Mark A. Febbraio

Muscle-derived interleukin-6: mechanisms for activation and possible biological roles

It has recently been demonstrated that the marked increase in the systemic concentration of cytokine interleukin‐6 (IL‐6) seen with exercise originates from the contracting limb and that skeletal muscle cells per se are the likely source of the production. This review summarizes the possible mechanisms for activation and biological consequences of muscle‐derived IL‐6. It appears that intramuscular IL‐6 is stimulated by complex signaling cascades initiated by both calcium (Ca2+) ‐dependent and ‐independent stimuli. It also seems likely that skeletal muscle produces IL‐6 to aid in maintaining metabolic homeostasis during periods of altered metabolic demand such as muscular exercise or insulin stimulation. It may do so via local and/or systemic effects. This review also explores the efficacy that IL‐6 may be used as a therapeutic drug in treating metabolic disorders such as obesity, type 2 diabetes, and atherosclerosis.—Febbraio, M. A., Pedersen, B. K. Muscle‐derived interleukin 6: mechanisms for activation and possible biological roles. FASEB J. 16, 1335–1347 (2002)

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’.

Exercise increases serum Hsp72 in humans

Abstract Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87 ± 0.24 and 1.02 ± 0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

Effects of heat stress on physiological responses and exercise performance in elite cyclists

This study examined the effect of heat stress on physiological responses and exercise performance in elite road cyclists. Eleven members of the Australian National Road Cycling Squad completed two 30 min cycling time-trials in an environmental chamber set at either 32 degrees C, (HT) or 23 degrees C (NT) with a relative humidity of 60% in each circumstance. The trials were separated by two days, with six subjects performing HT first. Power output was 6.5% lower (P<0.05) during HT compared with NT. Mean skin temperature and sweat rate were higher (P<0.05) in HT compared with NT. In contrast, rectal temperature was remarkably similar throughout each trial. During the first 10 min of exercise in HT when power output was not different between trials, blood lactate was higher (P<0.05), and blood pH lower (P<0.05). In contrast, during the last 10 min of exercise when power output was reduced (P<0.05), blood lactate was lower (P<0.05), and pH higher (P<0.05), in HT. These data indicate that heat stress is associated with a reduced power output during self-paced exercise in highly trained men. This decrease in performance appears to be associated with factors associated with body temperature rather than metabolic capacity.

The metabolic role of IL-6 produced during exercise: is IL-6 an exercise factor?

For most of the last century, researchers have searched for a muscle contraction-induced factor that mediates some of the exercise effects in other tissues such as the liver and the adipose tissue. It has been called the work stimulus, the work factor or the exercise factor. In the search for such a factor, a cytokine, IL-6, was found to be produced by contracting muscles and released into the blood. It has been demonstrated that IL-6 has many biological roles such as: (1) induction of lipolysis; (2) suppression of TNF production; (3) stimulation of cortisol production. The IL-6 gene is rapidly activated during exercise, and the activation of this gene is further enhanced when muscle glycogen content is low. In addition, carbohydrate supplementation during exercise has been shown to inhibit the release of IL-6 from contracting muscle. Thus, it is suggested that muscle-derived IL-6 fulfils the criteria of an exercise factor and that such classes of cytokines could be termed myokines.

Carbohydrate ingestion attenuates the increase in plasma interleukin‐6, but not skeletal muscle interleukin‐6 mRNA, during exercise in humans

The present study was undertaken to examine the effects of exercise and carbohydrate (CHO) ingestion on interleukin‐6 (IL‐6) gene expression in skeletal muscle and plasma IL‐6 concentration. Seven moderately trained men completed 60 min of exercise at a workload corresponding to each individuals lactate threshold on four randomised occasions. Two trials were conducted on a bicycle ergometer (Cyc) and two on a running treadmill (Run) either with (CHO) or without (Con) the ingestion of a CHO beverage throughout the exercise. Muscle biopsies were obtained from the vastus lateralis before and immediately after exercise and IL‐6 gene expression in these samples was determined using real‐time PCR. In addition, venous blood samples were collected at rest, and after 30 min during and at the cessation of exercise. These samples were analysed for plasma IL‐6. Irrespective of exercise mode or CHO ingestion, exercise resulted in a 21 ± 4‐fold increase (P < 0.01; main exercise effect) in IL‐6 mRNA expression. In contrast, while the mode of exercise did not affect the exercise‐induced increase in plasma IL‐6, CHO ingestion blunted (P < 0.01) this response. These data demonstrate that CHO ingestion attenuates the plasma IL‐6 concentration during both cycling and running exercise. However, because IL‐6 mRNA expression was unaffected by CHO ingestion, it is likely that the ingestion of CHO during exercise attenuates IL‐6 production by tissues other than skeletal muscle.

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.

Exercise induces hepatosplanchnic release of heat shock protein 72 in humans

Physical exercise results in the appearance of heat shock protein (HSP) 72 in the circulation that precedes any increase in gene or protein expression in contracting skeletal muscle. In rodents, exercise increases liver HSP72 expression and the hepatosplanchnic viscera are known to release many acute phase proteins. In the present study, we tested the hypothesis that the splanchnic tissue beds release HSP72 during exercise. Seven male subjects performed 120 min of semi‐recumbent cycling at 62 ± 2 % of maximal oxygen uptake. Blood samples were obtained simultaneously from a brachial artery, a femoral vein and the hepatic vein prior to and at 30, 60 and 120 min of exercise. Leg blood flow (LBF) was measured by thermodilution in the femoral vein, and hepatosplanchnic blood flow (HBL) was measured using indocyanine green dye. Net leg and net hepatosplanchnic HSP72 balance were calculated as the product of LBF and femoral venous‐arterial HSP72 difference and the product of HBF and hepatic venous‐arterial HSP72 difference, respectively. Arterial plasma HSP72 was only detected in one subject at rest but progressively appeared in the arterial samples throughout exercise such that at 120 min it was detected in all subjects (0.88 ± 0.35 pg l−1; P < 0.05 compared with rest). The contracting muscle did not, however, contribute to this increase since there was no difference in the femoral venous‐arterial HSP72 concentration at any time. Rather, the increase in arterial HSP72 was accounted for, at least in part, by release from the hepatosplanchnic viscera with values increasing (P < 0.05) from undetectable levels at rest to 5.2 ± 0.2 pg min−1 after 120 min. These data demonstrate that the splanchnic tissues release HSP72 during exercise and this release is responsible, in part, for the elevated systemic concentration of this protein during exercise.