The challenge arising with this hypothesis

The challenge arising with this hypothesis is to identify the stimuli that occur at different concentrations depending on the exercise intensity. One possible candidate is Lactate (La). It has long been considered a metabolic waste product and the cause of decrease in muscle pH and hence muscle fatigue. This idea has changed massively in the past. Now it is known that La is rather an intermediate of glucose metabolism, acting as an energy substrate and a gluconeogenic precursor. It has also been termed Lactormon for its signalling properties, inducing gene expression necessary for skeletal muscle kv1.3 inhibitor (Brooks et al., 2008) as Hashimoto et al. could demonstrate that La increases MCT1 and PGC1α mRNA content (Hashimoto et al., 2007). Further investigations by the same group led to the conclusion that most likely these effects are mediated by reactive oxygen species (ROS) as hydrogen peroxide (H2O2) has been shown to increase and that the adaptations caused are most likely generated via a vast oxygen-radical sensitive network and subsequent mitogen activated protein kinase (MAPK) signalling (Hashimoto et al., 2007).
Materials and methods
Results
Discussion The primary novel and most important finding of this study is that La induces withdrawal from the cell cycle and early differentiation of C2C12 cells, but delays late differentiation in a timely and dose-dependent manner. In cell culture experiments permanent withdrawal from the cell cycle (as necessary for early differentiation) is due to serum deprivation (Yaffe and Saxel, 1977b; Kitzmann et al., 1998) leading to changes in cell cycle regulators (Kitzmann and Fernandez, 2001). Hence, one would expect a reduction in proliferating cells after the medium switch. However, data from both the Ki67-analysis as well as the BrdU assay show that proliferation rates in control cells are much higher. Therefore we conclude that La enhances the changes in cell cycle regulation induced by serum withdrawal. Furthermore, a delay of the appearance of differentiation markers is reported here. Pax7 and Myf5 are increased with La treatment, whereas markers for the late differentiation phase (myogenin and MHC) are decreased indicating persistence of early differentiation phase of C2C12 cells after 5days of La treatment. Hence, our data suggests that La is additionally a potent regulator of gene expression during C2C12 myogenesis. The second novel finding is that La induces oxidative stress within C2C12 myoblasts. Our observations clearly demonstrate that 2h incubation with 20mM La DM induces 8-epi-PGF2α levels to rise. Adding AA, NAc, or LA to the 20mM La DM leads to a reduction of 8-epi-PGF2α levels, additionally establishing that ROS formation by La can be reversed by the use of antioxidants. Interestingly, the areas around the nuclei were mostly affected by oxidative stress where mitochondrial density is highest within the cell. We therefore additionally argue that La leads to increased ROS formation mostly within the mitochondrial membranes. This notion is supported by our finding that AA and NAc showed the highest efficacy of ROS scavenging. AA and NAc, in contrast to LA, unfold their direct antioxidative capacity mainly in the mitochondrion (Banaclocha, 2001; Nordberg and Arnér, 2001; Moreira et al., 2007; Mandl et al., 2009; Gillissen, 2011). Both substances were able to reduce the oxidative stress even below control levels. The mechanism by which LA deploys its antioxidative capacity remains elusive, but it has been described to act by the upregulation of the antioxidative enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (Cat) (Yu et al., 2013) which suggests a time-delayed, and hence a weakened effect for the time period observed. Nonetheless, further experiments are necessary to provide clear evidence of the idea that mitochondrial ROS generation is essentially elevated by La. The La-induced formation of ROS is controversially discussed in the literature. On the one hand, La was shown to be a capable scavenger of ROS in the absence of cells (Anbar and Neta, 1967; Groussard et al., 2000; Lampe et al., 2009). Whereas this finding was shown to be also present in cultured hepatocytes (Groussard et al., 2000), a protective effect was not established in neuronal precursor cells (Lampe et al., 2009). In contrast, other studies demonstrated that La increases ROS formation. One report describes the La-dependent enhancement of hydroxyl radical generation by the Fenton-reaction in a cell-free model (Ali et al., 2000). Hashimoto et al. (2007) showed an increased production of H2O2 in L6 myoblasts by 20mM La incubation indicating increased oxidative stress which is in agreement with our findings. Furthermore, the results for act. Casp-3 imply that La is able to induce apoptotic events, i.e. cellular stress. Whereas no data on the ability of La to induce the apoptotic pathway in C2C12 exists, ROS have been described to be able to initiate programmed cell death in this type of cells (Nishida et al., 2007; Gilliam et al., 2012; Lee et al., 2013). Our results unambiguously show that with La treatment, the oxidative stress increases within the cells. Therefore the rise in cleaved act. Casp-3 within cells is not surprising and provides further evidence for the conclusion that La induces cellular stress, marked by the increased generation of ROS.