Michael Svensson

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.

Predictive Metabolomics Evaluation of Nutrition-Modulated Metabolic Stress Responses in Human Blood Serum During the Early Recovery Phase of Strenuous Physical Exercise

We have investigated whether postexercise ingestion of carbohydrates in combination with proteins generates a different systemic metabolic response, as compared to the sole ingestion of carbohydrate or water, in the early recovery phase following exercise. In addition, metabolic patterns related to fitness level were studied together with individual responses to nutritional modulation. Twenty-four male subjects were exposed to 90 min of ergometer-cycling. Each participant was subject to four identical test-sessions, including ingestion of one of four beverages (water, low-carbohydrate beverage, high-carbohydrate beverage, and low-carbohydrate-protein beverage (LCHO-P)) immediately after cycling. Blood was collected at six time points, one pre- and five postexercise. Extracted blood serum was subject to metabolomic characterization by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS). Data was processed using hierarchical multivariate curve resolution (HMCR), and multivariate statistical analysis was carried out using orthogonal partial least-squares (OPLS). Predictive metabolomics, including predictive HMCR and OPLS classification, was applied to ensure efficient sample processing and validation of detected metabolic patterns. Separation of subjects in relation to ingested beverage was detected and interpreted. Pseudouridine was suggested as a novel marker for pro-anabolic effect following LCHO-P ingestion, which was supported by the detected decrease of the catabolic marker 3-methylhistidine. Separation of subjects according to fitness level was achieved, and nutritional modulation by LCHO-P was shown to improve the metabolic status of less fit subjects in the recovery phase. In addition, the potential of the methodology for detection of early signs of insulin resistance was also demonstrated.

Allantoin formation and urate and glutathione exchange in human muscle during submaximal exercise.

Seven males performed two exhaustive cycling bouts (EX1 and EX2) at a work-rate of 90% of maximal oxygen uptake, separated by 60 min. During EX1 there was a significant accumulation of urate (from 0.16 +/- 0.02 to 0.27 +/- 0.03 micromol/kg d.w.) and allantoin (from 0.39 +/- 0.05 to 0.69 +/- 0.14 micromol/kg d.w.) in the muscle. An uptake of urate was observed in early recovery from EX1 (0-9 min: 486 +/- 136 micromol; p <.05). There was no exchange of total glutathione or cysteine over the muscle either during or after exercise, and muscle and plasma total glutathione remained unaltered (p <.05). The glycogen levels were lowered by 40% at the onset of EX2, yet the level of oxidative stress in EX1 and EX2 was similar as evidenced by a similar increase in muscle allantoin in both exercise bouts. The data suggest that urate is utilized as antioxidant in human skeletal muscle and that reactive oxygen species are formed in muscle during intense submaximal exercise. No net exchange of glutathione appears to occur over the muscle either at rest, during exercise or in recovery. Moreover, when an exhaustive exercise bout is repeated with lowered glycogen levels, the level of oxidative stress is not different than that of the first bout.

Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1.

Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lack the apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility.

No evidence of an intracellular lactate shuttle in rat skeletal muscle

The concerted view is that cytosolic pyruvate is transferred into mitochondria and after oxidative decarboxylation further metabolized in the tricarboxylic acid cycle. Recently this view has been challenged. Based on experimental evidence from rat skeletal muscle it has been concluded that mitochondria predominantly oxidize lactate in vivo and that this constitutes part of an ‘intracellular lactate shuttle’. This view appears to be gaining acceptance in the scientific community and due to its conceptual importance, confirmation by independent experiments is required. We have repeated the experiments in mitochondria isolated from rat soleus muscle. Contrary to the previously published findings we cannot find any mitochondrial respiration with lactate. Analysis of lactate dehydrogenase (LDH) by spectrophotometry demonstrated that the activity in the mitochondrial fraction was only 0.7 % of total activity. However, even when external LDH was added to mitochondria, there were no signs of respiration with lactate. In the presence of conditions where lactate is converted to pyruvate (external additions of both LDH and NAD+), mitochondrial oxygen consumption increased. Furthermore, we provide theoretical evidence that direct mitochondrial lactate oxidation is energetically unlikely. Based on the present data we conclude that direct mitochondrial lactate oxidation does not occur in skeletal muscle. The presence of an ‘intracellular lactate shuttle’ can therefore be questioned.

Activation of FXII during haemodialysis

This study was designed to provide information on the kinetics of FXIIa activity and C3d release during haemodialysis. Dialysis was performed twice in 9 stable patient for 240 min with the same conditions except for a change in dialysate sodium profile (using a linear sodium programme starting at 138 and ending at 148 mmol/l or vice versa). Using paired statistics there was a significant (p < 0.02) increase in both C3d release and the FXIIa activity. The sodium profiles did not alter the outcome. The increase in FXIIa activity, which is maximal at the end of dialysis, is continuous, unlike the C3d release, which is maximal within 15 min of dialysis and then levels off. Both interactions are induced by the blood membrane contact. These results indicate that the FXIIa activation is not strictly coupled to the activation of the complement system.

Differentiation-specific alterations to glutathione synthesis in and hormonally stimulated release from human skeletal muscle cells

Muscle atrophy and cachexia are associated with many human diseases. These catabolic states are often associated with the loss of glutathione (GSH), which is thought to contribute to the induction of oxidative stress within the muscle. Glutathione synthesis and secretary characteristics were studied in human skeletal muscle myoblasts and myotube‐like cells derived from the myoblasts by growth factor restriction. Differentiation was associated with a shift in the sulfur amino acid precursor specificity for synthesis of GSH from cystine to cysteine, as well as loss in ability to use extracellular glutathione and activation of methionine use. The thiol drug N‐acetylcysteine was also shown to be an effective precursor irrespective of the state of differentiation. Additionally, myoblasts and myotube cultures were shown to secrete GSH continually, but only the differentiated cells responded to stress hormones such as glucagon, vasopressin, and phenylephrine, by increased secretion of the tripeptide. The data suggest that the skeletal muscle cells may provide an important hormonally regulated extra‐hepatic source of systemic GSH and also shed light on the mechanisms of accelerated turnover of GSH operating during strenuous muscle activity and trauma. The data may also provide biochemical rationales for the nutritional and/or pharmacological manipulation of GSH with sulfur amino acid precursors during the treatment of muscle‐specific oxidative stress and atrophy.

Evaluation of 2‐D DIGE for skeletal muscle: Protocol and repeatability

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel‐to‐gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two‐dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 18×24 cm gels, scanned with a Typhoon™ 9410 laser scanner and analysed in the DeCyder™ software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1–2.4 %). When setting the significance level to 99 %, no false‐positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3–11 and MW 10–200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.

Benefits of a Paleolithic diet with and without supervised exercise on fat mass, insulin sensitivity, and glycemic control: a randomized controlled trial in individuals with type 2 diabetes

Means to reduce future risk for cardiovascular disease in subjects with type 2 diabetes are urgently needed.

Validated and predictive processing of gas chromatography-mass spectrometry based metabolomics data for large scale screening studies, diagnostics and metabolite pattern verification.

The suggested approach makes it feasible to screen large metabolomics data, sample sets with retained data quality or to retrieve significant metabolic information from small sample sets that can be verified over multiple studies. Hierarchical multivariate curve resolution (H-MCR), followed by orthogonal partial least squares discriminant analysis (OPLS-DA) was used for processing and classification of gas chromatography/time of flight mass spectrometry (GC/TOFMS) data characterizing human serum samples collected in a study of strenuous physical exercise. The efficiency of predictive H-MCR processing of representative sample subsets, selected by chemometric approaches, for generating high quality data was proven. Extensive model validation by means of cross-validation and external predictions verified the robustness of the extracted metabolite patterns in the data. Comparisons of extracted metabolite patterns between models emphasized the reliability of the methodology in a biological information context. Furthermore, the high predictive power in longitudinal data provided proof for the potential use in clinical diagnosis. Finally, the predictive metabolite pattern was interpreted physiologically, highlighting the biological relevance of the diagnostic pattern.