. Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling. Am J Physiol Endocrinol Metab 290: E154 -E162, 2006. First published August 23, 2005 doi:10.1152/ajpendo.00330.2005.-Insulinstimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser 645 , Ser 649 , Ser 653 , Ser 657 ) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. "Insulin resistance" is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose. glucose transporter 4; protein kinase B; glycogen synthase kinase-3; phosphorylation; glucose metabolism; glycolytic flux; rat SKELETAL MUSCLE IS PARTICULARLY IMPORTANT in maintaining blood glucose homeostasis, as 70 -90% of insulin-stimulated glucose uptake occurs in skeletal muscle, where it is incorporated into and stored as glycogen (37). The glycogen concentration in skeletal muscles is limited, and a feedback mechanism exists. It has been 40 years since the classic study of Danforth (8), which showed that increased glycogen concentration reduced glycogen synthase activation in both the presence and absence of insulin. More recently, studies have shown that glycogen concentration also influences glucose uptake in skeletal muscles (2,9,17,18).Among the most important functions of insulin are stimulation of glucose uptake and activation of glycogen synthesis (35). Insulin stimulates glucose uptake in insulin responsive tissues by promoting translocation of the facilitative glucose t...