ON THE SIGNIFICANCE OF SERUM DILUTION AND CORTISOL ANTAGONISM IN THE RAT FAT PAD BIOASSAY OF INSULIN

ABSTRACT The dilution effect of normal human serum was readily detectable at small dilutions (1:3, 1:4) and was present to about the same extent both in males and females. It was found to be significantly greater in elderly as compared to younger subjects. Adrenalectomized rats had a significantly lower SILA level in undiluted serum than normal rats. Alloxan diabetic rats had a SILA level in undiluted serum, of about one third of the SILA level in normal rats. In contrast to normal human serum, any dilution effect of normal rat serum was detectable only at very high dilutions (1:27). The same pattern was found when serum from adrenalectomized and alloxan diabetic rats were assayed. The dilution effect in human serum is considered to be due mainly to a species difference. The smallest concentration of cortisol which produced a significant suppression of glucose utilization (as measured on the 14CO2 production from Glucose-1-14C) by rat epididymal fat pads in vitro, in the absence of insulin, was of the order of 0.0025 μg per ml (7 × 10−9 m). A linear log dose-response relationship was found between 0.0025 and 0.25 μg per ml and thus was well within the physiological range. Physiological concentrations of cortisol significantly suppressed the effect of submaximal concentrations of insulin on the 14CO2 production from Glucose-1-14C by the epididymal adipose tissue. A maximal insulin concentration abolished this cortisol effect. However, an extremely high concentration of cortisol (25 μg per ml) still inhibited the effect of 10 U insulin per ml to a slight extent. Addition of puromycin (5 × 10−5 m) abolished the suppressive effect of a physiological concentration of cortisol on insulin stimulated 14CO2 formation from Glucose-1-14C. The mechanism of the observed antagonism between insulin and cortisol at physiological concentrations is assumed to be of the competitive type mediated not by cortisol itself, but by a protein, the formation of which is induced by the action of cortisol.