Claus Brandt

The Role of Exercise-Induced Myokines in Muscle Homeostasis and the Defense against Chronic Diseases

Chronic inflammation is involved in the pathogenesis of insulin resistance, atherosclerosis, neurodegeneration, and tumour growth. Regular exercise offers protection against type 2 diabetes, cardiovascular diseases, colon cancer, breast cancer, and dementia. Evidence suggests that the protective effect of exercise may to some extent be ascribed to the antiinflammatory effect of regular exercise. Here we suggest that exercise may exert its anti-inflammatory effect via a reduction in visceral fat mass and/or by induction of an anti-inflammatory environment with each bout of exercise. According to our theory, such effects may in part be mediated via muscle-derived peptides, so-called “myokines”. Contracting skeletal muscles release myokines with endocrine effects, mediating direct anti-inflammatory effects, and/or specific effects on visceral fat. Other myokines work locally within the muscle and exert their effects on signalling pathways involved in fat oxidation and glucose uptake. By mediating anti-inflammatory effects in the muscle itself, myokines may also counteract TNF-driven insulin resistance. In conclusion, exercise-induced myokines appear to be involved in mediating both systemic as well as local anti-inflammatory effects.

Exercise Induces a Marked Increase in Plasma Follistatin: Evidence That Follistatin Is a Contraction-Induced Hepatokine

Follistatin is a member of the TGF-β super family and inhibits the action of myostatin to regulate skeletal muscle growth. The regulation of follistatin during physical exercise is unclear but may be important because physical activity is a major intervention to prevent age-related sarcopenia. First, healthy subjects performed either bicycle or one-legged knee extensor exercise. Arterial-venous differences were assessed during the one-legged knee extensor experiment. Next, mice performed 1 h of swimming, and the expression of follistatin was examined in various tissues using quantitative PCR. Western blotting assessed follistatin protein content in the liver. IL-6 and epinephrine were investigated as drivers of follistatin secretion. After 3 h of bicycle exercise, plasma follistatin increased 3 h into recovery with a peak of 7-fold. No net release of follistatin could be detected from the exercising limb. In mice performing a bout of swimming exercise, increases in plasma follistatin as well as follistatin mRNA and protein expression in the liver were observed. IL-6 infusion to healthy young men did not affect the follistatin concentration in the circulation. When mice were stimulated with epinephrine, no increase in the hepatic mRNA of follistatin was observed. This is the first study to demonstrate that plasma follistatin is increased during exercise and most likely originates from the liver. These data introduce new perspectives regarding muscle-liver cross talk during exercise and during recovery from exercise.

Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth

Regular physical activity protects against the development of breast and colon cancer, since it reduces the risk of developing these by 25-30%. During exercise, humoral factors are released from the working muscles for endocrinal signaling to other organs. We hypothesized that these myokines mediate some of the inhibitory effects of exercise on mammary cancer cell proliferation. Serum and muscles were collected from mice after an exercise bout. Incubation with exercise-conditioned serum inhibited MCF-7 cell proliferation by 52% and increased caspase activity by 54%. A similar increase in caspase activity was found after incubation of MCF-7 cells with conditioned media from electrically stimulated myotubes. PCR array analysis (CAPM-0838E; SABiosciences) revealed that seven genes were upregulated in the muscles after exercise, and of these oncostatin M (OSM) proved to inhibit MCF-7 proliferation by 42%, increase caspase activity by 46%, and induce apoptosis. Blocking OSM signaling with anti-OSM antibodies reduced the induction of caspase activity by 51%. To verify that OSM was a myokine, we showed that it was significantly upregulated in serum and in three muscles, tibialis cranialis, gastronemius, and soleus, after an exercise bout. In contrast, OSM expression remained unchanged in subcutaneous and visceral adipose tissue, liver, and spleen (mononuclear cells). We conclude that postexercise serum inhibits mammary cancer cell proliferation and induces apoptosis of these cells. We suggest that one or more myokines secreted from working muscles may be mediating this effect and that OSM is a possible candidate. These findings emphasize that role of physical activity in cancer treatment, showing a direct link between exercise-induced humoral factors and decreased tumor cell growth.

Erythropoietin Over-Expression Protects against Diet-Induced Obesity in Mice through Increased Fat Oxidation in Muscles

Erythropoietin can be over-expressed in skeletal muscles by gene electrotransfer, resulting in 100-fold increase in serum EPO and significant increases in haemoglobin levels. Earlier studies have suggested that EPO improves several metabolic parameters when administered to chronically ill kidney patients. Thus we applied the EPO over-expression model to investigate the metabolic effect of EPO in vivo. At 12 weeks, EPO expression resulted in a 23% weight reduction (P<0.01) in EPO transfected obese mice; thus the mice weighed 21.9±0.8 g (control, normal diet,) 21.9±1.4 g (EPO, normal diet), 35.3±3.3 g (control, high-fat diet) and 28.8±2.6 g (EPO, high-fat diet). Correspondingly, DXA scanning revealed that this was due to a 28% reduction in adipose tissue mass. The decrease in adipose tissue mass was accompanied by a complete normalisation of fasting insulin levels and glucose tolerance in the high-fat fed mice. EPO expression also induced a 14% increase in muscle volume and a 25% increase in vascularisation of the EPO transfected muscle. Muscle force and stamina were not affected by EPO expression. PCR array analysis revealed that genes involved in lipid metabolism, thermogenesis and inflammation were increased in muscles in response to EPO expression, while genes involved in glucose metabolism were down-regulated. In addition, muscular fat oxidation was increased 1.8-fold in both the EPO transfected and contralateral muscles. In conclusion, we have shown that EPO when expressed in supra-physiological levels has substantial metabolic effects including protection against diet-induced obesity and normalisation of glucose sensitivity associated with a shift to increased fat metabolism in the muscles.

LIF is a contraction-induced myokine stimulating human myocyte proliferation

The cytokine leukemia inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of myoblasts. We hypothesized that LIF is a contraction-induced myokine functioning in an autocrine fashion to activate gene regulation of human muscle satellite cell proliferation. Skeletal muscle LIF expression, regulation, and action were examined in two models: 1) young men performing a bout of heavy resistance exercise of the quadriceps muscle and 2) cultured primary human satellite cells. Resistance exercise induced a ninefold increase in LIF mRNA content in skeletal muscle, but LIF was not detectable in plasma of the subjects. However, electrically stimulated cultured human myotubes produced and secreted LIF, suggesting that LIF is a myokine with local effects. The well established exercise-induced signaling molecules PI3K, Akt, and mTor contributed to the regulation of LIF in cultured human myotubes as chemical inhibition of PI3K and mTor and siRNA knockdown of Akt1 were independently sufficient to downregulate LIF. Human myoblast proliferation was increased by recombinant exogenous LIF and decreased by siRNA knockdown of the endogenous LIF receptor. Finally, the transcription factors JunB and c-Myc, which promote myoblast proliferation, were induced by LIF in cultured human myotubes. Indeed, both JunB and c-Myc were also increased in skeletal muscle following resistance exercise. Our data suggest that LIF is a contraction-induced myokine, potentially acting in an autocrine or paracrine fashion to promote satellite cell proliferation.

Plasma and Muscle Myostatin in Relation to Type 2 Diabetes

Objective Myostatin is a secreted growth factor expressed in skeletal muscle tissue, which negatively regulates skeletal muscle mass. Recent animal studies suggest a role for myostatin in insulin resistance. We evaluated the possible metabolic role of myostatin in patients with type 2 diabetes and healthy controls. Design 76 patients with type 2 diabetes and 92 control subjects were included in the study. They were matched for age, gender and BMI. Plasma samples and biopsies from the vastus lateralis muscle were obtained to assess plasma myostatin and expression of myostatin in skeletal muscle. Results Patients with type 2 diabetes had higher fasting glucose (8.9 versus 5.1 mmol/L, P<0.001), plasma insulin (68.2 versus 47.2 pmol/L, P<0.002) and HOMA2-IR (1.6 versus 0.9, P<0.0001) when compared to controls. Patients with type 2 diabetes had 1.4 (P<0.01) higher levels of muscle myostatin mRNA content than the control subjects. Plasma myostatin concentrations did not differ between patients with type 2 diabetes and controls. In healthy controls, muscle myostatin mRNA correlated with HOMA2-IR (r = 0.30, P<0.01), plasma IL-6 (r = 0.34, P<0.05) and VO2 max (r = −0.26, P<0.05), however, no correlations were observed in patients with type 2 diabetes. Conclusions This study supports the idea that myostatin may have a negative effect on metabolism. However, the metabolic effect of myostatin appears to be overruled by other factors in patients with type 2 diabetes.

Interleukin-18 activates skeletal muscle AMPK and reduces weight gain and insulin resistance in mice

Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the α-isoform of the IL-18 receptor (IL-18R−/−) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R−/− mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.

Exercise-induced liver chemokine CXCL-1 expression is linked to muscle-derived interleukin-6 expression

Non‐technical summary  Exercise is known to stimulate the production of various exercise factors including the well‐described muscle‐derived interleukin‐6 (IL‐6). We show that exercise causes a massive expression of the chemokine CXCL‐1 in serum, in skeletal muscle and especially in the liver. Furthermore we find that this exercise‐induced liver CXCL‐1 expression is regulated by IL‐6 and that muscle‐derived IL‐6 is capable of stimulating liver CXCL‐1 expression. Such knowledge of the regulation of exercise factors contributes to the understanding of how the liver and muscle communicate in response to exercise.

IL-6 regulates exercise and training-induced adaptations in subcutaneous adipose tissue in mice.

Aim:  The aim of this study was to test the hypothesis that IL‐6 regulates exercise‐induced gene responses in subcutaneous adipose tissue in mice.

Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response to LIF. The mRNA and protein expressions of LIF and its receptor (LIFR) were measured in skeletal muscle biopsies from healthy individuals and patients with type 2 diabetes by use of qPCR and Western blot. LIF signaling and response were studied following administration of recombinant LIF and siRNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts from diabetic patients. Nonetheless, in the diabetic myoblasts, LIF-induced phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 was impaired. The deficient response to LIF administration in the diabetic myoblasts was further emphasized by a lack of increase in LIF-stimulated cell proliferation and a decreased LIF-stimulated induction of the proliferation-promoting factors cyclin D1, JunB, and c-myc. SOCS3 protein was upregulated in diabetic myoblasts, and knockdown of SOCS3 rescued LIF-induced gene expression in diabetic myoblasts, whereas neither STAT1 or STAT3 signaling nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function is compromised in diabetes.