Modulation of Stress and Anabolic Signalling Pathways by Whey Protein Isolate in C2C12 Cells Under Exercise-Mimetic Conditions

Background/Objectives: Skeletal muscle adaptation to metabolic stress involves a coordinated regulation of inflammatory, bioenergetic, and anabolic signalling pathways. This study aimed to investigate the potential role of whey protein isolate (WPI; commercial name: Volapure) as a modulator of cellular responses to stress in an in vitro model of exercise-mimetic stress over time. Methods: Murine C2C12-differentiated cells were exposed to an Exercise–Mimetic Mix (ExM) to reproduce key biochemical features of muscle stress. Cells were treated with WPI (1 mg/mL) using Pre-exposure (Pre-ExM) and Post-exposure (Post-ExM) protocols at 8 and 24 h. Multiple endpoints were assessed, including cell viability, reactive oxygen species (ROS) production, cytokine release (TNF-α, IL-6, IL-17), intracellular signalling pathways (p38 MAPK, ERK, AMPK, mTOR), bioenergetic markers (ATP, glycogen, lactate), protein synthesis (OPP incorporation), and Ca2+/Mg2+ fluxes. Results: ExM exposure induced a stress phenotype characterised by increased oxidative and inflammatory markers, impaired bioenergetic status, and reduced anabolic signalling. WPI was associated with modulation of these responses, reducing ROS and pro-inflammatory cytokines, restoring ATP and glycogen levels, and changes in ERK and mTOR-related signalling. The Post-ExM protocol showed greater modulation compared to the Pre-ExM approach, particularly at 24 h. WPI was also associated with the normalisation of ExM-altered Ca2+/Mg2+ fluxes. These findings should be interpreted as associative rather than causal. Conclusions: WPI was associated with modulation of key pathways involved in cellular adaptation to metabolic stress, supporting recovery of bioenergetic balance and anabolic signalling in C2C12 cells. These findings suggest a potential role for WPI in influencing cellular responses to metabolic stress, supporting recovery of bioenergetic balance and anabolic signalling in C2C12-differentiated-cells. However, further studies are required to confirm the translational relevance of these observations.