EFFECTS OF ENHANCED NITRIC OXIDE BIOAVAILABILITY ON EXERCISE TOLERANCE IN DIFFERENT CONDITIONS

Nitric oxide (NO) is a signaling molecule that influences different aspects of the cellular homeostasis and regulates several physiological processes. It is normally produced endogenously starting from aminoacid L-arginine but recent evidences suggest that it can be also increased through the ingestion of food rich in inorganic nitrate (mainly green leafy vegetables and beetroot). Indeed, ingested inorganic nitrate (NO3-), after been absorbed, can be converted in the oral cavity to nitrite (NO2-) and be finally reduced into NO in the blood. This alternative NO3- - NO2- - NO pathway seems facilitated in condition of low O2 availability (ischemia and hypoxia) and low pH. In the last 10 years, several studies have been conducted to investigate the effects of dietary NO3- supplementation on skeletal muscle function, since NO regulates microvascular blood flow, muscle contractile proprieties, glucose homeostasis, intracellular calcium handling, and mitochondrial respiration. Dietary NO3- supplementation is likely to elicit a positive outcome when testing endurance exercise capacity in healthy subjects and enhance exercise tolerance in patients. However, there are still unresolved questions about the mechanisms utilized by inorganic NO3- to affect skeletal muscle functions, the possible potentiating effect of hypoxic conditions and the effectiveness of this intervention in other disease populations. My PhD projects tried to address some of these questions. The first study aimed to examine the effects of increased NO bioavailability on contraction economy. One of the most interesting effects of dietary NO3- supplementation is the reduction of muscle O2 consumption (V ̇O_2) at a given exercise intensity, since the V ̇O_2 requested to carry out a specific exercise is generally a fixed amount regardless of sex, age and training status of the subject involved in the exercise. A lower V ̇O_2 during moderate intensity exercise has been demonstrated both in healthy subjects and disease patients after dietary NO3- supplementation. The mechanistic basis is not clear yet, and this effect has been related to reduced ATP cost of contraction and/or an enhanced mitochondrial coupling efficiency. In our study, we evaluated skeletal muscle contraction economy following NO2- infusion in hypoxic condition. Contractions of the in-vivo isolated canine muscle were obtained by direct nerve stimulation. Muscle blood flow was kept constantly high by pump-perfusion. O2 consumption during exercise was assessed directly by Fick method. Mitochondrial respiration rates were evaluated by high-resolution respirometry from muscle biopsies. In hypoxic conditions, but in the presence of constant and normal convective O2 delivery, NO2- infusion did not affect canine skeletal muscle oxidative metabolism. These evidences suggest that the effects of increased NO availability on muscle contraction efficiency in hypoxia, if present, are likely not attributable to changes in mitochondrial respiratory efficiency. The second study aimed to investigate the effects of increased NO bioavailability on the physiological responses to exercise after prolonged permanence at altitude. Data from Tibetan population living at altitude from generations suggest that changes in NO bioavailability may contribute to hypoxia acclimatization and their enhanced exercise efficiency. Moreover, recent studies have demonstrated that NO3- supplementation can limit exercise impairment following exposure to acute hypoxia. However, it is not known if Caucasian subjects exposed to several days of hypobaric hypoxia can benefits from increased NO bioavailability. Thus, we investigated the ergogenic effects of dietary NO3- supplementation on exercise performance at different intensities during a prolonged permanence at altitude. Cycling and arm-cranking exercises were performed in order to test possible different effects of NO3- supplementation in relation to a different muscle fibers recruitment pattern. Dietary NO3- supplementation reduced O2 cost during moderate-intensity exercise both in cycling and arm-cranking. In cycle-ergometer exercise this effect was dependent from aerobic fitness level of the subjects, in accordance to previous results obtained in normoxia. Moreover, dietary NO3- supplementation enhanced severe-intensity exercise tolerance, suggesting that dietary NO3- supplementation can be a valid ergogenic aid to counteract exercise intolerance at altitude. Finally, in the third study we evaluated the possible ergogenic effects of dietary NO3- supplementation in obese subjects. Literature shows that NO3- can exert significant positive effects on exercise tolerance and, as a consequence, quality of life of patients with an impaired skeletal muscle oxidative metabolism such as chronic heart failure, chronic obstructive pulmonary diseases and peripheral arterial disease patients. Obese patients are characterized by a higher O2 cost of exercise, and therefore a reduced exercise tolerance during constant work-rate exercise compared with healthy subjects. We evaluated the effects of beetroot juice (BR, rich in NO3-) supplementation on the main physiological variables associated with exercise tolerance in obese adolescents. We observed a significant increase in plasma NO3- concentration after BR supplementation. The O2 cost of moderate-intensity exercise was not different in BR condition versus placebo, whereas, during severe-intensity exercise, signs of a reduced amplitude of the O2 uptake slow component were observed in BR, in association with a significantly longer time to exhaustion. Thus, exercise intolerance of obese adolescents, at least at severe-intensity, can be attenuated by short-term dietary NO3- supplementation. This intervention can be a useful aid to counteract early fatigue and reduced physical activity in this at-risk population. Overall, the studies carried out during my PhD extend the current knowledge about dietary NO3- supplementation on physiological responses to exercise, starting from a mechanistic investigation in isolated canine muscle up to the evaluation of the ergogenic benefits of this intervention on exercise tolerance in obese adolescents.