Fig. 5: Valine-metabolite, 3-hydroxyisobutyrate drives basal glucose uptake and glucotoxicity-mediated impairment of myotube insulin signaling.

A qPCR of glucose transporters and stress markers in C2C12 mouse myotubes after treatment with 2 mM Val or 3-HIB in presence of 250 µM PA for 24-h normalized to BSA control with B2m as reference gene (n = 4). B Representative western blot of insulin stimulated (15 min) AKT phosphorylation in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to BSA control (n = 4). C Relative basal glucose uptake in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to PA (n = 8). D Relative insulin stimulated (30 min) glucose uptake in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to PA (n = 8). E–G qPCR of glucose transporter and stress markers in C2C12 mouse myotubes after treatment with 2 mM Val or 3-HIB in presence of 250 µM PA and 25 nM BAY-876 (Bay) for 24 h normalized to PA-Ctl with B2m as reference gene (n = 4). H Representative western blot of insulin-stimulated (15 min) AKT phosphorylation in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB in presence of 25 nM BAY-876 normalized to PA control (n = 4). Fold change in Western blots (B, H) refers to insulin-induced change normalized to BSA control (B) or PA control (H). Data are shown as mean + SEM except for western blot quantification where only mean of fold change is indicated below representative blots. *p < 0.05; **p < 0.01; ***p < 0.001 compared to respective control and. ^#p < 0.05 comparing Ctl to Bay treated groups. Different letters represent significant difference between groups in Western blots. BSA bovine serum albumin, PA palmitic acid, Bay BAY-876.