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Am J Physiol Endocrinol Metab 288: E133–E142, 2005.

First published September 21, 2004; doi:10.1152/ajpendo.00379.2004.

Malonyl-CoA and carnitine in regulation of fat oxidation


in human skeletal muscle during exercise
Carsten Roepstorff,1 Nils Halberg,1 Thore Hillig,1 Asish K. Saha,2 Neil B. Ruderman,2
Jørgen F. P. Wojtaszewski,1 Erik A. Richter,1 and Bente Kiens1
1
The Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences,
University of Copenhagen, Copenhagen, Denmark; and 2Diabetes Research Unit, Departments of Medicine, Physiology,
and Biophysics, Section of Endocrinology, Boston University School of Medicine, Boston, Massachusetts
Submitted 16 August 2004; accepted in final form 17 September 2004

Roepstorff, Carsten, Nils Halberg, Thore Hillig, Asish K. Saha, inner mitochondrial membrane is preceded by the transfer of
Neil B. Ruderman, Jørgen F. P. Wojtaszewski, Erik A. Richter, the acyl moiety to carnitine, a process catalyzed by carnitine
and Bente Kiens. Malonyl-CoA and carnitine in regulation of fat palmitoyltransferase 1 (CPT-1), an enzyme located in the outer
oxidation in human skeletal muscle during exercise. Am J Physiol mitochondrial membrane (23). Two mechanisms for the intra-
Endocrinol Metab 288: E133–E142, 2005. First published September
21, 2004; doi:10.1152/ajpendo.00379.2004.—Intracellular mecha-
cellular regulation of CPT-1 have been proposed; one involves
nisms regulating fat oxidation were investigated in human skeletal malonyl-CoA, and the other involves the cytosolic concentra-
muscle during exercise. Eight young, healthy, moderately trained men tion of carnitine.
performed bicycle exercise (60 min, 65% peak O2 consumption) on Malonyl-CoA is an intermediate in the de novo synthesis of
two occasions, where they ingested either 1) a high-carbohydrate diet fatty acids (FA) and an allosteric inhibitor of CPT-1 (14).
(H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to Studies in rat muscle suggest that changes in the glucose
alter muscle glycogen content as well as to induce, respectively, low supply and energy expenditure of the muscle cell regulate the
and high rates of fat oxidation. Leg fat oxidation was 122% higher concentration of malonyl-CoA, in keeping with its need to
during exercise in L-CHO than in H-CHO (P ⬍ 0.001). In keeping generate ATP from fat oxidation (28, 32, 33). Two types of
with this, the activity of ␣2-AMP-activated protein kinase (␣2-AMPK) regulation of malonyl-CoA have been described: one involving
was increased twice as much in L-CHO as in H-CHO (P ⬍ 0.01) at
60 min of exercise. However, acetyl-CoA carboxylase (ACC)␤ Ser221
the cytosolic concentration of citrate and the other involving
phosphorylation was increased to the same extent (6-fold) under the 5⬘-AMP-activated protein kinase (AMPK) (28, 32). Cytosolic
two conditions. The concentration of malonyl-CoA was reduced 13% citrate is both an allosteric activator of acetyl-CoA carboxylase
by exercise in both conditions (P ⬍ 0.05). Pyruvate dehydrogenase (ACC), the key enzyme governing malonyl-CoA synthesis, and
activity was higher during exercise in H-CHO than in L-CHO (P ⬍ a substrate for the malonyl-CoA precursor, cytosolic acetyl-
0.01). In H-CHO only, the concentrations of acetyl-CoA and acetyl- CoA. In rodents and humans, high glucose availability at rest
carnitine were increased (P ⬍ 0.001), and the concentration of free has been shown to elevate the cytosolic citrate concentration
carnitine was decreased (P ⬍ 0.01), by exercise. The data suggest that and consequently the muscle malonyl-CoA concentration (3,
a decrease in the concentration of malonyl-CoA, secondary to ␣2- 27, 32, 33). This is likely the mechanism whereby fat oxidation
AMPK activation and ACC inhibition (by phosphorylation), contrib- is inhibited by high glucose availability in resting humans (3,
utes to the increase in fat oxidation observed at the onset of exercise
regardless of muscle glycogen levels. They also suggest that, with
27). AMPK in turn regulates the concentration of malonyl-
high muscle glycogen, the availability of free carnitine may limit fat CoA by phosphorylating and inhibiting ACC (28) and possibly
oxidation during exercise, due to its increased use for acetylcarnitine by activating malonyl-CoA decarboxylase (MCD), the major
formation. enzyme regulating malonyl-CoA turnover in muscle (34). Dur-
ing muscle contractions in rodents, AMPK is activated and
acetylcarnitine; acetyl coenzyme A; pyruvate dehydrogenase activity; ACC is phosphorylated and consequently inhibited, leading to
adenosine 5⬘-monosphosphate-activated protein kinase; acetyl coen-
zyme A carboxylase
a decrease in muscle malonyl-CoA concentration, which may
induce the increase in fat oxidation at the onset of contraction
(28). In contrast, several human studies have failed to show a
FAT AND CARBOHYDRATE are the main sources of fuel for ATP reduction in the muscle malonyl-CoA concentration during
synthesis in human skeletal muscle. The ratio between fat and moderate-intensity exercise with normal muscle glycogen
carbohydrate oxidation during exercise depends on preexercise stores when fat oxidation is increased from resting levels (7,
substrate levels and exercise duration and intensity (12, 40, 42, 17, 18). Still, with low muscle glycogen content, there is a
44). However, the intracellular determinants of the use of these higher AMPK activation during exercise (44), which may
fuels are unclear. decrease the malonyl-CoA level and thereby cause the higher
It has been suggested that the fat oxidation rate in human fat oxidation seen during exercise with low vs. high muscle
skeletal muscle during exercise is regulated intracellularly at glycogen (44). So far, the muscle malonyl-CoA concentration
the entry of long-chain fatty acyl (LCFA)-CoA into the mito- has never been measured in humans during submaximal exer-
chondria (31, 36, 37). The transport of LCFA-CoA across the cise with low muscle glycogen content.

Address for reprint requests and other correspondence: C. Roepstorff, The


Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, The costs of publication of this article were defrayed in part by the payment
Dept. of Human Physiology, Universitetsparken 13, DK-2100 Copenhagen Ø, of page charges. The article must therefore be hereby marked “advertisement”
Denmark (E-mail: croepstorff@ifi.ku.dk). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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E134 MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION

Carnitine is a substrate for CPT-1. Consequently, the avail- MATERIALS AND METHODS
ability of cytosolic carnitine may limit fat oxidation. Another
Subjects. Eight young, healthy, moderately trained men [age, 26 ⫾
function of carnitine is to buffer accumulated acetyl-CoA in a 1 yr; height, 1.80 ⫾ 0.02 m; body mass, 76 ⫾ 3 kg; body mass index,
reaction catalyzed by carnitine acetyltransferase (CAT) (26). It 23.5 ⫾ 0.7 kg/m2; body fat, 14.6 ⫾ 1.9%; peak O2 consumption
has been proposed that, under exercise conditions with high (V̇O2 peak), 4.1 ⫾ 0.2 l/min; V̇O2 peak/body mass, 53.7 ⫾ 1.6
glycolytic flux and therefore excess formation of acetyl-CoA ml 䡠 kg⫺1 䡠 min⫺1] were recruited to participate in the study. All sub-
compared with its utilization by the tricarboxylic acid cycle, jects were nonsmokers and were engaged in 4 – 6 h/wk of regular
carnitine serves to buffer the excess acetyl-CoA, which leaves exercise training such as running, bicycling, and resistance training.
less carnitine available to CPT-1 (40, 43). By this mechanism, Before inclusion into the study, subjects were fully informed about the
carnitine may play a role in adjusting the rate of fat oxidation nature and possible risks of the study and gave their written consent.
The study was approved by the Copenhagen Ethics Committee (KF-
inversely to the rate of carbohydrate oxidation. Accordingly, in 01-078/01) and carried out in accordance with the code of ethics of the
humans it was shown that fat oxidation and muscle carnitine World Medical Association (Declaration of Helsinki II). The results
concentration were tightly coupled during incremental exer- presented in this study are part of a larger study on the regulation of
cise: with increasing exercise intensity, they both decreased, lipid metabolism during exercise in human skeletal muscle that has, in
while, on the other hand, carbohydrate oxidation increased (40). minor part, been published previously (30, 31).
The present study investigated intracellular mechanisms to Preexperimental testing. All subjects initially performed an incre-
regulate fat oxidation in response to altered carbohydrate mental exercise test on a bicycle ergometer (Monark 839 Electronic
Ergometer; Monark Exercise Sweden) to determine peak O2 uptake
availability in human skeletal muscle during submaximal ex-
(V̇O2 peak). Respiratory measurements were carried out with the Doug-
ercise. Healthy male subjects performed 60 min of submaximal las bag technique. Percent body fat was measured by dual-energy
bicycle exercise with either high (H-CHO) or low (L-CHO) X-ray absorptiometry (DEXA; Lunar, Madison, WI; DPX-IQ v. 4.6.6).
preexercise muscle glycogen. During the two exercise proto- Experimental design. Subjects underwent two experimental proto-
cols, with expected similar energy output but marked differ- cols separated by 2–3 wk. In both protocols, subjects completed a bout
ences in the relative fat and carbohydrate oxidation, we measured of glycogen-depleting bicycling followed by a controlled diet for the
in the same setup the malonyl-CoA, carnitine, acetylcarnitine, and rest of the day (Fig. 1, day 1). In one protocol the controlled diet
acetyl-CoA concentrations, the pyruvate dehydrogenase (PDH) consisted primarily of fat (L-CHO), and in the other protocol primar-
ily of carbohydrate (H-CHO) (see Glycogen depletion bout). The next
and AMPK activity, and the ACC␤ Ser221 phosphorylation in
day, subjects performed a bicycle exercise test for 60 min at ⬃65%
skeletal muscle to examine the intramuscular mechanisms that V̇O2 peak (Fig. 1, day 2). The order of the L-CHO and H-CHO
regulate the ratio between fat and carbohydrate oxidation protocols was randomized and stratified.
during exercise. To rule out marked differences between H- Dietary control. On the 3 days preceding the first protocol, subjects
CHO and L-CHO conditions in blood-borne substrate and determined their habitual energy and nutrient intake by a self-reported
hormone levels and their possible confounding effects on the dietary record. All food and beverage intakes were weighed to the
interpretations of the results, we allocated a light meal 4.5 h accuracy of 1 g and recorded. Subsequently, the energy intake and
before exercise and infused glucose intravenously during ex- composition of the habitual diet were calculated by means of a
computer database (Dankost 2000; Danish Catering Center, Copen-
ercise to keep the arterial blood glucose concentrations nearly
hagen, Denmark). On the 3 days preceding the second protocol,
constant and similar between the two conditions. By this subjects followed a diet identical to the one ingested before the first
approach, the levels of several other circulating metabolites protocol.
and hormones also were kept nearly similar between H-CHO Glycogen depletion bout. On the day of the glycogen depletion
and L-CHO. bout, subjects arrived at the laboratory at 7:30 AM. All subjects

Fig. 1. Schematic diagram showing the experimental


protocol. Each protocol consisted of 2 experimental
days. Day 1: glycogen depletion and dietary manipu-
lation. Day 2: exercise experiment. All subjects com-
pleted 2 protocols [high (H-CHO) and low (L-CHO)
preexercise muscle glycogen].

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MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION E135
abstained from any heavy physical activity on the day before the Breath samples. Expired air in the Douglas bags was processed,
glycogen depletion bout. A breakfast meal was served that contained and respiratory exchange ratio (RER) was calculated as described
25% of each subject’s daily energy intake and consisted of 30% previously (29).
energy (30 E%) as fat, 55 E% as carbohydrate, and 15 E% as protein. Blood samples. Blood O2 and CO2 concentrations were deter-
At 10 AM, subjects initiated the glycogen depletion bicycle bout, mined, and leg respiratory quotient (RQ) was calculated as described
which consisted of alternating periods of continuous and intermittent previously (29). Leg fat and carbohydrate oxidation rates were calcu-
exercise with a few short bouts of arm cranking in between, as lated by the following equations using the nonprotein RQ and then
previously described (31). The glycogen depletion bout lasted for expressed (in kJ/min) using standard caloric equivalents for fat and
3– 4.5 h and was well tolerated by all subjects. To determine the carbohydrate, respectively (20)
muscle glycogen concentration after the glycogen depletion protocol, fat oxidation rate (g/min) ⫽ 1.695 䡠 V̇O2 ⫺ 1.701 䡠 V̇CO2
a muscle biopsy was obtained under local anesthesia from the vastus
lateralis muscle by the needle biopsy technique. For the following 6 h, carbohydrate oxidation rate (g/min) ⫽ 4.585 䡠 V̇CO2 ⫺ 3.226 䡠 V̇O2
subjects were allowed to move freely around at the laboratory. Meals
were served immediately after the biopsy procedure and 1, 2, 3.5, and where V̇O2 is O2 consumption, and V̇CO2 is CO2 production. Blood
5 h after completion of the glycogen depletion bout. Then subjects glucose and lactate concentrations were measured automatically
were given a light snack to ingest at 11 PM and left the laboratory. (ABL510; Radiometer Medical, Copenhagen, Denmark). Plasma FA
The controlled diet during the recovery period consisted of 85 E% fat, concentrations were measured by a colorimetric commercial assay kit
(Wako Chemicals) performed by a COBAS FARA autoanalyzer
2 E% carbohydrate, and 13 E% protein in L-CHO and 8 E% fat, 80
(COBAS FARA 2, Roche Diagnostic Switzerland). Concentrations of
E% carbohydrate, and 12 E% protein in H-CHO. The total energy
insulin (Pharmacia Insulin Radioimmunoassay 100; Pharmacia &
intake during this period was calculated as the individual daily energy
Upjohn Diagnostics, Uppsala, Sweden) as well as epinephrine and
intake (DEI), as reported from the dietary record, minus the breakfast norepinephrine (KatCombi Radioimmunoassay; Immuno-Biological
(25% DEI) plus the estimated extra energy consumption during the Laboratories, Hamburg, Germany) in plasma were determined by
bicycle exercise to secure that subjects were in energy balance. During radioimmunoassay.
the glycogen depletion bout and for the rest of the day, subjects were Muscle biopsies. The biopsies were quick-frozen (⬍10 s after being
allowed to drink water ad libitum. obtained) in liquid nitrogen while still in the biopsy needle and stored
Exercise experimental protocol. Subjects arrived at the laboratory at ⫺80°C for subsequent biochemical analysis. Seventy-five to eighty
at 7:30 AM having abstained from any physical activity during the milligrams wet weight were freeze dried and dissected free of all
morning. A light breakfast was served that contained 10% of the visible adipose tissue, connective tissue, and blood under a micro-
individual DEI and consisted of 4 E% fat, 78 E% carbohydrate, and 18 scope. The dissected muscle fibers were pooled and then divided into
E% protein. After 30 min of rest in the supine position, Teflon subpools for the respective analyses. Malonyl-CoA content and PDH
catheters were inserted under local anesthesia into the femoral artery activity in its active form (PDHa) were measured on 25 and 10 mg of
and the contralateral femoral vein with a thermistor probe inserted wet muscle tissue, respectively, which were not freeze dried.
into the vein as described previously (29). During the second protocol, Muscle glycogen. The glycogen concentration was determined on 2
femoral catheters were inserted in opposite legs. A venous catheter mg dry wt by a fluorometric method (13).
was inserted into an antecubital vein for infusion of glucose. Muscle lysates. Muscle lysates were prepared from 6 mg dry wt of
Subjects then rested in the supine position. At 12 PM, femoral freeze-dried and dissected muscle tissue as described previously (10).
venous blood flow was measured by the thermodilution technique, Due to the limited amount of tissue, muscle lysates were only
using bolus injections of 5 ml of ice-cold sterile saline (1). Then, prepared from biopsies obtained at rest and at 60 min of exercise.
expired air was collected in a Douglas bag, and blood samples were Western blotting. Phosphorylation of ␣-AMPK Thr172 and ACC␤
obtained in duplicate for determination of resting blood concentra- Ser221 was detected by Western blotting on muscle lysates. The
tions. A muscle biopsy was obtained from the vastus lateralis muscle. lysates were boiled in Laemmli buffer before being subjected to
Then subjects initiated a 60-min bicycle exercise test at ⬃65% SDS-PAGE and immunoblotting. Primary phosphospecific antibodies
V̇O2 peak. Subjects exercised at the same absolute workload in L-CHO were rabbit anti-␣-AMPK Thr172-phos (Upstate Biotechnology, Lake
and H-CHO groups. A variable continuous infusion of glucose into an Placid, NY) and rabbit anti-ACC␣ Ser79-phos. Secondary antibody
antecubital vein was initiated after 1 min of exercise and continued was horseradish peroxidase-conjugated anti-rabbit IgG (DAKO,
throughout exercise to keep the blood glucose concentration constant Glostrup, Denmark). Antigen-antibody complexes were visualized
and similar to the basal resting level. The infusion rate of glucose was using enhanced chemiluminescence (ECL⫹; Amersham Biosciences)
evaluated by frequent arterial blood sampling (every 5 min) and and quantified by a Kodak Image Station E440CF (Kodak, Glostrup,
Denmark). The anti-␣-AMPK Thr172-phos antibody detected a single
subsequent rapid analysis of the glucose concentration. Every 10 min
band at ⬃63 kDa as expected. With the phosphospecific ACC␣ Ser79
during exercise, expired air was collected in a Douglas bag, blood was
antibody, which probably recognizes the equivalent Ser221 in human
drawn from the femoral artery and vein, and femoral venous blood
ACC␤ in the phosphorylated state, a single band was detected at
flow was measured by the thermodilution technique with continuous ⬃260 kDa as expected.
infusion of ice-cold sterile saline (1). After 30 min of bicycling, AMPK activity. ␣-Isoform-specific AMPK activity was determined
subjects shortly terminated exercise, and a biopsy was obtained from in immunoprecipitates from muscle lysates as described previously
the vastus lateralis muscle (⬍15 s of rest). Exercise was continued (44). Briefly, immunoprecipitates were prepared from 200 ␮g of
within 120 s. After completion of the 60-min bicycle test, another muscle lysate protein using anti-␣1- or anti-␣2-antibodies as described
biopsy was obtained from the vastus lateralis muscle (⬍15 s of rest). previously (46) (kindly donated by Dr. D. G. Hardie, University of
All biopsies were obtained through separate incisions spaced at least Dundee, Dundee, Scotland). AMPK activity was measured in the
5 cm apart. Heart rate was monitored throughout the experiment with immunoprecipitates using SAMS-peptide (HMRSAMSGLHLVKRR,
a Polar Vantage XL heart rate monitor (Polar Electro Finland), and 200 ␮M) as previously described (45).
blood pressure was measured through the femoral arterial catheter Malonyl-CoA. The muscle malonyl-CoA concentration was mea-
connected to a pressure transducer (Spectramed P23XL, Statham). sured as described previously (15), modified to human muscle tissue
During the first protocol, subjects were offered water ad libitum, and (7). In short, 25 mg of muscle tissue (wet wt) were homogenized in
their water intake was recorded. During the second protocol, subjects 250 ␮l of perchloric acid (10%) and neutralized with NaOH. Malonyl-
repeated their water intake from the first protocol. CoA was determined in the neutralized muscle homogenates by

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E136 MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION

measuring malonyl-CoA-dependent incorporation of [3H]acetyl-CoA Fat and carbohydrate oxidation. RER and leg RQ did not
into palmitate catalyzed by FA synthase (kindly donated by Dr. J. differ significantly from each other in either condition. At rest,
Knudsen, University of Southern Denmark). Each sample was deter- RER was 0.76 ⫾ 0.02 and 0.83 ⫾ 0.03 and leg RQ was 0.75 ⫾
mined in duplicate. The coefficient of variation (CV) between three 0.01 and 0.85 ⫾ 0.03 in L-CHO and H-CHO, respectively.
single determinations on the same muscle extract was 18%, and the
Within the first 10 min of exercise, RER increased (P ⬍ 0.01)
CV between two different muscle extracts, determined in duplicate,
from the same biopsy was 11%.
to 0.82 ⫾ 0.02 and 0.91 ⫾ 0.01 in L-CHO and H-CHO,
PDHa. Due to limited amount of muscle tissue, PDHa activity was respectively, and leg RQ increased (P ⬍ 0.01) to 0.85 ⫾ 0.02
only determined on the biopsies obtained at rest and at 60 min of and 0.93 ⫾ 0.02 in L-CHO and H-CHO, respectively. No
exercise. The frozen muscle tissue (10 mg) was homogenized in 300 further significant changes were seen in RER or leg RQ during
␮l of buffer (pH 7.8) containing 200 mM sucrose, 50 mM KCl, 5 mM exercise. There was a main effect of condition on RER and on
MgCl2, 5 mM EGTA, 50 mM Tris 䡠 HCl, 50 mM NaF, 5 mM leg RQ, both being lower in L-CHO than in H-CHO (P ⬍ 0.001).
dichloroacetate, and 0.1% Triton X-100. Subsequently, PDHa activity The fat oxidation rate across the leg did not differ signifi-
was determined by a radioisotopic assay first described by Constantin- cantly between L-CHO and H-CHO at rest (Fig. 2). In H-CHO
Teodosiu et al. (6) and modified by Putman et al. (25). The CV the leg fat oxidation rate increased (P ⬍ 0.05) from rest to
between five single determinations on the same muscle extract was exercise, and an even larger increase (P ⬍ 0.001) was seen in
17%, and the CV between four different muscle extracts from the L-CHO. The leg fat oxidation rate differed significantly be-
same biopsy determined in duplicate was 17%.
tween L-CHO and H-CHO at all exercise time points (P ⬍
Acetyl-CoA, acetylcarnitine, and free carnitine. Six milligrams dry
weight of freeze-dried and dissected muscle tissue were extracted with 0.001). The leg carbohydrate oxidation rate increased (P ⬍
0.6 M perchloric acid and neutralized with 2 M KHCO3. Acetyl-CoA, 0.001) from rest to exercise in both conditions. A main effect
acetylcarnitine, and free carnitine concentrations were determined by of condition was observed in the leg carbohydrate oxidation
radioisotopic assays (4). The CV between five single determinations rate (P ⬍ 0.01).
on the same muscle extract was 3.9, 3.1, and 5.8% for acetyl-CoA, Blood metabolite concentrations. At rest, the arterial con-
acetylcarnitine, and free carnitine, respectively. The CV between four centration of plasma FA was 674 ⫾ 110 and 596 ⫾ 112 ␮M in
different muscle extracts, determined in duplicate, from the same L-CHO and H-CHO, respectively. It decreased (P ⬍ 0.05) at
biopsy was 11.9, 8.1, and 6.2% for acetyl-CoA, acetylcarnitine, and initiation of exercise to 468 ⫾ 61 and 334 ⫾ 34 ␮M at 10 min
free carnitine, respectively. Recovery of acetyl-CoA, acetylcarnitine, in L-CHO and H-CHO, respectively, after which it increased
and carnitine added to muscle extracts was 90, 96, and 99%, respec- (P ⬍ 0.05) continuously during exercise to 703 ⫾ 58 ␮M in
tively.
L-CHO and 618 ⫾ 58 ␮M in H-CHO at 90 min. A main effect
Statistics. Data are presented as means ⫾ SE. For variables inde-
pendent of time, a paired t-test was performed to test for differences
of condition was observed on arterial plasma FA concentration,
between L-CHO and H-CHO. For variables measured before and after it being slightly higher in L-CHO than in H-CHO (P ⬍ 0.01).
exercise as well as variables measured before and during exercise, a The arterial blood glucose concentration at rest was 4.9 ⫾
two-way analysis of variance (ANOVA), with repeated measures for 0.2 and 5.1 ⫾ 0.1 mM in L-CHO and H-CHO, respectively
the time and protocol factors, was performed to test for differences (NS). During exercise, it was kept nearly constant because of
between protocols or changes due to time. When a significant main the intravenous glucose infusion.
effect of time was found, significant pairwise differences were per- Circulating hormones. At rest, the arterial plasma insulin
formed using Tukey’s post hoc test. Unless otherwise stated, a concentration was 6.6 ⫾ 1.1 and 6.5 ⫾ 0.6 ␮U/ml in L-CHO
probability of 0.05 was used as the level of significance. and H-CHO, respectively (NS). In both conditions, the arterial
plasma insulin concentration decreased (P ⬍ 0.001) continu-
RESULTS ously during exercise to 3.4 ⫾ 0.2 and 4.2 ⫾ 0.3 ␮U/ml at 60
min of exercise in L-CHO and H-CHO, respectively. The
Workload. During the 60-min bicycle exercise test, subjects
exercised at 180 ⫾ 7 W in both conditions. In L-CHO the
systemic V̇O2 averaged 2.8 ⫾ 0.1 l/min during exercise,
whereas in H-CHO the systemic V̇O2 averaged 2.5 ⫾ 0.1 l/min
during exercise (P ⬍ 0.001). The %V̇O2 peak during the bicycle
exercise test averaged 68 ⫾ 1 and 62 ⫾ 1% in L-CHO and
H-CHO, respectively (P ⬍ 0.001).
Intravenous glucose infusion. The glucose infusion rate
during exercise averaged 39 ⫾ 5 and 15 ⫾ 6 ␮mol䡠kg body
mass⫺1 䡠min⫺1 in L-CHO and H-CHO, respectively (P ⬍ 0.01).
Cardiovascular parameters. Heart rate was 62 ⫾ 2 and 56 ⫾
2 beats/min (bpm) at rest in L-CHO and H-CHO, respectively
(P ⬍ 0.05), and increased (P ⬍ 0.001) at initiation of exercise
to a plateau level averaging 163 ⫾ 5 and 152 ⫾ 6 bpm during
exercise in L-CHO and H-CHO (P ⬍ 0.001). At rest, femoral
venous blood flow was 0.51 ⫾ 0.09 and 0.41 ⫾ 0.02 l/min in
L-CHO and H-CHO, respectively [not significant (NS)]. At Fig. 2. Leg fat and carbohydrate (CHO) oxidation rates at rest and during 60
onset of exercise, femoral venous blood flow increased (P ⬍ min of bicycle exercise at 65% peak O2 consumption (V̇O2 peak) with L-CHO
or H-CHO. *Main effect of condition on CHO oxidation, P ⬍ 0.01. #Fat
0.001) to 6.5 ⫾ 0.4 and 6.1 ⫾ 0.4 l/min in L-CHO and H-CHO, oxidation different from L-CHO, P ⬍ 0.001. †CHO oxidation different from
respectively (NS), and remained at this level throughout exer- exercise in both conditions, P ⬍ 0.001. ‡Fat oxidation different from exercise
cise. in L-CHO (P ⬍ 0.001) and H-CHO (P ⬍ 0.05).

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MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION E137
arterial plasma insulin concentration was lower in L-CHO than Table 1. ␣-AMPK Thr172 phosphorylation and activity
in H-CHO at 20, 30, 50, and 60 min of exercise (P ⬍ 0.05).
Rest 60 min of Exercise
At rest, the arterial plasma epinephrine concentration was
0.60 ⫾ 0.10 and 0.54 ⫾ 0.08 nM in L-CHO and H-CHO, L-CHO H-CHO L-CHO H-CHO
respectively (NS). At initiation of exercise, it increased (P ⬍ ␣-AMPK Thr 172
0.001) to 1.79 ⫾ 0.19 and 1.54 ⫾ 0.19 nM at 10 min in L-CHO phosphorylation, AU 8⫾1 6⫾1 33⫾11*† 15⫾4
and H-CHO, respectively, whereafter a further continuous ␣1-AMPK activity, pmol䡠mg
increase (P ⬍ 0.001) occurred to 2.92 ⫾ 0.49 and 2.05 ⫾ 0.31 prot⫺1䡠min⫺1‡ 2.7⫾0.4 1.7⫾0.1 3.4⫾1.0 2.6⫾0.4
nM at 60 min of exercise in L-CHO and H-CHO, respectively. ␣2-AMPK activity, pmol䡠mg
prot⫺1䡠min⫺1 1.5⫾0.1 1.4⫾0.1 5.1⫾1.1*† 3.1⫾0.6§
During exercise, the arterial plasma epinephrine concentration
did not differ significantly between L-CHO and H-CHO. Values are means ⫾ SE. ␣-AMPK Thr phosphorylation and activity in
172

The arterial plasma norepinephrine concentration at rest was the vastus lateralis muscle before and at 60 min of bicycle exercise at 65%
peak O2 consumption with low (L-CHO) or high (H-CHO) preexercise muscle
1.22 ⫾ 0.29 and 1.49 ⫾ 0.29 nM in L-CHO and H-CHO, glycogen. AMPK, AMP-activated protein kinase; CHO, carbohydrate; AU,
respectively (NS). At onset of exercise, it increased (P ⬍ arbitrary units; prot, protein. *Different from H-CHO, P ⬍ 0.01. †Different
0.001) and averaged 14.88 ⫾ 1.39 and 11.04 ⫾ 0.87 nM in from rest, P ⬍ 0.01. ‡Borderline-significant main effect of exercise, P ⫽ 0.06.
L-CHO and H-CHO, respectively, during exercise. The arterial §Borderline significantly different from rest, P ⫽ 0.06.
plasma norepinephrine concentration was higher in L-CHO
than in H-CHO at 10 min (P ⬍ 0.05) and at 20, 40, 50, and 60
min (P ⬍ 0.01). ␣1-AMPK activity in the vastus lateralis muscle did not
Muscle glycogen. The glycogen concentration in the vastus differ significantly between conditions at rest or at 60 min of
lateralis muscle immediately after the glycogen depletion bout exercise (Table 1). A borderline-significant main effect of
was 35 ⫾ 10 and 84 ⫾ 42 mmol/kg dry wt in L-CHO and exercise was observed in ␣1-AMPK activity, which increased
H-CHO, respectively (NS) (Fig. 3). After the following dietary from rest to 60 min of exercise (P ⫽ 0.06).
manipulation and immediately before the 60-min bicycle ex- ␣2-AMPK activity was similar between L-CHO and H-CHO
ercise test, the glycogen concentration had increased (P ⬍ at rest (NS) (Table 1). In L-CHO a 238% increase (P ⬍ 0.01)
0.001) to 197 ⫾ 21 and 504 ⫾ 25 mmol/kg dry wt in L-CHO occurred from rest to 60 min of exercise, whereas the exercise-
and H-CHO, respectively. During the first 30 min of exercise, induced 119% increase in ␣2-AMPK activity in H-CHO only
a significant decrease (P ⬍ 0.01) was observed in the muscle reached borderline statistical significance (P ⫽ 0.06). At 60
glycogen concentration in both conditions, but the rate of min of exercise, ␣2-AMPK activity was 62% higher in L-CHO
glycogen breakdown was 46% higher in H-CHO than in than in H-CHO (P ⬍ 0.01).
L-CHO (P ⬍ 0.05). From 30 to 60 min of exercise, the ACC␤ Ser221 phosphorylation did not differ significantly
glycogen concentration decreased further (P ⬍ 0.001) in H- between L-CHO and H-CHO at rest or at 60 min of exercise,
CHO but not in L-CHO (NS). At rest and at 30 and 60 min of but it increased (P ⬍ 0.001) approximately sixfold from rest to
exercise, the glycogen concentration in the vastus lateralis exercise irrespective of condition (Fig. 4A).
muscle was lower in L-CHO than in H-CHO (P ⬍ 0.001). Malonyl-CoA. At rest the malonyl-CoA concentration in the
AMPK and ACC. ␣-AMPK Thr172 phosphorylation in the vastus lateralis muscle did not differ significantly between
vastus lateralis muscle at rest did not differ significantly be- L-CHO (0.32 ⫾ 0.03 ␮mol/kg wet wt) and H-CHO (0.30 ⫾
tween H-CHO and L-CHO (Table 1). From rest to 60 min of 0.03 ␮mol/kg wet wt) (Fig. 4B). From rest to 30 min of
exercise, a 306% increase (P ⬍ 0.01) occurred in ␣-AMPK exercise, there was a 13% decrease (P ⬍ 0.05) in the muscle
Thr172 phosphorylation in L-CHO, whereas no significant malonyl-CoA concentration irrespective of condition. The
change was observed in H-CHO. At 60 min of exercise, lower malonyl-CoA concentration persisted throughout exer-
␣-AMPK Thr172 phosphorylation was 126% higher (P ⬍ 0.01) cise (0 vs. 60 min, P ⬍ 0.05). During exercise there was no
in L-CHO than in H-CHO. significant effect of condition on the muscle malonyl-CoA
concentration.
PDH. At rest PDHa activity was lower in L-CHO (0.17 ⫾
0.06 mmol䡠min⫺1 䡠kg wet wt⫺1) than in H-CHO (0.52 ⫾ 0.10
mmol䡠min⫺1 䡠kg wet wt⫺1) (P ⬍ 0.01) (Fig. 5). In both
conditions, PDHa activity was higher after 60 min of exercise
than at rest (P ⬍ 0.001). After 60 min of exercise, PDHa
activity was still lower in L-CHO (0.86 ⫾ 0.12 mmol䡠min⫺1 䡠
kg wet wt⫺1) than in H-CHO (1.48 ⫾ 0.21 mmol䡠min⫺1 䡠kg
wet wt⫺1) (P ⬍ 0.01).
Acetyl-CoA. The resting acetyl-CoA concentration in the
vastus lateralis muscle did not differ significantly between
L-CHO (18.8 ⫾ 2.6 ␮mol/kg dry wt) and H-CHO (13.5 ⫾ 3.0
␮mol/kg dry wt) (Fig. 6A). In L-CHO it was ⬃35% lower (P ⬍
0.05) during exercise than at rest, whereas in H-CHO it was
Fig. 3. Glycogen concentration in the vastus lateralis muscle after glycogen ⬃70% higher at 30 min (P ⬍ 0.001) and at 60 min (P ⬍ 0.01)
depletion and before and at 30 and 60 min of bicycle exercise at 65% V̇O2 peak.
d.w., Dry weight. *Different from L-CHO, P ⬍ 0.001. #Different from rest and of exercise than at rest. The muscle acetyl-CoA concentration
30 and 60 min, P ⬍ 0.001. Different from rest, †P ⬍ 0.01 and ††P ⬍ 0.001. was lower in L-CHO than in H-CHO at 30 min (49%, P ⬍
‡Different from 30 min, P ⬍ 0.001. 0.001) and 60 min (45%, P ⬍ 0.01) of exercise.
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E138 MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION

tion rates. The latter was accomplished by manipulating the


muscle glycogen content to high (H-CHO) or low (L-CHO)
levels before a 60-min bicycle exercise bout at 65% V̇O2 peak.
It was previously suggested that low preexercise muscle
glycogen content might be associated with high AMPK activity
in rodents during muscle contraction in vitro (8) and in humans
during exercise (44). However, in the latter study, circulating
epinephrine was also dependent on muscle glycogen content
(44), wherefore the possible effect of muscle glycogen on
␣2-AMPK activity may have been confounded by adrenergic
stimulation of ␣2-AMPK. Adrenergic stimulation of AMPK
has been demonstrated in 3T3-L1 adipocytes (47) and has also
been reported for rat skeletal muscle (19). In the present study,
circulating epinephrine did not differ significantly between
conditions, and, still, ␣2-AMPK activity during exercise was
markedly higher in L-CHO than in H-CHO (Table 1). This
suggests that altered muscle glycogen alone can influence
␣2-AMPK activity in human skeletal muscle during exercise
independently of adrenergic stimulation, in agreement with
studies in rodent muscle (8). On this basis, AMPK could be a
likely candidate mediating the effect of muscle glycogen on fat
oxidation through regulation of ACC activity and malonyl-
CoA formation, as suggested earlier (34, 41). However, the
similar ACC␤ Ser221 phosphorylation and malonyl-CoA con-
centration between L-CHO and H-CHO in the present study
(Fig. 4) suggest that the effect of muscle glycogen on fat
oxidation occurs primarily by other mechanisms in human
Fig. 4. Acetyl-CoA carboxylase (ACC) phosphorylation and malonyl-CoA skeletal muscle during exercise.
concentration in the vastus lateralis muscle at rest and during 60 min of bicycle AMPK Thr172 phosphorylation closely paralleled ␣2-AMPK
exercise at 65% V̇O2 peak with L-CHO or H-CHO. A: ACC␤ Ser221 phosphor-
ylation. Arb units, arbitrary units. #Main effect of exercise, P ⬍ 0.001. B: activity in the present study (Table 1), indicating that the
malonyl-CoA concentration. w.w., Wet weight. †Different from rest, P ⬍ 0.05. markedly higher ␣2-AMPK activity during exercise in L-CHO
than in H-CHO was due to a higher stimulatory effect on
AMPK by an upstream AMPK kinase that phosphorylated
Carnitine and acetylcarnitine. At rest, the acetylcarnitine ␣2-AMPK on Thr172. However, altered muscle glycogen did
concentration in the vastus lateralis muscle was higher in not influence the large increase in ACC␤ Ser221 phosphoryla-
L-CHO (8.1 ⫾ 0.9 mmol/kg dry wt) than in H-CHO (4.4 ⫾ 1.0 tion seen with exercise (Fig. 4A), suggesting that allosteric
mmol/kg dry wt) (P ⬍ 0.01) (Fig. 6B). In L-CHO it did not regulation of AMPK, which is not detected in the AMPK
change significantly from rest to exercise, whereas it increased activity assay, may have overridden the covalent regulation of
(P ⬍ 0.001) ⬃138% in H-CHO. At 30 and 60 min of exercise, AMPK by an upstream AMPK kinase. This is in contrast to a
the muscle acetylcarnitine concentration was ⬃37% lower in previous study in which ␣2-AMPK activity and ACC␤ Ser221
L-CHO than in H-CHO (P ⬍ 0.01). phosphorylation were closely associated during exercise with
The free carnitine concentration in the vastus lateralis mus- low vs. high muscle glycogen (44). The major difference
cle at rest was lower in L-CHO (16.1 ⫾ 1.4 mmol/kg dry wt) between that study (44) and the present study was that, in the
than in H-CHO (20.2 ⫾ 1.3 mmol/kg dry wt) (P ⬍ 0.01) (Fig. present study, subjects ingested a preexercise meal and had
6C). In L-CHO it did not change significantly from rest to
exercise, whereas it decreased (P ⬍ 0.001) ⬃46% in H-CHO.
The muscle free carnitine concentration was higher in L-CHO
than in H-CHO at 30 min (55%, P ⬍ 0.001) and 60 min (43%,
P ⬍ 0.01) of exercise.
DISCUSSION

The intracellular mechanisms regulating the ratio between


fat and carbohydrate oxidation in human skeletal muscle dur-
ing exercise have not yet been fully clarified. A likely site of
regulation of fat oxidation is CPT-1 at the entry of LCFA-CoA
into mitochondria. The present study is the first to simulta-
neously measure several factors related to the possible role of
malonyl-CoA and carnitine in regulation of fat oxidation at
Fig. 5. Pyruvate dehydrogenase activity in the active form (PDHa) in the
CPT-1 in humans during exercise. Exercise was performed vastus lateralis muscle at rest and at 60 min of bicycle exercise at 65% V̇O2 peak
under two conditions at comparable work loads but with with L-CHO or H-CHO. *Main effect of condition (L-CHO vs. H-CHO), P ⬍
marked differences in the relative fat and carbohydrate oxida- 0.01. #Main effect of exercise, P ⬍ 0.001.

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MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION E139
as spatial dissociation of AMPK and ACC or phosphatase
activity acting on ACC␤ Ser221. Those factors may in some
cases render ACC␤ Ser221 phosphorylation a not-so-optimal
predictor of in vivo ␣2-AMPK activity. Altogether, the present
study suggests that during exercise, covalent regulation of
␣2-AMPK on Thr172 is glycogen dependent, but that ACC␤
Ser221 phosphorylation does not parallel ␣2-AMPK activity
when circulating glucose and epinephrine levels are standard-
ized. This is supported by findings in isolated contracting
rodent skeletal muscle with high and low muscle glycogen
levels, where differences in ␣2-AMPK activity did not translate
into differences in ACC activity (8).
Despite markedly different ␣2-AMPK activity during exer-
cise between H-CHO and L-CHO (Table 1), ACC␤ Ser221
phosphorylation and malonyl-CoA concentration did not differ
significantly between L-CHO and H-CHO (Fig. 4). Still, the fat
oxidation rate was higher during exercise with low muscle
glycogen even though 160% more glucose was infused intra-
venously under this condition. This suggests that the AMPK-
ACC-malonyl-CoA pathway is not likely to mediate the regu-
lation of fat oxidation by muscle glycogen availability in
human skeletal muscle during prolonged exercise. This con-
clusion is also supported by other human exercise models
where no association between malonyl-CoA content and fat
oxidation rate was obtained in skeletal muscle during pro-
longed moderate-intensity exercise (17) or during graded-
intensity exercise (7, 18). However, compartmentalization of
malonyl-CoA may occur in skeletal muscle, i.e., concentrations
of malonyl-CoA in the vicinity of CPT-1 may vary without any
detectable changes in the measurement of total muscle malo-
nyl-CoA concentration. In resting human skeletal muscle,
changes in malonyl-CoA concentration have occurred with
opposite changes in fat oxidation. Thus a significant negative
correlation was obtained between muscle malonyl-CoA con-
tent and fat oxidation rate in healthy middle-aged men during
a two-step euglycemic-hyperinsulinemic clamp (3). Further-
more, in healthy, young individuals, the inhibition of fat
oxidation induced by hyperglycemia with hyperinsulinemia
occurred together with a threefold increase in muscle malonyl-
CoA concentration (27). These results suggest that, in resting
human skeletal muscle, the downregulation of fat oxidation by
Fig. 6. Acetyl-CoA, acetylcarnitine, and carnitine concentrations in the vastus insulin may be mediated by malonyl-CoA.
lateralis muscle at rest and at 30 and 60 min of bicycle exercise at 65% V̇O2 peak
with L-CHO or H-CHO. A: acetyl-CoA. B: acetylcarnitine. C: carnitine.
The absolute increase in fat oxidation rate that occurred from
Different from L-CHO, *P ⬍ 0.01 and **P ⬍ 0.001. Different from rest, #P ⬍ rest to exercise in both conditions in the present study (Fig. 2)
0.05, †P ⬍ 0.01, and ‡P ⬍ 0.001. was associated with increased ACC␤ Ser221 phosphorylation
and decreased malonyl-CoA concentration (Fig. 4). This sug-
gests that inactivation of ACC␤ by AMPK and the consequent
glucose infused intravenously during exercise, which resulted decrease in malonyl-CoA concentration from rest to exercise
in similar blood glucose and epinephrine levels between con- may have relieved malonyl-CoA inhibition of fat oxidation in
ditions. In the previous study, blood glucose and plasma both conditions (Fig. 7). There was a tendency (P ⫽ 0.10) for
epinephrine concentrations differed markedly between condi- malonyl-CoA to decrease more from rest to exercise in L-CHO
tions (44). It follows that, in the present study, the different than in H-CHO. This could have been due to lower availability
rates of intravenous glucose infusion in H-CHO and L-CHO of cytosolic acetyl-CoA, the precursor of malonyl-CoA, in
leading to similar levels of circulating glucose and epinephrine L-CHO than in H-CHO. Alternatively, the activity of MCD,
between H-CHO and L-CHO may have contributed to the the enzyme catalyzing the turnover of malonyl-CoA, may have
dissociation between measured ␣2-AMPK activity and ACC␤ differed between the two conditions. It has been suggested that
Ser221 phosphorylation. If so, the in vivo ␣2-AMPK activity in AMPK phosphorylates and activates MCD during muscle con-
the present study may have been better reflected by the ACC␤ traction (19, 34). Consequently, the higher ␣2-AMPK activity
Ser221 phosphorylation state than by the in vitro ␣2-AMPK in L-CHO than in H-CHO might have induced higher malonyl-
activity. Alternatively, ACC␤ Ser221 phosphorylation may CoA turnover in L-CHO via an effect on MCD. The tendency
have been affected by factors other than AMPK activity, such toward a more pronounced decrease in muscle malonyl-CoA
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E140 MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION

Fig. 7. Metabolic events that may determine the fat oxidation rate in response to muscle glycogen availability in human skeletal muscle during exercise. A:
individuals with high muscle glycogen. Exercise activates glycogenolysis, glycolysis, and PDH, leading to markedly increased mitochondrial generation of
acetyl-CoA and, secondarily, acetylcarnitine. The latter results in a decrease in mitochondrial free carnitine and, consequently, cytosolic carnitine. Whole tissue
malonyl-CoA levels are reduced by exercise due to increased phosphorylation (inactivation) of ACC by AMP-activated protein kinase (AMPK), which is
activated moderately by exercise. The combination of reduced free carnitine content and reduced malonyl-CoA content leads to a moderate increase in fat
oxidation rate from rest to exercise. B: individuals with low muscle glycogen. The increases in glycogenolytic and glycolytic rates and PDH activity by exercise
are less than in individuals with high glycogen. Consequently, mitochondrial acetyl-CoA and acetylcarnitine generation appears to be lower, and no decrease in
whole tissue free carnitine content occurs from rest to exercise. Whole tissue malonyl-CoA levels are reduced by exercise due to increased phosphorylation
(inactivation) of ACC by AMPK, which is activated markedly by exercise. The reduction in malonyl-CoA may be slightly more pronounced compared with
high-glycogen individuals because of the lower concentration of acetyl-CoA, the source from which malonyl-CoA is formed by ACC. The combination of
unchanged free carnitine content and reduced malonyl-CoA content leads to a marked increase in fat oxidation rate from rest to exercise. Muscle concentration
of pyruvate was not measured in the present study but was previously shown to increase more from rest to exercise with high than with low muscle glycogen
(25). The muscle concentration of citrate, the source of the acetyl-CoA from which malonyl-CoA is generated (35), was not measured in the present study but
was previously shown not to differ during exercise with high and low muscle glycogen, although an increase with exercise occurred (11). AcCoA, acetyl-CoA;
MaCoA, malonyl-CoA; FACoA, fatty acyl-CoA; CAT, carnitine acetyltransferase; CPT, carnitine palmitoyltransferase-1/2 system; AMPK, 5⬘-AMP-activated
protein kinase; CL, citrate lyase; ⫹, activating effect; ⫼, inhibiting effect.

by exercise in L-CHO than in H-CHO may have contributed to carnitine is ⬃0.5 mM at pH 7.4 in human skeletal muscle (14).
the difference between conditions in fat oxidation rate during Nevertheless, partitioning of free carnitine between the cytosol
exercise (Fig. 7). However, the increase in absolute fat oxida- and the mitochondrial matrix makes it very difficult to estimate
tion rate by exercise was much more marked in L-CHO than in the absolute carnitine concentration in the vicinity of CPT-1.
H-CHO. Therefore, factors other than malonyl-CoA seem to be Furthermore, a drop in muscle pH during high-intensity exer-
more important in “fine tuning” the fat oxidation rate during cise (2, 39) leads to an increase in the Km of CPT-1 for
prolonged exercise with different muscle glycogen stores. carnitine (16). Thus carnitine may still be a potent regulator of
Availability of carnitine, a necessary substrate for CPT-1, CPT-1 during exercise. In support, the skeletal muscle free
could be one such factor. The importance of carnitine in fat carnitine concentration as well as the fat oxidation rate was
oxidation is evidenced by the fact that, in the pathology of lipid lower during prolonged moderate-intensity exercise in H-CHO
storage myopathy, a marked reduction in skeletal muscle than in L-CHO in the present study (Figs. 2 and 6C). Also, in
carnitine content by 85% was associated with a 75% reduction several previous human studies, similar alterations in muscle
in palmitate and oleate oxidation in rectus femoris muscle free carnitine content and fat oxidation rate occurred in parallel
homogenates (9). On the other hand, the usual fluctuations in during exercise. Thus muscle carnitine content and fat oxida-
free carnitine content in skeletal muscle of healthy humans tion rate increased to a similar extent during graded-intensity
between 1 and 4 mM are not expected to influence CPT-1 exercise (5, 40). During bicycle exercise at 75% V̇O2 peak to
activity, since it has been reported that the Km of CPT-1 for exhaustion with either high or low muscle glycogen, both
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MUSCLE MALONYL-COA, CARNITINE, AND FAT OXIDATION E141
muscle carnitine content and fat oxidation rate were markedly antibodies. We are grateful to Dr. Henriette Pilegaard, University of Copen-
higher with low muscle glycogen (25). Taken together, the hagen, Denmark, for help on the PDHa assay. We acknowledge the skilled
technical assistance of Irene Bech Nielsen, Betina Bolmgren, and Winnie
present and previous studies suggest a relation between muscle Taagerup.
free carnitine availability and fat oxidation rate in human
skeletal muscle during exercise. GRANTS
Protein oxidation was not measured in the present study, This study was supported by The Danish National Research Foundation
and, consequently, absolute rates of fat and carbohydrate oxi- (grant no. 504-14), The Copenhagen Muscle Research Centre, The Novo
dation across the leg were calculated using the nonprotein Nordisk Research Foundation, The Danish Diabetes Association, a Research
respiratory quotient (20). Protein oxidation may have differed and Technological Development Project (QLG1-CT-2001-01488) funded by
the European Commission, The Danish Sports Research Council, The Danish
between L-CHO and H-CHO and, in that case, would have Medical Research Council, and a Hallas Møller Fellowship from The Novo
slightly confounded the fat and carbohydrate oxidation rates Nordisk Foundation (J. F. P. Wojtaszewski).
reported in Fig. 2. However, during moderate-intensity exer-
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