[gibala, cochran et al, 2014] intermittent and continuous high-intensity exercise induce similar...

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

21

) at MCMASTER UNIV on February 16, 2014ep.physoc.orgDownloaded from Exp Physiol (

2

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


5

, 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


6

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


7

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


8

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


9

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


10

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


11

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


12

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


13

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


14

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


16

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


17

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


19

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

References 422

Akimoto T, Pohnert SC, Li P, et al. (2005). Exercise stimulates Pgc-1alpha transcription in 423 skeletal muscle through activation of the p38 MAPK pathway. J.Biol.Chem. 280, 19587-19593. 424 Bassett DR,Jr & Howley ET (2000). Limiting factors for maximum oxygen uptake and 425 determinants of endurance performance. Med.Sci.Sports Exerc. 32, 70-84. 426

Birk JB & Wojtaszewski JF (2006). Predominant alpha2/beta2/gamma3 AMPK activation during 427 exercise in human skeletal muscle. J.Physiol. 577, 1021-1032. 428

Bishop DJ, Granata C, Eynon N (2013). Can we optimise the exercise training prescription to 429 maximize improvements in mitochondrial function and content? Biochimica et Biophysica Acta. 430 In press. 431

Borg GA (1974). Perceived exertion. Exerc.Sport Sci.Rev. 2, 131-153. 432

Boyd JC, Simpson CA, Jung ME & Gurd BJ (2013). Reducing the intensity and volume of 433 interval training diminishes cardiovascular adaptation but not mitochondrial biogenesis in 434 overweight/obese men. PLoS One 8, e68091. 435

Burgomaster KA, Cermak NM, Phillips SM, Benton CR, Bonen A & Gibala MJ (2007). 436 Divergent response of metabolite transport proteins in human skeletal muscle after sprint interval 437 training and detraining. Am.J.Physiol.Regul.Integr.Comp.Physiol. 292, R1970-6. 438

Burgomaster KA, Howarth KR, Phillips SM, et al. (2008). Similar metabolic adaptations during 439 exercise after low volume sprint interval and traditional endurance training in humans. J.Physiol. 440 586, 151-160. 441

Burgomaster KA, Hughes SC, Heigenhauser GJ, Bradwell SN & Gibala MJ (2005). Six sessions 442 of sprint interval training increases muscle oxidative potential and cycle endurance capacity in 443 humans. J.Appl.Physiol. 98, 1985-1990. 444

Calvo JA, Daniels TG, Wang X, et al. (2008). Muscle-specific expression of PPARgamma 445 coactivator-1alpha improves exercise performance and increases peak oxygen uptake. 446 J.Appl.Physiol. 104, 1304-1312. 447

Cochran AJ, Little JP, Tarnopolsky MA & Gibala MJ (2010). Carbohydrate feeding during 448 recovery alters the skeletal muscle metabolic response to repeated sessions of high-intensity 449 interval exercise in humans. J.Appl.Physiol. 108, 628-636. 450

Edge J, Eynon N, McKenna MJ, Goodman CA, Harris RC & Bishop DJ (2013). Altering the rest 451 interval during high-intensity interval training does not affect muscle or performance 452 adaptations. Exp.Physiol. 98, 481-490. 453


21

Egan B, Carson BP, Garcia-Roves PM, et al. (2010). Exercise intensity-dependent regulation of 454 peroxisome proliferator-activated receptor coactivator-1 mRNA abundance is associated with 455 differential activation of upstream signalling kinases in human skeletal muscle. J.Physiol. 588, 456 1779-1790. 457

Fan M, Rhee J, St-Pierre J, et al. (2004). Suppression of mitochondrial respiration through 458 recruitment of p160 myb binding protein to PGC-1alpha: modulation by p38 MAPK. Genes Dev. 459 18, 278-289. 460

Geng T, Li P, Okutsu M, et al. (2010). PGC-1alpha plays a functional role in exercise-induced 461 mitochondrial biogenesis and angiogenesis but not fiber-type transformation in mouse skeletal 462 muscle. Am.J.Physiol.Cell.Physiol. 298, C572-9. 463

Gibala MJ, Little JP, van Essen M, et al. (2006). Short-term sprint interval versus traditional 464 endurance training: similar initial adaptations in human skeletal muscle and exercise 465 performance. J.Physiol. 575, 901-911. 466

Handschin C, Chin S, Li P, et al. (2007). Skeletal muscle fiber-type switching, exercise 467 intolerance, and myopathy in PGC-1alpha muscle-specific knock-out animals. J.Biol.Chem. 282, 468 30014-30021. 469

Jager S, Handschin C, St-Pierre J & Spiegelman BM (2007). AMP-activated protein kinase 470 (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. 471 Proc.Natl.Acad.Sci.U.S.A. 104, 12017-12022. 472

Larsen S, Nielsen J, Hansen CN, et al. (2012). Biomarkers of mitochondrial content in skeletal 473 muscle of healthy young human subjects. J.Physiol. 590, 3349-3360. 474

Little JP, Safdar A, Wilkin GP, Tarnopolsky MA & Gibala MJ (2010). A practical model of low-475 volume high-intensity interval training induces mitochondrial biogenesis in human skeletal 476 muscle: potential mechanisms. J.Physiol. 588, 1011-1022. 477

Perry CG, Heigenhauser GJ, Bonen A & Spriet LL (2008). High-intensity aerobic interval 478 training increases fat and carbohydrate metabolic capacities in human skeletal muscle. 479 Appl.Physiol.Nutr.Metab. 33, 1112-1123. 480

Pogozelski AR, Geng T, Li P, et al. (2009). P38gamma Mitogen-Activated Protein Kinase is a 481 Key Regulator in Skeletal Muscle Metabolic Adaptation in Mice. PLoS One 4, e7934. 482

Puigserver P, Rhee J, Lin J, et al. (2001). Cytokine stimulation of energy expenditure through 483 p38 MAP kinase activation of PPARgamma coactivator-1. Mol.Cell 8, 971-982. 484

Talanian JL, Galloway SD, Heigenhauser GJ, Bonen A & Spriet LL (2007). Two weeks of high-485 intensity aerobic interval training increases the capacity for fat oxidation during exercise in 486 women. J.Appl.Physiol. 102, 1439-1447. 487


22

Timmons JA (2011). Variability in training-induced skeletal muscle adaptation. J.Appl.Physiol. 488 110, 846-853. 489

Tjonna AE, Leinan IM, Bartnes AT, et al. (2013). Low- and high-volume of intensive endurance 490 training significantly improves maximal oxygen uptake after 10-weeks of training in healthy 491 men. PLoS One 8, e65382. 492

Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M & Holloszy JO (2000). Activation of 493 AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. 494 J.Appl.Physiol. 88, 2219-2226. 495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511


23

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

531

532

533


24

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.

534

535

536


25

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.

537

538

539


26

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.

540


27

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

28

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

567


INT CONT0

200

400

600 PRE

POST *

A)M

us

cle

Gly

co

ge

n(m

mo

l/kg

d.w

.)

INT CONT0

20

40

60

80B)

Mu

sc

le L

ac

tate

(mm

ol/k

g d

.w.)


INT CONT0.0

0.5

1.0

1.5

2.0

PRE

POST*

A) INT CONT

PRE POST PRE POST

p-p38

t-p38

p3

8 M

AP

KP

ho

sp

ho

ryla

tio

n (

a.u

.)

INT CONT0.00

0.02

0.04

0.06

0.08

0.10

B) INT CONT

PRE POST PRE POST

p-ACC

AC

C P

ho

sp

ho

ryla

tio

n (

a.u

.)


INT CONT0.0

0.1

0.2

0.3 PRE

POST*P

GC

-1a

mR

NA

(a

.u.)


PRE-TR POST-TR0

1

2

3

4C

S M

ax

ima

l A

cti

vit

y(m

mo

lk

g p

rote

in-1

min

-1)


Com

plex

I ND

UFA9

Com

plex

II S

ubun

it 70

kDa

Com

plex

III C

ore

Prot

ein

2

Com

plex

IV S

ubun

it IV a

Com

plex

V A

TP S

ynth

ase

0

1

2

3

4PRE-TRPOST-TR

*

Pro

tein

Co

nte

nt

(a.u

.)


PRE-TR POST-TR0

20

40

60 *V

O2

pe

ak (

ml k

g b

w-1

min

-1)


PRE-TR POST-TR0

10

20

30 *T

ime

to

co

mp

lete

25

0 k

J (

min

)


[gibala, cochran et al, 2014] intermittent and continuous high-intensity exercise induce similar...

Documents