Utilizing a modification of the rat diaphragm technic, studies of the mechanism of action of the albumin-associated antagonist were carried out using human fraction V. It was demonstrated that the antagonist could not be washed off of the diaphragm, that prior exposure of the diaphragm to insulin did not fully protect it from subsequent antagonism of the insulin effect and that, although antagonism was proportional to albumin concentration, large excesses of insulin could not completely overcome small concentrations of the antagonist. The intracellular accumulation of AIB was inhibited by the antagonistic albumin but not by a nonantagonistic one (bovine fraction V). Both albumins depressed radioactive glycine incorporation into muscle protein. The antagonist was shown to inhibit glucose uptake much more than glycogen synthesis.
These data indicate that the albumin-associated antagonist primarily blocks insulin effects on transport by either an extremely strong binding to the muscle cell or an irreversible alteration of the diaphragm's response to insulin without binding.