Abstract
Regular exercise has emerged as one of the best therapeutic strategies to prevent and treat type-2-diabetes. Exercise-induced changes in the muscle secretome, consisting of myokines and metabolites, may underlie the inter-organ communication between muscle and other organs. To investigate this crosstalk, we developed an in vitro system in which mouse C2C12 myotubes underwent electric pulse stimulation (EPS) to induce contraction. Subsequently the effects of EPS-conditioned media (EPS-CM) on hepatocytes were investigated. Here, we demonstrate that EPS-CM induces Metallothionein 1/2 and Slc30a2 gene expression and reduces Cyp2a3 gene expression in rat hepatocytes. When testing EPS-CM that was generated in the absence of C2C12 myotubes (non-cell EPS-CM) no decrease in Cyp2a3 expression was detected. However, similar inductions in hepatic Mt1/2 and Slc30a2 expression were observed. Non-cell EPS-CM were also applied to C2C12 myotubes and compared to C2C12 myotubes that underwent EPS: here changes in AMPK phosphorylation and myokine secretion largely depended on EPS-induced contraction. Taken together, these findings indicate that EPS can alter C2C12 myotube function and thereby affect gene expression in cells subjected to EPS-CM (Cyp2a3). However, EPS can also generate non-cell-mediated changes in cell culture media, which can affect gene expression in cells subjected to EPS-CM too. While EPS clearly represents a valuable tool in exercise research, care should be taken in experimental design to control for non-cell-mediated effects.
Highlights
Over the past few decades, the prevalence of obesity and its associated complications, type 2 diabetes and cardiovascular disease, have increased to a great extent[1,2]
Direct effects of electric pulse stimulation (EPS) on C2C12 myotube function (AMPK phosphorylation, myokine secretion) were mainly caused by EPS-induced contraction and not due to EPS-induced changes in cell culture media. These findings indicate that EPS clearly represents a valuable tool in exercise research, but care should be taken in experimental design to control for non-cell-mediated effects
The relatively mild induction of Il-6 secretion was largely due to EPS-induced contraction (Fold change 3.7 vs 1.2; Fig. 5D). These findings indicate that EPS can induce cell-dependent changes in C2C12 myotubes (AMPK phosphorylation, myokine secretion), but can generate non-cell-mediated changes in cell culture media, which can affect gene expression in cells subjected to EPS-Conditioned media (CM) (e.g. Mt1/2 and Slc30a2 in hepatocytes)
Summary
Over the past few decades, the prevalence of obesity and its associated complications, type 2 diabetes and cardiovascular disease, have increased to a great extent[1,2]. Cultured myotubes (mouse or human) have been used in several studies to identify myokines, often subjected to electric pulse stimulation (EPS) to induce contraction and thereby mimic exercise[12,13] Such approaches have for example resulted in the identification of Leukemia Inhibitory Factor (LIF)[14], pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4)[12] as exercise-induced myokines. At least one of these metabolites, β -aminoisobutyric acid (BAIBA), may function in an endocrine fashion, as it has been suggested to induce browning of white adipose tissue and increase β -oxidation in the liver[16] While these recent studies provide proof-of-principle that myokines and/or muscle-derived metabolites can mediate the crosstalk between exercising muscle and distant target organs (especially white adipose tissue8,9,16), effects on the liver were not always investigated. These findings indicate that EPS clearly represents a valuable tool in exercise research, but care should be taken in experimental design to control for non-cell-mediated effects
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