Department of Internal Medicine and Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Endurance training increases fatty acid oxidation (FAO) and skeletal
muscle oxidative capacity. However, the source of the additional fat
and the mechanisms for increasing FAO capacity in muscle are not clear.
We measured whole body and regional lipolytic activity and whole body
and plasma FAO in six lean women during 90 min of bicycling exercise
(50% pretraining peak O2 consumption) before and after 12 wk of endurance training. We also assessed skeletal muscle content of
peroxisome proliferator-activated receptor- (PPAR
) and its target
proteins that regulate FAO [medium-chain and very long chain acyl-CoA
dehydrogenase (MCAD and VLCAD)]. Despite a 25% increase in whole body
FAO during exercise after training (P < 0.05), training did
not alter regional adipose tissue lipolysis (abdominal: 0.56 ± 0.26 and 0.57 ± 0.10 µmol · 100 g
1
· min
1; femoral: 0.13 ± 0.07 and 0.09 ± 0.02 µmol · 100 g
1 · min
1),
whole body palmitate rate of appearance in plasma (168 ± 18 and
150 ± 25 µmol/min), and plasma FAO (554 ± 61 and 601 ± 45 µmol/min). However, training doubled the levels of muscle
PPAR
, MCAD, and VLCAD. We conclude that training increases the use
of nonplasma fatty acids and may enhance skeletal muscle oxidative capacity by PPAR
regulation of gene expression.
exercise; lipolysis; fatty acid; intramuscular triglyceride; stable isotopes
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INTRODUCTION |
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ENDURANCE TRAINING increases the use of endogenous fat as a fuel during exercise (13). The source of the additional triglyceride oxidized after training is controversial and may be affected by gender. Results from a recent study performed in women suggest that training increases the availability and oxidation of plasma fatty acids derived from adipose tissue triglycerides (8). In contrast, data from a study performed mostly in men (24) found that training did not increase the oxidation of plasma fatty acids, suggesting that the increase in fat oxidation after training is derived from intramuscular triglycerides. However, differences in preexercise feeding protocols between studies may have influenced the metabolic response to exercise and confounded potential gender comparisons. In the study involving men (24), exercise was performed in the postabsorptive state (after subjects fasted overnight), whereas in the study performed in women (8), exercise was performed 4-6 h after a meal, which can have a strong influence on substrate metabolism (27). The effects of training on whole body and regional lipolytic activity and fat oxidation during exercise in women during postabsorptive conditions (12 h fast) have not been studied.
The increase in fat oxidation during exercise induced by training is
associated with an increase in skeletal muscle fatty acid oxidative
capacity. Endurance training increases muscle levels of the
mitochondrial enzymes involved in -oxidation (26),
tricarboxylic acid cycle (TCA) activity (14), and
oxidative phosphorylation (12). However, little is known
about the mechanisms responsible for the increase in mitochondrial
proteins induced by training. Most mitochondrial proteins are encoded
by nuclear genes, and their expression is regulated by nuclear
transcription factors. The results of recent studies indicate that
peroxisome proliferator-activated receptor-
(PPAR
), a member of
the nuclear receptor transcription factor superfamily, plays a critical
role in the expression of genes involved in mitochondrial fatty acid
oxidation (1, 10). However, the effect of
endurance training on skeletal muscle PPAR
and its target fatty acid
oxidative enzymes has not been evaluated in either laboratory animals
or humans.
The purpose of this study was to determine the effect of endurance
exercise training on whole body regional adipose tissue and skeletal
muscle fatty acid metabolism during exercise in women after an
overnight fast. Specifically, we evaluated 1) whole body plasma fatty acid availability and uptake, 2) the relative
contribution of plasma and nonplasma fatty acids to total energy
production, and 3) regional (abdominal and femoral
subcutaneous adipose tissue) lipolytic activity during exercise
performed at the same absolute intensity before and after 12-14 wk
of endurance training. In addition, we assessed the effect of training
on skeletal muscle levels of mitochondrial enzymes involved in fatty
acid oxidation [medium-chain and very long-chain acyl-CoA
dehydrogenase (MCAD and VLCAD, respectively)] and the TCA cycle
[citrate synthase (CS)]. Levels of PPAR were also characterized in
skeletal muscle before and after training to determine whether this
regulatory factor might be involved in the known increase in
mitochondrial oxidative capacity during exercise training.
Our results indicate that the increase in whole body fat oxidation
after training is accompanied by an induction in levels of PPAR and
its target proteins, but not by an increase in adipose tissue lipolysis
or plasma fatty acid availability, uptake, and oxidation. These
findings suggest that alterations in lipid metabolism that occur with
endurance training are localized to skeletal muscle and may involve the
nuclear receptor PPAR
in these metabolic adaptations to training.
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METHODS |
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Subjects.
Six lean, premenopausal women (28 ± 3 yr of age) participated in
this study (Table 1). No subjects were
taking any medications or smoked tobacco, and none had evidence of
medical illness after a comprehensive examination, which included a
history and physical examination, routine blood tests, and an
electrocardiogram. All subjects were weight stable for 2 mo before
the study. No subjects were involved in any regular exercise program
for
6 mo before the study, and none had ever been endurance-trained
athletes. All experimental protocols were performed within the first 2 wk of the follicular phase of their menstrual cycle. Written informed consent was obtained before participation in the study, which was
approved by the Human Studies Committee and the General Clinical Research Center (GCRC) Scientific Advisory Committee of Washington University School of Medicine.
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Preliminary testing.
Peak aerobic capacity was measured before and after 12-14 wk of
endurance training. Peak oxygen consumption
(O2 peak) was measured with a Vmax 29 metabolic cart (SensorMedics, Yorba Linda, CA) during upright cycle
ergometer exercise. The protocol consisted of a 4-min warm-up, after
which the work rate was progressively increased every minute until at
least two of the following three criteria were met: 1) a
leveling off of the rate of oxygen consumption (
O2) despite increases in workload,
2) respiratory exchange ratio
1.15, and 3)
attainment of age-predicted maximal heart rate.
Experimental protocol. Subjects were admitted to the GCRC at Washington University School of Medicine on four occasions: twice before and twice after 12-14 wk of endurance training. At 1900 on each day of admission, subjects ingested a standard meal (60% carbohydrate, 25% fat, and 15% protein) containing 12 kcal/kg body weight. Subjects were randomized to perform an exercise study with isotope tracer infusion (i.e., exercise-infusion trial) or an exercise study without tracer infusion (i.e., background trial) the following morning. One week later, subjects were readmitted to the GCRC to perform the other study (exercise-infusion or background trial).
The exercise-infusion trials began at ~0700, after subjects had fasted overnight (12 h). Catheters were placed into a forearm vein for isotope infusion and into a radial artery for blood sampling while subjects were lying in bed. Blood and breath samples were taken to determine background substrate isotopic enrichments. Subjects then moved from bed to the chair of a cycle ergometer (Ergometrics 800, Ergoline, Germany) that was modified for recumbent cycling. At ~0815 (75 min before exercise), a priming dose (1.05 µmol/kg) of [1-13C]bicarbonate was given, and a constant infusion (0.035 µmol · kgMuscle biopsies.
Muscle tissue samples were obtained before and after training ~1 wk
after the exercise studies. After subjects fasted overnight, ~100 mg
of tissue were obtained from the vastus lateralis muscle by needle
biopsy (3). Samples were immediately frozen in liquid nitrogen and stored at 70°C. The pretraining sample was obtained
7 days after the pretraining exercise study but before the start of
exercise training. The posttraining sample was obtained exactly 3 days
after the last exercise training bout.
Exercise training. After the pretraining studies were completed, all subjects participated in an endurance training program, which consisted of cycling on an ergometer for 35-45 min, 4 days/wk, for 12-14 wk under direct supervision. Exercise intensity was based on the percentage of each subject's maximal heart rate (HRmax), determined during the initial aerobic capacity test. During the first 6 wk of the training period, exercise intensity was increased progressively from 70 to 85% HRmax, and exercise duration was increased from 35 to 45 min. To prevent weight loss during the 12- to 14-wk training period, energy balance was maintained by feeding the subjects a defined liquid formula supplement (Ensure, Ross Laboratories, Columbus, OH) at the end of each training session to replenish the calories expended during exercise. We have previously shown that this refeeding regimen prevented changes in body weight and body composition in lean men who completed 16 wk of endurance training (15).
Posttraining studies. All studies performed before training were repeated after 12-14 wk of exercise training. During the exercise studies, each subject exercised at the same absolute intensity before and after training. This allowed us to compare the absolute rates of substrate oxidation before and after training, as well as the relative contribution of plasma fatty acids, nonplasma fatty acids, and carbohydrate to total energy expenditure. The posttraining exercise studies and muscle biopsy were performed exactly 3 days after the end of exercise training. To ensure a 3-day rest interval before each study, subjects resumed training for several days after each procedure until all trials were completed. Therefore, the total duration of training ranged from 12 to 14 wk.
Analytical procedures. Plasma insulin concentration was measured by radioimmunoassay (antibody raised to porcine insulin; coefficient of variation = 11.8; Linco Research, St. Louis, MO). Plasma catecholamine concentrations were determined by a radioenzymatic method (34). Plasma fatty acid concentrations were quantified by gas chromatography by adding heptadecanoic acid to plasma as an internal standard (39). Microdialysis glycerol concentration was measured using a CMA 600 Microdialysis Analyzer (CMA Microdialysis).
The tracer-to-tracee ratio (TTR) for plasma palmitate was determined by gas chromatography-mass spectrometry (GC-MS) with an MSD 5971 system (Hewlett-Packard, Palo Alto, CA) with capillary column (30). Acetone was used to precipitate plasma proteins, and hexane was used to extract plasma lipids. Free fatty acids were converted to their methyl esters with iodomethane and isolated by using solid phase extraction cartridges. Samples were dried in a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY), reconstituted in heptane, and transferred to auto sampler vials for GC-MS analysis. Ions at mass-to-charge ratios 270.2 and 271.2, produced by electron impact ionization, were selectively monitored. The ratio of 13CO2 to 12CO2 in expired breath was determined by isotope ratio mass spectrometry (IRMS; Sira II, dual-inlet triple collector, VG Fisons, Cheshire, UK) as described previously (35). Briefly, CO2 was isolated from breath by passing the sample through a series of traps to remove water vapor, nitrogen, and oxygen. The purified sample was then ionized by electron bombardment and repelled past a series of focusing lenses toward the detector. The ratio of masses 45 and 44 were determined, representing 13CO2 and 12CO2, respectively. Skeletal muscle protein content of PPARCalculations. Steady-state substrate concentrations and TTRs were achieved during basal conditions, so basal palmitate rate of appearance (Ra) in plasma and disappearance from plasma (Rd) were calculated using Steele's equation for steady-state conditions (36). During exercise, however, the non-steady-state equation of Steele (36) was used to calculate palmitate Ra and Rd. The effective volume of distribution for palmitate was estimated to be 50 ml/kg body weight.
The oxidation of plasma fatty acids was calculated as (35)
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Statistical analysis.
A two-way ANOVA (training × time) with Tukey's post hoc analysis
was used to test significance of differences for palmitate Ra, palmitate Rd, and the rates of abdominal
and femoral subcutaneous adipose tissue glycerol release. A two-way
ANOVA (anatomical site × time) with Tukey's post hoc analysis
was used to assess the significance of differences in glycerol release
between abdominal and femoral regions. Values for pre- and posttraining
regional glycerol kinetics were combined, because there was no effect
of endurance training on regional lipolysis. Student's
t-test for independent samples was used to test the
significance of differences for all of the other parameters. A value of
P 0.05 was considered to be statistically significant.
All data are expressed as means ± SE.
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RESULTS |
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Twelve weeks of endurance exercise training increased aerobic
capacity by 15% (P < 0.05) but did not alter body
weight (Table 1). Subjects cycled at the same absolute workload before
and after training (51 ± 4 W), which elicited similar rates of
O2 (Table 1). However, mean HR response
during exercise was >10% lower after training (Table 1)
(P < 0.05).
Plasma hormone and substrate concentrations.
Exercise increased plasma epinephrine and norepinephrine concentrations
and reduced plasma insulin concentration compared with resting values
(all P < 0.05) (Table
2). Endurance training reduced the plasma
epinephrine response to exercise (P < 0.05) but did
not significantly alter plasma norepinephrine or insulin concentrations (Table 2). Basal plasma fatty acid concentration was almost identical before and after training (0.45 ± 0.02 and 0.44 ± 0.01 µmol/ml, respectively). Although exercise
increased (P < 0.05) plasma fatty acid concentration
above basal values, endurance training did not affect fatty acid
concentration during exercise (0.72 ± 0.04 and 0.67 ± 0.06 µmol/ml, mean concentration during the 60- to 90-min period of
exercise before and after training, respectively).
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Plasma lipid kinetics.
Plasma palmitate Ra and Rd increased more than
twofold during exercise (P < 0.05) (Fig.
1). Endurance training did not alter palmitate or total fatty acid kinetics during the last 30 min of
exercise (fatty acid Ra: 589 ± 65 and 545 ± 107 µmol/min; fatty acid Rd: 576 ± 62 and 537 ± 104 µmol/min, before and after training, respectively). The
contribution of palmitate to total plasma fatty acids during exercise
also remained the same before and after training (26 ± 1 and
25 ± 1%, respectively).
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Regional lipolysis.
Glycerol release from abdominal subcutaneous adipose tissue increased
progressively during exercise, whereas glycerol release from femoral
subcutaneous adipose tissue did not change (Fig. 2). Training did not alter the rate of
glycerol release from either abdominal or femoral adipose tissue sites.
During the last 25 min of exercise (65-90 min), the rate of
glycerol release was greater in the abdominal than in the femoral
region (P < 0.05).
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Substrate oxidation.
The mean rate of whole body fatty acid oxidation during the final 30 min of exercise was 25% greater after than before training (1,024 ± 114 and 789 ± 62 µmol/min, respectively; P < 0.05). However, endurance training did not affect the rate of plasma
fatty acid oxidation (554 ± 61 and 601 ± 45 µmol/min,
before and after training, respectively; Fig.
3) or the mean calculated acetate
correction factor (0.80 ± 0.01 and 0.80 ± 0.01). Therefore,
the increase in total fatty acid oxidation after training was due to an
increase in the oxidation of nonplasma fatty acids. Training also
caused a decrease in the relative contribution of carbohydrate to total energy production from 58 ± 2 to 47 ± 4% during the last
30 min of exercise (P < 0.05; Fig. 3).
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PPAR and fatty acid oxidative enzymes in skeletal muscle.
As shown in Fig. 4, endurance exercise
training resulted in a twofold increase in the mean immunodetectable
levels of MCAD, VLCAD, and CS in skeletal muscle (all P < 0.05). Mean skeletal muscle PPAR
protein content was also twofold
higher (P < 0.05) in trained compared with untrained
skeletal muscle (Fig. 5). Therefore, the
increase in enzymes involved in fatty acid oxidation was associated with an increase in PPAR
.
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DISCUSSION |
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An important adaptation to endurance training is a shift in
skeletal muscle energy metabolism from carbohydrate to fat and increased skeletal muscle mitochondrial capacity to oxidize fatty acids
during exercise. However, the source of the increased fat used during
exercise and the mechanism for mitochondrial fatty acid oxidative
enzyme induction remain unclear. In the present study, we found that
12-14 wk of endurance exercise training in lean women caused a
25% increase in total fat oxidation during exercise. The additional
fat oxidized during exercise was derived from nonplasma fatty acids
because plasma fatty acid availability, uptake, and oxidation did not
change. It is likely that the majority of these nonplasma fatty acids
originated from intramuscular triglycerides (IMTG), because plasma
triglycerides are probably not an important fuel during exercise
(28). In addition, we found that endurance training
increased skeletal muscle protein content of PPAR and its target
mitochondrial fatty acid oxidative enzymes. These data suggest that the
adaptations in lipid metabolism that occur with endurance exercise
training are intrinsic to skeletal muscle.
The effect of training on fatty acid kinetics in our female subjects is consistent with results reported previously in men (24, 31) but not in women (8). Data reported by Friedlander et al. (8) suggest that endurance training increases plasma fatty acid availability and oxidation during exercise in women. In contrast, we found that training increased the oxidation of nonplasma fatty acids during exercise. The reason(s) for the discrepancy between our findings and those reported by Friedlander et al. are unclear, but they may be related to differences in the contribution of palmitate to total plasma fatty acids rather than differences in palmitate kinetics itself. In fact, palmitate Ra (i.e., availability) during exercise was the same before and after training in both studies. However, Friedlander et al. found that the percentage of total plasma fatty acids derived from palmitate decreased after training, which caused an increase in the calculated rate of plasma fatty acid availability and oxidation. In contrast, the percentage of total plasma fatty acids derived from palmitate remained the same in our subjects. Another possible factor that may have caused differences between studies is the feeding protocol used before the exercise infusion protocol. The interval between feeding and an exercise bout can have a considerable effect on substrate metabolism during exercise (27). The subjects in our study performed the exercise-infusion protocol after they fasted overnight (12 h), whereas the subjects studied by Friedlander et al. ingested a meal 4-6 h before their exercise study.
It is possible that our calculated values for the rate of plasma fatty
acid oxidation during the last 30 min (60-90 min) of exercise
slightly overestimated the true values. In calculating plasma fatty
acid oxidation rates, we used an acetate correction factor established
during 50-60 min of exercise to estimate the amount of palmitate
tracer lost to label fixation (35). However, acetate
recovery increases with time (25), so it is possible that
we underestimated the acetate correction factor in our subjects during
the 60- to 90-min period of exercise, which would cause an artifactual
increase in plasma fatty acid oxidation rates. Indeed, our observation
that nearly all fatty acids cleared from plasma during exercise were
oxidized supports this possibility. An overestimation of plasma fatty
acid oxidation suggests that the rate of nonplasma fatty acid oxidation
may have been greater than we reported. However, this potential error
does not affect our conclusion that endurance training does not alter
plasma fatty acid oxidation rates during exercise. Acetate
recovery depends on the rate of O2
(35), and our subjects were exercising at the same
absolute intensity (i.e., same rate of
O2) before and after training.
We assumed that IMTG, rather than plasma triglycerides, provided the additional source of nonplasma fatty acids oxidized during exercise after training. Although our tracer methods cannot distinguish between triglyceride sources, it is unlikely that fatty acids derived from circulating triglycerides were responsible for the increase in fat oxidation, because plasma triglycerides are normally not an important source of energy production during exercise (28). Human skeletal muscle contains ~300 mmol of triglyceride (6), representing nearly 2,500 kcal of potential energy, which can serve as a considerable source of fuel during exercise. Moreover, the use of IMTG during exercise is efficient, because fatty acids released during lipolysis of IMTG are in close proximity to their site of oxidation (muscle mitochondria) and do not require transport from an extramuscular depot. By measuring IMTG content in muscle samples taken before and after exercise, Hurley et al. (16) found that 12 wk of endurance training doubled the use of IMTG during exercise. In our study, we also found that 12-14 wk of endurance training nearly doubled nonplasma fatty acid oxidation during exercise, presumably from IMTG. Nonetheless, an increased use of IMTG after endurance training is controversial because of conflicting results from some studies (8, 19), which may be related to differences in exercise protocols between studies, variability in IMTG concentration determined by the muscle biopsy technique, and differences in the interval between the last exercise bout and the experimental trial.
The increase in lipolysis during endurance exercise is largely due to
catecholamine-mediated stimulation of adipocyte -adrenergic receptors (2). In the present study, we found that the
mobilization of adipose tissue triglycerides during exercise was
heterogeneous (2, 11, 38).
Lipolytic rate increased progressively in abdominal, but not femoral,
adipose tissue during exercise in our subjects. Therefore, it is likely
that most of the fatty acids released into the circulation during
exercise were derived from upper-body rather than lower-body
subcutaneous fat. This observation is consistent with and extends the
data reported by Arner et al. (2), who found that adipose
tissue interstitial glycerol concentration increased more in abdominal
than in femoral sites during cycle ergometer exercise. Our results are
also consistent with studies demonstrating regional differences in
lipolytic sensitivity to catecholamines in vivo (11) and
in vitro (38). It is likely that regional heterogeneity in
catecholamine-mediated lipolysis of adipose tissue triglycerides is
determined by regional variations in
2- and
-adrenergic receptor affinity and density (38).
The results of the present study demonstrate that endurance training does not affect the lipolytic response to moderate-intensity exercise in either upper (abdominal) or lower (femoral) subcutaneous adipose tissue in lean women. Therefore, the similar values we observed in whole body fatty acid kinetics during exercise before and after training were caused by similar upper- and lower-body regional lipolytic rates rather than a combination of increased and decreased rates of lipolysis within different adipose tissue sites. Measurement of regional lipolytic activity by microdialysis has limitations, because it requires accurate measurement of ATBF and appropriate assessment of glycerol recovery. Although we measured blood flow in the identical contralateral adipose tissue region of each microdialysis probe by use of 133Xe clearance, it is possible that this may not accurately reflect nutritive blood flow at the exact site of each probe. We also assumed that 100% of interstitial glycerol was recovered by each probe, because others have demonstrated near 100% recovery when using the same probes and same perfusion rate (32). Therefore, incomplete glycerol recovery would affect our assessment of interstitial glycerol concentration. Although these limitations of the microdialysis method can be difficult to correct, they do not affect our qualitative conclusions regarding regional glycerol release, because the potential errors should be similar before and after training.
This is the first study to demonstrate that endurance exercise training
increases PPAR content in skeletal muscle. PPAR
regulates the
expression of genes encoding several key muscle enzymes involved in
fatty acid oxidation, including MCAD (10) and VLCAD
(1), which also increased in our subjects after training. Our data are consistent with the results obtained in a dog model, which
found that chronic electrical stimulation of latissimus dorsi muscle
increased muscle PPAR
content and MCAD gene expression (5). Although measurement of cellular protein content and
gene expression does not necessarily reflect enzyme activity, these observations suggest that PPAR
may be an important component of the
adaptive response to endurance training by transducing physiological
signals related to exercise training to the expression of nuclear genes
encoding in skeletal muscle mitochondrial fatty acid oxidation enzymes.
In fact, PPAR
could increase the production of mitochondrial fatty
acid oxidative enzymes without changing cellular PPAR
content.
Unsaturated long-chain fatty acids, which are released from adipose
tissue during exercise, serve as ligands for PPAR
and stimulate
PPAR
-activated gene transcription (20). Therefore,
increase in muscle PPAR
content and the intermittent increase in
fatty acid delivery to muscle during exercise training may both be
important factors in enhancing muscle fatty acid oxidative capacity.
Studies performed in PPAR
-deficient mice found that PPAR
is also
critical for the adaptation to other physiological conditions that
increase the demand for long-chain fatty acid oxidation, such as
fasting (18, 23) and high-fat feeding
(18).
The mechanism responsible for the training-induced increase in PPAR
in skeletal muscle is not known. One possibility is that fatty acids
regulate their own oxidation by serving as a PPAR
ligand and by
stimulating PPAR
expression directly. Indeed, fatty acids have been
shown to increase hepatic PPAR mRNA expression (37). In
addition, glucocorticoids, which are released during prolonged
exercise, are a potent stimulator of PPAR
mRNA induction in cultured
hepatocytes (22). Moreover, the effects of elevated fatty
acid concentration in combination with the synthetic glucocorticoid dexamethasone on PPAR expression are synergistic (37).
Therefore, regular exposure to elevated plasma glucocorticoids, such as
cortisol, together with a high rate of fatty acid flux during endurance training, may increase PPAR
mRNA expression and content in skeletal muscle.
In summary, we found that 12-14 wk of endurance exercise training
in lean women increased skeletal muscle oxidative capacity and the
oxidation of fatty acids during exercise. The increase in skeletal
muscle PPAR content and its target fatty acid oxidative enzymes
after training suggests a candidate gene regulatory pathway that is
activated by training. The additional fatty acids oxidized during
exercise after training were derived from nonplasma sources, possibly
from IMTG stores. Therefore, the adaptations in fat metabolism that
occur in response to endurance training are localized to skeletal
muscle: endurance training may increase the mobilization of endogenous
triglyceride present within working muscles and increase mitochondrial
oxidative enzyme content to enhance skeletal muscle fatty acid
oxidative capacity.
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ACKNOWLEDGEMENTS |
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We thank Renata Braudy and the nursing staff of the General Clinical Research Center for help in performing the experimental protocols, Dr. Guohong Zhao for technical assistance, and the study subjects for participating in this study.
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FOOTNOTES |
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This study was supported by the National Institutes of Health Grants DK-37948, DK-09749-01A1, DK-45416, RR-00036 (General Clinical Research Center), RR-00954 (Mass Spectrometry Resource), AG-00078 (Institutional National Research Service Award), AG-13629 (Claude Pepper Older American Independence Center), and DK-56341 (Clinical Nutrition Research Unit).
Address for reprint requests and other correspondence: S. Klein, Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8127, St. Louis, MO 63110-1093.
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.
Received 29 November 1999; accepted in final form 21 March 2000.
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