A Molecular and Functional Investigation of the Anabolic Effect of an Essential Amino Acids’ Blend Which Is Active In Vitro in Supporting Muscle Function

<b>Background/Objectives</b>: Essential amino acids' (EAAs) biological effects depend on both gastrointestinal stability and intestinal bioavailability. A commercially available EAA blend has previously shown to be highly bioaccessible and able to inhibit the DPP-IV enzyme both directly and at a cellular level following simulated digestion in vitro. In light with this consideration, the present study aimed to evaluate the intestinal in vitro bioavailability of GAF subjected to INFOGEST digestion (iGAF) and to investigate the metabolic effects of its bioavailable fraction on muscle cells using an integrated Caco-2/C2C12 co-culture model. <b>Methods</b>: Differentiated Caco-2 cell lines were treated with iGAF, and amino acid transport was quantified by ion-exchange chromatography. The basolateral fraction containing bioavailable EAAs was used to treat differentiated C2C12 myotubes for 24 h. Western blot analyses were performed to assess the activation of anabolic and metabolic pathways, including mTOR, Akt, GSK3, AMPK and GLUT-4. <b>Results</b>: More than 50% of each EAA present in iGAF crossed the Caco-2 monolayer, with BCAAs and phenylalanine particularly enriched in the basolateral fraction. Exposure of C2C12 myotubes to the bioavailable iGAF stimulated mTORC1 activation and increased the phosphorylation of Akt and GSK3, indicating an enhanced anabolic response. At a cellular level, iGAF also elevated the p-AMPK/AMPK ratio, suggesting activation of energy-sensing pathways. Moreover, GLUT4 protein levels and glucose uptake were significantly increased. <b>Conclusions</b>: The study focuses exclusively on a cellular model, and results suggested that iGAF is highly bioavailable in vitro and that its absorbed fraction activates key anabolic and metabolic pathways of skeletal muscle cells, enhancing both protein synthesis signaling and glucose utilization in vitro.