Fig. 8.

Schematic model of the regulation of Nrf2 and Keap1 signalling in human skeletal muscle immediately after incremental exercise to exhaustion in normoxia and severe hypoxia. Under basal conditions, Keap1 continuously targets Nrf2 for ubiquitination and degradation, allowing for minimal levels of Nrf2. The production of RONS during exhaustive exercise stimulates the activation of AMPK and CaMKII. Concomitantly, CaMKII acts indirectly as an upstream AMPK activator (by an unknown mechanism). AMPK promotes the increase of Nrf2 levels by two main mechanisms. Firstly, by phosphorylating p62 at Ser³⁴⁹, which stimulates the p62-mediated degradation of Keap1 via autophagy; secondly, by phosphorylating and blocking GSK3-β, which activates β-TrCP (an E3 ubiquitin-protein ligase) which tags Nrf2 for proteasomal degradation (not measured here). RONS may also activate PKCδ which phosphorylates Nrf2 at its Serine 40 promoting its nuclear translocation and genes transactivation. The lowered levels of Keap1 and the reduced amount of p62 observed here are suggestive of co-degradation following exercise. Overall, the augmented Nrf2 total and phosphorylated protein expression together with the rise in the Nrf2-to-Keap1 ratio elicited by exhausting exercise should be sufficient to enhance the Nrf2-mediated antioxidant response. A central role of Catalase is manifested by a remarkable increase in its protein content following exercise, which was exacerbated during exercise in severe acute hypoxia, likely as a response to increased H₂O₂ production, by superoxide dismutases. This process is facilitated in hypoxia due to the upregulation of SOD1. No acute changes in SOD2 protein expression were observed. Most changes evoked by the exhaustive exercise bout were almost entirely reverted to baseline in less than 60 s by an O₂-dependent mechanism. Activating/inhibiting actions are represented by blue/red connecting lines (dashed if the effect is indirect). Changes on cellular locations are presented with black dashed lines. The arrows and symbols depicted inside dashed grey boxes and located beside the specific markers illustrate the overall protein expression changes in this investigation, as follows: Thin arrows in green (phosphorylated form) and black (total form) depict the overall direction of the outcomes (increase/decrease) for the particular muscle protein; thick arrows in darker green represent the overall effect on stimulation/inhibition of Nrf2 signalling; the symbol § indicates a significant difference between the biopsies taken 60 s after the end of the exercise, i.e., between the legs recovering with and without ischaemia. A differential modulation due to F_IO₂ is illustrated by the presence of arrows in red (normoxia) and blue (severe hypoxia). The size of each arrow is commensurate with the magnitude of the change. Abbreviations not defined in the text: ARE, antioxidant response element; OS: oxidative stress. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)