Both insulin and hyperglycemia can effectively suppress hepatic glucose output (HGO). We examined whether insulin and hyperglycemia specifically suppress liver net glycogen breakdown in a rat model in which glycogen is the major source of HGO. We further examined whether insulin and hyperglycemia act by similar or distinct enzymatic mechanisms. HGO, the rate of net glycogen loss, and glycogen phosphorylase and synthase activities were measured in fed, anesthetized rats infused with saline or insulin (7 mU/min/kg) while either maintaining plasma glucose at basal (7.8 +/- 0.2 mmol/L, euglycemic clamp [EC]) or at 10 mmol/L above basal (18 +/- 0.4 mmol/L, hyperglycemic clamp [HC]). During the basal period, the rate of HGO in each group was comparable to the rate of net glycogen breakdown, averaging 76 +/- 9 and 75 +/- 5 mumol/min/kg, respectively. Thus glycogen breakdown appeared to be a major source of ongoing HGO. Over the last 60 minutes of the experimental period, the rate of glycogenolysis averaged 69 +/- 8 mumol/min/kg in saline-treated rats; this could account for about 80% of the total HGO. During both EC and HC studies, HGO was suppressed (5.5 +/- 3 and -3.6 +/- 10 mumol/min/kg, respectively; P < .001 for each). Net glycogen breakdown decreased by 50% in EC rats (P < .05) and ceased in HC rats (P < .001). Glycogen synthase was predominantly in the active form in all three experimental groups (87% +/- 2%, 89% +/- 2%, and 95% +/- 3% in saline, EC, and HC rats, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)