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

Maria Ntessalen, Nathan Ek 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*

*Corresponding author for this work

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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.

Original languageEnglish
Pages (from-to)79-89
Number of pages11
JournalThe American Journal of Clinical Nutrition
Volume111
Issue number1
Early online date10 Oct 2019
DOIs
Publication statusPublished - Jan 2020

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Nitrites
Nitrates
Sodium Nitrite
Skeletal Muscle
Pyruvic Acid
Oxidoreductases
ATP Translocases Mitochondrial ADP
Nitrite Reductases
Exercise
Mediterranean Diet
Molybdenum
Insurance Benefits
Dietary Supplements
Heme
Sodium Chloride
Coronary Artery Bypass
Drinking Water
Vegetables
Coronary Artery Disease
Carrier Proteins

Keywords

  • nitrate
  • nitrite
  • mitochondria
  • uncoupling protein
  • pyruvate dehydrogenase

Cite this

Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans. / Ntessalen, Maria; Procter, Nathan Ek; Schwarz, Konstantin; Loudon, Brodie L; Minnion, Magdalena; Fernandez, Bernadette O; Vassiliou, Vassilios S; Vauzour, David; Madhani, Melanie; Constantin-Teodosiu, Dumitru; Horowitz, John D; Feelisch, Martin; Dawson, Dana; Crichton, Paul G; Frenneaux, Michael P.

In: The American Journal of Clinical Nutrition, Vol. 111, No. 1, 01.2020, p. 79-89.

Research output: Contribution to journalArticle

Ntessalen, M, Procter, NE, Schwarz, K, Loudon, BL, Minnion, M, Fernandez, BO, Vassiliou, VS, Vauzour, D, Madhani, M, Constantin-Teodosiu, D, Horowitz, JD, Feelisch, M, Dawson, D, Crichton, PG & Frenneaux, MP 2020, 'Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans', The American Journal of Clinical Nutrition, vol. 111, no. 1, pp. 79-89. https://doi.org/10.1093/ajcn/nqz245
Ntessalen, Maria ; Procter, Nathan Ek ; Schwarz, Konstantin ; Loudon, Brodie L ; Minnion, Magdalena ; Fernandez, Bernadette O ; Vassiliou, Vassilios S ; Vauzour, David ; Madhani, Melanie ; Constantin-Teodosiu, Dumitru ; Horowitz, John D ; Feelisch, Martin ; Dawson, Dana ; Crichton, Paul G ; Frenneaux, Michael P. / Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans. In: The American Journal of Clinical Nutrition. 2020 ; Vol. 111, No. 1. pp. 79-89.
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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.",
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author = "Maria Ntessalen and Procter, {Nathan Ek} and Konstantin Schwarz and Loudon, {Brodie L} and Magdalena Minnion and Fernandez, {Bernadette O} and Vassiliou, {Vassilios S} and David Vauzour and Melanie Madhani and Dumitru Constantin-Teodosiu and Horowitz, {John D} and Martin Feelisch and Dana Dawson and Crichton, {Paul G} and Frenneaux, {Michael P}",
note = "Supported by Medical Research Council program grant MRC G1001340 (to M Madhani, M Feelisch, and MP Frenneaux). We thank Lesley Cheyne for their contributions to the present study. The authors’ responsibilities were as follows—VSV, M Madhani, JDH, MF, DD, MPF: designed the research; MN, NEKP, KS, BLL, M Minnion, BOF, DV, DC-T, PGC: conducted the research; DV: provided essential materials; MN, NEKP, M Minnion, BOF, DC-T, MF, PGC: analyzed the data; MN, NEKP, PGC, MPF: wrote the paper; MPF: had primary responsibility for the final manuscript; and all authors: read and approved the final manuscript. None of the authors reported a conflict of interest related to the study.",
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T1 - Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans

AU - Ntessalen, Maria

AU - Procter, Nathan Ek

AU - Schwarz, Konstantin

AU - Loudon, Brodie L

AU - Minnion, Magdalena

AU - Fernandez, Bernadette O

AU - Vassiliou, Vassilios S

AU - Vauzour, David

AU - Madhani, Melanie

AU - Constantin-Teodosiu, Dumitru

AU - Horowitz, John D

AU - Feelisch, Martin

AU - Dawson, Dana

AU - Crichton, Paul G

AU - Frenneaux, Michael P

N1 - Supported by Medical Research Council program grant MRC G1001340 (to M Madhani, M Feelisch, and MP Frenneaux). We thank Lesley Cheyne for their contributions to the present study. The authors’ responsibilities were as follows—VSV, M Madhani, JDH, MF, DD, MPF: designed the research; MN, NEKP, KS, BLL, M Minnion, BOF, DV, DC-T, PGC: conducted the research; DV: provided essential materials; MN, NEKP, M Minnion, BOF, DC-T, MF, PGC: analyzed the data; MN, NEKP, PGC, MPF: wrote the paper; MPF: had primary responsibility for the final manuscript; and all authors: read and approved the final manuscript. None of the authors reported a conflict of interest related to the study.

PY - 2020/1

Y1 - 2020/1

N2 - 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.

AB - 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.

KW - nitrate

KW - nitrite

KW - mitochondria

KW - uncoupling protein

KW - pyruvate dehydrogenase

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DO - 10.1093/ajcn/nqz245

M3 - Article

C2 - 31599928

VL - 111

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EP - 89

JO - The American Journal of Clinical Nutrition

JF - The American Journal of Clinical Nutrition

SN - 0002-9165

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ER -