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. 2015 Sep 15;593(18):4275-84.
doi: 10.1113/JP271219.

Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise

Andrew Philp  1   2 , Simon Schenk  3 , Joaquin Perez-Schindler  1 , D Lee Hamilton  4 , Leigh Breen  1   5 , Erin Laverone  2 , Stewart Jeromson  4 , Stuart M Phillips  5 , Keith Baar  2
Affiliations

Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise

Andrew Philp et al. J Physiol. .

Abstract

The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min(-1) ; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg(-1) ) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg(-1) ) of l-[ring-(13) C6 ]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation.

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Figures

Figure 1
Figure 1
Differential effect of rapamycin on MyoPS and MitoPS following endurance exercise A, endurance exercise rapidly increased MyoPS in the vehicle group, with this response being sustained during the 6 h recovery period. By contrast, rapamycin blocked MyoPS 0.5 h post-exercise. B, MitoPS was significantly increased post-exercise in both the vehicle and rapamycin groups. C, area under the curve analysis demonstrated that rapamycin suppressed MyoPS without affecting MitoPS. All data are expressed as the mean ± SEM, normalized to vehicle-basal. *Significantly different from vehicle-basal. §Significantly different from vehicle 30 min post-exercise. ΦSignificantly different from vehicle (P < 0.05; n = 4 per group).
Figure 2
Figure 2
Endurance exercise and rapamycin treatment reduce mTORC1 signalling in skeletal muscle A, mTORSer2448 was unchanged at any timepoint during the 6 h recovery period. B, S6K1Thr389 phosphorylation was reduced in the vehicle and rapamycin groups post-exercise. C, endurance exercise reduced S6K1 kinase activity in the vehicle group and rapamycin blocked S6K1 activity throughout. D, 4E-BP1Thr37/46 phosphorylation was significantly reduced during the 6 h recovery period in the vehicle group, whereas rapamycin blocked phosphorylation independent of exercise. E, S6Ser235/236 phosphorylation was reduced at 0.5 and 3 h post-exercise in the vehicle group, and completely blunted in the rapamycin group. F, S6Ser240/244 phosphorylation was reduced at 3 h post-exercise in the vehicle group, and completely blunted in the rapamycin group. All data expressed as the mean ± SEM, normalized to vehicle-basal. *Significantly different from vehicle-basal. §Significantly different from vehicle group at the relevant timepoint (P < 0.05; n = 4 per group).
Figure 3
Figure 3
PGC-1α gene expression and associated signalling is increased post-exercise following rapamycin administration A, endurance exercise increased PGC-1α gene expression in both the vehicle and rapamycin groups post-exercise. Rapamycin amplified the post-exercise response, with PGC-1α gene expression being significantly higher at each timepoint post-exercise compared to vehicle. B, PDK4 gene expression followed a similar pattern, increasing at 3 and 6 h post-exercise in the vehicle group, with this response being enhanced by rapamycin treatment, being significantly higher at 3 and 6 h post-exercise in the rapamycin group. C, TFAM followed a similar trend, increasing 6 h post-exercise in the vehicle group, and throughout the recovery period in the rapamycin group. All data expressed as the mean ± SEM, normalized to vehicle-basal. *Significantly different from vehicle-basal. §Significantly different from vehicle group at the relevant timepoint (P < 0.05; n = 4 per group).
Figure 4
Figure 4
Endurance exercise has no effect on AMPK activity or downstream signalling AMPK α1 (A) and AMPK α2 (B) activity were unchanged at any timepoint during the 6 h recovery period. In parallel, AMPKThr172(C) and ACCSer79 (D) phosphorylation were unchanged at any timepoint during the 6 h recovery period (n = 4 per group).
Figure 5
Figure 5
REDD1 but not eEF2 or ERK1/2 is rapidly induced following endurance exercise ERK1/2Thr202/Tyr204(A) and eEF2Thr56 (B) phosphorylation were unchanged at any timepoint during the 6 h recovery period. By contrast, REDD1 induction (C) occurred rapidly post-exercise in the vehicle group, increasing 3-fold, 0.5 h post-exercise, and remaining elevated 3 h post-exercise, before returning to baseline 6 h post-exercise. REDD1 induction did not occur in the rapamycin group. *Significantly different from vehicle-basal. §Significantly different from rapamycin group at the relevant timepoint (P < 0.05; n = 4 per group).
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