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Am J Physiol Endocrinol Metab 294: E408-E415, 2008. First published November 27, 2007; doi:10.1152/ajpendo.00403.2007
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Effect of sex differences on human MEF2 regulation during endurance exercise

Kristian Vissing,1 Sean L. McGee,2 Carsten Roepstorff,3 Peter Schjerling,4,5 Mark Hargreaves,2 and Bente Kiens3

1Department of Sport Science, University of Aarhus, Aarhus, Denmark; 2Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia; 3The Copenhagen Muscle Research Center, Department of Human Physiology, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen; 4The Copenhagen Muscle Research Center, Department of Molecular Muscle Biology, Rigshospitalet, Copenhagen; and 5Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Submitted 26 June 2007 ; accepted in final form 21 November 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% VO2peak in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-{alpha} ({alpha}AMPK) Thr172 (P < 0.001), histone deacetylase 5 (HDAC5) Ser498 (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC1{alpha}) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1{alpha} on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.

skeletal muscle; myogenic regulatory factor; AMP-activated protein kinase; histone deacetylase 5; peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha}

WOMEN AND MEN DIFFER in several aspects of muscle metabolism. During exercise, relative fat oxidation is slightly higher in women (11, 14, 31, 41) and intracellular triacylglycerol (IMTG) net hydrolysis is markedly higher in women than in men (33, 40). In a search of possible explanations for this sex-specific substrate selection in skeletal muscle during exercise, we have previously examined the effect of sex on the mRNA and protein expression and found several targets involved in muscle lipid metabolism to be higher in women than in men (17, 32). The underlying regulatory factors behind sex differences in lipid metabolism in skeletal muscle during exercise are still to be determined but may include sex differences in the effect of signaling and transcriptional regulatory events on metabolism in the basal resting state and/or as an accumulated response of physical activity.

At the level of transcription, various transcription factors have been suggested to contribute in regulating the expression of phenotype-specific metabolic genes in the adaptive response to exercise (39). Transcription factor binding sites have been observed to differ between promoter regions of phenotype-specific genes (5, 25), which suggests that several different muscle transcription factors could be collectively involved in the regulation of sex-specific metabolic properties.

Regulation through myocyte enhancer factor 2 (MEF2) transcription factor isoforms constitutes one feasible explanation for the development of specific metabolic properties. Accordingly, in a MEF2A knockout model, the expression of several lipid binding proteins and mitochondrial proteins was significantly decreased (26). MEF2 transcription factors have been shown able to dimerize with the myogenic regulatory factor (MRF) transcription factor family members (e.g., MyoD, myogenin, and MRF4) and thereby induce and/or enhance gene transcription irrespective of whether the specific target gene promoter region contains a MRF and/or a MEF2 consensus binding sequence. Thus MRF transcription factors also may be implicated in regulation of specific metabolic properties. For instance, somatic transfer and overexpression of one MRF transcription factor, myogenin, has been elegantly demonstrated to induce an increased expression of oxidative enzymes in skeletal muscle fibers of adult mice (10).

Upstream signaling control of MEF2 and MRF transcription factors constitutes another level of regulation that may differ between sexes. Metabolic perturbations during endurance exercise have been shown to elicit changes in AMP/ATP levels and induce downstream signaling through 5'-AMP-activated protein kinase (AMPK) (12, 16, 18). In a recent study, we demonstrated that such AMPK response to exercise was greater in men than in women during prolonged moderate-intensity exercise (33). AMPK activity has been demonstrated to activate downstream p38 mitogen-activated protein kinase (MAPK), which is then thought to positively regulate peroxisome proliferator-activated receptor (PPAR)-{gamma} coactivator 1{alpha} (PGC1{alpha}) as well as specific MEF2 transcription factors (1, 2, 18, 48, 49). PGC1{alpha} itself is believed to serve as a coactivator of several transcription factors, including the MEF2 transcription factors (29). PGC1{alpha} mRNA and protein expression is reported to increase in response to endurance exercise in humans (28, 34), and PGC1{alpha} is suggested to enhance MEF2 activation through an association between phosphorylated PGC1{alpha} and MEF2 in the nucleus (22, 24, 30). The mechanism stimulating MEF2-mediated transcription is balanced by an inhibitory mechanism supported by the ability of class II histone deacetylases (HDACs; in this context, specifically HDAC5) to condense chromatin and prevent MEF2-mediated transcription (24). Once phosphorylated, HDAC5 is exported from the nucleus, which mediates de-repression of MEF2. Recent reports suggest that AMPK phosphorylates HDAC5 (23).

Thus the acute regulation of MEF2-mediated transcription in response to endurance exercise could be partly regulated by 1) AMPK phosphorylation of HDAC5, leading to de-repression of MEF2; 2) activation of MEF2 through direct phosphorylation; and 3) PGC1{alpha} coactivating action on MEF2. Regarding the first of these proposed mechanisms, it may appear contradictive that AMPK activation during exercise was shown to be greater in men than in women (33), which should, in theory, then lead to greater MEF2 activation and thus greater induction of lipid metabolic genes in men than in women. However, it is important to realize that AMPK would exert its action on HDAC5 in the nucleus, whereas the sex difference in AMPK activation during exercise was in total crude muscle. Activation of AMPK in the nucleus may not necessarily mirror the average response in the whole muscle cell. Thus sex comparison of the above-mentioned pathway components has not been investigated in the basal state or in response to endurance exercise but may contribute to the understanding of differences between men and women in regulation of energy fuel selection during prolonged exercise.

The primary purpose of the present study, therefore, was to compare MEF2 activation between men and women through the AMPK-HDAC5-PGC1{alpha} signaling pathway in the basal resting state and in response to acute exercise. A secondary purpose included screening for MEF2 and MRF transcription factor candidates that may exert different roles between sexes at rest and/or during exercise.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Nine women and eight men were recruited to participate in the study (Table 1). All subjects were young, healthy nonsmokers and participated on a regular basis (3–4 h/wk) in leisure time activities such as running, cycling, strength training, and different game sports. Body mass index was in the range of 20–25 kg/m2. Maximal oxygen uptake (VO2max) amounted to 40–55 ml O2·min–1·kg body mass–1 in the female subjects and 50–65 ml O2·min–1·kg body mass–1 in the male subjects. Maximal oxygen uptake expressed per kilogram of lean body mass was similar between female and male subjects.


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  		Table 1. Subject characteristics

 
Furthermore, habitual physical activity level and exercise training history were similar between sexes (Table 1). All female subjects were eumenorrheic with a menstrual cycle length between 28 and 35 days and did not take any oral contraception. In the female subjects, preexperimental testing as well as the main experiments were carried out during the midfollicular phase of the menstrual cycle (days 7–10 from first day of the menstrual cycle).

Before volunteering, subjects were given full oral and written information about the course of the study and possible risks associated with participation. Written consent was obtained from each subject. The study was approved by The Copenhagen Ethics Committee (KF-01-046/02) and conformed to the code of ethics of the World Medical Association (Declaration of Helsinki II). The results presented in this study are part of a larger study on sex differences in intramuscular metabolic regulation during exercise, minor parts of which have been published previously (31, 33).

Preexercise protocol. For determination of VO2max, all subjects performed an incremental exercise test on a Monark Ergomedic 839E bicycle ergometer (Monark, Varberg, Sweden). In addition, they filled out a questionnaire regarding habitual physical activity and exercise training. Body composition was determined by hydrostatic weighing (37) with a correction for residual lung volume, measured using the oxygen dilution method (19). The determination of body composition was carried out after a 4-h fasting period.

Diet. During the 8 days preceding the main exercise experiment, all subjects consumed an isoenergetic diet containing 65 energy percent (E%) carbohydrate, 20 E% fat, and 15 E% protein. The constituents of the diet were weighed at the laboratory to 1-g accuracy and delivered to the subjects. The amount of energy to be consumed was individually determined based on guidelines from the World Health Organization (FAO/WHO/UNU 1985) (47). To ensure that subject's body weight remained unchanged during the 8-day dietary regimen, energy consumption during the experimental diet was adjusted after 4 days based on daily monitoring of body weight.

Exercise protocol. The subjects arrived at the laboratory at 0830 after an overnight fast. Subjects had abstained from exercise training for 36 h before the trial. The subjects rested in the supine position for ~30 min. A muscle biopsy was then obtained from the vastus lateralis muscle under local anesthesia of the skin and fascia. The biopsy was frozen in liquid nitrogen and stored at –80°C until further use. Immediately thereafter, the subjects initiated a 90-min bicycle exercise bout at 60% VO2max on a Monark Ergomedic 839E bicycle ergometer. Exercise modality, duration, and intensity were chosen to provide an exercise stimulus known to activate lipid metabolism and to activate the AMPK-HDAC5-MEF2 signaling pathway in men (22, 23). Expired air was collected in Douglas bags during exercise at 10, 20, 30, 45, 60, 75, and 90 min. Immediately after completion of exercise, another biopsy from the vastus lateralis muscle was obtained and frozen as described above. The postexercise biopsy was obtained from the opposite leg compared with the preexercise biopsy. During the experiment, subjects were offered water ad libitum.

Breath samples. Expired volumes of air in the Douglas bags were measured with a chain-suspended Collins spirometer, and a small sample of mixed expiratory air was analyzed for O2 (Servomex S-3A) and CO2 (Beckman LB2). The respiratory exchange ratio (RER) was calculated as the ratio between pulmonary CO2 excretion and O2 uptake. Whole body fat oxidation rate was calculated from the following equation including the nonprotein respiratory quotient and then expressed in kilojoules per kilogram of lean body mass using a standard caloric equivalent for fat (27): fat oxidation rate (g/min) = 1.695VO2 – 1.701VCO2.

Protein extraction. Nuclear and whole cell proteins were extracted from ~40–50 mg of wet muscle tissue as previously described (22). Protein concentration was determined using the bicinchoninic acid method.

Immunoprecipitation. Immunoprecipitation of MEF2 from the nuclear fraction was performed as previously described (22). Briefly, 500 µg of nuclear protein were made up to 500 µl in immunoprecipitation wash buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 5 mM Na-pyrophosphate, 1 mM DTT, and 1 mM PMSF). Samples were precleared with 50 µl of protein A-Sepharose beads (Amersham Biosciences, Castle Hill, Australia) before being incubated with 2 µl of anti-MEF2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Samples were again incubated with 50 µl of protein A-Sepharose beads (Amersham Biosciences) for 2 h while rotating at 4°C. The Sepharose-bound immune complex was pelleted by centrifugation and washed four times with 1 ml of immunoprecipitation wash buffer. Immune complexes were then resolved by SDS-PAGE and probed with antibodies recognizing phosphorylated Thr residues.

Immunoblotting. Immunoblotting was performed as previously described (23). Briefly, proteins were resolves by SDS-PAGE and transferred to nitrocellulose membranes and then blocked in skim milk for 60 min. Membranes were incubated in primary antibodies overnight before incubation in secondary antibodies. Antibody binding was viewed by incubation in enhanced chemiluminescence substrate (GE Healthcare) and exposure to a Chemidoc XRS system (Bio-Rad, Hercules, CA). Bands were identified and quantified using Quantity One 1D analysis software (Bio-Rad).

Antibodies. A MEF2 antibody (C21) detecting all MEF2 isoforms was purchased from Santa Cruz Biotechnology. Phospho-Thr antibodies were purchased from Cell Signaling (Danvers, MA). Antibodies recognizing AMPK phosphorylated at Thr172 and HDAC5 phosphorylated at Ser498 were generated as previously described (8).

mRNA isolation and quantification. Approximately 20–25 mg of wet muscle tissue were used for RNA extraction. RNA was extracted using the guanidinium thiocyanate-phenol-chloroform extraction method previously described by Chomczynski and Sacchi (7).

Extracted RNA was mixed with formaldehyde loading buffer, loaded as 200 ng/well on a denaturing formaldehyde agarose gel, and run at 7 V/cm for 65 min. After electrophoresis, the gel was stained in SYBR green II RNA gel stain (Cambrex) and examined on a Molecular Imager FX scanner (Bio-Rad) to verify RNA integrity.

The RNA content of the gel was transferred to a Positive nylon membrane (Appligene) using osmocapillary blotting with 25 mM NaOH. mRNA was quantified by Northern blotting using cloned PCR products as previously described (15). In brief, probe templates were amplified for MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, PGC1{alpha}, and GAPDH from human muscle cDNA using the Accuprime Taq PCR (Invitrogen) and specific primers from MWG Biotech (Table 2).


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  		Table 2. PCR and oligo probe information

 
PCR products were then inserted into the pBlueScript SKII (+) phagemid SmaI site and transformed into DH5{alpha} Escherichia coli cells. From these plasmids, M13 primers were used to generate double-stranded probe templates with a biotinylated sense strand. A {alpha}-32P-tagged antisense strand was synthesized from the sense strand, followed by retrieval of the antisense strand and measurement of the radioactive activity. The 28S rRNA probe was generated by end-tagging a 28S complementary oligo (Table 2) using a T4 polynucleotide kinase and [{gamma}-32P]ATP.

Probes were added as 2 x 10–6 cpm/ml and hybridized to the membranes during 50°C overnight rotation. The membranes were then washed at high-stringency conditions, followed by exposure on phosphor screens. Signal was captured by a phosphoimager and quantified using Quantity One (Bio-Rad) software. Hybridization with 28S probe was performed using essentially the same protocol, except preincubation and incubation were conducted at 42°C.

Statistical analyses. All RNA data were normalized to 28S rRNA and log-transformed. All other data were not log-transformed. All statistics were analyzed using SigmaStat and Microsoft Excel. Data are means ± SE. For variables independent of time, Student's t-test was performed to test for differences in basal levels between women and men. For variables measured before and after exercise or before and during exercise, a two-way analysis of variance (ANOVA), with repeated measures for the time factor, was performed to test for sex differences or changes due to time. When a significant interaction between the effects of sex and time was found, significant pairwise differences were detected using the Student-Newman-Keuls post hoc test. Correlation analysis was performed by Pearson product moment. mRNA data are presented as geometric means ± back-transformed SE. Protein data are presented as arbitrary units. For all statistical tests, P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Workload. The average workload during the 90-min bicycle exercise trial was 132 ± 6 and 174 ± 7 W in women and men, respectively (P < 0.001). Pulmonary VO2 during exercise averaged 1.9 ± 0.1 and 2.6 ± 0.1 l O2/min in women and men, respectively (P < 0.001). Expressed per kilogram of lean body mass (LBM), the VO2 averaged 38.7 ± 1.1 and 37.7 ± 0.6 ml O2·kg LBM–1·min–1 during exercise in women and men, respectively (not significant; NS). Relative exercise intensity averaged 60 ± 1% VO2peak in both women and men.

RER and fat oxidation. The RER at rest was 0.74 ± 0.03 and 0.71 ± 0.02 in women and men, respectively (NS). RER was significantly lower in women than in men at 60 and 90 min of exercise (P < 0.05). Whole body fat oxidation (per kg LBM) was similar in women and men at rest (NS). Fat oxidation was higher (P < 0.05) in women than in men during the last hour of exercise. For further details, see Roepstorff et al. (33).

Protein expression and phosphorylation. MEF2 total protein did not differ between sexes and did not change in response to exercise (Fig. 1A). MEF2 Thr phosphorylation increased ~30% during exercise irrespective of sex (P < 0.01; Fig. 1B). HDAC5 Ser498 phosphorylation increased by ~40% in men and ~95% in women (P < 0.001). A tendency toward an interaction between effects of sex and time was observed (P = 0.079; Fig. 1C). Nuclear {alpha}AMPK Thr172 phosphorylation increased by ~50% irrespective of sex (P < 0.001; Fig. 1D).


Figure 1
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  		Fig. 1. Total myocyte enhancer factor 2 (MEF2) protein, nuclear MEF2 Thr phosphorylation, histone deacetylase 5 (HDAC5) Ser498 phosphorylation, and nuclear AMP-activated protein kinase-{alpha} ({alpha}AMPK) Thr172 phosphorylation in women and men before and after 90 min of bicycle exercise (60% VO2peak). Results are shown for total MEF2 (A) MEF2 Thr phosphorylation (B), HDAC5 Ser498 phosphorylation (C), and {alpha}AMPK Thr172 phosphorylation (D). Data are presented as arbitrary units. Results of 2-way ANOVA (sex, time, and sex x time interaction) are shown at top right of each panel.

 
mRNA expression. Two splice variants were expressed for all measured MEF2 mRNA isoforms (Fig. 2). No significant sex differences were evident before exercise. In response to exercise, the MEF2A long splice variant (~6.0 kb) exhibited an interaction between effects of sex and time (P < 0.05) with an upregulation only in women (~30%, P < 0.05; Fig. 2A). The MEF2A short splice variant (~3.5 kb) exhibited an upregulation during exercise in both men and women (~20%, P < 0.05; Fig. 2B). MEF2C long (~1.8 kb) tended to be upregulated (P = 0.054) and MEF2C (~0.9 kb) short was significantly upregulated during exercise (P < 0.05) with no significant sex differences (Fig. 2, C and D). Both MEF2D long (~3.4 kb) and short (~1.4 kb) were upregulated during exercise (P < 0.05), an effect that was mainly accomplished by an upregulation in the order of 25–35% in men. The responses of MEF2D long and short in men were not significantly different from those in women, although a tendency toward an interaction between effects of sex and time was observed for MEF2D long (P = 0.11; Fig. 2, E and F).


Figure 2
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  		Fig. 2. MEF2 isoform mRNA expression in women and men before and after 90 min of bicycle exercise (60% VO2peak). mRNA response is shown for MEF2A large (long) and small (short) splice variants (A and B), MEF2C large and small splice variants (C and D), and MEF2D large and small splice variants (E and F). Data presented are geometric means ± back-transformed SE, shown as arbitrary units. Results of 2-way ANOVA (sex, time, and sex x time interaction) are shown at top right of each panel. *P < 0.05, different from before exercise within a specific sex.

 
For MRF transcription factors, no sex differences were evident before exercise. Upregulation in response to exercise was observed for MyoD (~50%, P < 0.001), myogenin (10–25%, P < 0.05), and MRF4 (20–30%, P < 0.01), with no significant differences between women and men (Fig. 3, A–C).


Figure 3
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  		Fig. 3. Myogenic regulatory factor (MRF) mRNA expression in women and men before and after 90 min of bicycle exercise (60% VO2peak). mRNA response is shown for MyoD (A), myogenin (B), and MRF4 (C). Data presented are geometric means ± back-transformed SE, shown as arbitrary units. Results of 2-way ANOVA (sex, time, and sex x time interaction) are shown at top right of each panel.

 
PGC1{alpha} mRNA content before exercise did not differ significantly between women and men. PGC1{alpha} mRNA content was upregulated during exercise (up to 85%, P < 0.05), with no significant difference between sexes (Fig. 4).


Figure 4
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  		Fig. 4. Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC1{alpha}) mRNA expression in women and men before and after 90 min of bicycle exercise (60% VO2peak). Data are geometric means ± back-transformed SE, shown as arbitrary units. Results of 2-way ANOVA (sex, time, and sex x time interaction) are shown at top right of each panel.

 
We chose 28S rRNA for normalization of our mRNA targets of interest. To validate this choice, we also measured and normalized another unrelated and commonly used housekeeping gene, GAPDH. We did observe a tendency toward minor upregulation of GAPDH relative to 28S during exercise (results not shown), which could theoretically result from an upregulation of GAPDH, a downregulation of 28S, or a combination of both. However, GAPDH upregulation in response to the present exercise intervention seems to be a more reasonable scenario compared with downregulation of 28S. Therefore, we consider that the normalization procedure used is the most valid one.

Correlation analyses. In an attempt to find potential factors to explain the sex differences in upregulation of MEF2A long and MEF2D long mRNA by exercise, we tested whether changes in MEF2A or MEF2D mRNA during exercise correlated with the relative area of type I muscle fibers [for further details on muscle fiber type composition, see Roepstorff et al. (33)]. No significant correlations were found (change in MEF2A vs. type I area%: r = 0.355, P = 0.194; change in MEF2D vs. type I area%: r = –0.170, P = 0.546).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of the present study were that 1) nuclear {alpha}AMPK Thr172, HDAC5 Ser498, and nuclear MEF2 Thr phosphorylation were similar at rest and increased equally in response to exercise in men and women and that 2) MEF2A mRNA expression increased in response to exercise exclusively in women, whereas MEF2D mRNA expression tended to increase only in men. These findings appeared together with findings of a greater relative fat oxidation or a lower relative carbohydrate oxidation in women compared with men during acute bicycle exercise bouts of 90 min at 60% VO2peak.

A higher fat oxidation as well as IMTG net hydrolysis was previously observed in women compared with matched men when submaximal exercise was performed at the same relative workload (11, 14, 33, 40, 41). These sex differences in substrate utilization were previously observed to be accompanied by enhanced gene expression of several proteins involved in lipid metabolism (17). It could be speculated that the underlying regulation of such a sex-specific substrate utilization pattern is attributed to sex-specific signaling through MEF2. However, in the current study, no significant sex differences were observed in protein signaling through an AMPK-HDAC-MEF2 pathway at rest or during exercise. Sex differences were observed for MEF2A and MEF2D mRNA expression, but these differences were very modest and remain to be further investigated.

The primary purpose of the present study was to compare MEF2 activation between men and women through the AMPK-HDAC5-PGC1{alpha} signaling pathway. We found no exercise or sex effects on total MEF2 protein. Nuclear MEF2 Thr phosphorylation increased in response to exercise, indicating that MEF2 was activated, but no difference in nuclear MEF2 Thr phosphorylation was observed between sexes at rest or during exercise.

It has been reported that MEF2 repressor proteins like HDACs repress dimerization between MEF2 and its transcriptional coactivators (22). In the present study, exercise induced an increase in HDAC5 Ser498 phosphorylation. No significant sex differences were observed, although HDAC5 Ser498 phosphorylation tended to increase more in response to exercise in women (95%) than in men (40%) (P = 0.079).

AMPK is believed to be an important signaling molecule in regulating muscle metabolism during exercise, and HDAC5, PGC1{alpha}, and MEF2 transcription factors are all thought to be AMPK targets (1, 2, 12, 16, 48, 49). In the present study, we observed an exercise-induced increase in nuclear AMPK phosphorylation in response to exercise that did not differ between sexes. This may appear to contradict previous results from the same study, in which we showed greater exercise-induced increase in AMPK phosphorylation and {alpha}2AMPK activity in crude muscle homogenate in men than in women (33). Since the nuclear AMPK phosphorylation and the whole muscle AMPK phosphorylation were measured in the same subjects, the discrepancy cannot be explained from differences in the study cohorts or in the study protocol. Although we have no clear-cut explanation for this, one possibility could be that the nuclear increase in AMPK phosphorylation in women was simply not observed because it was exceeded by a larger pool of extranuclear nonphosphorylated AMPK when measured in whole muscle. Another possibility could be that in men compared with women, a relatively larger subpool of the phosphorylated AMPK locates to the nucleus, with the remaining pool locating to extranuclear compartments.

In any case, our data seem to support the hypothesis that exercise-induced AMPK phosphorylates HDAC5 in the nucleus to release HDAC5 from an HDAC5-MEF2 complex, leading to translocation of HDAC5 out of the nucleus. The resulting de-repression of MEF2 allows its dimerization with coactivators of transcription and thus its transcriptional activity (24). One coactivator strongly believed to enhance MEF2 transcriptional activity is PGC1{alpha} (18). An increased association between PGC1{alpha} and MEF2 has been observed previously in response to a single bout of endurance exercise similar to that in the present study (22). The observed increase in PGC1{alpha} mRNA in the present study could therefore indicate an increased PGC1{alpha} signaling requirement as a response to exercise. Altogether, the present findings support the hypothesis that AMPK regulates MEF2-mediated transcription through de-repression from HDAC5 and perhaps through the induction of coactivator PGC1{alpha}. However, and in contrast to our hypothesis, these signaling events appeared to be equal between sexes and therefore could not explain sex differences in exercise-induced substrate utilization.

Our studies on signaling to MEF2 did not include analysis of specific molecular transcription factor isoforms of the signaling molecules. Accordingly, sex-specific regulation through specific transcription factor isoforms or homologs could not be excluded. To gain indicatory information for future studies on this matter, a secondary objective of the present study was, therefore, to screen for sex differences in mRNA expression of selected MEF2 transcription factor isoforms and MRF transcription factor homologs at rest and during exercise.

MEF2A, MEF2C, and MEF2D isoforms were included in our analysis. Alternative splicing of all three MEF2 genes has previously been reported but not extensively examined in exercise studies in humans (3, 21). We detected two different splice variants for all three MEF2 isoforms. In the basal resting state, no sex differences were observed for either MEF2 isoforms or splice variants. For one MEF2A splice variant, a minor but significant upregulation was observed in response to exercise only in women, whereas one of the MEF2D splice variants exhibited a tendency to a higher exercise-induced expression in men. MEF2A and MEF2D have previously been observed to be induced by electrical stimulation or exercise at both mRNA and protein levels (36, 38), but sex-specific responses has not been reported previously. Since MEF2 expression is higher in type I than in type II muscle fibers and the women in the present study were known to have a higher proportion of type I muscle fibers than the men (33), we addressed whether the observed sex-specific responses in MEF2A and MEF2D mRNA were quantitatively related to fiber type composition. This was not the case, suggesting that other yet unknown mechanisms lie behind the sex-specific MEF2A and MEF2D mRNA response to exercise.

mRNA expression of MRF family members MyoD, myogenin, and MRF4 have previously been observed to increase in response to acute endurance exercise (43–46). In the present study we observed MyoD, myogenin, and MRF4 to be upregulated immediately at termination of exercise, but with no expression changes between sexes.

The magnitude of expression changes does not in itself allow for solid conclusions. One limitation for interpretation of the mRNA data is the lack of an extended time course of investigation postexercise. Thus we can only speculate on the magnitude of potential sex differences in mRNA expression during recovery. Also, it should be taken into consideration that mRNA data have been demonstrated to depend on the choice of normalization RNA, since induced changes for normalization RNA will potentially lead to inverse changes for the mRNA targets of interest (9, 20, 35). By normalizing against 28S rRNA, we observed some minor regulation of the supposedly unregulated housekeeping gene GAPDH, which could in principle result from upregulation of GAPDH, downregulation of 28S, or the combination of both. Since we find GAPDH more likely to undergo exercise-induced regulation, we chose 28S for normalization. However, we cannot entirely exclude that 28S does in fact undergo exercise-induced regulation and that 28S downregulation has led to seemingly greater induction of our target genes.

Our data on MEF2 signaling do not provide proof of our hypothesis that sex-specific signaling through a MEF2 pathway can explain sex differences in lipid oxidation during exercise. Yet other molecular pathways may be involved in regulation of lipid metabolism and help to explain sex differences. Calmodulin and/or calcineurin signaling through, for example, calmodulin-dependent protein kinases (CaMK), nuclear factor of activated T cells (NFAT), and MEF2 constitutes other possible regulatory pathways. Although calcineurin does not seem to induce mitochondrial biogenesis or enzyme adaptation in response to short periods of training (13, 42), such pathways have been more strongly believed to be involved in muscle phenotype remodeling toward slow/oxidative type I fibers in adaptation to long-term endurance-type stimulation (18). In this regard it is interesting that the female subjects of our study exhibited a ~23% higher proportion of type I fibers than their male counterparts (33), a finding supported by other studies (6, 40). Thus sex differences in substrate utilization during exercise may be attributed to a higher percentage of type I muscle fibers in women compared with men so that sex differences in substrate utilization are actually explained by sex differences in fiber type composition. Another alternative explanation of sex differences in fuel partitioning during exercise could relate to endocrine regulation of the female sex hormones estrogen and progesterone or to circulating catecholamines. Both estrogen and epinephrine are hormones known to have profound effects on lipid metabolism, and concentrations of both these hormones differ between women and men during exercise (14, 31, 40). It is, however, not yet revealed how acute increases of such hormones might directly influence cell signaling to induce sex differences in substrate choice (4). To explain the preference in women for lipid metabolism, it has been speculated, but not yet demonstrated, that women might exhibit higher catecholamine-sensitivity or that the combined impact of female sex hormone and catecholamine responses in women might favor lipid metabolism during exercise (4).

In conclusion, we provide support for the hypothesis that MEF2 regulation is under the upstream control of a signaling pathway involving AMPK, HDAC5, and PGC1{alpha} during the acute regulatory response to endurance exercise. This regulation does not seem to differ between sexes. The significance of minor sex differences in MEF2A and MEF2D mRNA expression remains to be further investigated.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
S. L. McGee is an National Health and Medical Research Council of Australia Peter Doherty Fellow (400446). B. Kiens and C. Roepstorff were supported by the Commission of the European Communities (contract no. LSHM-CT-2004-005272 EXGENESIS). B. Kiens was supported by the Copenhagen Muscle Research Center, The Ministry of Food, Agriculture and Fisheries, and the Danish Agency for Science, Technology and Innovation. P. Schjerling was supported by the NovoNordisk Foundation, the Copenhagen Muscle Research Center, Hovedstadens Sygehusfaellesskab, and the Medical Faculty at the University of Copenhagen.


   ACKNOWLEDGMENTS
 
We thank Prof. Bruce Kemp for the HDAC5 phosphospecific antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Vissing, Dept. of Sports Science, Univ. of Aarhus, Dalgas Ave. 4, DK-8000 Aarhus C, Denmark (e-mail: vissing{at}idraet.au.dk)

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
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akimoto T, Pohnert SC, Li P, Zhang M, Gumbs C, Rosenberg PB, Williams RS, Yan Z. Exercise stimulates Pgc-1{alpha} transcription in skeletal muscle through activation of the p38 MAPK pathway. J Biol Chem 280: 19587–19593, 2005.[Abstract/Free Full Text]
  2. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, Wackerhage H. Selective activation of AMPK-PGC-1{alpha} or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19: 786–788, 2005.[Abstract/Free Full Text]
  3. Brand NJ. Myocyte enhancer factor 2 (MEF2). Int J Biochem Cell Biol 29: 1467–1470, 1997.[CrossRef][Web of Science][Medline]
  4. Braun B, Horton T. Endocrine regulation of exercise substrate utilization in women compared to men. Exerc Sport Sci Rev 29: 149–154, 2001.[CrossRef][Medline]
  5. Calvo S, Venepally P, Cheng J, Buonanno A. Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements. Mol Cell Biol 19: 515–525, 1999.[Abstract/Free Full Text]
  6. Carter SL, Rennie CD, Hamilton SJ, Tarnopolsky. Changes in skeletal muscle in males and females following endurance training. Can J Physiol Pharmacol 79: 386–392, 2001.[CrossRef][Web of Science][Medline]
  7. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[Web of Science][Medline]
  8. Clark SA, Chen ZP, Murphy KT, Aughey RJ, McKenna MJ, Kemp BE, Hawley JA. Intensified exercise training does not alter AMPK signaling in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E737–E743, 2004.[Abstract/Free Full Text]
  9. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344: 141–143, 2005.[CrossRef][Web of Science][Medline]
  10. Ekmark M, Gronevik E, Schjerling P, Gundersen K. Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer. J Physiol 548: 259–269, 2003.[Abstract/Free Full Text]
  11. Friedlander AL, Casazza GA, Horning MA, Buddinger TF, Brooks GA. Effects of exercise intensity and training on lipid metabolism in young women. Am J Physiol Endocrinol Metab 275: E853–E863, 1998.[Abstract/Free Full Text]
  12. Frosig C, Jorgensen SB, Hardie DG, Richter EA, Wojtaszewski JF. 5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E411–E417, 2004.[Abstract/Free Full Text]
  13. Garcia-Roves PM, Huss J, Holloszy JO. Role of calcineurin in exercise-induced mitochondrial biogenesis. Am J Physiol Endocrinol Metab 290: E1172–E1179, 2006.[Abstract/Free Full Text]
  14. Horton TJ, Pagliassotti MJ, Hobbs K, Hill JO. Fuel metabolism in men and women during and after long-duration exercise. J Appl Physiol 85: 1823–1832, 1998.[Abstract/Free Full Text]
  15. Jonsdottir IH, Schjerling P, Ostrowski K, Asp S, Richter EA, Pedersen BK. Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528: 157–163, 2000.[Abstract/Free Full Text]
  16. Jorgensen SB, Richter EA, Wojtaszewski JF. Role of AMPK in skeletal muscle metabolic regulation and adaptation in relation to exercise. J Physiol 574: 17–31, 2006.[Abstract/Free Full Text]
  17. Kiens B, Roepstorff C, Glatz JF, Bonen A, Schjerling P, Knudsen J, Nielsen JN. Lipid-binding proteins and lipoprotein lipase activity in human skeletal muscle: influence of physical activity and gender. J Appl Physiol 97: 1209–1218, 2004.[Abstract/Free Full Text]
  18. Koulmann N, Bigard AX. Interaction between signalling pathways involved in skeletal muscle responses to endurance exercise. Pflügers Arch 452: 125–139, 2006.[CrossRef][Web of Science][Medline]
  19. Lundsgaard C, Van Slyke DD. Studies of lung volume. I: relaxation between thorax size and lung volume in normal adults. J Exp Med 27: 65–85, 1917.
  20. Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, Parise G, Tarnopolsky MA. Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18: 226–231, 2004.[Abstract/Free Full Text]
  21. Martin JF, Miano JM, Hustad CM, Copeland NG, Jenkins NA, Olson EN. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing. Mol Cell Biol 14: 1647–1656, 1994.[Abstract/Free Full Text]
  22. McGee SL, Hargreaves M. Exercise and myocyte enhancer factor 2 regulation in human skeletal muscle. Diabetes 53: 1208–1214, 2004.[Abstract/Free Full Text]
  23. McGee SL, Howlett KF, Starkie RL, Cameron-Smith D, Kemp BE, Hargreaves M. Exercise increases nuclear AMPK {alpha}2 in human skeletal muscle. Diabetes 52: 926–928, 2003.[Abstract/Free Full Text]
  24. McKinsey TA, Zhang CL, Olson EN. MEF2: a calcium-dependent regulator of cell division, differentiation and death. Trends Biochem Sci 27: 40–47, 2002.[CrossRef][Web of Science][Medline]
  25. Nakayama M, Stauffer J, Cheng J, Banerjee-Basu S, Wawrousek E, Buonanno A. Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements. Mol Cell Biol 16: 2408–2417, 1996.[Abstract]
  26. Naya FJ, Black BL, Wu H, Bassel-Duby R, Richardson JA, Hill JA, Olson EN. Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nat Med 8: 1303–1309, 2002.[CrossRef][Web of Science][Medline]
  27. Peronnet F, Massicotte D. Table of nonprotein respiratory quotient: an update. Can J Sport Sci 16: 23–29, 1991.[Web of Science][Medline]
  28. Pilegaard H, Saltin B, Neufer PD. Exercise induces transient transcriptional activation of the PGC-1{alpha} gene in human skeletal muscle. J Physiol 546: 851–858, 2003.[Abstract/Free Full Text]
  29. Puigserver P. Tissue-specific regulation of metabolic pathways through the transcriptional coactivator PGC1-{alpha}. Int J Obes(Lond) 29, Suppl 1: S5–S9, 2005.[CrossRef]
  30. Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O'Malley B, Spiegelman BM. Activation of PPAR{gamma} coactivator-1 through transcription factor docking. Science 286: 1368–1371, 1999.[Abstract/Free Full Text]
  31. Roepstorff C, Donsmark M, Thiele M, Vistisen B, Stewart G, Vissing K, Schjerling P, Hardie DG, Galbo H, Kiens B. Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise. Am J Physiol Endocrinol Metab 291: E1106–E1114, 2006.[Abstract/Free Full Text]
  32. Roepstorff C, Schjerling P, Vistisen B, Madsen M, Steffensen CH, Rider MH, Kiens B. Regulation of oxidative enzyme activity and eukaryotic elongation factor 2 in human skeletal muscle: influence of gender and exercise. Acta Physiol Scand 184: 215–224, 2005.[CrossRef][Web of Science][Medline]
  33. Roepstorff C, Thiele M, Hillig T, Pilegaard H, Richter EA, Wojtaszewski JF, Kiens B. Higher skeletal muscle {alpha}2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise. J Physiol 574: 125–138, 2006.[Abstract/Free Full Text]
  34. Russell AP, Feilchenfeldt J, Schreiber S, Praz M, Crettenand A, Gobelet C, Meier CA, Bell DR, Kralli A, Giacobino JP, Deriaz O. Endurance training in humans leads to fiber type-specific increases in levels of peroxisome proliferator-activated receptor-gamma coactivator-1 and peroxisome proliferator-activated receptor-{alpha} in skeletal muscle. Diabetes 52: 2874–2881, 2003.[Abstract/Free Full Text]
  35. Schjerling P. The importance of internal controls in mRNA quantification. J Appl Physiol 90: 401–402, 2001.[Free Full Text]
  36. Silva JL, Giannocco G, Furuya DT, Lima GA, Moraes PA, Nachef S, Bordin S, Britto LR, Nunes MT, Machado UF. NF-{kappa}B, MEF2A, MEF2D and HIF1-a involvement on insulin- and contraction-induced regulation of GLUT4 gene expression in soleus muscle. Mol Cell Endocrinol 240: 82–93, 2005.[CrossRef][Web of Science][Medline]
  37. Siri WE. The gross composition of the body. Adv Biol Med Phys 4: 239–280, 1956.[Medline]
  38. Smith JA, Collins M, Grobler LA, Magee CJ, Ojuka EO. Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo. Am J Physiol Endocrinol Metab 292: E413–E420, 2007.[Abstract/Free Full Text]
  39. Spangenburg EE, Booth FW. Molecular regulation of individual skeletal muscle fibre types. Acta Physiol Scand 178: 413–424, 2003.[CrossRef][Web of Science][Medline]
  40. Steffensen CH, Roepstorff C, Madsen M, Kiens B. Myocellular triacylglycerol breakdown in females but not in males during exercise. Am J Physiol Endocrinol Metab 282: E634–E642, 2002.[Abstract/Free Full Text]
  41. Tarnopolsky LJ, MacDougall JD, Atkinson SA, Tarnopolsky MA, Sutton JR. Gender differences in substrate for endurance exercise. J Appl Physiol 68: 302–308, 1990.[Abstract/Free Full Text]
  42. Terada S, Nakagawa H, Nakamura Y, Muraoka I. Calcineurin is not involved in some mitochondrial enzyme adaptations to endurance exercise training in rat skeletal muscle. Eur J Appl Physiol 90: 210–217, 2003.[CrossRef][Web of Science][Medline]
  43. Vissing K, Andersen JL, Harridge SD, Sandri C, Hartkopp A, Kjaer M, Schjerling P. Gene expression of myogenic factors and phenotype-specific markers in electrically stimulated muscle of paraplegics. J Appl Physiol 99: 164–172, 2005.[Abstract/Free Full Text]
  44. Vissing K, Andersen JL, Schjerling P. Are exercise-induced genes induced by exercise? FASEB J 19: 94–96, 2005.[Abstract/Free Full Text]
  45. Walters EH, Stickland NC, Loughna PT. The expression of the myogenic regulatory factors in denervated and normal muscles of different phenotypes. J Muscle Res Cell Motil 21: 647–653, 2000.[CrossRef][Web of Science][Medline]
  46. Walters EH, Stickland NC, Loughna PT. MRF-4 exhibits fiber type- and muscle-specific pattern of expression in postnatal rat muscle. Am J Physiol Regul Integr Comp Physiol 278: R1381–R1384, 2000.[Abstract/Free Full Text]
  47. World Health Organization. Energy and protein requirements: report of a joint FAO/WHO/UNU Expert Consultation. Geneva: WHO, 1985.
  48. Xi X, Han J, Zhang JZ. Stimulation of glucose transport by AMP-activated protein kinase via activation of p38 mitogen-activated protein kinase. J Biol Chem 276: 41029–41034, 2001.[Abstract/Free Full Text]
  49. Zhao M, New L, Kravchenko VV, Kato Y, Gram H, di Padova F, Olson EN, Ulevitch RJ, Han J. Regulation of the MEF2 family of transcription factors by p38. Mol Cell Biol 19: 21–30, 1999.[Abstract/Free Full Text]




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