Mitochondrial efficiency : focus on dietary nitrate, hypoxia and exercise

Abstract: Metabolic efficiency affects weight control, generation of heat, exercise performance and health. The tiny radical nitric oxide (NO) targets several cellular components that can influence metabolic efficiency. NO is produced endogenously from L-arginine and molecular oxygen by specific NO-synthases. In addition, extensive research during the past two decades shows that the inorganic anions nitrate and nitrite, which are oxidation products from endogenous NO generation, can be reduced back to NO and other nitrogen oxides. Apart from reflecting endogenous NOS-activity, circulating nitrate and nitrite are dependent on dietary intake, where green leafy vegetables in particular contain high amounts of nitrate. Circulating nitrate is actively taken up by the salivary glands and excreted in saliva. Oral commensal bacteria then reduce salivary nitrate into nitrite, which after swallowing and effective uptake in the gut, reaches the systemic circulation. In blood and tissues several enzymatic and non-enzymatic pathways are able to reduce nitrite to NO. These pathways are potentiated under acidic and hypoxic conditions. The nitratenitrite- NO pathway is considered a back-up system during conditions when NO-synthases are failing. Prior to the inception of this thesis, our group had shown that dietary nitrate was able to decrease oxygen cost during exercise and we wanted to further explore its metabolic effects With this background, we investigated the underlying mechanisms behind the oxygen sparing effect of dietary nitrate during exercise (Paper I). Moreover, we explored the effects of dietary nitrate on muscular function in mice (Paper II), oxygen consumption in a human model of global hypoxia (Paper III) and on resting metabolic rate in humans (Paper IV). In the last two papers we wanted to investigate if metabolic efficiency can affect exercise tolerance in hypoxia (Paper V) and how cytochrome c oxidase (COX) subunit IV isoform composition affects resting metabolic rate (Paper VI). Respirometric analysis of isolated mitochondria from healthy humans revealed that dietary nitrate improves mitochondrial efficiency (P/O ratio) and this effect correlated strongly with the reduction in oxygen consumption during cycling ergometry. In addition we found respirometric support for less uncoupling which was supported by reduced expression of adenine nucleotide transporter (ANT), a major determinant of proton conductance (Paper I). In muscle from mice fed for 7 days with nitrate, electric stimulation led to increased contractile force and speed of force development in fast twitch muscle compared to controls. This was accompanied by higher Ca2+ levels and increased expression of the Ca2+- handling proteins dihydropyridine receptor and calsequestrin-1 (Paper II). In the human model of global hypoxia a reduction in arterial oxygen saturation was achieved during prolonged breath-holding by experienced free divers after nitrate or placebo. In contrast to our hypothesis, nitrate during resting apnea increased pulmonary oxygen uptake, reduced arterial oxygen saturation and shortened maximal breathholding time. This was probably related to a NO-mediated attenuation of the oxygen conserving diving response as showed by less bradycardia and indications of an attenuation of the increase in blood pressure after nitrate (Paper III). In healthy humans we could demonstrate that dietary nitrate reduces resting metabolic rate by 4% and that acute administration of nitrite in vitro reduces respiration by 40% in primary myotubes from the same individuals (Paper IV). We found that healthy subjects with a high metabolic efficiency in normoxia had higher tolerance to exercise in hypoxia. Interestingly, these subjects acutely reduced their metabolic efficiency during hypoxia in order to maintain power output. On the other hand, isolated mitochondria, which work in the lower efficiency range, acutely increased their efficiency during a steady state hypoxic challenge in order to maintain ATP production (Paper V). There is a largely unexplained variation in resting metabolic rate between seemingly similar individuals. We found that the inter-individual variation in resting metabolic rate seems to depend on the composition of COX subunit IV isoforms. We could show that COX IV-2 isoform is present in human skeletal muscle and that a high COX IV-2/COX IV-1 ratio showed a strong negative correlation to resting metabolic rate. Moreover, concurrent overexpression of COX IV-2 and knock down of COX IV-1 in primary human myotubes significantly reduced basal cell respiration and ROS generation without affecting the COX activity (Paper VI). In conclusion, this thesis demonstrates profound effects of dietary nitrate on mitochondrial efficiency, muscle function and metabolism. In addition, metabolic efficiency plays a role in exercise tolerance during hypoxia and adapts to obtain optimal power. Finally, mitochondrial COX subunit IV isoform composition seems to affect resting metabolic rate. The physiological, therapeutic and nutritional aspects of these findings create a platform for further studies on dietary nitrate, mitochondrial function and metabolism.