The complex study design allowed us to probe
The complex study design allowed us to probe the interaction between the three factors, whilst addressing the core question of whether PPARα is necessary for the protective effects of nitrate on metabolism in hypoxic muscle. Central to this study was the statistical approach, which permitted us to examine interactions between nitrate, hypoxia and PPARα expression, and address the main hypothesis . The three-way ANOVA represented a relatively conservative approach, and may have resulted in type II errors, particularly when considering the effect of a single stressor on an outcome, e.g. the effect of PPARα knockout on mitochondrial fatty LY3009120 oxidation capacity. Prior to commencing the study, we considered an alternative approach of separately examining wild-type and PPARα−/− mice using two two-way ANOVAs, but rejected this approach because it fails to test the main hypothesis. As with our previous studies [14,46], the precise control of nitrate intake was achieved with the use of a standardised quality-controlled diet and deionised water. A possible confounding factor was the negative effect of hypoxia on food and water intake, and thus body weight. This was most pronounced during the first week of hypoxic exposure, with water intake returning to normal in the second week and food intake recovering to match that of normoxic mice by the final week. The relatively long duration of the study was therefore a strength, since stabilisation of water intake meant that nitrate intake was identical between the supplemented groups for the final three weeks. A possible limitation of this study is the exclusive use of male mice, indeed dimorphic differences in the response to nitrate supplementation have been noted in human subjects . Finally, our previous studies on nitrate supplementation in hypoxia have largely used rats, owing to the similarity in NO production rates  and circulating nitrate/nitrite levels between rats and humans , however it was necessary to use genetically-manipulated mice in this study in order to understand the role of PPARα. In agreement with previous work on human muscle from our lab , hypoxia elicited metabolic responses in mouse soleus consistent with a decreased capacity for oxidative metabolism. The relative switch in abundance of myosin heavy chain isoforms away from type I MyHC towards type II MyHC, is consistent with a preference for glycolytic energy metabolism over oxidative phosphorylation, and this was paralleled by a lower activity of citrate synthase in hypoxic mouse soleus. Citrate synthase is a purported marker of mitochondrial content in human skeletal muscle , and fell in the muscle of lowlanders during an ascent of Everest alongside a loss of mitochondrial volume density . Citrate synthase activity was also lower in the soleus of PPARα−/− mice compared with wild-type mice. There was no difference in levels of PGC1α between hypoxic and normoxic mice though, suggesting that any changes in mitochondrial abundance in this study were not as a result of decreased PGC1α expression. Further effects of hypoxia on oxidative metabolism were seen upon examination of mitochondrial respiratory capacity, including decreased OXPHOS respiration per unit mass of tissue in the presence of succinate (S-pathway via complex II), palmitoyl-carnitine and pyruvate, and lower LEAK state respiration with palmitoyl-carnitine. In previous studies from our group on rat soleus, we did not see any effect of hypoxia on mass-corrected respiration rates in permeabilised fibres despite some changes in metabolic gene expression, enzyme activities and muscle metabolite concentrations [10,14]. This may highlight a difference in the effect of hypoxia on rats and mice, but may have been due to the shorter duration of hypoxic exposure used previously (2 weeks). The lower respiratory rates we report here in hypoxic mouse soleus may have resulted from a loss of mitochondrial content per se, as suggested by lower citrate synthase activities. Succinate-supported OXPHOS capacity also fell in the muscle of lowlanders acclimatising to high-altitude though , whilst protein levels of succinate dehydrogenase were seen in a separate group of subjects undertaking the same ascent to Everest Base Camp . Meanwhile, octanoyl-carnitine supported respiration rates declined in the muscle of lowlanders at high-altitude . Moreover, in the present study protein levels of CPT1 were found to be lower in hypoxic mouse muscle than in normoxic mice, perhaps contributing to a lower fatty acid oxidation capacity, whilst CPT1B expression fell in human muscle at altitude . Expression of CPT1B is controlled by PPARα, and our results may indicate a hypoxia-driven suppression of PPARα transcriptional activity, although notably in the present study hypoxic exposure resulted in lower CPT1 levels in both wild-type and transgenic mice, in addition to the negative effect of PPARα knockout.