*2.2. Cell Signaling*

Among the key intracellular signals influencing skeletal muscle adaptations to endurance training are changes in the AMP:ATP ratio, contraction-induced changes in mechanical strain, increased calcium flux, an increase in RONS, and the availability of endogenous CHO and FFA [7,8,74]. Nutritional intake has the potential to modify signaling across several of these pathways, primarily related to energy sensing and nutrient availability.

#### 2.2.1. Energy Sensing and the AMP-Activated Protein Kinase

The 5 AMP-activated protein kinase (AMPK) is a cellular energy sensor that regulates cellular and whole-body energy balance by inhibiting ATP-consuming pathways and activating ATP-producing pathways [75]. Activation of AMPK can lead to a range of metabolic adaptations including increases in glucose uptake, glycolytic flux, fat oxidation, and mitochondrial biogenesis [76]. The degree of AMPK activation during exercise can be influenced by exercise intensity [77], training status [78], muscle glycogen [79], and nutrient availability [80].

When starting exercise with normal muscle glycogen levels, studies that have shown a blunting effect of CHO ingestion on AMPKα2 activity [9,81] have been at lower intensities than those showing no di fferences between CHO-fed and fasted-state exercise [11,82]. Conversely, exercise that is undertaken with low, compared with normal muscle glycogen levels, has resulted in greater increases in the activity of AMPKα2 following 1 h of steady-state endurance exercise at 65–70% VO2max [83–85], but similar increases in AMPK activity and/or phosphorylation were seen following both exhaustive and non-exhaustive high-intensity exercise undertaken with high and low muscle glycogen levels [86–88]. Therefore, ingesting CHO before exercise may dampen AMPK activity during low but not high-intensity exercise, and an intensity threshold may exist below which CHO ingestion could blunt AMPK signaling.

The CHO content of the pre-exercise meal size could also influence molecular signaling. Compared with exercising in the fasted state, consumption of <70 g CHO prior to exercise had no e ffect [11,82] or even increased [10] skeletal muscle AMPK signaling following exercise compared with exercise performed in the fasted state. In contrast, ingesting 130–160 g of CHO before exercise reduced the exercise-induced increases in AMPKThr172 phosphorylation [89], with the phosphorylation of acetyl-CoA carboxylase (ACC) decreased [36] or unaffected [89]. When pooling a number of studies together, non-significant correlations can be observed between the exercise-induced increases in AMPK-α2 activation and CHO intake before exercise (Figure 7). Future studies that are designed to examine the relationships between meal size, exercise type and intensity, and AMPK activity are warranted.

Interpretation of the research comparing pre-exercise nutrition choices on AMPK activity during exercise is complicated by the small number of studies available, training status of participants, and specific markers being reported. For example, AMPK-α2 activity during exercise is reduced by short- and longer-term endurance training, making it difficult to compare between trained and untrained subjects [78,90,91]. Additionally, some studies report the phosphorylation of AMPKThr172, which reflects phosphorylation of both AMPK-α1 and -α2 subunits and may be less sensitive for detecting changes in AMPK activity that are only occurring in the -α2 subunit that is more responsive to exercise [81,82,86]. Further complicating interpretation of the available literature, several studies have shown a blunting effect of CHO ingestion on AMPK-α2 activity or AMPKThr172 phosphorylation, ye<sup>t</sup> similar increases in phosphorylation of ACC, a downstream substrate of AMPK [81,83,92]. Similar increases in PGC-1α mRNA expression following HIIT performed with low or high CHO availability have also been reported, despite phosphorylation of ACC being reduced by high CHO availability [36,93]. Furthermore, despite an attenuation of exercise-induced AMPK activation when ingesting CHO during a single bout of exercise [81], no differences in training adaptations were observed following 10 weeks of training with or without CHO ingestion during exercise [94]. These apparent discrepancies could be due to crosstalk between signaling pathways and/or the wide variability in exercise-induced changes in mRNA expression [95] and highlight the importance of looking at longer-term changes in mitochondrial content or function rather than acute changes in specific proteins.
