Filip J. Larsen

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.

Effects of dietary nitrate on oxygen cost during exercise.

Aim:  Nitric oxide (NO), synthesized from l‐arginine by NO synthases, plays a role in adaptation to physical exercise by modulating blood flow, muscular contraction and glucose uptake and in the control of cellular respiration. Recent studies show that NO can be formed in vivo also from the reduction of inorganic nitrate (NO3−) and nitrite (NO2−). The diet constitutes a major source of nitrate, and vegetables are particularly rich in this anion. The aim of this study was to investigate if dietary nitrate had any effect on metabolic and circulatory parameters during exercise.

Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice

The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate–nitrite–NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.

Dietary nitrate reduces maximal oxygen consumption while maintaining work performance in maximal exercise.

The anion nitrate-abundant in our diet-has recently emerged as a major pool of nitric oxide (NO) synthase-independent NO production. Nitrate is reduced stepwise in vivo to nitrite and then NO and possibly other bioactive nitrogen oxides. This reductive pathway is enhanced during low oxygen tension and acidosis. A recent study shows a reduction in oxygen consumption during submaximal exercise attributable to dietary nitrate. We went on to study the effects of dietary nitrate on various physiological and biochemical parameters during maximal exercise. Nine healthy, nonsmoking volunteers (age 30+/-2.3 years, VO(2max) 3.72+/-0.33 L/min) participated in this study, which had a randomized, double-blind crossover design. Subjects received dietary supplementation with sodium nitrate (0.1 mmol/kg/day) or placebo (NaCl) for 2 days before the test. This dose corresponds to the amount found in 100-300 g of a nitrate-rich vegetable such as spinach or beetroot. The maximal exercise tests consisted of an incremental exercise to exhaustion with combined arm and leg cranking on two separate ergometers. Dietary nitrate reduced VO(2max) from 3.72+/-0.33 to 3.62+/-0.31 L/min, P<0.05. Despite the reduction in VO(2max) the time to exhaustion trended to an increase after nitrate supplementation (524+/-31 vs 563+/-30 s, P=0.13). There was a correlation between the change in time to exhaustion and the change in VO(2max) (R(2)=0.47, P=0.04). A moderate dietary dose of nitrate significantly reduces VO(2max) during maximal exercise using a large active muscle mass. This reduction occurred with a trend toward increased time to exhaustion implying that two separate mechanisms are involved: one that reduces VO(2max) and another that improves the energetic function of the working muscles.

Roles of dietary inorganic nitrate in cardiovascular health and disease

Inorganic nitrate from dietary and endogenous sources is emerging as a substrate for in vivo generation of nitric oxide (NO) and other reactive nitrogen oxides. Dietary amounts of nitrate clearly have robust NO-like effects in humans, including blood pressure reduction, inhibition of platelet aggregation, and vasoprotective activity. In animal models, nitrate protects against ischaemia-reperfusion injuries and several other types of cardiovascular disorders. In addition, nitrate most surprisingly decreases whole body oxygen cost during exercise with preserved or even enhanced maximal performance. Oxidative stress and reduced NO bioavailability are critically linked to development of hypertension and other forms of cardiovascular diseases. Mechanistically, a central target for the effects of nitrate and its reaction products seems to be the mitochondrion and modulation of oxidative stress. All in vivo effects of nitrate are achievable with amounts corresponding to a rich intake of vegetables, which are particularly rich in this anion. A theory is now emerging suggesting nitrate as an active component in vegetables contributing to the beneficial health effects of this food group, including protection against cardiovascular disease and type-2 diabetes.

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.

Inorganic nitrite stimulates pancreatic islet blood flow and insulin secretion.

Reactive nitrogen and oxygen species have been proposed to be involved in control of insulin release from the pancreatic β cell. Recent evidence suggests that the supposedly inert anions nitrate and nitrite are metabolized in blood and tissues to form nitric oxide (NO) and other bioactive nitrogen oxides. Here we present evidence for a novel stimulatory role of nitrite in influencing pancreatic islet physiology via a dual mechanism, involving both indirect enhancement (through microcirculation redistribution) and direct insulinotropic effects on the β cell. In rats, intraperitoneal injection of sodium nitrite increased pancreatic islet blood flow by 50% and serum insulin concentrations by 30%, while whole pancreatic blood flow and glycemia remained unaffected. Nitrite also dose dependently enhanced insulin secretion from rat β cells in vitro under nonstimulatory glucose concentrations. This effect was not mimicked by nitrate and was abolished by the guanylyl cyclase (GC) inhibitor ODQ and the NO scavenger cPTIO. It was also mimicked by a cyclic GMP agonist (8-CPT-cGMP) and a classical NO donor (NONOate). Interestingly, a reactive oxygen species scavenger (vitamin E analog, Trolox) abolished the insulin secretion induced by nitrite. We conclude that nitrite exerts dual stimulatory effects on pancreatic islet function, including enhancement of islet blood flow and subsequent insulin secretion in vivo and direct stimulation of insulin release in vitro. The insulinotropic effect of nitrite is cGMP-dependent and involves formation of reactive nitrogen and oxygen species.

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.