Binding and degradation of 125I-labelled insulin by isolated rat fat cells

Abstract

1. 1. Isolated rat fat cells were incubated at 37°C with 125I-labelled insulin and separated from the incubation medium by centrifugation through dinonyl phthalate. At low 125I-labelled insulin concentrations (7 · 10 −11−7 · 10 −10 M) the uptake of 125I-labelled insulin by fat cells reache equilibrium after 45 min of incubation. The concentration dependence of equilibrium binding of 125I-labelled insulin showed that one part of the binding could be saturated, whereas another part was proportional to the concentration in the range investigated (7 · 10 −12−7 · 10 −6 M). The saturable part of the binding of 125I-labelled insulin could be explained by a reaction with a cellular receptor resulting in the formation of a receptor-insulin complex. The dissociation constant of the receptor-insulin complex was determined as 3 · 10 −9 M and the amount of receptors as 8 · 10 −10 moles/lcell, or approximately 5 · 10 4 per cell. The rate constants of dissociation and association were 7 · 10 −2 min and 2.5 · 10 7 min −1 · M −1, respectively. Neither vasopressin, glucagon nor des-B23-B30-octapeptide insulin, in concentrations of 7 · 10 −7 M, reduced the binding of 125I-labelled insulin, whereas proinsulin inhibited the binding with an inhibition constant of 1.3 · 10 −7 M. 2. 2. 125I-labelled insulin in the medium was degraded by fat cells. The Michaelis constant of the degrading enzyme system was 3.5 · 10 −74 M and the maximal velocity was 45 nmoles/min per cell. Proinsulin was degraded less rapidly than insulin and the degradation of 125I-labelled insulin was inhibited by proinsulin (7.3 · 10 −7 M), whereas glucagon (6 · 10 −7 M) had no effect. 3. 3. Binding of 125I-labelled insulin and lipogenesis from [2- 3h]glucose were measured on the same cells. Fat cells with a dissociation constant of binding of 3 · 10 10−9 M exhibited half-maximal stimulation of lipogenesis at a 125I-labelled insulin concentration of about 7 · 10 −11 M. 4. 4. The results show that the principle binding of insulin by fat cells occurs to a group of receptors with a K d of 3 · 10 −9 M and that the degradation of insulin and the receptor binding are processes independent of each other. It is suggested that the stimulatory effect of insulin on lipogenesis from glucose in fat cells is mediated by these receptors and that the half-maximal effect of insulin is obtained when about 2% of the receptors are occupied.