Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle

Abstract

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282% of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292%, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336%, P = 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647%, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292%, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2 alpha phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2 alpha. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.

Figure 1. PKB phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of PKB on Ser-473 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data expressed as a percentage of sedentary (Sed) controls (means ± s.e.m.). Time points are in minutes. *P < 0.05vs. controls (n = 7-10 per group).

Figure 2. mTOR phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of mTOR on Ser-2448 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data expressed as a percentage of sedentary controls (means ± s.e.m.). n = 7-10 per group.

Figure 3. 4E-BP1 phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of 4E-BP1 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data are calculated as percent γ-form and are expressed as a percentage of sedentary controls (means ± s.e.m.). ‡P < 0.01vs. controls. *P < 0.05vs. controls. n = 7-10 per group.

Figure 4. eIF4E to eIF4G association in response to acute resistance exercise in rat skeletal muscle

eIF4E to eIF4G association was determined in the gastrocnemius by immunoprecipitating eIF4E with a monoclonal antibody. eIF4G was assessed by immunoblot analysis and results were normalized to the total eIF4E in the immunoprecipitation. A, representative immunoblot. B, data expressed as a percentage of sedentary controls (means ± s.e.m.). *P < 0.05vs. controls (n = 7-10 per group).

Figure 5. ODC activity in response to acute resistance exercise in rat skeletal muscle

ODC activity was determined by measuring the release of ¹⁴ CO₂ from l-[1-¹⁴ C]ornithine. Data are expressed as arbitrary units (means ± s.e.m.). n = 5 per group.

Figure 6. S6K1 phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of S6K1 on Thr-389 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data expressed as a percentage of sedentary controls (means ± s.e.m.). n = 7-10 per group.

Figure 7. Ribosomal protein S6 phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of S6 on Ser-235-236/240-244 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data expressed as a percentage of sedentary controls (means ± s.e.m.). *P < 0.05vs. controls (n = 7-10 per group).

Figure 8. eIF2B activity in response to acute resistance exercise in rat skeletal muscle

The guanine nucleotide exchange activity of eIF2B in skeletal muscle was measured by the exchange of [³H]GDP bound to eIF2 for non-radioactively labelled GDP. Data are expressed as picomoles of GDP exchanged per minute (means ± s.e.m.). n = 5 per group.

Figure 9. eIF2α phosphorylation in response to acute resistance exercise in rat skeletal muscle

Phosphorylation of eIF2 α on Ser-51 was determined in the gastrocnemius using Western blot analysis. A, representative immunoblot. B, data are expressed as a percentage of sedentary controls (means ± s.e.m.). n = 7-10 per group.