Insulin Binding to Isolated Human Adipocytes

Abstract

In order to study the binding of insulin to insulin-sensitive human tissue, we isolated adipocytes from surgically obtained human fat tissue. Our data demonstrate that insulin readily and specifically binds to isolated adipocytes. The time course of this reaction indicates that steady state binding conditions occur at forty-five minutes, with a subsequent decline in binding later. There is no appreciable insulin or receptor degradation before forty-five minutes, suggesting a true equilibrium. After forty-five minutes the decline in binding can be accounted for by insulin plus receptor degradation. At 3 × 10−11 M 125-I-insulin and 2 × 105 cells per milliliter, 1.8 to 2.4 per cent of the 125-I-insulin was bound. The specificity of this binding reaction is demonstrated by the high concentrations of thyroid stimulating hormone and human growth hormone that are without effect on binding, while proinsulin and desalanine insulin inhibit binding in proportion to their biologic activity. On the other hand, binding can be readily inhibited by porcine insulin at physiologic concentrations, i.e. binding is 13 per cent inhibited at 1 ng. per milliliter and 50 per cent inhibited at 8.6 ng. per milliliter. The kinetic behavior of this reaction can be approximated by Scatchard analysis, which indicates 50,000 high affinity sites per cell with a dissociation constant (Kd) of 1.9 × 10−9 M per liter and 250,000 low affinity sites per cell with a Kd of 8 × 10−9 M per liter. In conclusion, these studies demonstrate and characterize the binding of insulin to normal isolated human adipocytes; they indicate that the study of insulin-adipocyte binding is a possible means to gain further insight into the mechanism of altered response to insulin in human disease states.