Figure 5. Effect of conditioned medium (CM) from insulin sensitive subjects and IR subjects on Akt and GSK3β phosphorylation in differentiated human myotubes.

Control denotes phosphorylation in the absence of CM, with phosphorylation level in the absence of insulin set at 1. Data are reported as mean ± s.d. for control subjects (Crohn’s patients denoted as control subjects), while individual values are reported for each IR subject. Each bar is the mean of experiments in triplicate. Panel A: 15 min incubation of human myotubes from insulin sensitive subjects with 25 µg/ml of CM proteins increased ⁴⁷³Ser Akt phosphorylation level in the absence of insulin, with a stronger effect of medium prepared from IR subjects than from insulin sensitive subjects (P<0.05 vs. control subjects). The stimulatory effect of 100 nM insulin in control was increased in the presence of CM from IR subjects (P<0.01 vs. control subjects). Blots are representative western blots of ⁴⁷³Ser Akt phosphorylation and total Akt amounts. Panel B: 15 min incubation of human myotubes from insulin sensitive subjects with 25 µg/ml of CM proteins increased ⁹Ser GSK3β phosphorylation level in the absence of insulin, with a stronger effect of medium prepared from IR subjects than from insulin sensitive subjects (P<0.05 vs. control subjects). The incremental stimulatory effect of 100 nM insulin over basal was reduced mainly in the presence of CM from IR subjects. Blot is a representative western blot of ⁹Ser GSK3β phosphorylation.