GLUCAGON BINDING TO RECEPTORS ON THE SURFACE OF CHICKEN ADIPOCYTES

Adipocytes isolated from abdominal fat of chickens were used 1) to demonstrate that [I-125]-tyr(10)-glucagon binds with all the characteristics of a hormone-receptor interaction and 2) to determine whether glucagon binds to one or more receptor sites. Binding of [I-125]-tyr(10)-glucagon was characterized at 12 degrees C, a temperature found to completely inhibit internalization of the radioligand. Culturing adipocytes increased radioligand binding by 60 to 100% and, therefore, fat cells were incubated for 72 h before characterizing binding. Binding of [I-125]-tyr(10)-glucagon to chicken adipocytes reached steady-state, was reversible, was specific for glucagon, was saturable, and increased linearly as a function of fat cell concentration. Both kinetic and steady-state experiments indicated that [I-125]-tyr(10)-glucagon bound to two sites on chicken adipocytes. Equilibrium dissociation constants (K-d) of 54 +/- 16 pM and 3.3 +/- 1.3 nM were obtained for [I-125]-tyr(10)-glucagon, whereas K-d of .49 and 81 nM were calculated for mammalian glucagon. There were 4,831 +/- 1,057 high-affinity receptors and 200,780 +/- 63,404 low-affinity receptors per adipocyte. Thus, only 2.3% of the glucagon binding sites were of high affinity. In addition to higher binding affinity, [I-125]-tyr(10)-glucagon stimulated glycerol release from chicken adipocytes with greater potency than porcine glucagon. Therefore, it was concluded that [I-125]-tyr(10)-glucagon was a supra-agonist that bound to high- and low-affinity receptors on the surface of chicken adipocytes with all the characteristics of a hormone-receptor interaction.