Stimulation of glycogen synthesis by heat shock in L6 skeletal-muscle cells: regulatory role of site-specific phosphorylation of glycogen-associated protein phosphatase 1

Abstract

Recent evidence suggests that glycogen-associated protein phosphatase 1 (PP-1G) is essential for basal and exercise-induced glycogen synthesis, which is mediated in part by dephosphorylation and activation of glycogen synthase (GS). In the present study, we examined the potential role of site-specific phosphorylation of PP-1G in heat-shock-induced glycogen synthesis. L6 rat skeletal-muscle cells were stably transfected with wild-type PP-1G or with PP-1G mutants in which site-1 (S1) Ser48 and site-2 (S2) Ser67 residues were substituted with Ala. Cells expressing wild-type and PP-1G mutants, S1, S2 and S1/S2, were examined for potential alterations in glycogen synthesis after a 60min heat shock at 45°C, followed by analysis of [14C]glucose incorporation into glycogen at 37°C. PP-1G S1 mutation caused a 90% increase in glycogen synthesis on heat-shock treatment, whereas the PP-1G S2 mutant was not sensitive to heat stress. The S1/S2 double mutant was comparable with wild-type, which showed a 30% increase over basal. Heat-shock-induced glycogen synthesis was accompanied by increased PP-1 and GS activities. The highest activation was observed in S1 mutant. Heat shock also resulted in a rapid and sustained Akt/ glycogen synthase kinase 3β (GSK-3β) phosphorylation. Wortmannin blocked heat-shock-induced Akt/GSK-3β phosphorylation, prevented 2-deoxyglucose uptake and abolished the heat-shock-induced glycogen synthesis. Muscle glycogen levels regulate GS activity and glycogen synthesis and were found to be markedly depleted in S1 mutant on heat-shock treatment, suggesting that PP-1G S1 Ser phosphorylation may inhibit glycogen degradation during thermal stimulation, as S1 mutation resulted in excessive glycogen synthesis on heat-shock treatment. In contrast, PP-1G S2 Ser phosphorylation may promote glycogen breakdown under stressful conditions. Heat-shock-induced glycogenesis appears to be mediated via phosphoinositide 3-kinase/Akt-dependent GSK-3β inactivation as well as phosphoinositide 3-kinase-independent PP-1 activation.