INTRODUCTION

Obesity is one of the main public health challenges in industrialized countries and the number of people affected by this condition is growing from year to year. Taking into consideration children and adolescents (2–19 yr), the percentage of people with obesity in the United States is an alarming 46.9%. Obesity during childhood is associated with an excess in body mass (BM) in adulthood, and among adults obesity is a significant risk factor for several chronic diseases and, conditions, including cardiovascular diseases, diabetes, and cancer (26).

Patients with obesity show a significant exercise intolerance and an impairment in physical activities of daily living because of a generalized sense of fatigue, which negatively affects their quality of life and may lead to severe deconditioning. The reduced exercise tolerance is associated with a higher O₂ cost for submaximal exercise (30, 33, 37), a lower gas exchange threshold, slower adjustment of the fundamental component of oxygen consumption (V̇o₂) kinetics (30), and a more pronounced amplitude of the slow component of V̇o₂ kinetics during heavy intensity exercise (30). These impairments are attributable to cardiovascular (33), respiratory (31, 32), and skeletal muscle factors (23, 30). In a vicious circle, the impaired exercise tolerance and the associated reduced level of physical activity worsen obesity and interfere with interventions (such as exercise prescription) aimed at its control.

Patients with obesity show an impaired endogenous nitric oxide (NO) synthesis, in particular in the presence of the metabolic syndrome (34). The mechanisms seem to be related to an increased generation of reactive oxygen species, an impaired function of NO synthase (NOS), and to decreased insulin signaling (34). More specifically, the inhibition of NOS enzyme activity in obesity is prevalent in the endothelium (20), but it can be also found in skeletal muscle (36), suggesting a possible shortage of NO bioavailability at the skeletal muscle level.

NO is produced endogenously from the oxidation of l-arginine in a reaction catalyzed by a family of NOS enzymes (l-arginine-NOS-NO pathway) (19). An alternative pathway of NO synthesis is, however, present. The ingestion of foods rich in nitrate ($N{O}_3^{-}$), like green leafy vegetables and beetroot, can increase NO bioavailability through the conversion of $N{O}_3^{-}$ to nitrite ($N{O}_2^{-}$) in the entero-salivary circulation, and the subsequent conversion of $N{O}_2^{-}$ to NO by peripheral enzymes or proteins (such as deoxygenated hemoglobin and myoglobin) after $N{O}_2^{-}$ has entered the systemic circulation (19).

Recent evidence suggests that an enhanced NO bioavailability can positively affect exercise tolerance through a variety of mechanisms (14). For example, an increased NO bioavailability may improve the efficiency of mitochondrial respiration, as evaluated by the amount of oxygen consumed per ATP produced (P/O ratio) (22); ameliorate muscle metabolic perturbations found during exercise, which may decrease the ATP cost of force production (1); and increase skeletal muscle microvascular blood flow, at least toward type 2 fibers, thereby improving the intramuscular matching between O₂ delivery and O₂ utilization and increasing microvascular Po₂ and peripheral O₂ diffusion (9). Thus, an increased oxidative efficiency (i.e., a lower O₂ requirement leading to a lower pulmonary V̇o₂ for the same work rate) could be of utmost interest in patients with obesity who typically show an increased O₂ cost of exercise (ΔV̇o₂/Δ work rate, where Δ represents change in) (33, 37), which is partly attributable to an increased O₂ cost of breathing (31, 32).

The aim of the present study was to evaluate the effects of short-term (6 days) dietary $N{O}_3^{-}$ supplementation on physiological parameters associated with exercise tolerance in a group of obese adolescents. We hypothesized that in this population $N{O}_3^{-}$ supplementation would lead to an enhanced exercise tolerance by reducing the O₂ cost of exercise in different intensity domains and by increasing the time to exhaustion during severe-intensity constant work rate (CWR) exercise. These results would extend the ergogenic effects of dietary $N{O}_3^{-}$ supplementations previously demonstrated in healthy subjects (2, 29) and in patients with peripheral arterial disease (18), chronic obstructive pulmonary disease (8), and chronic heart failure (42, 43) to patients with obesity.

MATERIALS AND METHODS

RESULTS

Adherence and safety.

All participants completed the intervention and follow-up testing. Adherence to BR supplementation, estimated by returned bottle count, and consumption log was 100%. There were no adverse events related to the supplementation, except temporarily red urine and red stool.

Plasma nitrate levels.

Plasma [$N{O}_3^{-}$] was significantly higher in BR (108.4 ± 37.2 μM) compared with PLA (15.4 ± 5.0 μM) (P < 0.0001) (Fig. 1). Higher values in BR were observed in all patients. The increase in plasma [$N{O}_3^{-}$] was inversely related to the patients’ BM (r² = 0.63; P = 0.0072).

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Incremental exercise.

Peak values of the main respiratory, cardiovascular, and metabolic variables obtained at exhaustion during incremental exercise are shown in Table 1. HR values reached ~85% of maximal predicted HR. GET occurred at 59 ± 9% of maximal work rate and 63 ± 9% of V̇o_2peak.

Table 1. Peak values of the main respiratory, cardiovascular, and metabolic variables determined at exhaustion during incremental exercise

Work Rate,W	V̇o2, l/min	V̇o2, ml·kg−1·min−1	V̇co2, l/min	R	VT, liters	fR, breaths/min	V̇e, l/min	$PE{T}_{O_2}$, mmHg	$PE{T}_{C{O}_2}$, mmHg	HR, beats/min	GET, W	GET, l/min	RPE, (0–10)
174 ± 44	2.33 ± 0.68	24.4 ± 5.5	2.71 ± 0.81	1.16 ± 0.07	1.71 ± 0.41	39 ± 6	66.7 ± 17.0	103.1 ± 5.1	45.8 ± 5.2	174 ± 10	100 ± 21	1.46 ± 0.36	7.9 ± 2.4

Values are mean (± SD). fR, breathing frequency; GET, gas exchange threshold; HR, heart rate; $PE{T}_{C{O}_2}$, end-tidal CO₂ partial pressure; $PE{T}_{O_2}$, end-tidal O₂ partial pressure; R, gas exchange ratio; RPE, rating of perceived exertion; V̇e, pulmonary ventilation; V̇co₂, carbon dioxide output; V̇o₂, oxygen consumption; V_T, tidal volume.

Moderate-intensity CWR exercise.

All patients completed the imposed 6 min of exercise. Breath-by-breath values obtained in the two repetitions of the exercise were time aligned and then superimposed for each subject. Mean values of the main physiological variables determined in both BR and PLA during the last 30 s of moderate-intensity CWR exercises are presented in Table 2. No significant differences were found for any of the variables between the two conditions. Work rate was set to be identical in each patient in the two conditions, and the value corresponded to ~45% of peak work rate. HR, expressed as percentage of maximal HR reached during the incremental exercise, was 73 ± 8% and 73 ± 9% in PLA and BR, respectively (no significant difference). Mean values of V̇o₂ calculated at different time points (last 3 min and last 1 min) were not different between BR (1.35 ± 0.23 and 1.35 ± 0.22 l/min, respectively) and PLA (1.32 ± 0.25 and 1.32 ± 0.25, l/min, respectively), suggesting that steady state was reached. The O₂ cost of cycling was 13.3 ± 1.7 ml·min⁻¹·W⁻¹ in BR and 12.9 ± 1.1 ml·min^-1·W⁻¹ in PLA.

Table 2. Main respiratory, cardiovascular, and metabolic variables determined at the end of the moderate- and severe-intensity CWR exercises after PLA and BR supplementation

	Work Rate	V̇o2, l/min	V̇o2, ml·kg−1·min−1	V̇o2, %peak	V̇co2, l/min	R	V̇e, l/min	HR, beats/min	HR, %peak
Moderate
PLA	77 ± 13	1.32 ± 0.21	14.2 ± 1.3	59 ± 12	1.29 ± 0.17	0.98 ± 0.06	34.3 ± 6.2	124 ± 12	73 ± 8
BR	77 ± 13	1.34 ± 0.21	14.6 ± 2.2	60 ± 12	1.30 ± 0.15	0.97 ± 0.06	34.1 ± 4.6	124 ± 12	73 ± 9
Severe
PLA	142 ± 38	2.33 ± 0.54	24.7 ± 4.8	100 ± 24	2.57 ± 0.56	1.10 ± 0.07	73.8 ± 18.0	169 ± 16	96 ± 9
BR	142 ± 38	2.22 ± 0.56	23.1 ± 4.5	95 ± 25	2.45 ± 0.58	1.10 ± 0.04	69.9 ± 16.6	172 ± 10	103 ± 4

Values are mean (± SD). BR, beetroot juice; CWR, constant work rate; HR, heart rate; PLA, placebo; R, gas exchange ratio; V̇e, pulmonary ventilation; V̇co₂, carbon dioxide output; V̇o₂, oxygen consumption.

Figure 2 shows the time courses of V̇o₂ obtained every 30 s during the moderate-intensity CWR exercise in both PLA and BR. The time-courses of the variable in the two conditions were comparable and no statistical differences were observed between the values obtained in the two groups at any given time.

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Severe-intensity CWR exercise.

Mean values of the main physiological variables determined in BR and PLA during the last 30 s of CWR severe-intensity CWR exercises are shown in Table 2. Work rate was designed to be the same in each patient in the two conditions and corresponded to 83 ± 4% of peak work rate. HR was 96 ± 9% of peak HR in PLA and 103 ± 4% of peak HR in BR. No patients in PLA and only one patient in BR completed the 15-min exercise. Time to exhaustion was significantly increased (by 23%) by $N{O}_3^{-}$ supplementation (561 ± 198 s and 457 ± 101 s, in BR and PLA, respectively, P = 0.0143) (Fig. 3). In three of the patients, time to exhaustion did not increase following $N{O}_3^{-}$ supplementation. No correlation was observed between the increase in plasma ($N{O}_3^{-}$) and time to exhaustion (r² = 0.13; P = 0.30). The O₂ cost of cycling (14.1 ± 0.9 ml·min⁻¹·W⁻¹ in BR vs. 14.9 ± 1.3 ml·min⁻¹·W⁻¹ in PLA) was not different following $N{O}_3^{-}$ administration (P = 0.38). No correlation was observed between the increase in plasma ($N{O}_3^{-}$) and O₂ cost of cycling (r² = 0.38; P = 0.10). Figure 4 (left) shows the time courses of V̇o₂ in the two conditions. To obtain this figure, individual V̇o₂ values were grouped for discrete time intervals, which were determined at discrete percentages of the individual total exercise time. Mean absolute individual values were calculated for the x and y variables for each time interval and these data were utilized to obtain the mean group values shown in Figure 4. Until 180 s of exercise, the exponential time courses of V̇o₂ in the two conditions were substantially superimposed. After 180 s, the time courses of V̇o₂ followed a linear increase without reaching a steady state. The slopes of the regression lines, calculated from the 3rd minute to the end of exercise were significantly different from zero (P < 0.0001). The rate of V̇o₂ increase, calculated as ΔV̇o₂ (V̇o₂ at end-exercise minus V̇o₂ at 3 min) divided by time (time at end-exercise minus 3 min), was significantly lower in BR versus PLA (29.3 ± 18.1 vs. 60.9 ± 18.1 ml/min², P = 0.0002) (Fig. 4, right).

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DISCUSSION

The main result of the present study was that in adolescents with obesity, short-term (6 days) dietary $N{O}_3^{-}$ supplementation enhanced exercise tolerance by significantly increasing the time to exhaustion during severe-intensity CWR exercise. The increased exercise tolerance was associated with a less pronounced slow component of pulmonary V̇o₂ kinetics. However, $N{O}_3^{-}$ supplementation had no effects on other physiological variables determined during moderate-intensity CWR exercise.

Obesity, also in adolescence, is usually associated with reduced cardiorespiratory fitness and exercise intolerance (30, 33). Whereas V̇o_2peak, expressed in absolute units (e.g., l/min), is usually normal compared with normal-weight controls, when it is normalized per unit of BM (ml·kg⁻¹·min⁻¹) values in obese patients are ~40% lower than in controls (23, 30). Other factors contributing to the impaired exercise tolerance in obesity include a higher O₂ cost for submaximal exercise (30, 33, 37), which is at least in part attributable to an increased O₂ cost of breathing (31, 32), a lower GET, slower adjustment of the fundamental component of V̇o₂ kinetics (30), and a more pronounced amplitude of the slow component of V̇o₂ kinetics during heavy-intensity exercise (30). At least in part, these impairments can be overcome by exercise training (24), including training specifically directed toward respiratory muscles (31).

In the present study, we evaluated the effects of a 6-day dietary $N{O}_3^{-}$ supplementation on a group of adolescents with obesity and we observed an improved exercise tolerance after the intervention. More specifically, time to exhaustion during severe-intensity CWR exercise, which represents a classic parameter for the evaluation of exercise tolerance, was significantly increased (by ~25%) after dietary $N{O}_3^{-}$ supplementation. Figure 4 shows that the linear increase in V̇o₂ from 200 s to the end of the exercise was characterized by a significantly lower slope in BR than in PLA. This represents clear evidence that the “excess V̇o₂” (11, 16), was reduced by $N{O}_3^{-}$ supplementation. The term excess V̇o₂ is usually utilized to indicate the progressive increase in V̇o₂ associated with the “slow component” of the V̇o₂ kinetics, that is the V̇o₂ in excess above that determined as the asymptote of the fundamental component. Because work rate is constant, this excess V̇o₂ indicates a reduced efficiency of muscle contraction. Considering the close interconnections between the loss of efficiency and fatigue, and ultimately exercise tolerance, during severe-intensity exercise (11, 16), it is not surprising that the lower slope of V̇o₂ as a function of time in BR was associated with (or was responsible for) a longer time to fatigue and an enhanced exercise tolerance, as demonstrated in previous works on healthy subjects (6, 29). Interestingly, in BR the time to exhaustion did not improve in 3 of 10 subjects, whereas it was remarkably higher in 5 of 10 subjects. This heterogeneity of results has been already reported by other authors both in healthy subjects (28) and in patients (43), and it has been shown to be correlated with changes of plasma [$N{O}_3^{-}$] or [$N{O}_2^{-}$] after supplementation. In our subjects, we did not observe any correlation between time to exhaustion and plasma [$N{O}_3^{-}$]. However, in BR the rate of V̇o₂ increase during severe-intensity exercise was reduced, suggesting less oxidative inefficiency, in all subjects.

The improved exercise tolerance of the present study confirms similar findings obtained in healthy subjects (2, 29) and in disease populations (8, 18, 42), such as patients with peripheral arterial disease (18), patients with chronic obstructive pulmonary diseases (8), and heart failure patients (43). Similar results have also been shown by different interventions aimed to increase NO bioavailability. For example, Sperandio et al. (35) demonstrated that sildenafil consumption in chronic heart failure patients improved muscle oxygenation and increased exercise capacity.

In the present study, dietary $N{O}_3^{-}$ supplementation did not affect physiological responses to moderate-intensity CWR exercise. In this exercise domain the O₂ cost of cycling was not significantly different in BR (12.9 ± 1.1 ml·min⁻¹·W⁻¹) versus PLA (13.3 ± 1.7 ml·min⁻¹·W⁻¹), being in both conditions substantially higher than the values usually observed in subjects with normal weight (~9–10 ml·min⁻¹·W⁻¹) (37). This confirms our previous data (30–32). Thus, in this exercise domain, $N{O}_3^{-}$ did not have any effect on the V̇o₂ kinetics. These results are in accordance to the results of some other studies (4, 6).

The lack of ergogenic effects of BR supplementation in the moderate intensity domain does not limit the relevance of the results of this study. The effects of BR supplementation during high-intensity exercise is indeed noteworthy, considering that the latter is considered a very effective intervention for weight reduction and improved health status in obesity (38).

The question could be raised if the $N{O}_3^{-}$ dose utilized in the present study was enough to induce significant changes in $N{O}_2^{-}$ and NO bioavailability. Whereas NO is too labile to be determined (13), in the $N{O}_3^{-}$ → $N{O}_2^{-}$ → NO pathway $N{O}_2^{-}$ would obviously be “closer” to the physiological relevant molecule (NO). Plasma [$N{O}_2^{-}$] could not be determined in the present study. Ample previous experimental evidence, however, demonstrates that following dietary $N{O}_2^{-}$ supplementation the increases in plasma [$N{O}_2^{-}$] and [$N{O}_2^{-}$] are closely correlated (13). In the present study, the daily $N{O}_3^{-}$ dose (5 mmol) was toward the low end of the efficacy spectrum (40, 41), particularly considering that we were treating patients with obesity with an elevated BM. The treatment, however, was continued for 6 days, i.e. for a period substantially longer than that adopted in previous “acute” supplementation studies (18, 25). The end result was that in all patients of the present study plasma [$N{O}_3^{-}$] substantially increased after the supplementation. On average the plasma [$N{O}_3^{-}$] increase was approximately sevenfold, reaching values quite similar to or even higher than those obtained in previous studies (13, 17) in which dietary $N{O}_3^{-}$ supplementation proved to be effective. Moreover, although in the present study plasma [$N{O}_3^{-}$] was inversely correlated with BM, no correlation was observed between plasma [$N{O}_3^{-}$] and the main outcome measure, the time to exhaustion during severe-intensity exercise.

Another issue we should take into consideration relates to the recruitment pattern of muscle fibers. It is known that additional fibers, mainly type 2 fibers, are recruited with time during heavy- and severe-intensity exercise in temporal association with the slow component of the V̇o₂ kinetics (16). Although it has been previously demonstrated that additional recruitment of fibers is not necessary for the development of the slow component (44), in several experimental conditions the slow component may be linked to a shift in fiber-type recruitment (15). BR supplementation seems to be more effective in type 2 compared with type 1 fibers (16). Indeed, the reduction of $N{O}_2^{-}$ to NO, mainly responsible of the ergogenic effects of $N{O}_3^{-}$ supplementation, seems to be augmented in conditions of low pH and Po₂, typical conditions associated with heavy- and severe-intensity exercise, in which type 2 fibers are predominantly activated. In rats running at 70% of V̇o_2peak blood flow was predominantly elevated in fast twitch muscle after BR ingestion (9). Thus, it is plausible that in presence of lower concentrations of NO, heavy- and severe-intensity domains of exercise could exacerbate tissue hypoxia within type 2 muscle fibers, resulting in a lower “metabolic stability” and fatigue development (10). Good metabolic stability during exercise is associated with smaller perturbations of the levels of metabolites related to muscle inefficiency and fatigue (10), such as decreases (compared with rest) in phosphocreatine, and increases in P_i, ADP-free, AMP-free, and IMP-free, etc. (10). Therefore, good metabolic stability is directly associated with a greater exercise tolerance. However, when intramuscular NO bioavailability is increased (as after $N{O}_3^{-}$ supplementation) the improved matching between O₂ delivery and O₂ uptake could improve “metabolic stability,” reduce metabolic inefficiency and fatigue, and improve enhance exercise tolerance (10).

Physical training of patients with obesity usually involves activities on cycle-ergometers because of the reduced load on hip, ankle, and knee joints compared with walking or running. However, cycling requires a lower metabolic demand compared with the other exercises, and this may limit the weight-loss process. Thus, future interventional studies to be carried out in patients with obesity should evaluate the ergogenic aid of dietary $N{O}_3^{-}$ supplementation during walking and running. The effects of dietary $N{O}_3^{-}$ supplementation on voluntary physical activities would also be an interesting topic of investigation. In this study, we did not evaluate the effects of $N{O}_3^{-}$ supplementation on blood pressure. Inorganic $N{O}_3^{-}$ and BR consumption are associated with a significant reduction in systolic and diastolic blood pressure (e.g., 2, 17); this effect would be particularly useful in subjects at high risk for cardiovascular diseases, such as and overweight patients or patients with obesity. Finally, in this study, we increased NO bioavailability by BR supplementation providing an amount of 100 kcal per day (proteins 4 g, carbohydrates 21 g, fats 0.1 g, fibers 0.6 g, and sodium 0.3 g). Although relatively small, this amount could become a problem during long-term supplementation in the obese population. Future studies should therefore consider other sources of $N{O}_3^{-}$ for chronic supplementations, such as nitrate-rich vegetables, as previously utilized in healthy subjects (28).

GRANTS

The work was partially supported by Progetti di Ricerca Corrente, Istituto Auxologico Italiano, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, Milan and Piancavallo (Project Reference Code: 01C407–2014; acronym: SUPNITESEROB) and by Progetto di interesse Invecchiamento CNR WP3.7.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.R., S.P., M.M., D.S., F.A., A.D.C., G.T., A.M.J., A.S., and B.G. conceived and designed research; L.R., S.P., D.S., A.V., F.A., A.D.C., and G.T. performed experiments; L.R., S.P., D.S., and A.V. analyzed data; L.R., S.P., M.M., D.S., A.V., A.M.J., A.S., and B.G. interpreted results of experiments; L.R. and S.P. prepared figures; L.R., S.P., and B.G. drafted manuscript; L.R., S.P., M.M., D.S., A.V., F.A., A.D.C., G.T., A.M.J., A.S., and B.G. edited and revised manuscript; L.R., S.P., M.M., D.S., A.V., F.A., A.D.C., G.T., A.M.J., A.S., and B.G. approved final version of manuscript.