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In recent years the carbohydrate metabolism of the isolated rat diaphragm has been widely studied (1)(2)(3)(4)(5)(6)(7)(8). Using this preparation, Gemmill (2, 3) succeeded in demonstrating in vitro the stimulating effect of insulin on the glucose utilization and glycogen synthesis of an isolated muscle. It has since been shown that a quantitative relationship exists between the amount of insulin present in the incubation medium and its effect on the glucose metabolism of the diaphragm (5, 6). Furthermore, it was found that the diaphragms of young rats are sensitive to extremely small concentrations of insulin (9). This investigation was intended to determine if the glucose metabolism of the isolated rat diaphragm could be used to demonstrate the presence of insulin in blood serum and to determine the concentration of this hormone in blood. and cooled. The muscle tissue, having been dried between filter paper, is weighed and the amount of glucose is determined in the medium (11). The difference in glucose utilization per 100 mg. of wet diaphragm between the flask containing insulin and the control flask indicates the effect of the added insulin. METHODSIn other experiments, four flasks containing the quarterdiaphragms of eight rats are used. Each diaphragm is cut in four pieces and the four quarters are distributed among the four flasks. Here, each flask contains eight quarter-diaphragms of eight different rats. This procedure is chosen so that the variation in glucose consumption and in sensitivity to insulin between individual diaphragms will be neutralized. The four flask technique enables us to compare the glucose utilization in glucose buffer solutions of three different insulin concentrations with the glucose utilization in a control vessel containing only glucose buffer. Generally speaking, the glucose utilization of hemi-diaphragms in buffer solutions without insulin is lower than that of quarter-diaphragms (on an equal weight basis), but the effect of added insulin is usually more evident when hemi-diaphragms are used. The glucose utilization of the diaphragm increases even further when it is divided into more fragments, but the effect of insulin becomes progressively less. For this reason, pieces of diaphragm smaller than a quarter-size have not been used in this work. Figure 1, a concentration action curve, illustrates the effect of different concentrations of a sample of purified insulin (expressed as units/ml. of the medium) on the glucose utilization (in mg./100 mg. wet tissue) of the rat diaphragm. The graph illustrates that, as the insulin concentration is increased, the insulin effect increases until, at about 107' units/ml., it tends to become more or less constant. In the region of lower concentrations, it was found that amounts as low as 5 X 10' still produced a significant increase in glucose utilization. The sample of pure insulin used in these experiments contained 28 units/mg.2 Assuming a molecular weight of insulin of 48,000, a concentration of 5 X 10' units/ml. is equal to 4.5 X 10 mo...

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