Charlotte Keller

IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin

Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.

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.

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

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.

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.

Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise

Interleukin‐6 (IL‐6) is produced by many different cell types. Human skeletal muscles produce and release high amounts of IL‐6 during exercise; however, the cell source of origin in the muscle is not known. Therefore, we studied the protein expression of IL‐6 by immunohistochemistry in human muscle tissue from biopsies obtained at time points 0, 3, 4.5, 6, 9, and 24 h in relation to 3 h of bicycle exercise performed by healthy young males (n=12) and in resting controls (n=6). The IL‐6 expression was clearly increased after exercise and remained high even by 24 h, relative to pre‐exercise or resting individuals. The IL‐6 immunostainings of skeletal muscle cells were homogeneous and without difference between muscle fiber types. The IL‐6 mRNA peaked immediately after the exercise, and, in accordance, the IL‐6 protein expression within muscle cells was most pronounced around 3 h post‐exercise. However, the finding that plasma IL‐6 concentration peaked in the end of exercise indicates a high turnover of muscle‐derived IL‐6. In conclusion, the finding of marked IL‐6 protein expression exclusively within skeletal muscle fibers following exercise demonstrates that skeletal muscle fibers of all types are the dominant cell source of exercise‐induced release of IL‐6 from working muscle.

Interleukin-6 Regulation of AMP-Activated Protein Kinase: Potential Role in the Systemic Response to Exercise and Prevention of the Metabolic Syndrome

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

Acute interleukin‐6 administration does not impair muscle glucose uptake or whole‐body glucose disposal in healthy humans

The cytokine interleukin (IL)‐6 has recently been linked with type 2 diabetes mellitus and has been suggested to affect glucose metabolism. To determine whether acute IL‐6 administration affects whole‐body glucose kinetics or muscle glucose uptake, 18 healthy young men were assigned to one of three groups receiving a high dose of recombinant human IL‐6 (HiIL‐6; n= 6), a low dose of IL‐6 (LoIL‐6; n= 6) or saline (Con; n= 6) infused into one femoral artery for 3 h. The stable isotope [6,6‐2H2] glucose was infused into a forearm vein throughout the 3 h infusion period and for a further 3 h after the cessation of infusion (recovery) to determine endogenous glucose production and whole‐body glucose disposal. Infusion with HiIL‐6 and LoIL‐6 resulted in a marked (P < 0.05) increase in systemic IL‐6 concentration throughout the 3 h of infusion (mean arterial plasma [IL‐6]s of 319 and 143 pg ml−1 for HiIL‐6 and LoIL‐6, respectively), followed by a rapid decline (P < 0.05) during the recovery period. Subjects experienced clinical symptoms such as shivering and discomfort during HiIL‐6 administration, but were asymptomatic during LoIL‐6 administration. In addition, only HiIL‐6 elevated (P < 0.05) plasma adrenaline (epinephrine). IL‐6 infusion, irrespective of dose, did not result in any changes to endogenous glucose production, whole‐body glucose disposal or leg‐ glucose uptake. These data demonstrate that acute IL‐6 administration does not impair whole‐body glucose disposal, net leg‐glucose uptake, or increase endogenous glucose production at rest in healthy young humans.