Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise

Lee J Wylie, Ji Won Park, Anni Vanhatalo, Stefan Kadach, Matthew I Black, Zdravko Stoyanov, Alan N Schechter, Andrew M Jones, Barbora Piknova, Lee J Wylie, Ji Won Park, Anni Vanhatalo, Stefan Kadach, Matthew I Black, Zdravko Stoyanov, Alan N Schechter, Andrew M Jones, Barbora Piknova

Abstract

Key points: Nitric oxide (NO), a potent vasodilator and a regulator of many physiological processes, is produced in mammals both enzymatically and by reduction of nitrite and nitrate ions. We have previously reported that, in rodents, skeletal muscle serves as a nitrate reservoir, with nitrate levels greatly exceeding those in blood or other internal organs, and with nitrate being reduced to NO during exercise. In the current study, we show that nitrate concentration is substantially greater in skeletal muscle than in blood and is elevated further by dietary nitrate ingestion in human volunteers. We also show that high-intensity exercise results in a reduction in the skeletal muscle nitrate store following supplementation, likely as a consequence of its reduction to nitrite and NO. We also report the presence of sialin, a nitrate transporter, and xanthine oxidoreductase in human skeletal muscle, indicating that muscle has the necessary apparatus for nitrate transport, storage and metabolism.

Abstract: Rodent skeletal muscle contains a large store of nitrate that can be augmented by the consumption of dietary nitrate. This muscle nitrate reservoir has been found to be an important source of nitrite and nitric oxide (NO) via its reduction by tissue xanthine oxidoreductase. To explore if this pathway is also active in human skeletal muscle during exercise, and if it is sensitive to local nitrate availability, we assessed exercise-induced changes in muscle nitrate and nitrite concentrations in young healthy humans, under baseline conditions and following dietary nitrate consumption. We found that baseline nitrate and nitrite concentrations were far higher in muscle than in plasma (∼4-fold and ∼29-fold, respectively), and that the consumption of a single bolus of dietary nitrate (12.8 mmol) significantly elevated nitrate concentration in both plasma (∼19-fold) and muscle (∼5-fold). Consistent with these observations, and with previous suggestions of active muscle nitrate transport, we present western blot data to show significant expression of the active nitrate/nitrite transporter sialin in human skeletal muscle. Furthermore, we report an exercise-induced reduction in human muscle nitrate concentration (by ∼39%), but only in the presence of an increased muscle nitrate store. Our results indicate that human skeletal muscle nitrate stores are sensitive to dietary nitrate intake and may contribute to NO generation during exercise. Together, these findings suggest that skeletal muscle plays an important role in the transport, storage and metabolism of nitrate in humans.

Keywords: exercise; nitrate; nitric oxide; nitrite; skeletal muscle.

Conflict of interest statement

Competing interests

Alan N. Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation. The other authors declare that they have no conflicts of interest.

© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Figures

Figure 1.. Schematic overview of the experimental…
Figure 1.. Schematic overview of the experimental protocol
Participants completed this protocol on two occasions, with nitrate-rich beetroot juice (NIT) or nitrate-depleted beetroot juice (placebo, PLA) supplementation in a randomized manner with repeated measures. Blood and muscle samples were collected at baseline, 120 min post-supplement ingestion, and immediately after a high-intensity bout of exercise (post-exercise).
Figure 2.. Baseline nitrate ( A )…
Figure 2.. Baseline nitrate (A) and nitrite (B) concentrations were higher in skeletal muscle (filled bars) than in plasma (open bars)
All available baseline data from both PLA and NIT arms were used (n = 24). **Difference of nitrate or nitrite in muscle from plasma values (P < 0.01).
Figure 3.. The concentration of nitrate and…
Figure 3.. The concentration of nitrate and nitrite in plasma (A and C, respectively) and muscle (B and D, respectively), measured pre-supplement (Pre) and 2 h after (Post) ingestion of dietary nitrate (NIT) or a placebo (PLA) supplement
Ingestion of nitrate significantly elevated the concentrations of nitrate in plasma and muscle, and the concentrations of nitrite in plasma. **Difference from pre-supplement (P < 0.01).
Figure 4.. Nitrate concentration in plasma (…
Figure 4.. Nitrate concentration in plasma (A) and muscle (B), and nitrite concentration in plasma (C) and muscle (D), measured immediately before (Pre) and after (Post) a high-intensity exercise bout, following the consumption of either a placebo (PLA) or dietary nitrate (NIT) supplement
The exercise bout significantly reduced muscle nitrate and plasma nitrite concentrations. *Difference from pre-exercise (P < 0.05). **Difference from pre-exercise (P < 0.01).
Figure 5.. Western blots of the neuronal…
Figure 5.. Western blots of the neuronal nitic oxide synthase (NOS-1), xanthine oxidoreductase (XOR), aldehyde oxidase (AO) and the nitrate transporter, sialin, in human skeletal muscle tissue of each of thirteen individual participants
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Note that all proteins were present in human skeletal muscle, and that there was large inter-individual variation in all four proteins, most notably in sialin and XOR.

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