The Uptake and Inactivation of Native Insulin by Isolated Fat Cells

With dilute suspensions of rat epididymal adipose tissue cells, glucose utilization was estimated by the conversion of glucose-14C to 14CO2 and 14C-labeled lipids. The maximal effect of insulin was observed with a glucose concentration of 1.25 mm or less. The same rate of glucose utilization was observed with and without insulin when the glucose concentration was 80 mm. Glucose utilization was stimulated with 1 microunit of insulin per ml, and insulin concentrations above 25 microunits per ml produced no further stimulation. The rate of glucose utilization was linear for 2 hours when the insulin concentration was 3 microunits per ml. With 5 microunits of insulin per ml, the glucose utilized in 2 hours did not increase when the volume of the incubation medium was doubled. The stimulatory effect of insulin was reversed by washing the cell suspension or by the addition of anti-insulin serum. These findings indicated that (a) a dilute suspension of fat cells did not inactivate insulin rapidly enough to lower the insulin concentration of the incubation medium significantly, and (b) the continued presence of insulin was required for a continuation of its biological effect. With a 30 times more concentrated suspension of fat cells, the change in immunoreactive insulin concentration of the incubation medium was measured. A rapid phase of insulin uptake occurred within the first minute, was linearly related to insulin concentration up to 10,000 microunits per ml, was not temperature-sensitive, and was abolished by a 30-sec exposure to 10-3 m maleimide. A subsequent phase of insulin uptake was slower, temperature-sensitive, and not abolished by maleimide. Cells previously incubated with insulin showed undiminished insulin uptake when reincubated in fresh medium. When insulin-treated cells were washed, no immunologically or biologically active insulin was released. It was concluded that (a) the rapid phase of insulin uptake may be the hormone-cell interaction which is necessary for the action of insulin to be manifest, and (b) a study of the factors which influence this process may lead to a better understanding of the mechanism of hormone action.