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

Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark 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 (glucose i ) 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 (glucose i ). muscle; cell culture; creatine kinase; glycogen metabolism; cell differentiation