Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2014/01/10/jn.113.18399 6.DCSupplemental.html
The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Acute Dietary Protein Intake Restriction Is Associated with Changes in Myostatin Expression after a Single Bout of Resistance Exercise in Healthy Young Men1,2
3 NUTRIM School for Nutrition, Toxicology, and Metabolism, Maastricht University, Maastricht, The Netherlands; 4Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada; and 5School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, UK
Abstract Skeletal muscle satellite cells (SCs) play an important role in the myogenic adaptive response to exercise. It remains to be established whether nutrition plays a role in SC activation in response to exercise. In the present study, we assessed whether dietary protein alters the SC response to a single bout of resistance exercise. Twenty healthy young (aged 21 6 2 y) males were randomly assigned to consume a 4-d controlled diet that provided either 1.2 g protein kg body weight21 d21 [normal protein diet (NPD)] or 0.1 g protein kg body weight21 d21 [low protein diet (LPD)]. On the second day of the controlled diet, participants performed a single bout of resistance exercise. Muscle biopsies from the vastus lateralis were collected before and after 12, 24, 48, and 72 h of post-exercise recovery. SC content and activation status were determined using immunohistochemistry. Protein and mRNA expression were determined using Western blotting and reverse transcription polymerase chain reaction. The number of myostatin + SCs decreased significantly at 12, 24, and 48 h (range, 214 to 249%; P < 0.05) after exercise cessation, with no differences between groups. Although the number of myostatin + SCs returned to baseline in the type II fibers on the NPD after 72 h of recovery, the number remained low on the LPD. At the 48 and 72 h time points, myostatin protein expression was elevated (86 6 26% and 88 6 29%, respectively) on the NPD (P < 0.05), whereas it was reduced at 72 h (236 6 12% compared with baseline) in the LPD group (P < 0.05). This study demonstrates that dietary protein intake does not modulate the post-exercise increase in SC content but modifies myostatin expression in skeletal muscle tissue. This trial was registered at clinicaltrials.gov as NCT01220037. J. Nutr. 144: 137–145, 2014.
Introduction Skeletal muscle stem cells, also known as satellite cells (SCs)7, are thought to play an important role in the myogenic adaptive response to exercise. SCs reside in a niche between the basal lamina and the sarcolemma of their associated muscle fiber. 1 Author disclosures: T. Snijders, L. B. Verdijk, B. R. McKay, J. S. J. Smeets, J. van Kranenburg, B. B. B. Groen, G. Parise, P. Greenhaff, and L. J. C. van Loon, no conflicts of interest. 2 Supplemental Figures 1–3 and Supplemental Table 1 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. 6 Present address: Department of Human Movement Sciences, Faculty of Health, Medicine, and Life Sciences, Maastricht University, Maastricht, The Netherlands. 7 Abbreviations used: BW, body weight; Ct, threshold cycle; DAPI, 4#,6-diamidino-2-fenylindool; En%, energy percent; LPD, low protein diet; MRF, myogenic regulatory factor; MyoD, myogenic differentiation; NPD, normal protein diet; SC, satellite cell; 1RM, one-repetition maximum. * To whom correspondence should be addressed. E-mail: L.vanLoon@ maastrichtuniversity.nl.
Normally, SCs remain in a quiescent state, but, in response to exercise and/or muscle damage, SCs can become activated and subsequently proliferate and differentiate. SCs differentiate to form new myofibers, fuse with existing fibers, or return to a quiescent state, replenishing the resident pool of SCs through selfrenewal (1). The progression of SCs through the myogenic program is mediated by the expression of different myogenic regulatory factors (MRFs) [e.g., myogenic differentiation (MyoD), myogenic factor-5, myogenin, and Mrf4]. The activation, proliferation, and differentiation processes of the SCs represent important regulatory steps that ultimately determine their fate in supporting skeletal muscle fiber adaptation. In addition to the MRFs, myostatin has been identified as a key regulator in the SC response to exercise and/or muscle damage. In animal muscle, reduced myostatin expression has been shown to result in substantial muscle hypertrophy (2,3). More recent work by McKay et al. (4) suggests that, in humans, myostatin is a strong negative regulator of the SC myogenic response to an acute bout
ã 2014 American Society for Nutrition. Manuscript received August 19, 2013. Initial review completed September 25, 2013. Revision accepted November 17, 2013. First published online December 4, 2013; doi:10.3945/jn.113.183996.
137
Downloaded from jn.nutrition.org at UNIVERSITÄTSBIBLIOTHEK HANNOVER (TIB/UB) on January 10, 2015
Tim Snijders,3,6 Lex B. Verdijk,3,6 Bryon R. McKay,4 Joey S.J. Smeets,3,6 Janneau van Kranenburg,3,6 Bart B.B. Groen,3,6 Gianni Parise,4 Paul Greenhaff,5 and Luc J.C. van Loon3,6*
Participants and Methods Participants. Twenty healthy male volunteers (aged 21 6 2 y; height, 1.82 6 0.01 m; bodyweight, 78 6 2 kg) participated in this study (Table 1). All participants were recreationally active, which was defined as participating in noncompetitive sporting activities being performed less than three times a week. Participants were informed about the nature and risks of the experimental procedures before their written consent was obtained. The study was approved by the Medical Ethical Committee of the Maastricht University Medical Centre and complied with the guidelines set out in the Declaration of Helsinki. Exclusion criteria were defined that would preclude successful participation in the exercise session, which included (silent) cardiac or peripheral vascular disease and orthopedic limitations. After inclusion, all participants were randomly assigned to consume either a controlled NPD or LPD. Body composition (fat and fat-free mass) was determined by dual energy x-ray absorptiometry scan (Hologic). Lean mass and percentage body fat were determined on a whole-body level and for specific regions (e.g., trunk and legs). In addition, all participants performed a familiarization trial to become experienced with the research protocol and the equipment. Proper lifting technique was demonstrated and then practiced by the participants for each of the 2 lower-limb exercises (leg press and leg extension). Subsequently, maximal strength (one-repetition maximum [1RM]) was estimated by using the multiple repetitions testing procedure during 2 separate visits (19). All strength tests were completed at least 2 wk before the start of the experimental protocol. Protocol. At 0800 h, after 24 h of the controlled diet, participants reported to the laboratory after an overnight fast. After 30 min of supine 138
Snijders et al.
TABLE 1 Participant characteristics of healthy young men who performed a single bout of resistance-type exercise and consumed a normal or low protein diet for 4 d1
Age, y Anthropometrics Height, m Weight, kg BMI, kg/m2 Body composition Whole-body lean mass, kg Leg lean mass, kg Leg fat, % 1RM muscle strength, kg Leg press Leg extension
NPD group
LPD group
22 6 1
21 6 1
1.82 6 0.02 75.9 6 1.8 23.0 6 0.6
1.83 6 0.03 79.2 6 3.0 23.7 6 0.8
58.4 6 17.2 21.1 6 0.4 15.1 6 1.7
59.7 6 18.8 22.3 6 0.9 16.9 6 1.8
214 6 7 137 6 4
235 6 9 138 6 5
1 Values are means 6 SEMs, n = 10 per group. LPD, low protein diet group; NPD, normal protein diet group; 1RM, one-repetition maximum.
rest, a muscle biopsy was taken from the musculus vastus lateralis. Thereafter, participants were provided with a standardized breakfast. Next, participants performed a single bout of resistance exercise, consisting of 2 different exercises. Participants performed six sets of 10 repetitions at 75% 1RM on the horizontal leg press machine (Technogym) and six sets of 10 repetitions at 75% 1RM on the leg extension machine (Technogym). A resting period of 2 min between sets was allowed. The entire protocol required ;40 min to complete. All participants were verbally encouraged during the test to complete the entire protocol. Before and after the exercise session, a 5–10 min warm up/cooling down was performed on a cycle ergometer. At the end of the exercise protocol the participants rested for 3 h in a supine position. At 1230 h, participants received a standardized lunch, after which participants were provided with a standardized dinner to be consumed at 1800 h that evening. On the same day, participants reported back to the laboratory at 2030 h for a second muscle biopsy sample collection (t = 12 h). Next, muscle biopsies were taken in an overnight fasted state exactly 24, 48, and 72 h after the start of the single exercise bout. During the 72 h post-exercise recovery, all participants adhered to either a standardized NPD (13 En% protein) or LPD (
The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Acute Dietary Protein Intake Restriction Is Associated with Changes in Myostatin Expression after a Single Bout of Resistance Exercise in Healthy Young Men1,2
3 NUTRIM School for Nutrition, Toxicology, and Metabolism, Maastricht University, Maastricht, The Netherlands; 4Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada; and 5School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, UK
Abstract Skeletal muscle satellite cells (SCs) play an important role in the myogenic adaptive response to exercise. It remains to be established whether nutrition plays a role in SC activation in response to exercise. In the present study, we assessed whether dietary protein alters the SC response to a single bout of resistance exercise. Twenty healthy young (aged 21 6 2 y) males were randomly assigned to consume a 4-d controlled diet that provided either 1.2 g protein kg body weight21 d21 [normal protein diet (NPD)] or 0.1 g protein kg body weight21 d21 [low protein diet (LPD)]. On the second day of the controlled diet, participants performed a single bout of resistance exercise. Muscle biopsies from the vastus lateralis were collected before and after 12, 24, 48, and 72 h of post-exercise recovery. SC content and activation status were determined using immunohistochemistry. Protein and mRNA expression were determined using Western blotting and reverse transcription polymerase chain reaction. The number of myostatin + SCs decreased significantly at 12, 24, and 48 h (range, 214 to 249%; P < 0.05) after exercise cessation, with no differences between groups. Although the number of myostatin + SCs returned to baseline in the type II fibers on the NPD after 72 h of recovery, the number remained low on the LPD. At the 48 and 72 h time points, myostatin protein expression was elevated (86 6 26% and 88 6 29%, respectively) on the NPD (P < 0.05), whereas it was reduced at 72 h (236 6 12% compared with baseline) in the LPD group (P < 0.05). This study demonstrates that dietary protein intake does not modulate the post-exercise increase in SC content but modifies myostatin expression in skeletal muscle tissue. This trial was registered at clinicaltrials.gov as NCT01220037. J. Nutr. 144: 137–145, 2014.
Introduction Skeletal muscle stem cells, also known as satellite cells (SCs)7, are thought to play an important role in the myogenic adaptive response to exercise. SCs reside in a niche between the basal lamina and the sarcolemma of their associated muscle fiber. 1 Author disclosures: T. Snijders, L. B. Verdijk, B. R. McKay, J. S. J. Smeets, J. van Kranenburg, B. B. B. Groen, G. Parise, P. Greenhaff, and L. J. C. van Loon, no conflicts of interest. 2 Supplemental Figures 1–3 and Supplemental Table 1 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. 6 Present address: Department of Human Movement Sciences, Faculty of Health, Medicine, and Life Sciences, Maastricht University, Maastricht, The Netherlands. 7 Abbreviations used: BW, body weight; Ct, threshold cycle; DAPI, 4#,6-diamidino-2-fenylindool; En%, energy percent; LPD, low protein diet; MRF, myogenic regulatory factor; MyoD, myogenic differentiation; NPD, normal protein diet; SC, satellite cell; 1RM, one-repetition maximum. * To whom correspondence should be addressed. E-mail: L.vanLoon@ maastrichtuniversity.nl.
Normally, SCs remain in a quiescent state, but, in response to exercise and/or muscle damage, SCs can become activated and subsequently proliferate and differentiate. SCs differentiate to form new myofibers, fuse with existing fibers, or return to a quiescent state, replenishing the resident pool of SCs through selfrenewal (1). The progression of SCs through the myogenic program is mediated by the expression of different myogenic regulatory factors (MRFs) [e.g., myogenic differentiation (MyoD), myogenic factor-5, myogenin, and Mrf4]. The activation, proliferation, and differentiation processes of the SCs represent important regulatory steps that ultimately determine their fate in supporting skeletal muscle fiber adaptation. In addition to the MRFs, myostatin has been identified as a key regulator in the SC response to exercise and/or muscle damage. In animal muscle, reduced myostatin expression has been shown to result in substantial muscle hypertrophy (2,3). More recent work by McKay et al. (4) suggests that, in humans, myostatin is a strong negative regulator of the SC myogenic response to an acute bout
ã 2014 American Society for Nutrition. Manuscript received August 19, 2013. Initial review completed September 25, 2013. Revision accepted November 17, 2013. First published online December 4, 2013; doi:10.3945/jn.113.183996.
137
Downloaded from jn.nutrition.org at UNIVERSITÄTSBIBLIOTHEK HANNOVER (TIB/UB) on January 10, 2015
Tim Snijders,3,6 Lex B. Verdijk,3,6 Bryon R. McKay,4 Joey S.J. Smeets,3,6 Janneau van Kranenburg,3,6 Bart B.B. Groen,3,6 Gianni Parise,4 Paul Greenhaff,5 and Luc J.C. van Loon3,6*
Participants and Methods Participants. Twenty healthy male volunteers (aged 21 6 2 y; height, 1.82 6 0.01 m; bodyweight, 78 6 2 kg) participated in this study (Table 1). All participants were recreationally active, which was defined as participating in noncompetitive sporting activities being performed less than three times a week. Participants were informed about the nature and risks of the experimental procedures before their written consent was obtained. The study was approved by the Medical Ethical Committee of the Maastricht University Medical Centre and complied with the guidelines set out in the Declaration of Helsinki. Exclusion criteria were defined that would preclude successful participation in the exercise session, which included (silent) cardiac or peripheral vascular disease and orthopedic limitations. After inclusion, all participants were randomly assigned to consume either a controlled NPD or LPD. Body composition (fat and fat-free mass) was determined by dual energy x-ray absorptiometry scan (Hologic). Lean mass and percentage body fat were determined on a whole-body level and for specific regions (e.g., trunk and legs). In addition, all participants performed a familiarization trial to become experienced with the research protocol and the equipment. Proper lifting technique was demonstrated and then practiced by the participants for each of the 2 lower-limb exercises (leg press and leg extension). Subsequently, maximal strength (one-repetition maximum [1RM]) was estimated by using the multiple repetitions testing procedure during 2 separate visits (19). All strength tests were completed at least 2 wk before the start of the experimental protocol. Protocol. At 0800 h, after 24 h of the controlled diet, participants reported to the laboratory after an overnight fast. After 30 min of supine 138
Snijders et al.
TABLE 1 Participant characteristics of healthy young men who performed a single bout of resistance-type exercise and consumed a normal or low protein diet for 4 d1
Age, y Anthropometrics Height, m Weight, kg BMI, kg/m2 Body composition Whole-body lean mass, kg Leg lean mass, kg Leg fat, % 1RM muscle strength, kg Leg press Leg extension
NPD group
LPD group
22 6 1
21 6 1
1.82 6 0.02 75.9 6 1.8 23.0 6 0.6
1.83 6 0.03 79.2 6 3.0 23.7 6 0.8
58.4 6 17.2 21.1 6 0.4 15.1 6 1.7
59.7 6 18.8 22.3 6 0.9 16.9 6 1.8
214 6 7 137 6 4
235 6 9 138 6 5
1 Values are means 6 SEMs, n = 10 per group. LPD, low protein diet group; NPD, normal protein diet group; 1RM, one-repetition maximum.
rest, a muscle biopsy was taken from the musculus vastus lateralis. Thereafter, participants were provided with a standardized breakfast. Next, participants performed a single bout of resistance exercise, consisting of 2 different exercises. Participants performed six sets of 10 repetitions at 75% 1RM on the horizontal leg press machine (Technogym) and six sets of 10 repetitions at 75% 1RM on the leg extension machine (Technogym). A resting period of 2 min between sets was allowed. The entire protocol required ;40 min to complete. All participants were verbally encouraged during the test to complete the entire protocol. Before and after the exercise session, a 5–10 min warm up/cooling down was performed on a cycle ergometer. At the end of the exercise protocol the participants rested for 3 h in a supine position. At 1230 h, participants received a standardized lunch, after which participants were provided with a standardized dinner to be consumed at 1800 h that evening. On the same day, participants reported back to the laboratory at 2030 h for a second muscle biopsy sample collection (t = 12 h). Next, muscle biopsies were taken in an overnight fasted state exactly 24, 48, and 72 h after the start of the single exercise bout. During the 72 h post-exercise recovery, all participants adhered to either a standardized NPD (13 En% protein) or LPD (
