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The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

You JS, Anderson GB, Dooley MS, Hornberger TA - Dis Model Mech (2015)

Bottom Line: Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation.Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size.Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus

Rapamycin exacerbates immobilization-induced decreases in protein synthesis and muscle mass. Mice were subjected to unilateral hindlimb immobilization for 3 or 7 days (IM+), or a non-immobilized control condition (IM− or CNT), and received an acute (day 3) or chronic (day 7) administration of rapamycin (RAP+) or the vehicle (RAP− or VEH) as described in the Materials and Methods. At 30 min prior to the collection of the EDL muscles, mice were injected with puromycin. The muscles were (A) subjected to western blot analysis for phosphorylated (P) (T389) and total (T) p70 and P (S240/244)- and T-S6, (B) analyzed for the muscle weight (MW) to body weight (BW) ratio, (C,D) subjected to immunohistochemistry for laminin to obtain the cross-sectional area (CSA) (≥120 fibers per muscle), or (E,F) subjected to western blot analysis for puromycin-labeled peptides (i.e. protein synthesis) and ubiquitylated proteins, respectively. The values in A, E and F were expressed relative to the values obtained in the time-matched IM−/RAP− (A) or CNT/VEH groups (E,F). All values are presented as the mean (+s.e.m. in graphs, n=3-12 muscles per group). * versus the time- and drug-matched IM− or CNT groups, # versus the time- and mobility-matched RAP− or VEH groups, P≤0.05.
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DMM019414F2: Rapamycin exacerbates immobilization-induced decreases in protein synthesis and muscle mass. Mice were subjected to unilateral hindlimb immobilization for 3 or 7 days (IM+), or a non-immobilized control condition (IM− or CNT), and received an acute (day 3) or chronic (day 7) administration of rapamycin (RAP+) or the vehicle (RAP− or VEH) as described in the Materials and Methods. At 30 min prior to the collection of the EDL muscles, mice were injected with puromycin. The muscles were (A) subjected to western blot analysis for phosphorylated (P) (T389) and total (T) p70 and P (S240/244)- and T-S6, (B) analyzed for the muscle weight (MW) to body weight (BW) ratio, (C,D) subjected to immunohistochemistry for laminin to obtain the cross-sectional area (CSA) (≥120 fibers per muscle), or (E,F) subjected to western blot analysis for puromycin-labeled peptides (i.e. protein synthesis) and ubiquitylated proteins, respectively. The values in A, E and F were expressed relative to the values obtained in the time-matched IM−/RAP− (A) or CNT/VEH groups (E,F). All values are presented as the mean (+s.e.m. in graphs, n=3-12 muscles per group). * versus the time- and drug-matched IM− or CNT groups, # versus the time- and mobility-matched RAP− or VEH groups, P≤0.05.

Mentions: Our observation that immobilization induces the activation of mTOR signaling was unexpected, but it was not entirely surprising because recent studies have shown that denervation of the sciatic nerve, which induces neurogenic atrophy, also results in the activation of mTOR signaling (Quy et al., 2013; Tang et al., 2014). However, the functional role of mTOR activation in neurogenic atrophy is not entirely clear because, during denervation, the activation of mTOR signaling not only increases protein synthesis, but it can also increase protein degradation through a negative feedback inhibition of the anti-catabolic PI3K-PKB signaling pathway (Harrington et al., 2005; Quy et al., 2013; Stitt et al., 2004; Tang et al., 2014). Thus, we set out to define the role that mTOR activation plays in immobilization-induced atrophy. To accomplish this, we first performed an experiment in which mice were treated with rapamycin during the period of immobilization. As shown in Fig. 2A, rapamycin effectively inhibited the increase in p70S6k (T389) phosphorylation that was observed in the extensor digitorum longus (EDL) muscles after 3 and 7 days of immobilization. In these analyses, we also examined the phosphorylation status of the ribosomal S6 protein, which is a downstream substrate of p70S6k (Ferrari et al., 1991). As expected, immobilization induced an increase in S6 (S240/244) phosphorylation and rapamycin significantly reduced the phosphorylation of these sites. Moreover, we found that immobilization induced an increase in the total amount of the S6 protein and this event was completely inhibited by rapamycin. Finally, and most importantly, our results demonstrated that rapamycin exacerbates the reductions in muscle mass and fiber size that occur after 7 days of immobilization (Fig. 2B-D).Fig. 2.


The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

You JS, Anderson GB, Dooley MS, Hornberger TA - Dis Model Mech (2015)

Rapamycin exacerbates immobilization-induced decreases in protein synthesis and muscle mass. Mice were subjected to unilateral hindlimb immobilization for 3 or 7 days (IM+), or a non-immobilized control condition (IM− or CNT), and received an acute (day 3) or chronic (day 7) administration of rapamycin (RAP+) or the vehicle (RAP− or VEH) as described in the Materials and Methods. At 30 min prior to the collection of the EDL muscles, mice were injected with puromycin. The muscles were (A) subjected to western blot analysis for phosphorylated (P) (T389) and total (T) p70 and P (S240/244)- and T-S6, (B) analyzed for the muscle weight (MW) to body weight (BW) ratio, (C,D) subjected to immunohistochemistry for laminin to obtain the cross-sectional area (CSA) (≥120 fibers per muscle), or (E,F) subjected to western blot analysis for puromycin-labeled peptides (i.e. protein synthesis) and ubiquitylated proteins, respectively. The values in A, E and F were expressed relative to the values obtained in the time-matched IM−/RAP− (A) or CNT/VEH groups (E,F). All values are presented as the mean (+s.e.m. in graphs, n=3-12 muscles per group). * versus the time- and drug-matched IM− or CNT groups, # versus the time- and mobility-matched RAP− or VEH groups, P≤0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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DMM019414F2: Rapamycin exacerbates immobilization-induced decreases in protein synthesis and muscle mass. Mice were subjected to unilateral hindlimb immobilization for 3 or 7 days (IM+), or a non-immobilized control condition (IM− or CNT), and received an acute (day 3) or chronic (day 7) administration of rapamycin (RAP+) or the vehicle (RAP− or VEH) as described in the Materials and Methods. At 30 min prior to the collection of the EDL muscles, mice were injected with puromycin. The muscles were (A) subjected to western blot analysis for phosphorylated (P) (T389) and total (T) p70 and P (S240/244)- and T-S6, (B) analyzed for the muscle weight (MW) to body weight (BW) ratio, (C,D) subjected to immunohistochemistry for laminin to obtain the cross-sectional area (CSA) (≥120 fibers per muscle), or (E,F) subjected to western blot analysis for puromycin-labeled peptides (i.e. protein synthesis) and ubiquitylated proteins, respectively. The values in A, E and F were expressed relative to the values obtained in the time-matched IM−/RAP− (A) or CNT/VEH groups (E,F). All values are presented as the mean (+s.e.m. in graphs, n=3-12 muscles per group). * versus the time- and drug-matched IM− or CNT groups, # versus the time- and mobility-matched RAP− or VEH groups, P≤0.05.
Mentions: Our observation that immobilization induces the activation of mTOR signaling was unexpected, but it was not entirely surprising because recent studies have shown that denervation of the sciatic nerve, which induces neurogenic atrophy, also results in the activation of mTOR signaling (Quy et al., 2013; Tang et al., 2014). However, the functional role of mTOR activation in neurogenic atrophy is not entirely clear because, during denervation, the activation of mTOR signaling not only increases protein synthesis, but it can also increase protein degradation through a negative feedback inhibition of the anti-catabolic PI3K-PKB signaling pathway (Harrington et al., 2005; Quy et al., 2013; Stitt et al., 2004; Tang et al., 2014). Thus, we set out to define the role that mTOR activation plays in immobilization-induced atrophy. To accomplish this, we first performed an experiment in which mice were treated with rapamycin during the period of immobilization. As shown in Fig. 2A, rapamycin effectively inhibited the increase in p70S6k (T389) phosphorylation that was observed in the extensor digitorum longus (EDL) muscles after 3 and 7 days of immobilization. In these analyses, we also examined the phosphorylation status of the ribosomal S6 protein, which is a downstream substrate of p70S6k (Ferrari et al., 1991). As expected, immobilization induced an increase in S6 (S240/244) phosphorylation and rapamycin significantly reduced the phosphorylation of these sites. Moreover, we found that immobilization induced an increase in the total amount of the S6 protein and this event was completely inhibited by rapamycin. Finally, and most importantly, our results demonstrated that rapamycin exacerbates the reductions in muscle mass and fiber size that occur after 7 days of immobilization (Fig. 2B-D).Fig. 2.

Bottom Line: Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation.Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size.Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus