Regulation of glycogen accumulation in L6 myotubes cultured under optimized differentiation conditions

Abstract

The differentiation of the L6 myogenic cell line was enhanced by the addition of dexamethasone, retinoic acid, insulin-like growth factor I (IGF-I), and creatine. Spontaneous contractions appeared from day 10 or 11 and persisted to day 14 or 15. Glucose transport was increased by insulin (100 nM) and IGF-I (5 nM) by ∼60%. The highest level of glycogen was measured in myotubes differentiated under the influence of a combination of 5 nM dexamethasone, 100 nM retinoic acid, 5 nM IGF-I, and 10 mM creatine with glucose as substrate. The glycogen accumulation rate was constant from 0 to 2 h of incubation and decreased gradually to zero at 4 h. From 0 to 0.5 h of the glycogen accumulation, the glycogen synthase a(GS a) activity was 30–35% of the total activity, with a subsequent gradual decline to 2.5% after 6 h. The glycogen phosphorylase a(GPh a) activity was constant at ∼80% from 0 to 0.5 h, increasing to ∼100% after 6 h. The activity ratio of GS a to GPh a decreased about sixfold without significant change in the rate of glycogen accumulation. This indicates that factors other than phosphorylation/dephosphorylation play a decisive role in the regulation of glycogen metabolism in L6 myotubes. Intracellular glucose (glucosei) and glucose 6-phosphate (G-6- P) may be such factors. The observed values of these parameters may in fact explain an activation of GS a(G-6- P) and an inhibition of GPh a(glucosei).