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Nitric Oxide, Sports Nutrition and Muscle Building
C H A P T E R
26 Nitric Oxide, Sports Nutrition and Muscle Building Lawrence J. Druhan The Levine Cancer Institute, Carolinas Healthcare System, Charlotte, NC, USA
INTRODUCTION Nitric oxide (NO) is a naturally occurring diatomous free radical, consisting of one atom of nitrogen and one atom of oxygen. In the atmosphere, NO is formed by combustion, and is typically thought of as a noxious substance, being involved in ozone layer depletion, the formation of acid rain, and air pollution. Conversely, organic synthesis of NO is a critical component in many physiological processes. Prokaryotic synthesis of NO is part of the nitrogen cycle, during which atmospheric nitrogen is reduced to ammonium which can then be used for the production of the nitrogen-containing molecules, DNA, RNA and proteins, necessary for life. During the denitrification step of the nitrogen cycle, NO is generated from the nitrogen oxides produced by living organisms as a step in the completion of the nitrogen cycle. The physiological function of NO was unknown prior to the landmark discoveries leading to the 1998 Nobel Prize in medicine. While investigating the mechanism of action of organic nitrates, which were known to induce relaxation of blood vessels, it was found that these drugs functioned by generating NO and that the generated NO increased the level of cyclic GMP in the vascular smooth muscle cells [1,2]. Subsequently, it was shown that a substance produced within the endothelium, termed endothelial derived relaxation factor (EDRF), activated the generation of the cGMP within the smooth muscle cells [3], and it was speculated that EDRF was NO. Subsequent experiments demonstrated that EDRF was indeed NO [4,5] and that an enzyme within the endothelium generated the NO from arginine [6,7]. These ground-breaking studies ushered in the era of NO signaling.
Nutrition and Enhanced Sports Performance. DOI: http://dx.doi.org/10.1016/B978-0-12-396454-0.00026-6
As a biological signaling molecule NO has many advantages: it is freely diffusible through biologic membranes and it has a short half life. Since the initial observation that NO is responsible for signaling between the endothelium and the vascular smooth muscle to induce vessel relaxation, NO signaling has been demonstrated in an extremely large range of physiological process; indeed every major organ system in the human body is either directly or indirectly affected by NO signaling. As such, it is not surprising that NO signaling has also been found to be involved in the physiology of skeletal muscle: in function, growth and repair. The following discussion will be directed toward the molecular mechanisms governed by NO in skeletal muscle, with an emphasis on potential nutritional modifications to augment the natural signaling processes and with an eye toward how these dietary modifications could and/ or do affect muscle growth.
THE NITRIC OXIDE SYNTHASES Enzymatic production of nitric oxide in humans is accomplished by the nitric oxide synthase family of enzymes. The three members of this family are neuronal NOS (NOS1 or nNOS), inducible NOS (NOS2 or iNOS) and endothelial NOS (NOS3 or eNOS). The conventional naming of the NOS enzymes implies cellular specificity; however, these enzymes were named according to the tissues/cells from which they were originally identified, and each of these enzymes has since been found in a much wider range of cells/tissues. The NOS enzymes all catalyze the same reaction: the conversion of arginine and oxygen to citrulline and NO using NADPH as the source for reducing equivalents. Additionally, the NOS
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enzymes share the same general structure, and utilize the same cofactors; however, they each have been found to elicit unique physiological functions. The production of varied function from the enzymatic generation of an identical product is possible because each of the three NOS enzymes differ in cellular and subcellular location and in regulation of activity [8]. Additionally, the activities of both nNOS and eNOS are positively regulated by increases in cellular calcium, whereas iNOS activity is calcium independent. Moreover, these enzymes are regulated by posttranslational modifications, including phosphorylation, acylation, and glutathionylation [9 12]. All three NOS isoforms have been identified in skeletal muscle. The principal NOS in skeletal muscle is nNOS [13]. Skeletal muscle contains an alternatively spliced variant of nNOS, nNOSμ. This splice variant is found in the cytoplasm and found localized to the membrane of the skeletal muscle via an association with the dystrophin protein complex [14], and mutations that cause human muscular dystrophy have been found to down-regulate nNOS activity and reduce muscle blood flow [15]. nNOSμ has been found to regulate skeletal muscle blood flow, and its activity can attenuate the activation of vasoconstriction to ensure adequate blood flow to the muscle during exercise [16,17]. Another nNOS splice variant, nNOSβ, has been found to be localized to the Golgi [18]. nNOSβ was shown to affect skeletal muscle force production. Expression of eNOS has been identified in skeletal muscle; however, the expression level is low. eNOS has been found to be associated with the vascular endothelium in skeletal muscle and with skeletal muscle mitochondria [19,20]. Although iNOS is generally not expressed in healthy human adult skeletal muscle, iNOS expression can be increased under some conditions [21]. Exercise has been found to both induce NO production and alter the expression of the various NOS isoforms. In humans the expression of nNOSμ has been shown to be increased by exercise [22]. In this study the authors demonstrated that nNOSμ was higher in endurance trained athletes compared with sedentary controls. Moreover, they showed that in sedentary subjects that underwent an exercise regimen, nNOSμ protein level was significantly increased. There was no detectable change in either eNOS or iNOS. Therefore, it is clear that the NOS family of enzymes is critical to the function of skeletal muscle, both at rest and during exercise.
THE NITRATE NITRITE NO PATHWAY Nitrite and nitrate are formed in the body via the oxidation of NOS-derived NO. Additionally, these
inorganic nitrogenous compounds are natural components of many healthy foods as well as being used as preservatives in many processed foods. Excess exposure to these compounds has been identified as carcinogenic [23,24]; however, there is increasing evidence that nitrite and nitrate are not just toxic byproducts but rather part of an intrinsic NO-recycling system that has physiologically relevant activity [25]. In addition to the recycling of NOS-derived NO, this pathway can directly convert inorganic nitrite and nitrate to NO in a NOS-independent fashion. The NOS-independent generation of NO (and indeed the recycling of NOS-derived NO) functions through the action of an entero-salivary system. In this system, dietary nitrate is absorbed in the gastrointestinal tract and transported to the saliva glands via the circulatory system. Bacteria in the saliva glands convert the nitrate to nitrite via the action of nitrate reductase enzymes. The nitrite rich saliva is then swallowed and either converted to NO in the stomach or absorbed in the GI tract and passed into the circulation. The nitrite in circulation can be converted to NO via enzymatic nitrite reduction in a number of different tissues, including skeletal muscle [26,27]. Thus, once in circulation, nitrite can act as an endocrine-like carrier of NO activity. The tissue metabolism of nitrite has been shown to be oxygen and pH dependent [26,28]. When the tissue oxygen concentration (or pH) is high, nitrite is preferentially oxidized to nitrate; however, at low oxygen tension, nitrite is converted to NO. Thus the nitritedependent generation of NO in skeletal muscle could be dependent upon the metabolic state of the tissue: under resting conditions the nitrite would be converted to nitrate and recycled whereas under strenuous exercise, when oxygen is limited (and pH is decreased), nitrite would be converted to NO. Somewhat surprisingly, however, dietary nitrate has been shown to result in lower oxygen demand during submaximal exercise [29]. Thus it seems that, even when tissue oxygen levels are not limiting, and the tissue pH is not significantly altered, nitrite can still affect skeletal muscle function.
SKELETAL MUSCLE FUNCTIONS MEDIATED BY NO The cardiovascular function of NO in humans has been well characterized [30,31]. However, NO has also been found to affect a wide range of functions within skeletal muscle, including the regulation of muscle function, metabolism, and growth. It has been shown that NO regulates muscle force production [32]. In activated rat diaphragm, inhibition of NOS decreased maximum velocity of shortening, loaded shortening
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SKELETAL MUSCLE FUNCTIONS MEDIATED BY NO
velocity, and power production. These affects were reversed by addition of an inorganic NO donor; however the NO donor had no affect alone. Thus, the endogenous generation of NO from NOS regulates muscle contraction. In regard to NO regulation of skeletal muscle metabolism, both oxygen consumption and glucose uptake are known to be regulated by NO [33 35], although, during exercise, it seems that the prevailing mechanism by which NO regulates skeletal muscle metabolism is via glucose homeostasis [35]. While these mechanisms could indirectly affect muscle growth, NO is also involved in the regulation of processes that are directly involved. These processes include transcription of skeletal muscle proteins, activation/proliferation of muscle satellite cells, and skeletal muscle blood flow.
Transcription of Skeletal Muscle Proteins and Activation of Satellite Cells Muscle growth during exercise has been mainly attributed to increased protein transcription in the myofibrils and to the activity of satellite cells. Nitric oxide has been shown to be directly involved in muscle hypertrophy and fiber type transition observed in chronically loaded muscle in a rodent model [36]. Although this work did not examine the involvement of satellite cells, it was shown that load-induced protein synthesis (as indicated by muscle mass) was inhibited in animals treated with a NOS inhibitor, indicating that NO plays a role in muscle protein synthesis. These authors went on to demonstrate that this NOS-dependence of the observed muscle hypertrophy was not due to an alteration of growth factor induction or activation/proliferation of satellite cells [37]. Rather, the NOS-dependent component of the observed loadinduced hypertrophy was due to an increase in mRNA for both actin and type 1 myosin heavy chain, indicating that endogenous production of NO was involved in the increase in synthesis of contractile proteins. Atrophy associated with functional unloading of muscle has been shown to be attenuated by the administration of arginine [38]. This study demonstrated that the decrease in myosin heavy chain type 1 associated with muscle unloading was diminished by the addition of arginine; however, this is likely a combination of alterations in protein synthesis and protein degradation found in unload-induced muscle atrophy [38,39]. Satellite cells are myocyte stem cells with the ability to generate new muscle fibers and to provide new myonuclei during muscle growth [40]. Moreover, increases in satellite cells have been observed in humans undergoing resistance training [41]. Thus, both myofibril protein synthesis and satellite cell
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number/activation are important for muscle growth not only in pathological settings (such as muscular dystrophy) but also in healthy subjects [42,43]. Nitric oxide has been shown to both activate satellite cells and to regulate protein synthesis. NO has been shown to stimulate the proliferation of satellite cells and to maintain the reserve pool of them [44]. Additionally, NO has been shown to activate satellite cells to enter the cell cycle for the production of new muscle [45,46]. These results (among others) have led to the hypothesis that therapies to increase muscle NO would be beneficial in pathophysiological settings that would benefit from increased muscle repair/growth, such as the muscular dystrophies [42]. Increase in muscle mass during resistance training has been shown to involve a number of mechanisms [47], including the NO-dependent mechanisms described above. Moreover, NO is known to be produced in working muscle [48]. Taken together, and with the above studies, these data indicate that it is plausible that increasing NO during resistance training by nutritional manipulation would increase the muscle growth response via stimulation of satellite cell proliferation/activation and/or by increasing protein synthesis in the myofibril. However, there are no studies to date directly examining this possibility in humans.
Mitochondrial Biogenesis Mitochondrial biogenesis is necessary both during mitosis and to respond to the energy needs of developed cells. As such, this process is necessary for muscle growth. The biogenesis of mitochondria is a complex process involving the synthesis of mitochondrial proteins and DNA, the synthesis and import of nuclear encoded proteins, and the synthesis and import of lipids. This process is governed by a large and complex system involving many cellular proteins and transcription factors [49]. In skeletal muscle the process of mitochondrial biogenesis is initiated when the energy demand of the myocyte exceeds the capacity of the resident mitochondria. This energy imbalance can occur under a number of conditions, including exercise [50]. The energy imbalance triggers a signaling cascade that initiates mitochondrial biogenesis. This signaling cascade is thought to be initiated by the activation of protein kinases, including AMP kinase (AMPK), which is activated in response to the increase in ATP consumption, and calcium-dependent kinases which are activated in response to changes in intracellular calcium. Activation of these kinase enzymes induces the expression of a family of transcription factors termed the peroxisome proliferator-
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activated receptor gamma coactivator (PGC-1) family. This family of transcription factors includes PCG-1α, PGC-1β, and PGC-1α-related coactivator (PRC). All of these transcription factors have been implicated in mitochondrial biosynthesis; however, it is PCG-1α that is thought to be the most significant member of this family in regard to the metabolic regulation of this process. Over-expression of PCG-1α demonstrates a significant increase in mitochondrial biogenesis accompanied by increased exercise capacity [51], while deletion of PGC-1α blunts the observed exercise-induced increase in mitochondrial enzymes [52]. Thus, PGC-1α has been referred to as the master regulator of mitochondrial biogenesis. Nitric oxide has been found to stimulate mitochondrial biogenesis in many tissues, including skeletal muscle [53]. In vitro experiments in myoblasts and myotubes have demonstrated that NO donors increase mitochondrial biogenesis [54 56]. Studies have demonstrated that the NO-induced increase in mitochondria was via a cGMP-dependent mechanism, which led to the activation of PCG-1α and its downstream targets [57], and that this process resulted in functional mitochondria [56]. Moreover, it was demonstrated that AMPK, which activates PCG1-α, is regulated by NO [58] and that NO and AMPK act synergistically to regulate PCG-1α. The NOS involvement in this NO-driven process is likely tied to the known calcium dependence of both nNOS and eNOS. Indeed, NOS has been implicated in mitochondrial biogenesis [59]. It was demonstrated that mice deficient in eNOS had decreased PCG-1α mRNA and decreased mitochondria [56], while mice deficient in nNOS seem to have decreased mitochondria at basal conditions [60]. Thus it was concluded that, while eNOS plays a positive role in the regulation of mitochondrial biogenesis, nNOS plays a negative role, at least in mice. However, together pharmacological and genetic evidence indicates that neither eNOS nor nNOS seem to play a role in exerciseinduced increase in mitochondrial biogenesis [59,61]. Thus it was concluded that, although NO does play a role in mitochondrial biogenesis under basal conditions, the observed increase in NO produced during exercise is not necessary for the observed exerciseinduced increase in mitochondria [35]. Arginine supplementation as a means to induce mitochondrial biogenesis in skeletal muscle has not been tested in a healthy human population. However, in patients with type II diabetes, mitochondrial density in skeletal muscle is decreased [62], and long-term treatment of type II diabetics with arginine supplementation has demonstrated increased benefit of hypocaloric diet and exercise [63]. Thus it is possible, given the discussion above, that the long-term treatment of humans with arginine, at least in the diabetic setting,
increases mitochondrial biogenesis in the skeletal muscle [64], and this increase in skeletal muscle mitochondria would be beneficial in regard to increasing muscle mass.
Skeletal Muscle Blood Flow Given the known vasodilatory action of NO, it is not surprising that nitric oxide has been found to be involved in the regulation of skeletal muscle blood flow [65]. It has been demonstrated that infusion of the NOS inhibitor l-NMMA into resting human subjects decreased forearm blood flow. Moreover, the response to acetylcholine, which is known to activate endothelial NO production, was blunted whereas the response to an NO-donating drug was not inhibited [66]. This NO-dependent regulation of skeletal muscle blood flow has also been observed during recovery after exercise [67]. Some authors have also observed that muscle blood flow during exercise is also regulated by NO [68,69]. However, there are issues with when and how these reports were measuring blood flow, and the accumulating evidence indicates that NOS activity is not the driving factor regulating skeletal blood flow during exercise [67,70 72]. Indeed, a recent study using positron emission tomography indicated that NOS inhibition decreased resting muscle blood flow but did not decrease blood flow during exercise [73]. These authors did, however, demonstrate inhibition of skeletal muscle blood flow during exercise by a combination of NOS and COX2 inhibition. Thus, while NOS-dependent NO alone does not regulate exerciseinduced changes in skeletal muscle blood flow, it can do so in synergy with other factors. The stimulation of NOS-derived NO has also been tested in regard to the regulation of skeletal blood flow. Bolus oral consumption of 10 g of arginine failed to alter skeletal blood flow, either at rest or during exercise [74]. Additionally, although plasma arginine level was increased, there was no increase in plasma markers for increased NO synthesis. This observation could be unique to oral administration of arginine or dose dependent, as 30 g bolus intravenous arginine was found to decrease blood pressure and peripheral resistance, while 6 g of arginine (either IV or PO) did not induce a significant change [75]. However, in a recent study, acute administration of 6 g of arginine did increase muscle blood volume during recovery after exercise, but failed to increase markers of increased NO production [76]. Thus it seems clear that oral administration of arginine does not regulate muscle blood flow, but this lack of arginine-induced alteration cannot per se be attributed to any change in NOS-dependent NO generation. Although NOS-
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NUTRITIONAL MODIFICATION OF SKELETAL MUSCLE HYPERTROPHY
independent NO has not specifically been tested in regard to skeletal muscle blood flow in humans, administration of nitrate (which is converted to nitrite and then NO) has been shown to increase skeletal muscle blood volume [77], and dietary nitrate has been shown to regulate skeletal blood flow in rodents [78]. In summary, skeletal muscle blood flow is regulated directly by NO at rest and after exercise. However, during exercise, the contribution of NO to this regulation is less pronounced, but does have synergistic affects. Attempts to alter skeletal muscle blood flow via the consumption of potential NO-generating substances have not shown significant changes [79,80].
NUTRITIONAL MODIFICATION OF SKELETAL MUSCLE HYPERTROPHY Since the identification of NO as a critical regulator of many physiological processes, there has been much interest in using nutritional modification to augment NO production for both the improvement of healthy humans and in the treatment of several pathophysiologic conditions [64,81 84], and these studies have shown varied results, both positive and negative. Moreover, it is clear that NO-directed supplementation can affect skeletal muscle hypertrophy and ameliorate some pathophysiologic conditions involving skeletal muscle dysfunction in animals [42]. Additionally, administration of an NO donor in combination with an anti-inflammatory drug has been demonstrated to be safe and have some efficacy in the treatment of muscular dystrophies [85]. However, while it is clear that NO signaling has the potential to positively regulate muscle growth, there have not been any peer-reviewed studies that directly examine if augmentation of NO signaling by dietary supplementation regulates muscle hypertrophy in humans.
Nutritional Supplementation for Enhanced Performance in Humans There have been many studies examining the effect of NO-directed nutritional supplements on exercise performance in humans [86,87]. However, these studies use a wide range of supplements, have significantly variable duration of treatment, use variable exercise protocols, measure different outcomes, and have a variable subject population. Thus, it is not surprising that, whereas some studies indicated positive effects, an approximately equal number showed no effect. Attempts have been made to stratify these studies in order to draw some conclusions. Alvares et al. examined the differing ergogenic effects of acute versus
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chronic supplementation of arginine [86]. Of the five acute (treatment of less than 3 days prior to testing) studies reviewed, three demonstrated a significant increase in performance; of the eight chronic (treatment of 10 days to 6 months) studies, four demonstrated significant increases. It should be noted that in some studies the supplements used to increase circulating arginine levels included other components: glycine, α-ketoisocaproic acid, aspartate, α-ketoglutarate, and creatine. Moreover, none of the studies tested for markers of increased NO activity. Alvares et al. concluded that, while the supplements are well tolerated, there is not enough evidence to recommend the use of nutritional supplementation of arginine as an aid in exercise performance in healthy individuals. A more recent and general review of the effects of NO-supplementation on exercise performance has come to similar conclusions [87]. In this review, Bescos et al. cite 42 studies that examined how various NOrelated substances affect performance, including not only supplements aimed at increasing circulating arginine levels, but also those aimed at increasing circulating nitrite levels. Again, only approximately half of the studies reviewed that reported performance data indicated an increase in performance in response to supplementation. Bescos et al. draw several conclusions. The potential benefit of arginine supplementation is variable based on the training status of the individual, with benefits being seen in moderately and untrained subjects but little effect in well trained athletes, and this benefit in general relies on the combination of arginine with other substances. Citrulline supplementation alone does not enhance performance. Nitrate supplementation effectively enhances exercise performance in untrained or moderately trained subjects, but the limited data indicate that this affect is not seen in endurance-trained athletes. Thus it is clear that while attempts to augment NO signaling can improve exercise performance in general, this improvement is not seen in all settings. Moreover, it is impossible in many of the published studies to dissect out the effects of the supplementation on muscle hypertrophy (which would increase muscle strength and thus exercise performance) from the other physiological effects of augmented NO signaling. Indeed, neither of these reviews addressed the potential for the enhancement of skeletal muscle hypertrophy by NO-supplementation in detail.
Nutritional Supplementation for Enhanced Muscle Hypertrophy in Humans As previously mentioned, there are no studies specifically examining the influences of NO-related nutritional supplements on muscle hypertrophy in humans.
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However, there are studies that examine how muscle strength is affected in response to these supplements. Given that muscle strength correlates with muscle cross-sectional area [88], increases in muscle strength can thus be used as a surrogate marker for muscle hypertrophy. Additionally, it is clear that the initiation of muscle hypertrophy requires continuous muscle stimulation. Indeed, muscle hypertrophy in response to resistance exercise is virtually nonexistent during the first weeks of training [47]. Therefore, studies that examine the effects of the chronic administration of NO-related nutritional supplements on muscle strength can provide some data regarding whether skeletal muscle hypertrophy can be affected by nutritional supplementation of NO signaling. Campbell et al. examined how chronic supplementation with l-arginine α-ketoglutarate (AAG) affected strength gain during an 8-week training protocol [89]. Administration of AAG significantly increased plasma arginine levels. The addition of AAG supplementation to a standardized exercise protocol over 8 weeks significantly increased muscle strength as assessed by a 1RM bench press test. From these data, it can be concluded that this supplement has augmented the exercise-induced muscle hypertrophy. Interestingly, there was no difference in body composition, muscle endurance, or aerobic capacity. It has been argued, rightly, that AAG can have affects outside its potential augmentation of NO signaling, because α-ketoglutarate could increase TCA cycle flux and initiate glutamate sparing [90]. As such, it cannot be conclusively concluded that it is solely the potential increase in NO signaling produced by AAG that elicits the effects observed. However, acute administration of AAG failed to alter muscle strength as measured by 1RM [90]. Thus the strength gain observed in the chronic supplementation model is not due to acute NO effects on muscle contractile function, bolstering the supposition that AAG supplementation did augment exerciseinduced muscle hypertrophy. Another study indicates that nutritional supplementation of NO signaling cannot in and of itself induce skeletal muscle hypertrophy [91]. Fricke et al. demonstrated that, in postmenopausal women, oral administration of l-arginine (18 g per day for 6 months) increased peak jump force, and they speculated that this supplementation could prevent the decline in muscle force observed in this population. However, they found no statistically significant changes in jump power, grip force, muscle cross-sectional area, or fat area. Thus, in this population, arginine supplementation does not affect muscle hypertrophy. Perhaps arginine supplementation alone cannot induce muscle hypertrophy, but it can augment exercise-induced hypertrophy.
CONCLUSION It is clear that NO regulates many processes in skeletal muscle and that alterations in NO signaling, both pharmacologic and nutritional, can alter the response of skeletal muscle. These observations can be used to make a compelling argument for the use of nutritional substances in the augmentation of NO signaling to elicit beneficial effects in skeletal muscle strength, growth and performance. However, there is still not a clear consensus on whether any of the nutritional substances tested in rigorous controlled settings are truly eliciting an effect and whether any observed effect is truly due to an alteration in NO signaling, especially in regard to muscle growth. That notwithstanding, many studies have provided evidence that nutritional supplements aimed at altering NO signaling can be beneficial. More work is needed to definitively prove not only if NO supplementation can be beneficial to specific skeletal muscle processes, but also to better define optimal substance type, dosage, and treatment protocols.
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26 Nitric Oxide, Sports Nutrition and Muscle Building Lawrence J. Druhan The Levine Cancer Institute, Carolinas Healthcare System, Charlotte, NC, USA
INTRODUCTION Nitric oxide (NO) is a naturally occurring diatomous free radical, consisting of one atom of nitrogen and one atom of oxygen. In the atmosphere, NO is formed by combustion, and is typically thought of as a noxious substance, being involved in ozone layer depletion, the formation of acid rain, and air pollution. Conversely, organic synthesis of NO is a critical component in many physiological processes. Prokaryotic synthesis of NO is part of the nitrogen cycle, during which atmospheric nitrogen is reduced to ammonium which can then be used for the production of the nitrogen-containing molecules, DNA, RNA and proteins, necessary for life. During the denitrification step of the nitrogen cycle, NO is generated from the nitrogen oxides produced by living organisms as a step in the completion of the nitrogen cycle. The physiological function of NO was unknown prior to the landmark discoveries leading to the 1998 Nobel Prize in medicine. While investigating the mechanism of action of organic nitrates, which were known to induce relaxation of blood vessels, it was found that these drugs functioned by generating NO and that the generated NO increased the level of cyclic GMP in the vascular smooth muscle cells [1,2]. Subsequently, it was shown that a substance produced within the endothelium, termed endothelial derived relaxation factor (EDRF), activated the generation of the cGMP within the smooth muscle cells [3], and it was speculated that EDRF was NO. Subsequent experiments demonstrated that EDRF was indeed NO [4,5] and that an enzyme within the endothelium generated the NO from arginine [6,7]. These ground-breaking studies ushered in the era of NO signaling.
Nutrition and Enhanced Sports Performance. DOI: http://dx.doi.org/10.1016/B978-0-12-396454-0.00026-6
As a biological signaling molecule NO has many advantages: it is freely diffusible through biologic membranes and it has a short half life. Since the initial observation that NO is responsible for signaling between the endothelium and the vascular smooth muscle to induce vessel relaxation, NO signaling has been demonstrated in an extremely large range of physiological process; indeed every major organ system in the human body is either directly or indirectly affected by NO signaling. As such, it is not surprising that NO signaling has also been found to be involved in the physiology of skeletal muscle: in function, growth and repair. The following discussion will be directed toward the molecular mechanisms governed by NO in skeletal muscle, with an emphasis on potential nutritional modifications to augment the natural signaling processes and with an eye toward how these dietary modifications could and/ or do affect muscle growth.
THE NITRIC OXIDE SYNTHASES Enzymatic production of nitric oxide in humans is accomplished by the nitric oxide synthase family of enzymes. The three members of this family are neuronal NOS (NOS1 or nNOS), inducible NOS (NOS2 or iNOS) and endothelial NOS (NOS3 or eNOS). The conventional naming of the NOS enzymes implies cellular specificity; however, these enzymes were named according to the tissues/cells from which they were originally identified, and each of these enzymes has since been found in a much wider range of cells/tissues. The NOS enzymes all catalyze the same reaction: the conversion of arginine and oxygen to citrulline and NO using NADPH as the source for reducing equivalents. Additionally, the NOS
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enzymes share the same general structure, and utilize the same cofactors; however, they each have been found to elicit unique physiological functions. The production of varied function from the enzymatic generation of an identical product is possible because each of the three NOS enzymes differ in cellular and subcellular location and in regulation of activity [8]. Additionally, the activities of both nNOS and eNOS are positively regulated by increases in cellular calcium, whereas iNOS activity is calcium independent. Moreover, these enzymes are regulated by posttranslational modifications, including phosphorylation, acylation, and glutathionylation [9 12]. All three NOS isoforms have been identified in skeletal muscle. The principal NOS in skeletal muscle is nNOS [13]. Skeletal muscle contains an alternatively spliced variant of nNOS, nNOSμ. This splice variant is found in the cytoplasm and found localized to the membrane of the skeletal muscle via an association with the dystrophin protein complex [14], and mutations that cause human muscular dystrophy have been found to down-regulate nNOS activity and reduce muscle blood flow [15]. nNOSμ has been found to regulate skeletal muscle blood flow, and its activity can attenuate the activation of vasoconstriction to ensure adequate blood flow to the muscle during exercise [16,17]. Another nNOS splice variant, nNOSβ, has been found to be localized to the Golgi [18]. nNOSβ was shown to affect skeletal muscle force production. Expression of eNOS has been identified in skeletal muscle; however, the expression level is low. eNOS has been found to be associated with the vascular endothelium in skeletal muscle and with skeletal muscle mitochondria [19,20]. Although iNOS is generally not expressed in healthy human adult skeletal muscle, iNOS expression can be increased under some conditions [21]. Exercise has been found to both induce NO production and alter the expression of the various NOS isoforms. In humans the expression of nNOSμ has been shown to be increased by exercise [22]. In this study the authors demonstrated that nNOSμ was higher in endurance trained athletes compared with sedentary controls. Moreover, they showed that in sedentary subjects that underwent an exercise regimen, nNOSμ protein level was significantly increased. There was no detectable change in either eNOS or iNOS. Therefore, it is clear that the NOS family of enzymes is critical to the function of skeletal muscle, both at rest and during exercise.
THE NITRATE NITRITE NO PATHWAY Nitrite and nitrate are formed in the body via the oxidation of NOS-derived NO. Additionally, these
inorganic nitrogenous compounds are natural components of many healthy foods as well as being used as preservatives in many processed foods. Excess exposure to these compounds has been identified as carcinogenic [23,24]; however, there is increasing evidence that nitrite and nitrate are not just toxic byproducts but rather part of an intrinsic NO-recycling system that has physiologically relevant activity [25]. In addition to the recycling of NOS-derived NO, this pathway can directly convert inorganic nitrite and nitrate to NO in a NOS-independent fashion. The NOS-independent generation of NO (and indeed the recycling of NOS-derived NO) functions through the action of an entero-salivary system. In this system, dietary nitrate is absorbed in the gastrointestinal tract and transported to the saliva glands via the circulatory system. Bacteria in the saliva glands convert the nitrate to nitrite via the action of nitrate reductase enzymes. The nitrite rich saliva is then swallowed and either converted to NO in the stomach or absorbed in the GI tract and passed into the circulation. The nitrite in circulation can be converted to NO via enzymatic nitrite reduction in a number of different tissues, including skeletal muscle [26,27]. Thus, once in circulation, nitrite can act as an endocrine-like carrier of NO activity. The tissue metabolism of nitrite has been shown to be oxygen and pH dependent [26,28]. When the tissue oxygen concentration (or pH) is high, nitrite is preferentially oxidized to nitrate; however, at low oxygen tension, nitrite is converted to NO. Thus the nitritedependent generation of NO in skeletal muscle could be dependent upon the metabolic state of the tissue: under resting conditions the nitrite would be converted to nitrate and recycled whereas under strenuous exercise, when oxygen is limited (and pH is decreased), nitrite would be converted to NO. Somewhat surprisingly, however, dietary nitrate has been shown to result in lower oxygen demand during submaximal exercise [29]. Thus it seems that, even when tissue oxygen levels are not limiting, and the tissue pH is not significantly altered, nitrite can still affect skeletal muscle function.
SKELETAL MUSCLE FUNCTIONS MEDIATED BY NO The cardiovascular function of NO in humans has been well characterized [30,31]. However, NO has also been found to affect a wide range of functions within skeletal muscle, including the regulation of muscle function, metabolism, and growth. It has been shown that NO regulates muscle force production [32]. In activated rat diaphragm, inhibition of NOS decreased maximum velocity of shortening, loaded shortening
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SKELETAL MUSCLE FUNCTIONS MEDIATED BY NO
velocity, and power production. These affects were reversed by addition of an inorganic NO donor; however the NO donor had no affect alone. Thus, the endogenous generation of NO from NOS regulates muscle contraction. In regard to NO regulation of skeletal muscle metabolism, both oxygen consumption and glucose uptake are known to be regulated by NO [33 35], although, during exercise, it seems that the prevailing mechanism by which NO regulates skeletal muscle metabolism is via glucose homeostasis [35]. While these mechanisms could indirectly affect muscle growth, NO is also involved in the regulation of processes that are directly involved. These processes include transcription of skeletal muscle proteins, activation/proliferation of muscle satellite cells, and skeletal muscle blood flow.
Transcription of Skeletal Muscle Proteins and Activation of Satellite Cells Muscle growth during exercise has been mainly attributed to increased protein transcription in the myofibrils and to the activity of satellite cells. Nitric oxide has been shown to be directly involved in muscle hypertrophy and fiber type transition observed in chronically loaded muscle in a rodent model [36]. Although this work did not examine the involvement of satellite cells, it was shown that load-induced protein synthesis (as indicated by muscle mass) was inhibited in animals treated with a NOS inhibitor, indicating that NO plays a role in muscle protein synthesis. These authors went on to demonstrate that this NOS-dependence of the observed muscle hypertrophy was not due to an alteration of growth factor induction or activation/proliferation of satellite cells [37]. Rather, the NOS-dependent component of the observed loadinduced hypertrophy was due to an increase in mRNA for both actin and type 1 myosin heavy chain, indicating that endogenous production of NO was involved in the increase in synthesis of contractile proteins. Atrophy associated with functional unloading of muscle has been shown to be attenuated by the administration of arginine [38]. This study demonstrated that the decrease in myosin heavy chain type 1 associated with muscle unloading was diminished by the addition of arginine; however, this is likely a combination of alterations in protein synthesis and protein degradation found in unload-induced muscle atrophy [38,39]. Satellite cells are myocyte stem cells with the ability to generate new muscle fibers and to provide new myonuclei during muscle growth [40]. Moreover, increases in satellite cells have been observed in humans undergoing resistance training [41]. Thus, both myofibril protein synthesis and satellite cell
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number/activation are important for muscle growth not only in pathological settings (such as muscular dystrophy) but also in healthy subjects [42,43]. Nitric oxide has been shown to both activate satellite cells and to regulate protein synthesis. NO has been shown to stimulate the proliferation of satellite cells and to maintain the reserve pool of them [44]. Additionally, NO has been shown to activate satellite cells to enter the cell cycle for the production of new muscle [45,46]. These results (among others) have led to the hypothesis that therapies to increase muscle NO would be beneficial in pathophysiological settings that would benefit from increased muscle repair/growth, such as the muscular dystrophies [42]. Increase in muscle mass during resistance training has been shown to involve a number of mechanisms [47], including the NO-dependent mechanisms described above. Moreover, NO is known to be produced in working muscle [48]. Taken together, and with the above studies, these data indicate that it is plausible that increasing NO during resistance training by nutritional manipulation would increase the muscle growth response via stimulation of satellite cell proliferation/activation and/or by increasing protein synthesis in the myofibril. However, there are no studies to date directly examining this possibility in humans.
Mitochondrial Biogenesis Mitochondrial biogenesis is necessary both during mitosis and to respond to the energy needs of developed cells. As such, this process is necessary for muscle growth. The biogenesis of mitochondria is a complex process involving the synthesis of mitochondrial proteins and DNA, the synthesis and import of nuclear encoded proteins, and the synthesis and import of lipids. This process is governed by a large and complex system involving many cellular proteins and transcription factors [49]. In skeletal muscle the process of mitochondrial biogenesis is initiated when the energy demand of the myocyte exceeds the capacity of the resident mitochondria. This energy imbalance can occur under a number of conditions, including exercise [50]. The energy imbalance triggers a signaling cascade that initiates mitochondrial biogenesis. This signaling cascade is thought to be initiated by the activation of protein kinases, including AMP kinase (AMPK), which is activated in response to the increase in ATP consumption, and calcium-dependent kinases which are activated in response to changes in intracellular calcium. Activation of these kinase enzymes induces the expression of a family of transcription factors termed the peroxisome proliferator-
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activated receptor gamma coactivator (PGC-1) family. This family of transcription factors includes PCG-1α, PGC-1β, and PGC-1α-related coactivator (PRC). All of these transcription factors have been implicated in mitochondrial biosynthesis; however, it is PCG-1α that is thought to be the most significant member of this family in regard to the metabolic regulation of this process. Over-expression of PCG-1α demonstrates a significant increase in mitochondrial biogenesis accompanied by increased exercise capacity [51], while deletion of PGC-1α blunts the observed exercise-induced increase in mitochondrial enzymes [52]. Thus, PGC-1α has been referred to as the master regulator of mitochondrial biogenesis. Nitric oxide has been found to stimulate mitochondrial biogenesis in many tissues, including skeletal muscle [53]. In vitro experiments in myoblasts and myotubes have demonstrated that NO donors increase mitochondrial biogenesis [54 56]. Studies have demonstrated that the NO-induced increase in mitochondria was via a cGMP-dependent mechanism, which led to the activation of PCG-1α and its downstream targets [57], and that this process resulted in functional mitochondria [56]. Moreover, it was demonstrated that AMPK, which activates PCG1-α, is regulated by NO [58] and that NO and AMPK act synergistically to regulate PCG-1α. The NOS involvement in this NO-driven process is likely tied to the known calcium dependence of both nNOS and eNOS. Indeed, NOS has been implicated in mitochondrial biogenesis [59]. It was demonstrated that mice deficient in eNOS had decreased PCG-1α mRNA and decreased mitochondria [56], while mice deficient in nNOS seem to have decreased mitochondria at basal conditions [60]. Thus it was concluded that, while eNOS plays a positive role in the regulation of mitochondrial biogenesis, nNOS plays a negative role, at least in mice. However, together pharmacological and genetic evidence indicates that neither eNOS nor nNOS seem to play a role in exerciseinduced increase in mitochondrial biogenesis [59,61]. Thus it was concluded that, although NO does play a role in mitochondrial biogenesis under basal conditions, the observed increase in NO produced during exercise is not necessary for the observed exerciseinduced increase in mitochondria [35]. Arginine supplementation as a means to induce mitochondrial biogenesis in skeletal muscle has not been tested in a healthy human population. However, in patients with type II diabetes, mitochondrial density in skeletal muscle is decreased [62], and long-term treatment of type II diabetics with arginine supplementation has demonstrated increased benefit of hypocaloric diet and exercise [63]. Thus it is possible, given the discussion above, that the long-term treatment of humans with arginine, at least in the diabetic setting,
increases mitochondrial biogenesis in the skeletal muscle [64], and this increase in skeletal muscle mitochondria would be beneficial in regard to increasing muscle mass.
Skeletal Muscle Blood Flow Given the known vasodilatory action of NO, it is not surprising that nitric oxide has been found to be involved in the regulation of skeletal muscle blood flow [65]. It has been demonstrated that infusion of the NOS inhibitor l-NMMA into resting human subjects decreased forearm blood flow. Moreover, the response to acetylcholine, which is known to activate endothelial NO production, was blunted whereas the response to an NO-donating drug was not inhibited [66]. This NO-dependent regulation of skeletal muscle blood flow has also been observed during recovery after exercise [67]. Some authors have also observed that muscle blood flow during exercise is also regulated by NO [68,69]. However, there are issues with when and how these reports were measuring blood flow, and the accumulating evidence indicates that NOS activity is not the driving factor regulating skeletal blood flow during exercise [67,70 72]. Indeed, a recent study using positron emission tomography indicated that NOS inhibition decreased resting muscle blood flow but did not decrease blood flow during exercise [73]. These authors did, however, demonstrate inhibition of skeletal muscle blood flow during exercise by a combination of NOS and COX2 inhibition. Thus, while NOS-dependent NO alone does not regulate exerciseinduced changes in skeletal muscle blood flow, it can do so in synergy with other factors. The stimulation of NOS-derived NO has also been tested in regard to the regulation of skeletal blood flow. Bolus oral consumption of 10 g of arginine failed to alter skeletal blood flow, either at rest or during exercise [74]. Additionally, although plasma arginine level was increased, there was no increase in plasma markers for increased NO synthesis. This observation could be unique to oral administration of arginine or dose dependent, as 30 g bolus intravenous arginine was found to decrease blood pressure and peripheral resistance, while 6 g of arginine (either IV or PO) did not induce a significant change [75]. However, in a recent study, acute administration of 6 g of arginine did increase muscle blood volume during recovery after exercise, but failed to increase markers of increased NO production [76]. Thus it seems clear that oral administration of arginine does not regulate muscle blood flow, but this lack of arginine-induced alteration cannot per se be attributed to any change in NOS-dependent NO generation. Although NOS-
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independent NO has not specifically been tested in regard to skeletal muscle blood flow in humans, administration of nitrate (which is converted to nitrite and then NO) has been shown to increase skeletal muscle blood volume [77], and dietary nitrate has been shown to regulate skeletal blood flow in rodents [78]. In summary, skeletal muscle blood flow is regulated directly by NO at rest and after exercise. However, during exercise, the contribution of NO to this regulation is less pronounced, but does have synergistic affects. Attempts to alter skeletal muscle blood flow via the consumption of potential NO-generating substances have not shown significant changes [79,80].
NUTRITIONAL MODIFICATION OF SKELETAL MUSCLE HYPERTROPHY Since the identification of NO as a critical regulator of many physiological processes, there has been much interest in using nutritional modification to augment NO production for both the improvement of healthy humans and in the treatment of several pathophysiologic conditions [64,81 84], and these studies have shown varied results, both positive and negative. Moreover, it is clear that NO-directed supplementation can affect skeletal muscle hypertrophy and ameliorate some pathophysiologic conditions involving skeletal muscle dysfunction in animals [42]. Additionally, administration of an NO donor in combination with an anti-inflammatory drug has been demonstrated to be safe and have some efficacy in the treatment of muscular dystrophies [85]. However, while it is clear that NO signaling has the potential to positively regulate muscle growth, there have not been any peer-reviewed studies that directly examine if augmentation of NO signaling by dietary supplementation regulates muscle hypertrophy in humans.
Nutritional Supplementation for Enhanced Performance in Humans There have been many studies examining the effect of NO-directed nutritional supplements on exercise performance in humans [86,87]. However, these studies use a wide range of supplements, have significantly variable duration of treatment, use variable exercise protocols, measure different outcomes, and have a variable subject population. Thus, it is not surprising that, whereas some studies indicated positive effects, an approximately equal number showed no effect. Attempts have been made to stratify these studies in order to draw some conclusions. Alvares et al. examined the differing ergogenic effects of acute versus
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chronic supplementation of arginine [86]. Of the five acute (treatment of less than 3 days prior to testing) studies reviewed, three demonstrated a significant increase in performance; of the eight chronic (treatment of 10 days to 6 months) studies, four demonstrated significant increases. It should be noted that in some studies the supplements used to increase circulating arginine levels included other components: glycine, α-ketoisocaproic acid, aspartate, α-ketoglutarate, and creatine. Moreover, none of the studies tested for markers of increased NO activity. Alvares et al. concluded that, while the supplements are well tolerated, there is not enough evidence to recommend the use of nutritional supplementation of arginine as an aid in exercise performance in healthy individuals. A more recent and general review of the effects of NO-supplementation on exercise performance has come to similar conclusions [87]. In this review, Bescos et al. cite 42 studies that examined how various NOrelated substances affect performance, including not only supplements aimed at increasing circulating arginine levels, but also those aimed at increasing circulating nitrite levels. Again, only approximately half of the studies reviewed that reported performance data indicated an increase in performance in response to supplementation. Bescos et al. draw several conclusions. The potential benefit of arginine supplementation is variable based on the training status of the individual, with benefits being seen in moderately and untrained subjects but little effect in well trained athletes, and this benefit in general relies on the combination of arginine with other substances. Citrulline supplementation alone does not enhance performance. Nitrate supplementation effectively enhances exercise performance in untrained or moderately trained subjects, but the limited data indicate that this affect is not seen in endurance-trained athletes. Thus it is clear that while attempts to augment NO signaling can improve exercise performance in general, this improvement is not seen in all settings. Moreover, it is impossible in many of the published studies to dissect out the effects of the supplementation on muscle hypertrophy (which would increase muscle strength and thus exercise performance) from the other physiological effects of augmented NO signaling. Indeed, neither of these reviews addressed the potential for the enhancement of skeletal muscle hypertrophy by NO-supplementation in detail.
Nutritional Supplementation for Enhanced Muscle Hypertrophy in Humans As previously mentioned, there are no studies specifically examining the influences of NO-related nutritional supplements on muscle hypertrophy in humans.
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However, there are studies that examine how muscle strength is affected in response to these supplements. Given that muscle strength correlates with muscle cross-sectional area [88], increases in muscle strength can thus be used as a surrogate marker for muscle hypertrophy. Additionally, it is clear that the initiation of muscle hypertrophy requires continuous muscle stimulation. Indeed, muscle hypertrophy in response to resistance exercise is virtually nonexistent during the first weeks of training [47]. Therefore, studies that examine the effects of the chronic administration of NO-related nutritional supplements on muscle strength can provide some data regarding whether skeletal muscle hypertrophy can be affected by nutritional supplementation of NO signaling. Campbell et al. examined how chronic supplementation with l-arginine α-ketoglutarate (AAG) affected strength gain during an 8-week training protocol [89]. Administration of AAG significantly increased plasma arginine levels. The addition of AAG supplementation to a standardized exercise protocol over 8 weeks significantly increased muscle strength as assessed by a 1RM bench press test. From these data, it can be concluded that this supplement has augmented the exercise-induced muscle hypertrophy. Interestingly, there was no difference in body composition, muscle endurance, or aerobic capacity. It has been argued, rightly, that AAG can have affects outside its potential augmentation of NO signaling, because α-ketoglutarate could increase TCA cycle flux and initiate glutamate sparing [90]. As such, it cannot be conclusively concluded that it is solely the potential increase in NO signaling produced by AAG that elicits the effects observed. However, acute administration of AAG failed to alter muscle strength as measured by 1RM [90]. Thus the strength gain observed in the chronic supplementation model is not due to acute NO effects on muscle contractile function, bolstering the supposition that AAG supplementation did augment exerciseinduced muscle hypertrophy. Another study indicates that nutritional supplementation of NO signaling cannot in and of itself induce skeletal muscle hypertrophy [91]. Fricke et al. demonstrated that, in postmenopausal women, oral administration of l-arginine (18 g per day for 6 months) increased peak jump force, and they speculated that this supplementation could prevent the decline in muscle force observed in this population. However, they found no statistically significant changes in jump power, grip force, muscle cross-sectional area, or fat area. Thus, in this population, arginine supplementation does not affect muscle hypertrophy. Perhaps arginine supplementation alone cannot induce muscle hypertrophy, but it can augment exercise-induced hypertrophy.
CONCLUSION It is clear that NO regulates many processes in skeletal muscle and that alterations in NO signaling, both pharmacologic and nutritional, can alter the response of skeletal muscle. These observations can be used to make a compelling argument for the use of nutritional substances in the augmentation of NO signaling to elicit beneficial effects in skeletal muscle strength, growth and performance. However, there is still not a clear consensus on whether any of the nutritional substances tested in rigorous controlled settings are truly eliciting an effect and whether any observed effect is truly due to an alteration in NO signaling, especially in regard to muscle growth. That notwithstanding, many studies have provided evidence that nutritional supplements aimed at altering NO signaling can be beneficial. More work is needed to definitively prove not only if NO supplementation can be beneficial to specific skeletal muscle processes, but also to better define optimal substance type, dosage, and treatment protocols.
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