The results of the present study support our hypothesis, indicating that NO-Shotgun®
supplementation in conjunction with a 28 days of heavy resistance training, is effective at increasing fat-free mass, muscle strength and mass, myofibrillar protein content, and markers of satellite cell activation, while having no effect on whole blood and serum clinical safety markers in untrained males. Our results agree with previously reported studies that resistance training, when performed in conjunction with creatine [24
], whey protein and leucine [36
], and HMB [37
] is effective at improving body composition, muscle strength and mass and markers of satellite cell activation.
We observed both NO and PL to significantly increase total body mass (P = 0.001). Additionally, fat-free mass was increased in both groups, and the 4.75% increase in NO was significantly greater than the 1.69% increase in PL. These findings are similar to results observed after 12 wk of heavy resistance training and creatine supplementation, where fat-free mass was increased 9.44% in the creatine group and 1.84% in the carbohydrate placebo group [24
]. In addition, 10 wk of heavy resistance training and whey protein and amino acid supplementation resulted in increases in fat-free mass of 5.62% compared to increases of 2.70% for carbohydrate placebo [34
Relative to muscle strength, we observed NO to increase in bench press and leg press strength by 8.82% and 18.40%, respectively, compared to the respective increases in bench press and leg press strength of 0.74% and 10.30% for PL. However, only bench press was significantly greater for NO compared to PL (p = 0.003). Our observed increases in muscle strength are supported by previous studies which demonstrated heavy resistance training, when combined with creatine [24
], protein and amino acids (34), and whey protein and leucine [24
] to improve strength levels when compared to placebo. However, it should be noted that NO-Shotgun®
contains beta-alanine, which has been shown to possibly potentiate the effects of creatine. A recent study has shown that 10 wk of resistance training combined with creatine and beta-alanine produced superior gains in strength and fat-free mass compared to creatine or placebo [39
In the present study, we also showed that after 28 days of heavy resistance training and supplementation NO underwent increases in myofibrillar protein of 70.39% that were significantly greater than the 26.34% increase in PL (p < 0.001), and that the increases for NO were significantly different than PL (p = 0.014). This is a similar pattern of response from longer-term studies where creatine supplementation, in conjunction with 12 wk of resistance training, resulted in a 57.92% increase in myofibrillar protein content when compared to a maltodextrose placebo group, which only increased 11.62% [24
]. In addition, 10 wk of heavy resistance training combined with a protein and amino acid supplement resulted in a 25.03% increase in myofibrillar protein compared to 10.54% for a carbohydrate placebo [34
We have demonstrated 28 days of heavy resistance training to increase serum IGF-1 by 9.34% and 8.58%, respectively for NO and PL; however, there was no difference between groups. Treating C2
myoblasts with creatine has been shown to increase the expression of the IGF-1 peptide [40
]. A positive relationship has been reported between IGF-1 peptide and total DNA content in muscle during resistance exercise due to satellite cell proliferation stimulated by the locally produced IGF-1 [7
]. However, while the IGF-I peptide expressed in skeletal muscleincreases muscular protein synthesis and stimulates differentiation of proliferating satellite cells [14
], it is unclear whether increases in hepatically-derived circulating IGF-1 has any direct effect on muscle hypertrophy. We have previously shown that 10 wk of heavy resistance training combined with a daily supplement containing whey/casein protein and free amino acids increased circulating IGF-1 levels, while also increasing muscle strength and mass [34
]. Additionally, 16 wk of resistance training has been shown to increase circulating IGF-1 levels [42
]. However, 12 wk of heavy resistance training has been shown to increase muscle strength and mass without any corresponding increases in circulating IGF-1 [43
]. Increases in muscle hypertrophy independent of increases in circulating IGF-1 can possibly be explained by a recent study using a liver IGF-1 deficient mouse model, which involves a reduction in serum IGF-1 of approximately 80% [44
]. After 16 wk of resistance training, the IGF-1-deficient mice and control mice exhibited equivalent gains in muscle strength, suggesting that performance and recovery in response to resistance training is normal even when there is a severe deficiency in circulating IGF-1.
HGF is a growth factor bound to an extracellular matrix in skeletal muscle [45
] that is capable of activating quiescent satellite cells [46
]. Serum HGF levels have been shown to increase 24 hr following a single bout of eccentric exercise [47
]. In cultured satellite cells, mechanical stretch has been shown to induce the activity of nitric oxide synthase and increase nitric oxide production, which was associated with increases in HGF [48
]. In the present study, for serum HGF we observed PL to decrease 8.71% with training, whereas NO increased 47.42%. Based on the fact that NO-Shotgun®
contains arginine, an alleged mediator of nitric oxide synthesis, our results may be partially explained on the premise that nitric oxide mediates the release of HGF, and that nitric oxide synthase activity is increased with satellite cell activation.
Skeletal muscle markers of satellite cell activation examined in this study were phospoyrlated c-met (the proto-oncogene receptor for HGF), total DNA, and the MRFs (MyoD, Myf5, MRF-4, and myogenin). While circulating levels of HGF were increased for NO, skeletal muscle phosphorylated c-met was also increased for NO from resistance training by 118.55% (p = 0.019), with a strong trend for NO to be significantly greater than PL (p = 0.067). Increases in the phosphorylation of the HGF receptor, c-met, may be indicative of a possible increase in satellite cell activation. Since HGF levels increased significantly for NO, an increase in the c-met receptor would likely allow for increased binding of HGF.
Resistance training can increase the number of satellite cells and increase myonuclei in the myofiber [11
]. However, it has been shown that 16 wk of heavy resistance training combined with creatine supplementation augments satellite cell activation, as evidenced by increases in skeletal muscle mean fiber and area myonuclear number to a much greater extent to whey protein or resistance training alone [28
]. Furthermore, the creatine group was shown to have the greatest increase in maximal isometric quadriceps contraction strength. Relative to results for the whey protein group, it was shown to undergo greater increases in skeletal muscle mean fiber area and myonuclear number and isokinetic quadriceps strength when compared to the control group.
In the present study, we did not directly assess satellite cell or myonuclear number. Rather, we assessed markers that are considered to be valid indicators of increased satellite cell activation. In so doing, both groups underwent increases in all MRFs with heavy training. However, Myo-D and MRF-4 showed significantly greater increases in NO than PL. For NO, Myo-D increased by 70.91%, MRF-4 increased by 56.24%, myf5 increased by 54.38%, and myogenin increased by 71.17%, while PL only increased Myo-D increased by 11.53%, MRF-4 increased by 11.24%, myf5 increased by 19.45%%, and myogenin increased by 28.15%. This is a noteworthy result, as MyoD and Myf5 are believed to be involved in satellite proliferation, and myogenin and MRF-4 are involved in satellite cell differentiation [17
]. Therefore, our results suggest that NO may have been undergoing a greater amount of satellite cell proliferation and differentiation, as indicated by elevated levels of MyoD and MRF-4, respectively.
We have demonstrated in the present study that total DNA content for NO was increased 88.75%, whereas PL was only increased 4.67% with training (p = 0.011), and the increases observed in NO were significantly greater than PL (p = 0.041). During muscle hypertrophy, myonuclei increase sequentially [49
] as satellite cells proliferate, fuse with muscle fibers and donate their nuclei, and increase myonuclear number [50
]. Consequently, increases in myonuclear number and sarcoplasmic volume are proportional and the myocyte myonuclear domain remains constant, thereby resulting in no appreciable change in DNA/protein and subsequent maintenance in the myonuclear domain. Conversely, because an increase in myonuclear number expands the quantity of DNA available for gene expression and subsequent protein synthesis, the additional myonuclei will facilitate skeletal muscle hypertrophy, thereby resulting in a decrease in DNA/protein as more muscle protein is synthesized from fewer myocytes/DNA [51
]. Nuclei within mature muscle fibers are mitotically inactive [52
]; therefore, an increase in skeletal muscle DNA content is indicative of myogenically-induced satellite cell activation.
We observed the increases in myofibrillar protein and total DNA content to occur in both groups; however, while DNA/protein was decreased in PL, it was maintained in NO. Both groups also underwent increase increases in the MRFs and phosphorylated c-met, but the increases were greater for NO. This scenario is conceivably attributed to increases in satellite cell activation due to the premise that initial muscle fiber hypertrophy can expand the myonuclear domain as existing myonuclei increase their protein synthesis to support moderate increases in sarcoplasmic volume [12
]. However, once a certain limit in the myonuclear domain is reached, further myofiber hypertrophy may only occur as a result of satellite cell activation and the subsequent addition of new myonuclei [42
]. Based on our results for the markers of myogenesis and the maintenance of the myonuclear domain, the present data suggest that the muscle hypetrophy occurring in response to 28 days of heavy resistance exercise combined with NO-Shotgun®
supplementation appears to be more effective at promoting the myogenic activation of satellite cells than resistance exercise combined with a carbohydrate placebo. IGF-I activates phosphatidylinositol-3 kinase (PI3K) resulting in downstream phosphorylation of Akt [30
]. Creatine supplementation has also been shown to enhance the differentiation of myogenic C2
cells by activating the p38 MAPK pathway, as the activation of p38 and the transcription factor, myocyte enhancer factor 2 (MEF-2) were increased [29
]. The p38 MAPK pathway is an important signaling pathway responsible for up-regulating the expression of various sarcomeric genes in response to mechanical overload. The Akt/mTOR pathway is an important pathway involved in up-regulating translational activity en route to increases in muscle protein synthesis. The Akt/mTOR pathway was activated in C2
myoblasts treated with creatine, as Akt, mTOR, and p70S6 kinase activity were elevated [29
]. The Akt/mTOR pathway can also be activated by leucine [30
Supplemental leucine leads to increased levels of α-ketoisocaproate (KIC) [31
], which inhibits the activity of the branched-chain keto-acid dehydrogenase (BCKDH) complex, thereby blunting BCAA oxidation [32
] and muscle proteolysis [54
] during heavy resistance exercise. It has been shown that 14 days of KIC and beta-hydroxy-beta-methylbutyrate (HMB) supplementation reduced signs and symptoms of exercise-induced muscle damage in untrained males following a single bout of eccentrically-biased resistance exercise [55
]. Furthermore, BCAA ingested prior to 60 min of cycling exercise at 50% of maximal work capacity has been shown to attenuate exercise-induced skeletal muscle proteolysis [56
In regard to clinical safety measures, all of the whole blood and serum markers assessed remained within normal clinical ranges throughout the duration of the study. As a result, we observed no significant differences between PL and NO, indicating NO-Shotgun®
to have no deleterious effects with regard to the whole blood and serum variables we assessed. The NO-Shotgun®
supplement contains a number of different compounds with many of these having little to no clinical safety data available. However, there are safety data available for creatine. Creatine is well-tolerated in most individuals in short-term studies [57
]. Nevertheless, idiosyncratic effects may occur when large amounts of an exogenous substance containing an amino group are consumed, with the consequent increased load on the liver and kidneys [58
]. Therefore, concerns have been raised regarding the long-term safety of creatine supplementation. To date, however, studies consisting of durations of nine wk to five yr have not found clinically significant deviations from normal values in renal, hepatic, and cardiac safety markers in healthy individuals [58
NO-Shotgun® is a nutritional supplement that contains a synergistic blend of compounds, such as creatine, leucine, KIC, and arginine which have been shown in previous studies to be effective at increasing muscle strength and mass, myofibrillar protein content, muscle protein synthesis, and satellite cell activation. Based on the results presented herein, it is difficult to conclude whether any one compound or the additive and/or synergistic effects of various compounds contained in NO-Shotgun® were responsible for eliciting the effects we observed to occur. Therefore, we conclude that 28 days resistance training, when supplemented with NO-Shotgun®, has no negative effects on the clinical safety markers assessed, while effectively increasing muscle strength and mass, myofibrillar protein content, and stimulating increases in myogenic markers indicative of satellite cell activation.