Increased muscle carnitine
after increased contractile
palmitoyltransferase
activity
II mRNA
ZHEN YAN, STANLEY
SALMONS,
JONATHAN
JARVIS, AND FRANK W. BOOTH
Department
of Physiology
and Cell Biology, University
of Texas-Houston
Health Science Center,
Houston,
Texas 77030; and Department
of Human Anatomy and Cell Biology, The University
of Liverpool, Liverpool L69 3BX, United Kingdom
Yan, Zhen, Stanley
Salmons,
Jonathan
Jarvis,
and
Frank W. Booth. Increased muscle carnitine palmitoyltransferase II mRNA after increased contractile
activity. Am. J.
Physiol. 268 (Endocrinol.
Metab. 31): E277-E281,
1995.The capacity of skeletal muscle to oxidize fatty acids increases
with endurance
training.
The oxidation
of long-chain
fatty
acids occurs in mitochondria
and is initiated by a carnitinedependent transport
step in which three enzymes help fatty
acyl groups enter the matrix compartment.
The purpose of
this study was to determine whether pretranslational
regulation of one of these three enzymes, carnitine palmitoyltransferase II (CPT II), as estimated from the level of CPT II mRNA,
plays a role in the doubling of CPT activity in skeletal muscle
of rats subjected to daily 2-h bouts of running on treadmills (P.
A. Mole, L. B. Oscai, and J. 0. Holloszy. J. Clin. Invest. 50:
2323-2330,
1971). After 100 min/day of running
on motordriven treadmills
for 2 wk, CPT II mRNA in the plantaris
muscle was unchanged when normalized
per unit of extracted
RNA but was 50% higher (P < 0.05) over sedentary controls
when normalized
per unit of muscle wet weight. To test
whether
additional
contractile
activity would make CPT II
mRNA even higher, continuous
indirect electrical stimulation
was imposed on the tibialis anterior muscles. After 9 days of
chronic stimulation,
CPT II mRNA was 63, 221, and 137%
greater than control (P < 0.001) when normalized
to extracted
RNA, muscle wet weight, and whole muscle, respectively,
compared with the muscle in the control rats. These data
indicate that pretranslational
regulation
of CPT II occurs in
response to increased contractile activity in skeletal muscle.
free fatty acid oxidation;
skeletal muscle; messenger ribonucleic acid; pretranslational
regulation;
mitochondria;
treadmill running; chronic stimulation
BECOMES
A MORE
important source of energy for
submaximal exercise after endurance training (4). For
example, a two-legged cycling test revealed a more
pronounced metabolism of fatty acids in the trained
than in the nontrained leg during submaximal exercise
in subjects who had trained with a one-leg bicycle
ergometer (7). Henriksson (7) suggested that, during
submaximal exercise, the trained leg oxidized more fatty
acid because of an increased muscle oxidative capacity,
since blood flow was similar in the two legs. Saltin and
Astrand (24) indicated the complexity of pinpointing the
rate-limiting step that produces increased fatty oxidation as a result of endurance training. They suggested
that any or all of the following reported adaptations,
which occur in trained skeletal muscle, could account for
an enhanced oxidation of fatty acids by the trained
skeletal muscle: 1) an increase in the activities of
enzymes of the citric acid cycle, fatty acid oxidation, and
electron transport; 2) an elevation of carnitine and of
FAT
0193~1849/95
$3.00
Copyright
o 1995
carnitine transporters for fatty acids; 3) an increase in
the concentration of fatty acid-binding proteins that
transfer fatty acids through the cytoplasm; and 4) a
decrease in the fiber volume supplied by capillaries.
Some of the processes related to these steps do not show
changes in mRNA. For example, 7 days of treadmill
running did not alter the level of acyl-CoA synthetase
mRNA and lipoprotein lipase mRNA in the gastrocnemius muscle of rats (27). On the other hand, lipoprotein
lipase activity was increased in the white and red
portions of the vastus lateralis muscle immediately after
an acute bout of swimming (13). To our knowledge, little
other information is available on the response of mRNAs
for proteins involved in fatty oxidation in skeletal muscle
after training.
The mitochondrial oxidation of long-chain fatty acids
is initiated by a carnitine-dependent
transport step
whereby fatty acyl groups gain entry to the matrix
compartment by the actions of three enzymes: carnitine
palmitoyltransferase
I and II (CPT I and CPT II,
respectively) and carnitine-acylcarnitine translocase (18).
CPT II is located in the inner membrane of mitochondria and splits acylcarnitine into acyl-CoA and carnitine.
Chronic electrical stimulation has been found to result
in a threefold increase in the consumption of fatty acids
by contracting muscles (10) and increase the levels of
mRNA encoding several enzymes in the citric acid cycle
and electron transport chain (22), but little information
is available on the response of mRNAs of proteins
involved in fatty acid oxidation. The model of chronic
stimulation can provide information as to the extent of
muscle plasticity (22). The aim of this study was to
determine whether increased contractile activity produced by chronic stimulation would increase CPT II
mRNA and to compare the magnitude of such response
to that induced by treadmill training.
MATERIALS
AND
METHODS
Animals.
Female Sprague-Dawley
rats (Harlan, 100-120 g
and 125-149 g body wt for the treadmill running and chronic
stimulation
studies, respectively) were housed in temperaturecontrolled
quarters
(21°C) with a 12:12-h light-dark
cycle.
Animals were provided with water and chow (Harlan Teklab)
ad libitum.
Experimental
protocols were approved by the
University of Texas Health Science Center at Houston Institutional Animal Welfare Committee. Where indicated, anesthesia [a mixture of ketamine (54 mg/ml), xylazine (2.2 mg/ml),
and acepromazine
(3.5 mg/ml)]
was injected into the right
gluteus muscle (1.4 ml/kg).
Seventeen rats in this study were
Treadmill
running.
preacclimatized
to treadmill
running
by performing
lo-min
daily runs for 3 wk on an 8” inclined motor-driven
treadmill.
The treadmill speed was gradually increased from 0.07 m/s at
the American
Physiological
Society
E277
E278
TRAINING
INCREASES
the start of preacclimatization
to 0.45 m/s at the end of
preacclimatization.
Nine rats that avoided the rear of the
running
compartment
the most were selected for endurance
training,
which consisted of running
100 min/day
(in 3
separate sessions of 33.3 min) at a speed of 0.36-0.45
m/s on
an 8” incline for 14 days. The remaining
eight rats were
assigned to the control group and remained
in their cages
during the endurance
training.
At the end of the 14-day
training
period, plantaris muscles were taken from anesthetized rats.
Chronic electrical stimulation.
Electrodes and devices were
implanted into five rats for chronic stimulation
as described by
Mayne et al. (17). The deep peroneal nerve of the left hindlimb
was exposed from the lateral side in anesthetized
rats under
aseptic conditions. A battery-powered
implantable
stimulator
(23) was implanted in the abdominal
cavity, and two stainless
steel electrodes were secured N 0.5 cm apart with sutures
under the deep peroneal nerve, which supplies motor fibers to
the muscle in the anterior
compartment
of the hindlimb
including the tibialis anterior muscle. The same procedure was
performed
for the right hindlimb
except that the electrodes
were not connected to a stimulator.
Stimulation
was commenced 2 days later. Rectangular
pulses (0.2 ms) were applied
continuously
at 10 Hz for 24 h/day. Tibialis anterior muscles
were dissected from anesthetized
rats at the end of 9 days of
stimulation.
RNA isolation.
Designated
muscles were dissected and
quickly frozen with liquid-nitrogen-cooled
tongs and stored at
-80°C. Before being homogenized,
N 100 mg (wet wt) muscle
was powdered in a mortar containing
enough liquid nitrogen
to prevent the sample from thawing.
RNA was extracted
(extracted RNA) by the RNAzol (Biotecx Laboratories,
Houston, TX) protocol (3). After homogenization
in 3.5 ml RNAzol
B, ice-cold chloroform
(3.5 ml) was added to the homogenate,
and the mixture was kept on ice for 15 min. After centrifugation at 12,000 g for 15 min at 4”C, the extracted RNA was
precipitated
with isopropanol,
and the RNA concentrations
were determined
spectrophotometrically
at 260 nm wavelength.
Riboprobe
generation.
The CPT II cDNA cloned in pBSIIKS+ (Stratagene)
was generously
supplied by Dr. J. D.
McGarry
(31). It was digested with Nde I and used as a
template to synthesize antisense RNA. A 448nucleotide
antisense RNA was synthesized with an in vitro transcription
kit
(Promega)
labeled with [32P]CTP (>400
Ci/mmol;
Amer-
CPT
II MRNA
sham). The specific radioactivity
of the riboprobe was 108-log
counts . min l (cpm) Fg DNA?
Northern blot anaZysis. Extracted RNA (20 kg) was electrophoresed in formaldehyde-agarose
gels and transferred
to
nylon membranes by capillary blotting (25). Ethidium
bromide
staining of gels indicated equivalent amounts of 18s and 28s
rRNA in each lane. RNA was immobilized
by ultraviolet
cross-linking
(Stratolinker),
after which the membranes were
prehybridized
at 60°C for 15 min in hybridization
buffer
containing
6~ SSPE (pH 7.0), 50% formamide,
5 x Denhart’s
solution, 0.5% sodium dodecyl sulfate, and 0.1 mg/ml denatured salmon sperm DNA. Hybridizations
were performed at
60°C for 12-14 h in the hybridization
buffer with the addition
of lo6 cpm/ml [32P]CPT II riboprobe. A specific band appeared
at the 2580-nucleotide
position upon autoradiography,
established with 18s and 28s rRNA markers. Esser et al. (6) have
reported that CPT II mRNA contains - 2,500 bases.
Total RNA quantification.
To estimate
CPT II mRNA
concentration
per gram of wet weight in muscles, total RNA
was determined
according
to Munro
and Fleck (2 1). The
concentration
of CPT II mRNA per gram of wet weight was
calculated
by multiplying
CPT II mRNA per microgram
extracted RNA by total RNA per milligram
wet weight (2).
CPT II mRNA per whole muscle was calculated by multiplying
CPT II mRNA per milligram
wet weight by muscle wet weight.
Statistics. An unpaired
Student’s t-test was employed for
comparisons
between treadmill
trained and control plantaris
muscles. A one-way analysis of variance was performed
for
comparisons
among control, contralateral
control, and stimulated tibialis anterior
muscles in the chronic stimulation
study. When significant
differences among groups were detected, a Newman-Keuls
test was performed
to determine
differences among experimental
groups. P < 0.05 was accepted as significant for all statistical analyses.
l
RESULTS
After 14 days of treadmill running, CPT II mRNA per
unit of wet weight in the plantaris muscles was increased 50% (P < 0.05; Table 1; Fig. 1). No significant
difference in the percentage of CPT II mRNA per unit of
extracted RNA was noted.
In comparisons between the tibialis anterior muscle of
the stimulated leg and a separate control rat, CPT II
Table 1. CPT II mRNA levels in skeletal muscle of rats that have undergone either 14 days of treadmill running
or 9 days of chronic stimulation
Treadmill
Control
CPT II mRNA
IOD/ kg RNA
IOD/mg
wet wt
IOD/muscle
Total RNA, pg/mg
Muscle wt, g
wet wt
Running
(Plantaris)
(n = 15)
Trained
(n = 18)
0.33 + 0.03
0.32 + 0.03
ND
1.00 t 0.05
ND
0.41+ 0.03
0.48 + 0.05’”
ND
1.15 + 0.06’”
ND
Chronic
%Change
vs. control
+25
+50
ND
+15
ND
Control
(72 = 7)
0.40
0.43
190
1.08
0.44
k
+
+
+
+
0.02
0.02
14
0.03
0.02
Stimulation
Contralateral
control
(n = 4)
0.19
0.26
110
1.27
0.42
+
+
+
+
k
0.04-j0.07
32’”
0.07*
0.02
(Tibialis
Anterior)
Stimulated
(n = 5)
0.65
1.38
451
2.112
0.33
t 0.057$
iz O.l4”f$
rf: 27”f$
0.117
* 0.03’“§
%Change
vs. control
+63
+221
+137
+95
-25
Values are means + SE; IZ, no. of rats. ND, not determined.
Control,
set of animals
separate
from treatment
group; contralateral
control,
sham-operated
nonstimulated
limb of treatment
group. Integrated
optical density
(IOD) units were obtained
from image analysis (BioImage;
Millipore)
of -2580-nucleotide
band on autoradiographs
of Northern
blot analysis
of carnitine
palmitoyltransferase
(CPT)
II mRNA.
Normalization
of CPT II mRNA per unit of muscle wet weight was done by multiplying
CPT II mRNA/pg
extracted
RNA by total RNA/mg
muscle wet weight (2). Normalization
of CPT II mRNA per whole muscle was calculated
by multiplying
CPT II mRNA/mg
wet weight by muscle
wet weight. See MATERIALS
AND METHODS
for procedures
to extract
RNA and total RNA. *P < 0.05 from control rats. -f-P < 0.001 from control
rats. $P < 0.001 from contralateral
control muscles. $P < 0.05 from contralateral
control muscles.
TRAINING
Trained
INCREASES
Control
f------m
i
i
28s -
/
18s -
:
I
Fig. 1. Northern
blot analysis of carnitine
palmitoyltransferase
(CPT)
II mRNA
in rat plan&is
muscle after 14 days of treadmill
running
training
(100 miniday).
32P-labeled
antisense
RNA was synthesized
in
vitro from CPT II cDNA and hybridized
to 20 pg extracted
RNA (see
MATERIALS
AND METHODS)
from plantaris
muscles of trained
and control
rats. Positions
of 28s and 18s rRNA
are shown. Relative
integrated
optical density
of specific bands was determined
( - 2580 nucleotides
indicated
by arrow;
see Table 1 for results).
mRNA
tracted
Because
higher
mRNA
was 63% (P < 0.001) greater per unit of exRNA in the stimulated
muscle (Table 1; Fig. 2).
total RNA per gram of wet weight was 51%
(P < 0.01) in the stimulated
muscle, CPT II
per gram of muscle wet weight and per whole
18sFig. 2. Northern
blot analysis of CPT II mRNA in rat tibialis anterior
muscle after 9 days of chronic
nerve stimulation
(10 Hz, 24 h/day).
32P-labeled
antisense
RNA was synthesized
in vitro from CPT II cDNA
and hybridized
to 20 pg extracted
RNA (see MATERIALS
AND METHODS)
from
chronic
stimulated
and sham-operated
contralateral
control
(contra
control)
muscle
and from
unoperated
control
rat tibialis
anterior
muscle.
Relative
integrated
optical density
of specific bands
was determined
( - 2580 nucleotides
indicated
by arrow;
see Table 1
for results).
CPT
II MRNA
E279
muscle were 221 and 137% greater, respectively,
than in
the sedentary control rat (P < 0.001).
Interestingly,
significant changes occurred in muscles
of the nonstimulated
contralateral
leg. Comparisons
of
these muscles with muscles from the unoperated
unstimulated
control group revealed decreases
of 52%
(P < O.OOl), 40% (not significant),
and 42% (P < 0.05)
in CPT II mRNA per unit of extracted RNA, in CPT II
mRNA per unit of muscle wet weight, and in CPT II
mRNA per whole muscle, respectively
(Table 1). Contralateral effects from chronic stimulation
have previously
been observed for alterations
in the compositions
of
myosin protein isoforms and Ca2+-adenosinetriphosphatase (ATPase) isoform mRNAs (see Ref. 15 and references therein).
Leberer et al. (15) indicate that the
reasons for these contralateral
effects during chronic
stimulation
are unknown,
but they speculate that the
effect on the contralateral
side could be caused by reflex
activity from the stimulated
side or by unusual longterm positions of the animal with altered loads on the
contralateral
dorsiflexors.
DISCUSSION
The novel observation
in these studies is that increased contractile
activity was associated with greater
level of CPT II mRNA.
CPT II is one of the three
enzymes involved in the transfer
of long-chain
fatty
acids from the cytoplasm to the mitochondrial
matrix. A
pretranslational
response of these enzymes to increased
contractile
activity
in skeletal muscle has not been
reported previously.
The observation
of higher CPT II
mRNA suggests an increase in CPT II transcription,
an
increase in CPT II mRNA processing,
an increase in
CPT II mRNA stability,
or any combination
of these
three possibilities.
Previously,
Mole et al. (19) reported that the capacity
of gastrocnemius
and quadriceps muscles for oxidizing
palmitate, oleate, linoleate, palmityl CoA, and palmityl
carnitine doubled in rats that had undergone a 12-wk
program of treadmill running in which the rats were
running for 2 h/day at the end of training. Two important consequences
of a training-induced
increase in the
capacity for oxidizing
fatty acids may be improved
submaximal
exercise endurance and body fat reduction.
Aerobic training
increases
mitochondrial
density in
skeletal muscles, which then oxidize more fatty acids
during submaximal
low-intensity
exercise; as a result,
the duration of work to exhaustion
is prolonged during
exercise (see Refs. 8 and 24 and references
therein).
Endurance
training affects fatty acid mobilization
and
oxidation independently.
Issekutz
et al. (11) have suggested that supply of fatty acids from adipose tissue is
rate-limiting
for fatty acid utilization.
However,
Kiens
et al. (12) later reported that the trained leg of a subject
has a greater uptake of free fatty acid (FFA) at a given
delivery of FFA than that observed in the contralateral
nontrained
leg, which implies that endurance training
can enhance the process of extraction
of the supplied
fatty acids by the muscles. Thus, as Saltin and Astrand
(24) have stated, it is not yet possible to identify the
relative
role of the various
adaptations
that could
E280
TRAINING
INCREASES
explain the shift toward greater fatty acid oxidation
after training. Nonetheless,
the increase in fatty utilization that results from endurance exercise training has
health benefits. Despres et al. (5) have shown a greater
loss of abdominal fat than midthigh adipose tissue in
obese women after 14 mo of training that consisted of
aerobic exercise for 90 min each day, 4-5 times/wk.
Shimomura et al. (27) have reported a decrease in
mesenteric, but not subcutaneous, fat in rats that run 1
h/day for 7 days. Preferential loss of abdominal fat is
beneficial because a high accumulation of abdominal fat
is associated with an increased risk of coronary heart
disease (14).
The higher level of CPT II mRNA indicates that
contractile activity exerts pretranslational control for
CPT II synthesis. The absolute increase in CPT II
mRNA concentration was about six times higher in
chronically stimulated tibialis anterior muscles than in
run-trained plantaris muscles. Results from various
laboratories have similarly shown that nuclear-encoded
mRNAs undergo a greater percentage increase for mitochondrial proteins in chronic stimulation than run
training. Previous reports have indicated that chronic
stimulation leads to increased levels of mRNAs for
nuclear encoded mitochondrial proteins: P-subunit of
FIATPase mRNA increases by 165% (29), citrate synthase mRNA increases by 240-600%
(1, 26), cytochrome b mRNA increases by 564% (29, 30), and
subunits III and VIc of cytochrome oxidase mRNA
increase by lOO-200% (9,29). The present results show
a 221% higher CPT II mRNA concentration in chronically stimulated muscle. In contrast to chronic electrical
stimulation, most studies based on endurance training
by treadmill running have been unable to demonstrate
an increase in the mRNA of the nuclear-encoded mitochondrial proteins in rat skeletal muscles. Acyl-CoA
synthetase mRNA (27), 5’-aminolevulinate
synthase
mRNA (28), and subunits 111 and W of cytochrome
oxidase mRNA (28) have been found not to increase
after training by running on treadmills. However, a
number of studies do report increases in mRNAs for
mitochondrial proteins with treadmill running. CPT II
mRNA does increase by 50% (Table l), cytochrome c
mRNA increases by 18-56% (20), and cytochrome oxidase subunit III and WmRNAs increase 100% (16) after
treadmill training.
The present study demonstrates a pretranslational
regulation of CPT II mRNA in skeletal muscle by
contractile activity. This mitochondrial protein, which is
encoded by the nuclear genome, has an increased transcription, an increased mRNA processing, or an enhanced stability of mRNA as a result of the increase in
muscle contractile activity induced by treadmill running
and chronic electrical stimulation. Fiber recruitment in
treadmill running is often not of sufficient duration or
intensity to invoke a demonstrable alteration in all
mRNA concentrations for nuclear-encoded mitochondrial proteins. On the basis of the present results, it is
likely that the higher CPT II mRNA plays some role in
the previously reported adaptive increases in fatty acid
oxidation in endurance-trained skeletal muscle (12. 19).
CPT
II MRNA
We thank
Drs. Dennis McGarry
and Jeanie McMillin
for helpful
discussions
and for the carnitine
palmitoyltransferase
II cDNA. We
thank Mei-Hua
Liu for technical
assistance.
This research
was supported
by National
Institute
of Arthritis
and
Musculoskeletal
and Skin Diseases Grant AR-19393.
Present
address
for Z. Yan and S. Salmons:
Dept.
of Human
Anatomy
and Cell Biology,
The University
of Liverpool,
PO Box 147,
Liverpool
L69 3BX, United Kingdom.
Address for reprint
requests:
F. Booth, Dept. of Physiology
and Cell
Biology,
University
of Texas-Houston
Health
Science Center,
6431
Fannin
St., Rm. 4.100 MSB, Houston,
TX 77030.
Received
28 June
1994; accepted
in final
form
20 September
1994.
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