Tomas A. Schiffer

Dietary Inorganic Nitrate Improves Mitochondrial Efficiency in Humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

•  Dietary supplementation with inorganic nitrate has beneficial effects on skeletal muscle responses to exercise. •  Both mitochondrial and extra‐mitochondrial explanations have been proposed. •  Contractile force of fast‐twitch muscles was enhanced in mice supplemented with 1 mm NaNO3 in drinking water for 7 days. •  Myoplasmic free [Ca2+] during tetanic stimulation was increased in fast‐twitch muscles of nitrate‐supplemented mice and this was accompanied by increased expression of calsequestrin 1 and the dihydropyridine receptor. •  These results provide a new mechanism by which nitrate exerts beneficial effects on muscle function with applications to sports performance and a potential therapeutic role in conditions with muscle weakness.

Regulation of mitochondrial function and energetics by reactive nitrogen oxides

Endogenous nitric oxide (NO) generated from L-arginine by NO synthase regulates mitochondrial function by binding to cytochrome c oxidase in competition with oxygen. This interaction can elicit a variety of intracellular signaling events of both physiological and pathophysiological significance. Recent lines of research demonstrate that inorganic nitrate and nitrite, derived from oxidized NO or from the diet, are metabolized in vivo to form NO and other bioactive nitrogen oxides with intriguing effects on cellular energetics and cytoprotection. Here we discuss the latest advances in our understanding of the roles of nitrate, nitrite, and NO in the modulation of mitochondrial function, with a particular focus on dietary nitrate and exercise.

Mitochondrial oxygen affinity predicts basal metabolic rate in humans

The basal metabolic rate (BMR) is referred to as the minimal rate of metabolism required to support basic body functions. It is well known that individual BMR varies greatly, even when correcting for body weight, fat content, and thyroid hormone levels, but the mechanistic determinants of this phenomenon remain unknown. Here, we show in humans that mass‐related BMR correlates strongly to the mitochondrial oxygen affinity (p50mito; R2=0.66, P=0.0004) measured in isolated skeletal muscle mitochondria. A similar relationship was found for oxygen affinity and efficiency during constant‐load submaximal exercise (R2=0.46, P=0.007). In contrast, BMR did not correlate to overall mitochondrial density or to proton leak. Mechanistically, part of the p50mito seems to be controlled by the excess of cytochrome c oxidase (COX) protein and activity relative to other mitochondrial proteins. This is illustrated by the 5‐fold increase in p50mito after partial cyanide inhibition of COX at doses that do not affect maximal mitochondrial electron flux through the ETS. These data suggest that the interindividual variation in BMR in humans is primarily explained by differences in mitochondrial oxygen affinity. The implications of these findings are discussed in terms of a trade‐off between aerobic efficiency and power.—Larsen, F. J., Schiffer, T. A., Sahlin, K., Ekblom, B., Weitzberg, E., Lundberg, J. O. Mitochondrial oxygen affinity predicts basal metabolic rate in humans. FASEB J. 25, 2843‐2852 (2011). www.fasebj.org

Dietary nitrate reduces resting metabolic rate: a randomized, crossover study in humans

BACKGROUND Nitrate, which is an inorganic anion abundant in vegetables, increases the efficiency of isolated human mitochondria. Such an effect might be reflected in changes in the resting metabolic rate (RMR) and formation of reactive oxygen species. The bioactivation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species. OBJECTIVE We studied effects of inorganic nitrate, in amounts that represented a diet rich in vegetables, on the RMR in healthy volunteers. DESIGN In a randomized, double-blind, crossover study, we measured the RMR by using indirect calorimetry in 13 healthy volunteers after a 3-d dietary intervention with sodium nitrate (NaNO₃) or a placebo (NaCl). The nitrate dose (0.1 mmol · kg⁻¹ · d⁻¹) corresponded to the amount in 200-300 g spinach, beetroot, lettuce, or other vegetable that was rich in nitrate. Effects of direct nitrite exposure on cell respiration were studied in cultured human primary myotubes. RESULTS The RMR was 4.2% lower after nitrate compared with placebo administration, and the change correlated strongly to the degree of nitrate accumulation in saliva (r² = 0.71). The thyroid hormone status, insulin sensitivity, glucose uptake, plasma concentration of isoprostanes, and total antioxidant capacity were unaffected by nitrate. The administration of nitrite to human primary myotubes acutely inhibited respiration. CONCLUSIONS Dietary inorganic nitrate reduces the RMR. This effect may have implications for the regulation of metabolic function in health and disease.

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK−/− cardiomyocytes. Transmission electron microscopy of BK−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Effects of long-term dietary nitrate supplementation in mice

Background Inorganic nitrate (NO3-) is a precursor of nitric oxide (NO) in the body and a large number of short-term studies with dietary nitrate supplementation in animals and humans show beneficial effects on cardiovascular health, exercise efficiency, host defense and ischemia reperfusion injury. In contrast, there is a long withstanding concern regarding the putative adverse effects of chronic nitrate exposure related to cancer and adverse hormonal effects. To address these concerns we performed in mice, a physiological and biochemical multi-analysis on the effects of long-term dietary nitrate supplementation. Design 7 week-old C57BL/6 mice were put on a low-nitrate chow and at 20 weeks-old were treated with NaNO3 (1 mmol/L) or NaCl (1 mmol/L, control) in the drinking water. The groups were monitored for weight gain, food and water consumption, blood pressure, glucose metabolism, body composition and oxygen consumption until one group was reduced to eight animals due to death or illness. At that point remaining animals were sacrificed and blood and tissues were analyzed with respect to metabolism, cardiovascular function, inflammation, and oxidative stress. Results Animals were supplemented for 17 months before final sacrifice. Body composition, oxygen consumption, blood pressure, glucose tolerance were measured during the experiment, and vascular reactivity and muscle mitochondrial efficiency measured at the end of the experiment with no differences identified between groups. Nitrate supplementation was associated with improved insulin response, decreased plasma IL-10 and a trend towards improved survival. Conclusions Long term dietary nitrate in mice, at levels similar to the upper intake range in the western society, is not detrimental.

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55‐72%, resulting in inhibition of mitochondrial respiration by 50‐65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15‐72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6‐fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.—Larsen, F. J., Schiffer, T. A., Ørtenblad, N., Zinner, C., Morales‐Alamo, D., Willis, S. J., Calbet, J. A., Holmberg, H.‐C., Boushel, R. High‐intensity sprint training inhibits mitochondrial respiration through aconitase inactivation. FASEB J. 30, 417‐427 (2016). www.fasebj.org

Effects of dietary inorganic nitrate on static and dynamic breath-holding in humans

Inorganic nitrate has been shown to reduce oxygen cost during exercise. Since the nitrate-nitrite-NO pathway is facilitated during hypoxia, we investigated the effects of dietary nitrate on oxygen consumption and cardiovascular responses during apnea. These variables were measured in two randomized, double-blind, placebo-controlled, crossover protocols at rest and ergometer exercise in competitive breath-hold divers. Subjects held their breath for predetermined times along with maximum effort apneas after two separate 3-day periods with supplementation of potassium nitrate/placebo. In contrast to our hypothesis, nitrate supplementation led to lower arterial oxygen saturation (SaO(2), 77 ± 3%) compared to placebo (80 ± 2%) during static apnea, along with lower end-tidal fraction of oxygen (FETO(2)) after 4 min of apnea (nitrate 6.9 ± 0.4% vs. placebo 7.6 ± 0.4%). Maximum apnea duration was shorter after nitrate (329 ± 13 s) compared to placebo (344 ± 13 s). During cycle ergometry nitrate had no effect on SaO(2), FETO(2) or maximum apnea duration. The negative effects of inorganic nitrate during static apnea may be explained by an attenuated diving response.

Dietary nitrate markedly improves voluntary running in mice.

Nitrate supplementation is shown to increase submaximal force in human and mouse skeletal muscles. In this study, we test the hypothesis that the increased submaximal force induced by nitrate supplementation reduces the effort of submaximal voluntary running, resulting in increased running speed and distance. C57Bl/6N male mice were fed nitrate in the drinking water and housed with or without access to an in-cage running wheel. Nitrate supplementation in sedentary mice had no effect on endurance in a treadmill test, nor did it enhance mitochondrial function. However, after three weeks with in-cage running wheel, mice fed nitrate ran on average 20% faster and 30% further than controls (p<0.01). Compared to running controls, this resulted in ~13% improved endurance on a subsequent treadmill test (p<0.05) and increased mitochondrial oxidative capacity, as judged from a mean increase in citrate synthase activity of 14% (p<0.05). After six weeks with nitrate, the mice were running 58% longer distances per night. When nitrate supplementation was removed from the diet, the running distance and speed decreased to the control level, despite the improved endurance achieved during nitrate supplementation. In conclusion, low-frequency force improvement due to nitrate supplementation facilitates submaximal exercise such as voluntary running.