International Journal of Sports Physiology and Performance, 2015, 10, 139-146 http://dx.doi.org/10.1123/ijspp.2013-0475 © 2015 Human Kinetics, Inc.

www.IJSPP-Journal.com ORIGINAL INVESTIGATION

The Effect of Rate of Weight Reduction on Serum Myostatin and Follistatin Concentrations in Competitive Wrestlers Mohamad S. Motevalli, Vincent J. Dalbo, Reza S. Attarzadeh, Amir Rashidlamir, Patrick S. Tucker, and Aaron T. Scanlan Purpose: To evaluate anthropometric measures and serum markers of myostatin-pathway activity after different weight-reduction protocols in wrestlers. Methods: Subjects were randomly assigned to a gradual-weight-reduction (GWR) or rapid-weight-reduction (RWR) group. Food logs were collected for the duration of the study. Anthropometric measurements and serum samples were collected after an 8-h fast at baseline and after the weight-reduction intervention. Subjects reduced body mass by 4%. The GWR group restricted calories over 12 d, while the RWR group restricted calories over 2 d. A series of 2 × 5 repeated-measures (RM) ANOVAs was conducted to examine differences in nutrient consumption, while separate 2 × 2 RM ANOVAs were conducted to examine differences in anthropometric measures and serum markers. When applicable, Tukey post hoc comparisons were conducted. Significance for all tests was set at P < .05. Results: There were no between-groups differences for any anthropometric measure (P > .05). Subjects in both groups experienced a significant reduction in body mass, fat mass, lean mass, and percent body fat (P < .05). There were no between-groups differences in serum markers of myostatin-pathway activity (P > .05), but subjects in the RWR condition experienced a significant increase in serum myostatin (P < .01), a decrease in follistatin (P < .01), and an increase in myostatin-to-follistatin ratio (P < .001). Conclusion: Although there were no between-groups differences for any outcome variables, the serum myostatin-to-follistatin ratio was significantly increased in the RWR group, possibly signaling the early stages of skeletal-muscle catabolism. Keywords: fasting, caloric restriction, calorie restriction, athletes, weight loss The myostatin pathway functions as a negative regulator of skeletal-muscle growth and differentiation1 and is influenced by regulatory steps.2 While the basic control points of myostatinpathway activity have been previously described,2,3 it is important to note that the mature form of myostatin binds to activin IIB receptors, which initiate a signaling cascade that results in the regulation of gene transcription.4 Follistatin acts as a regulator of the myostatin pathway by binding to the active form of myostatin and preventing the binding of myostatin to the activin IIB receptor.5 In this regard, myostatin inhibition via myostatin knockout6,7 or in the presence an overexpressed follistatin transgene8 is associated with an increase in skeletal-muscle mass6–8 and reduced fat mass1,9,10 compared with wild-type counterparts. Conversely, increased myostatin concentrations have been found to promote the storage of adipose tissue.11,12 Body mass and body-mass reduction have been reported to influence myostatin-pathway biomarkers in animals13,14 and humans.15–18 Specifically, myostatin is increased in skeletal muscle and adipose tissue of obese mice,13 and, as expected, muscle and plasma concentrations of myostatin have been reported to be increased in obese humans.15 In mice, 2 days of food deprivation resulted in a significant decrease in body and muscle mass with a resultant increase in myostatin mRNA expression in fast-twitch Motevalli, Attarzadeh, and Rashidlamir are with the Dept of Physical Education and Sports Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. Dalbo, Tucker, and Scanlan are with the School of Medical and Applied Sciences, Central Queensland University, Rockhampton, Australia. Address author correspondence to Mohamad Motevalli at mohamad_motevali@ yahoo.com.

tibialis anterior fibers.14 Notably, obese humans who underwent biliopancreatic diversion17 or gastric bypass surgery18 experienced a significant decrease in fat mass17,18 and a significant increase in skeletal-muscle myostatin mRNA expression,17,18 likely related to a significant decrease of lean mass.17 Finally, follistatin in white adipose tissue was found to be more highly expressed in lean than in obese women; however, after long-term weight reduction, follistatin was increased in previously obese women, resulting in follistatin expression equivalent to that in lean women.16 Taken together, these results demonstrate a clear relationship between body mass and body-mass reduction on markers of myostatin-pathway activity. Body-mass reduction in sports with weight classes (eg, wrestling, boxing, judo, and mixed martial arts) has been an ongoing concern for health care professionals. The need for additional research examining safe body-mass-reduction protocols has been highlighted by deaths of college wrestlers19 and the recent influx of participants into grappling-based sports such as mixed martial arts. Despite the literature recommending a gradual reduction in body mass through moderate energy restriction,19,20 rapid body-massreduction methods such as fluid restriction, use of saunas, plastic suits, increasing exercise intensity and duration, food restriction, laxatives, and diuretics are commonly used by athletes, particularly when the sport-regulating body does not require the use of a minimal weight program.21,22 After rapid weight reduction, studies examining the effects of weight-loss strategies in athletes have indicated that the primary cause of the decrease in body mass is fluid loss, followed by catabolism of lean body mass23; when combined, these factors can negatively affect strength,24 power,25 performance,26 and metabolic rate.27 However, the primary cause of the decrease in body mass after a gradual weight-reduction protocol is a decrease in body fat.28 139

140  Motevalli et al

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Studies have reported myostatin13,14,17,18,29 or follistatin13,16 changes after body-mass reduction; however, the majority of those studies examined intramuscular alterations of myostatin in the form of mRNA13,14,17,18,29 or protein14 in animal models13,14 and obese humans.17,18 To date, only 1 study has examined the effects of a decrease in body mass on serum myostatin concentrations. That study used a mixed-gender model with significant variations in body mass and only 5 subjects.30 To our knowledge no study has examined serum follistatin concentrations after a decrease in body mass. Considering the inhibitory function of myostatin in skeletal muscle and its regulatory role in adipose tissue, we hypothesized that serum markers of myostatin-pathway activity would be influenced by the rate of body-mass reduction typically experienced by weight-class athletes. Therefore, the purpose of our investigation was to evaluate serum concentrations of myostatin, follistatin, the serum myostatin-to-follistatin ratio, and lean mass after different body-mass-reduction protocols in well-trained wrestlers.

Methods Subjects Well-trained wrestlers (N = 30; 22.5 ± 1.7 y, 78.3 ± 8.2 kg, 12.1% ± 2.7% body fat) voluntarily participated in this study. All research procedures were granted prior approval by an institutional human research ethics committee in accordance with the Helsinki Declaration. Inclusion criteria were as follows: no history of heart disease, no history of endocrine dysfunction, no supplement use other than a multivitamin within the 6 months previous to initiation of the study,

a body-fat percentage of 6.0% or greater at the start of testing, and being competitively overweight, which was defined as having a current body mass at least 4% heavier than the weight at which the subject normally competes. Procedures were explained to subjects, and written informed consent was obtained before participation. Subjects were then randomly assigned to a gradual-weight-reduction (GWR: n = 15; 22.3 ± 1.7 y, 79.6 ± 7.2 kg, 12.2% ± 2.6% body fat) or rapid-weight-reduction (RWR: n = 15; 22.7 ± 1.8 y, 77.1 ± 9.1 kg, 12.0% ± 2.9% body fat) group.

Research Design Overview.  An overview of the research design can be seen in Figure

1. Subjects were randomly assigned to a GWR or RWR group and were required to maintain a food diary for the duration of the study. Specially, subjects in each group completed a 10-day food diary to establish baseline energy and macronutrient consumption and an additional 12-day food diary to record energy and macronutrient consumption during the weight-reduction protocols.

Gradual Weight-Reduction Protocol.  After completion of the

initial 10-day food diary, subjects in the GWR group observed an 8-hour overnight fast and reported to the laboratory for baseline testing between 8 and 8:30 AM. Baseline testing (day 1) consisted of collecting demographic information, anthropometric measures, and 10 mL of blood from an antecubital vein. Subjects then began a 12-day weight-reduction protocol (days 1–12) consisting of a progressive caloric-restriction diet and returned to the laboratory between 8 and 8:30 AM on day 13 for posttesting, following identical procedures as at baseline testing.

Figure 1 — Schematic of study design. All testing occurred between 8 and 8:30 AM after a 12-hour overnight fast and avoidance of exercise. Daily food diaries were recorded from the initiation of the study in the GWR and RWR groups. Gray bars signify the start of an 8-hour fast; black bars signify a testing time point. Abbreviations: GWR, gradual weight reduction; RWR, rapid weight reduction.

Myostatin and Follistatin   141

Rapid Weight-Reduction Protocol.  After completion of the initial 10-day food diary, subjects in the RWR group continued to maintain a food diary and reported to the laboratory on day 10 between 8 and 8:30 AM for baseline testing after an 8-hour overnight fast. Baseline testing (day 10) consisted of collecting demographic information, anthropometric measures, and 10 mL of blood from an antecubital vein. Subjects then began a 48-hour weight-reduction protocol (days 11 and 12) consisting of self-regulated weight reduction and returned to the laboratory between 8 and 8:30 AM on day 13 for posttesting, following identical procedures as at baseline testing.

Science Co, Del Rio, TX, USA) concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer instructions. Posttest serum concentrations for myostatin and follistatin were expressed relative to changes in plasma volume using hematocrit and hemoglobin values according to equations of Dill and Costill.33

Statistical Analysis The Shapiro-Wilk statistic was performed on serum myostatin and follistatin data at each time point to assess the data for normality. Since all data were normally distributed, parametric statistics were used for all analyses. A series of separate 2 × 5 (group: RWR vs GWR; time: –10 d, days 1–4, days 5–8, days 9–10, and days 11–12) repeated-measures ANOVAs was used to examine differences in energy and macronutrient consumption. When applicable, group effects were examined using independent t tests at each time point and time effects were examined with separate 1-way ANOVAs with Tukey post hoc comparisons. Separate 2 × 2 (group: RWR vs GWR; time: baseline vs posttesting) repeated-measures ANOVAs were used to examine differences in anthropometric and serum markers. When applicable, group effects were examined using independent t tests at each time point and time effects were examined using separate dependent t tests. For each test, statistical significance was established at P < .05. SPSS for Windows (v. 20.0.0.1, IBM Corp, Armonk, NY, USA) was used for all statistical analyses.

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Weight-Reduction Protocols Subjects in the GWR group reduced body mass according to a 12-day diet developed by Rashid-Lamir and Ravasi.24 The GWR protocol can be seen in Table 1. Subjects in the RWR group reduced body mass 48 hours before the conclusion of the investigation by their own design without the use of diuretics or laxatives. The RWR group was designed to reflect traditional weight-reduction methods used by athletes in weight-dependent sports.22,31 Subjects in each group were required to reduce baseline body mass by 4% and were able to consume water ad libitum. Subjects in the GWR and RWR groups each participated in daily moderate physical training over the duration of the study. Training primarily consisted of specific technical training. However, subjects in the RWR condition were able to perform additional physical training during the weightreduction phase of their protocol. To make meaningful statistical comparisons, the body-mass-reduction protocols were segmented into the following phases: phase 1 (days 1–4), phase 2 (days 5–8), phase 3 (days 9–10), and phase 4 (days 11–12).

Results Anthropometric Measures

Anthropometric Measurements

Anthropometric measures can be seen in Table 2. There were no between-groups differences at baseline or posttesting for body mass, fat mass, lean mass, or percent body fat (P > .05). After the bodymass-reduction intervention, subjects in GWR and RWR groups all lost a significant amount of body mass, fat mass, lean mass, and percent body fat (P < .01).

A calibrated scale and stadiometer (Balas stand scale and stadiometer, IRI) were used to determine height and body mass. Triceps, subscapular, and abdominal skinfold thickness were measured on the right side of the body by a trained investigator using a calibrated caliper (Lafayette skinfold caliper 01127A, Lafayette, IN, USA). Body-fat percentage, fat mass, and lean mass were calculated using Wagner equations that were developed for wrestlers.32 The same investigator collected all anthropometric measures during the course of the investigation (coefficient of variation = .39%).

Energy and Macronutrient Consumption Energy and macronutrient consumption are presented in Table 3. There was a significant group × time interaction (P < .001), time effect (P < .001), and group effect (P < .001) for kilocalories, protein, fat, and carbohydrate consumption. Subjects in the GWR group consumed significantly less kilocalories, protein, fat, and carbohydrate during phase II (days 5–8), phase III (days 9–10), and phase IV (days 11–12) of the diet compared with baseline (P < .01). Subjects in the RWR group consumed significantly less kilocalories, protein, fat, and carbohydrate during phase IV of the diet compared with baseline (P < .001). In regard to between-groups differences, subjects in the GWR group consumed significantly less kilocalories and carbohydrate during phases II (days 5–8) and

Blood Sampling and Laboratory Measurements Before each blood collection, subjects observed an 8-hour overnight fast and were instructed to avoid exercise before reporting to the laboratory between 8 and 8:30 AM. Blood was collected from an antecubital vein using standard procedures. After clotting occurred, blood samples were centrifuged at 3000 RPM for 10 minutes. The serum was removed from the centrifuge and frozen at –70°C for later analysis. Serum myostatin (bound) (human myostatin, Glory Science Co, Del Rio, TX, USA) and follistatin (human follistatin, Glory

Table 1  Gradual Weight-Reduction Protocol, Caloric Decrease From Baseline Phase 1

Phase 2

Phase 3

Phase 4

Meal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 10

Day 11

Day 12

Lunch

10%

10%

10%

0%

20%

20%

20%

10%

30%

30%

30%

20%

Dinner

10%

10%

10%

0%

20%

20%

20%

10%

30%

30%

30%

20%

Note: There was no caloric decrease at breakfast over the intervention.

142  Motevalli et al

Table 2  Participant Anthropometrics, Mean ± SD Variable

Weight-reduction group

Baseline

Posttest

Within group

Between groups

Body mass (kg)

Gradual

79.6 ± 7.2

76.2 ± 6.9†

P < .001

Group × time = .22

Rapid

77.1 ± 9.1

73.9 ± 8.7*

P < .001

Group = .42 Time < .001

Fat mass (kg)

Gradual

9.7 ± 2.4

7.3 ± 1.4†

P < .001

Group × time < .001

Rapid

9.3 ± 2.6

8.4 ± 2.2*

P < .001

Group = .71 Time < .001

Lean mass (kg)

Gradual

69.8 ± 6.2

68.9 ± 6.2†

P = .004

Group × time < .001

Rapid

67.8 ± 8.2

65.6 ± 7.9*

P < .001

Group = .32

Gradual

12.2 ± 2.6

9.6 ± 1.7†

P < .001

Group × time < .001

Rapid

12.0 ± 2.9

11.3 ± 2.7*

P < .001

Time < .001 Body fat (%)

Group = .39

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Time < .001 †Significantly less than baseline in gradual weight reduction. *Significantly less than baseline in rapid weight reduction.

Table 3  Energy and Macronutrient Consumption, Mean ± SD Energy

Group

Baseline

1–4 d

5–8 d

9–10 d

11–12 d

Within group

Between groups

Kilocalories (/d)

GWR

2960 ± 367

2768 ± 347

2533 ± 319†‡

2234 ± 283†‡

2358 ± 294†‡

P < .05

G × T < .001

RWR

2825 ± 414

2794 ± 421

2843 ± 448

2814 ± 403

548 ± 110*

P < .001

Group < .001 Time < .001

Protein (g/d)

GWR

142.1 ± 18.8

134.1 ± 17.2

122.3 ± 15.9†

108.1 ± 15.0†‡

112.5 ± 14.8†‡

P < .05

G × T < .001

RWR

136.3 ± 22.3

133.7 ± 22.0

135.0 ± 20.7

133.1 ± 18.8

35.1 ± 7.0*

P < .001

Group < .001 Time < .001

Fat (g/d)

GWR

85.4 ± 10.3

79.3 ± 9.9

72.8 ± 9.0†

64.0 ± 7.5†‡

67.7 ± 8.4†‡

P < .01

G × T < .001

RWR

80.3 ± 12.7

77.9 ± 9.7

80.7 ± 13.6

80.4 ± 13.0

9.6 ± 4.0*

P < .001

Group < .001

GWR

403.2 ± 50.2

379.1 ± 47.6

346.7 ± 43.6†‡

306.8 ± 38.0†‡

322.5 ± 40.5†‡

P < .01

G × T < .001

RWR

389.4 ± 55.7

388.1 ± 60.0

395.0 ± 60.5

389.5 ± 55.3

80.2 ± 13.9*

P < .001

Group < .001

Time < .001 Carbohydrate (g/d)

Time < .001 Abbreviations: GWR, gradual weight reduction; RWR, rapid weight reduction; G, group; T, time. †Significantly less than baseline in GWR. *Significantly less than baseline in RWR. ‡Significant between-groups difference (P < .05).

III (days 9–10) of the diet and less fat and protein during phase III (days 9–10) of the diet than the RWR group (P < .05). Subjects in the RWR group consumed significantly less kilocalories, protein, fat, and carbohydrate than subjects in the GWR group during phase IV (days 11–12) of the diet (P < .05).

Serum Myostatin and Follistatin There was a significant group × time interaction for serum myostatin concentration (P < .01). Results indicate a main effect for time (P < .01). Further analyses revealed a significant increase in serum myostatin concentration over time in the RWR group (P < .01) (Figure 2[A]). There was a significant group × time interaction for serum follistatin concentration (P < .01). The main effect for time (P < .01) revealed a significant decrease in serum follistatin concentration in the RWR group (P < .01) (Figure 2[B]). There was a

significant group × time interaction for the ratio of serum myostatin to follistatin (P < .001). The main effect for time (P < .001) revealed a significant increase in the ratio of serum myostatin to follistatin in the RWR group (P < .001) (Figure 2[C]).

Discussion This was the first study to examine the effects of body-mass reduction on serum markers of myostatin-pathway activity in athletes. The primary findings from our study were as follows: a reduction in body mass of 4% resulted in a significant decrease in lean mass in the GWR and RWR groups. Moreover, serum myostatin was significantly increased and serum follistatin was significantly decreased, resulting in a significant increase in the serum myostatin-to-follistatin ratio in the RWR group, while serum myostatin, follistatin,

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Myostatin and Follistatin   143

Figure 2 — Serum markers of myostatin pathway activity, mean ± SD. (A) Serum myostatin concentrations (ng/mL). (B) Serum follistatin concentrations (ng/mL). (C) Ratio of serum myostatin to follistatin (ng/mL). Abbreviations: G, group; T, time; GWR, gradual weight reduction; RWR, rapid weight reduction. *Significantly less than baseline in RWR. §Significantly greater than baseline in RWR.

and the myostatin-to-follistatin ratio were unchanged in the GWR group. Taken together, these findings suggest that a more catabolic environment was evident in the RWR than the GWR group. Each of the weight-reduction protocols produced a 4% reduction in body mass over the course of the study. Specifically, body mass was decreased by 4.21% ± 0.34% in the GWR group and 4.10% ± 0.20% in the RWR group. Subjects in the GWR group gradually restricted calories at a rate of approximately 15%/d over a duration of 12 days, while subjects in the RWR group restricted calories at a rate of approximately 80%/d for 2 days. Each of the body-mass-reduction protocols yielded a significant decrease in body mass, fat mass, lean mass, and percent body fat, with no between-groups differences at baseline or after the weight-reduction intervention. It was surprising that lean mass in the GWR group

was significantly decreased despite the finding that protein was consumed at a rate of ~1.42 g · kg–1 · d–1 during the most calorically restricted phase of the body-mass-reduction protocol. Conversely, subjects in the RWR group only consumed ~0.47 g · kg–1 · d–1 of protein during the 2 days of severe caloric restriction and lost nonsignificantly greater amounts of lean mass than those in the GWR group (GWR –0.9 kg, RWR –2.2 kg). However, it is important to note that the nonsignificantly greater loss of lean mass in the RWR group may be the result of dehydration,34 which is in agreement with percent change values in plasma volume experienced by the GWR (4.73% ± 6.46%) and RWR (–6.87% ± 5.45%) groups, P < .001. The role of readily available protein to reduce skeletal-muscle wasting is supported by the finding that myostatin-binding protein FLRG is significantly increased in subjects who consumed 15 g of

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whey protein immediately before and after exercise, compared with a placebo group.35 Furthermore, it is highly likely that if the severe caloric-restriction protocol were maintained for a longer duration, significant between-groups differences would have occurred, a speculation that is supported by prior research14,23 and our serum markers of myostatin-pathway activity. Serum myostatin, serum follistatin, and the ratio of serum myostatin to follistatin were unchanged in the GWR group. Conversely, in the RWR group there was a significant increase in serum myostatin and a significant decrease in serum follistatin, resulting in a significant increase in the ratio of serum myostatin to follistatin. When considered in their entirety, these findings suggest that a more catabolic environment was present in the RWR than in the GWR group. A more pronounced catabolic environment would be expected in the RWR group, as these subjects were exposed to 2 days of severe caloric restriction (548 ± 110 kcal/d) and low protein consumption (~0.47 g · kg–1 · d–1). As a result, it is highly likely that significant between-groups differences would have emerged if subjects were required to lose greater amounts of body mass. To date, only 3 studies have examined the effects of body-mass reduction on myostatin in humans, and each study was statistically underpowered (n ≤ 6).17,18,30 Milan et al17 examined the mRNA expression of myostatin in skeletal muscle from 6 morbidly obese men and women before and 18 ± 2 months after biliopancreatic diversion. The mRNA expression of myostatin in skeletal muscle was found to be significantly decreased after a 38% and 64% reduction in body mass and fat mass, respectively. Park et al18 examined the mRNA expression of myostatin in skeletal muscle from 3 morbidly obese women before and approximately 12 months after gastric bypass surgery. Subjects reduced body mass by 44%, but data for fat mass and lean mass were not provided. Skeletal-muscle mRNA expression of myostatin was significantly decreased after gastric bypass surgery. The investigations by Milan et al17 and Park et al18 used surgical procedures designed to promote substantial body-weight reduction (38% and 44%, respectively) and reported a significant decrease in skeletal-muscle mRNA expression of myostatin after body-mass stabilization. Milan et al17 speculated that the decrease in skeletal-muscle mRNA expression of myostatin may serve as a mechanism against skeletal-muscle wasting, consistent with the findings of Dalbo et al.2 In the current study, serum myostatin concentration was significantly increased in the RWR group as subjects lost a significant amount of body mass and lean mass. Unlike the Milan et al17 and Park et al18 investigations, where the body mass of subjects had stabilized, in our study, subjects in the RWR group were currently experiencing a rapid decrease in body mass. Larsen et al30 examined the effects of a 15-hour and 40-hour fast on plasma myostatin and skeletal-muscle mRNA expression of myostatin in 6 college-age men and women. They reported no change in plasma myostatin or the mRNA expression of myostatin, atrogin-1, or MuRF-1, which are genes indicative of ubiquitin proteasome-pathway activity.36 Larsen et al30 were the first to examine the effects of caloric restriction on myostatin in humans, but it is difficult to make meaningful comparisons with our study. Specifically, Larsen et al30 had a small sample size, used a mixedgender model, had subjects with varying body mass (males 79.4 ± 6.7 kg, females 63.7 ± 5.0 kg) representing a significant difference between genders, did not monitor body-composition changes, and assessed baseline serum and skeletal-muscle mRNA expression of myostatin in a fed state. Nevertheless, they acknowledged that muscle atrophy occurs in response to protein malnourishment and fasting and suggested that longer periods of fasting are likely needed

to elicit alterations in the expression of myostatin. As a result of their findings Larsen et al30 speculated that 40 hours of fasting may not be enough to elicit catabolic processes resulting in skeletal-muscle atrophy. Results from our investigation found that 48 hours of severe caloric restriction was enough to elicit a catabolic response in the form of increased serum myostatin, decreased serum follistatin, and increased serum myostatin-to-follistatin ratio. It is important to note that the use of skinfolds during a state of dehydration likely decreased the accuracy of our body-composition results37 in participants in the RWR group. However, according to a position stand under the auspices of the International Olympic Committee Medical Commission, the accuracy of each body-composition method, including Bod Pod, dual–X-ray absorptiometry, and underwater weighing, would have also been negatively influenced by participants being in a dehydrated state.37 It is also our contention that the most important finding of our study is that the RWR protocol resulted in alterations of serum markers of myostatinpathway activity that are suggestive of a catabolic environment and may represent early signs of skeletal-muscle catabolism, which is supported by work conducted in animal models.38,39 Our study was the first to examine the effects of caloric restriction on serum markers of myostatin-pathway activity in an athletic population. Since there were no between-groups differences for body-composition variables (body mass, fat mass, lean mass, and percent body fat) or serum markers of myostatin-pathway activity (myostatin, follistatin, and ratio of myostatin to follistatin), we cannot conclude that the GWR protocol employed in our investigation is more effective than the RWR protocols typically employed by weight-restricted athletes. However, athletes in the current investigation were only required to reduce body mass by 4%, which is not extreme for experienced weight-dependent athletes. Moreover, although there were no significant between-groups differences, athletes in the RWR group were in a more catabolic state based on serum markers of myostatin-pathway activity than athletes in the GWR group. Athletes in the RWR group experienced an increase in serum myostatin, a decrease in serum follistatin, and an increase in the ratio of serum myostatin to follistatin, while no change over time was observed in any of the serum markers in the GWR athletes. As a result, if caloric restriction were carried out for a longer duration it is highly likely that significant between-groups differences would have been observed for changes in lean mass and serum markers of myostatin-pathway activity. It would be prudent for future investigations to examine the effects of extreme body-mass reduction in already-lean, elite athletes to examine the effects of competitive weight “cutting” on myostatin-pathway signaling. Furthermore, our findings highlight the need for future research to examine the effects of competitive weight-reduction protocols on intramuscular signaling pathways indicative of skeletal-muscle catabolism, as mRNA and protein expression provide more detailed information than serum markers regarding adaptations occurring in skeletal-muscle tissue.

Practical Applications To our knowledge no study has examined serum follistatin concentrations after a decrease in body mass. Considering the impact that myostatin has on skeletal muscle and adipose tissue, it is important to understand how serum markers of myostatin-pathway activity are influenced by the rate of body-mass reduction, an essential component of many weight-class sports. This investigation provides useful information regarding the activity of serum concentrations of myostatin, follistatin, ratio of serum myostatin to follistatin, and lean mass after 2 body-mass-reduction protocols commonly used by

Myostatin and Follistatin   145

wrestlers. This information is vital for the development of optimal training and dieting techniques in weight-class sports.

Conclusions It can be stated with confidence that the medically preferred gradual weight-reduction method is no more effective than 2 days of severe caloric restriction in preserving skeletal-muscle mass in male athletes reducing body mass by 4%. However, it is highly likely that if severe caloric restriction were carried out for longer durations or to reduce body mass by more than 4%, a gradual weight-reduction method would likely be more effective for maintaining lean mass.

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Acknowledgments We would like to thank the wrestlers for their participation in the study. We would also like to thank Col Samuel Trautman for his technical assistance and Greg Gass for his assistance with manuscript preparation.

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The effect of rate of weight reduction on serum myostatin and follistatin concentrations in competitive wrestlers.

To evaluate anthropometric measures and serum markers of myostatin-pathway activity after different weight-reduction protocols in wrestlers...
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