Palmitoylethanolamide (PEA) Regulates Cell Cycle Progression and Promotes an Anti-Inflammatory Transcriptomic Signature in C2C12 Skeletal Muscle Cells

Palmitoylethanolamide (PEA) is an endogenous lipid mediator with recognized immunomodulatory actions, yet its effects in skeletal muscle remain poorly defined. We investigated whether PEA influences myogenesis and profiled the acute transcriptomic response of differentiated C2C12 myotubes. Dose-tolerability assays identified reduced metabolic activity and viability at 100 µM PEA, so 10 µM was used for subsequent experiments. Across differentiation, PEA decreased myotube number (90.3 ± 10.6 vs 112.6 ± 10.1 in control) while increasing nuclear fusion index (37.8 ± 5.7% vs 30.7 ± 3.2%); myotube area was unchanged. In myoblasts, 24 h PEA exposure increased the proportion of cells in G0/G1 (48.2 ± 1.2% vs 42.3 ± 1.9%) with a concomitant reduction in S-phase (21.7 ± 1.2% vs 25.5 ± 1.2%), consistent with G1 arrest and reduced proliferative drive. RNA sequencing revealed a distinct transcriptional signature after 24 h PEA treatment, with 1,952 differentially expressed genes (1,028 upregulated; 924 downregulated, q < 0.05). Pathway-level changes encompassed cytokine–receptor interactions, chemokine, JAK–STAT, NF-κB, NOD-like and Toll-like receptor signalling. PEA downregulated NF-κB target/pro-inflammatory cytokine genes (Il1a, Il1b, Il12a, Il33, Tnfrsf19) while upregulating interferon-related and chemokine genes (e.g., Stat2, Oas1b, Gbp7, Cxcl14, Ccl5), indicating an anti-inflammatory/immune-priming profile. Notably, N-acylethanolamine acid amidase (Naaa), a lysosomal PEA hydrolase, was highly expressed and significantly induced, whereas fatty acid amide hydrolase (Faah) was low and unchanged, suggesting a muscle-specific bias toward NAAA-dependent PEA metabolism. Peroxisome proliferator-activated receptor-α (PPARα) was expressed at low levels and unaffected by PEA, indicating that the observed responses occur largely independent of canonical PPARα signalling. Together, these data show that PEA biases skeletal muscle toward a less proliferative but more fused and inflammation-resolving phenotype, accompanied by transcriptional reprogramming of immune pathways and preferential engagement of NAAA. While in vitro concentrations exceed reported plasma levels and mRNA changes may not fully predict protein/function, these findings provide the first comprehensive transcriptomic characterisation of PEA in myotubes and motivate in vivo studies to test whether PEA’s dual actions translate into benefits for muscle regeneration, adaptation, or anti-atrophy interventions.