Carbs <i>vs</i> . fats: Who takes first in skeletal muscle signalling?

Greater skeletal muscle activity during exercise increases cellular energy demand. The activity of AMP-activated protein kinase (AMPK), a molecule involved in energy sensing pathways, also increases during exercise, which turns on additional pathways. One of the molecules involved in the downstream effects of AMPK is acetyl-CoA carboxylase (ACC; Spaulding & Yan, 2022). Once phosphorylated by AMPK, ACC activity is reduced, resulting in increased fatty acid oxidation and fat utilization. Previous research indicates that high non-esterified fatty acid (NEFA) availability may increase levels of phosphorylated AMPK (pAMPK) and phosphorylated ACC (pACC) through calcium-dependent pathways (Anavi et al., 2010). Furthermore, the concentration of muscle glycogen could also have an effect on the activation of these molecules. Fasted-state exercise requires fatty acids and muscle glycogen to be utilized as an energy source, and NEFA concentration will be higher in a fasted state, as there is lower glucose availability. Carbohydrates can spare muscle glycogen and prevent the increase in fatty acid oxidation. Therefore, it is challenging to determine the independent effects of NEFA and muscle glycogen on AMPK and ACC signalling. However, Bradshaw et al. (2025) recently published a paper in The Journal of Physiology in which they cleverly isolated and examined the effects of muscle glycogen and NEFA availability on skeletal muscle cell signalling following an acute bout of aerobic exercise. To examine how substrate availability impacts AMPK and ACC activation, Bradshaw et al. (2025) studied three conditions: (1) exercising after an overnight fast (FAST), representing high fatty acid availability with low carbohydrate, insulin and glycogen availability; (2) exercise with carbohydrate intake (CARB), representing low fatty acid availability and high carbohydrate availability; and (3) fasted exercise, but with niacin ingestion (NIACIN). Niacin was used to decrease fatty acid mobilization in adipose tissue (Christie et al., 1996), resulting in lower NEFA concentrations, which subsequently decreases fat oxidation. It is critical to note that these three visits were performed in a randomized order. The proposed hypothesis was that reduced carbohydrate availability would be more important than high fatty acid availability for increasing AMPK signalling; in other words, exercising while fasted with niacin will result in greater AMPK signalling. The study included eight healthy males between 18 and 60 years old. Figure 1 supplied by Bradshaw et al. (2025) gives an overview of the study design. A preliminary visit was performed after an overnight fast. A primary objective of this visit was to determine lactate threshold 1 (LT1) on a cycle ergometer. LT1 was determined by an increase in lactate of 0.5 mmol L−1 above resting levels. After baseline, using a randomized, cross-over design, participants returned to the lab after an overnight fast on three separate occasions with an 11 ± 6 day washout period. Following the first biopsy, participants consumed either a placebo drink (FAST and NIACIN) or carbohydrate drink containing 1.6 g kg−1 body mass of maltodextrin (CARB). After 30 min of rest, participants consumed either a placebo tablet (CARB and FAST) or niacin tablets (NIACIN; 10 mg kg−1). Exercise began after another 30 min of rest. The exercise protocol required participants to cycle for 60 min at 95% of LT1. Every 15 min, 100 mL drinks were consumed of either 0.2 mg kg−1 maltodextrin or placebo. After 30 min of exercise, again participants consumed either a placebo or niacin supplement (0.5 mg kg−1). Breath and blood samples were taken every 15 min with another biopsy 5 min following the test. Specifically, the phosphorylation status of proteins AMPK and ACC were analysed through muscle western blotting. Gene expression was also analysed. The results of this study are intriguing. As expected, plasma NEFA concentration decreased slightly in all groups at the start of exercise. However, after 15 min of exercise, plasma NEFA was significantly higher in the FAST condition compared to NIACIN and CARB and remained higher through 60 min of cycling. Other variables, including plasma insulin, plasma glucose and plasma lactate, indicate Bradshaw et al. (2025) were successful in manipulating NEFA and carbohydrate availability. Moreover, the experimental design successfully provided conditions where the researchers could independently look at the effects of substrate utilization on metabolic pathways. As expected, fat oxidation was significantly higher in FAST compared to CARB but no significant differences were seen in CARB compared to NIACIN. Carbohydrate oxidation was significantly higher in CARB compared to FAST with no significant differences seen in CARB compared to NIACIN. The NIACIN condition reduced fatty acid availability during exercise. Respiratory exchange ratio (RER) was significantly higher in CARB than FAST with no further increase compared to NIACIN, indicating increased carbohydrate oxidation relative to fat. The observed increase in net muscle glycogen utilization in NIACIN relative to CARB is noteworthy. With lower amounts of free fatty acids circulating in the blood during a fasted state, this condition requires muscle glycogen to be utilized as fuel. Relative to rest, levels of AMPK were higher following exercise in all conditions, with no significant difference between conditions. However, there were differences in the downstream effect on ACC, as ACC phosphorylation was significantly lower in CARB compared to FAST and NIACIN following exercise. In summary, carbohydrate ingestion reduced net muscle glycogen utilization and exercise-induced ACC phosphorylation in comparison to NIACIN. Since conditions had similar NEFA concentrations, the lower ACC activation with carbohydrate ingestion is more likely to be due to an inhibitory effect of high carbohydrate availability. Furthermore, it is necessary to discuss the role of insulin during exercise. As seen in the CARB condition, increased concentrations of insulin will increase glucose uptake in the muscle cells via GLUT4 transporters. In contrast, NIACIN decreased fatty acid concentration but did not stimulate insulin secretion, as seen in the CARB condition. The increases in insulin concentrations were associated with decreased pACC and, consequently, decreased fat oxidation. The focus of this study was to determine if exercise-induced cell signalling in a fasted state was due to low carbohydrate or increased free fatty acid availability. The increase in muscle glycogen utilization in NIACIN was probably due to increased glycogenolysis. Again, greater amounts of pACC in FAST and NIACIN suggest AMPK activation may be due to low carbohydrate availability. As suggested by Zhang et al. (2014), a similar framework takes place with the activation of PDK4, a regulatory enzyme. Activation of this molecule, and subsequent deactivation of PDH, results in a decrease in aerobic glycolytic pathways and an increase in fat oxidation. Substrate utilization also reflects how these signalling pathways unfold, as Bradshaw et al. (2025) noted greater carbohydrate oxidation in CARB and NIACIN conditions. They observed an insignificant decrease in PDK4 in the niacin condition, which would also indicate that shift towards glycolysis. However, the lack of significant differences in total PDK4 or activated PDH begs further questioning. Additional research would help understand these signalling pathways and the correlations between PDK4 and substrate availability. While this study had several strengths, there are a few limitations and areas for further investigation. Notably, no women were included in this study; therefore, future studies should include men and women in order to evaluate potential sex differences. Additionally, it is unknown if acute exercise or carbohydrate availability is more impactful on AMPK activation. As mentioned, Bradshaw et al. (2025) did not see a significant difference in pAMPK between conditions, which may have occurred if exercise intensity was slightly higher. A higher intensity may be needed to activate other molecules in this signalling pathway. Future research should include minor intensity adjustments, as exercise itself may be a greater driver of AMPK activation. However, a counter argument is that large increases in exercise intensity may make it more difficult to evaluate the independent effect of substrate availability. Lastly, future studies should examine the health implications of these findings, as well as possible differences in exercise performance. It is possible that niacin could have other effects on gene expression and signalling pathways that have not been identified. In addition to decreasing fatty acid availability, niacin may have further effects on skeletal muscle metabolism. Another limitation could stem from the calories obtained during the carbohydrate condition. It is possible that different energy balances could have affected gene expression or cell signalling regardless of exercise. Finally, increasing the number of participants would improve external validity and, although burdensome, additional muscle biopsies could have provided further insight into substrate utilization. The novel findings from Bradshaw et al. (2025) suggest that a decrease in ACC phosphorylation may be more dependent on carbohydrate, rather than fatty acid, availability. Using niacin to control NEFA concentrations helped the researchers study AMPK signalling during fasted exercise. These findings will help guide future research examining metabolic signalling and can be applied to a sports performance or clinical setting. 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. None declared. Sole author. None. The author would like to thank Dr Daniel Craighead, University of Minnesota-Twin Cities, for his support and thoughtful feedback on this article.