Effects of prior exercise on components of insulin signalling in equine skeletal muscle

Abstract

Exercise is an important stimulus for the regulation of numerous metabolic functions in muscle, but there has been little investigation of the mechanism of exercise-enhanced glucose utilisation. To examine the effects of exercise on the signalling components Akt and glycogen synthase kinase-3 (GSK3) involved in insulin regulation of glycogen synthase (GS) activity. It was hypothesised that hyperinsulinaemia and prior exercise would alter the extent of Akt phosphorylation and GSK3beta deactivation and increase insulin stimulation of GS activity in skeletal muscle. Eight Standardbred horses completed a euglycaemic-hyperinsulinaemic clamp (EHC) either without prior exercise (NonEX) or at 0.5, 4 or 24 h post exercise (0.5 PostEX, 4 PostEX, and 24 PostEX, respectively). Muscle samples for determination of Akt and GSK3 serine phosphorylation, and GS fractional velocity (GS(FV)) were collected 5 min before (Pre-INS) and at the end (Post-INS) of each EHC (pre- and post insulin stimulation). Protein was separated by SDS-PAGE on polyacrylamide gels, transferred to nitrocellulose membranes and incubated with antibodies against phospho-Akt Ser473 and GSK3alpha/beta Ser21/9. GS(FV) was increased (P<0.05) 2-fold at 0.5 PostEX, 4 PostEX and 24 PostEX when compared to NonEX in Pre-INS and in Post-INS was increased 33 and 28% in NonEX and at 24PostEX (P<0.05), respectively, when compared to Pre-INS in these trials. Phospho-Akt Ser473 was increased at 0.5 PostEX only (P<0.05) in Pre-INS but increased in all trials in Post-INS. Phospho-GSK3alpha (Ser21) was also greater (P<0.05) at 0.5 PostEX in Pre-INS than in NonEX and 24 PostEX and in Post-INS increased (P<0.05) in NonEX and at 24 PostEX but not at 0.5 PostEX or 4 PostEX post exercise. There was no effect of time or treatment on GSK3, (Ser9) phosphorylation or on total GSK3. Elevated plasma insulin and prior exercise were associated with Akt activation and GSK3alpha deactivation (0.5 PostEX) indicating that an effect of exercise is evident in the early post exercise period and supporting a physiological role for GSK3alpha in the regulation of GS activity. The changes are consistent with insulin signalling downstream from these molecules.