In a previous paper (I), we reported a new phenomenon in the behavior of insulin: when the surviving isolated diaphragm of the rat is momentarily equilibrated (as little time as 10 seconds suffices) in a medium containing insulin and t, hen equilibrated aerobically with glucose, the extra synthesis of glycogen compared to controls not pretreated with insulin indicates that the metabolism of the tissue is being accelerated. The effect persists after prolonged washing of the diaphragm and, as shown by appropriate controls, is not due to insulin in solution. We concluded therefore that the insulin is bound to the tissues by forces not yet defined and in the bound form exerts its customary effect. Subsequently we reported (2, 3) that adipose tissue and lactating mammary tissue of the rat showed the same phenomenon and responded in their respective characteristic ways to the bound hormone. Our further experience since these publications has made it appear probable that the fixation of insulin to tissues is necessary for its physiological action and that the phenomenon is a central one in the problem of the action of insulin. For example, t. he action of the bound insulin on the glycogen synthesis of the ratI diaphragm is altered by the endocrine state of the experimental animal, being diminished in alloxan diabetes, increased following hypophysectomy, and decreased by the prior injection of pituitary and adrenal hormones.

In these early experiments we were limited to measuring the insulin bound by determining its extra met, abolic effects. While this method had the great advantage of specificity, it could never give absolute amounts, but only approximations of relative quantities of bound insulin. More important, as we have pointed out before, no answer as to whether the contrainsulin action of other hormones is due to inhibition of action of the bound insulin or to inhibition of binding could be obtained by the biological method. The direct and quantitative determination of the minute amounts of bound insulin could best be made by the use of isotopic