Binding of Insulin-131I by Isolated Frog Sartorius Muscles: RELATIONSHIP TO CHANGES IN PERMEABILITY TO SUGAR CAUSED BY INSULIN

Abstract

Abstract Isolated frog sartorius muscles were incubated with insulin at 19°, and changes in permeability to sugar were studied, with the use of 3-methylglucose as a model substrate. After incubation of muscles with different concentrations of insulin in the range of 20 to 500 or 1000 microunits per ml for 3½ hours, permeability attained a value that was approximately proportional to the logarithm of the concentration. No decrease of permeability occurred when muscles that had been exposed to insulin were later washed with Ringer's solution at 0°, but when washing was performed at 19°, the hormonal effect was reversed. In order to investigate the relationship of these findings to the binding of insulin, frog sartorius muscles were incubated with insulin-131I. More insulin-131I was found in incubated tissues than could be accounted for by their content of extracellular fluid, and this excess material has been referred to as insulin-131I. When muscles that had accumulated insulin-131I were washed with Ringer's solution at 0°, only the insulin-131I of the extracellular fluid was removed, and the accumulated material was retained without appreciable loss. Thus, when washing was conducted at 0°, a firm binding of insulin-131I to muscle was demonstrable. On the other hand, when washing was carried out at 19°, insulin-131I in the muscle was degraded to a form that was soluble in trichloracetic acid, and the amount that was retained fell progressively. Thus, degradation of insulin during washing at 19° may facilitate removal of the hormone from the cells and thereby permit reversal of the effect on permeability. At 19°, accumulation of insulin-131I was detectable soon after addition of the muscle to the incubation medium, but there was an absolute lag period of approximately 20 min before insulin began to enhance permeability to sugar. At 0°, accumulation of insulin-131I occurred almost as readily as at 19°, but permeability was not altered by insulin at this temperature during several hours of observation. Thus, the effect of insulin on permeability appears to involve a temperature-dependent reaction that occurs after initial binding of the hormone to the tissue. When studies of accumulation were carried out at different concentrations of insulin-131I, part of the accumulation process exhibited a high affinity for the hormone, and the tissue became saturated at a concentration of hormone, generally 1000 microunits per ml or less, which was sufficient to cause a maximal change in permeability to sugar. This type of accumulation has been referred to as specific in order to distinguish it from a concomitantly occurring nonspecific form which continued to increase in extent as the concentration of insulin-131I was raised to levels that greatly exceeded the physiological range.