Kinetics of insulin internalization and processing in adipocytes: Effects of insulin concentration

Abstract

We have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with ¹²⁵l-insulin at 37°C; and at frequent, early time points surface-bound and intracellular insulin were quantitated. Surface-bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4°C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration-dependent manner (P < .001). Thus, values for ke were 0.121 ± 0.006 min⁻¹ versus 0.074 ± 0.011 min⁻¹ at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration-dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of ¹²⁵l-insulin into the adipocyte interior, washing away surface-bound and extracellular insulin, and then monitoring the subsequent efflux of pre-loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insulin efflux is unaffected.