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Insulin and glucagon binding was studied in isolated rat hepatocytes, and the receptor occupancy was compared to the hormonal stimulation of amino acid transport. Binding was measured at 20 and 37 C in steady state conditions using 125I-labeled iodohormone and varying concentrations of unlabeled hormone. When measured at 20 C, insulin binding was compatible with either a two-site model: a high affinity (Kd ≅ 0.6 nM) and a low affinity (Kd ≅ 12 nM) site or one class of binding sites with negatively cooperative interactions. At 20 C, the rate of dissociation of bound 125Iiodoinsulin was enhanced in the presence of unlabeled insulin, suggesting negatively cooperative interactions among insulin receptors at this temperature. At 37 C, in contrast, insulin bound predominantly to a high affinity site (Kd ≅ 0.6 nM). These data indicate that at 37 C, the low affinity site is no longer present (or detectable), and/ or there is a marked decrease in cooperative interactions at this temperature. When insulin-stimulated α-aminoisobutyric acid influx was compared to receptor occupancy at 37 C, stimulation of amino acid transport paralleled receptor occupancy at this temperature, i.e. insulin binding to the high affinity site. These results suggest that insulin action in rat hepatocytes is initiated by the interaction of the hormone with a single class of noncooperative, high affinity binding sites. Glucagon binding involved one type of noncooperative binding sites (Kd ≅ 5 nM) at both 20 and 37 C. There was no direct proportionality between glucagon binding and glucagon stimulation of amino acid transport, with a half-maximal effect occurring at about 20% total receptor occupancy. The data indicate that the high coupling efficiency which has been reported for glucagon binding and proximal events of glucagon action (e.g. cAMP accumulation) is also observed for a more distal effect, such as stimulation of amino acid transport.
Fehlmann et al. (Wed,) studied this question.