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  • Once consumed nitrate is believed to be reduced in a


    Once consumed, nitrate is believed to be reduced in a stepwise fashion, firstly to nitrite (NO2−) and then to nitric oxide (NO) [23, 24], adding to the endogenous production of NO from the NO synthase-dependent oxidation of L-arginine [25]. NO is a diatomic free radical that acts as a signalling molecule and is regarded to be the chief mediator of the responses observed with nitrate supplementation [26]. NO has been shown to exert numerous biological effects in relation both to the delivery of O2, through the well characterised mediation of vascular tone [27,28], and the utilisation of O2 through effects upon mitochondrial function. Whilst some of the changes in tissue metabolism exerted by NO may occur secondary to altered haemodynamic function and improved O2 delivery [29], studies in cultured myocytes have shown direct effects of nitrate supplementation on fatty Artesunate metabolism via activation of the PPAR family of transcription factors by cyclic GMP (cGMP), a downstream mediator of NO activity. In vivo, nitrate supplementation thereby enhanced fatty acid oxidation capacity and mitochondrial content in the skeletal muscle of normoxic and hypoxic rodents, lowering intramuscular levels of long-chain fatty acid intermediates [14]. Elevated levels of NO metabolites have been seen in human populations native to high altitude regions in Tibet and the Andes [[30], [31], [32]], with selection of genetic variants in NO synthase genes [33]. In particular, Tibetans have high levels of exhaled NO [34] and circulating NO biomarkers, which are associated with enhanced forearm blood flow [32]. The haemodynamic effects of elevated NO production may therefore be protective at altitude, indeed in lowlanders circulating levels of nitrate, nitrite and cGMP increased during acclimatisation [35]. It is not clear though, whether the protective effects of nitrate on mitochondrial function and other aspects of energy metabolism occur at altitude, since PPARα expression and/or activity is downregulated in hypoxic skeletal muscle [6,10]. Moreover, in one Tibetan population, the Sherpa, selection for a variant in the gene encoding PPARα (PPARA) is associated with a lower capacity for skeletal muscle fatty acid oxidation compared with lowlanders, alongside improved mitochondrial efficiency, lower levels of oxidative stress and protection of muscle energetics at high altitude [6].
    Discussion In the context of chronic tissue hypoxia, oxidative metabolism is suppressed [3], thereby matching tissue oxygen demand to the diminished supply. A key player in this response in a number of tissues is PPARα [5,7,8,10], a master regulator of fatty acid oxidation [4]. Dietary supplementation with inorganic nitrate exerts haemodynamic effects [29] which can improve tissue oxygenation, and alleviated the metabolic consequences of hypoxia in the rat heart [46]. Nitrate also stimulates oxidative metabolism in skeletal muscle, acting via PPARα [14], therefore we sought to investigate whether the protective effects of nitrate in the context of hypoxia were independent of PPARα. In mouse soleus, hypoxia was associated with a fibre-type switch towards fast contracting fibres, with no effect of nitrate or PPARα expression. Citrate synthase activity was lower in the soleus with hypoxia and also with PPARα−/−, but in both cases this was prevented by dietary nitrate. Regarding mitochondrial respiration, LEAK state respiration with glutamate and malate and OXPHOS respiration with succinate were suppressed in hypoxic soleus. Meanwhile, fatty acid and pyruvate oxidation capacities were lower in hypoxic soleus, whilst nitrate reversed the hypoxia-induced decline in pyruvate (though not fatty-acid) oxidation. These effects on fatty acid and pyruvate oxidation occurred in a PPARα-independent manner. As expected, protein levels of LCAD and CPT1 were lower in PPARα−/− mice compared with wild-types, but CPT1 levels were also lower in hypoxic mice across both genotypes, with no effect of nitrate treatment.