The effects of various concentrations of trypsin on insulin-sensitive phosphodiesterase activity and insulin binding in isolated fat cells were investigated. The cells were treated at the lowest tracer insulin concentration (0.08 nM), at 24°C for 60 min. The specific binding of radioactive insulin to fat cells treated respective by with none, 10 μg/ml and 1 mg/ml of trypsin was 4.8%, 4.0% and 0.4%/2 × 10⁵ cells. Scatchard analysis indicated a decrease in the number of insulin receptors in fat cells treated with trypsin.
To measure the phosphodiesterase activity, isolated fat cells were treated with none, 10 μg/ml and 1 mg/ml trypsin at 37°C for 15 min, then incubated at 37°C for 10 min with or without insulin. A crude microsomal fraction prepared by differential centrifugation was assayed for the phosphodiesterase activity. In all but the basal activity, the enzyme activity in trypsin-treated fat cells was lower at all insulin concentrations. In fat cells treated with 10 μg/ml of trypsin, the dose-response curve of the phosphodiesterase activation by insulin shifted to the right, and half-maximum stimulation was obtained at 0.43 nM compared with 0.15 nM insulin concentration in the controls (p<0.001). This indicates a decrease in sensitivity to insulin. Responsiveness to insulin, expressed as a percentage of the basal enzyme activity, was also reuced to 200% in fat cells treated with 10 μg/ml of trypsin, compared with 260% in the controls (p<0.001). In fat cells treated with 1 mg/ml of trypsin, the phosphodiesterase activation by insulin disappeared.
These results indicate that changes in the binding of insulin to its specific receptors directly affect the subsequent biological action of insulin.