Distinguishing the effects of carbohydrate <i>vs</i> . lipid availability during exercise: Importance of glycogen depletion

The article ‘Isolating the effects of carbohydrate and lipid availability on exercise-induced skeletal muscle signalling in males’ by Bradshaw and colleagues addresses an important research gap on the independent effects of muscle glycogen and circulating free fatty acids on exercise-induced AMP-activated protein kinase (AMPK) signalling (Bradshaw et al., 2025). AMPK signalling plays a key role in physiological adaptations to endurance training; thus, identifying the specific stimulus responsible for increased AMPK signalling post-exercise has considerable implications for optimizing adaptations to endurance training. The study has many merits, including the creative use of niacin to reduce fatty acid availability in a low carbohydrate state and use of blood sampling and muscle biopsies to comprehensively analyse metabolic responses at the systemic and gene levels. We are writing to suggest considerations that we believe are important when interpreting these findings and for future work continuing this line of investigation. Participants completed exercise after three conditions: (i) an overnight fast intended to increase fatty acid availability and deplete carbohydrate availability (FAST), (ii) carbohydrate ingestion before and during exercise intended to increase carbohydrate availability and decrease fatty acid availability (CARB), and (iii) an overnight fast and niacin ingestion intended to decrease fatty acid availability and deplete carbohydrate availability (NIACIN). If the interventions successfully did what they were intended to do, one would expect pre-exercise muscle glycogen and net muscle glycogen utilization to be lower in the FAST and NIACIN conditions compared to the CARB condition. However, pre-exercise muscle glycogen did not appear different between any of the three conditions, and net muscle glycogen utilization was not different between the FAST and CARB conditions, as seen in Figure 4 of the paper (Bradshaw et al., 2025). The overnight fasting protocol does not appear to have sufficiently depleted muscle glycogen in the FAST and NIACIN conditions, which were intended to elicit low glycogen availability. It is worth noting that even 72 h of fasting decreases muscle glycogen by only 20–30% (Bak et al., 2018; Frank et al., 2013). Protein fold change data revealed that exercise-induced acetyl-CoA carboxylase (ACC) inactivation was significantly increased in both the FAST and NIACIN conditions compared with the CARB condition, leading the authors to conclude that ‘…exercise-induced ACC inactivation may be sensitive to carbohydrate availability and muscle glycogen utilization, independent of fatty acid availability’ (Bradshaw et al., 2025). Given that glycogen may not have been sufficiently depleted in the FAST and NIACIN conditions, we suggest cautious interpretation of the purportedly independent sensitivity of exercise-induced ACC inactivation to muscle glycogen utilization. Perhaps these data instead indicate the sensitivity of exercise-induced ACC inactivation to an overnight fast, which potentially caused minor dehydration that was rescued by the carbohydrate drink, or other effects of fasting per se, rather than the influence of diminished carbohydrate availability. Alternatively, greater ACC activation in the CARB condition could be the result of greater plasma insulin compared to the FAST and NIACIN conditions (Figure 2B of the paper), further supported by increased pAkt in the CARB condition (Figure 5 of the paper; Bradshaw et al., 2025). Future interventions used for studying this area could implement a muscle glycogen-depleting exercise session followed by caloric, but not carbohydrate, replenishment the day prior to the testing day (Cheng et al., 2020; Thomassen et al., 2025). Performing a niacin intervention on a population with McArdle's disease, another possible model for glycogen depletion, could further help to isolate the effect of glycogen availability independent of fat utilization (Andersen et al., 2009). Additionally, we request clarification on the carbohydrate supplementation protocol in the CARB intervention. The method schematized in Figure 1 of the paper depicts the initial carbohydrate drink dose at 1.6 mg/kg, followed by four doses at 0.2 mg/kg. The accompanying text, however, lists the initial dose at 1.6 g/kg but affirms the following doses at 0.2 mg/kg. Taking the average participant body mass as an example, a 75.4 kg individual would receive supplemental doses of 0.015 g of carbohydrate, a negligible amount. Such a small dose seems incompatible with the plasma glucose and insulin responses observed in Figure 2. We agree with the authors’ conclusion that acute exercise is the primary stimulus for AMPK activation and applaud their vigorous work to provide meaningful data. We close this letter by emphasizing the importance of the authors’ contribution in a largely unexplored area. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare they have no competing interests. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. No funding was received for this work.