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

The differentiation of the L6 myogenic cell line was enhanced by the additi on of dexamethasane, retinoic acid, insulin-like growth factor I (IGF-I), a nd creatine. Spontaneous contractions appeared from day 10 or 11 and persis ted to day 14 or 15. Glucose transport was increased by insulin (100 nM) an d IGF-I (5 nM) by similar to 60%. The highest level of glycogen was measure d in myotubes differentiated under the influence of a combination of 5 nM d examethasone, 100 nM retinoic acid, 5 nM IGF-I, and 10 mM creatine with glu cose 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 (GSa) activity was 30-35 % of the total activity, with a subsequent gradual decline to 2.5% after 6 h. The glycogen phosphorylase a (GPha) activity was constant at similar to 80% from 0 to 0.5 h, increasing to similar to 100% after 6 h. The activity ratio of GSa to GPha decreased about sixfold without significant change in the rate of glycogen accumulation. This indicates that factors other than p hosphorylation/dephosphorylation play a decisive role in the regulation of glycogen metabolism in L6 myotubes. Intracellular glucose (glucose(i)) and glucose 6-phosphate (G-6-P) may be such factors. The observed values of the se parameters may in fact explain an activation of GSa (G-6-P) and an inhib ition of GPha (glucose(i)).