Eight weeks of inorganic nitrate/nitrite supplementation improves aerobic exercise capacity and the gas exchange threshold in patients with type 2 diabetes

Abstract

Patients with type 2 diabetes mellitus (T2DM) have reduced exercise capacity, indexed by lower maximal oxygen consumption (V̇o_2max) and achievement of the gas exchange threshold (GET) at a lower % V̇o_2max. The ubiquitous signaling molecule nitric oxide (NO) plays a multifaceted role during exercise and, as patients with T2DM have poor endogenous NO production, we investigated if inorganic nitrate/nitrite supplementation (an exogenous source of NO) improves exercise capacity in patients with T2DM. Thirty-six patients with T2DM (10F, 59 ± 9 yr, 32.0 ± 5.1 kg/m², HbA1c = 7.4 ± 1.4%) consumed beetroot juice containing either inorganic nitrate/nitrite (4.03 mmol/0.29 mmol) or a placebo (0.8 mmol/0.00 mmol) for 8 wk. A maximal exercise test was completed before and after both interventions. V̇o_2max was determined by averaging 15-s data, whereas the GET was identified using the V-slope method and breath-by-breath data. Inorganic nitrate/nitrite increased both absolute (1.96 ± 0.67 to 2.07 ± 0.75 L/min) and relative (20.7 ± 7.0 to 21.9 ± 7.4 mL/kg/min, P < 0.05 for both) V̇o_2max, whereas no changes were observed following placebo (1.94 ± 0.40 to 1.90 ± 0.39 L/min, P = 0.33; 20.0 ± 4.2 to 19.7 ± 4.6 mL/kg/min, P = 0.39). Maximal workload was also increased following inorganic nitrate/nitrite supplementation (134 ± 47 to 140 ± 51 W, P < 0.05) but not placebo (138 ± 32 to 138 ± 32 W, P = 0.98). V̇o₂ at the GET (1.11 ± 0.27 to 1.27 ± 0.38L/min) and the %V̇o_2max in which GET occurred (56 ± 8 to 61 ± 7%, P < 0.05 for both) increased following inorganic nitrate/nitrite supplementation but not placebo (1.10 ± 0.23 to 1.08 ± 0.21 L/min, P = 0.60; 57 ± 9 to 57 ± 8%, P = 0.90) although the workload at GET did not achieve statistical significance (group-by-time P = 0.06). Combined inorganic nitrate/nitrite consumption improves exercise capacity, maximal workload, and promotes a rightward shift in the GET in patients with T2DM. This manuscript reports data from a registered Clinical Trial at ClinicalTrials.gov ID: NCT02804932.

NEW & NOTEWORTHY We report that increasing nitric oxide bioavailability via 8 wk of inorganic nitrate/nitrite supplementation improves maximal aerobic exercise capacity in patients with type 2 diabetes mellitus. Similarly, we observed a rightward shift in the gas exchange threshold. Taken together, these data indicate inorganic nitrate/nitrite may serve as a means to improve fitness in patients with type 2 diabetes mellitus.

INTRODUCTION

Aerobic exercise capacity (V̇o_2max) is a well-established, independent predictor of major adverse cardiovascular events in patients with type 2 diabetes mellitus (T2DM) (1). Alternatively, the gas exchange threshold (GET) is an index of aerobic fitness commonly used in clinical populations (2) that also has prognostic utility (3). Exercise capacity is influenced by several physiological systems with the myocardium, vasculature, and skeletal muscle being considered principal determinants (4). Indeed, patients with T2DM have impairments in each of these systems that contribute to their characteristically low exercise capacity (5). Numerous signaling pathways are involved with exercise (6), although the ubiquitously produced free radical nitric oxide (NO) has multimodal effects (7–9). As patients with T2DM have low levels of NO (10), augmenting their NO bioavailability may lead to commensurate improvements in exercise capacity.

Nearly 15 years ago, dietary inorganic nitrate was reported to increase circulating markers of NO bioavailability (11). Since this seminal discovery, the enterosalivary network responsible for inorganic nitrate-mediated increases in NO bioavailability has been described extensively (12). The effects of inorganic nitrate on exercise capacity have also been assessed in both healthy and clinical populations, with generally positive results, particularly in those with low endogenous NO production (13, 14); however, patients with T2DM are relatively understudied in this field. We recently reported that combined inorganic nitrate/nitrite supplementation increases blood flow to contracting skeletal muscle in patients with T2DM (15) although whether this translates to improved exercise capacity remains unknown. Thus, we tested the hypothesis that V̇o_2max and the GET would increase in patients with T2DM following 8 wk of inorganic nitrate/nitrite supplementation. As improvements in exercise capacity have been paralleled by commensurate change in glucose homeostasis (16), we also studied patients’ glycosylated hemoglobin (HbA1c) in addition to glucose and insulin in a fasted state as well as in response to an oral glucose challenge. Although preclinical models of T2DM report inorganic nitrate can improve glycemic management (17–19), this does not appear to translate to humans (20, 21); thus, we did not hypothesize any changes would occur in our cohort.

METHODS

Subjects

Forty-one patients with T2DM were recruited into the study as outlined previously (15, 22). Prior to data collection, all patients provided written informed consent for participation in this clinical trial (ClinicalTrials.gov ID: NCT02804932), which was approved by the University of Iowa’s Institutional Board and conformed to the 1964 Declaration of Helsinki along with its later amendments. All patients were ≥40 yr, <42 kg/m², abstained from tobacco use for >1 year, free of symptomatic coronary artery disease, and did not have diagnosed heart failure, renal impairments, and/or hypotension. Four patients did not complete study procedures due to scheduling difficulties or health concerns unrelated to the trial. Only one female patient was premenopausal and used a long-acting, implantable contraceptive; all postmenopausal females were not prescribed hormone therapy.

Experimental Overview

After consenting, patients were scheduled for presupplementation testing that consisted of a maximal exercise test and venous blood sampling, among other procedures described elsewhere (15, 22), which were repeated following 8 wk of daily inorganic nitrate/nitrite or placebo supplementation. Patients completed three visits before and then following their respective intervention (6 visits total). One day consisted of a maximal exercise test (in the afternoon, not fasted, with medications), another for an oral glucose tolerance test and biopsy of the vastus lateralis (in the morning, fasted, without medications), with the third to study the regulation of blood flow to contracting skeletal muscle (in the morning, fasted, without medications). All visits, on their respective side of the intervention, were completed within 3–7 days of one another. Patients were instructed to consume a light meal within 2 h of maximal exercise tests to reduce the risk of hypoglycemia and/or nausea and to refrain from strenuous activities and/or exercise on the day of testing. During the 48 h preceding venous blood sampling, patients were instructed to adhere to a low nitrate/nitrite diet (e.g., minimal to no green leafy vegetables or processed meat consumption). In addition, patients were asked to abstain from alcohol, caffeine, and vitamin/supplement consumption 24 h before fasting blood collection. Importantly, patients consumed their final interventional supplement the day before blood collection.

Maximal Exercise Testing

Patients completed their maximal exercise test on a cycle ergometer (Corival, Lode BV, The Netherlands) until volitional exhaustion. Prior to each test, expired gas analyzers were calibrated using compressed gas (16%O₂, 4%CO₂) and room air (∼21%O₂, ∼0.04%CO₂) with the pneumotachometer being calibrated via 3-L syringe over a spectrum of flow rates (3–300 L/min; TrueOne 2400, ParvoMedics Inc., Salt Lake City, UT). Calibrations were repeated until internal validation criteria were met. Heart rate and rhythm were continuously monitored using a 12-lead electrocardiogram by a board-certified physician. Blood pressure measurements were taken for safety but were not systematically recorded and are subsequently not reported in this manuscript. The testing protocol was identical for all patients and began with a 60-s rest period followed by 60 s of unloaded (0 W, warm-up) cycling that increased to 20 W for 60 s then 10 W/min until the test concluded; patients were encouraged to maintain a cadence of ≥60 rpm during their warm-up and throughout the test. Maximal effort was defined as achieving two or more of the following criteria: a respiratory exchange ratio (RER) ≥1.10, a rating of perceived exertion ≥18, 90% of age-predicted maximal heart rate estimated as 220 age. All patients prescribed a β-antagonist (n = 8) achieved both the RER and perceived exertion criteria during their exercise tests. V̇o_2max was defined as the mean of two or three 15-s average data points during the final minute of exercise. The GET was identified using the ratio of breath-by-breath carbon dioxide production (V̇co₂) to oxygen consumption (V̇o₂) data by four observers (2). All observers were blinded to the patient’s identity, time point (pre- vs. postsupplementation), and group randomization (nitrate/nitrite vs. placebo). The average coefficient of variation for all tests among observers was 4.7 ± 2.7% (0.0%–9.7%). After initial evaluations were complete, each observer re-identified the GET on 12 exercise tests resulting in intraobserver coefficients of variation averaging 6.6 ± 1.7% (4.1%–7.6%).

Blood Collection and Analyses

Chemiluminescence assays were used to quantify plasma nitrate and nitrate concentrations as detailed previously (23). Briefly, fasting samples were collected, centrifuged, with plasma being aliquoted and frozen at −80°C. Samples were analyzed within 30 min of thawing by their addition to vanadium(III) chloride in hydrochloric acid at 90°C (nitrate) and potassium iodide in room-temperature acetic acid (nitrite) using a nitric oxide analyzer (NOA 280i, Sievers Instruments, Boulder, CO). Whole venous blood samples were obtained while patients were in a fasted state before consuming a beverage containing 75 g of glucose. Additional blood samples were collected at 60 min and 120 min after consumption of the glucose-containing beverage to estimate insulin sensitivity. Area under the curve (AUC) was calculated for both glucose and insulin using the trapezoid equation. The Matsuda index (24) was also calculated. Whole blood samples collected for clinical measures (e.g., HbA1c) were analyzed using standard procedures by the University of Iowa Hospitals and Clinics.

Interventions

As described previously (15, 22), patients were assigned in a randomized, double-blinded parallel fashion to consume either beetroot juice containing 4.03 mmol of nitrate and 0.29 mmol of nitrite or ∼0.08 mmol of nitrate with no nitrite (placebo) once daily for 8 wk (Superbeets, HumanN, Inc., Austin, TX). Patients made their supplements daily by dissolving beetroot powder into water. The nitrate and nitrite content of both supplements was verified via high-performance liquid chromatography before the start of data collection.

Statistical Analyses

Data are presented as means ± standard deviation throughout the manuscript unless otherwise noted. Group data were compared using mixed-model, two-way repeated-measures analyses of variance with group (nitrate/nitrite, placebo) and time (pre, post) effects. If significant F-ratios were detected, post hoc comparisons were made using Tukey’s test. All statistical comparisons were made using SigmaPlot (Systat Software Inc., San Jose, CA) with an a priori significance level set at α < 0.05.

RESULTS

Study Population

Demographical data (e.g., body mass index, medication use) have been reported elsewhere (15). There were no between-group differences in pre- or postsupplementation age, body mass index, years with T2DM, prescription medications, nor comorbidities. In our prior publication, we reported inorganic nitrate/nitrite supplementation reduced systolic and mean arterial pressure but not diastolic blood pressure or heart rate; no changes were observed following placebo (22). Due to technical errors during exercise data extraction, V̇o_2max results are reported in 36 patients while GET data reflects 33 patients. There were no changes in body mass during either the inorganic nitrate (94.5 ± 15.3 to 95.0 ± 15.3 kg) or placebo (97.2 ± 17.4 to 97.0 ± 17.1 kg; beverage-by-time P = 0.25) interventions. In addition, subjects did not report any changes in medication use during either intervention.

Exercise Capacity

Following inorganic nitrate/nitrite supplementation, both absolute (Fig. 1A) and relative (Fig. 1B) V̇o_2max increased (P < 0.05), whereas no changes were observed following placebo (P = 0.31–0.38). The absolute V̇o₂ at GET increased following inorganic nitrate/nitrite supplementation (P < 0.05) but not placebo (P = 0.60, Fig. 2A). Similarly, the GET occurred at a higher %V̇o_2max after inorganic nitrate/nitrite supplementation (P < 0.05) but was unchanged following placebo (P = 0.90, Fig. 2B). Cardiopulmonary data and workload at both V̇o_2max and the GET are reported in Table 1. Inorganic nitrate/nitrite supplementation increased minute ventilation and workload at V̇o_2max relative to presupplementation values (P < 0.05 for both) with no change observed following placebo (P = 0.58 and 0.98, respectively). No other cardiopulmonary data changed at V̇o_2max following either intervention (group-by-time P = 0.05–0.99). Minute ventilation, V̇co₂, and heart rate at GET increased following inorganic nitrate/nitrite (P < 0.05 for all) but not placebo (P = 0.66, 0.45, and 0.62, respectively) with no other cardiopulmonary data achieving statistical significance (group-by-time P = 0.06–0.99).

Figure 1.

The absolute (A) and relative (B) maximal volume of oxygen consumption (V̇o_2max) following inorganic nitrate/nitrite as well as placebo supplementation. Group data are shown as circles (means ± standard error). Individual responses are shown as triangles with data from males indicated by filled shapes, whereas female data are empty. *P < 0.05 vs. Pre. Data were compared using a two-way repeated measures analysis of variance.

Figure 2.

The volume of oxygen consumption (V̇o₂) at the gas exchange threshold (GET, A) and percent (%) of maximal V̇o₂ (V̇o_2max) at which GET occurred (B) following inorganic nitrate/nitrite and placebo supplementation. Group data are shown as circles (means ± standard error). Individual responses are shown as triangles with data from males indicated by filled shapes, whereas female data are empty. *P < 0.05 vs. Pre. Data were compared using a two-way analysis of variance.

Table 1.

Data at maximal exercise capacity and gas exchange threshold

	Nitrate/Nitrite		Placebo		Two-Way RMANOVA
	Pre	Post	Pre	Post	Interaction	Supplement	Time
V̇o2max	n = 18		n = 18
Time, min	13.4 ± 4.7	13.7 ± 5.1	13.5 ± 2.9	13.5 ± 3.1	0.22	0.96	0.29
V̇co2, L/min	2.35 ± 0.79	2.36 ± 0.88	2.23 ± 0.48	2.16 ± 0.47	0.54	0.47	0.58
$\text{F}{\text{E}}_{\text{C}{\text{O}}_2}$, %	3.8 ± 0.4	3.7 ± 0.5	3.6 ± 0.5	3.6 ± 0.5	0.97	0.37	0.12
V̇e, L/min	75.7 ± 23.4	80.4 ± 27.5*	78.3 ± 20.2	77.3 ± 21.8	<0.05	0.98	0.19
RR, breaths/min	36 ± 6	37 ± 7	38 ± 6	38 ± 7	0.66	0.34	0.38
VT, L	2.10 ± 0.59	2.19 ± 0.68	2.06 ± 0.41	2.01 ± 0.38	0.06	0.51	0.64
$\text{F}{\text{E}}_{{\text{O}}_2}$, %	17.6 ± 0.5	17.6 ± 0.5	17.7 ± 0.6	17.7 ± 0.5	0.83	0.51	0.39
RER	1.16 ± 0.06	1.14 ± 0.06	1.15 ± 0.08	1.14 ± 0.10	0.73	0.72	0.13
HR, beats/min	152 ± 18	155 ± 15	153 ± 16	150 ± 18	0.05	0.64	0.99
Workload, W	134 ± 47	140 ± 51*	138 ± 32	138 ± 32	<0.05	0.97	<0.05
RPE	17 ± 3	18 ± 2	18 ± 2	18 ± 2	0.13	0.47	0.25
GET	n = 16		n = 17
Time, min	6.6 ± 2.6	7.6 ± 3.3	7.0 ± 2.2	6.7 ± 1.9	0.09	0.76	0.44
V̇co2, L/min	1.02 ± 0.29	1.17 ± 0.37*	1.00 ± 0.25	0.96 ± 0.19	<0.05	0.22	0.18
$\text{F}{\text{E}}_{\text{C}{\text{O}}_2}$, %	4.2 ± 0.5	4.2 ± 0.6	4.0 ± 0.5	3.9 ± 0.4	0.26	0.12	0.95
V̇e, L/min	30.5 ± 6.3	34.7 ± 8.8*	31.6 ± 6.5	31.0 ± 5.8	<0.05	0.58	0.07
RR, breaths/min	21 ± 4	22 ± 5	22 ± 5	23 ± 5	0.99	0.42	0.16
VT, L	1.55 ± 0.47	1.67 ± 0.55	1.50 ± 0.39	1.39 ± 0.29	0.07	0.24	0.96
$\text{F}{\text{E}}_{{\text{O}}_2}$, %	16.5 ± 0.5	16.5 ± 0.7	16.7 ± 0.5	16.7 ± 0.6	0.99	0.25	0.89
RER	0.91 ± 0.07	0.93 ± 0.08	0.91 ± 0.07	0.89 ± 0.05	0.25	0.37	0.97
HR, beats/min	108 ± 8	120 ± 14*†	113 ± 14	112 ± 15	<0.05	0.78	<0.05
Workload, W	61 ± 25	70 ± 34	65 ± 22	59 ± 20	0.06	0.72	0.72

Data are shown as means ± standard deviation. $\text{F}{\text{E}}_{\text{C}{\text{O}}_2}$, fractional expired carbon dioxide; $\text{F}{\text{E}}_{{\text{O}}_2}$, fractional expired oxygen; GET, gas exchange threshold; HR heart rate; RER, respiratory exchange ratio; RPE, rate of perceived exertion; RR, respiratory rate; V̇e, minute ventilation; V̇o_2max, maximal oxygen consumption; V̇co₂, volume of carbon dioxide production.

^*P < 0.05 vs. pre nitrate/nitrite; †P < 0.05 vs. post placebo.

Blood Chemistry

As reported elsewhere, inorganic nitrate/nitrite supplementation improved NO bioavailability as assessed via plasma nitrate and nitrite concentrations in this group of subjects (15). Fasting blood data, along with responses to the oral glucose challenge, before and following both interventions are shown in Table 2. There were no between-group differences observed for any of these data (group-by-time P = 0.12–0.98). One patient in the placebo group did not complete a preintervention glucose tolerance test; thus, these data reflect 17 subjects.

Table 2.

Clinical blood chemistry results

	Nitrate/Nitrite (n = 18)		Placebo (n = 17)		Two-Way RMANOVA
	Pre	Post	Pre	Post	Interaction	Supplement	Time
HbA1c	7.4 ± 1.4	7.4 ± 1.3	7.3 ± 1.4	7.3 ± 1.5	0.70	0.75	0.91
Glucose, mg/dL
Fasting	166 ± 50	170 ± 64	159 ± 40	160 ± 46	0.69	0.41	0.60
60 min	298 ± 69	308 ± 83	308 ± 70	311 ± 74	0.78	0.79	0.51
120 min	300 ± 97	295 ± 84	285 ± 72	279 ± 87	0.60	0.54	0.76
AUC	204 ± 75	200 ± 59	212 ± 72	211 ± 71	0.95	0.42	0.94
Insulin, mIU/L
Fasting	19.7 ± 14.3	21.2 ± 17.0	21.7 ± 14.7	23.5 ± 16.3	0.98	0.57	0.33
60 min	66.9 ± 66.0	59.1 ± 53.4	60.1 ± 32.8	63.9 ± 41.0	0.14	0.90	0.61
120 min	66.9 ± 64.5	61.4 ± 45.9	66.2 ± 43.2	66.3 ± 38.7	0.29	0.84	0.30
AUC	46.3 ± 60.2	39.1 ± 44.1	41.4 ± 26.7	41.6 ± 27.9	0.12	0.99	0.14
Matsuda Index	2.2 ± 1.3	2.3 ± 1.7	2.0 ± 1.4	1.9 ± 1.2	0.18	0.53	0.69

Data are shown as means ± standard deviation. AUC, area under the curve; HbA1c, glycosylated hemoglobin.

DISCUSSION

Data from the present study are the first to report 8 wk of inorganic nitrate/nitrite supplementation results in modest, but statistically significant, improvements in V̇o_2max (Fig. 1), which was accompanied by greater minute ventilation and maximal workload (Table 1), in patients with T2DM. We also observed a higher V̇o₂ at the GET (Fig. 2) coupled with higher V̇co₂, minute ventilation, and heart rate (Table 1). Although increases in exercise capacity can be paralleled by improved glucose regulation in this population (16), we did not observe changes in HbA1c, fasting blood glucose, or insulin nor patients’ responses to an oral glucose challenge (Table 2) supporting previous works (20, 21, 25). Taken together, our study reports that 8 wk of inorganic nitrate/nitrite supplementation improves maximal oxygen uptake, and promotes a rightward shift in the GET, in patients with T2DM.

Exercise is frequently prescribed to patients with T2DM due to the systemic benefits observed after a relatively short time span (weeks). In a meta-analysis, Boulé et al. (16) reported that patients with T2DM who completed an aerobic exercise training intervention for 8 wk or more demonstrated an 11.8% in V̇o_2max, which shared a stronger relationship to exercise intensity compared with exercise volume. Although studies indicate that training at higher exercise intensities elicits greater improvements in V̇o_2max (26), perceived intensity is a barrier to exercise adherence in patients with T2DM (27). To this point, data in Fig. 1 suggest that approximately half of the increase in V̇o_2max with higher-intensity exercise training can be obtained via inorganic nitrate/nitrite supplementation in patients with T2DM. It could also be postulated that inorganic nitrate/nitrite supplementation, with concomitant exercise training, would lead to more robust improvements in V̇o_2max relative to either intervention alone. Work from Santana et al. (28) supports this hypothesis whereby recreational runners who consumed inorganic nitrate (12.0 mmol) and completed a blended training protocol (sprint and distance running) improved performance on a 10-km time trial after 4 wk relative to exercise training with a placebo. As V̇o_2max was likely higher in subjects from Santana’s project (28) relative to the patients in our study, and that inorganic nitrate/nitrite provides greater benefits in populations with low NO bioavailability (12), patients with T2DM who complete a combined exercise/nitrate regimen would likely demonstrate a more significant increase in aerobic exercise capacity relative to either intervention independently. However, not all studies, particularly those in clinical populations (29), support this hypothesis.

As V̇o_2max is a composite measure guided by the synchronization of several physiological systems, identifying the mechanism(s) responsible for the findings shown in Fig. 1 is challenging. Recent data from the Shah group (30) indicates that both local (intracoronary) and systemic (intravenous) nitrite infusion improves left ventricular diastolic performance in patients undergoing an angiogram with normal systolic function. Importantly, left ventricular diastolic function predicts exercise capacity independent of demographical factors (31). Although these data were collected under resting conditions and over a short period of time (≤10 min) (30), they support the notion that prolonged inorganic nitrate/nitrite could have increased V̇o_2max in our cohort via improved myocardial performance. In addition to a central effect on the cardiovascular system, we have previously reported that inorganic nitrate/nitrite supplementation improves perfusion to contracting skeletal muscle (15), which is reduced in patients with T2DM (32–34). Taken together, these data suggest that increasing NO bioavailability improves both central and peripheral components of the cardiovascular system that likely contributed to our findings shown in Fig. 1.

Increased oxygen delivery to match metabolic demand is among the most fundamental determinants of V̇o_2max. However, there is compelling evidence that the GET is not a function of low PO₂, and augmenting blood flow (i.e., O₂ delivery) does not directly influence the GET per se (2). Rather, increasing blood flow to exercising skeletal muscle can result in greater lactate clearance, particularly during higher flow rates such as those observed during near maximal exertion (35), and may subsequently promote a rightward shift in the GET during whole body exercise. Along these lines, seminal studies show lactate is principally released as CO₂ (36) and increases ventilation during exercise via activation of the carotid bodies (37). Interestingly, we have reported that both acute (38) and prolonged inorganic nitrate/nitrite supplementation (23) reduces the hypoxic ventilatory response (i.e., carotid chemoreflex sensitivity) suggesting that minute ventilation at the GET should have been attenuated (if changed at all); rather, Table 1 reports a 14% increase. Although this finding can be interpreted as a second pathway for inorganic nitrate/nitrite to improve the buffering capacity for lactate (in addition to greater clearance), the mechanism of action appears counterintuitive. One potential explanation for this observation could be upregulation of monocarboxylate transporters (MCTs) on the surface of glomus cells responsible for introducing lactate into the carotid body (39). Indeed, T2DM is associated with dysfunction of MCTs (40) and preclinical data demonstrate sildenafil (a selective phosphodiesterase-5 inhibitor) augments exercise-induced MCT expression within skeletal muscle (41). Thus, inorganic nitrate/nitrite may have increased glomus cell responsiveness to lactate, which could manifest as greater minute ventilation at the GET and V̇o_2max. Regardless of the mechanism(s), we demonstrate that 8 wk of inorganic nitrate/nitrite supplementation improves the GET in patients with T2DM which may be a function of increased lactate clearance and/or ventilation.

Although NO is typically discussed within the cardiovascular domain, poor endogenous NO production is linked to metabolic disturbances in humans (42) suggesting inorganic nitrate/nitrite may improve glucose homeostasis in patients with T2DM. Data in eNOS^−/− mice reported beneficial effects of inorganic nitrate supplementation on body weight, visceral adiposity, triglycerides, fasting glucose, and HbA1c (17), which were later corroborated by studies using a T2DM model of Wistar rats (19). More recently, Cordero-Herrera et al. (18) found that the effects of metformin, the frontline treatment for T2DM, on insulin sensitivity and glucose homeostasis in mice with cardiometabolic disease were comparable with those observed with inorganic nitrate. Despite these promising preclinical studies, our data (Table 2), along with prior studies (20, 21, 25), unfortunately, indicate that these results do not translate to patients with T2DM. A distinguishing factor between preclinical and translational studies is the confounding pharmacotherapies in patients that are often excluded in rodent trials. That is, drugs used to treat T2DM may abolish the benefits of inorganic nitrate/nitrite on metabolic homeostasis as proposed by Bahadoran et al. (20); indeed, combined metformin-nitrate treatment did not improve metabolic outcomes to a greater extent than either intervention independently (18).

Despite the novelty of our findings, we recognize the following experimental limitations to this study. First, our active supplement contained both inorganic nitrate and nitrite; thus, we cannot ascertain whether the beneficial effects reported in this manuscript are attributable to one anion or the other. Regarding the experimental design, neither physical activity nor dietary patterns were assessed during this trial. It is therefore possible that changes in patients’ lifestyles may have influenced our findings. Similarly, the pre-exercise meal was not standardized for nutritional content nor the time of consumption. Thus, it is possible acute alterations in circulating substrate or NO bioavailability may have influenced our findings. Importantly, our previously reported data (15) suggests that patients assigned to the placebo were not partaking in a nitrate-rich diet as NO bioavailability did not differ between groups at baseline nor increase following the intervention. Although we previously reported resting blood pressure was reduced in this cohort (22), blood pressure was not assessed at regular intervals during exercise or at maximal work rate. Accordingly, a reduction in afterload at or near maximal workload may have contributed to the improved V̇o_2max observed following inorganic nitrate supplementation. Along these lines, inorganic nitrate/nitrite supplementation reduces the pressor response to skeletal muscle metaboreflex activation (43), which is exaggerated with T2DM (44). Therefore, attenuation of the skeletal muscle metaboreflex may have contributed to our findings shown in Fig. 1. Finally, submaximal exercise tolerance, either at the GET or an absolute workload, was not assessed in our study limiting our insight into the effects of inorganic nitrate/nitrite on critical speed and power in patients with T2DM.

Conclusions

Our study reports that both V̇o_2max (Fig. 1) and the GET (Fig. 2) are improved in patients with T2DM following 8 wk of inorganic nitrate/nitrite supplementation. The former was accompanied by increases in minute ventilation and workload, whereas the latter corresponded to increased minute ventilation, V̇co₂, and heart rate (Table 1). Clinically, Nylen et al. (45) found an increase in V̇o_2max of one metabolic equivalent (MET) corresponding to a 23% reduction in all-cause mortality over 7.8 years for patients with T2DM. Whereas a 0.33 MET increase was observed in the present study, the clinical significance of this finding is unclear. Despite these benefits, we did not observe concomitant changes in markers of glycemic management (Table 2). These data corroborate several studies reporting on the ergonomic effects of inorganic nitrate/nitrite supplementation (14) as well as two prior trials reporting no effect on glucose regulation (21, 25).

GRANTS

This study was supported by the American Diabetes Association 1-16-1CTS-015 (to D. P. Casey) with Core ESR facilities supported by the National Institutes of Health P30 CA086862.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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