Ingestion of a Multi-Ingredient Supplement Does Not Alter Exercise-Induced Satellite Cell Responses in Older Men

Ingestion of a Multi-Ingredient Supplement Does Not Alter Exercise-Induced Satellite Cell... Abstract Background Nutritional supplementation can have beneficial effects on body composition, strength, and function in older adults. However, whether the response of satellite cells can be altered by nutritional supplementation in older adults remains unknown. Objective We assessed whether a multi-ingredient protein-based supplement taken over a prolonged period of time could alter the muscle satellite cell response after exercise in older men. Methods Twenty-seven older men [mean ± SD age: 73 ± 1 y; mean ± SD body mass index (kg/m2): 28 ± 1] participated in a randomized double-blind experiment. Participants were randomly divided into an experimental (EXP) group (n = 13) who consumed a multi-ingredient protein-based supplement [30 g whey protein, 2.5 g creatine, 500 IU vitamin D, 400 mg Ca, and 1500 mg n–3 (ω-3) polyunsaturated fatty acids] 2 times/d for 7 wk or a control (CON; 22 g maltodextrin) group (n = 14). After 7 wk of supplementation, all participants performed a single resistance exercise session, and muscle biopsy samples were taken from the vastus lateralis before and 24 and 48 h after exercise. Immunohistochemistry was used to assess the change in type I and II muscle fiber satellite cell content and activation status of the cells. In addition, mRNA expression of the myogenic regulatory factors was determined by using reverse transcriptase–polymerase chain reaction. Results In response to the single bout of exercise, type I muscle fiber satellite cell content was significantly increased at 24 h (0.132 ± 0.015 and 0.131 ± 0.011 satellite cells/fiber in CON and EXP groups, respectively) and 48 h (0.126 ± 0.010 and 0.120 ± 0.012 satellite cells/fiber in CON and EXP groups, respectively) compared with pre-exercise (0.092 ± 0.007 and 0.118 ± 0.017 satellite cells/fiber in CON and EXP groups, respectively) muscle biopsy samples (P < 0.01), with no difference between the 2 groups. In both groups, we observed no significant changes in type II muscle fiber satellite cell content after exercise. Conclusion Ingesting a multi-ingredient protein-based supplement for 7 wk did not alter the type I or II muscle fiber satellite cell response during postexercise recovery in older men. This trial was registered at www.clinicaltrials.gov as NCT02281331. whey protein, aging, MyoD, creatine, muscle Introduction Sarcopenia, the progressive loss of skeletal muscle mass and strength with age (1), is associated with the development of functional impairments and increased risk of morbidity and mortality (2–4). Skeletal muscle satellite cells are indispensable for muscle fiber regeneration, repair, and growth (5, 6). Reduced muscle satellite cell number, function, or both occur with aging and have been suggested to play an important role in the development of sarcopenia, impaired muscle fiber adaptive response, or both during prolonged exercise training in older adults (7). Exercise training and nutritional supplementation are the 2 most extensively investigated anabolic strategies to counter the effects of sarcopenia. It has been well established that skeletal muscle mass, function, fiber size, and satellite cell content increase significantly in response to resistance exercise training (8–11). In contrast, mixed results have been reported for the impact of nutritional supplementation on muscle mass and function in older adults. Whereas some studies showed clear improvements in muscle mass or function with the ingestion of nutritional supplements (12–15), others did not (16–18). This discrepancy is likely explained by the timing, amount, or composition of the nutritional supplement. Heterogeneity of the need for multiple nutrients in older persons provides a rationale for a multi-ingredient supplement to yield a benefit across a larger group of older adults. Accordingly, we recently reported that a multi-ingredient protein-based supplement, independent of exercise training, can increase skeletal muscle mass and strength in healthy older adults (19). However, whether longer-term ingestion of a multi-ingredient protein-based nutritional supplement can mitigate the impairment in muscle satellite cell function in older adults remains unknown. A limited number of studies have reported on the impact of nutrition on muscle satellite cell content, function, or both. In vitro studies have shown that the administration of the essential amino acid leucine increases satellite cell proliferation (20) and Myogenic Differentiation (MyoD) expression (21). In addition, the nutrient-sensitive signaling protein mammalian target of rapamycin complex 1 has been reported to control the transition of quiescent satellite cells from the G0 to G(alert) phase (22, 23). We have previously shown that low (0.1 g protein ⋅ kg body weight−1 ⋅ d−1) compared with normal (1.1 g protein ⋅ kg body weight−1 ⋅ d−1) protein intake did not influence the muscle satellite cell response after exercise in healthy young men (24). Conversely, Farup et al. (25) reported an enhanced muscle satellite cell response when additional whey protein was supplemented during the first 48 h after an exercise bout in young men. Reidy et al. (26) reported that the ingestion of an essential amino acid supplement restored a normally impaired satellite cell activation response in older men; however, the improvement in satellite cell activation was not accompanied by a significant increase in satellite cell content. In that study (26), a limited sample size, sampling muscle at only a single postexercise time point, or the provision of the supplement only once postexercise may have obscured the observation of a significant increase in satellite cell content. Therefore, in the current study, we undertook a more extensive evaluation of whether a nutritional supplement, provided 2 times/d over a 7-wk period, can affect the satellite cell response after a single bout of exercise in older men. Methods Participants Twenty-seven healthy older men [mean ± SD age: 73 ± 1 y; weight: 84 ± 4 kg; mean ± SD BMI (kg/m2): 28 ± 1] were recruited to participate in a 7-wk nutritional intervention program. All participants had a BMI in the normal to overweight range and resting blood pressure <140/90 mm Hg. All participants showed normal cardiac function during a maximal exercise stress test. Exclusion criteria included smoking, diabetes, regular use of nonsteroidal anti-inflammatory drugs, use of statins, and history of chronic illness that would affect the results of the investigation. An oral-glucose-tolerance test was performed to exclude participants with type 2 diabetes. All subjects had not participated in any structured exercise program in the past year and were living independently. All participants were informed of the nature and possible risks of the experimental procedures before their written informed consent was obtained. The study was approved by the Hamilton Health Sciences Integrated Research Ethics Board and conformed to the guidelines outlined in the Declaration of Helsinki. Participants gave their informed written consent before their inclusion in the study. The study was part of a larger project investigating the impact of nutrition and exercise training on skeletal muscle mass and strength and metabolic health in older adults (19, 27). This trial was registered at www.clinicaltrials.gov as NCT02281331. Experimental outline An overview of the double-blind randomized study design with parallel groups is shown in Supplemental Figure 1. After inclusion into the study, participants were randomly assigned to an experimental (EXP) group in which they ingested a multi-ingredient protein-based supplement or to a control (CON) group who ingested a carbohydrate-based beverage. Supplements were consumed 2 times/d for 7 wk. Anthropometric measurements (weight, height), muscle strength [1 repetition maximum (1RM)], dietary intake (3-d food intake record), and body composition (DXA scan) were assessed before (between T = −2 and T = 0) and after (week 6; T = 6) the intervention period. Finally, in week 7 (T = 7), all participants performed a single bout of resistance exercise to assess the acute muscle satellite cell response; muscle biopsy samples were taken before exercise and at 24 and 48 h after exercise. Supplementation Participants were randomly assigned to an EXP group receiving a multi-ingredient protein-based supplement or a CON group receiving a carbohydrate-based placebo. The multi-ingredient supplement contained 30 g whey protein, 2.5 g creatine, 400 mg Ca, 500 IU vitamin D, 1 g carbohydrate, and 0.75 g n–3 FAs (which delivered 0.7 g EPA and 0.45 g DHA), providing a total of 210 kJ. The composition of the nutritional supplement was based on a previous publications showing isolated positive effects of protein (28), creatine (29), vitamin D (30), or n–3 FA (13) supplementation with or without exercise training on muscle mass and strength gains in older adults. The control supplement contained 22 g carbohydrate, providing 62 kJ. Beverages were masked for taste and smell. Participants ingested the drinks twice daily, once after breakfast and once in the evening before bedtime. All drinks were provided in a double-blinded manner. Supplementation was maintained during posttesting and through the 48-h exercise (T = 6 and T = 7) period. Single bout of resistance exercise All of the participants performed a single bout of resistance exercise after 7 wk of supplementation. The single bout of exercise consisted of 3 sets of 10 repetitions each at 65% of 1RM leg press, chest press, horizontal row, and leg extension (HUR). Exercise was performed under personal supervision. All participants were verbally encouraged during the exercise, and the final set of each exercise was performed to volitional failure. A resting period of 2 min between sets was allowed. Before and after the resistance exercise, a 5-min warm-up and cool-down were performed on a cycle ergometer. Habitual dietary intake and physical activity Before the onset and in week 6 (T = 6) of the nutritional intervention, participants recorded a 3-d dietary record (2 weekdays, 1 weekend day) to assess potential changes in daily food intake that might have occurred during the intervention. Food intake records were analyzed as described previously (19). All participants were instructed not to perform any vigorous physical activity 5 d before the postexercise acute satellite cell response measurement. Body composition Whole-body and regional lean soft tissue mass (i.e., fat-free and bone-free mass), fat mass, and bone mineral content were measured with the use of DXA (GE-Lunar iDXA; Aymes Medical) after a 10- to 12-h overnight fast, as described previously (19). Muscle strength Muscle strength was assessed by using 1RM strength tests for the following exercises: leg press, chest press, lateral pull-down, horizontal row, shoulder press, and leg extension (HUR), as described previously (19). Muscle biopsy Percutaneous needle biopsy samples were taken, after an overnight (∼10 h) fast, from the midportion of the vastus lateralis under local anesthetic with the use of a 5-mm Bergstrom needle adapted for manual suction. Subjects had not participated in any physical activity for ≥5 d before the collection of the resting muscle biopsy sample. Consecutive muscle biopsy sampling was alternated between legs. The leg in which the baseline biopsy sample was drawn from was randomized. Incisions for the repeated muscle biopsy sampling in the same leg (e.g., pre- and 48-h postexercise sample) were spaced by at ≥3 cm to minimize any effect of the previous biopsy. Upon excision, muscle samples were immediately mounted in optimal cutting temperature compound and frozen in liquid nitrogen–cooled isopentane. A second sample was snap-frozen directly in liquid nitrogen and stored at −80°C for further analyses. Immunohistochemistry Muscle cross-sections (7 µm) were prepared from unfixed optimal cutting temperature embedded samples, allowed to air dry for 30 min, and stored at −80°C. Slides were then stained with antibodies against Pax7 (neat; Developmental Studies Hybridoma Bank), MyoD (anti-MyoD1; clone 5.8A, 1:50; Dako), A4.951 [myosin heavy chain (MHC) type I, slow isoform, 1:1; Developmental Studies Hybridoma Bank], MHC-II (fast isoform, 1:1000; ab91506; Abcam), and laminin (anti-laminin; ab11575; Abcam). Secondary antibodies used were Pax7 (Alexa Fluor 488 or 594, 1:500; Invitrogen, Molecular Probes), MyoD (biotinylated secondary antibody, 1:200; Vector Canada; and streptavidin-594 fluorochrome, 1:500; Invitrogen, Molecular Probes), A4.951 (Alexa Fluor 488, 1:500), MHC-II (Alexa Fluor 647, 1:500), and laminin (Alexa Fluor 488, 647; 1:500). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; 1:20,000; Sigma-Aldrich) before cover slipping with fluorescent mounting media (Dako). Slides were viewed with a Nikon Eclipse Ti Microscope (Nikon Instruments) and equipped with a high-resolution Photometrics CoolSNAP HQ2 fluorescent camera (Nikon Instruments). Images were captured and analyzed by using Nikon NIS Elements AR 3.2 software (Nikon Instruments). All images were obtained with the 20× objective, and ≥200 muscle fibers/subject per time point were included in the analyses for satellite cell content and activation status (i.e., Pax7+/MyoD− or Pax7+/MyoD+), fiber cross-sectional area, and perimeter. The activation status of satellite cells was determined via the colocalization of Pax7+ and DAPI (Pax7+/MyoD) or the colocalization of Pax7, MyoD, and DAPI (i.e., Pax7+/MyoD+). Slides were blinded for both group and time point. All immunofluorescent analyses were completed in a blinded fashion. Quantitative real-time RT-PCR RNA was isolated from 15–25 mg muscle tissue with the use of the Trizol/RNeasy method, as described previously (31). All qPCR reactions were performed in duplicate in 25-µL volumes containing RT Sybr Green qPCR Master Mix (Qiagen Sciences), prepared with the epMotion 5075 Eppendorf automated pipetting system (Eppendorf), and carried out by using an Eppendorf Realplex2 Master Cycler epgradient (Eppendorf). Primers are listed in Supplemental Table 1. mRNA expression was calculated by using the 2−∆∆Ct method and expressed as fold change from pre (pre-exercise), as described previously (32). Briefly, Ct values were first normalized to the housekeeping gene GAPDH. GAPDH expression was not different from pre-exercise at any of the postexercise time points. Ct values normalized to GAPDH were expressed as ΔΔCt. Statistical analyses A samples calculation was performed based on our previously published data (33) and a clinical relevant difference of 51% (SD = 42%) between the EXP and CON groups in type II muscle fiber satellite cell content increase at 48 h after the single exercise session. Taking into consideration a drop-out rate of 10% during the experimental trial, the final number of participants who had to be included per group was 14. This sample size was determined with a power of 80% and a significance level of 5% (α = 0.05). Independent-samples t tests were used to identify baseline differences between the CON and EXP groups. A Levene's test for equality of variances was performed; in case of unequal variance, an independent-samples t test was performed for unequal variances. Nutritional supplementation–induced changes in body composition, 1RM muscle strength, and dietary intake over time were analyzed by using repeated-measures ANOVA with time (baseline compared with postexercise) as within-subject and treatment (EXP compared with CON) as between-subject factors. A separate repeated-measures ANOVA with time (baseline compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject and treatment (EXP compared with CON) as between-subject factors was used to analyze the effect of treatment on the acute satellite cell response. In case a fiber type × time or time × treatment interaction was observed, fiber type or groups were analyzed separately. Finally, a repeated-measures ANOVA was used with time (baseline compared with 24 h compared with 48h) as within-subject and treatment (EXP compared with CON) as between-subject factor to analyze the effect of treatment and the acute changes in mRNA expression. In case of a time × treatment interaction, groups were analyzed separately. For all repeated-measures ANOVAs, a Mauchly's test of sphericity was performed to assess equality of variances between groups. In case of unequal variance, a Greenhouse-Geisser correction was applied to test for within-subject effects. Data are expressed as means ± SEMs. Significance was accepted as P < 0.05. Statistical analysis was completed with the use of SPSS (version 23.0). Results Participant characteristics No significant differences were observed at baseline between the CON and EXP groups for age (74 ± 2 compared with 72 ± 2 y), weight (85.0 ± 3.6 compared with 80.1 ± 2.5 kg), or BMI (28.3 ± 0.9 compared with 27.8 ± 0.8), respectively. Body composition At baseline, no significant differences in whole-body or regional lean mass were observed between the groups. A significant time × treatment interaction was observed for whole-body (P < 0.01) and leg (P < 0.05) lean mass. Whole-body (baseline compared with post: 52.2 ± 1.4 compared with 53.4 ± 1.4 kg) and leg (baseline compared with post: 18.2 ± 0.5 compared with 18.6 ± 0.6 kg) lean mass increased significantly after the 6-wk intervention period in the EXP group (P < 0.05), whereas no changes were observed in whole-body (baseline compared with post: 55.3 ± 2.2 compared with 55.0 ± 2.1 kg) and leg (baseline compared with post: 19.4 ± 1.0 compared with 19.3 ± 1.0 kg) lean mass in the CON group. Muscle strength No significant difference in 1RM muscle strength for any of the exercises performed was observed between the CON and EXP groups at baseline. A significant time × treatment interaction was observed for total 1RM lower body (P < 0.05), total 1RM upper body (P < 0.05), and all 1RM exercise combined (P < 0.05). Total 1RM lower body (baseline compared with post: 101.5 ± 4.1 compared with 107.8 ± 4.1 kg), total 1RM upper body (baseline compared with post: 95.2 ± 4.4 compared with 103.5 ± 4.6 kg), and all 1RM exercise combined (baseline compared with post: 197.0 ± 7.8 compared with 211.4 ± 7.8 kg) increased significantly in the EXP group (P < 0.05), whereas no change in total 1RM lower body (baseline compared with post: 98.4 ± 8.8 compared with 99.1 ± 9.0 kg), total 1RM upper body (baseline compared with post: 97.2 ± 5.3 compared with 98.6 ± 5.5 kg), and all 1RM exercise combined (baseline compared with post: 195.6 ± 13.6 compared with 197.4 ± 11.8 kg) was observed in the CON group (Table 1). TABLE 1 Habitual dietary intake assessed by 3-d food intake record before and after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group) *Different from baseline, P < 0.05. CON group, control supplemented; EXP group, experimental supplemented; Post, after intervention. View Large TABLE 1 Habitual dietary intake assessed by 3-d food intake record before and after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group) *Different from baseline, P < 0.05. CON group, control supplemented; EXP group, experimental supplemented; Post, after intervention. View Large Habitual dietary intake At baseline, we observed no significant differences in percentage of energy intakes of carbohydrate, fat, and protein between the CON and EXP groups (Table 1). We did observe a main effect of treatment for total energy intake (P < 0.05), indicating that energy intake was significantly higher in the CON compared with the EXP group over the course of the entire intervention period. Although no change was observed in the CON group, protein intake increased significantly in the EXP group during the nutritional intervention period (P < 0.001; Table 1). Muscle fiber characteristics In the postsupplementation muscle biopsy sample, no significant difference in type I and type II muscle fiber–type distribution, size, myonuclear content/domain size, or satellite cell content was observed between the EXP and CON groups (Table 2). However, although a significant difference between type I and type II muscle fiber size, myonuclear content, and domain size was observed in the postsupplementation muscle biopsy sample of the CON group, this was not the case in the EXP group (Table 2). TABLE 2 Muscle fiber characteristics after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group). *Different from type I muscle fibers, P < 0.05. CON, control supplemented; EXP, experimental supplemented. View Large TABLE 2 Muscle fiber characteristics after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group). *Different from type I muscle fibers, P < 0.05. CON, control supplemented; EXP, experimental supplemented. View Large Single bout of exercise No difference in total exercise volume (weight × number of repetitions) performed during the single bout of resistance exercise was observed between the CON and EXP groups (4050 ± 200 compared with 3946 ± 363 kg × number of repetitions, respectively). Acute satellite cell response A significant fiber type × time interaction was observed (P < 0.01) for the acute changes in satellite cell content after exercise; therefore, type I and type II muscle fibers were analyzed separately. A significant main effect of time was observed (P < 0.01) for type I muscle fiber satellite cell content, with no difference between the 2 groups (Figure 1G). Post hoc analysis indicated that type I muscle fiber satellite cell content was significantly (P = 0.017) higher 24 h postexercise in both groups compared with the pre-exercise muscle biopsy sample. In contrast, type II muscle fiber satellite content did not change postexercise in either the EXP or the CON group (Figure 1G). FIGURE 1 View largeDownload slide Representative image of fiber-type–specific analyses of satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) MHC-I (green), laminin (green), MHC-II (yellow), Pax7 (red), DAPI (blue). (B) Pax7 (red), DAPI (blue). (C) MHC-II (yellow), DAPI (blue). (D) DAPI (blue) only. (E) MHC-I (green), laminin (green), Pax7 (red). (F) Pax7 (red) only. (G) The number of satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. *Satellite cells in images A–F. Values represent means ± SEMs. Labeled means within treatment group without a common letter differ, P < 0.05. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; Pre, before resistance exercise. FIGURE 1 View largeDownload slide Representative image of fiber-type–specific analyses of satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) MHC-I (green), laminin (green), MHC-II (yellow), Pax7 (red), DAPI (blue). (B) Pax7 (red), DAPI (blue). (C) MHC-II (yellow), DAPI (blue). (D) DAPI (blue) only. (E) MHC-I (green), laminin (green), Pax7 (red). (F) Pax7 (red) only. (G) The number of satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. *Satellite cells in images A–F. Values represent means ± SEMs. Labeled means within treatment group without a common letter differ, P < 0.05. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; Pre, before resistance exercise. The number of MyoD+ satellite cells per fiber was not different between the CON and EXP groups in the pre-exercise muscle biopsy sample (Figure 2E). A significant main effect of time (P < 0.05) was observed for the number of MyoD+ satellite cells in both groups. The number of MyoD+ satellite cells tended to be increased at 48 h of postexercise recovery in both groups (P = 0.09; Figure 2E). FIGURE 2 View largeDownload slide Representative images of mixed analyses of muscle MyoD+ satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) Laminin (yellow), DAPI (blue), Pax7 (green), MyoD (red). (B) Laminin (yellow), MyoD (red). (C) Laminin (yellow), Pax7 (green). (D) Laminin (yellow), DAPI (blue). (E) The number of MyoD+ satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. *MyoD+ satellite cells in images A–D. Values are means ± SEMs. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MyoD, Myogenic Differentiation; pre, before resistance exercise. FIGURE 2 View largeDownload slide Representative images of mixed analyses of muscle MyoD+ satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) Laminin (yellow), DAPI (blue), Pax7 (green), MyoD (red). (B) Laminin (yellow), MyoD (red). (C) Laminin (yellow), Pax7 (green). (D) Laminin (yellow), DAPI (blue). (E) The number of MyoD+ satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. *MyoD+ satellite cells in images A–D. Values are means ± SEMs. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MyoD, Myogenic Differentiation; pre, before resistance exercise. mRNA expression No significant time × treatment interaction or main effects of time were observed for MRF4 and MYOGENIN mRNA expression in response to the single bout of exercise (Figure 3). A tendency for a time × treatment interaction was observed for Myogenin Factor 5 (MYF5) (P = 0.096) and MYOD (P = 0.076) mRNA expression, with no significant main effects of time (Figure 3B, C). FIGURE 3 View largeDownload slide Changes in MRF4 (A), MYF5 (B), MYOD (C), and MYOGENIN (D) mRNA expression before and after 24 and 48 h of a single bout of resistance exercise in healthy older men. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. Values are means ± SEMs; EXP (n = 13) and CON (n = 14). Data are expressed as fold-changes from pre, calculated by using –2∆∆Ct, with genes of interest normalized to GAPDH. CON, control; EXP, experimental; MRF4, Myogenic Regulatory Factor 4; MYF5, Myogenin Factor 5; MYOD, Myogenic Differentiation; Pre, before resistance exercise. FIGURE 3 View largeDownload slide Changes in MRF4 (A), MYF5 (B), MYOD (C), and MYOGENIN (D) mRNA expression before and after 24 and 48 h of a single bout of resistance exercise in healthy older men. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. Values are means ± SEMs; EXP (n = 13) and CON (n = 14). Data are expressed as fold-changes from pre, calculated by using –2∆∆Ct, with genes of interest normalized to GAPDH. CON, control; EXP, experimental; MRF4, Myogenic Regulatory Factor 4; MYF5, Myogenin Factor 5; MYOD, Myogenic Differentiation; Pre, before resistance exercise. Discussion We discovered that 7 wk of twice-daily ingestion of a multi-ingredient protein-based supplement increased skeletal muscle mass and strength in healthy older men. However, the nutritional supplement did not alter muscle satellite cell function in response to a single bout of resistance exercise in older adults. Previously, we showed the beneficial effect of a multi-ingredient protein-based supplement, independent of exercise training, on muscle mass and strength in a larger group of healthy older men (19). Although no changes were observed in the CON group, lean tissue mass and muscle strength increased significantly after 6 wk of nutritional supplementation in the EXP group. It is important to note that these effects were observed despite habitual protein intake exceeding the RDA of 0.8 g protein ⋅ kg body weight−1 ⋅ d−1 in our study participants. Although a significant increase in leg lean mass was observed in the EXP group during the 6-wk intervention period, we report no significant difference in type I and type II muscle fiber size between the groups after supplementation (Table 2). Interestingly, however, there was a significant difference between type I and type II muscle fiber size, myonuclear content, and domain size in the postsupplementation muscle biopsy sample in the CON group, which was not the case in the EXP group (Table 2). The lack of difference between the type I and II fibers in the EXP group may indicate a small degree of type II muscle fiber hypertrophy after supplementation in the EXP group; however, this is speculation because we lack a presupplement biopsy sample, which would be required to make this a firmer conclusion. Together, these results indicate the potential of a multi-ingredient protein-based supplement as an effective intervention to counter the negative effects of age-related sarcopenia. Skeletal muscle satellite cells play a key role in muscle fiber regeneration, repair, and hypertrophy (5, 6). However, we have previously shown that satellite cell content as well as function decline with aging in humans, particularly in the type II muscle fibers (9, 34, 35). Although type I and type II muscle fiber satellite cell content and activation status increase significantly in response to a single bout of resistance exercise in healthy young men, this is not the case for type II muscle fibers in healthy older men (34–36). A decline in muscle satellite cell function with increasing age has been hypothesized to play an important role in the development of sarcopenia, impaired muscle adaptive response to anabolic stimuli (i.e., exercise training), or both (7). To investigate whether the beneficial effect of the nutritional supplement on muscle strength and lean tissue mass was also accompanied by improved satellite cell function during postexercise recovery, all participants performed a single bout of resistance exercise after supplementation. The bout of exercise was performed 1 wk after the postsupplementation period strength testing. Importantly, all participants continued to consume the supplement until 48 h after exercise. In other words, the supplement was ingested within 1 h of completing the exercise session as well as before going to sleep at night after the bout of exercise. First, we showed that ingesting a multi-ingredient protein-based supplement 2 times/d for 7 wk does not change resting type I and type II muscle fiber satellite cell content, number of MyoD+ satellite cells, or basal mRNA expression of the different myogenic regulatory factors. In the CON group, the number of mixed-muscle MyoD+ satellite cells increased significantly in response to a single bout of resistance exercise. In addition, although type I muscle fiber satellite cell content increased significantly, there was no change in type II muscle fiber satellite cell content during the 48-h postexercise time course in the CON group. These results are consistent with our previous findings (34–36). However, more importantly, we observed no difference in the acute change in MyoD+ satellite cells or type I and type II muscle fiber satellite cell content after a single bout of exercise between the CON and EXP groups. Likewise, no significant difference in myogenic regulatory factor mRNA expression was observed after the bout of exercise between the EXP and CON groups. Only a tendency for a time × treatment interaction was observed for MYF5 (P = 0.096) and MYOD (P = 0.076) mRNA expression. However, whether a (relatively small) potentially differential response in MYF5 and MYOD mRNA expression during postexercise recovery holds any physiologic relevance for the muscle adaptive response in older men is questionable, especially because it was not accompanied by a difference in satellite cell activation status or content at any postexercise time point. The current study appears to be in contrast with the recent publication of Reidy et al. (26), which reported an improved satellite cell activation response in older men when 10 g essential amino acids was ingested 1 h after a single bout of resistance exercise. However, in this study, no significant difference in the increase in type II muscle fiber satellite cell content was observed with supplementation at 24 h after the single exercise session. The authors of that study argued that the limited sample size, 14 subjects (9 in the experimental and 5 in the control group), may have prevented them from detecting a significant change in postexercise satellite cell content. Conversely, with greater statistical power we observed no differences between CON and EXP groups. In addition, it is important to note that in the study of Reidy et al. (26) the satellite cell response was assessed at only 1 postexercise time point, which may have further impaired their ability to detect significant changes in satellite cell content. The present study represents a more extensive evaluation including 2 (24-h and 48-h) postexercise time points. However, the most important difference between the 2 studies is that our participants ingested a multi-ingredient protein-based supplement over a prolonged period (2 times/d for 7 wk) before the single bout of exercise was performed (including supplementation ingestion twice during post−exercise recovery), whereas Reidy et al. (26) provided a single 10-g dose of essential amino acids 1 h after exercise. Although habituation to the supplement may have occurred in the present study, we believe that our study design provides a more practical evaluation of whether nutritional supplementation in older adults results in benefit to muscle satellite cell function. We can only speculate on the possibility of an improved exercise-induced acute muscle satellite cell activation response at an earlier time point during the supplementation intervention. Whether this may also explain the observed difference in the increase in lean mass and muscle strength observed between the 2 groups remains to be elucidated. We conclude that ingesting a multi-ingredient protein-based supplement does not alter the type I or II muscle fiber satellite cell function after a single bout of resistance exercise in healthy older men. Acknowledgments The authors’ responsibilities were as follows—TS, KEB, SMP, and GP: designed the research; TS, KEB, and DAK: conducted the research; TS, JPN, NIS, and NM: performed the muscle biopsy analyses; TS and KEB: analyzed the data; TS, SMP, and GP: wrote the manuscript; TS: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by a funding from the Labarge Optimal Aging Initiative from McMaster University (to GP) and a Canadian Institutes of Health Research (CIHR) grant (MOP-123296) to SMP. KEB was supported by a CIHR Canada Graduate Scholarship (CGS-D). Author disclosures: TS, KEB, JPN, NIS, NM, DAK, and GP, no conflicts of interest. SMP reports receipt of competitive grant support, travel expenses, and honoraria for speaking received from the US National Dairy Council. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental Figure 1 and Supplemental Table 1 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Abbreviations used: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; MyoD, Myogenic Differentiation; 1RM, 1-repetition maximum. References 1. Cao L , Morley JE . 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Nutrition Oxford University Press

Ingestion of a Multi-Ingredient Supplement Does Not Alter Exercise-Induced Satellite Cell Responses in Older Men

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Oxford University Press
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© 2018 American Society for Nutrition.
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0022-3166
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1541-6100
D.O.I.
10.1093/jn/nxy063
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Abstract

Abstract Background Nutritional supplementation can have beneficial effects on body composition, strength, and function in older adults. However, whether the response of satellite cells can be altered by nutritional supplementation in older adults remains unknown. Objective We assessed whether a multi-ingredient protein-based supplement taken over a prolonged period of time could alter the muscle satellite cell response after exercise in older men. Methods Twenty-seven older men [mean ± SD age: 73 ± 1 y; mean ± SD body mass index (kg/m2): 28 ± 1] participated in a randomized double-blind experiment. Participants were randomly divided into an experimental (EXP) group (n = 13) who consumed a multi-ingredient protein-based supplement [30 g whey protein, 2.5 g creatine, 500 IU vitamin D, 400 mg Ca, and 1500 mg n–3 (ω-3) polyunsaturated fatty acids] 2 times/d for 7 wk or a control (CON; 22 g maltodextrin) group (n = 14). After 7 wk of supplementation, all participants performed a single resistance exercise session, and muscle biopsy samples were taken from the vastus lateralis before and 24 and 48 h after exercise. Immunohistochemistry was used to assess the change in type I and II muscle fiber satellite cell content and activation status of the cells. In addition, mRNA expression of the myogenic regulatory factors was determined by using reverse transcriptase–polymerase chain reaction. Results In response to the single bout of exercise, type I muscle fiber satellite cell content was significantly increased at 24 h (0.132 ± 0.015 and 0.131 ± 0.011 satellite cells/fiber in CON and EXP groups, respectively) and 48 h (0.126 ± 0.010 and 0.120 ± 0.012 satellite cells/fiber in CON and EXP groups, respectively) compared with pre-exercise (0.092 ± 0.007 and 0.118 ± 0.017 satellite cells/fiber in CON and EXP groups, respectively) muscle biopsy samples (P < 0.01), with no difference between the 2 groups. In both groups, we observed no significant changes in type II muscle fiber satellite cell content after exercise. Conclusion Ingesting a multi-ingredient protein-based supplement for 7 wk did not alter the type I or II muscle fiber satellite cell response during postexercise recovery in older men. This trial was registered at www.clinicaltrials.gov as NCT02281331. whey protein, aging, MyoD, creatine, muscle Introduction Sarcopenia, the progressive loss of skeletal muscle mass and strength with age (1), is associated with the development of functional impairments and increased risk of morbidity and mortality (2–4). Skeletal muscle satellite cells are indispensable for muscle fiber regeneration, repair, and growth (5, 6). Reduced muscle satellite cell number, function, or both occur with aging and have been suggested to play an important role in the development of sarcopenia, impaired muscle fiber adaptive response, or both during prolonged exercise training in older adults (7). Exercise training and nutritional supplementation are the 2 most extensively investigated anabolic strategies to counter the effects of sarcopenia. It has been well established that skeletal muscle mass, function, fiber size, and satellite cell content increase significantly in response to resistance exercise training (8–11). In contrast, mixed results have been reported for the impact of nutritional supplementation on muscle mass and function in older adults. Whereas some studies showed clear improvements in muscle mass or function with the ingestion of nutritional supplements (12–15), others did not (16–18). This discrepancy is likely explained by the timing, amount, or composition of the nutritional supplement. Heterogeneity of the need for multiple nutrients in older persons provides a rationale for a multi-ingredient supplement to yield a benefit across a larger group of older adults. Accordingly, we recently reported that a multi-ingredient protein-based supplement, independent of exercise training, can increase skeletal muscle mass and strength in healthy older adults (19). However, whether longer-term ingestion of a multi-ingredient protein-based nutritional supplement can mitigate the impairment in muscle satellite cell function in older adults remains unknown. A limited number of studies have reported on the impact of nutrition on muscle satellite cell content, function, or both. In vitro studies have shown that the administration of the essential amino acid leucine increases satellite cell proliferation (20) and Myogenic Differentiation (MyoD) expression (21). In addition, the nutrient-sensitive signaling protein mammalian target of rapamycin complex 1 has been reported to control the transition of quiescent satellite cells from the G0 to G(alert) phase (22, 23). We have previously shown that low (0.1 g protein ⋅ kg body weight−1 ⋅ d−1) compared with normal (1.1 g protein ⋅ kg body weight−1 ⋅ d−1) protein intake did not influence the muscle satellite cell response after exercise in healthy young men (24). Conversely, Farup et al. (25) reported an enhanced muscle satellite cell response when additional whey protein was supplemented during the first 48 h after an exercise bout in young men. Reidy et al. (26) reported that the ingestion of an essential amino acid supplement restored a normally impaired satellite cell activation response in older men; however, the improvement in satellite cell activation was not accompanied by a significant increase in satellite cell content. In that study (26), a limited sample size, sampling muscle at only a single postexercise time point, or the provision of the supplement only once postexercise may have obscured the observation of a significant increase in satellite cell content. Therefore, in the current study, we undertook a more extensive evaluation of whether a nutritional supplement, provided 2 times/d over a 7-wk period, can affect the satellite cell response after a single bout of exercise in older men. Methods Participants Twenty-seven healthy older men [mean ± SD age: 73 ± 1 y; weight: 84 ± 4 kg; mean ± SD BMI (kg/m2): 28 ± 1] were recruited to participate in a 7-wk nutritional intervention program. All participants had a BMI in the normal to overweight range and resting blood pressure <140/90 mm Hg. All participants showed normal cardiac function during a maximal exercise stress test. Exclusion criteria included smoking, diabetes, regular use of nonsteroidal anti-inflammatory drugs, use of statins, and history of chronic illness that would affect the results of the investigation. An oral-glucose-tolerance test was performed to exclude participants with type 2 diabetes. All subjects had not participated in any structured exercise program in the past year and were living independently. All participants were informed of the nature and possible risks of the experimental procedures before their written informed consent was obtained. The study was approved by the Hamilton Health Sciences Integrated Research Ethics Board and conformed to the guidelines outlined in the Declaration of Helsinki. Participants gave their informed written consent before their inclusion in the study. The study was part of a larger project investigating the impact of nutrition and exercise training on skeletal muscle mass and strength and metabolic health in older adults (19, 27). This trial was registered at www.clinicaltrials.gov as NCT02281331. Experimental outline An overview of the double-blind randomized study design with parallel groups is shown in Supplemental Figure 1. After inclusion into the study, participants were randomly assigned to an experimental (EXP) group in which they ingested a multi-ingredient protein-based supplement or to a control (CON) group who ingested a carbohydrate-based beverage. Supplements were consumed 2 times/d for 7 wk. Anthropometric measurements (weight, height), muscle strength [1 repetition maximum (1RM)], dietary intake (3-d food intake record), and body composition (DXA scan) were assessed before (between T = −2 and T = 0) and after (week 6; T = 6) the intervention period. Finally, in week 7 (T = 7), all participants performed a single bout of resistance exercise to assess the acute muscle satellite cell response; muscle biopsy samples were taken before exercise and at 24 and 48 h after exercise. Supplementation Participants were randomly assigned to an EXP group receiving a multi-ingredient protein-based supplement or a CON group receiving a carbohydrate-based placebo. The multi-ingredient supplement contained 30 g whey protein, 2.5 g creatine, 400 mg Ca, 500 IU vitamin D, 1 g carbohydrate, and 0.75 g n–3 FAs (which delivered 0.7 g EPA and 0.45 g DHA), providing a total of 210 kJ. The composition of the nutritional supplement was based on a previous publications showing isolated positive effects of protein (28), creatine (29), vitamin D (30), or n–3 FA (13) supplementation with or without exercise training on muscle mass and strength gains in older adults. The control supplement contained 22 g carbohydrate, providing 62 kJ. Beverages were masked for taste and smell. Participants ingested the drinks twice daily, once after breakfast and once in the evening before bedtime. All drinks were provided in a double-blinded manner. Supplementation was maintained during posttesting and through the 48-h exercise (T = 6 and T = 7) period. Single bout of resistance exercise All of the participants performed a single bout of resistance exercise after 7 wk of supplementation. The single bout of exercise consisted of 3 sets of 10 repetitions each at 65% of 1RM leg press, chest press, horizontal row, and leg extension (HUR). Exercise was performed under personal supervision. All participants were verbally encouraged during the exercise, and the final set of each exercise was performed to volitional failure. A resting period of 2 min between sets was allowed. Before and after the resistance exercise, a 5-min warm-up and cool-down were performed on a cycle ergometer. Habitual dietary intake and physical activity Before the onset and in week 6 (T = 6) of the nutritional intervention, participants recorded a 3-d dietary record (2 weekdays, 1 weekend day) to assess potential changes in daily food intake that might have occurred during the intervention. Food intake records were analyzed as described previously (19). All participants were instructed not to perform any vigorous physical activity 5 d before the postexercise acute satellite cell response measurement. Body composition Whole-body and regional lean soft tissue mass (i.e., fat-free and bone-free mass), fat mass, and bone mineral content were measured with the use of DXA (GE-Lunar iDXA; Aymes Medical) after a 10- to 12-h overnight fast, as described previously (19). Muscle strength Muscle strength was assessed by using 1RM strength tests for the following exercises: leg press, chest press, lateral pull-down, horizontal row, shoulder press, and leg extension (HUR), as described previously (19). Muscle biopsy Percutaneous needle biopsy samples were taken, after an overnight (∼10 h) fast, from the midportion of the vastus lateralis under local anesthetic with the use of a 5-mm Bergstrom needle adapted for manual suction. Subjects had not participated in any physical activity for ≥5 d before the collection of the resting muscle biopsy sample. Consecutive muscle biopsy sampling was alternated between legs. The leg in which the baseline biopsy sample was drawn from was randomized. Incisions for the repeated muscle biopsy sampling in the same leg (e.g., pre- and 48-h postexercise sample) were spaced by at ≥3 cm to minimize any effect of the previous biopsy. Upon excision, muscle samples were immediately mounted in optimal cutting temperature compound and frozen in liquid nitrogen–cooled isopentane. A second sample was snap-frozen directly in liquid nitrogen and stored at −80°C for further analyses. Immunohistochemistry Muscle cross-sections (7 µm) were prepared from unfixed optimal cutting temperature embedded samples, allowed to air dry for 30 min, and stored at −80°C. Slides were then stained with antibodies against Pax7 (neat; Developmental Studies Hybridoma Bank), MyoD (anti-MyoD1; clone 5.8A, 1:50; Dako), A4.951 [myosin heavy chain (MHC) type I, slow isoform, 1:1; Developmental Studies Hybridoma Bank], MHC-II (fast isoform, 1:1000; ab91506; Abcam), and laminin (anti-laminin; ab11575; Abcam). Secondary antibodies used were Pax7 (Alexa Fluor 488 or 594, 1:500; Invitrogen, Molecular Probes), MyoD (biotinylated secondary antibody, 1:200; Vector Canada; and streptavidin-594 fluorochrome, 1:500; Invitrogen, Molecular Probes), A4.951 (Alexa Fluor 488, 1:500), MHC-II (Alexa Fluor 647, 1:500), and laminin (Alexa Fluor 488, 647; 1:500). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; 1:20,000; Sigma-Aldrich) before cover slipping with fluorescent mounting media (Dako). Slides were viewed with a Nikon Eclipse Ti Microscope (Nikon Instruments) and equipped with a high-resolution Photometrics CoolSNAP HQ2 fluorescent camera (Nikon Instruments). Images were captured and analyzed by using Nikon NIS Elements AR 3.2 software (Nikon Instruments). All images were obtained with the 20× objective, and ≥200 muscle fibers/subject per time point were included in the analyses for satellite cell content and activation status (i.e., Pax7+/MyoD− or Pax7+/MyoD+), fiber cross-sectional area, and perimeter. The activation status of satellite cells was determined via the colocalization of Pax7+ and DAPI (Pax7+/MyoD) or the colocalization of Pax7, MyoD, and DAPI (i.e., Pax7+/MyoD+). Slides were blinded for both group and time point. All immunofluorescent analyses were completed in a blinded fashion. Quantitative real-time RT-PCR RNA was isolated from 15–25 mg muscle tissue with the use of the Trizol/RNeasy method, as described previously (31). All qPCR reactions were performed in duplicate in 25-µL volumes containing RT Sybr Green qPCR Master Mix (Qiagen Sciences), prepared with the epMotion 5075 Eppendorf automated pipetting system (Eppendorf), and carried out by using an Eppendorf Realplex2 Master Cycler epgradient (Eppendorf). Primers are listed in Supplemental Table 1. mRNA expression was calculated by using the 2−∆∆Ct method and expressed as fold change from pre (pre-exercise), as described previously (32). Briefly, Ct values were first normalized to the housekeeping gene GAPDH. GAPDH expression was not different from pre-exercise at any of the postexercise time points. Ct values normalized to GAPDH were expressed as ΔΔCt. Statistical analyses A samples calculation was performed based on our previously published data (33) and a clinical relevant difference of 51% (SD = 42%) between the EXP and CON groups in type II muscle fiber satellite cell content increase at 48 h after the single exercise session. Taking into consideration a drop-out rate of 10% during the experimental trial, the final number of participants who had to be included per group was 14. This sample size was determined with a power of 80% and a significance level of 5% (α = 0.05). Independent-samples t tests were used to identify baseline differences between the CON and EXP groups. A Levene's test for equality of variances was performed; in case of unequal variance, an independent-samples t test was performed for unequal variances. Nutritional supplementation–induced changes in body composition, 1RM muscle strength, and dietary intake over time were analyzed by using repeated-measures ANOVA with time (baseline compared with postexercise) as within-subject and treatment (EXP compared with CON) as between-subject factors. A separate repeated-measures ANOVA with time (baseline compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject and treatment (EXP compared with CON) as between-subject factors was used to analyze the effect of treatment on the acute satellite cell response. In case a fiber type × time or time × treatment interaction was observed, fiber type or groups were analyzed separately. Finally, a repeated-measures ANOVA was used with time (baseline compared with 24 h compared with 48h) as within-subject and treatment (EXP compared with CON) as between-subject factor to analyze the effect of treatment and the acute changes in mRNA expression. In case of a time × treatment interaction, groups were analyzed separately. For all repeated-measures ANOVAs, a Mauchly's test of sphericity was performed to assess equality of variances between groups. In case of unequal variance, a Greenhouse-Geisser correction was applied to test for within-subject effects. Data are expressed as means ± SEMs. Significance was accepted as P < 0.05. Statistical analysis was completed with the use of SPSS (version 23.0). Results Participant characteristics No significant differences were observed at baseline between the CON and EXP groups for age (74 ± 2 compared with 72 ± 2 y), weight (85.0 ± 3.6 compared with 80.1 ± 2.5 kg), or BMI (28.3 ± 0.9 compared with 27.8 ± 0.8), respectively. Body composition At baseline, no significant differences in whole-body or regional lean mass were observed between the groups. A significant time × treatment interaction was observed for whole-body (P < 0.01) and leg (P < 0.05) lean mass. Whole-body (baseline compared with post: 52.2 ± 1.4 compared with 53.4 ± 1.4 kg) and leg (baseline compared with post: 18.2 ± 0.5 compared with 18.6 ± 0.6 kg) lean mass increased significantly after the 6-wk intervention period in the EXP group (P < 0.05), whereas no changes were observed in whole-body (baseline compared with post: 55.3 ± 2.2 compared with 55.0 ± 2.1 kg) and leg (baseline compared with post: 19.4 ± 1.0 compared with 19.3 ± 1.0 kg) lean mass in the CON group. Muscle strength No significant difference in 1RM muscle strength for any of the exercises performed was observed between the CON and EXP groups at baseline. A significant time × treatment interaction was observed for total 1RM lower body (P < 0.05), total 1RM upper body (P < 0.05), and all 1RM exercise combined (P < 0.05). Total 1RM lower body (baseline compared with post: 101.5 ± 4.1 compared with 107.8 ± 4.1 kg), total 1RM upper body (baseline compared with post: 95.2 ± 4.4 compared with 103.5 ± 4.6 kg), and all 1RM exercise combined (baseline compared with post: 197.0 ± 7.8 compared with 211.4 ± 7.8 kg) increased significantly in the EXP group (P < 0.05), whereas no change in total 1RM lower body (baseline compared with post: 98.4 ± 8.8 compared with 99.1 ± 9.0 kg), total 1RM upper body (baseline compared with post: 97.2 ± 5.3 compared with 98.6 ± 5.5 kg), and all 1RM exercise combined (baseline compared with post: 195.6 ± 13.6 compared with 197.4 ± 11.8 kg) was observed in the CON group (Table 1). TABLE 1 Habitual dietary intake assessed by 3-d food intake record before and after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group) *Different from baseline, P < 0.05. CON group, control supplemented; EXP group, experimental supplemented; Post, after intervention. View Large TABLE 1 Habitual dietary intake assessed by 3-d food intake record before and after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 CON group EXP group P Baseline Post Baseline Post Time Treatment Interaction Total energy, MJ/d 10.3 ± 0.6 10.3 ± 0.7 8.6 ± 0.5 8.3 ± 0.4 NS <0.05 NS Total energy including supplement, MJ/d 10.3 ± 0.6 11.0 ± 0.7* 8.6 ± 0.5 9.2 ± 0.4* <0.05 <0.05 NS Carbohydrates, % of energy 48 ± 4 47 ± 3 50 ± 2 48 ± 2 NS NS NS Fat, % of energy 32 ± 3 33 ± 3 29 ± 2 32 ± 2 NS NS NS Alcohol, % of energy 3 ± 1 3 ± 1 4 ± 1 3 ± 1 NS NS NS Protein, % of energy 17 ± 1 17 ± 1 17 ± 1 17 ± 1 NS NS NS Protein intake, g/d 103 ± 8 106 ± 11 86 ± 8 84 ± 8 NS NS NS Protein intake, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 NS NS NS Protein intake including supplement, g · kg−1 · d−1 1.2 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.7 ± 0.1* <0.001 <0.001 <0.001 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group) *Different from baseline, P < 0.05. CON group, control supplemented; EXP group, experimental supplemented; Post, after intervention. View Large Habitual dietary intake At baseline, we observed no significant differences in percentage of energy intakes of carbohydrate, fat, and protein between the CON and EXP groups (Table 1). We did observe a main effect of treatment for total energy intake (P < 0.05), indicating that energy intake was significantly higher in the CON compared with the EXP group over the course of the entire intervention period. Although no change was observed in the CON group, protein intake increased significantly in the EXP group during the nutritional intervention period (P < 0.001; Table 1). Muscle fiber characteristics In the postsupplementation muscle biopsy sample, no significant difference in type I and type II muscle fiber–type distribution, size, myonuclear content/domain size, or satellite cell content was observed between the EXP and CON groups (Table 2). However, although a significant difference between type I and type II muscle fiber size, myonuclear content, and domain size was observed in the postsupplementation muscle biopsy sample of the CON group, this was not the case in the EXP group (Table 2). TABLE 2 Muscle fiber characteristics after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group). *Different from type I muscle fibers, P < 0.05. CON, control supplemented; EXP, experimental supplemented. View Large TABLE 2 Muscle fiber characteristics after 6 wk of ingesting a multi-ingredient protein-based supplement in healthy older men1 Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* Fiber type CON group EXP group Fiber type distribution, % fiber  I 41 ± 3 39 ± 2  II 59 ± 3* 61 ± 2* Muscle fiber size, µm2  I 6900 ± 630 6656 ± 468  II 5055 ± 454* 5913 ± 576 Myonuclear content, n/fiber  I 3.8 ± 0.2 3.8 ± 0.2  II 3.4 ± 0.2* 3.6 ± 0.3 Myonuclear domain, µm2  I 1774 ± 111 1745 ± 89  II 1480 ± 62* 1642 ± 100 Satellite cell content, n/fiber  I 0.092 ± 0.007 0.118 ± 0.017  II 0.061 ± 0.006* 0.077 ± 0.013* 1Values are means ± SEMs; n = 14 (CON group) or n = 13 (EXP group). *Different from type I muscle fibers, P < 0.05. CON, control supplemented; EXP, experimental supplemented. View Large Single bout of exercise No difference in total exercise volume (weight × number of repetitions) performed during the single bout of resistance exercise was observed between the CON and EXP groups (4050 ± 200 compared with 3946 ± 363 kg × number of repetitions, respectively). Acute satellite cell response A significant fiber type × time interaction was observed (P < 0.01) for the acute changes in satellite cell content after exercise; therefore, type I and type II muscle fibers were analyzed separately. A significant main effect of time was observed (P < 0.01) for type I muscle fiber satellite cell content, with no difference between the 2 groups (Figure 1G). Post hoc analysis indicated that type I muscle fiber satellite cell content was significantly (P = 0.017) higher 24 h postexercise in both groups compared with the pre-exercise muscle biopsy sample. In contrast, type II muscle fiber satellite content did not change postexercise in either the EXP or the CON group (Figure 1G). FIGURE 1 View largeDownload slide Representative image of fiber-type–specific analyses of satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) MHC-I (green), laminin (green), MHC-II (yellow), Pax7 (red), DAPI (blue). (B) Pax7 (red), DAPI (blue). (C) MHC-II (yellow), DAPI (blue). (D) DAPI (blue) only. (E) MHC-I (green), laminin (green), Pax7 (red). (F) Pax7 (red) only. (G) The number of satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. *Satellite cells in images A–F. Values represent means ± SEMs. Labeled means within treatment group without a common letter differ, P < 0.05. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; Pre, before resistance exercise. FIGURE 1 View largeDownload slide Representative image of fiber-type–specific analyses of satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) MHC-I (green), laminin (green), MHC-II (yellow), Pax7 (red), DAPI (blue). (B) Pax7 (red), DAPI (blue). (C) MHC-II (yellow), DAPI (blue). (D) DAPI (blue) only. (E) MHC-I (green), laminin (green), Pax7 (red). (F) Pax7 (red) only. (G) The number of satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) and fiber type (type I compared with type II) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. *Satellite cells in images A–F. Values represent means ± SEMs. Labeled means within treatment group without a common letter differ, P < 0.05. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; Pre, before resistance exercise. The number of MyoD+ satellite cells per fiber was not different between the CON and EXP groups in the pre-exercise muscle biopsy sample (Figure 2E). A significant main effect of time (P < 0.05) was observed for the number of MyoD+ satellite cells in both groups. The number of MyoD+ satellite cells tended to be increased at 48 h of postexercise recovery in both groups (P = 0.09; Figure 2E). FIGURE 2 View largeDownload slide Representative images of mixed analyses of muscle MyoD+ satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) Laminin (yellow), DAPI (blue), Pax7 (green), MyoD (red). (B) Laminin (yellow), MyoD (red). (C) Laminin (yellow), Pax7 (green). (D) Laminin (yellow), DAPI (blue). (E) The number of MyoD+ satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. *MyoD+ satellite cells in images A–D. Values are means ± SEMs. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MyoD, Myogenic Differentiation; pre, before resistance exercise. FIGURE 2 View largeDownload slide Representative images of mixed analyses of muscle MyoD+ satellite cell number before and 24 and 48 h after performing a single bout of resistance exercise in healthy older men. (A) Laminin (yellow), DAPI (blue), Pax7 (green), MyoD (red). (B) Laminin (yellow), MyoD (red). (C) Laminin (yellow), Pax7 (green). (D) Laminin (yellow), DAPI (blue). (E) The number of MyoD+ satellite cells expressed per fiber in the EXP (n = 13) and CON (n = 14) groups. *MyoD+ satellite cells in images A–D. Values are means ± SEMs. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48 h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MyoD, Myogenic Differentiation; pre, before resistance exercise. mRNA expression No significant time × treatment interaction or main effects of time were observed for MRF4 and MYOGENIN mRNA expression in response to the single bout of exercise (Figure 3). A tendency for a time × treatment interaction was observed for Myogenin Factor 5 (MYF5) (P = 0.096) and MYOD (P = 0.076) mRNA expression, with no significant main effects of time (Figure 3B, C). FIGURE 3 View largeDownload slide Changes in MRF4 (A), MYF5 (B), MYOD (C), and MYOGENIN (D) mRNA expression before and after 24 and 48 h of a single bout of resistance exercise in healthy older men. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. Values are means ± SEMs; EXP (n = 13) and CON (n = 14). Data are expressed as fold-changes from pre, calculated by using –2∆∆Ct, with genes of interest normalized to GAPDH. CON, control; EXP, experimental; MRF4, Myogenic Regulatory Factor 4; MYF5, Myogenin Factor 5; MYOD, Myogenic Differentiation; Pre, before resistance exercise. FIGURE 3 View largeDownload slide Changes in MRF4 (A), MYF5 (B), MYOD (C), and MYOGENIN (D) mRNA expression before and after 24 and 48 h of a single bout of resistance exercise in healthy older men. A repeated-measures ANOVA with time (pre compared with 24 h compared with 48h) as within-subject factor and treatment (EXP compared with CON) as between-subject factor was used to analyze the effect of treatment on the acute satellite cell response. Values are means ± SEMs; EXP (n = 13) and CON (n = 14). Data are expressed as fold-changes from pre, calculated by using –2∆∆Ct, with genes of interest normalized to GAPDH. CON, control; EXP, experimental; MRF4, Myogenic Regulatory Factor 4; MYF5, Myogenin Factor 5; MYOD, Myogenic Differentiation; Pre, before resistance exercise. Discussion We discovered that 7 wk of twice-daily ingestion of a multi-ingredient protein-based supplement increased skeletal muscle mass and strength in healthy older men. However, the nutritional supplement did not alter muscle satellite cell function in response to a single bout of resistance exercise in older adults. Previously, we showed the beneficial effect of a multi-ingredient protein-based supplement, independent of exercise training, on muscle mass and strength in a larger group of healthy older men (19). Although no changes were observed in the CON group, lean tissue mass and muscle strength increased significantly after 6 wk of nutritional supplementation in the EXP group. It is important to note that these effects were observed despite habitual protein intake exceeding the RDA of 0.8 g protein ⋅ kg body weight−1 ⋅ d−1 in our study participants. Although a significant increase in leg lean mass was observed in the EXP group during the 6-wk intervention period, we report no significant difference in type I and type II muscle fiber size between the groups after supplementation (Table 2). Interestingly, however, there was a significant difference between type I and type II muscle fiber size, myonuclear content, and domain size in the postsupplementation muscle biopsy sample in the CON group, which was not the case in the EXP group (Table 2). The lack of difference between the type I and II fibers in the EXP group may indicate a small degree of type II muscle fiber hypertrophy after supplementation in the EXP group; however, this is speculation because we lack a presupplement biopsy sample, which would be required to make this a firmer conclusion. Together, these results indicate the potential of a multi-ingredient protein-based supplement as an effective intervention to counter the negative effects of age-related sarcopenia. Skeletal muscle satellite cells play a key role in muscle fiber regeneration, repair, and hypertrophy (5, 6). However, we have previously shown that satellite cell content as well as function decline with aging in humans, particularly in the type II muscle fibers (9, 34, 35). Although type I and type II muscle fiber satellite cell content and activation status increase significantly in response to a single bout of resistance exercise in healthy young men, this is not the case for type II muscle fibers in healthy older men (34–36). A decline in muscle satellite cell function with increasing age has been hypothesized to play an important role in the development of sarcopenia, impaired muscle adaptive response to anabolic stimuli (i.e., exercise training), or both (7). To investigate whether the beneficial effect of the nutritional supplement on muscle strength and lean tissue mass was also accompanied by improved satellite cell function during postexercise recovery, all participants performed a single bout of resistance exercise after supplementation. The bout of exercise was performed 1 wk after the postsupplementation period strength testing. Importantly, all participants continued to consume the supplement until 48 h after exercise. In other words, the supplement was ingested within 1 h of completing the exercise session as well as before going to sleep at night after the bout of exercise. First, we showed that ingesting a multi-ingredient protein-based supplement 2 times/d for 7 wk does not change resting type I and type II muscle fiber satellite cell content, number of MyoD+ satellite cells, or basal mRNA expression of the different myogenic regulatory factors. In the CON group, the number of mixed-muscle MyoD+ satellite cells increased significantly in response to a single bout of resistance exercise. In addition, although type I muscle fiber satellite cell content increased significantly, there was no change in type II muscle fiber satellite cell content during the 48-h postexercise time course in the CON group. These results are consistent with our previous findings (34–36). However, more importantly, we observed no difference in the acute change in MyoD+ satellite cells or type I and type II muscle fiber satellite cell content after a single bout of exercise between the CON and EXP groups. Likewise, no significant difference in myogenic regulatory factor mRNA expression was observed after the bout of exercise between the EXP and CON groups. Only a tendency for a time × treatment interaction was observed for MYF5 (P = 0.096) and MYOD (P = 0.076) mRNA expression. However, whether a (relatively small) potentially differential response in MYF5 and MYOD mRNA expression during postexercise recovery holds any physiologic relevance for the muscle adaptive response in older men is questionable, especially because it was not accompanied by a difference in satellite cell activation status or content at any postexercise time point. The current study appears to be in contrast with the recent publication of Reidy et al. (26), which reported an improved satellite cell activation response in older men when 10 g essential amino acids was ingested 1 h after a single bout of resistance exercise. However, in this study, no significant difference in the increase in type II muscle fiber satellite cell content was observed with supplementation at 24 h after the single exercise session. The authors of that study argued that the limited sample size, 14 subjects (9 in the experimental and 5 in the control group), may have prevented them from detecting a significant change in postexercise satellite cell content. Conversely, with greater statistical power we observed no differences between CON and EXP groups. In addition, it is important to note that in the study of Reidy et al. (26) the satellite cell response was assessed at only 1 postexercise time point, which may have further impaired their ability to detect significant changes in satellite cell content. The present study represents a more extensive evaluation including 2 (24-h and 48-h) postexercise time points. However, the most important difference between the 2 studies is that our participants ingested a multi-ingredient protein-based supplement over a prolonged period (2 times/d for 7 wk) before the single bout of exercise was performed (including supplementation ingestion twice during post−exercise recovery), whereas Reidy et al. (26) provided a single 10-g dose of essential amino acids 1 h after exercise. Although habituation to the supplement may have occurred in the present study, we believe that our study design provides a more practical evaluation of whether nutritional supplementation in older adults results in benefit to muscle satellite cell function. We can only speculate on the possibility of an improved exercise-induced acute muscle satellite cell activation response at an earlier time point during the supplementation intervention. Whether this may also explain the observed difference in the increase in lean mass and muscle strength observed between the 2 groups remains to be elucidated. We conclude that ingesting a multi-ingredient protein-based supplement does not alter the type I or II muscle fiber satellite cell function after a single bout of resistance exercise in healthy older men. Acknowledgments The authors’ responsibilities were as follows—TS, KEB, SMP, and GP: designed the research; TS, KEB, and DAK: conducted the research; TS, JPN, NIS, and NM: performed the muscle biopsy analyses; TS and KEB: analyzed the data; TS, SMP, and GP: wrote the manuscript; TS: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by a funding from the Labarge Optimal Aging Initiative from McMaster University (to GP) and a Canadian Institutes of Health Research (CIHR) grant (MOP-123296) to SMP. KEB was supported by a CIHR Canada Graduate Scholarship (CGS-D). Author disclosures: TS, KEB, JPN, NIS, NM, DAK, and GP, no conflicts of interest. SMP reports receipt of competitive grant support, travel expenses, and honoraria for speaking received from the US National Dairy Council. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental Figure 1 and Supplemental Table 1 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Abbreviations used: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; EXP, experimental; MHC, myosin heavy chain; MyoD, Myogenic Differentiation; 1RM, 1-repetition maximum. References 1. Cao L , Morley JE . 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of NutritionOxford University Press

Published: Jun 7, 2018

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