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Mini Review ARTICLE

Front. Physiol., 29 July 2015 | https://doi.org/10.3389/fphys.2015.00211

On the mechanism by which dietary nitrate improves human skeletal muscle function

  • 1School of Biomedical and Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth University, Plymouth, UK
  • 2Department of Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK
  • 3Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, University of Exeter, Exeter, UK

Inorganic nitrate is present at high levels in beetroot and celery, and in green leafy vegetables such as spinach and lettuce. Though long believed inert, nitrate can be reduced to nitrite in the human mouth and, further, under hypoxia and/or low pH, to nitric oxide. Dietary nitrate has thus been associated favorably with nitric-oxide-regulated processes including blood flow and energy metabolism. Indeed, the therapeutic potential of dietary nitrate in cardiovascular disease and metabolic syndrome—both aging-related medical disorders—has attracted considerable recent research interest. We and others have shown that dietary nitrate supplementation lowers the oxygen cost of human exercise, as less respiratory activity appears to be required for a set rate of skeletal muscle work. This striking observation predicts that nitrate benefits the energy metabolism of human muscle, increasing the efficiency of either mitochondrial ATP synthesis and/or of cellular ATP-consuming processes. In this mini-review, we evaluate experimental support for the dietary nitrate effects on muscle bioenergetics and we critically discuss the likelihood of nitric oxide as the molecular mediator of such effects.

Introduction

Inorganic nitrate (NO3) has long been considered an undesirable food component and pollutant of drinking water as nitrosation of secondary amines may produce carcinogenic N-nitrosamines (Magee and Barnes, 1956). However, the evidence that NO3 causes human cancers is weak and dietary NO3, as e.g. found in beetroot and spinach, has instead been linked to many physiological benefits (Gilchrist et al., 2010). Humans do not only get NO3 from their diet as it is also generated endogenously (Tannenbaum et al., 1978) by oxidation of nitric oxide (NO) formed canonically via the L-arginine/NO synthase pathway (Moncada and Higgs, 1993). Importantly, inorganic NO3 can be reduced to nitrite (NO2) and then NO, which offers an additional path of mammalian NO production that, unlike the canonical route, is independent of oxygen (O2) (Lundberg et al., 2008). NO is widely believed to mediate the benefits of NO3 (Lundberg et al., 2009) including protection against cardiovascular disease (Omar and Webb, 2014) and the metabolic syndrome (Carlström et al., 2010). It has recently been found that dietary NO3 lowers the O2 cost of human exercise as less respiratory activity is required for a set rate of skeletal muscle work (Larsen et al., 2007, 2010; Bailey et al., 2009, 2010). This finding is interesting as it challenges exercise physiology dogma that the steady-state O2 consumption for any individual is immutable at a given sub-maximal workload irrespective of age, fitness, diet, or pharmacological intervention (Poole and Richardson, 1997). This mini-review aims to evaluate the mechanistic understanding of NO3 effects on skeletal muscle function.

O2 Cost of Human Exercise

In a seminal publication, Larsen and colleagues reported that a 3-day supplementation with 0.1 mmol·kg−1 NaNO3· day−1 lowered pulmonary O2 uptake (V˙O2) by ~3–5% in humans completing sub-maximal cycling exercise (Larsen et al., 2007). Bailey and co-workers subsequently observed a 5% lower V˙O2 during low-intensity cycling exercise and a 16% improvement in the tolerable duration of high-intensity exercise over days 3–6 of a 6-day supplementation period with 5.6 mmol NO3· day−1, administered as 500 mL NO3-rich beetroot juice· day−1 (Bailey et al., 2009). Importantly, NO3-depleted beetroot juice does not improve exercise economy and performance (Lansley et al., 2011) eliminating antioxidants and polyphenols (Wootton-Beard and Ryan, 2011) as exclusive “active ingredients”. NO3-induced improvements have been observed in humans completing walking, running, cycling, rowing and kayaking exercise, and positive responses arise both acutely, i.e., 1–3 h after NO3 ingestion, and after prolonged NO3 supplementation over 3–15 days (Table 1). Acute (2.5 h post-ingestion) lowering of V˙O2 during low-intensity exercise is progressively greater at 4.2, 8.4, and 16.8 mmol NO3, whereas high-intensity exercise tolerance is unaffected by 4.2 mmol NO3, but acutely improved to a similar extent by 8.4 and 16.8 mmol (Wylie et al., 2013). Therefore, short-term supplementation (≥3 days) with at least 5 mmol NO3· day−1, or acute ingestion of at least 8.4 mmol NO3, might represent an effective dietary intervention to improve the economy and performance of human locomotion, at least in healthy, moderately fit adults (Porcelli et al., 2014). Since effects on resting V˙O2 are equivocal (Bailey et al., 2010; Kelly et al., 2013b; Larsen et al., 2014), NO3 benefits may be exclusive to contracting skeletal muscles.

TABLE 1
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Table 1. Dietary nitrate improves the economy and/or performance of human locomotion.

Physiological NO3 effects appear muscle-fiber-type-specific as evidenced by improved perfusion (Ferguson et al., 2014) and calcium handling (Hernández et al., 2012) of murine fast-twitch type II but not slow-twitch type I muscle. Consistent with this, NO3 benefit on V˙O2 adjustment following the onset of exercise and on tolerance to high-intensity exercise is relatively large when the contribution of type II muscle fibers to force production is increased in human skeletal muscle (Breese et al., 2013; Bailey et al., 2015). These preferential physiological effects may relate to the comparably low microvascular PO2 in resting and contracting type II muscle (McDonough et al., 2005). Indeed, NO3 improves exercise economy and performance in hypoxia (Masschelein et al., 2012; Muggeridge et al., 2014) more markedly than in normoxia (Kelly et al., 2014). Importantly, NO3 attenuates the degree of exercise intolerance and the slowing of PCr recovery kinetics in hypoxia to the levels seen in normoxia (Vanhatalo et al., 2011). It thus appears that exercise economy and performance benefit most from NO3 when muscle O2 availability is low.

Although the majority of studies in healthy adults observe NO3-improved exercise economy and/or performance, the effects are attenuated in well-trained endurance athletes (Bescós et al., 2012; Peacock et al., 2012; Christensen et al., 2013; Boorsma et al., 2014; Hoon et al., 2014; Lane et al., 2014), inconsistent in diseased populations (Berry et al., 2014; Kerley et al., 2015; Leong et al., 2015; Shepherd et al., 2015; Zamani et al., 2015), and possibly different in aging humans (Kelly et al., 2013b). More generally, there is evidence of distinct NO3 responders and non-responders in many studies. The relative efficacy of dietary NO3 effects on skeletal muscle thus appears variable, which underscores the need for detailed mechanistic understanding. To aid such understanding, it is important to ascertain how the human body processes dietary NO3.

Molecular Fate of Dietary NO3

When humans eat NO3-rich food, NO3 is converted to NO2 by nitrate reductases in commensal bacteria that reside in the posterior part of the tongue (Duncan et al., 1995). Salivary NO2 is rapidly protonated in the acidic environment of the stomach resulting in the formation of NO and other reactive nitrogen species (RNS) including nitrogen dioxide (NO2), nitrous acid (HNO2), and dinitrogen trioxide (N2O3) (Benjamin et al., 1994; Lundberg et al., 1994; Lundberg and Weitzberg, 2013). NO3 ingestion increases plasma NO2 levels in human subjects (e.g., Lundberg and Govoni, 2004; Webb et al., 2008; Bailey et al., 2009; Vanhatalo et al., 2010; Kelly et al., 2013a). Possibly catalyzed by xanthine oxidase (Zhang et al., 1998; Li et al., 2003) and/or deoxyhaemoglobin (Cosby et al., 2003; Gladwin et al., 2004; Gladwin and Kim-Shapiro, 2008), NO2 is reduced to NO under conditions of low oxygen tension (Figure 1). Other sites of NO2 reductase activity include cytochrome c (Basu et al., 2008) and mitochondrial respiratory complexes III (Kozlov et al., 1999) and IV (Castello et al., 2006). However, mammalian NO2 reductase activity has only been shown in vitro and in animal models (Feelisch et al., 2008; Jansson et al., 2008), and under low PO2 (Li et al., 2001; Feelisch et al., 2008) and low pH (Modin et al., 2001). Generally, the low pKa of NO2 (3.34, Oxtoby and Nachtrieb, 1996) limits its physiological reduction, which is an inefficient process per se (Li et al., 2008) and thus requires high NO2 concentrations. Indeed, at physiological NO2 levels (see below), even hypoxic red blood cells do not liberate significant NO (Bryan et al., 2004). O2 competitively inhibits NO2 reduction by xanthine oxidase (Li et al., 2004) and oxygenated haem effectively scavenges free NO (Feelisch et al., 2008). NO may be converted to N2O3 that in turn may react with free thiols to generate S-nitrosothiols (Hess et al., 2005) via an S-nitrosation reaction (Figure 1). NO is also able to modify proteins through nitrosylation, e.g., via reaction with the haem of myoglobin (Ignarro, 1991). NO furthermore binds, in a reversible and O2-competitive manner, to the haem of cytochrome c oxidase, and in an O2-independent way, to the enzyme's active site copper (Giulivi et al., 2006; Brown and Borutaite, 2007; Cooper and Giulivi, 2007). Peroxynitrite (ONOO) arising from the reaction of NO with the superoxide anion radical may undergo a nitration reaction with tyrosine residues to form 3-nitrotyrosine (Figure 1Radi, 2004). Importantly, tyrosine-containing proteins are also nitrated in a myeloperoxidase-catalyzed reaction using NO2 and hydrogen peroxide (Marquez and Dunford, 1995).

FIGURE 1
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Figure 1. Putative mechanism by which dietary NO3 may lower the O2 cost of human exercise. Dietary NO3 increases plasma NO3 and NO2 levels thus improving efficiency of skeletal muscle ATP supply by oxidative phosphorylation and/or of ATP turnover. Effects on the bioenergetics of skeletal muscle cells may be direct or indirect through formation of NO. Shown reactions are examples of RNS-induced protein modifications (see text for details).

Physiological NO2 levels range from 50 to 500 nM in human plasma (Bryan et al., 2004; Dejam et al., 2005; Feelisch et al., 2008) and a mean concentration of 12 μM has been measured in human skin (Mowbray et al., 2009). In rodents, NO2 concentration varies substantially between tissues, from below quantifiable limit in the liver, heart and lung to as high as 2 μM in kidney and 3.7 μM in lymph nodes; NO3 varies from 1 μM in the kidney to 50 μM in the aorta (Garcia-Saura et al., 2010). In humans, the ingestion of 10 mg NaNO3·kg−1 has been shown to increase mean plasma NO3 within 90 min from 30 to 432 μM and mean plasma NO2 from 123 to 392 nM (Lundberg and Govoni, 2004). Similarly, 500 mL beetroot juice containing 45 mM NO3 on average raises mean plasma NO3 to 380 μM and NO2 to 600 nM within 30 min and 3 h of ingestion, respectively (Webb et al., 2008). Plasma NO3 and NO2 reach peak concentrations, respectively, 1–2 and 2–3 h post-ingestion, and NO3 gradually returns to its base level after about 24 h (Ender et al., 1964; McKnight et al., 1997; Webb et al., 2008; Wylie et al., 2013). Mammalian tissue NO2 and NO3 both have in vivo half-lives of tens of minutes (Bryan et al., 2005). The half-life of NO2 in whole human blood is only about 110 sec (Kelm, 1999) as it is rapidly oxidized to NO3; the half-life of NO3 in blood is 5–8 h (Wagner et al., 1983; McKnight et al., 1997). About 60% of ingested NO3 is excreted by the kidneys (Green et al., 1981; Wagner et al., 1983).

The reliability of commonly reported NO3 and NO2 values very much depends on the assays used to measure these inorganic anions. The modified Griess reaction using sulfanilamide and N-1-napthylethylenediamine dihydrochloride is a frequently used assay for measuring NO2 (Tsikas, 2007). Plasma NO3 concentrations are readily determined using this spectrophotometric assay following NO3 reduction to NO2 by cadmium (Green et al., 1982) or vanadium salts (Miranda et al., 2001). However, the Griess test lacks the sensitivity to probe the nanomolar NO2 levels present in human plasma. Ozone-based chemiluminescence is a preferred method of detection, which often involves deproteinisation of plasma samples by zinc sulfate precipitation before analysis (Higuchi and Motomizu, 1999). NO2 measurement by chemiluminescence usually involves acetic acid/sodium iodide-mediated reduction to NO, which then reacts with ozone to produce a chemiluminescence signal (Bateman et al., 2002). NO3 can also be measured this way by reduction to NO via reflux of the sample in vanadium chloride at 95°C. Confounding the NO3/NO2 literature, in some assays NO3 is reduced to NO2 by bacterial nitrate reductases (Sun et al., 2003) whose activity varies from batch to batch. Confusing matters further, mere “NOx” values are reported to denote the sum of NO2 and NO3 levels. A last analytical note concerns the use of NO3-depleted beetroot juice as placebo control in in vivo studies (see Section O2 Cost of Human Exercise). It is important for experiments involving human participants to use a placebo juice that looks, tastes, and smells the same as the “real thing”. A placebo that meets these criteria can be prepared by passing beetroot juice through a Purolite a520e anion exchange column, which effectively and selectively removes NO3 (Gilchrist et al., 2013).

Skeletal Muscle Bioenergetics

Dietary NO3 benefits on the O2 cost of exercise likely arise from increased efficiency of ATP synthesis and/or of skeletal muscle work (Figure 1). Indeed, NO3 increases the rate of human skeletal muscle PCr recovery after exercise in hypoxia suggesting an augmented maximum rate of oxidative ATP synthesis (Vanhatalo et al., 2011), and lowers the ATP cost of contractile force production (Bailey et al., 2010). These in vivo studies confirm that NO3 indeed affects skeletal muscle bioenergetics, but they do not disclose the underlying molecular mechanism. In vitro experiments with C2C12 myocytes show that beetroot juice provokes mitochondrial biogenesis and modestly increases basal cellular respiration without affecting respiratory capacity and proton leak (Vaughan et al., 2014). These observations indicate improved mitochondrial coupling efficiency as beetroot juice has increased the proportion of total O2 consumption coupled to ATP synthesis. Although the C2C12 experiments lack an appropriate NO3-depleted beetroot juice control (see above), increased coupling efficiency of oxidative phosphorylation agrees with data reported by Larsen et al. (2011), who show that skeletal muscle mitochondria isolated from NO3-supplemented subjects exhibit higher respiratory control and P/O ratios (defined in Brand and Nicholls, 2011) than mitochondria from non-supplemented controls, and that increases in P/O ratio correlate with NO3-induced decreases in whole-body O2 uptake during exercise. This increased efficiency of ATP synthesis in isolated mitochondria, however, emerges from decreased respiration linked to mitochondrial proton leak, not from stimulated O2 uptake coupled to phosphorylation (Larsen et al., 2011). NO3-lowered proton leak coincides with decreases in adenine nucleotide translocase protein and, to a lesser extent, uncoupling protein-3 (Larsen et al., 2011). It should be emphasized that these mitochondrial carriers do not necessarily contribute to proton leak (Nedergaard and Cannon, 2003; Vozza et al., 2014) and that leak is expected to account for little skeletal muscle respiration at low protonmotive force (Affourtit and Brand, 2006), i.e., the bioenergetic state attained during exercise. Dietary NO3 also lowers the apparent affinity of mitochondrial respiration for O2, an effect that is reproduced in vitro—acutely and pH-dependently—by NO2 (Larsen et al., 2011). Lowered affinity is attributed to an NO-induced rise in the apparent Km of cytochrome c oxidase for O2 (Larsen et al., 2011) but, inconsistently, NO2 does not affect mitochondrial respiration or efficiency (Larsen et al., 2011) like NO is expected to (Brown and Borutaite, 2007). Apparent mitochondrial respiratory affinity for O2 depends strongly on the extent to which respiration is controlled by the enzyme reacting with O2 (Affourtit et al., 2001)—control of cytochrome c oxidase over O2 consumption may well have been affected by NO2 and pH, and also by dietary NO3-induced mitochondrial changes.

It remains to be demonstrated convincingly whether or not dietary NO3 effects in skeletal muscle are mediated by NO. Nitrite reductase activity requires high NO2 levels and exceptionally low PO2 and pH (see Section Molecular Fate of Dietary NO3) that may indeed manifest in the ischaemic heart (Brown and Borutaite, 2007; Hendgen-Cotta et al., 2010), but are unlikely in healthy muscle. In contracting muscle, myoglobin O2 saturation remains as high as 50% (Takakura et al., 2015) and although globins indeed exhibit nitrite reductase activity at this saturation (Huang et al., 2005), cytoplasmic NO will likely be scavenged by oxymyoglobin (Hendgen-Cotta et al., 2010). Even if O2 were sufficiently low for NO2 reduction in exercising muscle, we stress that dietary NO3 intake remodels skeletal muscle bioenergetics in the hours to days before exercise (see Section O2 Cost of Human Exercise), i.e., when the muscles are at rest. Importantly, NO2 also modulates cell signaling independently of NO in hypoxia and normoxia (Bryan et al., 2005). NO2 activates AMPK in rat aortic smooth muscle cells thus stimulating mitochondrial biogenesis, and increasing coupling efficiency and cellular respiratory control (Mo et al., 2012). NO2 activates PKA in cultured cardiomyocytes, stimulating mitochondrial fusion and again increasing cellular respiratory control (Pride et al., 2013). In both systems, NO2 improves efficiency of oxidative ATP synthesis without apparent effect on proton leak, which agrees with the beetroot juice effects on C2C12 respiration (Vaughan et al., 2014). NO2 also activates PKA in cultured adipocytes, increasing mitochondrial fusion, and stimulating glucose uptake (Khoo et al., 2014). Moreover, NO2 increases proliferation of muscle (Totzeck et al., 2014) and epithelial cells (Wang et al., 2012).

RNS can modify proteins (see Section Molecular fate of dietary NO3) and may thus improve mitochondrial coupling efficiency in various ways, e.g., by increasing proton translocation to electron transfer stoichiometries of respiratory complexes (cf. Clerc et al., 2007). By definition (Brand and Nicholls, 2011), coupling efficiency benefits from decreased proton leak and increased phosphorylation-linked respiration, as indeed reported by (Larsen et al., 2011) and (Vaughan et al., 2014), respectively. System-kinetic modeling furthermore suggests that substrate oxidation capacity, which is dependent on fuel and O2 availability, correlates positively with coupling efficiency (Affourtit and Brand, 2009). Dietary NO3 may thus improve efficiency of muscle ATP synthesis, at least in part, by increasing expression of glucose transporters (Jiang et al., 2014) and/or by raising insulin availability (Nyström et al., 2012).

Dietary NO3 increases the contractile force of fast-twitch mouse muscle by improving calcium handling (Hernández et al., 2012) suggesting the efficiency of ATP-demanding contraction may have increased. To our knowledge, no other data are available on the mechanism by which dietary NO3 affects ATP turnover. However, NO3 supplementation may also alter efficiency of other ATP-consumers and, importantly, the relative importance of dietary NO3 effects on skeletal muscle ATP supply and ATP turnover remains unclear. A systems-level functional analysis of cellular energy metabolism (cf. Brand, 1998) may shed light on these issues. Using myocytes isolated from human muscle biopsies (Nisr and Affourtit, 2014) the relative effects of RNS on ATP-generating and ATP-consuming fluxes—linked through the cell's phosphorylation potential (Figure 1)—may be identified and quantified in an unbiased manner. A challenge of such in vitro analysis will be the approximation of physiologically meaningful conditions, in particular the O2 tensions and energy demands that prevail during the development of dietary NO3 benefits.

Conclusion

The striking benefit of dietary NO3 on the O2 cost of exercise is of obvious interest to athletes (Jones, 2014), but may also well impact on the quality of life of aging people suffering from muscle weakness and exercise intolerance. To rationally evaluate translational potential, our mechanistic understanding of dietary NO3 benefits on human skeletal muscle needs to be improved. By integrating biochemistry and physiology, and studying subjects at different age, it will be important to demonstrate which reactive nitrogen species mediate dietary NO3 effects at the cellular level, disclose all effects of nitrogen species on myocellular bioenergetics, confirm if they are direct or indirect via action on other tissues, and quantify the relative importance of these effects.

Author Contributions

CA wrote Sections Introduction, Skeletal Muscle Bioenergetics, and Conclusion, and produced the table and figure; SB and AJ wrote Section O2 Cost of Human Exercise; MS and PW wrote Section Molecular Fate of Dietary NO3. All authors edited and approved the entire manuscript.

Conflict of Interest Statement

The development of a placebo beetroot juice preparation in PGW's laboratory was supported by James White Drinks Ltd. Otherwise, we confirm that this mini-review was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

Research in the authors' laboratories is currently supported by the Medical Research Council (New Investigator Research Grant G1100165/1 to CA), Gatorade Sports Science Institute (Grants PEP-1330 and PEP-1420 to AJ), the Exeter Leukaemia Fund (Grant ST-06354 to AJ), the Dunhill Medical Trust (Grant R269/1112 to AJ) and the NIHR Exeter Clinical Research Facility (PW and MS). The views and opinions shown within this paper are those of the authors and do not necessarily represent those of the NIHR, NHS or the Department of Health.

Abbreviations

AMPK, AMP-activated kinase; PCr, phosphocreatine; PKA, protein kinase A; PO2, partial oxygen tension; RNS, reactive nitrogen species; V˙O2, pulmonary oxygen uptake.

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Keywords: dietary nitrate, nitrite, nitric oxide, oxygen cost of human exercise, cellular bioenergetics, skeletal muscle mitochondria, coupling efficiency of oxidative phosphorylation, ATP turnover

Citation: Affourtit C, Bailey SJ, Jones AM, Smallwood MJ and Winyard PG (2015) On the mechanism by which dietary nitrate improves human skeletal muscle function. Front. Physiol. 6:211. doi: 10.3389/fphys.2015.00211

Received: 23 June 2015; Accepted: 14 July 2015;
Published: 29 July 2015.

Edited by:

Gilles Gouspillou, Université du Québec à Montréal, Canada

Reviewed by:

Sruti Shiva, University of Pittsburgh, USA
Emanuele Marzetti, Catholic University of the Sacred Heart, Italy

Copyright © 2015 Affourtit, Bailey, Jones, Smallwood and Winyard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Charles Affourtit, School of Biomedical and Healthcare Sciences, Plymouth University, Portland Square Building, Drake Circus, PL4 8AA, Plymouth, UK, charles.affourtit@plymouth.ac.uk