Background: Specific molecular pathways control the distinct metabolic responses of human skeletal muscle to feeding. While 24-h transcriptomic analyses of skeletal muscle demonstrate robust diurnal rhythmicity, that is sensitive to divergent feeding patterns, phosphorylation signalling across the day is less well defined. This is notable as phosphorylation events are typically more transient in order to govern the rapid changes in cellular metabolism required to respond to changes in nutritional states. It therefore remains unclear how distinct feeding patterns impose structured temporal organisation on the muscle phospho-proteome.
Aims/Objectives: This study is an exploratory analysis to characterise the 24-hour temporal dynamics of the human skeletal muscle phospho-proteome under varying patterns of controlled nasogastric nutrient delivery (continuous versus dual‑bolus feeding).
Methods: Following 7-days of lifestyle standardisation 18 participants (16M/2F; Age: 26 ± 9 y; BMI: 23.5 ± 2.3 kg×m-2; Resting Metabolic Rate; 1842 ± 229 kcal×d-1) underwent a 24‑hour experimental period receiving enteral nutrition either continuously (n = 9) or as two discrete bolus ‘meals’ (0800 and 2000 h; n = 9), with skeletal muscle biopsies collected every 4 hours for 24 hours (starting at midday). Phospho-proteomic profiling was performed using label-free mass spectrometry, followed by data preprocessing and missingness filtering (≥25 % and ≥70 % presence thresholds). Data were normalised on a per-phospho-site basis, and temporal behaviour was analysed using unsupervised clustering of scaled phospho-site profiles. Cluster assignments were compared between feeding conditions to assess changes in temporal organisation, and pathway enrichment analysis was performed to provide biological context to identified patterns.
Results: Unsupervised clustering identified six distinct temporal phosphorylation profiles per condition across ~10,500 phospho-sites, with the bolus condition characterised by a greater number of transient peaks and troughs relative to the more stable profiles observed under continuous feeding. Phospho-sites that exhibited clear time‑specific patterns under bolus feeding became less temporally distinct under continuous feeding. Gene ontology analysis indicated that changes in temporal patterns between bolus and continuous feeding were associated with processes related to muscle development and adaptation, with additional enrichment of glycolysis and NADH regeneration.
Summary: These findings suggest that divergent feeding patterns drive coordinated, time‑specific phosphorylation responses in skeletal muscle over a 24-h period, whereas continuous feeding leads to less defined temporal organisation of metabolic signalling. This highlights a potential role for feeding pattern in shaping muscle metabolic and adaptive responses.