Insulin stimulation of amino acid uptake in rat diaphragm. Relationship to protein sythesis.

1. Puromycin dihydrochloride inhibited uptake of CYaminoisobutyric acid (AID), 1-aminocyclopentane-I-carboxylic acid, and a, y-diaminobutyric acid by intact diaphragm muscle from male rats weighing 60 to 90 g when the antibiotic was added to the system in vitro 120 and 180 min before the addition of the substrate. 2. In the absence of insulin, this inhibitory response was also produced by cycloheximide, but not by the aminonucleoside of puromycin or actinomycin D. In the presence of insulin, AIB uptake was inhibited by actinomycin D and the aminonucleoside of puromycin as well as by puromycin dihydrochloride and cycloheximide, the degree of inhibition paralleling inhibition of r,-lysine incorporation into protein. 3. The antibiotics shown to inhibit amino acid transport did not change tissue water spaces, oxygen consumption, the concentration of cr-amino nitrogen in the incubation medium, or the diffusion constant for AIB (KD). 4. In diaphragm muscle from rats weighing 60 to 90 g, incubation with puromycin for 180 min reduced subsequent stimulation of AIB transport by insulin. In muscle from animals weighing 220 to 250 g, stimulation by insulin was completely abolished by puromycin. 5. These studies show that puromycin inhibits the synthesis of a rapidly turning over protein or proteins involved in the transport of AIR The results suggest that insulin stimulates amino acid transport in two distinct ways: by increasing the affinity of the carrier mechanism for amino acids; and by initiating the synthesis of a specific protein or proteins which enhances transport.