| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Institute of Exercise and Sport Sciences and 2Laboratory of Cellular and Molecular Biology, Copenhagen Muscle Research Centre, August Krogh Institute, University of Copenhagen, 2100 Copenhagen, Denmark
Submitted 12 March 2003 ; accepted in final form 15 May 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-subunit mRNA level
was approximately threefold higher (P < 0.01) at 0, 1, and 3 h
after exercise, relative to the preexercise resting level. After 3-5 h of
recovery in the untrained state, pyruvate dehydrogenase kinase 4 and
hexokinase II mRNA levels were elevated 13-fold (P < 0.001) and
6-fold (P < 0.01), respectively. However, after the training
period, only pyruvate dehydrogenase kinase 4 mRNA levels were elevated
(P < 0.05) during the recovery period. No changes in resting mRNA
levels were observed as a result of training. In conclusion, cellular
adaptations to high-intensity exercise training may, in part, be induced by
transcriptional regulation. After training, the transcriptional response to an
exercise bout at a given workload is diminished. sodium-potassium-adenosine 5'-triphosphatase; lactate/hydrogen ion transport; messenger ribonucleic acid; regulation of substrate utilization; glycolysis
High-intensity exercise demands a high-glycolytic flux, and a typical adaptation to high-intensity training is increased activity of various glycolytic enzymes (32). After high-intensity exercise training, phosphofructokinase (PFK) activity is increased (9, 14, 31, 33, 40), whereas endurance training reduces PFK activity (10, 39). These findings underline the general concept of training specificity. It has been shown that endurance exercise leads to a transient increase in hexokinase II (HKII) transcription and mRNA levels in human skeletal muscle (29, 30), but it is unknown whether high-intensity intermittent exercise has the same impact on transcriptional regulation of the HKII gene. This does seem possible based on the results from endurance exercise studies and the fact that HKII activity normally increases after high-intensity training (21, 22). A possible effect of increased HKII activity during recovery from high-intensity exercise may be an increased rate of glycogen replenishment, because blood glucose is the major precursor for glycogen replenishment after high-intensity exercise (4). Another effect of endurance exercise, related to regulation of substrate choice, is an increase in pyruvate dehydrogenase kinase 4 (PDK4) mRNA levels and transcription (30). An increased amount of PDK4 during recovery from exercise is probably a mechanism to ensure muscle glycogen replenishment after exercise via inactivation of the PDH complex. Because of the rapid glycogen breakdown during high-intensity exercise (3), PDK4 expression may also increase after high-intensity exercise to ensure glycogen replenishment.
Accumulation of mRNA as a result of repeated training sessions has been suggested to be a general model for cellular adaptations to exercise-training (25, 41). It is unclear, however, whether such an adaptation occurs with regular training and whether a trained muscle responds in the same manner as an untrained muscle to a given work performed.
Thus the present study was designed to test the hypotheses that 1) an acute bout of high-intensity exercise induces a transient increase in transcription of genes related to regulation of metabolism and ion homeostasis; 2) several weeks of high-intensity exercise training lead to elevated level of mRNA at rest; and 3) transient increases in mRNA after a high-intensity exercise bout are reduced when the exercise is performed at the same absolute workload after a training period due to the reduced stress on the muscle cells.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Design. High-intensity intermittent training was performed with one leg for 5.5 wk. Before and after the training period, one training session was carried out at the same absolute workload. Changes in mRNA levels were followed in the recovery phase from these two training sessions.
Training. The leg to be trained was selected randomly. The
subjects performed one-legged knee-extensor exercise in the supine position,
permitting the exercise to be confined to the quadriceps muscle
(1). The subjects were
familiarized with the one-legged knee-extensor model at least three times
before the training started. The training intensity corresponded to 
150%
of the leg maximal oxygen uptake. The training intensity was adjusted every
second week to maintain the same relative workload throughout the training
period. The training period lasted 5.6 ± 0.5 wk and consisted of 23.2
± 1.5 training sessions. Each training session consisted of a 5-min
warm-up at 10 W, 5-min rest, and 15 exercise intervals composed of kicking for
1 min at the predetermined workload followed by 3-min rest. Training was
conducted three times per week in weeks 1-2, four times per week in
weeks 3-4, and five times per week for the remainder of the period.
After the training period, an incremental test was performed with the control
and the trained leg separately. The power output was initially 40 W, and
increased 10 W every second minute until exhaustion. Time to exhaustion was
10.6 ± 0.7 min for the trained leg and 8.2 ± 0.7 min for the
untrained leg (P < 0.05).
Experimental days. On the experimental days, the exercise protocol was the same as used for the training sessions, and the workload was the same before and after the training period. Muscle samples were obtained from vastus lateralis muscle under local anesthesia with the use of the percutaneous needle biopsy technique with suction (5). Muscle biopsies were obtained at rest, immediately after exercise, and 3 h into the recovery period. Additional biopsies were obtained after 1, 5, and 24 h of recovery in the untrained state.
RNA isolation. RNA was isolated from 25-mg wet wt muscle
tissue by a modified guanidinium thiocyanate (GT)phenol-chloroform extraction
method adapted from Chomczynski and Sacchi
(8), as described previously
(30). Briefly, the samples
were homogenized in the GT solution. Extraction was performed by
centrifugation after adding NaOAc, pH 4.0, diethyl
pyrocarbonate-saturated-phenol, and chloroform-isoamyl-oxygen-hydrogen. RNA
was precipitated by centrifugation after adding isopropanol. Yeast tRNA was
added before precipitation to facilitate localization of the pellet. To
solubilize DNA, the pellet was incubated in 4 M lithium chloride. The
resulting pellet was resuspended in GT solution. A second isopropanol
precipitation was carried out, and the pellet was then rinsed with 75% EtOH
and resuspended in 2 µl of 0.1 mM EDTA containing nuclease-free water per
milligram original tissue.
Reverse transcription. The reverse transcription reaction was performed by using the Superscript II RNase H- system and oligo(dT) primers, as described by the manufacturer (Invitrogen, Carlsbad, CA). A volume of 11 µl of RNA was reverse transcribed, and the RT product was diluted in nuclease-free water to a total volume of 150 µl.
Real-time PCR. Specific primers and probes were designed for each of the mRNA sequences of interest (Table 1). The cDNA sequences were obtained from the National Center for Biotechnology Information and The Sanger Centre databases. Primers and probes were designed by use of Primer Express version 2.0 (Applied Biosystems). Probes were labeled with 6-carboxyfluorescein at the 5' end and 6-carboxy-N,N,N',N'-tetramethylrhodamine at the 3'end. Specificity of the obtained product sequence was confirmed by a search in the National Center for Biotechnology Information Blast database. It was verified that amplification of RNA samples not subjected to reverse transcription did not result in a detectable PCR product within the cycle numbers used for analysis of mRNA expression. GAPDH mRNA content was determined by using commercially available primers and probes (Applied Biosystems). Primer and probe optimization and validation of amplification efficiency were carried out. Validation of the different PCR product sizes was performed by electrophoresis (ethidium bromide containing 2.5% agarose gel). The ABI 7700 real-time PCR system was used for relative quantification. Each reaction was composed of 2.5 µl of the diluted cDNA and 12.5 µl of 2x TaqMan Universal MasterMix (AmpliTaq Gold DNA Polymerase, AmpErase uracil N-glycosylase, dNTPs with dUTP, buffer components, ROX as passive reference; Applied Biosystems). Primers, probe, and water were added to give a final reaction volume of 25 µl/well. Triple analysis was performed for each sample. Before PCR cycling, incubation at 50°C for 2 min followed by 95°C for 10 min was performed to activate the uracil N-glycosylase enzyme and AmpliTaq Gold enzyme, respectively. PCR cycling was performed by heating to 95°C for 15 s followed by 60°C for 60 s. A total of 40 cycles were completed.
|
Data analysis. All samples from each subject were analyzed simultaneously. The cycle at which the fluorescence signal rises above a manually chosen threshold level is defined as the threshold cycle. The average standard deviation in determination of threshold cycle within triplicates was 0.14 (0.01-0.20). All samples were analyzed for the content of the different target mRNA sequences and a reference mRNA sequence (GAPDH). An arbitrary concentration of each gene was calculated from external standard curves. The ratio of the target gene content to the content of the reference gene was calculated.
Statistics. Before the statistical analysis was performed, all targets to reference gene ratios were logarithmic transformed to obtain normal distributed data. Changes in mRNA levels after one acute exercise bout were tested by a one-way ANOVA for repeated measurements. When the untrained state was tested, samples obtained before and 0, 1, 3, 5, and 24 h after exercise were included. Samples obtained before, immediately after, and 3 h after exercise were included in the test of the trained state. A two-way ANOVA for repeated measurements was used to test if any interaction between training state and sample time was present and to test whether any difference in the mRNA response existed between the untrained and trained state. The level of significance was set to P < 0.05. Because of the logarithmic transformation for the statistical analyses, the data reported in the text and shown in Figs. 1 and 2 are the anti-logarithmic values (geometric mean) of the target gene to reference gene mRNA ratio, normalized to the ratio of the presample obtained in the untrained state. The 95% confidence intervals are reported and depicted on the graphs because of the antilogarithmic transformation.
|
|
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-subunit mRNA
approximately threefold after 0, 1, and 3 h of recovery, whereas no increases
were observed in the Na+-K+-ATPase 2-
and 
1-subunit mRNA content
(Fig. 1). After the training
period, no changes in Na+-K+-ATPase
1-, 2-, and 1-subunit
mRNA, as an effect of exercise, could be detected. No change in ATP-sensitive
K+ channel mRNA content was observed in either the untrained or the
trained state (Tables 2 and
3).
|
|
MCT1, MCT4, and NHE1. In neither the untrained nor the trained state did high-intensity intermittent exercise induce significant changes in MCT1, MCT4, or NHE1 mRNA levels during recovery (Tables 2 and 3).
PDK4, HKII, and PFK. After 3 and 5 h of recovery from
high-intensity intermittent exercise in the untrained state, PDK4 mRNA was
increased (P < 0.001) 13-fold, and HKII mRNA was increased
(P < 0.01) 6-fold (Fig.
2). In the trained state, high-intensity intermittent exercise
induced an increase (P < 0.05) in PDK4, but not (P =
0.07) in HKII mRNA (Fig. 2).
The resting level of PDK4 mRNA was the same in the untrained and trained
state. A higher level of PDK4 mRNA was apparent at 3 h of recovery in the
untrained state relative to the trained state (P < 0.05)
(Fig. 2). No changes could be
detected in PFK mRNA levels after the exercise bout, neither in the untrained
nor the trained state (Table
2).
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-subunit, HKII, and PDK4 mRNA content in untrained human
skeletal muscle; 2) no changes could be detected in mRNA resting
levels of the investigated genes as a result of training; and 3) of
the investigated transcripts, only PDK4 mRNA was elevated after exercise in
the trained state.
Effect of intense, repeated exercise on transcription. Our finding
of a high-intensity, exercise-induced increase in the
Na+-K+-ATPase 1-subunit mRNA level
indicates, for the first time, that expression of this catalytic subunit may
be regulated at the transcriptional level in human skeletal muscle. Our
results are supported by results from a study on rats that have been exercised
for 1 h on a treadmill, which led to an increase in
Na+-K+-ATPase 1-subunit mRNA, but not
2- and 1-subunit mRNA
(37). Thus exercise apparently
does not induce increases in Na+-K+-ATPase
2 and 1 mRNA levels. Nevertheless, the
expression of these subunits probably is regulated at the transcriptional
level. In rats, the thyroid state affects Na+-K+-ATPase
2 and 1 mRNA content, but not
1 mRNA content
(2,
13). Furthermore,
glucocorticoids induce increased transcription of
Na+-K+-ATPase 2- and
1-but not 1-subunits in rat skeletal muscle
(36). Thus different stimuli
and pathways apparently regulate the expression of the various subunits of the
Na+-K+-ATPase. It cannot be excluded that exercise for a
longer duration, with a larger muscle mass can lead to an increase in
Na+-K+-ATPase 2 and 1
mRNA as well, because this type of exercise results in an elevated plasma
cortisol level (19). The
mechanism causing the increase in transcription of the
Na+-K+-ATPase 1-subunit gene during
exercise is unknown. It may be that the contraction-induced increase in
intracellular Na+ caused by intense knee extensions
(35) is important in this
respect, as indicated by studies on rat kidney epithelial cells
(24).
Even though NHE1, MCT1, and MCT4 protein expression has been shown to increase with exercise training (16, 17, 28), we were unable to find any increases in NHE1, MCT1, and MCT4 mRNA. This could be due to posttranscriptional regulation of the expression of these proteins. It may also be due to insufficient power of the applied analyses (Table 3). Clearly, further studies are needed to clarify the role of transcriptional regulation of the expression of these proteins.
After high-intensity exercise, an increase in PDK4 mRNA occurred. This is
in concordance with results from human exercise studies, where an induction of
8- and 20-fold has been reported after prolonged exercise with an
isolated muscle group or whole body exercise, respectively
(29,
30). An increase in PDK4 mRNA
has been shown to correlate with an increased level of PDK4 protein, which
increases PDH kinase activity
(27) and thus reduces the
pyruvate to acetyl-CoA conversion in skeletal muscle
(7). This mechanism could be
responsible for the shift toward fat utilization in skeletal muscle during
recovery from exercise, allowing more efficient glycogen replenishment
(18).
Another enzyme that is key to glucose metabolism is HKII (38). We found an increase in HKII mRNA after high-intensity exercise training in the untrained state, which is in agreement with previous studies with the use of various exercise regimes (20, 29, 30). Previously, it has been shown that increases in the HKII mRNA levels correlate well with an increase in HKII protein content and activity (20, 26). Thus the increased HKII mRNA level may reflect the need for an increased glucose phosphorylation, both during and after exercise, thus ensuring substrate availability for glycolysis during exercise and for glycogen synthesis in the recovery period.
After exercise in both the trained and untrained state, PFK mRNA was unaltered. Previous studies on PFK activity after high-intensity sprint training have repeatedly found increases in PFK activity (9, 14, 31, 33, 40). Higher PFK activity is, therefore, expected after training in the present study, leading to the suggestion that changes in PFK activity after a training period are independent of changes in PFK mRNA. This is in agreement with the finding of equal amounts of PFK mRNA in control subjects and endurance athletes but lower PFK activity and protein content in endurance athletes (39).
Effect of training. Although transient increases in
Na+-K+-ATPase 1-subunit, HKII, and
PDK4 mRNA levels were apparent after exercise in the untrained state, training
did not result in increased mRNA resting levels for the transcripts
investigated in the present study. However, nearly all of the investigated
mRNA levels were, on average, nonsignificantly higher (Figs.
1 and
2,
Table 1) after the training
period (range: 1.1- to 2.1-fold). Thus we cannot exclude that a general
increase existed but was too modest to be detected. It is possible that a
higher training frequency, or longer training duration, could have lead to
detectable increases in, at least some, mRNA resting levels. Although no
changes could be detected in PDK4 mRNA resting levels after the training
period, a reduction existed in five of the six subjects. This finding
underlines that regulation of gene expression by exercise is gene
specific.
As a result of the training period, no changes were observed in
Na+-K+-ATPase 1-subunit mRNA after
exercise at the same work intensity. After high-intensity exercise in the
trained state, a reduced increase was also seen for HKII and PDK4 mRNA
(Fig. 2). The reduced induction
of HKII and PDK4 mRNA, and the reduction of PDK4 mRNA resting levels in five
subjects, may have been caused by higher levels of muscle glycogen
(29) as a result of the
training period.
It is possible that the mRNA changes during recovery from exercise would have been the same in the trained and untrained state, if exercise had been carried out at the same relative instead of the same absolute workload. Further studies are needed to clarify the role of exercise intensity for induction of gene expression after a period of training.
In summary, the present study shows that
Na+-K+-ATPase 1-subunit, PDK4, and
HKII mRNA are elevated after high-intensity exercise, implying that
transcriptional regulation of selected genes is important in relation to
adaptation to high-intensity training. Furthermore, a reduction or abolishment
of the increase in mRNA was found when subjects exercised at the same absolute
workload after a high-intensity training period. No change in resting mRNA
content as a consequence of intense exercise training was found for the
investigated transcripts.
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
2- and 2-isoforms. Am J Physiol
Cell Physiol 265:
C680-C687, 1993.
2- and 1-subunit abundance and
mRNA in rat skeletal muscle. Am J Physiol Cell Physiol
280: C509-C516,
2001.
This article has been cited by other articles:
|
|
 |
|
  K. De Bock, W. Derave, B. O. Eijnde, M. K. Hesselink, E. Koninckx, A. J. Rose, P. Schrauwen, A. Bonen, E. A. Richter, and P. Hespel Effect of training in the fasted state on metabolic responses during exercise with carbohydrate intake J Appl Physiol, April 1, 2008; 104(4): 1045 - 1055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Aughey, K. T. Murphy, S. A. Clark, A. P. Garnham, R. J. Snow, D. Cameron-Smith, J. A. Hawley, and M. J. McKenna Muscle Na+-K+-ATPase activity and isoform adaptations to intense interval exercise and training in well-trained athletes J Appl Physiol, July 1, 2007; 103(1): 39 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Phakdeekitcharoen, S. Phudhichareonrat, C. Pookarnjanamorakot, C. Kijkunasathian, N. Tubtong, W. Kittikanokrat, and P. Radinahamed Thyroid Hormone Increases mRNA and Protein Expression of Na+-K+-ATPase {alpha}2 and {beta}1 Subunits in Human Skeletal Muscles J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 353 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Murphy, A. C. Petersen, C. Goodman, X. Gong, J. A. Leppik, A. P. Garnham, D. Cameron-Smith, R. J. Snow, and M. J. McKenna Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2006; 290(2): R414 - R424. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Shearer, T. E. Graham, D. S. Battram, D. L. Robinson, E. A. Richter, R. J. Wilson, and M. Bakovic Glycogenin activity and mRNA expression in response to volitional exhaustion in human skeletal muscle J Appl Physiol, September 1, 2005; 99(3): 957 - 962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nordsborg, M. Thomassen, C. Lundby, H. Pilegaard, and J. Bangsbo Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R84 - R91. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang, A. Creer, B. Jemiolo, and S. Trappe Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle J Appl Physiol, May 1, 2005; 98(5): 1745 - 1752. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Fischer, P. Plomgaard, A. K. Hansen, H. Pilegaard, B. Saltin, and B. K. Pedersen Endurance training reduces the contraction-induced interleukin-6 mRNA expression in human skeletal muscle Am J Physiol Endocrinol Metab, December 1, 2004; 287(6): E1189 - E1194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. LeBlanc, S. J. Peters, R. J. Tunstall, D. Cameron-Smith, and G. J. F. Heigenhauser Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle J. Physiol., June 1, 2004; 557(2): 559 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. T. Murphy, R. J. Snow, A. C. Petersen, R. M. Murphy, J. Mollica, J. S. Lee, A. P. Garnham, R. J. Aughey, J. A. Leppik, I. Medved, et al. Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle J. Physiol., April 15, 2004; 556(2): 507 - 519. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
