1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes.
METHODS
In 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 D3 (1,25(OH)2D3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube.


RESULTS
After differentiation, myotubes were cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by L-[1-(13) C]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)2D3 receptor (VDR) were determined by qPCR. 1,25(OH)2D3 (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)2D3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)2D3.


CONCLUSIONS
1,25(OH)2D3 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.
Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, Clermont-Ferrand, France; INRA, UMR 1019, UNH, CRNH Auvergne, Clermont-Ferrand, France.
Jérôme
J
Salles
Audrey
A
Chanet
Christophe
C
Giraudet
Véronique
V
Patrac
Philippe
P
Pierre
Marion
M
Jourdan
Yvette C
YC
Luiking
Sjors
S
Verlaan
Carole
C
Migné
Yves
Y
Boirie
Stéphane
S
Walrand
D045930:Anabolic Agents / Q000494:pharmacology; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007930:Leucine / Q000494:pharmacology*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
D045930:Anabolic Agents; D007328:Insulin; D018167:Receptors, Calcitriol; C546843:mTOR protein, mouse; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D002117:Calcitriol; D007930:Leucine
1,25(OH)2-vitamin D3; Akt/mTOR pathway; C2C12 myotubes; Protein synthesis rate; Skeletal muscle