Effects of phenylarsine oxide on insulin-stimulated system A amino acid uptake in skeletal muscle

The role of vicinal sulfhydryls in the stimulation by insulin of system A amino acid uptake in mammalian skeletal muscle was investigated. Neutral amino acid uptake via system A carriers was assessed using the nonmetabolizable analogue α-(methylamino)isobutyric acid (MeAIB). Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal sulfhydryls, at 40 μM inhibited basal and insulin-stimulated (2 mU/ml) MeAIB uptake in rat epitrochlearis muscles by ~50% and ~80%, respectively. No significant changes in the ATP level or in the lactate-to-pyruvate ratio were observed. Both inhibitory effects were completely preventable by coincubation with dimercaptopropanol, a vicinal dithiol, indicating the effects were mediated specifically by interactions with vicinal sulfhydryls. Stimulation of MeAIB uptake by the insulin-mimicker vanadate (10 mM) or by insulin-like growth factor I (IGF-I, 20 nM) was also inhibited by 80-90% by PAO. Kinetic analysis showed that PAO decreased the apparent V(max) for basal and insulin-stimulated MeAIB uptake without altering the apparent K(m). MeAIB uptake already maximally stimulated by insulin was rapidly (half-time = ~10 min) reversed by the addition of PAO so that the rate of MeAIB uptake was the same as in muscles incubated throughout with insulin and PAO. These results implicate a major role for vicinal sulfhydryls in the stimulation by insulin of amino acid uptake via system A carriers in skeletal muscle and suggest that the site of action of PAO on this system is distal to the insulin receptor, possibly at the carrier molecule itself.