Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans

Maria Ntessalen, Nathan E K Procter, Konstantin Schwarz, Brodie L Loudon, Magdalena Minnion, Bernadette O Fernandez, Vassilios S Vassiliou, David Vauzour, Melanie Madhani, Dumitru Constantin-Teodosiu, John D Horowitz, Martin Feelisch, Dana Dawson, Paul G Crichton, Michael P Frenneaux, Maria Ntessalen, Nathan E K Procter, Konstantin Schwarz, Brodie L Loudon, Magdalena Minnion, Bernadette O Fernandez, Vassilios S Vassiliou, David Vauzour, Melanie Madhani, Dumitru Constantin-Teodosiu, John D Horowitz, Martin Feelisch, Dana Dawson, Paul G Crichton, Michael P Frenneaux

Abstract

Background: Inorganic nitrate, abundant in leafy green vegetables and beetroot, is thought to have protective health benefits. Adherence to a Mediterranean diet reduces the incidence and severity of coronary artery disease, whereas supplementation with nitrate can improve submaximal exercise performance. Once ingested, oral commensal bacteria may reduce nitrate to nitrite, which may subsequently be reduced to nitric oxide during conditions of hypoxia and in the presence of "nitrite reductases" such as heme- and molybdenum-containing enzymes.

Objective: We aimed to explore the putative effects of inorganic nitrate and nitrite on mitochondrial function in skeletal muscle.

Methods: Mice were subjected to a nitrate/nitrite-depleted diet for 2 wk, then supplemented with sodium nitrate, sodium nitrite, or sodium chloride (1 g/L) in drinking water ad libitum for 7 d before killing. Skeletal muscle mitochondrial function and expression of uncoupling protein (UCP) 3, ADP/ATP carrier protein (AAC) 1 and AAC2, and pyruvate dehydrogenase (PDH) were assessed by respirometry and Western blotting. Studies were also undertaken in human skeletal muscle biopsies from a cohort of coronary artery bypass graft patients treated with either sodium nitrite (30-min infusion of 10 μmol/min) or vehicle [0.9% (wt:vol) saline] 24 h before surgery.

Results: Neither sodium nitrate nor sodium nitrite supplementation altered mitochondrial coupling efficiency in murine skeletal muscle, and expression of UCP3, AAC1, or AAC2, and PDH phosphorylation status did not differ between the nitrite and saline groups. Similar results were observed in human samples.

Conclusions: Sodium nitrite failed to improve mitochondrial metabolic efficiency, rendering this mechanism implausible for the purported exercise benefits of dietary nitrate supplementation. This trial was registered at clinicaltrials.gov as NCT04001283.

Keywords: mitochondria; nitrate; nitrite; pyruvate dehydrogenase; uncoupling proteins.

Copyright © American Society for Nutrition 2019.

Figures

FIGURE 1
FIGURE 1
A schematic of the trial design (acute phase) for the “Effect of Nitrite on Cardiac Muscle and Blood Vessels in Patients Undergoing Coronary Artery Bypass Grafting Surgery” study.
FIGURE 2
FIGURE 2
Plasma and concentrations in mice after supplementation with sodium nitrate (n = 10–13 per group) or sodium nitrite (n = 6 per group) for 7 d. (A) Plasma concentrations in mice supplemented with sodium nitrate or sodium nitrite. (B) Plasma concentrations in mice supplemented with sodium nitrate or sodium nitrite.
FIGURE 3
FIGURE 3
Expression of UCP3 and AAC proteins in mouse skeletal muscle after 7 d dietary supplementation with sodium nitrate (n = 12 per group) or sodium nitrite (n = 7–8 per group). (A) UCP3 and (B) AAC expression were unchanged by sodium nitrate or sodium nitrite supplementation. AAC, ADP/ATP carrier protein; AU, arbitrary units; C, control; N, nitrate/nitrite; UCP, uncoupling protein.
FIGURE 4
FIGURE 4
Protein expression and phosphorylation status of PDH in mouse skeletal muscle in response to dietary sodium nitrate (n = 12 per group) or sodium nitrite (n = 8 per group) supplementation. Sodium nitrate, but not sodium nitrite, supplementation resulted in dephosphorylation of serines 293 and 300 of the PDH complex in mouse skeletal muscle. *,**Significant differences: *P < 0.01, **P < 0.001, compared with control. White bars, NaCl; black bars, NaNO3; gray bars, NaNO2. AU, arbitrary units; C, control; N, nitrate/nitrite; PDH, pyruvate dehydrogenase.
FIGURE 5
FIGURE 5
Respiratory activity of skeletal muscle mitochondria isolated from mice supplemented with sodium nitrate (n = 9 per group) for 7 d, as assessed by Seahorse XF24 analyzer. (A) Skeletal muscle mitochondrial respiration in response to dietary supplementation with sodium nitrate. (B) RCRs were unchanged in response to supplementation with sodium nitrate. (C) Mitochondrial leak was unchanged in response to sodium nitrate. No significant differences were found between treatment and control groups (at P < 0.05; independent t test). See the Methods section for further details. White bars, NaCl; black bars, NaNO3. AA, antimycin A; AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; RCR, respiratory control ratio; St2, state 2; St3*, state 3 (ADP at saturated concentrations).
FIGURE 6
FIGURE 6
Respiratory activity of skeletal muscle mitochondria isolated from mice supplemented with sodium nitrite (n = 5–6 per group) for 7 d, as assessed by Seahorse XF24 analyzer. (A) Skeletal muscle mitochondrial respiration in response to dietary supplementation with sodium nitrite. Nitrite supplementation resulted in decreased oligomycin-induced respiration when compared with control. *Significant differences: *P < 0.05. (B) RCRs were unchanged in response to supplementation with sodium nitrite. (C) Mitochondrial leak was unchanged in response to sodium nitrite. No significant differences were found between treatment and control groups (at P < 0.05; independent t test). See the Methods section for further details. White bars, NaCl; gray bars, NaNO2. AA, antimycin A; AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; RCR, respiratory control ratio; St2, state 2; St3*, state 3 (ADP at saturated concentrations).
FIGURE 7
FIGURE 7
Respiratory activity of skeletal muscle mitochondria isolated from mice supplemented with sodium nitrite (n = 8 per group) for 7 d, as evaluated by Clark-type oxygen electrode. (A) Skeletal muscle mitochondrial respiration in response to dietary supplementation with sodium nitrite. (B) RCRs were unchanged in response to supplementation with sodium nitrite. (C) Mitochondrial leak was unchanged in response to sodium nitrite. (D) Mitochondrial P:O ratios were unchanged in response to sodium nitrite. No significant differences were found between treatment and control groups (at P < 0.05; independent t test). See the Methods section for further details. White bars, NaCl; gray bars, NaNO2. AA, antimycin A; AU, arbitrary units; CATR, carboxyatractyloside; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; P:O, phosphate:oxygen; RCR, respiratory control ratio; St2, state 2; St3*, state 3 (ADP at saturated concentrations).
FIGURE 8
FIGURE 8
Expression of UCP3 and AAC in human skeletal muscle after infusion of sodium nitrite (n = 20) or placebo (n = 15). (A) Expression of UCP3 was unchanged in human skeletal muscle in response to sodium nitrite. (B) Expression of AAC1 was unchanged in human skeletal muscle in response to sodium nitrite. (C) Expression of AAC2 was unchanged in human skeletal muscle in response to sodium nitrite. (D) Expression and phosphorylation status of PDH were unchanged in human skeletal muscle in response to sodium nitrite. White bars, NaCl; gray bars, NaNO2. AAC, ADP/ATP carrier protein; AU, arbitrary units; C, control; N, nitrate/nitrite; PDH, pyruvate dehydrogenase; UCP, uncoupling protein.
FIGURE 9
FIGURE 9
Plasma concentrations of and in humans over time in response to a 30-min bolus injection of sodium nitrite or saline placebo (see the Methods section for details). (A) Plasma concentrations were significantly increased 6 h postinfusion in patients receiving intravenous sodium nitrite (F[1,18] = 9.44). **Significant differences: **P < 0.01. (B) Plasma concentrations were significantly increased immediately postinfusion in patients receiving intravenous sodium nitrite (F[1,32] = 27.342). **Significant differences: **P < 0.01. White circles, NaCl; gray circles, NaNO2.

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