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  • Echinomycin In line with our hypothesis dietary supplementat


    In line with our hypothesis, dietary supplementation with inorganic nitrate elicited protective effects on metabolism in hypoxic mouse soleus independently of PPARα. The hypoxia-driven effects of lower citrate synthase activity and lower mitochondrial respiration rates (LEAK rate with palmitoyl-carnitine and OXPHOS rate with pyruvate) were prevented in nitrate-supplemented mice irrespective of genotype. However, it should be noted that not all effects of hypoxia were prevented by nitrate supplementation in this study, perhaps owing to the severity of the hypoxic exposure used here in comparison with previous studies [14,46]. Mechanistically, nitrate is known to exert effects on tissue oxygen delivery through its role as a vasodilator [27,28], and improvements in tissue oxygenation via enhanced blood flow may underpin the PPARα-independent effects reported here. In addition, nitrate exerts effects on skeletal muscle metabolism via other transcription factors, including PPARβ/δ [14]. Indeed, it has been suggested that high levels of expression of PPARβ/δ compensate for the loss of PPARα in the skeletal muscle of PPARα−/− mice [52], and this may explain why mitochondrial fatty Echinomycin oxidative phosphorylation capacities were not lower in PPARα−/− mice compared with wild-type mice in this study. Moreover, the activation of PPARα expression by nitrate is brought about by the sequential reduction of nitrate to nitrite and NO and thence the production of cGMP at the tissue level. cGMP also acts a mediator in the downstream cellular response to natriuretic peptides, eliciting numerous metabolic effects, many of which are independent of PPARα [53]. For instance, cGMP is known to promote mitochondrial biogenesis in skeletal muscle, acting via PGC1α [54]. Since PGC1α levels were unaffected in mouse muscle by hypoxia or by ablation of PPARα, it is possible that prevention of the hypoxia driven loss of citrate synthase activity seen here was a result of PGC1α activation by cGMP, brought about via improved NO availability.
    Conclusions In conclusion, dietary nitrate is able to exert effects on skeletal muscle metabolism that counteract the effect of hypoxia, and this occurs independently of PPARα. Our work may hold implications for the use of nitrate in the treatment of diseases in which hypoxia is a factor and in which PPARα expression and/or transcriptional activity is attenuated. Moreover, our work suggests nitrate supplementation might offer benefits to lowlander subjects at high-altitude, in whom PPARα transcriptional activity is suppressed [6], and suggests that the high circulating nitrogen oxide levels seen in some Tibetan populations [32,34] might offer metabolic benefits despite lower PPARα expression in the muscles of some groups, e.g. the Sherpa [6]. The following are the supplementary data related to this article.
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    Introduction Protists, fungi and plants possess an auxiliary respiratory system in their mitochondria, which buffers metabolic stresses that arise from limitations on electron flow in the system of oxidative phosphorylation (OXPHOS). This alternative respiratory chain (aRC) comprises representatives of just two classes of enzyme: one or more alternative NADH dehydrogenases (NDX), that transfer electrons from NADH to an intermediate electron carrier, ubiquinone [1], and alternative oxidases (AOX), that complete electron transfer from ubiquinol directly to oxygen [2]. In terms of their net redox chemistry, NDX and AOX can functionally replace respiratory complex I (cI, NADH:ubiquinone oxidoreductase) and complexes III (cIII, ubiquinol:cytochrome c oxidoreductase) plus IV (cIV, cytochrome c oxidase) of the OXPHOS system, respectively. In contrast to the standard OXPHOS complexes of the mitochondrial respiratory chain (RC), the alternative enzymes have five distinct properties. First, they are each composed of a single polypeptide. Second, their reaction chemistry is non proton-motive, instead dissipating the released free energy as heat. Third, they are universally coded only by the nuclear genome. Fourth, they are refractory to the commonly used OXPHOS inhibitors. Finally, their biochemical properties limit their activity to metabolic conditions where they are functionally required, at least in the case of AOX. As a result, AOX may be considered a self-regulating enzyme that does not ‘short-circuit’ the OXPHOS system, except when the latter is already dysfunctional. In plants, the basis of this restriction is relatively well understood, in that the enzyme only becomes active when the substrate pool of reduced quinones accumulates to elevated levels, that would be considered abnormally high in non-photosynthetic organisms [3,4]. In effect, the enzyme displays a much higher K for ubiquinol than does cIII. This property has been assumed to apply to AOX from other taxa, but has not yet been formally demonstrated.