Nuclear receptors and myokines: mediators of exercise-induced skeletal muscle metabolism

Abstract

Skeletal muscle is a crucial organ in mediating (exercise-induced) beneficial health effects. In this thesis we gained important knowledge on the molecular biology of the muscle. With our focus on the muscle, we investigated the crosstalk with other organs, the regulation of myokines and the role of nuclear receptors. First, we used an exercise mimic, electric pulse stimulation (EPS), to investigate the effect of the exercise-induced muscle secretome on the liver. We subjected C2C12 myotubes to EPS and hepatocytes were treated with this contraction-induced secretome. We demonstrate that EPS can induce both cell-mediated and non-cell-mediated effects on hepatocytes. Direct effects of EPS on C2C12 myotube function were mainly caused by EPS-induced contraction and not due to EPS-induced changes in cell culture media. This indicates 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. Secondly, we assessed the importance of cellular context in Nur77-mediated gene regulation by comparing genome-wide DNA binding and gene expression profiles in C2C12 myotubes and RAW264.7 macrophages. These experiments revealed predominantly cell-type specific DNA binding of Nur77, with less than 16% of the Nur77 binding sites being shared between the two cell types. In addition, cell-type specific transcriptional regulation was observed, with genes differentially regulated in the same direction, in one cell-type only or even in an opposing manner. Newly identified Nur77 target genes include the genes encoding the myokines IL-15 and MCP-1. We suggest that besides Nur77 DNA binding itself, other mechanisms, including interplay with cell-specific factors, determine whether a potential Nur77 target gene is actually activated or repressed in a given cellular context. The third aim was to assess to what extent elevation of the myokine MCP-1 in muscle and elevated MCP-1 levels in plasma may be able to interfere with insulin signaling in skeletal muscle and cause insulin resistance. We made use of MCK-MCP-1 transgenic mice that specifically over-expresses MCP-1 in skeletal muscle. Our studies indicate that elevation of MCP-1 production in skeletal muscle and elevated MCP-1 levels in plasma promote inflammation in skeletal muscle but do not influence insulin signaling in skeletal muscle and have no effect on insulin sensitivity and glucose tolerance in lean and obese mice. Our data argue against MCP-1 promoting insulin resistance in skeletal muscle and raises questions about the impact of inflammation on insulin sensitivity in muscle. Last, we elucidated the impact of the first natural human LXRβ mutation on LXRβ function. By exome, sequencing, an alanine-to-threonine mutation in the LBD of LXRβ at the position 440 (LXRβ-A440T) was identified in a patient with several disorders (e.g. extremely fatty muscle). We demonstrate that this mutation results in a loss-of-function for classical LXRβ targets genes (Abcg1), while it also results in a gain-of-function of new targets genes (Gpr56) in C2C12 muscle cells.