Insulin-like Activity of Dilute Human Serum Assayed by an Isolated Adipose Cell Method

Abstract

A method for insulin assay using isolated adipose cells is described. Fat cells incubated in Krebs-Ringer bicarbonate buffer containing glucose (0.1 mg. per ml.) and albumin (5 per cent) converted glucose-l-14-C to Co2. Addition of insulin caused a maximal stimulation of about eight- to sixteen-fold of the Co2 production. The dose-response relationship exhibited a typical sigmoid shaped curve. The minimum detectable insulin concentration was about 2-5 μU. per ml.; the maximum response was obtained with insulin concentrations of about 300 to 600 μU. per ml. In the optimal range (about 20 to 160 μU. per ml.) the insulin concentrations were determined with a precision of about ±10 per cent (four replications). This method was applied to human serum. It appeared that dilute (1 per cent), dialyzed, fasting serum contained large amounts of “insulin-like” activity (about 20 to 70 μU. per ml.). The dose response relationship of dialyzed serum in a concentration less than about 2 per cent paralleled that of insulin. The increment in Co2 production with greater serum concentrations was less than the increment with greater insulin concentrations. The insulin-like activity of 1 per cent serum did not change after an intravenous glucose load; it was not suppressed by addition of insulin antibodies. Fractionation of serum by starch block electrophoresis located all of the activity in a broad peak in the (β-γ globulin region. Insulin added to serum was quantitatively recovered in a separate peak located in the slower moving component of the albumin fraction. When serum was subjected to gel nitration on Sephadex G 75 the “insulin-like” activity emerged with the total excluded fraction whereas insulin added to serum was retarded on the column. These properties were similar to those of the previously described “atypical” insulin. Its nature remained undefined.