Jørn Wulff Helge

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

•  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects. •  The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein, and complex I–IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres). •  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content. •  mtDNA was found to be a poor biomarker of mitochondrial content. •  Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.

Influence of pre-exercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes.

Transcription of metabolic genes is transiently induced during recovery from exercise in skeletal muscle of humans. To determine whether pre‐exercise muscle glycogen content influences the magnitude and/or duration of this adaptive response, six male subjects performed one‐legged cycling exercise to lower muscle glycogen content in one leg and then, the following day, completed 2.5 h low intensity two‐legged cycling exercise. Nuclei and mRNA were isolated from biopsies obtained from the vastus lateralis muscle of the control and reduced glycogen (pre‐exercise glycogen = 609 ± 47 and 337 ± 33 mmol kg−1 dry weight, respectively) legs before and after 0, 2 and 5 h of recovery. Exercise induced a significant (P < 0.05) increase (2‐ to 3‐fold) in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) and uncoupling protein 3 (UCP3) genes in the reduced glycogen leg only. Although PDK4, lipoprotein lipase (LPL) and hexokinase II (HKII) mRNA were elevated in the reduced glycogen leg before exercise, no consistent difference was found between the two legs in response to exercise. In a second study, six subjects completed two trials (separated by 2 weeks) consisting of 3 h of two‐legged knee extensor exercise with either control (398 ± 52 mmol kg−1 dry weight) or low (240 ± 38 mmol kg−1 dry weight) pre‐exercise muscle glycogen. Exercise induced a significantly greater increase in PDK4 transcription in the low glycogen (> 6‐fold) than in the control (< 3‐fold) trial. Induction of PDK4 and UCP3 mRNA in response to exercise was also signficantly higher in the low glycogen (11.4‐ and 3.5‐fold, respectively) than in the control (5.0‐ and 1.7‐fold, respectively) trial. These data indicate that low muscle glycogen content enhances the transcriptional activation of some metabolic genes in response to exercise, raising the possibility that signalling mechanisms sensitive to glycogen content and/or FFA availability may be linked to the transcriptional control of exercise‐responsive genes.

Caffeine ingestion does not alter carbohydrate or fat metabolism in human skeletal muscle during exercise

1 This study examined the effect of ingesting caffeine (6 mg kg−1) on muscle carbohydrate and fat metabolism during steady‐state exercise in humans. Young male subjects (n= 10) performed 1 h of exercise (70 % maximal oxygen consumption (V̇O2,max)) on two occasions (after ingestion of placebo and caffeine) and leg metabolism was quantified by the combination of direct Fick measures and muscle biopsies. 2 Following caffeine ingestion serum fatty acid and glycerol concentration increased (P≤ 0.05) at rest, suggesting enhanced adipose tissue lipolysis. 3 In addition circulating adrenaline concentration was increased (P≤ 0.05) at rest following caffeine ingestion and this, as well as leg noradrenaline spillover, was elevated (P≤ 0.05) above placebo values during exercise. 4 Caffeine resulted in a modest increase (P≤ 0.05) in leg vascular resistance, but no difference was found in leg blood flow. 5 Arterial lactate and glucose concentrations were increased (P≤ 0.05) by caffeine, while the rise in plasma potassium was dampened (P≤ 0.05). 6 There were no differences in respiratory exchange ratio or in leg glucose uptake, net muscle glycogenolysis, leg lactate release or muscle lactate, or glucose 6‐phosphate concentration. Similarly there were no differences between treatments in leg fatty acid uptake, glycerol release or muscle acetyl CoA concentration. 7 These findings indicate that caffeine ingestion stimulated the sympathetic nervous system but did not alter the carbohydrate or fat metabolism in the monitored leg. Other tissues must have been involved in the changes in circulating potassium, fatty acids, glucose and lactate.

The effect of graded exercise on IL-6 release and glucose uptake in human skeletal muscle.

In this study, the hypothesis that the release of interleukin (IL)‐6 from human muscle is linked to exercise intensity and muscle glucose uptake was investigated. In the overnight fasted state, seven healthy males performed knee extension exercise, kicking with both legs, each at 25 % of maximal power (Wmax) for 45 min (eliciting 23 ± 1 % of pulmonary maximal oxygen uptake, V̇O2,max) and then simultaneously with one leg at 65 % and the other leg at 85 % Wmax for 35 min (40 ± 1 % of pulmonary V̇O2,max). Blood was sampled from a femoral artery and both femoral veins, and blood flow was determined by thermodilution. Thigh plasma flow (0.15 ± 0.01, 1.4 ± 0.2, 2.0 ± 0.1 and 2.3 ± 0.2 l min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) and thigh oxygen uptake (0.02 ± 0.01, 0.27 ± 0.03, 0.48 ± 0.04 and 0.55 ± 0.05 l min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) increased with increasing exercise intensity (P < 0.05). Also, thigh IL‐6 release (0.4 ± 0.1, 1.3 ± 0.5, 1.5 ± 0.6 and 2.5 ± 0.7 ng min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) and thigh glucose uptake (0.05 ± 0.01, 0.3 ± 0.05, 0.75 ± 0.16, 1.07 ± 0.15 mmol min−1 thigh−1 at rest and 25 %, 65 % and 85 % Wmax, respectively) increased with increasing exercise intensity (P < 0.05). During the last 35 min of exercise, arterial catecholamine concentrations were higher (P < 0.05) than at rest and during low‐intensity exercise. During exercise, thigh IL‐6 release was positively related to both thigh glucose uptake (P < 0.001) and thigh glucose delivery (P < 0.005), but not to thigh glucose extraction. Thigh IL‐6 release was also positively related to arterial plasma adrenaline concentration. The pre‐exercise muscle glycogen concentration tended to correlate with the arteriovenous IL‐6 concentration difference at rest, and the postexercise glycogen concentration was inversely correlated with IL‐6 release during the final 35 min of exercise. In conclusion, the study indicates that IL‐6 release from human muscle is positively related to exercise intensity, arterial adrenaline concentration and muscle glucose uptake. This supports the hypothesis that IL‐6 may be linked to the regulation of glucose homeostasis during exercise. The observation of a relationship between IL‐6 release and muscle glycogen store both at rest and after exercise suggests that IL‐6 may act as a carbohydrate sensor.

Interaction of training and diet on metabolism and endurance during exercise in man.

1. Ten untrained young men ingested a carbohydrate‐rich diet (65 energy percent (E%) carbohydrate, T‐CHO) and ten similar subjects a fat‐rich diet (62 E% fat, T‐FAT) while endurance training was performed 3‐4 times a week for 7 weeks. For another 8th week of training both groups ingested the carbohydrate‐rich diet (T‐CHO and T‐FAT/CHO). 2. Maximal oxygen uptake increased by 11% (P < 0.05) in both groups after 7 and 8 weeks. Time to exhaustion at 81% of pre‐training maximal oxygen uptake increased significantly from a mean (+/‐ S.E.M.) of 35 +/‐ 4 min to 102 +/‐ 5 and 65 +/‐ 7 min in T‐CHO and T‐FAT, respectively, after 7 weeks (P < 0.05, T‐CHO vs. T‐FAT). After 8 weeks, endurance remained unchanged in T‐CHO but increased (P < 0.05) to 77 +/‐ 9 min in T‐FAT/CHO which, however, was still less (P < 0.05) than in T‐CHO. 3. Muscle glycogen breakdown rate during exercise was halved by endurance training equally in both T‐CHO and T‐FAT after 7 and 8 weeks, and muscle glycogen stores at exhaustion were not depleted in any group. 4. During exercise after 7 weeks, the respiratory exchange ratio (RER) was unchanged in T‐CHO (0.88 +/‐ 0.01) compared with pre‐training but decreased (P < 0.05) to 0.82 +/‐ 0.02 in T‐FAT. After 8 weeks, RER in both T‐CHO and T‐FAT/CHO was approximately 0.87. 5. During exercise, plasma noradrenaline concentration and heart rate were higher in T‐FAT than in T‐CHO both at 7 and at 8 weeks. 6. It is concluded that ingesting a fat‐rich diet during an endurance training programme is detrimental to improvement in endurance. This is not due to a simple lack of carbohydrate fuel, but rather to suboptimal adaptations that are not remedied by short‐term increased carbohydrate availability. Furthermore, the study suggests that the decrease in RER usually seen after training when exercising at the same absolute intensity as before training can be prevented by a carbohydrate‐rich diet.

Fat utilization during exercise: adaptation to a fat-rich diet increases utilization of plasma fatty acids and very low density lipoprotein-triacylglycerol in humans

1 This study was carried out to test the hypothesis that the greater fat oxidation observed during exercise after adaptation to a high‐fat diet is due to an increased uptake of fat originating from the bloodstream. 2 Of 13 male untrained subjects, seven consumed a fat‐rich diet (62% fat, 21% carbohydrate) and six consumed a carbohydrate‐rich diet (20% fat, 65% carbohydrate). After 7 weeks of training and diet, 60 min of bicycle exercise was performed at 68 ± 1% of maximum oxygen uptake. During exercise [1‐13C]palmitate was infused, arterial and venous femoral blood samples were collected, and blood flow was determined by the thermodilution technique. Muscle biopsy samples were taken from the vastus lateralis muscle before and after exercise. 3 During exercise, the respiratory exchange ratio was significantly lower in subjects consuming the fat‐rich diet (0.86 ± 0.01, mean ±s.e.m.) than in those consuming the carbohydrate‐rich diet (0.93 ± 0.02). The leg fatty acid (FA) uptake (183 ± 37 vs. 105 ± 28 μmol min−1) and very low density lipoprotein‐triacylglycerol (VLDL‐TG) uptake (132 ± 26 vs. 16 ± 21 μmol min−1) were both higher (each P < 0.05) in the subjects consuming the fat‐rich diet. Whole‐body plasma FA oxidation (determined by comparison of 13CO2 production and blood palmitate labelling) was 55‐65% of total lipid oxidation, and was higher after the fat‐rich diet than after the carbohydrate‐rich diet (13.5 ± 1.2 vs. 8.9 ± 1.1 μmol min−1 kg−1; P < 0.05). Muscle glycogen breakdown was significantly lower in the subjects taking the fat‐rich diet than those taking the carbohydrate‐rich diet (2.6 ± 0.5 vs. 4.8 ± 0.5 mmol (kg dry weight)−1 min−1, respectively; P < 0.05), whereas leg glucose uptake was similar (1.07 ± 0.13 vs. 1.15 ± 0.13 mmol min−1). 4 In conclusion, plasma VLDL‐TG appears to be an important substrate source during aerobic exercise, and in combination with the higher plasma FA uptake it accounts for the increased fat oxidation observed during exercise after fat diet adaptation. The decreased carbohydrate oxidation was apparently due to muscle glycogen sparing and not to diminished plasma glucose uptake.

Interleukin-18 in plasma and adipose tissue: effects of obesity, insulin resistance, and weight loss.

OBJECTIVE Interleukin (IL)-18 is associated with obesity, insulin resistance, and cardiovascular disease. The present study compared 1) IL-18 in adipocytes versus stromal vascular (SV) cells, 2) IL-18 in plasma and adipose tissue (AT) in obese versus lean subjects, and 3) IL-18 in plasma, AT, and skeletal muscle (SM) in obese subjects after weight loss. SUBJECTS AND METHODS At baseline, plasma and AT IL-18 in 23 obese subjects were compared with that in 12 lean subjects. The obese subjects were submitted to a 15-week life-style intervention (hypocaloric diet and daily exercise) after which plasma samples, AT, and SM biopsies were obtained. Analyses were performed by ELISA and RT-PCR respectively. RESULTS IL-18 expression in isolated adipocytes was approximately 2% of that in SV cells. Plasma IL-18 was higher in obese subjects (P < 0.001) and associated with insulin resistance (HOMA; P < 0.001). AT expression of IL-18, CD14, and CD68 was higher in obese (P < 0.01). The intervention reduced body weight (P < 0.001), plasma IL-18 (P < 0.001), and increased insulin sensitivity (HOMA; P < 0.05). AT and SM expression of IL-18 remained unchanged after the intervention. Changes in plasma IL-18 were associated with changes in insulin sensitivity (P < 0.05) but not with BMI or AT expression of IL-18. CONCLUSION Plasma IL-18 is associated with changes in insulin resistance and reduced after weight loss. AT expression of IL-18 is increased in obesity but not affected by weight loss, indicating that changes in plasma IL-18 are related to insulin resistance rather than changes in obesity per se.

Whole‐body fat oxidation determined by graded exercise and indirect calorimetry: a role for muscle oxidative capacity?

During whole‐body exercise, peak fat oxidation occurs at a moderate intensity. This study investigated whole‐body peak fat oxidation in untrained and trained subjects, and the presence of a relation between skeletal muscle oxidative enzyme activity and whole‐body peak fat oxidation. Healthy male subjects were recruited and categorized into an untrained (N=8, VO2max 3.5±0.1 L/min) and a trained (N=8, VO2max 4.6±0.2 L/min) group. Subjects performed a graded exercise test commencing at 60 W for 8 min followed by 35 W increments every 3 min. On a separate day, muscle biopsies were obtained from vastus lateralis and a 3 h bicycle exercise test was performed at 58% of VO2max. Whole‐body fat oxidation was calculated during prolonged and graded exercise from the respiratory exchange ratio using standard indirect calorimetry equations. Based on the graded exercise test, whole‐body peak fat oxidation was determined. The body composition was determined by DEXA.

Simvastatin Effects on Skeletal Muscle: Relation to Decreased Mitochondrial Function and Glucose Intolerance

OBJECTIVES Glucose tolerance and skeletal muscle coenzyme Q(10) (Q(10)) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9). BACKGROUND A prevalent side effect of statin therapy is muscle pain, and yet the basic mechanism behind it remains unknown. We hypothesize that a statin-induced reduction in muscle Q(10) may attenuate mitochondrial OXPHOS capacity, which may be an underlying mechanism. METHODS Plasma glucose and insulin concentrations were measured during an oral glucose tolerance test. Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers by high-resolution respirometry in a cross-sectional design. Mitochondrial content (estimated by citrate synthase [CS] activity, cardiolipin content, and voltage-dependent anion channel [VDAC] content) as well as Q(10) content was determined. RESULTS Simvastatin-treated patients had an impaired glucose tolerance and displayed a decreased insulin sensitivity index. Regarding mitochondrial studies, Q(10) content was reduced (p = 0.05), whereas mitochondrial content was similar between the groups. OXPHOS capacity was comparable between groups when complex I- and complex II-linked substrates were used alone, but when complex I + II-linked substrates were used (eliciting convergent electron input into the Q intersection [maximal ex vivo OXPHOS capacity]), a decreased (p < 0.01) capacity was observed in the patients compared with the control subjects. CONCLUSIONS These simvastatin-treated patients were glucose intolerant. A decreased Q(10) content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment.

Acute exercise improves motor memory: exploring potential biomarkers.

We have recently shown that a single bout of acute cardiovascular exercise improves motor skill learning through an optimization of long-term motor memory. Here we expand this previous finding, to explore potential exercise-related biomarkers and their association with measures of motor memory and skill acquisition. Thirty-two healthy young male subjects were randomly allocated into either an exercise or control group. Following either an intense bout of cycling or rest subjects practiced a visuomotor tracking task. Motor skill acquisition was assessed during practice and retention 1 h, 24 h and 7 days after practice. Plasma levels of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), epinephrine, norepinephrine, dopamine and lactate were analyzed at baseline, immediately after exercise or rest and during motor practice. The exercise group showed significantly better skill retention 24h and 7 days after acquisition. The concentration of all blood compounds increased significantly immediately after exercise and remained significantly elevated for 15 min following exercise except for BDNF and VEGF. Higher concentrations of norepinephrine and lactate immediately after exercise were associated with better acquisition. Higher concentrations of BDNF correlated with better retention 1 h and 7 days after practice. Similarly, higher concentrations of norepinephrine were associated with better retention 7 days after practice whereas lactate correlated with better retention 1h as well as 24 h and 7 days after practice. Thus, improvements in motor skill acquisition and retention induced by acute cardiovascular exercise are associated with increased concentrations of biomarkers involved in memory and learning processes. More mechanistic studies are required to elucidate the specific role of each biomarker in the formation of motor memory.