Abstract Glycogen synthase (GS), a key regulatory enzyme in glycogen synthesis, is controlled by multisite phosphorylation and allosteric regulation and is activated by insulin. This study investigated changes in GS activity and expression in hepatocytes isolated from rats under altered nutritional and diabetic conditions. Experiments were carried out in healthy rats fed a chow diet, rats on high simple sugar (60% of energy from fructose and sucrose) or high fat (46% of energy from fat) diet, and in rats with streptozotocin induced diabetes. In the presence of insulin, activated GS activity (GS I form) was increased by 89% in hepatocytes isolated from healthy rats. The stimulatory effect of insulin on GS activity and expression was blunted by cycloheximide and actinomycin treatment. In rats fed a high simple sugar or high fat diet, insulin stimulation of GS i in isolated hepatocytes was impaired and GS expression was significantly lower in rats fed the high fat diet in comparison to controls. GLUT-2 protein expression was significantly lowered by both the high fat and high simple sugar diets. In hepatocytes isolated from diabetic rats, total GS activity (GS t ) was lower than in hepatocytes from healthy animals. Insulin added to the incubation medium did not stimulate GS activity, demonstrating impaired sensitivity to insulin in diabetic rats. However, insulin administration significantly increased GS expression indicating that a defect in synthase phosphorylation may be responsible for impaired GS activity in the diabetic state. The results presented in this study further confirm that GS activity is affected by both dietary and hormonal factors which can be measured in a rat hepatocyte model.