ScopeIn recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)(2)-vitamin D-3 (1,25(OH)(2)D-3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. Methods and resultsAfter differentiation, myotubes were cultured in 1,25(OH)(2)D-3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by l-[1-C-13]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)(2)D-3 receptor (VDR) were determined by qPCR. 1,25(OH)(2)D-3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)(2)D-3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)(2)D-3. Conclusion1,25(OH)(2)D-3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes.