Ylva Hellsten

Effects of α-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

We tested the hypothesis that 5′AMP‐activated protein kinase (AMPK) plays an important role in regulating the acute, exercise‐induced activation of metabolic genes in skeletal muscle, which were dissected from whole‐body α2‐ and α1‐AMPK knockout (KO) and wild‐type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running increased α1‐AMPK kinase activity, phosphorylation (P) of AMPK, and acetyl‐CoA carboxylase (ACC)β in α2‐WT and α2‐KO muscles and increased α2‐AMPK kinase activity in α2‐WT. In α2‐KO muscles, AMPK‐P and ACCβ‐P were markedly lower compared with α2‐WT. However, in α1‐WT and α1‐KO muscles, AMPK‐P and ACCβ‐P levels were identical at rest and increased similarly during exercise in the two genotypes. The α2‐KO decreased peroxisome‐proliferator‐activated receptor γ coactivator (PGC)‐1α, uncoupling protein‐3 (UCP3), and hexokinase II (HKII) transcription at rest but did not affect exercise‐induced transcription. Exercise increased the mRNA content of PGC‐1α, Forkhead box class O (FOXO)1, HKII, and pyruvate dehydrogenase kinase 4 (PDK4) similarly in α2‐WT and α2‐KO mice, whereas glucose transporter GLUT 4, carnitine palmitoyltransferase 1 (CPTI), lipoprotein lipase, and UCP3 mRNA were unchanged by exercise in both genotypes. CPTI mRNA was lower in α2‐KO muscles than in α2‐WT muscles at all time‐points. In α1‐WT and α1‐KO muscles, running increased the mRNA content of PGC‐1α and FOXOl similarly. The α2‐KO was associated with lower muscle adenosine 5′‐triphosphate content, and the inosine monophosphate content increased substantially at the end of exercise only in α2‐KO muscles. In addition, subcutaneous injection of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐4‐ribofuranoside (AICAR) increased the mRNA content of PGC‐1α, HKII, FOXO1, PDK4, and UCP3, and α2‐KO abolished the AICAR‐induced increases in PGC‐1α and HKII mRNA. In conclusion, KO of the α2‐ but not the α1‐AMPK isoform markedly diminished AMPK activation during running. Nevertheless, exercise‐induced activation of the investigated genes in mouse skeletal muscle was not impaired in α1‐ or α2‐AMPK KO muscles. Although it cannot be ruled out that activation of the remaining α‐isoform is sufficient to increase gene activation during exercise, the present data do not support an essential role of AMPK in regulating exercise‐induced gene activation in skeletal muscle.

Adenosine Concentrations in the Interstitium of Resting and Contracting Human Skeletal Muscle

BACKGROUND Adenosine has been proposed to be a locally produced regulator of blood flow in skeletal muscle. However, the fundamental questions of to what extent adenosine is formed in skeletal muscle tissue of humans, whether it is present in the interstitium, and where it exerts its vasodilatory effect remain unanswered. METHODS AND RESULTS The interstitial adenosine concentration was determined in the vastus lateralis muscle of healthy humans via dialysis probes inserted in the muscle. The probes were perfused with buffer, and the dialysate samples were collected at rest and during graded knee extensor exercise. At rest, the interstitial concentration of adenosine was 220+/-100 nmol/L and femoral arterial blood flow (FaBF) was 0.19+/-0.02 L/min. When the subjects exercised lightly, at a work rate of 10 W, there was a markedly higher (1140+/-540 nmol/L; P<0.05) interstitial adenosine concentration and a higher FaBF (2.22+/-0.18 L/min; P<0.05) compared with at rest. When exercise was performed at 20, 30, 40, or 50 W, the concentration of adenosine was moderately greater for each increment, as was the level of leg blood flow. The interstitial concentrations of ATP, ADP, and AMP increased from rest (0.13+/-0.03, 0.07+/-0.03, and 0.07+/-0.02 micromol/L, respectively) to exercise (10 W; 2.00+/-1.32, 2.08+/-1.23, and 1.65+/-0.50 micromol/L, respectively; P<0.05). CONCLUSIONS The present study provides, for the first time, interstitial adenosine concentrations in human skeletal muscle and demonstrates that adenosine and its precursors increase in the exercising muscle interstitium, at a rate associated with intensity of muscle contraction and the magnitude of muscle blood flow.

Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.

The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher ( P < 0.05) in the trained than in untrained muscle ( n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower ( P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.

Xanthine oxidase in human skeletal muscle following eccentric exercise: a role in inflammation.

1. The present study tested the hypothesis that the level of xanthine oxidase is elevated in injured human skeletal muscle in association with inflammatory events. Seven male subjects performed five bouts of strenuous one‐legged eccentric exercise. Muscle biopsies from both the exercised and the control leg, together with venous blood samples, were obtained prior to exercise and at 45 min, 24, 48 and 96 h after exercise. The time courses of xanthine oxidase immunoreactivity and indicators of muscle damage and inflammation were examined. 2. The number of xanthine oxidase structures observed by immunohistological methods in the exercised muscle was up to eightfold higher than control from day 1 to day 4 after exercise (P < 0.05). The increase was attributed to an enhanced expression of xanthine oxidase in microvascular endothelial cells and an invasion of leucocytes containing xanthine oxidase. 3. The concentration of plasma interleukin‐6 was significantly higher 90 min after exercise than before exercise (P < 0.05) and remained higher than pre‐exercise levels throughout the 4 days. On day 4 the plasma creatine kinase activity was approximately 150‐fold higher (P < 0.05) than resting levels. 4. Despite the increase in xanthine oxidase in the muscle there were no detectable changes in the levels of muscle malondialdehyde or in plasma antioxidant capacity up to 4 days post‐exercise. 5. It is concluded that eccentric exercise leads to an increased level of xanthine oxidase in human muscle and that the increase is associated with secondary inflammatory processes. The increase in xanthine oxidase in the muscle occurs mainly in microvascular endothelial cells, but occurs also via infiltrating leucocytes containing xanthine oxidase. A role for leucocytes in xanthine oxidase induction in endothelium is proposed.

Effect of high intensity training on capillarization and presence of angiogenic factors in human skeletal muscle

The effect of intense training on endothelial proliferation, capillary growth and distribution of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was examined in human skeletal muscle. Two intermittent knee extensor training protocols (at ∼150% (Study 1) versus∼90% (Study 2) of leg V̇O2 max) were conducted. Muscle biopsies were obtained throughout the training periods for immunohistochemical assessment of capillarization, cell proliferation (Ki‐67‐positive cells), VEGF and bFGF. In Study 1, microdialysis samples were collected from the trained and untrained leg at rest and during exercise and added to endothelial cells to measure the proliferative effect. After 4 weeks of training there was a higher (P < 0.05) capillary‐to‐fibre ratio (Study 1: 2.4 ± 0.1 versus 1.7 ± 0.1) and number of Ki‐67‐positive cells (Study 1: 0.18 ± 0.05 versus 0.00 ± 0.01) than before training. Neither the location of proliferating endothelial cells nor capillarization was related to muscle fibre type. The endothelial cell proliferative effect of the muscle microdialysate increased from rest to exercise in both the untrained leg (from 262 ± 60 to 573 ± 87% of control perfusate) and the trained leg (from 303 ± 75 to 415 ± 108% of perfusate). VEGF and bFGF were localized in endothelial and skeletal muscle cells and training induced no changes in distribution. The results demonstrate that intense intermittent endurance training induces capillary growth and a transient proliferation of endothelial cells within 4 weeks, with a similar growth occurring around type I versus type II muscle fibres.

Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions.

We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.

AMP deamination and purine exchange in human skeletal muscle during and after intense exercise

1 The present study examined the regulation of human skeletal muscle AMP deamination during intense exercise and quantified muscle accumulation and release of purines during and after intense exercise. 2 Seven healthy males performed knee extensor exercise at 64.3 W (range: 50–70 W) to exhaustion (234 s; 191–259 s). In addition, on two separate days the subjects performed exercise at the same intensity for 30 s and 80 % of exhaustion time (mean, 186 s; range, 153–207 s), respectively. Muscle biopsies were obtained from m.v. lateralis before and after each of the exercise bouts. For the exhaustive bout femoral arterio‐venous concentration differences and blood flow were also determined. 3 During the first 30 s of exercise there was no change in muscle adenosine triphosphate (ATP), inosine monophosphate (IMP) and ammonia (NH3), although estimated free ADP and AMP increased 5‐ and 45‐fold, respectively, during this period. After 186 s and at exhaustion muscle ATP had decreased (P < 0.05) by 15 and 19 %, respectively, muscle IMP was elevated (P < 0.05) from 0.20 to 3.65 and 5.67 mmol (kg dry weight)−1, respectively, and muscle NH3 had increased (P < 0.05) from 0.47 to 2.55 and 2.33 mmol (kg d.w.)−1, respectively. The concentration of H+ did not change during the first 30 s of exercise, but increased (P < 0.05) to 245.9 nmol l−1 (pH 6.61) after 186 s and to 374.5 nmol l−1 (pH 6.43) at exhaustion. 4 Muscle inosine and hypoxanthine did not change during exercise. In the first 10 min after exercise the muscle IMP concentration decreased (P < 0.05) by 2.96 mmol (kg d.w.)−1 of which inosine and hypoxanthine formation could account for 30 %. The total release of inosine and hypoxanthine during exercise and 90 min of recovery amounted to 1.07 mmol corresponding to 46 % of the net ATP decrease during exercise or 9 % of ATP at rest. 5 The present data suggest that AMP deamination is inhibited during the initial phase of intense exercise, probably due to accumulation of orthophosphate, and that lowered pH is an important positive modulator of AMP deaminase in contracting human skeletal muscle in vivo. Furthermore, formation and release of purines occurs mainly after intense exercise and leads to a considerable loss of nucleotides.

Intense interval training enhances human skeletal muscle oxygen uptake in the initial phase of dynamic exercise at high but not at low intensities

The present study tested the hypothesis that intense interval training enhances human skeletal muscle blood flow and oxygen uptake (V̇  O 2 ) at the onset of dynamic exercise. We also investigated whether possible training effects were dependent on exercise intensity. Six habitually active males carried out 7 weeks of intermittent‐exercise one‐legged knee‐extensor training at an intensity corresponding to ∼150% of peak thigh V̇  O 2 on three to five occasions per week. After the training period, cardiovascular and metabolic measurements were performed during knee‐extensor exercise with the trained leg (TL) and the control leg (CL) for 10 min at intensities of 10 and 30 W, and also for 4 min at 50 W. Femoral venous blood flow was higher (P < 0.05) in TL than CL from 75 to 180 s at 30 W (∼75 s: 3.43 ± 0.20 versus 2.99 ± 0.18 l min−1) and from 40 to 210 s at 50 W (∼75 s: 5.03 ± 0.41 versus 4.13 ± 0.33 l min−1). Mean arterial pressure was not different between legs. Thus, thigh vascular conductance was higher (P < 0.05) in TL than CL from 35 to 270 s at 30 W and from 150 to 240 s at 50 W. Femoral arterial–venous (a‐v) O2 difference was higher (P < 0.05) in TL than CL from 20 to 70 s at 30 W, but not different between TL and CL at 50 W. Thigh V̇  O 2 was higher (P < 0.05) in TL than CL from 20 to 110 s at 30 W (∼45 s: 0.38 ± 0.04 versus 0.30 ± 0.03 l min−1), and from 45 to 240 s at 50 W (∼45 s: 0.64 ± 0.06 versus 0.44 ± 0.08 l min−1). No differences were observed between TL and CL during exercise at 10 W. The present data demonstrate that intense interval training elevates muscle oxygen uptake, blood flow and vascular conductance in the initial phase of exercise at high, but not at low, intensities.

Oxidation of urate in human skeletal muscle during exercise.

The purpose of the present study was to investigate whether high metabolic stress to skeletal muscle, induced by intensive exercise, would lead to an oxidation of urate to allantoin in the exercised muscle. Seven healthy male subjects performed short term (4.39 +/- 0.04 [+/-SE] min) exhaustive cycling exercise. Muscle samples were obtained from m. v. lateralis before and during the first few minutes after the exercise. Venous blood samples were obtained before and up to 45 min after the exercise. The concentration of urate in muscle decreased from a resting level of 0.26 +/- 0.023 to 0.084 +/- 0.016 mumol.g-1 w.w. (p < .05) during the exercise and then rapidly increased during recovery to reach the resting level within 3 min after exercise. The concentration of allantoin in the muscle increased from a resting value of 0.03 +/- 0.007 to 0.10 +/- 0.014 mumol.g-1 w.w. immediately after exercise (p < .05) and then decreased to 0.079 +/- 0.002 mumol.g-1 w.w. during the first 3 min after exercise (p < .05). Plasma urate levels increased slowly from 305 +/- 16 to 426 +/- 20 mumol.liter-1 at 45 min in recovery (p < .05). Plasma allantoin was 11.9 +/- 2.6 mumol.liter-1 at rest and by 5 min the level was more than twofold higher and remained elevated throughout recovery (p < .05). The present results indicate that urate is oxidized to allantoin in the muscle during exercise, probably due to generation of free radicals. Furthermore, the findings support the suggested importance of urate as a free radical scavenger in vivo.

Exercise-induced hyperaemia and leg oxygen uptake are not altered during effective inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester in humans

1 In the present study the highly potent nitric oxide synthase (NOS) inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) was intravenously infused and examined for its efficacy in inhibiting NOS activity and in altering blood flow and oxygen uptake in human skeletal muscle. 2 The plasma concentrations of l‐NAME and its active metabolite NG‐nitro‐l‐arginine (l‐NA), and the activity of NOS in skeletal muscle were measured in healthy male subjects (n= 6) before (control) and after 60 min of intravenous infusion of l‐NAME (4 mg kg−1). In another group of healthy males (n= 8), the physiological effects of l‐NAME were studied at rest, and during submaximal and exhaustive knee extensor exercise before (control) and 30 min after l‐NAME infusion (4 mg kg−1). 3 The plasma concentrations of l‐NAME and l‐NA were highest (8.4 ± 1.6 and 8.3 ± 0.8 μmol l−1) after 60 min of l‐NAME infusion. Ninety minutes later mainly l‐NA remained in plasma (5.1 ± 0.4 μmol l−1). Thirty minutes after l‐NAME infusion, the muscle l‐NA content was 38 ± 4 μmol (kg dry wt)−1 and muscle NOS activity was reduced by 67 ± 8 % (P < 0.05). 4 Leg blood flow and leg oxygen uptake during submaximal and exhaustive exercise were similar (P > 0.05) following l‐NAME infusion and in control. Blood flow during recovery was lower in the l‐NAME condition (P < 0.05). 5 In conclusion, the present study shows for the first time that systemic infusion of l‐NAME in humans causes a marked reduction in skeletal muscle NOS activity. Despite this attenuated NOS activity, exercise‐induced hyperaemia and oxygen uptake were unaltered. Thus, the data strongly suggest that NO is not essential for the regulation of blood flow or oxygen uptake in contracting human skeletal muscle.