Nitrate-induced improvements in exercise performance are coincident with exuberant changes in metabolic genes and the metabolome in zebrafish (Danio rerio) skeletal muscle

Abstract

Dietary nitrate supplementation improves exercise performance by reducing the oxygen cost of exercise and enhancing skeletal muscle function. However, the mechanisms underlying these effects are not well understood. The purpose of this study was to assess changes in skeletal muscle energy metabolism associated with exercise performance in a zebrafish model. Fish were exposed to sodium nitrate (60.7 mg/L, 303.5 mg/L, 606.9 mg/L), or control water, for 21 days and analyzed at intervals (5, 10, 20, 30, 40 cm/sec) during a two-hour strenuous exercise test. We measured oxygen consumption during an exercise test and assessed muscle nitrate concentrations, gene expression and the muscle metabolome before, during and after exercise. Nitrate exposure reduced the oxygen cost of exercise and increased muscle nitrate concentrations at rest, which were reduced with increasing exercise duration. In skeletal muscle, nitrate treatment upregulated expression of genes central to nutrient sensing (mtor), redox signaling (nrf2a) and muscle differentiation (sox6). In rested muscle, nitrate treatment increased phosphocreatine (P = 0.002), creatine (P =0.0005), ATP (P = 0.0008), ADP (P = 0.002), and AMP (P =0.004) compared to rested-control muscle. Following the highest swimming speed, concentration of phosphocreatine (P = 8.0 x 10-5), creatine (P =6.0 x 10-7), ATP (P = 2.0 x 10-6), ADP (P = 0.0002), and AMP (P =0.004) decreased compared to rested nitrate muscle. Our data suggests nitrate exposure in zebrafish lowers the oxygen cost of exercise by changing the metabolic programming of muscle prior to exercise and increasing availability of energy-rich metabolites required for exercise.

Nitrate exposure reprograms hepatic amino acid and nutrient sensing pathways prior to exercise: A metabolomic and transcriptomic investigation in zebrafish (Danio rerio)

“…A primary finding of our study is that 21 days of sub-chronic nitrate exposure significantly increased arginine bioavailability, sparing arginine and likely modulating endogenous NO metabolism. Similarly, to our previously published results in whole fish and zebrafish muscle, we observed a greater abundance of arginine in nitrate-treated liver at rest (Axton et al, 2019;Keller et al, 2021). Our data support that sub-chronic nitrate treatment may improve exercise performance, in part, by improving NO bioavailability, sparing arginine, and increasing indices of hepatic gluconeogenesis in the liver.…”

“…In addition, ketogenic amino acids are ultimately degraded to CO 2 in the TCA cycle and contribute to ATP production. As with our observations in whole zebrafish and zebrafish skeletal muscle (Axton et al, 2019;Keller et al, 2021), the effect of nitrate was most prominent at the rested condition and not at peak-exercise or post-exercise. A primary finding of our study is that 21 days of sub-chronic nitrate exposure significantly increased arginine bioavailability, sparing arginine and likely modulating endogenous NO metabolism.…”

“…Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise an improving the efficiency of ATP resynthesis in mitochondria, however the mechanisms for this effect are not well understood (Larsen et al, 2011;Affourtit et al, 2015). We demonstrated a dose-dependent effect of nitrate on exercise performance in zebrafish in two previously published studies (Axton et al, 2019;Keller et al, 2021). Using untargeted metabolomics technologies of whole zebrafish and zebrafish muscle, we attributed these nitrate-induced, performanceenhancing effects to changes in the metabolic programming of muscle prior to exercise by increasing the availability of energyproducing metabolites, including phosphocreatine and ATP, required for exercise.…”

“…Unlike our previous results in whole zebrafish, we did not observe a change in glucose-6-phosphate with nitrate exposure or exercise. In nitrate-treated skeletal muscle at rest, we observed an increase in glucose and glucose-6-phosphate, likely from increased glucose uptake in muscle ( Keller et al, 2021 ). It is important to note that we did not see changes in expression of genes central to gluconeogenesis including fpb1a and g6pd .…”

“…Our previous experimental analysis in whole zebrafish showed that 21 days of nitrate exposure reduced the oxygen cost of exercise, similar to humans ( Axton et al, 2019 ). Metabolomics analysis in whole zebrafish and zebrafish muscle revealed that this exercise performance effect was coincident with increased availability of metabolite fuels (i.e., ATP, glycolytic and tricarboxylic acid (TCA) intermediates, lactate and ketone bodies) in rested zebrafish ( Axton et al, 2019 ; Keller et al, 2021 ). These findings in whole fish were striking and led us to investigate organ-specific changes in metabolism specifically in the liver.…”