Because greater Akt substrate of 160 kDa (AS160) phosphorylation has been reported in insulin-stimulated skeletal muscles without improved Akt activation several hours post-exercise, we hypothesized that prior exercise would result in attenuated AS160 dephosphorylation in insulin-stimulated rat skeletal muscle. Epitrochlearis muscles were isolated from rats that were sedentary (SED) or exercised 3 h earlier (3 h post-exercise; 3hPEX). Paired muscles were incubated with [3H]-2-deoxyglucose

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... without insulin or with insulin. Lysates from other insulin-stimulated muscles from SED or 3hPEX rats were evaluated using AS160Thr642 and AS160Ser588 dephosphorylation assays. Prior exercise led to greater 2-DG uptake concomitant with greater AS160Thr642 phosphorylation and a non-significant trend (P=0.087) for greater AS160Ser588. Prior exercise also reduced AS160Thr642 and AS160Ser588 dephosphorylation rates. These results support the idea that attenuated AS160 dephosphorylation may favor greater AS160 phosphorylation post-exercise.