The roles of insulin, adrenal corticol hormones, and nutritional factors in the regulation of hepatic glycogen metabolism were investigated by means of fasting and refeeding normal and adrenalectomized (ADX) rats. More specifically, the hypothesis in question in this study is that certain hepatic phosphoprotein phosphatases are targets of insulin action in liver. In anesthetized rats, the hepatic glycogen concentration and the activities of hepatic glycogen synthase, glycogen synthase phosphatase, glycogen phosphorylase, and phosphorylase phosphatase were correlated with peripheral plasma glucose and immunoreactive insulin levels. Hepatic phosphatase activities were measured in (soluble) the high speed supernatant and smooth endoplasmic reticulum (SER). Fasting resulted in expected diminutions in circulating glucose and insulin levels and loss of hepatic glycogen. These changes were greater in ADX rats. The percentage of hepatic glycogen synthase in the active or I form increased with fasting in normal rats, but did not change in ADX rats. Hepatic synthase phosphatase activities were decreased in SER by fasting in both normal and ADX rats, but to a much greater extent in the latter; soluble synthase phosphatase was much less affected by fasting. The percentage of phosphorylase in the active or a form was significantly decreased in normal, but not ADX, rats. Phosphorylase phosphatase activities were not significantly changed by fasting in any of the subcellular fractions in normal liver, but were increased in the hepatic SER of ADX rats. Refeeding fasted rats for 2 and 6 h resulted in increased hepatic glycogen, activation of glycogen synthase, and increased circulating levels of both insulin and glucose. Refeeding also caused increases in SER-associated synthase phosphatase activity in ADX animals. SER phosphorylase phosphatase activities were significantly increased by refeeding in normal rats, but were decreased in ADX rats. Regression analysis of the data suggested statistically significant positive correlations between insulin levels and SER synthase phosphatase activity in ADX animals, on the one hand, and SER synthase phosphatase and the percentage of synthase in the I form, on the other. No statistically significant correlation between insulin levels and phosphorylase phosphatase activities could be demonstrated. These results are compatible with the hypothesis that glycogen synthase phosphatase activity in liver, especially that associated with SER, is subject to physiological regulation by circulating levels of insulin. In contrast, phosphorylase phosphatase activity seems to be much less influenced by changes in the circulating insulin level. The results are compatible with the proposition that SER-associated phosphoprotein phosphatases are physiologically relevant in the regulation of hepatic glycogen metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)