Enhanced skeletal muscle ribosome biogenesis, yet attenuated mTORC1 and ribosome biogenesis-related signalling, following short-term concurrent versus single-mode resistance training

Abstract

Combining endurance training with resistance training (RT) may attenuate skeletal muscle hypertrophic adaptation versus RT alone; however, the underlying mechanisms are unclear. We investigated changes in markers of ribosome biogenesis, a process linked with skeletal muscle hypertrophy, following concurrent training versus RT alone. Twenty-three males underwent eight weeks of RT, either performed alone (RT group, n = 8), or combined with either high-intensity interval training (HIT+RT group, n = 8), or moderate-intensity continuous training (MICT+RT group, n = 7). Muscle samples (vastus lateralis) were obtained before training, and immediately before, 1 h and 3 h after the final training session. Training-induced changes in basal expression of the 45S ribosomal RNA (rRNA) precursor (45S pre-rRNA), and 5.8S and 28S mature rRNAs, were greater with concurrent training versus RT. However, during the final training session, RT further increased both mTORC1 (p70S6K1 and rps6 phosphorylation) and 45S pre-rRNA transcription-related signalling (TIF-1A and UBF phosphorylation) versus concurrent training. These data suggest that when performed in a training-accustomed state, RT induces further increases mTORC1 and ribosome biogenesis-related signalling in human skeletal muscle versus concurrent training; however, changes in ribosome biogenesis markers were more favourable following a period of short-term concurrent training versus RT performed alone.

Figure 1

Overview of the role of mTORC1 signalling in promoting ribosome biogenesis following a single session of resistance exercise, and the potential effect of incorporating endurance training (i.e., performing concurrent training). Adapted with permission from. Ribosome biogenesis involves transcription of the 45S rRNA (ribosomal RNA) precursor (45S pre-rRNA) (A) mediated by RNA Polymerase I (Pol-I), processing of the 45S pre-rRNA into several smaller rRNAs (18S, 5.8S and 28S rRNAs) (B), assembly of these rRNAs and other ribosomal proteins into ribosomal subunits (40S and 60S) (C), and nuclear export of these ribosomal subunits into the cytoplasm^, (D). As well as regulating translational efficiency via downstream control of p70S6K (p70 kDa ribosomal protein subunit kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E binding protein 1) (E), mTORC1 is a key mediator of ribosome biogenesis by regulating transcription factors for genes encoding RNA Pol-I (and also RNA Pol-II and –III, which are not shown in figure). Transcription of the 45S pre-rRNA by RNA Pol-I requires a transcriptional complex including TIF-1A (transcription initiation factor 1A; also known as RRN5) and UBF (upstream binding factor), both of which are regulated by the mTORC1 pathway^, (F). Activation of AMPK is known to inhibit mTORC1 signalling in rodent skeletal muscle, and AMPK activation in skeletal muscle is traditionally associated with endurance-type exercise. However, whether signalling events initiated by endurance training, when performed concurrently with resistance training, have the potential to interfere with mTORC1-mediated regulation of ribosome biogenesis is currently unclear (G).

Figure 2

Expression of 45S pre-rRNA relative to the geometric mean of the expression of three housekeeping genes (HKG) (cyclophillin, β2M and TBP) before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h and 3 h after a single exercise bout performed post-training. Data presented are means ± SD and expressed relative to the PRE-T value for each corresponding group. *P < 0.05 vs. PRE-T, ^achange between PRE-T and POST-T substantially different vs. RT.

Figure 3

Expression of the mature rRNA transcripts 5.8S rRNA (A), 18S rRNA (C), and 28S rRNA (E), and rRNA transcripts bound to the 45S pre-RNA precursor: 5.8S rRNA (span) (B) 18S rRNA (span) (D) and 28S rRNA (span) (F) relative to the geometric mean of the expression of three housekeeping genes (HKG) (cyclophillin, β2M and TBP) before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h and 3 h after a single exercise bout performed post-training. Data presented are means ± SD and expressed relative to the PRE-T value for each corresponding group. *P < 0.05 vs. PRE-T, ^†P < 0.05 vs. POST-T, ^achange between PRE-T and POST-T substantially greater vs RT.

Figure 4

Phosphorylation of TIF-1A^Ser649 (A), UBF^Ser388 (B), and total protein content of cyclin D1 (C) before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h and 3 h after a single exercise bout performed post-training. Data presented are means ± SD and expressed relative to the PRE-T value for each corresponding group. *P < 0.05 vs. PRE-T, ^†P < 0.05 vs. POST-T. Change from POST-T substantially greater vs. ^eHIT+RT, ^fMICT+RT.

Figure 5

mRNA expression of TIF-1A (A), UBF (B), POLR1B (C), and cyclin D1 (D) relative to the geometric mean of the expression of three housekeeping genes (HKG) (cyclophillin, β2M and TBP) before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h and 3 h after a single exercise bout performed post-training. Data presented are means ± SD and expressed relative to the PRE value for each corresponding group. *P < 0.05 vs. PRE-T, ^†P < 0.05 vs. POST-T. Change from POST-T substantially greater vs. d = RT.

Figure 6

Phosphorylation of AMPK^Thr172 (A), ACC^Ser79 (B), mTOR^Ser2448 (C), p70S6K^Thr389 (D), rps6^Ser235/236 (E) and 4E-BP1^Thr36/47 (F) before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h and 3 h after a single exercise bout performed post-training. Data presented are means ± SD and expressed relative to the PRE value for each corresponding group. *P < 0.05 vs. PRE-T, ^†P < 0.05 vs. POST-T. Change from POST-T substantially greater vs. ^eHIT+RT, ^fMICT+RT.

Figure 7

Representative immunohistochemical images of muscle cross-sections obtained before (PRE) and after (POST) eight weeks of either RT alone (images (A) and (B), respectively), or RT combined with either high-intensity interval training (HIT+RT; images (C) and (D), respectively) or moderate-intensity continuous training (MICT+RT; images (E and F) respectively). Muscle fibre membranes are stained red, type I muscle fibres are stained green, and type II muscle fibres are unstained.

Figure 8

Study overview (A) and timelines for the final training session (B). Participants first completed 8 weeks of either resistance training (RT) alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT). For the final training session (B), participants completed the RE protocol alone (i) or after a 15-min recovery following the completion of either HIT (ii) or work-matched MICT (iii) cycling. Muscle biopsies were obtained from the vastus lateralis at rest before training, and immediately before beginning the final training session, and 1 h and 3 h after completion of RE.

Figure 9

Representative western blots for the phosphorylation (p-) and total protein content of signalling proteins before (PRE-T) and after (POST-T) eight weeks of either RT alone, or RT combined with either high-intensity interval training (HIT+RT) or moderate-intensity continuous training (MICT+RT), and 1 h (+1 h) and 3 h (+3 h) after a single exercise bout performed post-training. Cropped western blot images are displayed for clarity of presentation, and full-length western blot images are presented in supplementary information.