PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia

Abstract

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα^-/-) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O₂) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα^-/- mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Keywords: Fatty acids; Hypoxia; Metabolism; Muscle; Nitric oxide.

Fig. 1

Study design. Each stage took place within the ages shown ±4 d, and the length of each stage was identical for each mouse. The left section represents wild-type (PPARα^+/+) mice and the right section represents PPARα knockout (PPARα^−/−) mice. The number in brackets indicates the number of mice in each group. Chloride (0.7 mM NaCl) in distilled water ad libitum; Nitrate (0.7 mM NaNO₃) in distilled water ad libitum; Normoxia, 21% atmospheric O₂; Hypoxia, 10% atmospheric O₂. Background patterns reflect those used in box and whisker plots in subsequent figures.

Fig. 2

Body weights, nitrate intake and circulating haemoglobin. A) Body weights (n = 10–12 per group) on the first day of the week indicated, with dietary nitrate supplementation beginning on week 1 and hypoxic treatment beginning on week 2. Data represented as mean ± SEM. B) Cumulative nitrate (NO₃⁻) intake from food and water throughout the study. C) Haemoglobin concentration (g/L), immediately after sacrifice. Data are represented as minimum to maximum values, n = 6–12 per group. ***P < 0.001 nitrate effect (orange) or hypoxia effect (blue).

Fig. 3

Soleus muscle morphology and mitochondrial markers. A) Myosin heavy chain isoform type proportions, B) PGC1α protein levels, C) Citrate synthase activity in mouse soleus. Data presented as mean ± SEM in graph A and as minimum to maximum values in graphs B and C, n = 5–10 per experiment group. *P < 0.05, **P < 0.01 genotype effect (black) or hypoxia effect (blue).

Fig. 4

Mitochondrial respiratory capacities. A) LEAK state respiration (J_O2) through the N-pathway via Complex I with malate and glutamate (GM_L). B) OXPHOS state respiration (J_O2) through the N-pathway via Complex I with malate, glutamate and ADP (GM_P). C) OXPHOS state respiration (J_O2) through the S-pathway via Complex II with succinate and rotenone (S_P). Respiration rates are corrected to mass of soleus muscle fibres. Data are represented as minimum to maximum values, n = 6–10 per experiment group. *P < 0.05, **P < 0.01 genotype effect (black) or hypoxia effect (blue).

Fig. 5

Mitochondrial fatty acid and pyruvate oxidation capacities. A) LEAK state respiration (J_O2) with malate and palmitoyl carnitine (PalM_L). B) OXPHOS state respiration (J_O2) with malate and palmitoyl carnitine (PalM_P). C) OXPHOS state respiration (J_O2) through the N-pathway via Complex I with malate and pyruvate (PM_P). Respiration rates are corrected to mass of soleus muscle fibres. Data are represented as minimum to maximum values, n = 6–10 per experiment group. *P < 0.05, **P < 0.01, ***P < 0.001 genotype effect (black) or hypoxia effect (blue).

Fig. 6

Fatty acid oxidation enzyme protein levels. A) Levels of long chain acyl CoA dehydrogenase (LCAD) and B) carnitine palmitoyltransferase protein in soleus muscle. Data are represented as minimum to maximum values, n = 3–5 per experiment group. *P < 0.05 genotype effect (black) or hypoxia effect (blue).