1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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This study investigated the
effect of reduced free fatty acid (FFA) availability on pyruvate
dehydrogenase activation (PDHa) and carbohydrate metabolism during
moderate aerobic exercise. Eight active male subjects cycled for 40 min
at 55% O2 peak on two occasions.
During one trial, subjects ingested 20 mg/kg body mass of the
antilipolytic drug nicotinic acid (NA) during the hour before exercise
to reduce FFA. Nothing was ingested in the control trial (CON). Blood
and expired gas measurements were obtained throughout the trials, and
muscle biopsy samples were obtained immediately before exercise and at
5, 20, and 40 min of exercise. Plasma FFA were lower in the NA trial
(0.13 ± 0.01 vs. 0.48 ± 0.03 mM, P < 0.05), and the respiratory exchange ratio (RER) was increased with NA
(0.93 ± 0.01 vs. 0.89 ± 0.01, P < 0.05),
resulting in a 14.5 ± 1.8% increase in carbohydrate oxidation compared with CON. PDHa increased rapidly in both trials at exercise onset but was ~15% higher (P < 0.05) throughout
exercise in the NA trial (2.44 ± 0.19 and 2.07 ± 0.12 mmol · kg wet
muscle
1 · min
1 for NA
and CON at 40 min). Muscle glycogenolysis was 15.3 ± 9.6% greater in the NA trial vs. the CON trial but did not reach statistical significance. Glucose 6-phosphate contents were elevated
(P < 0.05) in the NA trial at 30 and 40 min of
exercise, but pyruvate and lactate contents were unaffected. These data
demonstrate that the reduction of exogenous FFA availability increased
the activation of PDH and carbohydrate oxidation during moderate
aerobic exercise in men. The increased activation of PDH was not
explained by changes in muscle pyruvate or the ATP/ADP ratio but may be
related to a decrease in the NADH/NAD+ ratio or an
epinephrine-induced increase in calcium concentration.
pyruvate dehydrogenase activity; nicotinic acid; carbohydrate and fat oxidation
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INTRODUCTION |
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FAT AND
CARBOHYDRATE are the primary fuels for exercise. The exact
mechanisms that dictate the relative contribution of each of these
substrates at a given exercise intensity are still not elucidated
(40, 46). Numerous laboratories, including our own, have examined the effects of increasing fat availability on whole
body and skeletal muscle carbohydrate metabolism (11, 14, 15, 17,
29, 30, 37, 43). The administration of Intralipid and heparin
has been commonly used to acutely increase plasma free fatty acid (FFA)
concentrations (17, 37, 43). The findings from our
laboratory (15, 29, 30) demonstrated that muscle glycogen
use was decreased between exercise power outputs of 40-85%
maximal oxygen uptake (O2 max) and that
pyruvate dehydrogenase activation (PDHa) was decreased at 40-65%
O2 max in the high-fat condition. This
decreased glycogenolysis, glycolysis, and carbohydrate oxidation
appeared to be mediated through reductions in the accumulation of free Pi, ADP, and AMP, important regulators of glycogen
phosphorylase and PDHa.
In contrast, relatively few studies have examined the effects of reduced fat availability on skeletal muscle carbohydrate metabolism during exercise. Previous human studies have shown that oral nicotinic acid (NA) supplementation decreases exogenous FFA availability and fat oxidation and increases carbohydrate oxidation, as indicated by an increased respiratory exchange ratio (RER) (6, 16, 19, 20, 22, 25, 28, 42). However, only Bergström et al. (3) examined muscle metabolism directly with muscle biopsies and showed increased glycogen depletion with NA ingestion. No studies have examined the effects of reduced FFA availability on the activation and regulation of PDH.
In our previous work (8, 29), increasing FFA availability decreased PDHa, possibly by increasing mitochondrial NADH and pyruvate at rest and during the 1st min of exercise. We argued that the elevated NADH stimulated PDH kinase (PDK) and that reduced pyruvate resulted in less PDK inhibition, together resulting in less activation of PDHa at the onset of exercise. It is currently unknown whether deceasing FFA availability through NA ingestion would do the opposite and activate PDH to a greater degree than in the control trial.
Therefore, the purpose of the present study was to administer NA to decrease FFA availability to the exercising muscle and examine whole body carbohydrate oxidation and, for the first time, the activation of PDH during moderate exercise. Several muscle metabolites believed to regulate PDHa were also measured in an attempt to explain any changes in PDHa. We hypothesized that NA ingestion would increase the activation of PDH and that increased pyruvate levels would explain the greater PDHa and reliance on carbohydrate metabolism.
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METHODS |
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Subjects.
Eight active male subjects volunteered to participate in this study. No
subject was taking any medications or engaging in aerobic exercise more
than two times per week. Their mean (±SE) age, weight, and
O2 max were 24.4 ± 1.3 yr,
75.5 ± 4.7 kg, and 53.8 ± 2.3 ml · kg
1 · min
1,
respectively. All subjects were informed of the experiment protocol, and the possible associated risks of the study were explained to
subjects both orally and in writing before written informed consent was
obtained. The ethics committees of the University of Guelph and
McMaster University approved the study.
Preexperimental protocol. Subjects performed a continuous and incremental test to exhaustion on a cycle ergometer (LODE Instrument, Groningen, The Netherlands). A 3-day dietary recall over 2 weekdays and 1 weekend day was used to assess their normal diet. Their calculated percent (±SE) macronutrient breakdown of carbohydrate, fat, and protein was 52 ± 2, 33 ± 1, and 15 ± 1%, respectively.
Subjects visited the laboratory on three occasions. On the first visit, subjects completed a practice ride for 40 min with NA supplementation and no blood or muscle sampling. The purposes of the practice trial were to familiarize the subject with the protocol, confirm the subject's tolerance to oral NA supplementation, and to confirm the exercise power output of ~55%Experimental protocol. During the two experimental trials, an indwelling catheter was inserted into an antecubital vein for blood sampling and was kept patent with an isotonic saline drip. Four incisions were made over the vastus lateralis muscle of one leg under local anesthesia (2% Lidocaine, no epinephrine) for later muscle biopsy sampling.
Subjects then consumed either NA or nothing (CON) in randomized fashion during the 60 min before exercise. Blood samples were obtained immediately before supplementation atDrug administration. During the NA trial, subjects ingested a total of 20 mg NA/kg body mass (BM) in three individual doses: 10 mg/kg BM 60 min before exercise, 5 mg/kg BM 30 min before exercise, and 5 mg/kg BM immediately before cycling, as previously described (19). NA was obtained from C. E. Jamieson, Toronto, Canada. The dosing period was spaced over 1 h to minimize adverse symptoms. All subjects experienced the normal side effects of NA ingestion, which included flushing (a reddening of the skin due to peripheral vasodilation over most of the body), tingling sensations, and a sensation of heat, which started ~15-20 min after the first dose and subsided during the final 30 min of rest before exercise. No subjects experienced stomach or gastrointestinal upset. Although we do not believe that these symptoms affected the whole body or skeletal muscle responses to exercise, we cannot completely rule this out.
Analyses. A small piece of frozen wet muscle (~10-15 mg) was removed from the larger muscle piece while submerged in liquid N2 for the determination of PDHa, as described by Putman et al. (36). The remainder of the muscle sample was freeze-dried, dissected free of all visible blood and connective tissue, and powdered for subsequent metabolite and glycogen analyses. An aliquot of freeze-dried muscle (~10-12 mg) was extracted with 0.5 M perchloric acid (PCA) containing 1 mM EDTA and neutralized with 2.2 M KHCO3. This extract was used for the measurement of creatine (Cr), phosphocreatine (PCr), ATP, lactate, and glucose 6-phosphate (G-6-P) by enzymatic spectrophotometric assays (2, 18) and acetyl-CoA and acetylcarnitine with radiometric measures (7). Pyruvate and citrate were analyzed fluorometrically (32). Muscle glycogen content was measured from a second aliquot of freeze-dried muscle (~4-6 mg) from resting and 40-min biopsy samples. All muscle measurements were normalized to the highest total Cr measured among the eight biopsies from each subject.
Venous whole blood was placed in a heparinized tube, and a portion was immediately deproteinized in a 1:5 ratio with 0.6% (wt/vol) PCA. This PCA extract was stored atCalculations.
Free ADP (ADPf) and AMP (AMPf) contents were
calculated by assuming equilibrium of the creatine kinase and adenylate
kinase reactions (13). Specifically, ADPf was
calculated using the measured ATP, Cr, and PCr values, an estimated
H+ concentration, and the creatine kinase constant of
1.66 × 109. The H+ concentration was
estimated from the measured lactate and pyruvate contents as described
by Sahlin et al. (38). AMPf was calculated from the estimated ADPf and measured ATP content using the
adenylate kinase equilibrium constant of 1.05. Free inorganic phosphate (Pif) was calculated by adding the estimated
resting free phosphate of 10.8 mmol/kg dry mass (dm)
(13) to the difference in PCr content ([PCr])
minus the accumulation of G-6-P between rest and the given
exercise time points.
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Statistics. All data are presented as means ± SE. A two-way repeated-measures ANOVA (treatment × time) was used to determine significant differences between treatments. When a significant F-ratio was obtained, post hoc analysis was completed using a Student-Newman-Keuls test. A single-tailed paired t-test was used to determine the net glycogen utilization between trials. Statistical significance was accepted at P < 0.05.
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RESULTS |
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Respiratory measures.
There were no differences in O2 over
time in either trial or between trials (Table
1). Percent
O2 peak in both trials
averaged 56.4 ± 0.6%. Ventilation was higher (P < 0.05; trial effect) in NA compared with CON (Table 1). RER was
significantly higher (0.93 ± 0.01 vs. 0.89 ± 0.01, P < 0.05) in the NA trial compared with control (Fig.
1). NA ingestion resulted in a 14.5 ± 1.8% increase (P < 0.05) in total carbohydrate
oxidation and a 32.6 ± 3.9% reduction (P < 0.05) in fat oxidation (Fig. 2).
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PDHa.
NA supplementation had no effect on resting PDHa (Fig.
3). PDHa increased to higher
levels in the NA trial throughout exercise (P < 0.05;
trial effect). Specifically, increases in the NA trial were ~10, 10, and 15% higher than CON at 5, 20, and 40 min of exercise,
respectively.
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Muscle metabolites.
PCr was similar at rest and decreased (P < 0.05) to
the same extent during exercise in both trials (Table
2). The muscle content of ATP was
unaffected by prior NA ingestion or exercise in both trials (Table 2).
ADPf and AMPf were not different between trials
at rest and increased (P < 0.05) similarly throughout
exercise (Table 2). Calculated free Pi (Pif)
increased (P < 0.05) during exercise but was not
different between trials (Table 2).
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Blood measurements.
Resting plasma glycerol and FFA were similar between trials before NA
ingestion (Fig. 4). At rest, plasma FFA
was lower in the NA trial following 30 and 60 min of supplementation
vs. CON and remained at very low levels during exercise. Once exercise commenced, plasma glycerol increased in both trials but to a higher level (P < 0.05) in the CON trial by 20 min and beyond
(Fig. 4). Plasma FFA increased (P < 0.05) in the CON
trial after exercise onset but remained below resting values
(P < 0.05) during exercise in the NA trial (Fig. 4).
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DISCUSSION |
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This study investigated the effect of reduced FFA availability on PDH and carbohydrate metabolism during moderate aerobic exercise. The administration of NA decreased plasma FFA to low levels at rest and during exercise. NA ingestion did not affect PDHa at rest, but acetylcarnitine was markedly reduced. In support of our first hypothesis, the reduced exogenous FFA availability resulted in increased carbohydrate oxidation and a greater PDHa during exercise. Contrary to our second hypothesis, the PDHa could not be explained by changes in muscle pyruvate or the ATP-to-ADP ratio (ATP/ADP) but may be related to a decreased mitochondrial NADH-to-NAD+ ratio (NADH/NAD+) or an epinephrine-induced increase in calcium concentration.
Reduced FFA availability increases PDHa. The novel finding of this study is that reduced FFA availability increased PDHa during moderate aerobic exercise. PDH is covalently regulated by PDH phosphatase, which dephosphorylates and activates PDH to its active, a form, and PDH kinase, which phosphorylates and inhibits PDH to its inactive, b form (24, 26, 39). In turn, several allosteric modulators regulate the activities of PDH phosphatase and PDK. Specifically, at rest, PDK is stimulated by high ratios of acetyl-CoA/CoA, ATP/ADP, and NADH/NAD+, keeping PDH mainly in the inactive, b form. Conversely, during exercise, PDK is inhibited by pyruvate and low ratios of ATP/ADP, and the phosphatase is acutely stimulated by Ca2+ moving PDH to the a form (for reviews see Refs. 24, 39).
The increase in PDHa in the NA trial cannot be explained by changes in pyruvate and the ATP/ADP ratio, as the exercise-induced changes were similar between trials. We were surprised by the lack of increase in muscle pyruvate in the NA trial, but the expected increase due to the increased glycolytic flux was either not present or too small to detect. Although there was a marginal increase in acetyl-CoA at the end of exercise in the NA treatment, which might suggest a downregulation of PDHa by the kinase, previous studies suggest that changes in PDHa during exercise are independent of the acetyl-CoA/CoA ratio (10, 23, 36). Our laboratory has previously shown that NADH/NAD+ is increased at rest and under conditions of increased fat availability, resulting in decreased PDHa (8, 29). Specifically, Odland et al. (29) estimated mitochondrial NADH content with the whole muscle technique when FFA availability was increased and found increased [NADH] at rest and in the 1st min of exercise and a decreased PDHa. This finding is supported by in vitro work, which suggests that PDK is stimulated by NADH and inhibited by NAD+ (35). In the present study, the NADH/NAD+ ratio may also have been a factor but was not measured. In this study, the reverse situation may have occurred, with decreasing FFA and decreasing mitochondrial [NADH] with this reduced redox state increasing PDHa by inhibiting the kinase. Regardless, there is still no consensus as to what role the redox state plays with regard to PDHa during exercise, and until there is a clearly established method to measure the mitochondrial redox state, these issues will not be resolved. Watt et al. (45) have suggested that PDHa may also be mediated by epinephrine-induced cAMP effects on either the phosphatase or the kinase through increases in mitochondrial calcium concentration. It is well established that increases in Ca2+, pyruvate, and ADP contribute to the activation of PDH at the onset of exercise (9, 23, 24, 31, 36). When epinephrine was added to the perfusion medium during in vitro work in isolated rat heart, higher intramitochondrial concentrations of Ca2+ (12) were reported as well as increased PDHa (27). Similarly, another study demonstrated increased sarcoplasmic calcium release byEffects of reduced FFA availability on whole body carbohydrate
oxidation.
The present dose of 20 mg/kg BM significantly reduced fat availability
to be oxidized by the working muscles. This NA dose resulted in reduced
FFA availability, and this reduced FFA availability increased whole
body carbohydrate oxidation by ~15% and decreased fat oxidation by
~30% during exercise. All of these findings are consistent with
previous NA studies, which have reported similar shifts in RER after NA
ingestion and during exercise for 30-60 min at similar power
outputs (~50-70% O2 max)
(3, 20, 22, 25, 28). Howlett et al. (22)
reported average RER values of 0.81 ± 0.01 in the control trial
and 0.88 ± 0.01 in the NA trial between 30 and 60 min of exercise
at a similar intensity of ~60% of
O2 max. It should be noted that that
study used female subjects, and their findings of slightly increased
carbohydrate metabolism under conditions of reduced FFA
availability may be due to a sex difference. It has been reported that
females rely more heavily on fat oxidation than males do at a given
exercise intensity, which may have resulted in their being more
affected by the NA (21, 41).
Reduced FFA availability on muscle glycogenolysis and glycolysis.
There exists only one other study in obtaining muscle biopsy samples
when exogenous FFA availability was reduced during exercise, and the
only muscle parameter measured was glycogen content (3). In that study, Bergström et al. (3) reported a
significant ~20% increase in net glycogen utilization in the NA
trial compared with controls after 45-60 min of exercise at a
O2 of ~1.1 l/min. In the present
study, we did not find a significant difference in glycogen depletion
between CON and NA treatments at a
O2 of
~2.2 l/min, even though there was a nonsignificant 15% increase in
muscle glycogen depletion in the NA trial (CON:
glycogen of ~150
glycosyl units vs. NA:
glycogen of ~173 glycosyl units, NS,
P = 0.063). It is likely that a significant effect
would have been found if we had extended our exercise trial length to
60 min, as in the trial of Bergström et al.
Acetyl group availability.
A novel finding of this investigation was that acetylcarnitine was
significantly reduced at rest following 60 min of NA ingestion. It was
expected that the NA ingestion would have no effect on resting
acetylcarnitine levels or would actually cause an increase. The
reduction in FFA availability should have increased the reliance on
carbohydrate in the resting muscle. Accordingly, an increased flux
through glycolysis leading to greater pyruvate production and an
overproduction of acetyl-CoA may lead to an increased production of
acetylcarnitine. This has been shown to occur at the onset of exercise
(23), when the production of pyruvate and acetyl-CoA is
severalfold higher than during rest. Interestingly, the present results
are in agreement with previous work from our laboratory (44), where a carbohydrate load (1 g
carbohydrate/kg BM) ingested 1 h before exercise also caused a
decrease in plasma FFA and resulted in reduced muscle acetylcarnitine
content at rest. The reason for this decrease is not readily apparent,
and the significance of this finding at rest remains to be elucidated
but does not appear to affect the subsequent response to exercise
(44). Acetylcarnitine normally functions as a buffer for
acetyl-CoA and is formed when there is an overproduction of acetyl
units in the muscle and is degraded to provide acetyl units when the
supply is reduced. The primary fuel source for energy at rest is
through fat oxidation, resulting in the production of acetyl-CoA units
via -oxidation. Possibly by limiting exogenous FFA availability,
during NA ingestion, inadequate acetyl units and reducing equivalents
(NADH) were being provided to meet the energy needs of the electron
transport chain in resting muscle. This, in turn, could have caused a
decreased resting acetylcarnitine level. A decreased resting pyruvate
level might have been expected during NA ingestion if it were limiting the production of acetyl-CoA through PDH, but this did not occur.
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ACKNOWLEDGEMENTS |
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We sincerely thank Drs. Graham Jones and Kieran Killian (McMaster University Hospital, Hamilton, ON, Canada) for expert medical assistance.
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FOOTNOTES |
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This study was supported by the Natural Sciences and Engineering Research Council of Canada (L. L. Spriet) and the Canadian Institute of Health Research (G. J. F. Heigenhauser).
Address for reprint requests and other correspondence: L. L. Spriet, Dept. of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, ON, N1G 2W1, Canada (E-mail: lspriet{at}uoguelph.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.
First published November 10, 2002;10.1152/ajpendo.00418.2002
Received 20 September 2002; accepted in final form 10 November 2002.
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