limb lactate oxidation. Additionally, tracer fractional extraction was highly variable during exercise, and we estimated muscle lactate uptake from ¹³CO₂ release across limbs. Variable tracer uptake data could be explained in part by [¹³C]lactate release into venous blood from glycogenolysis of ¹³C-labeled glycogen stored during rest. However, this is unlikely as the period of rest was too short to extensively label blood glucose (2) or muscle glycogen stores with [¹³C]glucose. Alternatively, we considered whether ¹³CO₂ release from active muscle could be due to isotopic equilibration in the TCA cycle during gluconeogenesis. For the present, we are confident ¹³CO₂ release across working muscle provides an acceptable minimal estimate of lactate uptake as decarboxylation of [3-¹³C]lactate tracer in working muscle is due to oxidation in the TCA cycle and not loss of label during gluconeogenesis because key enzymes of gluconeogenesis (e.g., phosphoenolpyruvate carboxykinase) are not expressed in skeletal muscle.

Finally, there has been controversy as to whether lactate tracers measure lactate turnover or pyruvate turnover, and, therefore, total carbohydrate oxidation. Concerns with using tracer lactate to quantitate turnover stem from studies (36) in which tracer lactate or pyruvate infusion resulted in similar lactate and pyruvate enrichments in blood after several minutes. However, the authors failed to appreciate that the lactate-pyruvate equilibrium in blood due to action of LDH in erythrocytes leaves almost all tracer as lactate, because plasma lactate-to-pyruvate ratio rises from 10 to 50 or more during exercise. Hence, infused lactate tracer remains in the blood as lactate.

More importantly, with regard to the use of tracers, our data suggest that lactate turnover does not measure pyruvate turnover because the ratio of whole body and leg lactate oxidation to total body and leg carbohydrate oxidation was always much less than 1 (Table 3). As already discussed, we obtained excellent correlations between lactate R_a and muscle _tot (Fig. 5A). Therefore, our data suggest that carbon-labeled lactate tracers can be used to quantitate blood lactate flux.

Conclusions