1 Department of Integrative
Biology, To evaluate the
hypothesis that endurance training increases intramuscular triglyceride
(IMTG) oxidation, we studied leg net free fatty acid (FFA) and glycerol
exchange during 1 h of cycle ergometry at two intensities before
training [45 and 65% of peak rate of oxygen consumption
(
exertion; free fatty acid; glucose; lactate; crossover concept; substrate shuttling
EFFECTS OF EXERCISE intensity and training state on
muscle lipid metabolism in humans are unclear, as the relatively few
studies have utilized different experimental approaches. Some
investigations have employed isotopic tracers and indirect calorimetry
(25, 29, 33), whereas others have utilized arteriovenous (a-v) balance
methods (17, 22, 35) and muscle biopsies (18, 22). In addition,
longitudinal and cross-sectional designs have been employed that have
tested trained and untrained subjects at either the same absolute or
relative exercise intensity, but rarely both. Additionally, almost all
studies have tested subjects after an overnight fast, in a
glycogen-depleted state, or both (1, 2, 14, 16, 17, 20, 22, 30), making
difficult comparisons to real life exercise, which is usually preceded
by resting and eating.
Using a-v measurements, some studies have shown significant free fatty
acid (FFA) uptake by resting limbs (14, 15, 35). In contrast, other
investigations report net limb FFA release at rest (2, 22, 30, 32). In
parallel, net glycerol release from resting limbs has been a consistent
observation (2, 14, 15, 22, 30, 32, 35). Although Hagenfeldt and Wahren (14) did not observe increased net limb FFA uptake in the transition from rest to exercise, most studies report increased net FFA uptake during exercise compared with rest (2, 22, 30, 32, 35). In addition,
some investigators report higher net glycerol release during exercise
compared with rest (2, 22, 30, 32, 35). This result is, however, not
without contradiction (14, 17).
To date, only three studies have examined training effects on working
limb FFA and glycerol uptake and release. Investigators using single
leg kicking protocols have found increased net FFA uptake at 2 (22) and
3 (35) h of exercise at the same absolute and relative exercise
intensity, respectively. Although one study has found increased net
glycerol release after 2 h of exercise (22), others have not (17, 35).
Recently, several groups of investigators used FFA isotope tracers and
indirect calorimetry to estimate the contribution of intramuscular
lipid stores to total lipid oxidation (25, 29, 33). Because the
difference between whole body FFA oxidation measured by indirect
calorimetry and tracer-measured FFA flux was greater after training,
the data were interpreted to mean that training increased reliance on
intramuscular triglycerides (IMTG; see Refs. 25 and 29).
The effect of training on IMTG use during exercise has also been
addressed by means of the muscle biopsy technique. Hurley et al. (18)
measured decreases in intramuscular lipid via muscle biopsies and
concluded that training produced greater reliance on intramuscular
lipid sources for oxidation. However, Kiens et al. (22) did not confirm
this result. Most recently, Kiens and Richter (23) reported unchanged
IMTG concentration after 90 min of exercise.
Because of controversies over the effects of exercise intensity and
training status on the roles played by IMTG and plasma FFA as fuels
during exercise, the present study was undertaken. Using a longitudinal
design and careful nutritional control, we evaluated the hypothesis
that endurance training increases intramuscular FFA oxidation at given
absolute and relative exercise intensities. Results of limb metabolite
a-v and respiratory quotient (RQ) measurements do not support the
hypothesis; results indicate that training does not appreciably change
reliance on IMTG or plasma FFA, which are of minor importance as fuels
for moderate and greater-intensity exercises.
Subjects
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
O2 peak)]
and after training [65% pretraining
O2 peak, same
absolute workload (ABT), and 65% posttraining
O2 peak, same
relative intensity (RLT)]. Nine male subjects (178.1 ± 2.5 cm, 81.8 ± 3.3 kg, 27.4 ± 2.0 yr) were tested before and after
9 wk of cycle ergometer training, five times per week at 75%
O2 peak.
The power output that elicited 66.1 ± 1.1% of
O2 peak before
training elicited 54.0 ± 1.7% after training due to a 14.6 ± 3.1% increase in
O2 peak. Training
significantly (P < 0.05) decreased
pulmonary respiratory exchange ratio (RER) values at ABT (0.96 ± 0.01 at 65% pre- vs. 0.93 ± 0.01 posttraining) but not RLT (0.95 ± 0.01). After training, leg respiratory quotient (RQ) was not
significantly different at either ABT (0.98 ± 0.02 pre- vs. 0.98 ± 0.03 posttraining) or RLT (1.01 ± 0.02). Net FFA uptake was
increased at RLT but not ABT after training. FFA fractional extraction
was not significantly different after training or at any exercise
intensity. Net glycerol release, and therefore IMTG lipolysis
calculated from three times net glycerol release, did not change from
rest to exercise or at ABT but decreased at the same RLT after
training. Muscle biopsies revealed minor muscle triglyceride changes
during exercise. Simultaneous measurements of leg RQ, net FFA uptake,
and glycerol release by working legs indicated no change in leg FFA
oxidation, FFA uptake, or IMTG lipolysis during leg cycling exercise
that elicits 65% pre- and 54% posttraining
O2 peak. Training
increases working muscle FFA uptake at 65%
O2 peak, but high RER
and RQ values at all work intensities indicate that FFA and
IMTG are of secondary importance as fuels in moderate and
greater-intensity exercise.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
O2 peak) of <45
ml · kg
1 · min
1.
Subjects were included in the study if they had <25% body fat, were
nonsmokers and diet and weight stable, had a 1-s forced expiratory volume (FEV1) of 70% or more,
and were injury and disease free as determined by physical examination.
This study was approved by the Committee for the Protection of Human
Subjects at Stanford University and the University of California,
Berkeley (CPHS 97-6-34).
Experimental Design
After preliminary screening and interviews, subjects performed two graded exercise tests to determine thePreliminary Testing
All exercise tests were performed on an electronically braked cycle ergometer (Monark Ergometric 829E). For determination ofTesting Protocol
The night preceding each experimental trial, subjects were admitted to the metabolic ward where they remained until testing was completed the following day. Subjects were fed a standardized dinner [1,174 kcal: 66% carbohydrate (CHO), 21% fat, 13% protein] that was replicated the night before each experimental trial. Later that evening, subjects ate a standardized snack (500 kcal: 53% CHO, 31% fat, and 16% protein) before retiring. Two subjects were tested per day with morning and afternoon testing randomly assigned to each subject and replicated for every trial. Morning procedures started at 7:00 AM, whereas preliminary afternoon procedures began at 1:00 PM. Each subject was tested at the same time of day throughout the study. Morning subjects ate a standardized pretrial meal (448 kcal: 72% CHO, 10% fat, 18% protein) at 6:00 AM, 1 h before procedures started, and 4.5-5 h before exercise. Afternoon subjects ate a standardized breakfast in the morning (729 kcal: 57% CHO, 33% fat, 10% protein) and the standardized pretrial meal at noon, again 1 h before procedures began.Catheterizations
After local lidocaine anesthesia, the femoral artery and vein of the same leg were cannulated using standard percutaneous techniques as previously described (37). A 5.1-Fr, 50-cm, Cordis arterial catheter (model 91100900; North American Medical Instrument) was inserted 25 cm and was positioned in the distal abdominal aorta via the femoral artery. A 6-Fr thermodilution venous catheter (model 93-135-6F; American Edwards Laboratories) was placed with the tip in the distal iliac vein through a venous sheath in the femoral vein 20 cm from the skin. Both catheters were sutured to the skin and further secured by an Ace bandage wrap. The external portions of each catheter were directed toward the hip for easy access during exercise. Alternate legs were used for both pretraining and posttraining testing. One subject experienced blood leaking from catheter placements during the beginning minutes of exercise at 65% pretraining and did not perform further exercise. Two different subjects did not receive a venous catheter for one of their trials. As a result, a sample size of 6-9 was used for calculations and comparisons.Blood Sampling
Blood temperature was obtained from a thermistor at the end of the venous thermodilution catheter immediately before blood sampling. Arterial and venous blood samples were drawn simultaneously and anaerobically over 5 s after 75 and 90 min of rest and at 5, 15, 30, 45, and 60 min of exercise. PO2, PCO2, and pH were measured within 30 min (ABL 300; Radiometer, Copenhagen, Denmark). Blood for FFA concentration ([FFA]) and glycerol concentration ([glycerol]) determination was immediately transferred to tubes containing EDTA, shaken, and placed on ice. After the final blood sample at the end of exercise, samples were centrifuged at 3,000 rpm for 10 min, and plasma was transferred to storage tubes and frozen atMuscle Sampling and Analysis
During preparation for blood sampling, one vastus lateralis was prepared for percutaneous needle biopsy. For each experimental trial, biopsies were taken from two locations separated by 1.5 cm: the distal site for preexercise sampling and the proximal site for immediate postexercise sampling. Right and left vastus muscles were alternated between trials. Biopsies taken at rest, and within 10 s of exercise cessation, were immediately plunged in liquid nitrogen and subsequently stored under liquid nitrogen or atHemodynamics
Heart rate and electrocardiogram (ECG) were continuously recorded and displayed using a three-lead ECG connected to a MacLab analog-to-digital converter (ADInstruments, Castle Hill, Australia) and were tracked on a Macintosh 7200/200 Power Mac computer (Apple Computer, Cupertino, CA). Arterial blood pressure was also continuously recorded and displayed using a Transpac pressure transducer (Baxter) positioned at the level of the heart connected to the MacLab system and was calibrated before every trial. Iliac venous blood flow was determined by the thermodilution technique using a cardiac output computer (model 9520; American Edwards Laboratories) with a 10-ml bolus injection of sterile saline cooled to 0°C via an ice slurry (American Edwards Laboratories-Set II, 93-520). Measurements were made in triplicate or quadruplicate during rest and exercise immediately after blood sampling. The validity and precautions associated with this technique have been described previously (3).Metabolite Analysis
FFAs were measured in duplicate using the Wako NEFA-C kit (Wako Chemicals, Richmond, VA). Glycerol was also measured in duplicate using a triglyceride kit (GPO-Trinder), and glucose was measured in duplicate using a hexokinase enzymatic kit, both from Sigma Chemical (St. Louis, MO). Lactate was measured in duplicate using the method of Gutmann and Wahlefeld (13) using lactate dehydrogenase.Training Protocol
All training was performed on stationary cycle ergometers 5 days/wk with workloads adjusted to the required intensity based on maximal exercise test results, and heart rates were recorded daily by personal trainers who had taken the exercise physiology course at the University of California, Berkeley. Subjects were asked to exercise 1 day/wk on their own in addition to cycle ergometry training so that total training was 6 days/wk. All subjects were exercising at 75% of theirCalculations
Leg RQ. Leg RQ was calculated from the ratio of venous-arterial CO2 content difference (v-aCO2) and arteriovenous O2 content difference (a-vO2)
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Blood CO2 content. Blood PCO2, PO2, pH, and hemoglobin (Hb) were measured on both arterial and venous samples and were used in the calculations by Douglas et al. (8) for determination of blood CO2 content (CCO2)
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Blood O2 content. Blood O2 content was calculated using hemoglobin concentration and oxygen saturation (SO2) determined from the equation of Nunn (27)
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Statistical Analyses
Average arterial glucose and lactate concentration from the last 30 min of exercise were analyzed using a one-factor ANOVA with repeated measures. Differences between groups and changes over time in concentration, a-v difference, net uptake/release of FFA and glycerol, and FFA fractional extraction were determined using a repeated-measures factorial ANOVA. Post hoc comparisons were made using Fisher's protected least significant difference. One-hour averages of leg and body FFA oxidation, IMTG lipolysis, and IMTG oxidation were analyzed using unpaired Student's t-tests. Muscle biopsy concentration differences were determined with paired Student's t-tests. Statistical significance was set atOther Measurements
In addition to parameters included here, systemic blood pressure, cardiac output, glucoregulatory hormones, muscle metabolites, glucose and lactate fluxes, and tissue exchange rates were measured. These results will be reported separately. ![]() |
RESULTS |
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Subject Characteristics
Anthropometric data for subjects pre- and posttraining are shown in Table 1. Subjects were weight stable throughout the study period, although absolute changes in percent body fat decreased significantly as determined by both skin-fold measurements (
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Muscle Triglyceride Concentrations
Resting quadriceps triglyceride concentrations decreased after training and tended to increase during exercise (Table 3). However, the increases in IMTG concentration were quantitatively small, and the significant increase during exercise at 45%
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Indirect Calorimetry
Mean steady-stateLeg Blood Flow
Leg blood flow increased from rest to exercise in all groups. Exercise intensity significantly increased leg blood flow throughout exercise before and after training, with higher intensities eliciting significantly greater blood flows (Table 2). Furthermore, training status affected blood flow, as the same absolute workload after training resulted in significantly higher leg blood flows (10.3%) across all time points compared with before training. Likewise, the same relative workload elicited 25.7% higher leg blood flows after training.Arterial Glucose and Lactate Concentrations
Arterial glucose concentrations did not change at rest after training, nor were average glucose concentrations different for the last 30 min of exercise before or after training regardless of exercise intensity (Fig. 1A). Resting lactate concentrations were also not significantly different before and after training (Fig. 1B). Lactate concentrations increased significantly from rest to exercise in all but the 45% pretraining intensities. Increasing exercise intensity significantly raised lactate concentrations compared with rest by 44.5% (45% pretraining) and 366% (65% pretraining) before training and by 159% (ABT) and 332% (RLT) after training. Training resulted in significantly lower lactate concentrations at the same absolute (55% decrease) and relative workloads (26% decrease).
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Arterial [FFA], [Glycerol], Net Uptake, and Release Across the Legs
FFA. Although there were no differences among exercise intensities, after an initial decline at exercise onset, arterial [FFA] increased over time (Fig. 2A). Negative limb [a-v] differences at rest indicated net FFA release, with a switch to positive [a-v] differences indicating net limb FFA uptake during exercise. FFA a-v differences were similar among exercise intensities, with mean values increasing over time (Fig. 2B). Fractional limb FFA extraction was not significantly different with training or exercise intensity, at 5.84% pretraining and 6.25% posttraining (Fig. 2C). Thus neither exercise intensity nor training status affected fractional FFA uptake. Mean net FFA uptake tended to be higher at 65% pretraining than 45% pretraining, although the difference was not significant (P = 0.06; Fig. 2D). After training at both ABT and RLT, mean net FFA uptake was significantly greater than 45% pretraining (P < 0.05). At ABT, mean net FFA uptake was not significantly different from 65% pretraining. However, at RLT, mean net FFA uptake was significantly greater than 65% pretraining, even though values were similar at 45 and 60 min, indicating that net FFA uptake increased more quickly at the beginning of exercise after training (Fig. 2D). There was a trend for increased mean net FFA uptake after training at RLT vs. ABT; however, the difference did not reach significance (P < 0.06).
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Glycerol. Arterial
[glycerol] increased significantly over time from resting
values of 0.07 mM for all groups to a 60-min exercise value of 0.20 mM
for all but the 45% pretraining trials (Fig.
3A).
Mean exercise [glycerol] for 45% pretraining was
significantly lower than the other three exercise intensities. Glycerol
v-a differences during exercise were significantly lower than at rest, falling from a value of 0.035 mmol to close to zero during exercise (Fig. 3B). Although there were no
significant changes over time during exercise, glycerol v-a during the
65% pretraining trial decreased to negative values such that the mean
v-a difference was significantly lower than 45% pretraining and ABT.
Net glycerol release did not change significantly from rest throughout
exercise, resulting in little net glycerol release during any of the
pretraining or posttraining trials (Fig.
3C). After training, mean exercise net glycerol release at RLT was significantly lower than during ABT,
with values from the last 30 min of exercise averaging from 0.019 mmol/min for RLT to 0.047 mmol/min for ABT. Net glycerol release data are consistent with literature values as shown in Fig.
4.
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Leg RQ. As reported by others (20, 28,
34), leg RQ varied more than pulmonary RER. Leg RQ was not
significantly different at rest before or after training (Fig.
5). Average exercise RQ was not different
from rest at 45%
O2 peak but was
increased significantly at 65%
O2 peak
pretraining. Exercise RQ was also not significantly different from rest
after training at either intensity. At the same absolute or relative
exercise intensity after training, there were no differences in leg RQ.
However, both posttraining exercise leg RQ values were significantly
higher then 45% pretraining.
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One-hour averages of leg FFA oxidation calculated from leg RQ and whole body FFA oxidation calculated from pulmonary RER are shown in Fig. 6. There were no significant differences in leg FFA oxidation during the two pretraining intensities (Fig. 6A). Posttraining, leg FFA oxidation was significantly lower (72%) at RLT compared with ABT (Fig. 6A). Whole body FFA oxidation at 65% pretraining was significantly lower than 45% pretraining (45%) and ABT (58%; Fig. 6B). Figure 6 also shows mean net FFA uptake and IMTG lipolysis calculated from net glycerol release data as described in METHODS. There was a tendency for greater mean net FFA uptake with increased exercise intensity pre- and posttraining (Fig. 6C); however, the differences did not reach significance (P < 0.06). Mean net FFA uptake was significantly greater at the same relative exercise intensity posttraining (P < 0.05). IMTG lipolysis was significantly decreased posttraining at RLT compared with ABT (P < 0.05; Fig. 6D).
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DISCUSSION |
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We evaluated the hypothesis that endurance training increases reliance on IMTG at given absolute and relative exercise intensities; our results do not support the hypothesis. Working limb net glycerol release, and, therefore, intramuscular (intralimb) lipolysis, were unaffected at either absolute or relative workloads after training. Furthermore, no other data source (i.e., whole body RER, leg RQ, net FFA uptake, or change in muscle triglyceride content) indicated blood-borne FFA or IMTG to be major energy sources during 1 h of sustained exercise regardless of training state or exercise intensity.
Training Adaptations
Our training program was successful in promoting significant metabolic adaptations (Tables 1 and 2, Fig. 1B). During our 9-wk training program, subjects significantly increasedNutritional Controls
Our experiments were designed to reveal effects of exercise and training on substrate utilization. We employed experimental and nutritional controls appropriate to represent practices and conditions typical in the population at large. For these reasons, we fed subjects to be weight stable and rested them the day before experimentation. Furthermore, we fed standardized, low-glycemic-index meals 4.5-5.5 h before exercise studies. Those efforts produced stable blood glucose levels during exercise, suggesting that subjects commenced exercise with normal liver glycogen reserves.After resting and feeding subjects before experiments, arterial [FFA] and [glycerol] rose during exercise (Figs. 2A and 3A). In parallel studies on different subjects, we measured glycerol and FFA kinetics in men and women during rest and exercise both before and after training and found increased FFA and glycerol rates of appearance in women (12) and men (Friedlander, A.L., G.A. Casazza, M.A. Horning, and G.A. Brooks, unpublished observations). These results are consistent and show that our experimental design permitted lipolysis to occur during exercise and made FFAs available to working muscle in excess of demand. However, glycogen, glucose, and lactate were preferred to FFAs and IMTG when blood glucose was stable and liver and muscle glycogen contents replete, as determined by leg RQ and mass balance. Thus the effects on substrate utilization that we observed are attributable to exercise intensity and endurance training and are not confounded by undernutrition, liver glycogen depletion, or hypoglycemia.
IMTG Concentration
Values for IMTG concentration are low compared with those of others (18, 22, 23). In this regard, we refer to the recent paper of Wendling et al. (36) who showed great interindividual variability in IMTG values. More important, degradation rates during exercise are similar to those reported by others (18, 22, 23). Similar to Kiens and Richter (23) and Kiens et al. (22), IMTG concentration did not decrease in our study after 1 h of exercise (Table 3), which corroborates our net glycerol release data suggesting that IMTG is not an important substrate for exercise when subjects are fed a meal 4-5 h before exercise.Glycerol Exchange
Our results of resting net limb glycerol release (0.05 ± 0.01 mmol/min) are similar to previously reported values that range from 0.01 to 0.041 mmol/min (1, 22, 31, 32; Fig. 4). Unlike our results, however, some investigators have found increased net glycerol release during exercise compared with rest (1, 32). Our results are similar to those of Henriksson (17) who found unchanged glycerol release during exercise in trained and untrained working legs. Only one study reported increased net glycerol release after training at the same absolute workload, but 2 h of exercise were required to produce the effect (22). Although in the report of Kiens et al. (22) increased net limb glycerol release implicated increased intramuscular lipolysis, muscle biopsies did not reveal significantly lower IMTG stores after one leg knee extension exercise. Therefore, increased net glycerol release after 2 h of exercise may not have represented degradation of only IMTG but rather may have included FFA from other adipose tissue stores. Moreover, Phillips et al. (29) and we (12 and Friedlander et al., unpublished observation) found unchanged whole body glycerol turnover at the same absolute and relative workload after training, consistent with unaltered IMTG utilization. Thus, in the aggregate, glycerol tracer (29), limb balance (Fig. 3C and Refs. 17, 22, 35), and muscle biopsy data for IMTG (Table 3 and Refs. 22, 23) are interpreted to mean training does not increase IMTG use during exercise.In contrast to results of net glycerol release and glycerol turnover studies, data from FFA turnover and indirect calorimetry, as well as muscle biopsies measuring IMTG, suggest that training may increase reliance on IMTG at a given absolute workload (18, 25, 29). Martin et al. (25) used the difference between whole body FFA oxidation calculated from pulmonary RER and isotopically determined FFA rate of disposal (Rd) to indirectly estimate IMTG utilization. Assuming the difference between these parameters represented intramuscular metabolism, they concluded that subjects utilized significantly more intramuscular lipid when evaluated at the same absolute workload after training.
To mimic the calculations of Martin et al. (25) and Phillips et al. (29), we used whole body fat oxidation (from RER) and limb net FFA uptake (from a-v difference) to estimate IMTG oxidation. Like them, our calculations also produced the apparent result of significantly increased (116%) IMTG oxidation at the same absolute intensity after training. Yet, using working limb net glycerol release to directly measure lipolysis, we found unchanged IMTG utilization after training at given absolute and relative exercise intensities. Our data provide evidence to suggest that methodologies employing whole body RER and FFA Rd overestimate IMTG oxidation (Fig. 6). Moreover, working muscle glycerol release rates calculated from indirect measures (29, 33) are 5- to 10-fold higher than directly measured net glycerol release rates reported for working limbs (Fig. 4). The indirect estimation of IMTG oxidation (29, 33) relies on the assumption that the difference between a parameter measured in breath (i.e., total lipid oxidation) and a parameter measured in blood (i.e., FFA Rd) represents exclusively events occurring in intracellular triglyceride depots in contracting muscle. This assumption lacks experimental basis and is at variance with our results. Thus disagreement over interpretation of results obtained in various investigations regarding intramuscular lipolysis may be due to assumptions inherent in the methodologies employed.
FFA Exchange
On resting subjects following a 12- to 14-h fast, most investigations report the legs to release FFA (2, 17, 22, 31, 32). Net FFA uptake during exercise is a consistent finding (2, 14, 15, 20, 22, 30, 32, 35), with increased net FFA uptake in trained subjects only after 2 (22) and 3 h (35) of leg kicking at the same absolute and relative intensity, respectively. Thus our limb FFA exchange data agree with literature values, as we found net limb FFA release at rest. During exercise, FFA were extracted on a net basis, with no effect of training at the same absolute workload during 1 h of cycling. Although it is an underestimate of fractional FFA extraction determined with tracers (35), fractional FFA extraction averaged 6.0% for untrained and 6.4% for trained subjects during exercise in our study, comparable to Kiens et al. (22) who observed 6-7% fractional extraction for the first hour of exercise in both trained and untrained subjects. Although there were trends suggesting elevated net FFA uptake with increasing exercise intensity (P < 0.06), similar to Hagenfeldt and Wahren (15), we did not find a significant difference in net FFA uptake when increasing exercise intensity before or after training (Figs. 2D and 6). In contrast to Jansson and Kaijser (20) who also compared trained and untrained subjects using cycle ergometry, we found increased net FFA uptake at the same relative intensity after training. Thus we report the only limb balance data using longitudinal comparisons indicating training-enhanced net FFA uptake at the same relative exercise intensity during cycle ergometry. Moreover, our and previous results from net limb balance (22) suggest that, after training, an exercising leg does not extract more FFA on a net basis after 1 h of exercise at a given workload.Only one report utilized a longitudinal design to compare FFA Rd at the same absolute and relative exercise intensity before and after training (12). Friedlander et al. (12) found slightly increased tracer-measured FFA Rd for a given power output and increased FFA Rd at the same relative exercise intensity after training. If whole body FFA kinetics respond similarly to working limb net FFA uptake, one might expect increased net FFA uptake at both the same absolute and relative exercise intensity after training. However, although we found increased net FFA uptake at RLT, we did not observe significantly different net FFA uptake or leg FFA oxidation at ABT compared with 65% pretraining (Fig. 6 and Table 2), suggesting that whole body tracer kinetics do not always parallel FFA metabolism of the working limb.
In our report, we were careful to state that we measured leg net metabolite exchange, although we made repeated inference to muscle metabolism. Although it is true that muscle comprised most of the tissue and during exercise received most blood flow in the limbs studied, possible contributions of other tissues cannot be ignored. For instance, subcutaneous and intermuscular adipose tissue could have and probably did experience lipolysis during limb exercise. Release of glycerol and FFA from nonmuscle adipose tissue could have minimized the possibility of measuring muscle FFA uptake. However, our results are likely due to intramuscular rather than subcutaneous adipose tissue effects because muscle mass predominates in the leg. Furthermore, the anatomy of venous drainage in the leg would result in glycerol and FFA from subcutaneous fat entering the femoral venous blood without opportunity for muscle exchange. Because glycerol release was close to nil during rest and exercise, we can tentatively conclude that lipolysis in both subcutaneous and intramuscular fat depots was negligible. Additionally, neither adipocytes nor myocytes are known to contain glycerol kinase (26); thus, it is unlikely that reesterification accounted for minimal leg net glycerol release. Moreover, we overlooked possible contributions of lipid delivery via lipoproteins. Although largely unstudied, lipoproteins could deliver significant quantities of fatty acid during exercise. However, for the present, we discount this possibility, because leg RQ was high, glycerol release was small, and glycerol [v-a] declined during exercise. Thus we conclude that no lipid stores (IMTG, lipoproteins, interstitial or subcutaneous adipose tissue) underwent active lipolysis during exercise. Because future work is necessary to confirm the validity of our technical approaches and assumptions, we have emphasized interpretation of results from several sources, including whole body RER, leg RQ, glycerol, and FFA exchange. To reiterate, none of these experimental approaches indicate increased IMTG utilization at either the same absolute or relative exercise intensity after training.
Leg RQ
There have been few studies investigating training and leg RQ, with divergent results. Henriksson (17) and Kiens et al. (22) found lower leg RQ after training at the same relative intensity during cycle ergometry, and absolute intensity during leg kicking, respectively. However, our results are similar to those of Saltin et al. (34) and Jansson and Kaijser (20) who found unchanged leg RQ's after training at a given workload during cycle ergometry. Reported differences may be associated with the mode of exercise, as power output during leg cycling is much greater than during kicking. Additionally, leg kicking does not alter the hormonal milieu characteristic of whole body exercise, which will influence substrate utilization. Thus our data, in addition to RQ values reported by others (20, 34), suggest that, unlike the whole body, leg substrate utilization does not change appreciably during cycle ergometry at a given workload after training.Although our results of leg RQ are consistent with literature values,
they do not match the pattern of limb FFA uptake observed during
posttraining trials at 65% new
O2 peak (Fig. 6). The discrepancy may be attributed to the assumption of complete FFA oxidation or algorithms for computing leg RQ from blood gases. However,
despite differences in the pattern of leg FFA oxidation determined from
leg RQ and net FFA uptake, it remains that both methods yield low
estimates of FFA oxidation during exercise. Additionally, after storage
of FFA uptake as IMTG has been accounted for (Table 3), oxidation of
the remaining FFA taken up during 65% posttraining
O2 peak would cause the
limb RQ to decrease only from 1.00 to 0.96. Thus our conclusion of
small fat oxidation by the exercising leg before and after training is
supported by both FFA and glycerol a-v differences and RQ measurements.
Tracers vs. Net Balance Studies
We interpret our results of lower whole body RER but unchanged leg RQ at a given exercise intensity to mean that inactive tissue and not active skeletal muscle increases fat oxidation after training (Table 2). Similarly, Odland et al. (28) studied subjects 2-3 h after a meal during cycle ergometry at 65%Differences between the present investigation showing a training effect increasing working limb FFA uptake (Figs. 2D and 6) and previous studies on men using tracers to estimate FFA Rd may be related more to the subject populations studied and details of protocol rather than physiology. For instance, in their report, Martin et al. (25) observed training to decrease FFA Rd for exercise at a given power output. However, in their study, circulating [FFA] were lower after training, whereas in our study arterial [FFA] rose to the same extent under all exercise conditions (Fig. 2A), and we measured increased net FFA uptake in working limbs at a given relative power output. Based on extensive biochemical studies demonstrating training to increase muscle capillarity, mitochondrial, and fatty acid binding protein contents, if blood flow and arterial [FFA] are maintained to working trained muscles, increased net FFA uptake is the expected result (J. O. Holloszy, personal communication).
In conclusion, results of the present study suggest that training does
not increase reliance on IMTG stores at either the same absolute or
relative exercise intensity when subjects are tested in energy and
nitrogen balance. Even though we observed a small increase in working
limb FFA at a power output eliciting 65% of
O2 peak after
training, the balance of muscle substrate utilization was changed
little by 9 wk of endurance cycle training. CHO-derived energy sources
were the main fuel sources for working skeletal muscle during prolonged
(1-h) moderate-intensity exercise, both before and after training.
Furthermore, accepting the present data using the mass balance
technique, along with other data obtained using indirect calorimetry
and isotope tracers, we conclude that, during exercise, CHO-derived
energy sources sustain working muscle while other tissues depend on
lipid oxidation.
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ACKNOWLEDGEMENTS |
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The investigators thank the subjects for participating in this study and complying with the training program. The assistance of the nursing staff and dietitians at the General Research, Education, and Clinical Center in the Palo Alto Veterans Affairs (VA) Health Care Center is appreciated. We also thank David Guido for performing muscle biopsies and Jacinda Mawson and for blood gas analysis. We thank the student trainers who were vital in subject training and transport. We greatly appreciate the help of Barry Braun in blood sampling during the VA trials. We also thank Champion Nutrition for the generous gift of Cytomax consumed by subjects after training.
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FOOTNOTES |
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This work was supported by National Institute of Health Grants AR-42906 and DK-19577.
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. §1734 solely to indicate this fact.
Address for reprint requests: G. A. Brooks, Exercise Physiology Laboratory, Dept. of Integrative Biology, 5101 Valley Life Sciences Bldg., Univ. of California, Berkeley, CA 94720-3140.
Received 23 February 1998; accepted in final form 28 September 1998.
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