1
Intermittent and continuous high-intensity exercise induce similar acute but different chronic 1
muscle training adaptations 2
Andrew J.R. Cochran1, Michael E. Percival1, Steven Tricarico1, Jonathan P. Little1, Naomi 3
Cermak1, Jenna B. Gillen1, Mark A. Tarnopolsky2, and Martin J. Gibala1 4
5
1Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, 6
Hamilton, Ontario, Canada; 2Department of Pediatrics and Medicine, Division of Neuromuscular 7
and Neurometabolic Disorders, McMaster University, McMaster University Medical Centre, 8
Hamilton, Ontario, Canada. 9
10
11 Correspondence: Martin J. Gibala, Ph.D. 12
Department of Kinesiology 13 McMaster University 14 1280 Main St. West 15 Hamilton, ON L8S 4K1 16 Canada 17 Phone: 905-525-9140 x23591 18 Fax: 905-523-6011 19 E-mail: [email protected] 20
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New Findings 22
What is the central question of this study? 23
How important is the interval in high-intensity interval training? 24
What is the main finding and its importance? 25
The intermittent nature of high-intensity interval training (HIIT) is important for 26
maximizing skeletal muscle adaptations to this type of exercise, at least when a relatively 27
small total volume of work is performed in an "all out" manner. The protein signalling 28
responses to an acute bout of HIIT were generally not predictive of training-29
induced outcomes. Nonetheless, a single session of exercise lasting
3
High-intensity interval training (HIIT) performed in an all-out manner (e.g., repeated Wingate 42
Tests) is a time-efficient strategy to induce skeletal muscle remodelling towards a more oxidative 43
phenotype. A fundamental question that remains unclear, however, is whether the intermittent or 44
pulsed nature of the stimulus is critical to the adaptive response. In Study 1, we examined 45
whether the activation of signalling cascades linked to mitochondrial biogenesis was dependent 46
on the manner in which an acute high-intensity exercise stimulus was applied. Subjects 47
performed either 4 x 30 s Wingate Tests interspersed with 4 min of rest (INT), or a bout of 48
continuous exercise (CONT) that was matched for total work (67 7 kJ) and which required ~4 49
min to complete as fast as possible. Both protocols elicited similar increases in markers of 50
AMPK and p38 MAPK activation, and PGC-1 mRNA expression (main effects for time, 51
P0.05). In Study 2, we determined whether 6 wk of the CONT protocol (3 d/wk) would 52
increase skeletal muscle mitochondrial content similar to what we have previously reported after 53
6 wk of INT. Despite similar acute signalling responses to the CONT and INT protocols, training 54
with CONT did not increase the maximal activity or protein content of a range of mitochondrial 55
markers. However, peak oxygen uptake (VO2peak) was higher after CONT training (45.7 5.4 to 56
48.3 6.5 mLkg-1min-1; p < 0.05) and 250 kJ time trial performance was improved (26:32 57
4:48 to 23:55 4:16 min:sec, p < 0.001) in our recreationally-active participants. We conclude 58
that the intermittent nature of the stimulus is important for maximizing skeletal muscle 59
adaptations to low-volume, all-out HIIT. Despite the lack of skeletal muscle mitochondrial 60
adaptations, our data show that a training program based on a brief bout of high-intensity 61
exercise, which lasted
4
Introduction 66
High-intensity interval training (HIIT) characterized by short bursts of relatively 67
intense exercise interspersed by periods of recovery within a given training session stimulates 68
mitochondrial biogenesis in skeletal muscle and remodelling towards a more oxidative 69
phenotype (Burgomaster, et al. , 2005, Gibala, et al. , 2006, Perry, et al. , 2008, Talanian, et al. , 70
2007). HIIT performed using brief all-out or supramaximal work efforts (e.g., repeated 71
Wingate Tests) appears to be a particularly potent training stimulus. For example, subjects who 72
trained three days per week using 4-6 x 30 sec bursts of all-out cycling interspersed by 4 min of 73
recovery (for a total of only 2-3 min of intense exercise within a ~20 min session), showed 74
metabolic adaptations including increased mitochondrial content that was similar to those who 75
performed 40-60 min of continuous moderate-intensity training per session, 5 d per week 76
(Burgomaster, et al. , 2008). It is therefore possible to stimulate rapid adaptations in skeletal 77
muscle that are comparable to traditional endurance training with a relatively small dose of HIIT, 78
provided the exercise stimulus is very intense and applied in an intermittent manner 79
(Burgomaster, et al. , 2008, Gibala, et al. , 2006). 80
Exercise-induced mitochondrial biogenesis is influenced by relative work intensity, 81
duration and volume, but the precise role of the various factors remains unclear. Using a 82
continuous exercise protocol, Egan et al. (Egan, et al. , 2010) showed that selected signalling 83
proteins linked to mitochondrial biogenesis were phosphorylated to a greater extent following 84
higher intensity exercise (~36 min at 80% VO2peak) compared to a work-matched bout of lower 85
intensity exercise (~70 min at 39% VO2peak). These data are consistent with the notion that higher 86
intensities may be more effective for stimulating mitochondrial biogenesis, at least when a 87
relatively large volume of exercise (~1700 kJ) is performed. In contrast, Boyd et al. (Boyd, et al. 88
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, 2013) recently reported that the increase in skeletal muscle mitochondrial content after 6 wk of 89
HIIT was similar when subjects trained three times per week using a protocol that consisted of 90
10 x 1 min cycling efforts at either 70% or 100% of peak power output (PPO). Finally, another 91
recent study by Edge et al. (Edge, et al. , 2013) considered the role of the rest interval in the 92
skeletal muscle adaptive response to HIIT. These authors found that 15 sessions of 6-10 x 2 min 93
efforts at ~90-110 of pre-training PPO increased muscle Na+-K+-ATPase content and 94
phosphocreatine resynthesis, however manipulating the rest period during training (such that 95
either 1 or 3 min of recovery was permitted between efforts, with total work matched between 96
groups), did not affect these changes. 97
Another fundamental question relates to the importance of the interval in HIIT, i.e., 98
whether the intermittent or pulsatile nature of this training strategy (and characteristic alternating 99
hard/easy pattern) is fundamental to the adaptive response. In the present investigation, we 100
sought to further investigate whether skeletal muscle adaptation to brief, all-out exercise was 101
dependent on the manner in which the stimulus was applied. In Study 1, we first examined the 102
acute response of selected signalling proteins we have examined previously in our all-out HIIT 103
model to determine whether exercise intermittency altered exercise-induced activation of 104
proteins involved in mitochondrial biogenesis. We hypothesized that an acute bout of low-105
volume all-out exercise would activate signalling cascades linked to mitochondrial biogenesis to 106
a similar extent, regardless of whether the exercise was performed in an intermittent (INT) or 107
continuous (CONT) manner. After establishing that both protocols elicited similar acute 108
signalling responses in the preliminary study, we subsequently conducted a 6 wk training study 109
(Study 2) to determine if training with the CONT protocol would induce skeletal muscle 110
adaptations similar to what we have previously shown after training with the INT protocol. We 111
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hypothesized that CONT training would elicit skeletal muscle adaptations including increased 112
mitochondrial content, similar to what we have previously shown after 6 wk of INT training 113
(Burgomaster, et al. , 2008). 114
115
Methods 116
Ethical approval 117
All experimental procedures were approved by the Hamilton Integrated Research Ethics 118
Board, and conformed in all respects with the Declaration of Helsinki. All subjects completed 119
routine medical screening and provided written informed consent prior to study participation. 120
Subjects 121
A total of 17 subjects volunteered to participate in the two studies (Table 1). Eight 122
subjects took part in the acute investigation (Study 1), which involved a repeated measures 123
design to evaluate the skeletal muscle metabolic response to an acute bout of high-intensity 124
exercise matched for total work but performed in an intermittent (INT) or continuous manner 125
(CONT). Nine subjects took part in the training study (Study 2), which examined skeletal muscle 126
remodelling in response to 6 wk of training using the CONT protocol. All subjects were young 127
healthy individuals who were habitually active but not specifically trained in any sport. 128
Study 1 - Acute Investigation 129
Pre-Experimental Procedures 130
VO2peak and peak aerobic power output (Wpeak) were initially determined during a ramp 131
protocol to volitional fatigue on an electromagnetically-braked cycle ergometer (Lode Excalibur 132
Sport, Groningen, the Netherlands) using an online gas collection system (Moxus modular 133
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oxygen uptake system, AEI technologies, Pittsburgh, PA, USA) as we have previously described 134
(Cochran, et al. , 2010). Specifically, participants began cycling for 2 min at 50 W, followed by a 135
progressive increase in power demand at the rate of 1 W every 2 sec. Thereafter, subjects 136
participated in a minimum of two familiarization trials on separate days using the same 137
electronically-braked cycle ergometer employed during the main phase of the study (Velotron, 138
RacerMate Inc., Seattle, WA) in order to become acquainted with the exercise protocols. Due to 139
the nature of the experimental design, all subjects performed the INT exercise protocol during 140
their first familiarization visit. This was necessary in order to determine the total amount of work 141
needing to be performed during the CONT exercise protocol for a given subject. 142
The INT protocol consisted of 4 x 30 sec all-out sprints, performed against a resistance 143
equivalent to 7.5% of body mass (i.e. repeated Wingate Tests), interspersed with 4 min of 144
recovery, as we have previously described (Burgomaster, et al. , 2005). A computer with 145
appropriate software (Velotron Wingate Software v1.0) was interfaced with the ergometer and 146
permitted the appropriate load to be applied for each subject. Total work output, peak power and 147
mean power were calculated and recorded by an online data acquisition system. 148
For the CONT protocol, subjects performed the same total volume of work as in the INT 149
exercise session, but as a single, continuous, all-out effort. The ergometer was interfaced with 150
software (Velotron Coaching Software v1.5) that linked power output directly to pedalling 151
cadence, while quantifying total work done in real-time. Subjects were instructed to complete 152
their designated amount of work as quickly as possible by maintaining the highest pedalling 153
cadence possible. Between 50 and 100 rpm, power output corresponded with a range of 75 to 154
500 W. Cycling was terminated immediately upon completion of the designated amount of work. 155
Experimental Trials 156
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The main experiment consisted of two trial days separated by at least one week. Trials 157
were conducted in a randomized, counterbalanced manner with half the subjects starting with the 158
INT protocol and the other half with the CONT protocol. Subjects were instructed to refrain 159
from exercise for 48 h prior to each experimental trial, and to avoid caffeine and alcohol for at 160
least 12 h before the trials. Subjects maintained individual food diaries for the 24 h period 161
preceding the first trial, and replicated their diet during the second trial. 162
On the day of each trial, subjects arrived at the laboratory in the morning, 60-90 min after 163
ingesting their habitual breakfast. Food records were collected and subjects then changed into 164
athletic apparel, and rested quietly until trial commencement. A resting needle muscle biopsy 165
sample was obtained from the vastus lateralis of one thigh under local anesthesia (1% xylocaine) 166
as previously described (Gibala, et al. , 2006). The muscle sample was immediately frozen in 167
liquid nitrogen and stored at -80 C until further analyses. After resting for another 10 min, the 168
subjects moved to the cycle ergometer and completed a standardized warm-up that consisted of 2 169
min of unloaded cycling followed by 5 min of rest. Subjects then performed the designated 170
exercise protocol. A second muscle biopsy was obtained immediately upon cessation of cycling, 171
and subjects were asked to provide a rating of perceived exertion for the overall exercise 172
protocol, using the Borg scale (Borg, 1974). Subjects then rested quietly in the laboratory for 3 173
h, at which point a third muscle biopsy was taken. The three biopsies for a given trial were 174
obtained from the same leg through separate incisions >2 cm apart. 175
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176
Study 2 - Training Study 177
Pre-Experimental Procedures 178
Subjects initially performed baseline VO2peak testing as described for Study 1. Thereafter, 179
subjects undertook a series of familiarization sessions in order to become accustomed to the 180
testing and training procedures. These sessions included a 250 kJ simulated cycling time trial 181
(TT), a 60 min steady-state session at ~65% VO2peak, and a practice training session which was 182
modelled after the CONT protocol employed in Study 1. TT familiarizations were repeated at 1 183
wk intervals until participants could not further improve beyond their previous session. 184
Consistency in performance during familiarizations were verified by t-test (p = 0.3), and the 185
latter of two similar results were taken as baseline TT performance. Subjects completed 24 h diet 186
records prior to each of these tests, and diets were replicated over the 24 h period preceding post-187
training tests. 188
250 kJ Time Trial. All chronic study participants were instructed to complete, as quickly 189
as possible, a simulated TT consisting of 250 kJ of total work. This test was performed on the 190
same electromagnetically-braked cycle ergometer (Velotron, RacerMate Inc., Seattle, WA) 191
interfaced with software (Velotron Coaching Software v1.5) as training at a standardized 192
gearing. Again, the cycle ergometer was programmed such that power outputs between 75 and 193
500 W were directly associated with pedalling rates, and subjects were instructed to maintain the 194
highest pedalling cadence possible. No feedback was given during the rides with the exception of 195
work remaining, and the test was terminated immediately upon the completion of 250 kJ. 196
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60 min steady-state ride at 65% VO2peak. Subjects cycled continuously for 60 min at an 197
intensity designed to elicit 65% of their peak oxygen uptake. The steady-state ride was 198
conducted on the same cycle ergometer as the VO2peak measurement (Lode Excalibur), and 199
respiratory measurements were made at specific 5 min intervals throughout exercise using the 200
same metabolic cart system described previously (Moxus oxygen uptake system, AEI). 201
Skeletal Muscle Biopsy. A resting skeletal muscle biopsy was taken approximately one 202
week following performance testing as described for Study 1. Subjects were instructed to record 203
their diet for the 24 h preceding the biopsy, while refraining from exercise for a minimum of 48 204
h, and abstaining from caffeine and alcohol for a minimum of 12 h pre-biopsy. Muscle samples 205
were immediately frozen under liquid nitrogen, and subsequently stored at -80C until further 206
analysis. Diets were replicated post-training, and a second resting biopsy was taken 72 h 207
following the last exercise training session. 208
Exercise training 209
Training was performed 3 d per week for 6 wks, for a total of 18 sessions to align directly 210
with our previous 6 wk INT study schedule. The training intervention was modelled after the 211
CONT protocol employed in Study 1 and each session consisted of a single bout of high-212
intensity cycling completed as quickly as possible. Based on our acute investigation and other 213
pilot work, mean power produced over the course of 4 Wingate tests interspersed with 4 min of 214
recovery in recreationally-active subjects averaged ~1.0 kJ per kg of body mass. Subjects were 215
therefore assigned an initial exercise training load that corresponded to 1.0 kJ per kg body 216
weight. Training load was subsequently increased to 1.25 kJ per kg body weight during the 217
second half of the 6 wk intervention in order to provide progression and maintain the duration of 218
the training session. Workload was self-selected and varied over the training session based on 219
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pedalling cadence, with a range of 50-100 rpm corresponding to ~75-500 W. During each 220
training session, heart rate was monitored and ratings of perceived exercise (RPE) scores were 221
obtained based on the Borg scale (Borg, 1974). 222
Post-training testing and procedures 223
Post-training procedures were identical in all respects to those conducted prior to training 224
onset, with the exception of order. Subjects first underwent a second resting skeletal muscle 225
biopsy ~72 h post-training. This time point was chosen to evaluate training-induced changes in 226
resting muscle. Steady-state, TT and VO2peak tests took place at 48 h intervals thereafter. Due to 227
scheduling difficulties and travel conflicts however, we could only obtain post-training VO2peak 228
measures on 6 of our 9 subjects. All subjects adhered to previously recorded diet records for the 229
24 h preceding each of biopsy and testing procedures. 230
Muscle Analysis 231
Western Blotting. Whole cell lysates were prepared by adding ~30 mg wet muscle to ice-232
cold RIPA buffer (50 mM HCL, 150 mM NaCl, 1 mM PMSF, 1% NP-40, 0.5% sodium 233
deoxycholate, and 0.1% SDS) containing protease (Complete Mini, Roche Applied Science, 234
Laval, PQ, Canada) and phosphatase inhibitors (PhosSTOP, Roche Applied Science, Laval, 235
PQ, Canada). Samples were minced and homogenized on ice (Pro 250, Pro Scientific, Oxford, 236
CT, USA), sonicated, and agitated end-over-end for 15 min at 4oC. Samples were then 237
centrifuged at 15,000 g for 5 min at 4oC. The pellet was then resuspended, and following a 238
second centrifugation at 15,000 g for 10 min, the supernatant was collected for subsequent 239
analysis. Homogenate protein concentrations were determined using a commercial, detergent-240
compatible, colorimetric assay (BCA protein assay, Pierce, Rockford, IL). Equal amounts of 241
protein (5-20 g, depending on the protein of interest) were then loaded onto 7.5-12.5% SDS-242
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PAGE gels and separated by electrophoresis for 2-2.5 hours at 100 V. Proteins were transferred 243
to nitrocellulose membranes for 1 hr at 100 V. Ponceau S staining was performed following the 244
transfer and was used to control for equal loading and transfer between lanes. Membranes were 245
blocked using a 5% fat-free milk or BSA solution in TBS-T at room temperature, and incubated 246
overnight with the appropriate primary antibodies diluted in a 3% fat-free milk or BSA in TBS-247
T, thereafter. For Study 1, primary antibodies targeting phospho-p38 MAPK, total-p38 MAPK, 248
phospho-acetyl-CoA carboxylase (ACC), were purchased from Cell Signaling Technology 249
(Beverly, MA). For study 2, primary antibodies targeted against 5 separate mitochondrial 250
protein markers including NDUFA9 (Mitosciences, MS111), Complex II 70 kDa subunit 251
(Mitosciences, MS204), Complex III Core 2 protein (Mitosciences, MS304), cytochrome c 252
oxidase subunit IV (COXIV; Mitosciences, MS408) and the ATP synthase subunit 253
(Mitosciences, MS507). We also probed nitrocellulose membranes against glucose transporter 4 254
(GLUT4; Millipore AB1345), and monocarboxylate transporters 1 and 4 (MCT1, Millipore 255
AB3538; MCT4, Millipore AB3316). Blots were incubated in the appropriate secondary 256
antibodies for 1 hour at RT, and visualized by chemiluminescence (Supersignal West Dura, 257
Pierce). Signal quantification was performed using NIH Image J software. 258
Real-time RT-PCR. Frozen wet muscle samples (~20 mg) were homogenized in TRIzol 259
reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated using the RNeasy Mini Kit in 260
conjunction with the RNase-Free DNase Set DNA digestion (Qiagen, Mississauga, ON, Canada). 261
RNA was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit 262
from Applied Biosystems (Carlsbad, CA), aliquoted, and stored at -80oC until further analysis. 263
RT-PCR reactions for PGC-1 mRNA expression were run using forward (5-CAT CAA AGA 264
AGC CCA GGT ACA-3) and reverse (5-GGA CTT GCT GAG TTG TGC ATA-3) primers in 265
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combination with SYBR green/ROX fluorescence chemistry (PerfeCTa, Quanta Biosciences). 266
Reactions were run on a thermal cycler (Applied Biosystems, Carlsbad, CA), and expression 267
levels were normalized to the housekeeper gene 2-microglobulin (Forward: 5-GGC TAT CCA 268
GCG TAC TCC AA-3; Reverse: 5-GAT GAA ACC CAG ACA CAT AGC A -3), which was 269
verified to be unchanged in response to our exercise interventions (data not shown). 270
Citrate synthase maximal activity. Approximately 20 mg of wet muscle was 271
homogenized using glass tissue pestles in 10 volumes of buffer containing 70 mM sucrose, 220 272
mM mannitol, 10 mM HEPES (pH 7.4), supplemented with protease inhibitors (Complete 273
Mini, Roche Applied Science, Laval, PQ, Canada). Citrate synthase (CS) maximal activity was 274
then quantified as we have described previously (Gibala, et al. , 2006, Little, et al. , 2010). 275
Homogenate protein content was determined via BCA method using a commercial assay (Pierce, 276
Rockford, IL, USA) and enzyme activity expressed as mmolkg protein-1hr-1 wet weight. 277
Statistical Analyses 278
Exercise data from Study 1 was analyzed via paired Student's t-tests, while all muscle 279
data from Study 1 was analyzed using a two-factor repeated-measured ANOVA, followed where 280
appropriate by a Tukeys HSD post hoc test. All data from Study 2 was analyzed using paired 281
Student's t-tests. The level of significance was set at P 0.05 for all analyses and all analyses 282
were conducted using SigmaStat 3.1 software (Systat Software, Chicago, IL). All data are 283
presented as means standard deviation (SD). 284
285
Results 286
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Acute Investigation 287
Performance data are presented in Table 2. Total work and ratings of perceived exertion 288
were not different between trials (p=0.71, and p=0.81, respectively). Peak power output and 289
mean power output, averaged over the four Wingate tests in the INT trial, was higher than the 290
respective values calculated for the CONT trial. Conversely, total exercise duration in the CONT 291
trial (~4 min) was approximately double that of the INT trial (2 min, i.e., 4 x 30s), although the 292
latter session required a total of 14 min including recovery between intervals. 293
Muscle glycogen content was reduced by ~25%, and muscle lactate concentration was 294
elevated ~10-fold after exercise, with no difference between protocols (main effects for time, 295
p
15
also unchanged after training (P 0.10), the one exception being cytochrome c oxidase subunit 4 310
(COXIV), which showed a 20% increase (p = 0.014; Figure 5). Similarly, the protein content of 311
GLUT4, MCT1 and MCT4 were unchanged after training (data not shown). 312
Peak oxygen uptake was increased by 6% after training (p < 0.05; Figure 6), while time 313
to complete 250 kJ of improved by ~9% (p < 0.001; Figure 7). There were no differences in 314
heart rate, respiratory exchange rate or ventilation during steady-state cycling at 65% of pre-315
training VO2peak before and after training (Table 3). 316
317
Discussion 318
The overriding goal of the present study was to determine whether the characteristic pulsed 319
nature of high-intensity interval exercise is critical to maximize adaptation to this type of 320
training. While training using brief intermittent bursts of all out exercise is a potent stimulus to 321
induce skeletal muscle remodelling towards a more oxidative phenotype (Burgomaster, et al. , 322
2008, Gibala, et al. , 2006), it is unclear if the alternating pattern of hard/easy effort is 323
fundamental to the training response. The results of our acute investigation (Study 1) showed 324
that both INT and CONT protocols elicited similar increases in signalling cascades linked to 325
mitochondrial biogenesis, including the protein phosphorylation of ACC and p38 MAPK and 326
PGC-1 mRNA expression. Despite this, a range of mitochondrial enzyme markers were 327
generally unchanged after 6 wk of training with the CONT protocol (Study 2). This finding is in 328
contrast to the robust increases in mitochondrial protein content and maximal enzyme activities 329
that we have repeatedly observed after 2-6 wk of the INT training protocol (Gibala et al., 2006, 330
Burgomaster et al., 2008). 331
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An obvious limitation of the present work was the lack of a direct comparison between 332
the CONT and INT protocols. With respect to our of measurements of skeletal muscle 333
adaptation, it has been proposed that CS is one of the most appropriate indicators of 334
mitochondrial content in human skeletal muscle, as it is highly correlated with gold-standard 335
measures of mitochondrial content made by electron microscopy (Larsen, et al. , 2012). 336
Interestingly, a recent review by Bishop et al (Bishop et al., 2013) suggested that training volume 337
is more important for increasing mitochondrial content than training intensity, which may in part 338
explain the lack of change in CS activity. CONT training also had no effect on other markers of 339
skeletal muscle adaptation including the protein content of GLUT4, MCT1 and MCT4, which we 340
have previously shown to be increased by INT training (Burgomaster, et al. , 2007). Overall, 341
these data suggest that the intermittent nature of the HIIT stimulus may be important for 342
maximizing skeletal muscle adaptations, at least when a relatively small total volume of high 343
intensity exercise is performed in an all out manner.. Additional studies with larger sample sizes 344
and more comprehensive assessment of physiological adaptation are warranted in order to 345
support or refute this hypothesis. 346
Despite the lack of change in most markers of skeletal muscle oxidative or metabolite 347
transport capacity, 6 wk of CONT training improved time to complete 250 kJ of work. While 348
numerous factors are involved in determining exercise performance (Coyle 2005), one factor that 349
may have contributed to the improved performance in the present study was an enhanced whole 350
body aerobic capacity (Bassett and Howley. , 2000), as reflected by the significant 6% increase 351
in VO2peak after training (despite being measured in only 6 of our 9 subjects). This observation 352
supports the idea that brief bouts of very intense exercise can improve cardiorespiratory fitness. 353
Tjonna and associates (Tjonna, et al. , 2013) recently reported a 10% improvement in VO2peak 354
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after 10 wk of training in which overweight but healthy subjects performed a single 4 min bout 355
of continuous exercise at an intensity that elicited 90% of maximal heart rate (HRmax), three 356
times per week. Subjects in that study performed a 10 min warm-up at 70% HRmax, followed 357
by 4 min at 90% HRmax and 5 min cool-down at 70% HRmax, for a total time commitment of 358
19 min (Tjonna, et al. , 2013). In the present study, subjects performed only 2 min of unloaded 359
cycling as a warm-up, and thus our data show that VO2peak can be enhanced by a training 360
protocol consisting of
18
that specific subunits of AMPK and p38 MAPK may play distinctive roles in the adaptive 378
response to exercise (Birk and Wojtaszewski. , 2006, Pogozelski, et al. , 2009) and therefore it is 379
possible that subtle differences in activation could not be resolved by our Western Blotting 380
techniques. Our data also highlights the need for studies examining both acute and training 381
responses within the same individuals. Indeed, we are unaware of any evidence reporting that 382
subject-to-subject variability in either AMPK, p38 MAPK or PGC-1 are correlated with 383
training adaptation in human muscle, and this has led some to question the purpose focusing so 384
much research upon upstream signalling events (Timmons, 2011). Furthermore, our findings 385
underscore that changes in mRNA expression do not necessarily confer a similar change in 386
functional protein or enzyme activity, and that relatively little is known at present regarding the 387
effects that different types of exercise may have on processes downstream from mRNA 388
expression in human skeletal muscle. These factors include mRNA stability and turnover, 389
protein translation, protein import and assembly, mitochondrial fusion/fission, and mitophagy. 390
Any combination of these processes may be responsible for the diversion between mRNA and 391
protein expressions. More work must be done to examine the effects that exercise intensity, 392
duration, and factors such as intermittency may have on the intervening biological processes 393
between mRNA content and functional protein expression. 394
In summary, we have shown that performing a given amount of work using an all-out 395
effort results in similar activation of signaling cascades linked to mitochondrial biogenesis, 396
regardless of whether the exercise is performed in an intermittent or continuous manner. Despite 397
similar acute signalling responses to the CONT and INT protocols, a range of mitochondrial 398
enzyme markers were generally unchanged after 6 wk of training with the CONT protocol, 399
which, although not measured in the present study, is in contrast to the robust increases we have 400
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previously reported after 2 and 6 wk of training with the INT protocol (Burgomaster, et al. , 401
2008, Gibala et al., 2006). Thus, the acute responses were not necessarily predictive of training-402
induced adaptations. Despite the lack of skeletal muscle mitochondrial adaptations, our data 403
show that a single session of exercise lasting
20
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Competing Interests 512
All authors declare no competing interests. 513
514
Author Contributions 515
Conception and design of the experiments: AJRC, MJG, JPL, JBG and MAT. Collection, 516
analysis and interpretation of the data: AJRC, MEP, ST, JPL, NC, JBG, MAT and MJG. Drafting 517
the article or revising it critically for important intellectual content: AJRC, MEP, ST, JPL, NC, 518
JBG, MAT and MJG. 519
520
Funding 521
This project was supported by operating grants from the Natural Sciences and Engineering 522
Research Council of Canada (NSERC) to MJG and MAT. AJRC was supported by a NSERC 523
PGS-D scholarship and JPL held a NSERC CGS-D scholarship. MP held NSERC CGS-M, while 524
NC held a NSERC PGS-D, and JG held NSERC CGS-M. The authors have no conflicts of 525
interest to declare. 526
527
Acknowledgements 528
We would like to thank our subjects for their commitment and effort, as well as Todd Prior, 529
Adeel Safdar, and Mahmood Akhtar for their technical assistance. 530
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Table 1. Subject characteristics for those completing acute INT versus CONT high-intensity exercise, and those completing 6 weeks of CONT-based training. Variable Acute Study Chronic Study Participants 8 men; 0 women 5 men; 4 women Age (years) 22 1 22 2 Weight (kg) 78 8 78 11 Height (cm) 181 5 173 9 VO2peak (mLkg-1min-1) 48 7 47 5 Values are mean S.D.
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Table 2. Performance characteristics for the acute INT and CONT high-intensity exercise sessions. INT CONT Total work (kJ) 66.8 6.8 67.0 6.8 Peak power output (W) 824 126 510 101* Mean power output (W) 557 90 281 46* Work duration (min:s) 2:00 0:00 4:02 0:26* Ratings of perceived exertion 18.1 1.2 18 1.8 Values are means SD, n = 8 subjects. INT, intermittent; CONT, continuous trial. *p 0.05 versus INT.
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Table 3. Cardiorespiratory data during cycling exercise at 65% VO2peak before and after 6 weeks of CONT-based training PRE-TR POST-TR Heart rate (beatsmin-1) 158 14 157 16 Respiratory Exchange Rate 0.87 0.04 0.86 0.02 Ventilation (Lmin-1) 55.6 8.1 54.2 6.2 VO2 (Lmin-1) 2.14 0.41 2.07 0.39 _______________________________________________________________________________________________________
Values are means SD, n = 9 subjects. PRE-TR, pre-training; POST-TR, post-training; VO2, oxygen uptake; CONT, continuous trial.
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Figure Legends 541
Figure 1. Muscle glycogen (A) and lactate (B) concentrations measured before (PRE) and after 542
(POST) performing ~67 kJ of work intermittently (INT) or continuously (CONT) at maximal 543
effort. Values are means SEM for 8 subjects. *P
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Figure 6. Peak oxygen uptake (VO2peak) relative to total body weight before (PRE-TR) and after 561
(POST-TR) 6 wks of low-volume, all-out CONT training. Values are means SD for 6 subjects. 562
*P0.05 vs pre-training. 563
Figure 7. Total time to complete 250 kJ of mechanical work before (PRE-TR) and after (POST-564
TR) 6 wks of low-volume, all-out CONT training. Values are means SD for 9 subjects. 565
*P0.001 vs pre-training. 566
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INT CONT0
200
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