Adam Steensberg

Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6.

1 Plasma interleukin (IL)‐6 concentration is increased with exercise and it has been demonstrated that contracting muscles can produce IL‐ The question addressed in the present study was whether the IL‐6 production by contracting skeletal muscle is of such a magnitude that it can account for the IL‐6 accumulating in the blood. 2 This was studied in six healthy males, who performed one‐legged dynamic knee extensor exercise for 5 h at 25 W, which represented 40% of peak power output (Wmax). Arterial‐femoral venous (a‐fv) differences over the exercising and the resting leg were obtained before and every hour during the exercise. Leg blood flow was measured in parallel by the ultrasound Doppler technique. IL‐6 was measured by enzyme‐linked immunosorbent assay (ELISA). 3 Arterial plasma concentrations for IL‐6 increased 19‐fold compared to rest. The a‐fv difference for IL‐6 over the exercising leg followed the same pattern as did the net IL‐6 release. Over the resting leg, there was no significant a‐fv difference or net IL‐6 release. The work was produced by 2.5 kg of active muscle, which means that during the last 2 h of exercise, the median IL‐6 production was 6.8 ng min−1 (kg active muscle)−1 (range, 3.96‐9.69 ng min−1 kg−1). 4 The net IL‐6 release from the muscle over the last 2 h of exercise was 17‐fold higher than the elevation in arterial IL‐6 concentration and at 5 h of exercise the net release during 1 min was half of the IL‐6 content in the plasma. This indicates a very high turnover of IL‐6 during muscular exercise. We suggest that IL‐6 produced by skeletal contracting muscle contributes to the maintenance of glucose homeostasis during prolonged exercise.

The Yo-yo Intermittent Recovery Test: Physiological Response, Reliability, and Validity

PURPOSE To examine the physiological response and reproducibility of the Yo-Yo intermittent recovery test and its application to elite soccer. METHODS Heart rate was measured, and metabolites were determined in blood and muscle biopsies obtained before, during, and after the Yo-Yo test in 17 males. Physiological measurements were also performed during a Yo-Yo retest and an exhaustive incremental treadmill test (ITT). Additionally, 37 male elite soccer players performed two to four seasonal tests, and the results were related to physical performance in matches. RESULTS The test-retest CV for the Yo-Yo test was 4.9%. Peak heart rate was similar in ITT and Yo-Yo test (189 +/- 2 vs 187 +/- 2 bpm), whereas peak blood lactate was higher (P < 0.05) in the Yo-Yo test. During the Yo-Yo test, muscle lactate increased eightfold (P < 0.05) and muscle creatine phosphate (CP) and glycogen decreased (P < 0.05) by 51% and 23%, respectively. No significant differences were observed in muscle CP, lactate, pH, or glycogen between 90 and 100% of exhaustion time. During the precompetition period, elite soccer players improved (P < 0.05) Yo-Yo test performance and maximum oxygen uptake ([OV0312]O(2max)) by 25 +/- 6 and 7 +/- 1%, respectively. High-intensity running covered by the players during games was correlated to Yo-Yo test performance (r = 0.71, P < 0.05) but not to [OV0312]O(2max) and ITT performance. CONCLUSION The test had a high reproducibility and sensitivity, allowing for detailed analysis of the physical capacity of athletes in intermittent sports. Specifically, the Yo-Yo intermittent recovery test was a valid measure of fitness performance in soccer. During the test, the aerobic loading approached maximal values, and the anaerobic energy system was highly taxed. Additionally, the study suggests that fatigue during intense intermittent short-term exercise was unrelated to muscle CP, lactate, pH, and glycogen.

Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content

In humans, the plasma interleukin 6 (IL‐6) concentration increases dramatically during low‐intensity exercise. Measurements across the working limb indicate that skeletal muscle is the source of IL‐6 production. To determine whether energy availability influences the regulation of IL‐6 expression during prolonged exercise, six male subjects completed two trials consisting of 180 min of two‐legged dynamic knee extensor with either normal or low (~60% of control) pre‐exercise muscle glycogen levels. Increases in plasma IL‐6 during exercise were significantly higher (P < 0.05) in the low‐glycogen (16‐fold) trial verses the control (10‐fold) trial. Transcriptional activation of the IL‐6 gene in skeletal muscle was also higher in the low‐glycogen trial; it increased by about 40‐fold after 90 min of exercise and about 60‐fold after 180 min of exercise. Muscle IL‐6 mRNA followed a similar but delayed pattern, increasing by more than 100‐fold in the low‐glycogen trial and by about 30‐fold in the control trial. These data demonstrate that exercise activates transcription of the IL‐6 gene in working skeletal muscle, a response that is dramatically enhanced when glycogen levels are low. These findings also support the hypothesis that IL‐6 may be produced by contracting myofibers when glycogen levels become critically low as a means of signaling the liver to increase glucose production.

Muscle-derived interleukin-6: possible biological effects

Interleukin‐6 (IL‐6) is produced locally in working skeletal muscle and can account for the increase in plasma IL‐6 during exercise. The production of IL‐6 during exercise is related to the intensity and duration of the exercise, and low muscle glycogen content stimulates the production. Muscle‐derived IL‐6 is released into the circulation during exercise in high amounts and is likely to work in a hormone‐like fashion, exerting an effect on the liver and adipose tissue, thereby contributing to the maintenance of glucose homeostasis during exercise and mediating exercise‐induced lipolysis. Muscle‐derived IL‐6 may also work to inhibit the effects of pro‐inflammatory cytokines such as tumour necrosis factor α. The latter cytokine is produced by adipose tissue and inflammatory cells and appears to play a pathogenetic role in insulin resistance and atherogenesis.

Interleukin‐6 production in contracting human skeletal muscle is influenced by pre‐exercise muscle glycogen content

1 Prolonged exercise results in a progressive decline in glycogen content and a concomitant increase in the release of the cytokine interleukin‐6 (IL‐6) from contracting muscle. This study tests the hypothesis that the exercise‐induced IL‐6 release from contracting muscle is linked to the intramuscular glycogen availability. 2 Seven men performed 5 h of a two‐legged knee‐extensor exercise, with one leg with normal, and one leg with reduced, muscle glycogen content. Muscle biopsies were obtained before (pre‐ex), immediately after (end‐ex) and 3 h into recovery (3 h rec) from exercise in both legs. In addition, catheters were placed in one femoral artery and both femoral veins and blood was sampled from these catheters prior to exercise and at 1 h intervals during exercise and into recovery. 3 Pre‐exercise glycogen content was lower in the glycogen‐depleted leg compared with the control leg. Intramuscular IL‐6 mRNA levels increased with exercise in both legs, but this increase was augmented in the leg having the lowest glycogen content at end‐ex. The arterial plasma concentration of IL‐6 increased from 0.6 ± 0.1 ng l−1 pre‐ex to 21.7 ± 5.6 ng l−1 end‐ex. The depleted leg had already released IL‐6 after 1 h (4.38 ± 2.80 ng min−1 (P < 0.05)), whereas no significant release was observed in the control leg (0.36 ± 0.14 ng min−1). A significant net IL‐6 release was not observed until 2 h in the control leg. 4 This study demonstrates that glycogen availability is associated with alterations in the rate of IL‐6 production and release in contracting skeletal muscle.

Searching for the exercise factor: is IL-6 a candidate?

For years the search for the stimulus that initiates and maintains the change of excitability or sensibility of the regulating centers in exercise has been progressing. For lack of more precise knowledge, it has been called the ‘work stimulus’, ‘the work factor’ or ‘the exercise factor’. In other terms, one big challenge for muscle and exercise physiologists has been to determine how muscles signal to central and peripheral organs. Here we discuss the possibility that interleukin-6 (IL-6) could mediate some of the health beneficial effects of exercise. In resting muscle, the IL-6 gene is silent, but it is rapidly activated by contractions. The transcription rate is very fast and the fold changes of IL-6 mRNA is marked. IL-6 is released from working muscles into the circulation in high amounts. The IL-6 production is modulated by the glycogen content in muscles, and IL-6 thus works as an energy sensor. IL-6 exerts its effect on adipose tissue, inducing lipolysis and gene transcription in abdominal subcutaneous fat and increases whole body lipid oxidation. Furthermore, IL-6 inhibits low-grade TNF-α-production and may thereby inhibit TNF-α-induced insulin resistance and atherosclerosis development. We propose that IL-6 and other cytokines, which are produced and released by skeletal muscles, exerting their effects in other organs of the body, should be named ‘myokines’.

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.

Muscle-derived interleukin-6: Lipolytic, anti-inflammatory and immune regulatory effects

Interleukin-6 (IL-6) is produced locally in working skeletal muscle and can account for the exercise-induced increase in plasma IL-6. The transcription rate for IL-6 in muscle nuclei isolated from muscle biopsies during exercise is very high and is enhanced further when muscle glycogen content is low. Furthermore, cultured human primary muscle cells can increase IL-6 mRNA when incubated with the calcium ionophore ionomycin and it is likely that myocytes produce IL-6 in response to muscle contraction. The biological roles of muscle-derived IL-6 have been investigated in studies in which human recombinant IL-6 was infused in healthy volunteers to mimic closely the IL-6 concentrations observed during prolonged exercise. Using stable isotopes, we have demonstrated that physiological concentrations of IL-6 induce lipolysis. Although we have yet to determine the precise biological action of muscle-derived IL-6, our data support the hypothesis that the role of IL-6 released from contracting muscle during exercise is to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery during exercise. In addition, IL-6 inhibits low-level TNF-α production, and IL-6 produced during exercise probably inhibits TNF-α-induced insulin resistance in peripheral tissues. Hence, IL-6 produced by skeletal muscle during contraction may play an important role in the beneficial health effects of exercise

Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle

To test the hypothesis that a decrease in intramuscular glycogen availability may stimulate heat shock protein expression, seven men depleted one leg of muscle glycogen the day before performing 4–5 h of exhaustive, two‐legged knee extensor exercise at 40 % of leg peak power output. Subjects then rested for a further 3 h. Muscle biopsies were obtained from the depleted and control leg before, immediately after and 3 h into recovery from exercise. These samples were analysed for muscle glycogen, and HSP72 gene and protein expression. In addition, catheters were placed in one femoral artery and both femoral veins and blood was sampled from these catheters prior to exercise and at 1 h intervals during exercise and into recovery for the measurement of arterial‐venous differences in serum HSP72. Plasma creatine kinase (CK) was also measured from arterial blood samples. Pre‐exercise muscle glycogen content was 40 % lower in the depleted compared with the control leg and this difference was maintained throughout the experiment (P < 0.05; main treatment effect). Neither HSP72 gene nor protein expression was different pre‐exercise. However, both HSP72 gene and protein increased (P < 0.05) post‐exercise in the depleted leg, but not in the control leg. Exercise did not increase plasma CK concentrations and we were unable to detect HSP72 in the serum of any samples. These results demonstrate that while acute, concentric exercise is capable of increasing HSP72 in human skeletal muscle, it does so only when glycogen is reduced to relatively low levels. Hence, our data suggest that HSP72 protein expression is related to glycogen availability. In addition, because CK did not increase and we found no evidence of HSP72 in the venous effluent, our data suggest that skeletal muscle is impermeable to HSP72.

Glucose ingestion attenuates interleukin-6 release from contracting skeletal muscle in humans

To examine whether glucose ingestion during exercise affects the release of interleukin‐6 (IL‐6) from the contracting limb, seven men performed 120 min of semi‐recumbent cycling on two occasions while ingesting either 250 ml of a 6.4 % carbohydrate (GLU trial) or sweet placebo (CON trial) beverage at the onset of, and at 15 min intervals throughout, exercise. Muscle biopsies obtained before and immediately after exercise were analysed for glycogen and IL‐6 mRNA expression. Blood samples were simultaneously obtained from a brachial artery and a femoral vein prior to and during exercise and leg blood flow was measured by thermodilution in the femoral vein. Net leg IL‐6 release, and net leg glucose and free fatty acid (FFA) uptake, were calculated from these measurements. The arterial IL‐6 concentration was lower (P < 0.05) after 120 min of exercise in GLU, but neither intramuscular glycogen nor IL‐6 mRNA were different when comparing GLU with CON. However, net leg IL‐6 release was attenuated (P < 0.05) in GLU compared with CON. This corresponded with an enhanced (P < 0.05) glucose uptake and a reduced (P < 0.05) FFA uptake in GLU. These results demonstrate that glucose ingestion during exercise attenuates leg IL‐6 release but does not decrease intramuscular expression of IL‐6 mRNA.