1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and 3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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The increase in skeletal
muscle pyruvate dehydrogenase kinase (PDK) activity was measured in
skeletal muscle of six healthy males after a eucaloric
high-fat/low-carbohydrate (HF/LC; 5% carbohydrate, 73% fat, and 22%
protein of total energy intake) diet compared with a standardized
prediet (50% carbohdyrate, 30% fat, and 21% protein). Biopsies were
obtained from the vastus lateralis muscle after 3 days on the prediet
(day 0) and after 1, 2, and 3 days of the HF/LC diet. Intact
mitchondria were extracted from fresh muscle and analyzed for PDK
activity and Western blotting of PDK2 and PDK4 protein. A second biopsy
was taken at each time point and frozen for Northern blot analysis of
PDK2 and PDK4 mRNAs. PDK activity increased in a linear fashion over
the 3-day HF/LC diet and was significantly higher than control by 1 day. PDK activity was 0.09 ± 0.03, 0.18 ± 0.05, 0.30 ± 0.07, and 0.37 ± 0.09 min1 at 0, 1, 2, and 3 days, respectively. PDK4 protein and mRNA increased maximally by
day 1, and PDK2 protein and mRNA were unaffected by the
HF/LC diet. Resting respiratory exchange ratios decreased after 1 day
of the HF/LC diet (from 0.79 ± 0.02 to 0.72 ± 0.02) and
remained depressed throughout the 3-day dietary intervention (0.68 ± 0.01). The immediate shift to fat utilization was accompanied by
increased blood glycerol,
-hydroxybutyrate, and plasma free fatty
acid concentrations. These results suggest that the continuing increase
in PDK activity over the 3-day HF/LC diet is not due to increasing PDK
protein beyond 1 day. This could be due to the contribution of another
isoform to the total PDK activity or to a continual increase in PDK4 or
PDK2 specific activity.
pyruvate dehydrogenase kinase 2; pyruvate dehydrogenase kinase 4; respiratory exchange ratio; pyruvate dehydrogenase activity; mitochondria
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INTRODUCTION |
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PYRUVATE DEHYDROGENASE (PDH) is a multienzyme complex that catalyzes the conversion of pyruvate to acetyl-coenzyme A. The reaction is the first irreversible step in the oxidation of carbohydrate-derived carbon and regulates the entry of carbohydrate into the tricarboxylic acid cycle. PDH activity is tightly regulated by reversible phosphorylation and dephosphorylation reactions catalyzed by an intrinsic kinase and phosphatase (22, 23, 35, 40). Phosphorylation of E1 catalytic subunits of PDH by PDH kinase (PDK) causes inactivation of the enzyme, whereas PDH phosphatase removes phosphate and returns the enzyme to its active form (PDHa) (22, 23). The relative activities of the phosphatase and PDK determine the proportion of the complex in the active form.
During rest, PDH is acutely regulated by the allosteric regulation of the kinase and phosphatase by intramitochondrial effectors. High ATP-to-ADP, NADH-to-NAD+, and acetyl-CoA-to-CoASH ratios and low pyruvate concentrations favor PDK activation and decreased PDH activity (9, 12, 35, 40). During exercise, PDH phosphatase is accelerated by increasing Ca2+ concentrations, allowing for activation of the PDH complex (32).
In addition to this acute regulation, rodent studies have demonstrated an adaptive increase in PDK activity after prolonged periods of carbohydrate restriction (e.g., starvation, diabetes) (16, 19, 34) or increased dietary fat (27). The increase in PDK persists even after a rigorous mitochondrial preparation, indicating that enhanced activity was not simply the result of effector accumulation (the result of increased reliance on fat fuel) but reflected a stable adaptation or covalent modification.
By virtue of its size, skeletal muscle is the major site of whole body glucose disposal. Overall regulation of carbohydrate disposal by skeletal muscle is regulated by a combination of glucose uptake and phosphorylation, storage as glycogen, and oxidation through PDH. With starvation and a prolonged high-fat diet, decreased flux through PDH serves a protective role, helping to preserve scant carbohydrate stores. However, in diabetes, the decrease in carbohydrate oxidation due to increased PDK activity can exacerbate the clinical dilemma of decreased skeletal muscle glucose disposal.
A recent study from our laboratory (29) demonstrated that human skeletal muscle PDK activity increased three- to fivefold when subjects consumed a high fat/low carbohydrate (HF/LC) diet. Increased activity was observed as early as 3 days on the diet, and the effect persisted until 6 days. That study did not investigate whether the increase in PDK activity occurred before 3 days or whether it was the result of increased protein synthesis or a stable increase in the specific activity.
There are currently four known isoforms of PDK (13, 31), which differ significantly in their tissue distribution (6, 13) as well as in their specific activity and sensitivity to effectors (6). Therefore, the relative concentrations of the various isoforms at any given time will affect the way in which the kinase responds to acute changes in intramitochondrial concentrations of effectors and determine the proportion of the complex that is active. The tissue distribution of the four isoforms is also different between rats and humans, with all four isoforms present in human skeletal muscle but only PDK1, PDK2, and PDK4 detected in rat skeletal muscle (6, 13). Early studies in rodent models indicated that PDK4 was the only isoform contributing to increased activity in heart (42) and skeletal muscle (41) with diabetes and starvation. However, more recently, an increase in PDK2 was observed in mixed fast-twitch muscle after a prolonged, 48-h fast and a 28-day HF diet (14, 36). Only one study has examined PDK isoform expression in human skeletal muscle in response to increased reliance on fat fuel, associated with insulin resistance in the Pima Indians of Arizona (1, 20). Those subjects had higher levels of both PDK2 and PDK4 mRNA in skeletal muscle of insulin-resistant individuals compared with their nondiabetic counterparts (25). However, no studies to date have investigated the early response to an increased reliance on fat fuel through dietary manipulation, and currently there are no human studies that link PDK activity to isoform protein and mRNA.
The initial purpose of this study was to examine the time course of the increase in PDK activity during the first 3 days of a HF/LC diet. We hypothesized that there would be a gradual increase in PDK activity over the 3 days, significantly different by 1 or 2 days. The second purpose was to characterize the changes in PDK protein and mRNA of the most abundant isoforms in human skeletal muscle (PDK2 and PDK4). Our goal was to determine whether the increase in PDK activity over 3 days on a HF/LC diet was the result of an increase in abundance of one or both of the PDK isoforms. We hypothesized that PDK4 expression would increase in concert with the increases in PDK activity and that PDK2 expression would be unaltered during the short-term dietary intervention.
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METHODS |
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Subjects.
Six male university students volunteered for this study. They were in
good health with a mean age of 22.0 ± 0.4 yr (range 19-25
yr), weight of 82.5 ± 6.6 kg (range 70-105 kg), and height of 187 ± 5 cm (range 173-201 cm). Three of the subjects were
very active (aerobic activity >8 h/wk), two were moderately active (aerobic activity ~4 h/wk), and one was less active (~1 h/wk), as
determined by activity questionnaires. The mean relative maximal oxygen
uptake (O2 max) was 51 ± 4 ml · kg
1 · min
1 (range
32-57.8
ml · kg
1 · min
1). Three
additional subjects volunteered for a less invasive study of the
changes in the respiratory exchange ratio (RER). Similar to the other
subjects, all were recreationally active and participated in some form
of aerobic activity
3 times/wk. These three subjects were 22 yr of
age, with an average weight of 72.3 kg (68-80 kg) and height of
180 cm (174-187 cm). Subjects were informed of the risks of the
procedures before giving informed consent, and the study was approved
by the ethics committees of the University of Guelph and McMaster University.
Study design.
Subjects provided a 3-day diet record of their regular (REG) diet. An
individualized eucaloric prediet [PRE; only minor changes were made to
achieve 50% carbohydrate (CHO), 30% fat, and 20% protein] and a
HF/LC diet (5% CHO, 73% fat, and 22% protein) were designed and
prescribed for each subject (Fig. 1). The
eucaloric HF/LC diet was designed without including fish or flaxseed
products, which would be high in n-3 fatty acids, because these have
been shown to influence PDK gene expression in animal tissue
(11). The dietary regimens were outlined in detail for the
subjects, and they were given instructions concerning the importance of consuming the diets as prescribed and recording everything they ate or
drank. Food was purchased and packaged for each subject. Before the
actual study, the subjects reported to the lab to determine O2 max. The subjects then reported to
the laboratory four times. They began the PRE diet 3 days before
reporting to the laboratory for the first time (day 0). In
every case, the subjects arrived in the laboratory 1-1.5 h after a
breakfast that adhered to the diet they were consuming the night before
(PRE or HF/LC diet). On day 0, two biopsies were taken from
the vastus lateralis muscle as previously described (3).
They immediately began the HF/LC diet. Subjects continued with their
normal daily activities but refrained from formal exercise sessions
during the diet intervention. They returned to the laboratory on
days 1, 2, and 3 of the HF/LC diet for two
biopsies on each day.
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Processing of muscle samples. Intact mitochondria were extracted from fresh muscle (~75-80 mg) for the analysis of PDK activity and for Western blotting for the PDK isoforms. A second biopsy (~50-100 mg) was frozen in liquid nitrogen for Northern blotting for the mRNA of the PDK isoforms.
Mitochondrial extraction. Intact mitochondria were extracted by differential centrifugation as previously described (17, 26, 29). Briefly, minced muscle was homogenized using a glass-on-glass Potter homogenizer in 20 volumes of a buffer containing (in mM) 100 KCl, 40 Tris · HCl, 10 Tris base, 5 magnesium sulfate, 1 EDTA, and 1 ATP (pH 7.5). The supernatant was retained after centrifugation at 700 g for 10 min, and a crude mitochondrial pellet was extracted with centrifugation at 14,000 g (10 min). The pellet was washed, resuspended, and pelleted twice (7,000 g, 10 min) in 10 volumes of (in mM) 100 KCl, 40 Tris · HCl, 10 Tris base, 1 magnesium sulfate, 0.1 EDTA, and 0.25 ATP (pH 7.5). The first wash buffer included 1% (wt/vol) bovine serum albumin, and the second was protein free. The final mitochondrial pellet was resuspended in a volume corresponding to 1 µl/mg fresh muscle extracted. The final buffer contained (in mM) 220 sucrose, 70 mannitol, 10 Tris · HCl, and 1 EDTA (pH 7.4). All procedures were carried out at 0-4°C. Unless specifically stated, all chemicals were obtained from Sigma Chemical (St. Louis, MO).
Incubation of mitochondria for PDK activity. The final mitochondrial suspension (50 µl) was diluted with 250 µl of buffer containing 10 µM carbonyl cyanide m-chlorophenylhydrazone, 20 mM Tris · HCl, 120 mM KCl, 2 mM EGTA, and 5 mM potassium (pH 7.4) and incubated for 20 min at 30°C, driving ATP concentration to zero and causing complete conversion of PDH to the active form as previously described (10). Mitochondria were pelleted at 7,000 g for 10 min and stored in liquid nitrogen for later analysis of PDK.
PDK activity.
PDK activity was determined as previously described (29).
Briefly, the mitochondrial pellet was resuspended in ~300 µl of a
buffer containing 30 mM KH2PO4, 5 mM EGTA, 5 mM
dithiothreitol, 25 µg/ml oligomycin B, 1.0 mM tosyl-lysyl
chloromethyl ketone, 0.1% (wt/vol) Triton X-100, and 1%
(wt/vol) bovine serum albumin (pH 7.0), and freeze thawed twice to
ensure that all mitochondria were broken. The suspension was warmed to
30°C, and two aliquots of the suspension were diluted 1:1 in a buffer
containing (in mM) 200 sucrose, 50 potassium chloride, 5 magnesium
chloride, 5 EGTA, 50 Tris · HCl, 50 sodium fluoride, and 5 dichloroacetate and 0.1% (wt/vol) Triton X-100 (pH 7.8) for later
analysis of PDH activity. This point represents "zero-time" or
"total PDH." Magnesium ATP was added to the remaining suspension to
bring the concentration to 0.3 mM, and timed samples were taken every
20-30 s for 3-5 min (depending on PDK activity) as previously
described (10, 29, 38). For our method, however, the
samples were diluted 1:1 in the previously described sodium
fluoride-dichloroacetate buffer to lock the PDHa activity through
inhibition of the phosphatase and kinase, respectively. The samples
were stored on ice for analysis of PDHa activity by radioisotopic
measurement as described previously (8, 30). PDK activity
is reported as the apparent first-order rate constant of the
inactivation of PDH (min1), or the slope of ln
{%[PDHa activity (with ATP addition)]/total PDH (without ATP
addition)} vs. time (10, 38). There was no appreciable
loss of activity in the absence of ATP over the 3- to 5-min experiment.
Calculation of recovery and quality of mitochondrial preparation. Citrate synthase (CS) activities on the total muscle homogenate (CShomog) and mitochondrial suspensions were measured as previously described (29, 33). Briefly, a small volume of the mitochondrial suspension was diluted 20-fold with the final sucrose and mannitol buffer and divided into two fractions. Extramitochondrial CS (CSem) was measured in the intact mitochondrial preparation, and CS activity in the total suspension (CSts) was measured after the preparation was frozen and thawed twice to open mitochondria. Triton (0.1%) was included in the cuvette for measurement of CSts and CShomog.
Recovery of intact mitochondria was calculated as
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Western blotting. Mitochondria were diluted to a final protein concentration of 1 µg/µl in 50 mM Tris · HCl, pH 6.8, containing 2% (wt/vol) SDS, 0.1 M dithiothreitol, 0.1% (wt/vol) bromophenol blue, 10% (vol/vol) glycerol, 1 mM benzamidine, 0.1 mg/ml trypsin inhibitor, 1 µg/ml aprotinin, 0.1 mM tosyl-lysyl phenylmethyl ketone, 1 µM leupeptin, and 1 µM pepstatin A. Samples were solubilized by boiling for 5 min and then cooled on ice for 5 min. Standard SDS-PAGE was performed with a 4% stacking and 10% separating gel (5 µg mitochondrial protein/lane). Electroblotting and immunodetection were performed as previously described (42). Polyclonal antisera against recombinant PDK2 and PDK4 had previously been tested for cross-reactivity (42). Antibody-antigen complexes were visualized with 125I-labeled protein A (ICN Pharmaceuticals, Irvine, CA) followed by autoradiography. Relative densities were quantified using Northern Eclipse from Empix Imaging (Mississauga, ON, Canada), and results were normalized to the day 0 (or control) value for each subject.
Northern blotting. Total RNA was extracted from frozen muscle by use of a BIO101 FastRNA Kit-Green (La Jolla, CA). Total RNA (6-8 µg) was loaded onto 1% denaturing agarose gel and run with 10 mM MOPS, 4 mM sodium acetate, and 0.5 mM EDTA buffer (pH 7.0) at 75 V for 4 h. Gels were visualized under ultraviolet light, and then RNA was blotted using capillary action with a 20× standard sodium citrate solution overnight onto a Nytran supercharged membrane (Schleicher & Schuell, Keene, NH). cDNAs for PDK2 and PDK4 have been described previously (6, 42). The individual cDNAs were labeled using [32P]ATP and [32P]CTP (Amersham Pharmacia Biotech, Piscataway, NJ) and the random primed DNA labeling kit (Boehringer Mannheim, Montreal, QC, Canada). Stratagene QuickHyb solution and protocol were used for hybridization with the labeled probe, except that hybridization was performed for 2 h at 68°C (Stratagene Cloning Systems, La Jolla, CA). After blots were washed according to the method prescribed for Statagene QuickHyb, autoradiographs were developed using Kodak X-omat film (Rochester, NY). Relative densities were quantified using Northern Eclipse from Empix Imaging.
Resting RER.
The three participants underwent the diets as described for the major
study and reported to the laboratory at least 4 h postprandial on
days 0, 1, 2, and 3 of the HF/LC diet. Blood was
sampled on days 0 and 3 as previously described.
Subjects were asked to lie in a supine position in a quiet, darkened
room for 30 min. At this time, their expired gases were analyzed for
volume and oxygen and carbon dioxide concentration for 15 min (model
2900, SensorMedics, Yorba Linda, CA). These measurements were used to
calculate oxygen consumption (O2) and
carbon dioxide production (
CO2) and the average resting RER
(
CO2/
O2).
Diet analysis and statistics. REG, PRE, and HF/LC diets were analyzed using Nutripro (West Publishing, Salem, OR) and then reported as the mean analysis for 3 days for REG, PRE, and HF/LC diets.
Results were analyzed using a one-way ANOVA (time) with repeated measures. A Fisher protected least significant difference post hoc test was used to compare means. Significance was accepted at P < 0.05. ![]() |
RESULTS |
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Diet analysis. The results of the diet analysis for the REG, PRE, and HF/LC diets demonstrated that the eucaloric diets were maintained. Total energy intakes were 13,180 ± 1,151, 13,560 ± 1,053, and 13,308 ± 974 kJ for the REG, PRE, and HF/LC diets, respectively. The PRE diet adhered closely to the target values of an average North American mixed diet (49.3 ± 0.5% CHO, 29.8 ± 0.5% fat, and 20.8 ± 0.4% protein) and did not vary greatly from the REG diet. The HF/LC diet was severely restricted in CHO (4.9 ± 0.2%) and very high in fat (73.2 ± 0.9%) but had similar protein content compared with the PRE diet (22.5 ± 0.3%).
Blood measurements.
Plasma FFA and whole blood -hydroxybutyrate were significantly
elevated after 1 day on the HF/LC diet and remained elevated at
days 2 and 3 (Table
1). Blood glycerol levels were
significantly elevated on days 2 and 3 on the
HF/LC diet. Plasma insulin decreased by ~55% on days
1-3, but the high day 0 variability precluded statistical significance. Blood lactate and glucose were unchanged by
the 3-day HF/LC diet.
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Resting RER.
Resting RER decreased from 0.79 ± 0.02 on day 0 to
0.72 ± 0.02 on day 1 and remained depressed on
days 2 and 3 (0.68 ± 0.01) on the HF/LC
diet (Fig. 2).
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PDK activity and isoforms.
Mean PDK activity was elevated as early as 1 day on the HF/LC diet and
continued to increase up to 3 days in a linear fashion (Fig.
3A). The individual data (Fig.
3B) showed that PDK increased in all subjects, with a large
variability between subjects.
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DISCUSSION |
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A major finding of this study was that PDK activity increased as early as 1 day on a HF/LC diet. The increase in PDK activity was linear throughout the 3 days, gradually increasing in all subjects. The increase in PDK activity was accompanied by an increase in PDK4 protein and mRNA, but PDK2 protein and mRNA were unchanged throughout the 3 days. The increase in PDK4 protein and mRNA was maximal after 1 day. Therefore, the continuing increase in activity could be due to the contribution of another isoform or to changes in the specific activity of PDK2 or PDK4. This provides evidence of a dissociation of the protein and mRNA concentration of PDK4 from the total enzyme activity and underscores the need for measurement of PDK activity, because increased mRNA levels alone are not adequate to quantitatively predict changes in all situations. Although a few studies report changes in PDK isoform mRNA, this study and our previous study (29) are the only reported measures of human skeletal muscle PDK activity.
Fuel utilization in response to HF/LC diet. The experimental diet chosen in this study was extreme, with both a very high fat content (73 vs. 30%) and a very low CHO content (5 vs. 50%) compared with the average Western diet. The decrease in resting RER by 1 day indicated a rapid shift in fuel utilization from a mix of CHO and fat to almost complete fat oxidation. This was consistent with data from a previous study that used a similar HF/LC diet (72% of energy provided by fat) for 5 days and demonstrated a decrease in resting RER to 0.71 (18). However, the previous work did not examine the time course during the 5-day diet to determine how rapidly such a shift would occur. Our data do not preclude the possibility that the fuel switch happened even earlier, within the first 24 h.
The shift to reliance on fat metabolism was correlated with the increased plasma FFA, glycerol, andTime course of increased PDK activity and PDK isoform expression.
PDK activity increased rapidly during the HF/LC diet and was
significantly different from control by day 1. This
correlated with the change in RER and the shift to fat metabolism.
Although PDK2 protein and mRNA were unaltered throughout the diet, PDK4 protein and mRNA were also increased maximally in 1 day. However, PDK
activity continued to increase gradually in a linear fashion over the
3-day period. When the data from the present study and a previous study
(29) from this laboratory were combined, PDK activity
increased linearly throughout the first 6 days of the HF/LC diet (Fig.
7). This suggests that either another PDK
isoform was contributing to the continuous increase in PDK activity or there was an increase in PDK specific activity. The binding of the
kinase to the PDH complex has been shown to stimulate activity; therefore, one possibility is that the specific activity of newly synthesized PDK4 (or loosely associated PDK2) could be gradually enhanced as it is assembled and bound into the complex (24, 43). The concept of gradual incorporation of PDK is supported by
a recent study on PDK activity in mitochondria from 5-day septic rats
(39). That work examined the increase in PDK activity in both the complex-bound fraction and the loosely associated "free" PDK fraction. Those results demonstrated that PDK activity was significantly increased in both the fractions, threefold in the PDK
fraction that was intrinsic to the complex and almost twofold in the
free PDK fraction (39). Although protein was not measured, it was clear that not all of the PDK protein was immediately
incorporated into the complex. Therefore, this could account for the
discrepancy in the present study between the time course of increased
total mitochondrial PDK4 protein and the total PDK activity.
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Rate of increase of PDK activity and the role of insulin
sensitivity.
Although PDK activity increased in all six subjects, there was
considerable variability in the rate of increase during the first 3 days of the HF/LC diet. The subjects with the greatest increase in PDK
activity in the first 3 days were those individuals who were more
physically active (as suggested by their prestudy activity
questionnaire). There was a strong correlation between a subject's
physical activity level and the change in ()PDK activity in
the first 3 days of the dietary intervention (Fig.
8). However, this correlation disappeared
by 6 days (Fig. 8). This suggests that the early rate of
response to the diet is sensitive to a subject's aerobic
capacity but that the overall change in PDK activity over 6 days was
similar for all individuals, regardless of training status. The
mechanism for this is unclear. Decreasing insulin levels (or a
decreased response to insulin) and increased skeletal muscle fat
metabolism have both been implicated as important regulators of PDK
activity and expression (7, 25, 27, 28, 37). It has been
suggested previously that there is an increase in insulin sensitivity
with training through some postreceptor event (5). Because
there was no correlation between a subject's activity level and the
decrease in plasma insulin by 1 or 3 days of this study, it is possible
that trained individuals were more sensitive to the similar decrease in
circulating insulin concentration, resulting in a larger early increase
in PDK activity.
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Rate of increase of PDK activity and the role of increased fat
metabolism.
Training also increases skeletal muscle fat metabolism; therefore, it
is also possible that the increased FFA metabolism in the active
individuals stimulated the more rapid increase in PDK activity. The
family of peroxisomal proliferator-activated receptors (PPARs) has
evoked considerable interest as the possible link between FFAs,
metabolic regulation, and gene expression. Rat work has demonstrated
increased skeletal muscle PDK4 expression with PPAR agonist, and it
is possible that PPAR
may participate as a link between increased
fat metabolism and decreased carbohydrate oxidation by downregulating
PDH (41). PPAR
is similarly implicated in the
upregulation of many fat-metabolizing enzymes and could serve as a
generalized coordinator of metabolism (21). Recent work
has demonstrated that PPAR
increases with exercise training, highlighting the possibility that increased concentrations of PPAR
could contribute to the rapid increase in PDK activity observed in fit
individuals (15). It is clear that these possibilities require further investigation.
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ACKNOWLEDGEMENTS |
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The technical assistance of Linda Van Ostaaijen is gratefully acknowledged.
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FOOTNOTES |
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This study was supported by operating grants from the Natural Sciences and Engineering Research, and Medical Research Councils of Canada, and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47844). S. J. Peters was supported by an Ontario Graduate Scholarship and the Gatorade Sport Science Institute.
Address for reprint requests and other correspondence: S. J. Peters, Dept. of Physical Education, Faculty of Applied Health Sciences, Brock University, St. Catharines, ON, Canada L2S 3A1 (E-mail: speters{at}arnie.pec.brocku.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 April 2001; accepted in final form 19 July 2001.
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