Kent Sahlin

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.

The time course of phosphorylcreatine resynthesis during recovery of the quadriceps muscle in man.

SummaryThe time course of phosphorylcreatine (PC) resynthesis in the human m. quadriceps femoris was studied during recovery from exhaustive dynamic exercise and from isometric contraction sustained to fatigue. The immediate postexercise muscle PC content after either form of exercise was 15–16% of the resting muscle content. The time course of PC resynthesis during recovery was biphasic exhibiting a fast and a slow recovery component. The half-time for the fast component was 21–22 s but this accounted for a smaller fraction of the total PC restored during recovery from the isometric contraction than after the dynamic exercise. The half-time for the slow component was in each case more than 170 s. After 2 and 4 min recovery the total amounts of PC resynthesized after the isometric exercise were significantly lower than from the dynamic exercise.Occlusion of the circulation to the quadriceps completely abolished the resynthesis of PC. Restoration of resynthesis occurred only after release of occlusion.

Lactate content and pH in muscle samples obtained after dynamic exercise

SummaryAnalyzes were made on muscle samples taken from the lateral part of the m. quadriceps femoris of man (lactate, pyruvate, and pH) on venous blood (lactate, pyruvate) and on capillary blood (pH). Samples were taken at rest, immediately after termination of dynamic exercise and during 20 min recovery from exhaustive dynamic exercise.Muscle pH decreased from 7.08 at rest to 6.60 at exhaustion. Decrease in muscle pH was linearly related to muscle content of lactate + pyruvate. The relationship was slightly different from what has been obtained after isometric exercise and this difference was ascribed to acid-base exchange with the blood during dynamic exercise.Lactate content was highly elevated in muscle after exercise and the concentration was 2–3 times higher than in blood. Pyruvate content was, however, only slightly higher than that at rest. During recovery lactate content of muscle decreased exponentially with respect to time, whereas pyruvate content increased. The half-time of lactate decrease was 9.5 min. From the lactate dehydrogenase equilibrium relative values on NADH/NAD ratio could be calculated. It was found that NADH/NAD was highly increased after exercise and that it had not returned to the basal value after 20 min recovery.

Mitochondrial Respiration Is Decreased in Skeletal Muscle of Patients With Type 2 Diabetes

We tested the hypothesis of a lower respiratory capacity per mitochondrion in skeletal muscle of type 2 diabetic patients compared with obese subjects. Muscle biopsies obtained from 10 obese type 2 diabetic and 8 obese nondiabetic male subjects were used for assessment of 3-hydroxy-Acyl-CoA-dehydrogenase (HAD) and citrate synthase activity, uncoupling protein (UCP)3 content, oxidative stress measured as 4-hydroxy-2-nonenal (HNE), fiber type distribution, and respiration in isolated mitochondria. Respiration was normalized to citrate synthase activity (mitochondrial content) in isolated mitochondria. Maximal ADP-stimulated respiration (state 3) with pyruvate plus malate and respiration through the electron transport chain (ETC) were reduced in type 2 diabetic patients, and the proportion of type 2X fibers were higher in type 2 diabetic patients compared with obese subjects (all P < 0.05). There were no differences in respiration with palmitoyl-l-carnitine plus malate, citrate synthase activity, HAD activity, UCP3 content, or oxidative stress measured as HNE between the groups. In the whole group, state 3 respiration with pyruvate plus malate and respiration through ETC were negatively associated with A1C, and the proportion of type 2X fibers correlated with markers of insulin resistance (P < 0.05). In conclusion, we provide evidence for a functional impairment in mitochondrial respiration and increased amount of type 2X fibers in muscle of type 2 diabetic patients. These alterations may contribute to the development of type 2 diabetes in humans with obesity.

Metabolic Factors in Fatigue

SummaryThe supply of energy is of fundamental importance for the ability to sustain exercise. The maximal duration of exercise is negatively related to the relative intensity both during dynamic and static exercise. Since exercise intensity is linearly related to the rate of energy utilisation this suggests that energetic deficiency plays a major role in the aetiology of muscle fatigue. Charac-teristic metabolic changes in the muscle are generally observed at fatigue — the pattern being different after short term exercise (lactate accumulation and phosphocreatine depletion) from after prolonged exercise at moderate intensity (glycogen depletion). A common metabolic denominator at fatigue during these and many other conditions is a reduced capacity to generate ATP and is expressed by an increased catabolism of the adenine nucleotide pool in the muscle fibre. Transient increases in ADP are suggested to occur during energetic deficiency and may be the cause of fatigue. Experimental evidence from human studies demonstrate that near maximal power output can be attained during acidotic conditions. Decreases in muscle pH is therefore unlikely to affect the contractile machinery by a direct effect. However, acidosis may interfere with the energy supply possibly by reducing the glycolytic rate, and could by this mechanism be related to muscle fatigue.

The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle.

1 The role of phosphorylcreatine (PCr) and creatine (Cr) in the regulation of mitochondrial respiration was investigated in permeabilised fibre bundles prepared from human vastus lateralis muscle. 2 Fibre respiration was measured in the absence of ADP (V̇0) and after sequential additions of submaximal ADP (0.1 mm ADP, V̇submax), PCr (or Cr) and saturating [ADP] (V̇max). 3 V̇ submax increased by 55% after addition of saturating creatine (P < 0.01; n= 8) and half the maximal effect was obtained at 5 mm[Cr]. In contrast, V̇submax decreased by 54% after addition of saturating phosphorylcreatine (P < 0.01; n= 8) and half the maximal effect was obtained at 1 mm[PCr]. V̇max was not affected by Cr or PCr. 4 V̇ submax was similar when PCr and Cr were added simultaneously at concentrations similar to those in muscle at rest (PCr/Cr = 2) and at low‐intensity exercise (PCr/Cr = 0.5). At conditions mimicking high‐intensity exercise (PCr/Cr = 0.1), V̇submax increased to 60% of V̇max (P < 0.01) vs. rest and low‐intensity exercise). 5 Eight of the subjects participated in a 16 day Cr supplementation programme. Following Cr supplementation, V̇0 decreased by 17% (P < 0.01) vs. prior to Cr supplementation), whereas ADP‐stimulated respiration (with and without Cr or PCr) was unchanged. 6 For the first time evidence is given that PCr is an important regulator of mitochondrial ADP‐stimulated respiration. Phosphorylcreatine decreases the sensitivity of mitochondrial respiration to ADP whereas Cr has the opposite effect. During transition from rest to high‐intensity exercise, decreases in the PCr/Cr ratio will effectively increase the sensitivity of mitochondrial respiration to ADP. The decrease in V̇0 after Cr supplementation indicates that intrinsic changes in membrane proton conductance occur.

Mitochondrial function and antioxidative defence in human muscle: effects of endurance training and oxidative stress

1 The influence of endurance training on oxidative phosphorylation and the susceptibility of mitochondrial oxidative function to reactive oxygen species (ROS) was investigated in skeletal muscle of four men and four women. Mitochondria were isolated from muscle biopsies taken before and after 6 weeks of endurance training. Mitochondrial respiration was measured before and after exposure of mitochondria to exogenous ROS (H2O2+ FeCl2). 2 Endurance training increased peak pulmonary O2 uptake (V̇O2,peak) by 24 % and maximal ADP‐stimulated mitochondrial oxygen consumption (state 3) by 40 % (P < 0.05). Respiration in the absence of ADP (state 4), the respiratory control ratio (RCR = state 3/state 4) and the ratio between added ADP and consumed oxygen (P/O) remained unchanged by the training programme. 3 Exposure to ROS reduced state 3 respiration but the effect was not significantly different between pre‐ and post‐training samples. State 4 oxygen consumption increased after exposure to ROS both before (+189 %, P < 0.05) and after training (+243 %, P < 0.05) and the effect was significantly higher after training (P < 0.05, pre‐ vs. post‐training). The augmented state 4 respiration could in part be attenuated by atractyloside, which indicates that ADP/ATP translocase was affected by ROS. The P/O ratio in ROS‐treated mitochondria was significantly lower (P < 0.05) compared to control conditions, both before (−18.6 ± 2.2 %) and after training (−18.5 ± 1.1 %). 4 Muscle activities of superoxide dismutase (mitochondrial and cytosolic), glutathione peroxidase and muscle glutathione status were unaffected by training. There was a positive correlation between muscle superoxide dismutase activity and age (r= 0.75; P < 0.05; range of age 20–37 years), which may reflect an adaptation to increased generation of ROS in senescent muscle. The muscle glutathione pool was more reduced in subjects with high activity of glutathione peroxidase (r= 0.81; P < 0.05). 5 The influence of short‐term training on mitochondrial oxygen consumption has for the first time been investigated in human skeletal muscle. The results showed that maximal mitochondrial oxidative power is increased after endurance training but that the efficiency of energy transfer (P/O ratio) remained unchanged. Antioxidative defence was unchanged after training when expressed relative to muscle weight. Although this corresponds to a reduced antioxidant protection per individual mitochondrion, the sensitivity of aerobic energy transfer to ROS was unchanged. However, the augmented ROS‐induced non‐coupled respiration after training indicates an increased susceptibility of mitochondrial membrane proton conductance to oxidative stress.

Cycling efficiency in humans is related to low UCP3 content and to type I fibres but not to mitochondrial efficiency

The purpose of this study was to investigate the hypothesis that cycling efficiency in vivo is related to mitochondrial efficiency measured in vitro and to investigate the effect of training status on these parameters. Nine endurance trained and nine untrained male subjects (, respectively) completed an incremental submaximal efficiency test for determination of cycling efficiency (gross efficiency, work efficiency (WE) and delta efficiency). Muscle biopsies were taken from m. vastus lateralis and analysed for mitochondrial respiration, mitochondrial efficiency (MEff; i.e. P/O ratio), UCP3 protein content and fibre type composition (% MHC I). MEff was determined in isolated mitochondria during maximal (state 3) and submaximal (constant rate of ADP infusion) rates of respiration with pyruvate. The rates of mitochondrial respiration and oxidative phosphorylation per muscle mass were about 40% higher in trained subjects but were not different when expressed per unit citrate synthase (CS) activity (a marker of mitochondrial density). Training status had no influence on WE (trained 28.0 ± 0.5, untrained 27.7 ± 0.8%, N.S.). Muscle UCP3 was 52% higher in untrained subjects, when expressed per muscle mass (P < 0.05 versus trained). WE was inversely correlated to UCP3 (r=−0.57, P < 0.05) and positively correlated to percentage MHC I (r= 0.58, P < 0.05). MEff was lower (P < 0.05) at submaximal respiration rates (2.39 ± 0.01 at 50%) than at state 3 (2.48 ± 0.01) but was neither influenced by training status nor correlated to cycling efficiency. In conclusion cycling efficiency was not influenced by training status and not correlated to MEff, but was related to type I fibres and inversely related to UCP3. The inverse correlation between WE and UCP3 indicates that extrinsic factors may influence UCP3 activity and thus MEff in vivo.

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

Effects of acute and chronic endurance exercise on mitochondrial uncoupling in human skeletal muscle

Mitochondrial proteins such as uncoupling protein 3 (UCP3) and adenine nucleotide translocase (ANT) may mediate back‐leakage of protons and serve as uncouplers of oxidative phosphorylation. We hypothesized that UCP3 and ANT increase after prolonged exercise and/or endurance training, resulting in increased uncoupled respiration (UCR). Subjects were investigated with muscle biopsies before and after acute exercise (75 min of cycling at 70% of ) or 6 weeks endurance training. Mitochondria were isolated and respiration measured in the absence (UCR or state 4) and presence of ADP (coupled respiration or state 3). Protein expression of UCP3 and ANT was measured with Western blotting. After endurance training , citrate synthase activity (CS), state 3 respiration and ANT increased by 24, 47, 40 and 95%, respectively (all P < 0.05), whereas UCP3 remained unchanged. When expressed per unit of CS (a marker of mitochondrial volume) UCP3 and UCR decreased by 54% and 18%(P < 0.05). CS increased by 43% after acute exercise and remained elevated after 3 h of recovery (P < 0.05), whereas the other muscle parameters remained unchanged. An intriguing finding was that acute exercise reversibly enhanced the capacity of mitochondria to accumulate Ca2+(P < 0.05) before opening of permeability transition pores. In conclusion, UCP3 protein and UCR decrease after endurance training when related to mitochondrial volume. These changes may prevent excessive basal thermogenesis. Acute exercise enhances mitochondrial resistance to Ca2+ overload but does not influence UCR or protein expression of UCP3 and ANT. The increased Ca2+ resistance may prevent mitochondrial degradation and the mechanism needs to be further explored.