Beetroot juice supplementation reduces whole body oxygen consumption but does not improve indices of mitochondrial efficiency in human skeletal muscle (2024)

• Oral consumption of nitrate (NO₃⁻) in beetroot juice has been shown to decrease the oxygen cost of submaximal exercise; however, the mechanism of action remains unresolved.

• We supplemented recreationally active males with beetroot juice to determine if this altered mitochondrial bioenergetics.

• Despite reduced submaximal exercise oxygen consumption, measures of mitochondrial coupling and respiratory efficiency were not altered in muscle.

• In contrast, rates of mitochondrial hydrogen peroxide (H₂O₂) emission were increased in the absence of markers of lipid or protein oxidative damage.

• These results suggest that improvements in mitochondrial oxidative metabolism are not the cause of beetroot juice‐mediated improvements in whole body oxygen consumption.

Abbreviations

ANT

adenine nucleotide translocase

BRJ

beetroot juice

IMF

intermyofibrillar mitochondria

PDH

pyruvate dehydrogenase

PmFB

permeabilized muscle fibre bundles

P/O ratio

ADP consumed/oxygen consumed

RCR

respiratory control ratio

ROS

reactive oxygen species

SOD

superoxide dismutase

SS

subsarcolemmal mitochondria

UCP3

uncoupling protein 3

Ethics approval

Thirteen healthy, recreationally active males (23.6± 0.6 years, 79.4±1.5kg, V˙O2 peak 49.6±1.4ml⁻¹ min⁻¹kg⁻¹) were initially recruited for this study, of which 10 underwent biopsy trials both prior to, and following, supplementation. Prior to participation, written informed consent was obtained from all subjects. All procedures were approved by the Research Ethics Boards of the University of Guelph (Guelph, Ontario) and McMaster University (Hamilton, Ontario), and conform to the Declaration of Helsinki.

Pre‐experimental tests

Prior to supplementation, subjects underwent a continuous incremental cycling test to exhaustion on an electronically braked cycle ergometer (Lode, Groningen, the Netherlands) for determination of pulmonary V˙O2 peak, as well as a 20min cycle test with 10min at a workload corresponding to 50% V˙O2 peak followed by an immediate step increase to a workload corresponding to 70% V˙O2 peak for an additional 10min. For familiarization, subjects performed a practice trial of the 20min test, which also served to confirm subjects were at the correct workloads. Trials were separated by 48h. Ventilation and expired oxygen and carbon dioxide concentrations were measured throughout all tests using a metabolic measurement system (MOXUS Modular VO₂ System, AEI Technologies, Pittsburgh, PA, USA), and the last 5min at each power output was used for analysis (Fig. ​(Fig.11).

Study design

Following the completion of pre‐experimental tests (≤48h) and an overnight fast, 10 subjects had four biopsies taken from the vastus lateralis as previously described (Bergstrom, 1975). A small portion of the first biopsy was taken and immediately used for the preparation of permeabilized muscle fibres (PmFB; described below). The remaining portion was combined with the second and third biopsies for the isolation of mitochondrial subfractions. The fourth biopsy was flash‐frozen for Western blotting. Due to limited tissue yield, mitochondrial isolations and Western blotting could only be performed on a subset of five participants. Following the biopsies, subjects began supplementing with concentrated BRJ (2×6.5mmol NO₃⁻/70ml Beet It Sport; James White Drinks, Ipswich, UK; taken twice daily for a total daily intake of ∼26mmol NO₃⁻) for 7days in an effort to maintain elevated blood plasma NO₃⁻ and NO₂⁻ levels. On day5 of the supplementation period, subjects repeated the submaximal cycling exercise at 50 and 70% of V˙O2 peak. On the morning of day7 after an overnight fast, subjects consumed 2×Beet It Sport shots 90min prior to having four biopsies taken from the vastus lateralis of the contralateral leg for post‐supplementation measurements. Subjects were asked to refrain from consuming foods rich in nitrates for the duration of the study, and to abstain from the use of antibacterial mouthwash, as this has been shown to attenuate the conversion of nitrate to nitrite by commensal bacteria (Govoni etal. 2008).

Preparation of permeabilized fibres

A small portion of each biopsy was placed in ice‐cold BIOPS (50mm MES, 7.23mm K₂EGTA, 2.77mm CaK₂EGTA, 20mm imidazole, 0.5mm DTT, 20mm taurine, 5.77mm ATP, 15mm PCr, and 6.56mm MgCl₂.H₂O; pH7.1) and separated under a microscope into bundles using fine‐tipped forceps as described previously (Perry etal. 2012). Fibre bundles were then treated with 30μgml⁻¹ saponin for 30min at 4°C, then either washed for 15min in MiR05 respiration buffer (0.5mm EGTA, 10mm KH₂PO₄, 110mm sucrose, and 1mgml⁻¹ fatty acid‐free bovine serum albumin (BSA); pH 7.1) for respiration analysis, or buffer Z (105mm K‐Mes, 30mm KCl, 1mm EGTA, 10mm K₂HPO₄, 5mm MgCl₂.H₂O, 0.005mm pyruvate, 0.002mm malate with 5mgml⁻¹ fatty acid‐free BSA; pH7.4) for measurements of H₂O₂ emission as described below.

Mitochondrial isolation

Mitochondrial subsarcolemmal (SS) and intermyofibrillar (IMF) populations were isolated by differential centrifugation as described previously (Campbell etal. 2004) with minor modifications (Matravadia etal. 2014). Muscle was quickly minced and diluted in 1mlmg⁻¹ isolation buffer (100mm sucrose, 100mm KCl, 50mm Tris HCl, 1mm KH₂PO₄, 0.1mm EGTA, 2mgml⁻¹ fatty acid‐free BSA, 1mm ATP; pH7.4) and hom*ogenized using a Polytron with a Teflon head. SS mitochondria underwent centrifugation at 800g for 10min and the supernatant was pelleted at 10,000g for 10min before being washed in Mg²⁺‐absent MiR05 buffer (0.5mm EGTA, 60mm potassium lactobionate, 10mm KH₂PO₄, 20mm HEPES, 110mm sucrose, 1mgml⁻¹ BSA; pH7.1), and spun again at 10,000g for 10min. After the initial 800g spin, the IMF mitochondrial pellet was resuspended 10‐fold in isolation buffer and treated with 0.025mlg⁻¹ protease for 5min before being diluted 10‐fold in isolation buffer and spun again at 5000g for 5min. The IMF pellet was resuspended in isolation buffer and pelleted at 800g for 10min after which the supernatant was pelleted at 10,000g for 10min before being washed in Mg²⁺‐absent MiR05 buffer, and spun again at 10,000g for 10min. The final pellets for both SS and IMF isolations were resuspended in Mg²⁺‐absent MiR05 buffer prior to respiration, H₂O₂ emission and membrane potential measurements. Protein content was quantified spectrophotometrically using bicinchroninic acid.

Mitochondrial respiration

Measurements of O₂ consumption were performed in MiR05 respiration medium on prepared permeabilized fibres using an Oxygraph‐2K (Oroboros Instruments, Innsbruck, Austria) at 37°C in the presence of 25μm blebbistatin as previously described (Perry etal. 2011). Mitochondrial kinetics in PmFB were analysed using three separate titration protocols. ADP kinetics were analysed in the presence and absence of 20mm creatine. Both ADP titrations were initiated with 2mm malate and 10mm pyruvate, and ADP was titrated at various concentrations. Pyruvate kinetics were determined in the presence of 5mm ADP, 5mm malate, and 20mm creatine while pyruvate was titrated in at various concentrations. Glutamate (10mm) and 10mm succinate were added following the pyruvate and ADP titrations to determine maximum mitochondrial respiration. Finally, 10μm cytochrome c was added, with <10% increase in respiration in all measurements indicating outer mitochondrial membrane integrity. All fibres were recovered from the respirometer, freeze‐dried, and weighed for normalization to muscle bundle weight. The apparent K_m for pyruvate and ADP were determined as previously described (Perry etal. 2012).

In isolated mitochondria, respiration experiments were performed at 25°C as described previously (Lally etal. 2013). State IV respiration was determined in the presence of 5mm pyruvate and 2mm malate. To determine P/O ratios, 50μm ADP was added followed by two boluses of 100μm ADP. The change in oxygen concentration in the chamber following each of the two 100μm ADP boluses was averaged and used for the calculation of P/O ratios. Maximal state III respiration was determined by the addition of 5mm ADP, with 10mm glutamate and 10mm succinate added to determine maximal complex I and maximal complex I+II respiration, respectively.

H₂O₂ emission

Mitochondrial H₂O₂ emission in PmFB was measured in buffer Z (105mm K‐MES, 30mm KCl, 1mm EGTA, 10mm K₂HPO₄, 5mm MgCl₂.H₂O, 0.005mm pyruvate, 0.002mm malate with 5mgml⁻¹ fatty acid‐free BSA; pH7.4) fluorometrically (Lumina; Thermo Scientific, Waltham, MA, USA) at 37°C in the presence of 25μm blebbistatin. Cuvettes contained 10μgml⁻¹ oligomycin, 10μm Amplex Red (Invitrogen, Carlsbad, CA, USA), 0.5Uml⁻¹ horseradish peroxidase, and in both the presence and absence of 40Uml⁻¹ superoxide dismutase (SOD). In isolated mitochondria, H₂O₂ emission was measured in MiR05 at 25°C in cuvettes containing 10μgml⁻¹ oligomycin, 10μm Amplex Red (Invitrogen), 0.5Uml⁻¹ horseradish peroxidase, and 40Uml⁻¹ SOD. The reaction for both PmFB and isolated mitochondria was initiated by the addition of 10mm succinate. The rate of H₂O₂ emission was calculated from the slope (absorbance/minutes) after subtracting the background, from a standard curve established with the same reaction conditions. In PmFB, H₂O₂ rates were normalized to fibre dry weight as performed previously (Anderson etal. 2009), and in isolated mitochondria were normalized to protein content.

Membrane potential

Mitochondrial membrane potential was measured fluorometrically at 25°C in cuvettes filled with MiR05 and 2μm Safranine‐O as described previously (Goo etal. 2013), with some modifications. Briefly, mitochondria were added to cuvettes followed by the addition of 10mm pyruvate and 5mm malate (state IV), 2.5mm ADP (state III), 10mm glutamate (maximal complex I), and 10mm succinate (maximal complex I+II). Mitochondria were uncoupled by the addition of 2μl 2,4‐dinitrophenol (DNP), followed by the addition of Safranine‐O (2×1μm) for calibration of the fluorescence signal. Membrane potential was then estimated using the Nernst equation:

where R is the universal gas constant, F is Faraday's constant, T is absolute temperature, and z is the valency (+1) of Safranine.

Western blotting

Western blotting was performed on whole muscle hom*ogenate using methods described previously (Herbst etal. 2014). Samples were loaded equally for α‐tubulin (Abcam, Cambridge, UK), UCP3 (Millipore, Billerica, MA, USA), ANT1 (Mitosciences, Eugene, OR, USA), ANT2 (Abcam), SOD2 (Abcam), catalase (Abcam), 4‐hydroxynonenal (4HNE; Alpha Diagnostics, Antonio, TX, USA), pyruvate dehydrogenase (PDH) subunit E1α (Molecular Probes, Eugene, OR, USA), oxidative phoshorylation (OXPHOS; Mitosciences), and for the Oxyblot protein oxidation detection kit (Millipore) and N3‐nitrosylation (Cayman Chemical, Ann Arbor, MI, USA). Proteins were separated by SDS‐polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and were incubated in blocking solution, primary antibody, and the corresponding secondary antibody as specified by the supplier. All samples were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability, and all gels were loaded with 10μg protein. Linearity of signal was confirmed from 5–15μg of protein. Membrane proteins were detected by enhanced chemiluminesence (Perkin Elmer, Woodbridge, ON, Canada) and quantified by densitometry (Alpha Innotech Fluorchem HD2; Fisher Scientific, Ottawa, ON, Canada).

Plasma analyses

Five separate subjects repeated the supplementation protocol for the collection of blood plasma. Subjects reported to the lab following an overnight fast, and had blood samples taken immediately prior to consuming their first bolus of BRJ. Samples were then taken again 1.5 and 3h after ingestion. This process was repeated again in the evening, and on day6 of the supplementation period. Blood samples (8ml) were collected in EDTA‐containing tubes and centrifuged at 1000g for 10min at 4°C. Plasma aliquots were frozen and stored at −80°C for subsequent analysis of plasma nitrate (NO₃⁻) and nitrite (NO₂⁻) by chemiluminescence using a Sievers gas‐phase chemiluminescence NO analyser (Sievers NOA 280i; Analytix, Durham, UK) (Lundberg & Govoni, 2004).

Statistics

A two‐way analysis of variance was used to detect differences between day 1, day 6, and sampling time‐points for blood plasma NO₃⁻ and NO₂⁻ measurements. If significance was detected, a Fischer LSD post hoc test was applied. A Student's paired ttest (two‐way) was used to detect differences between pre‐ and post‐supplementation data in submaximal exercise trials, and in isolated mitochondria and permeabilized muscle fibre experiments. Significance was determined at P<0.05 with confidence intervals ≥95%.

Whole body oxygen consumption and energy expenditure

We first characterized the pulmonary V˙O2 responses during exercise to ensure that the supplementation with BRJ coincided with the expected metabolic effect. Oxygen consumption was unaltered while exercising at 50% V˙O2 peak (Fig.2A). In contrast, oxygen consumption while exercising at 70% V˙O2 peak decreased ∼100ml following BRJ consumption, representing ∼3% of oxygen consumption (Fig.2B). It is worth noting that of the 13 participants tested in the exercise trials, 8 showed a decrease in pulmonary V˙O2 at 50% (Fig.2A), and 12 of 13 subjects decreased at 70% (Fig.2B). Despite the change in oxygen consumption at 70% V˙O2 peak, there was no change in respiratory exchange ratio (Pre, 0.99±0.01; Post, 0.99±0.01), indicating that there was no change in fuel preference following BRJ consumption.

Respiration in permeabilized fibres

Given the change in oxygen consumption, we next aimed to determine if BRJ supplementation altered various indices of mitochondrial bioenergetics. Supplementation did not alter leak respiration (non‐ADP‐stimulated respiration; Fig.3A) or maximal substrate‐supported respiration (Fig.3A). In support of a lack of change in maximal respiration, the content of various electron transport chain proteins and PDH was not altered (Fig.3B and C). A previous paper found that sodium nitrate supplementation decreased ANT protein content (Larsen etal. 2011), and therefore we next examined if BRJ altered mitochondrial ADP sensitivity. Titrating ADP in the presence of substrates revealed an expected Michaelis–Menten curve (insets in Fig.4A and D) that was utilized to estimate the ADP sensitivity (apparent K_m). BRJ supplementation did not alter maximal respiration (Fig.4B and E) or the apparent K_m (Fig.4C and E) in the presence or absence of creatine. In support of this, ANT1, ANT2 and UCP3 protein contents were unaltered following supplementation (Fig.4G and H). To further examine the potential effect of BRJ supplementation on electron transport function we determined the kinetic properties of pyruvate to stimulate respiration in the presence of saturating ADP concentrations. Unfortunately some of the permeabilized fibre bundles could not be recovered for dry weight assessments following respiration experiments, and therefore only kinetic properties were examined and not maximal respiration. Pyruvate displayed a typical Michaelis–Menten curve (Fig.5A); however, BRJ did not alter the apparent sensitivity of mitochondria to pyruvate (Fig.5B). Altogether these data suggest that BRJ, while decreasing oxygen consumption during exercise, does not improve mitochondrial respiratory measures in permeabilized muscle fibres.

Respiration and membrane potential in isolated mitochondria

Given the lack of change in mitochondrial respiration, in a subset of participants (n=5) both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subpopulations were isolated to directly examine mitochondrial coupling efficiencies. BRJ supplementation did not alter the P/O ratio (Fig.6A and B), or the respiratory control ratio (RCR) (Fig.6C and D) in SS or IMF mitochondria. Furthermore, there was no correlation between the measured coupling ratios in either subpopulation and the change in whole body V˙O2 at either power output (50%: R²=0.04 SS, 0.003 IMF; 70%; R²=0.008 SS, 0.119 IMF). In addition, changes in membrane potential (ΔΨ) were estimated fluorometrically using Safranine‐O. In both SS and IMF mitochondria, ΔΨ decreased following the addition of substrates and ADP (Fig.7A and B); however, there were no differences in either fraction following supplementation. These data further support the interpretation that a reduction in whole body oxygen consumption during exercise is independent of improvements in mitochondrial respiration/coupling.

H₂O₂ emission and oxidative stress

Given that NO can inhibit cytochrome oxidase (Bolaños etal. 1994; Brown & Cooper, 1994; Cleeter etal. 1994; Giuffrè etal. 2000), we also examined the possibility that BRJ could increase the propensity of mitochondria to produce ROS. In permeabilized fibres, maximal succinate‐induced mitochondrial H₂O₂ emission increased ∼50% following supplementation in the absence (Fig.8A) and presence (Fig.8B) of exogenous superoxide dismutase (SOD). The presence of exogenous SOD did not increase H₂O₂ emission (Fig.8A and B), suggesting the Qo site of complex III does not release superoxide into the intermembrane space or contribute to succinate‐induced ROS in humans. This supports the notion that reverse electron flow to complex I represents the majority of superoxide production in the presence of succinate. However, there was no correlation between the propensity to produce H₂O₂ and the change in whole body V˙O2 either with (R²=0.03 at 50%, 0.115 at 70%) or without (R²=0.08 at 50%, 0.209 at 70%) exogenous SOD. Within IMF mitochondria, which represent ∼80% of the mitochondria within muscle (Hoppeler etal. 1985; Ferreira etal. 2010), there was also a strong trend for an increase in H₂O₂ emission (P=0.07). However, there was no increase within SS mitochondria (Fig.8C and D). The increased propensity to produce ROS within permeabilized fibres did not result from a reduction in either catalase or SOD2 (Fig.8E and F) and did not manifest with overt oxidative stress, as indicated by a lack of change in lipid peroxidation (4HNE), protein carbonylation or nitrotyrosine residues in muscle hom*ogenates (Fig.8G and H).

Temporal effects of beetroot juice consumption on blood nitrate and nitrite concentrations

Despite the observed decrease in whole body V˙O2 and increase in muscle mitochondrial ROS emission, given the lack of response with respect to mitochondrial coupling we wanted to confirm that the dosing protocol resulted in increases in plasma NO₃⁻ and NO₂⁻. Therefore in separate participants we re‐ran the study to only measure plasma responses. In these individuals we characterized the blood NO₃⁻ and NO₂⁻ responses during the first and last full day of the supplementation period. In general, both NO₃⁻ (Fig.9A) and NO₂⁻ (Fig.9B) concentrations increased (P<0.05) within 90min, and remained elevated 2h following ingestion in the morning and in the evening. The basic response to BRJ consumption was not dramatically altered when measured following 6days of supplementation. However, NO₃⁻ levels were higher in the morning on day 6 (Fig.9A), such that NO₃⁻ was higher in the morning at all time points examined, in association with a strong trend (P=0.07) for an increase in NO₂⁻ (Fig.9B) 90min following ingestion. In the evening, NO₃⁻ was not dramatically different on day 6, while NO₂⁻ was lower 120min after the consumption of BRJ (Fig.9A and B). Therefore, the plasma responses indicate that the dosing protocol used in the current study was sufficient to maintain elevated plasma NO₃⁻ and NO₂⁻ concentrations throughout the experiment.

Limitations

A limitation of this study was the time delay between measurements, as whole body oxygen consumption, muscle biopsies and the collection of blood were taken on different days. While we could have performed the exercise trials on the same experimental day as the biopsies, out of necessity this would dictate that the exercise and biopsies occurred at different times following the consumption of the BRJ (i.e. biopsies at 90min and exercise 120–150min post‐consumption). Instead, we opted to standardize the time that all measurements were taken following the final morning's dose of BRJ, which dictated that measurements were taken on different days. This decision was made as we are unaware of data to indicate that the response to BRJ would be different between days 5 and 7; however, the acute response to beetroot juice is known to vary considerably across time. Nevertheless, the reader should be aware of the time delay between measures.

Unfortunately the subjects recruited for blood measurements represent a different cohort, and therefore should only be interpreted as proof‐of‐principle that the current dosing protocol resulted in the expected increase in plasma NO₂⁻ and NO₃⁻. Given the results of our blood measures, we are confident our dosing protocol was sufficient to result in a prolonged elevation in plasma NO₂⁻ and NO₃⁻ in the first cohort of subjects, and therefore the lack of an effect is not the result of our supplementation regime. This is further corroborated by our results at 70% V˙O2 peak exhibiting the well‐documented decrease in whole body V˙O2 following NO₃⁻ supplementation (Larsen etal. 2007, 2011; Bailey etal. 2009, 2010; Vanhatalo etal. 2010; Lansley etal. 2011; Cermak etal. 2012). This shows that our dosing protocol is still eliciting an ergogenic effect, but the underlying mechanism is not an alteration in mitochondrial coupling, and thus lies elsewhere.

Another possible perceived limitation was the small sample size, particularly for the isolated mitochondria experiments. However, in order to detect a 5% change in mitochondrial coupling ratios with 80% power, for SS mitochondria alone we would need to recruit an additional 50 subjects. Furthermore, the results of our mitochondrial measurements support what was seen within the larger group in PmFB experiments. We therefore felt we could not ethically justify recruiting more subjects and performing biopsies just to increase the sample size when we did not anticipate seeing an effect, as the tissue requirement for isolated mitochondria is significant.

Conclusions

Altogether, in the current study we provide evidence that improvements in mitochondrial coupling efficiency are not required for the reduction in whole body oxygen utilization during exercise following BRJ supplementation in recreationally active subjects. Instead, we provide evidence that mitochondrial H₂O₂ is increased; however, it remains to be determined if this contributes to the observed reduction in V˙O2. This is in contrast to the results found following 3days of sodium nitrate supplementation (Larsen etal. 2011). This is surprising given that nitrate appears to be the active ingredient in both supplements, as a nitrate‐depleted beetroot juice placebo has been shown to have no effect on pulmonary responses to exercise in both moderate‐ and severe‐intensity exercise, as well as blood pressure (Lansley etal. 2011). It is therefore possible that these two supplements act through different mechanisms, or elicit effects beyond simply increasing dietary nitrate intake. Future work should compare both BRJ and sodium nitrate directly with respect to mitochondrial adaptations and H₂O₂ emission to further our understanding of how these supplements may alter metabolism.

Competing interests

None declared.

Author contributions

J.W. and G.P.H. designed experiments, interpreted the data, and wrote the manuscript. L.L.S. designed experiments and interpreted the data. J.W., A.L., G.J.F.H., J.M.G.S., L.B.V. and L.J.C.v.L. performed experiments. All authors edited the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was funded by The Natural Sciences and Engineering Research Council of Canada (G.P.H, 03656; L.L.S., 03996) and infrastructure was purchased with the assistance of the Canadian Foundation for Innovation/Ontario Research Fund (G.P.H., 25136).

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