The role of increased glucose transport in the hormonal regulation of glycogen synthase by insulin was investigated in 3T3-L1 adipocytes. Insulin treatment stimulated glycogen synthase activity 4 -5-fold in these cells. Cytosolic glycogen synthase levels decreased by 75% in response to insulin, whereas, conversely, the glycogenolytic agent isoproterenol increased cytosolic enzyme levels by 200%. Removal of extracellular glucose reduced glycogen synthase activation by 40% and completely blocked enzymatic translocation. Addition of 5 mM 2-deoxyglucose did not restore glycogen synthase translocation but did augment dephosphorylation of the protein by insulin. The translocation event could be reconstituted in vitro only by the addition of UDP-glucose to basal cell lysates. Amylase pretreatment of the extracts suppressed glycogen synthase translocation, indicating that the enzyme was binding to glycogen. Incubation of 3T3-L1 adipocytes with 10 mM glucosamine induced a state of insulin resistance, blocked the translocation of glycogen synthase, and inhibited insulinstimulated glycogen synthesis by 50%. Surprisingly, glycogen synthase activation by insulin was enhanced 4-fold, in part due to allosteric activation by a glucosamine metabolite. In vitro, glucosamine 6-phosphate and glucose 6-phosphate stimulated glycogen synthase activity with similar concentration curves. These results indicate that glucose metabolites have an impact on the regulation of glycogen synthase activation and localization by insulin.Insulin is responsible for the maintenance of blood glucose levels in a narrow physiological range. Under hyperglycemic conditions, insulin increases glucose uptake and stimulates the rate-limiting enzymes that regulate glucose oxidation and storage. In healthy subjects, 80 -90% of glucose disposal occurs in skeletal muscle, where it is primarily stored as glycogen. Insulin acutely stimulates glycogen synthesis through activation of glycogen synthase (GS) 1 and inactivation of glycogen phosphorylase. The coordinate increase in glucose uptake and regulation of glycogen metabolizing enzymes accounts for the marked stimulation of glycogen synthesis by insulin.GS, the rate-limiting enzyme in glycogen synthesis, is regulated allosterically and by covalent modification (1, 2). The protein is phosphorylated on six residues by a variety of kinases, which cumulatively inhibit its activity. Insulin-stimulated activation of glycogen targeted protein phosphatase-1 (PP1) results in the dephosphorylation of GS (3). The hormone can also produce the inactivation of glycogen synthase kinase-3 (GSK-3), resulting in the disinhibition of GS by preventing its phosphorylation (4 -6). Additionally, binding of glucose 6-phosphate (G6P) allosterically activates the enzyme and increases its susceptibility to dephosphorylation (7-9). The increase in glucose transport and phosphorylation caused by insulin results in elevated levels of intracellular G6P, producing further activation of GS.Although the signaling pathways involved the metabolic ...

A protocol was developed in 3T3-L1 adipocytes that resulted in the specific desensitization of glycogen synthase activation by insulin. Cells were pretreated for 15 min with 100 nM insulin, and then recovered for 1.5 h in the absence of hormone. Subsequent basal and insulininduced phosphorylation of the insulin receptor, IRS-1, MAPK, Akt kinase, and GSK-3 were similar in control and pretreated cells. Additionally, enhanced glucose transport and incorporation into lipid in response to insulin were unaffected. However, pretreatment reduced insulin-stimulated glycogen synthesis by over 50%, due to a nearly complete inhibition of glycogen synthase activation. Removal of extracellular glucose during the recovery period blocked the increase in glycogen levels, and restored insulin-induced glycogen synthase activation. Furthermore, incubation of pretreated 3T3-L1 adipocytes with glycogenolytic agents reversed the desensitization event. Separation of cellular lysates on sucrose gradients revealed that glycogen synthase was primarily located in the dense pellet fraction, with lesser amounts in the lighter fractions. Insulin induced glycogen synthase translocation from the lighter to the denser glycogen-containing fractions. Interestingly, insulin preferentially activated translocated enzyme while having little effect on the majority of glycogen synthase activity in the pellet fraction. In insulin-pretreated cells, glycogen synthase did not return to the lighter fractions during recovery, and thus did not move in response to the second insulin exposure. These results suggest that, in 3T3-L1 adipocytes, the translocation of glycogen synthase may be an important step in the regulation of glycogen synthesis by insulin. Furthermore, intracellular glycogen levels can regulate glycogen synthase activation, potentially through modulation of enzymatic localization.