Amino acids are important modulators of skeletal muscle metabolism, but their impact on glucose uptake by skeletal muscle remains unclear. To address the effect of an amino acid (AA) mixture consisting predominately of isoleucine on glucose uptake we first conducted a dose-response experiment, investigating how different concentrations of the AA mixture affect glucose uptake by isolated rat epitrochlearis muscle. In a subsequent experiment we examined how the AA mixture affects insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle. It was found that the AA mixture with as little as 0.5 mM Ile increases [H3]2-deoxy-D-glucose (2-DG) uptake by 76% compared to basal glucose uptake. The AA mixtures with 1, 2 or 4 mM Ile provided no significant additional effect. Next we combined the AA mixture consisting of 2 mM Ile, 0.012 mM Cys, 0.006 mM Val and 0.014 mM Leu with physiological levels (75 μU/ml, sINS) and maximally-stimulating levels (2 mU/ml, mINS) of insulin. The AA mixture only, sINS and mINS significantly increased 2-DG uptake compared to basal by 63, 79 and 298%, respectively. When the AA mixture was combined with sINS and mINS 2-DG uptake was further increased significantly by 26 and 14%, respectively. Western blotting analysis revealed that compared to basal the AA mixture increased AS160 phosphorylation, while phosphorylation of Akt and mTOR did not change. Combining the AA mixture with sINS resulted in no additional phosphorylation compared to sINS alone. Interestingly, addition of the AA mixture to mINS resulted in increased phosphorylation of mTOR, Akt and AS160 compared to mINS alone. Our results suggest that certain AAs (1) increase glucose uptake in the absence of insulin and (2) augment insulin-stimulated glucose uptake in an additive manner. These effects on glucose uptake appear to be mediated via a molecular pathway that is partially independent from the canonical insulin signaling cascade. / text