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A decline in mitochondrial function occurs in many conditions. A report in this issue (2011) shows that dietary inorganic nitrates enhance muscle mitochondrial efficiency. It is an attractive hypothesis that dietary changes enhance energy efficiency, but its potential application depends on long-term studies investigating net benefits versus adverse effects.
Mitochondrial are critical for oxidizing fuels (i.e., glucose, fatty acids, amino acids) and converting them to ATP, the chemical energy required for cellular functions. A vast body of literature demonstrates that mitochondria are responsive to a variety of environmental stimuli. For example, they are responsive to aerobic exercise (Holloszy and Coyle, 1984), insulin and amino acid infusion (Stump et al., 2003) and thyroid hormones (Short et al., 2007). In this issue of Cell Metabolism, Larsen et al. (2011) add to this body of literature by demonstrating that several facets of skeletal mitochondrial physiology are influenced by dietary intake of inorganic nitrates.
Larsen and co-workers present the findings from a placebo-controlled, cross-over study of nitrate supplementation in healthy humans. Dietary inorganic nitrate increased the capacity for ATP synthesis in mitochondria isolated from muscle biopsy tissue. This increase in ATP production capacity appears to occur in the absence of any increase in mitochondrial content. Although long-term studies are needed to rule out the influence of nitrates on mitochondrial biogenesis, these observations suggest that nitrates enhance mitochondrial coupling efficiency (Larsen et al., 2011). Mitochondrial efficiency is determined by a variety of factors such as the macronutrient source (e.g., glucose versus free-fatty acids) of electron flow into the cytochrome chain and various ways in which the transmembrane proton gradient is dissipated or uncoupled, liberating potential energy rather than coupling it to ATP synthesis (Figure 1). The influence of nitrates on mitochondrial coupling was thoroughly examined using high-resolution respirometry in isolated mitochondria. With nitrate supplementation, mitochondrial proton leak was reduced and P:O ratio was increased, supporting the notion that nitrates decrease energy “wastage”, effectively increasing the amount of ATP generated per unit of oxygen consumed.
Armed with this evidence from in vitro experiments, Larsen et al. next showed that whole-body oxygen cost during steady-state exercise decreases with nitrate supplementation while the mechanical work output to oxygen cost (Watt/VO2) concomitantly increases, providing in vivo confirmation of experiments in isolated mitochondria. Exercise economy can be affected by changes in mechanical efficiency, mitochondrial coupling efficiency or both. The results in the current study suggest that nitrate-induced improvements in exercise economy are due in part to enhanced mitochondrial coupling efficiency; this differs from another study that reported that such improvements were due to enhanced mechanical efficiency (and reduced ATP turnover)(Bailey et al., 2010). Further studies are needed to determine whether both of these occur simultaneously.
The current study provides strong evidence that short-term dietary nitrate supplementation enhances mitochondrial efficiency, decreases mitochondrial proton leak, and enhances exercise performance. While the underlying mechanisms are not clear, nitrate supplementation reduced the expression of adenine nucleotide translocase which liberates protons in the process of exchanging ADP and ATP across the inner mitochondrial membrane (Figure 1). Furthermore, Larsen et al. found a tendency for lower uncoupling protein 3 (UCP3) expression, which dissipates the mitochondrial proton gradient during transport of fatty acids (Figure 1). In contrast, increased mitochondrial biogenesis by triiodothyronine increases the expression of uncoupling proteins (Short et al., 2007) and may increase proton leak (Lebon et al., 2001) that will render ATP production inefficient. Results from the current paper suggest that nitrate may act directly on complex I and complex IV, possibly influencing the proton stoichiometry of these enzymes. Such potential mechanisms include modification of proteins (acetylation and phosphorylation), allosteric modulation and binding to cofactors.
The physiological relevance of nitrate’s effects on mitochondria is an important consideration. Although strong evidence supports the notion that whole-body oxygen cost may be reduced because of improved mitochondrial efficiency, additional studies are needed to rule out the possibility that nitrates may induce a shift toward non-oxidative pathways for cellular ATP synthesis. Such a shift may have negative consequences in tissues such as cardiac muscle, which relies heavily on oxidative metabolism to meet metabolic demands at rest and during exercise. However, extrapolation from skeletal muscle with mixed fiber types to myocardium with predominantly mitochondrial rich muscle fibers requires additional studies. The current studies were performed in young (25±1 yrs), lean, non-smoking and physically fit (VO2 peak 56±3 ml.kg−1.min−1) people. What would happen in older people with low VO2 peak and in people with potential risk of coronary artery disease? Will high nitrate containing diet offer an advantage in climbing high altitude where oxygen tension is low. It is known that nitrates can be shunted to NO (via salivary recycling) that may not only impact blood flow but also mitochondrial biogenesis (Nisoli et al., 2003) when administered on a long-term basis. The impact on blood flow could be an advantage in conditions such as chronic heart failure with low blood flow causing substantial cellular dysfunction and in hypertension. Future studies may also address the impact of nitrates on mitochondrial reactive oxygen species (ROS) production, which is considered to be an inverse function of membrane potential. It is difficult to ignore the possibility that improved coupling may lead to increased ROS production rates which may have an adverse effect on proteins, DNA and cellular functions.
The importance of the current study is the demonstration that mitochondrial efficiency can be enhanced by dietary manipulation. Many leafy green vegetables such as spinach, celery and green lettuce are rich in nitrates/nitrites. While these could have positive impact on cardiometabolic risk factors, other foods especially preservatives containing nitrates as well as drinking water containing high concentrations of nitrates are reported to be associated with high incidence of cancers. The current study is a short-term study and the full positive and potential negative impact of a high nitrate containing diet (as well as long-term nitrate supplementation) needs to be carefully evaluated by long term studies.
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