Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

Abstract

Background: The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles.

Results: In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2-10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2-16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3-13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2-18 fold in atrophic denervated muscle.

Conclusions: The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

Figure 1

Muscle weights. Muscle weights of 6-days denervated (Den) hypertrophic hemidiaphragm muscles and 6-days denervated atrophic anterior tibial muscles compared to innervated (Inn) controls. Mean values ± standard error of the mean. ***p < 0.001.

Figure 2

Akt and GSK-3β protein and phosphorylation levels in 6-days denervated hypertrophic hemidiaphragm muscle. Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated hypertrophic hemidiaphragm muscle (Den) compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated hemidiaphragm muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. **p < 0.01, ***p < 0.001.

Figure 3

4EBP1, p70S6K1 and rpS6 protein and phosphorylation levels in 6-days denervated hypertrophic hemidiaphragm muscle. Expression of 4EBP1, p70S6K1 and rpS6 total protein (t-4EBP1, a; t-p70S6K1, c and t-rpS6, e) and phosphorylated 4EBP1 (p-4EBP1) at S65 (b), phosphorylated p70S6K1 (p-p70S6K1) at T389 (d) and phosphorylated rpS6 (p-rpS6) at S235/236 (f) in 6-days denervated hypertrophic hemidiaphragm muscle (Den) compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated hemidiaphragm muscle sample was used as a reference sample and included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. **p < 0.01, ***p < 0.001.

Figure 4

Akt and GSK-3β protein and phosphorylation levels in 6-days denervated atrophic anterior tibial muscle. Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated atrophic anterior tibial (Den) muscle compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated anterior tibial muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

4EBP1, p70S6K1 and rpS6 protein and phosphorylation levels in 6-days denervated atrophic anterior tibial muscle. Expression of 4EBP1, p70S6K1 and rpS6 total protein (t-4EBP1, a; t-p70S6K1, c and t-rpS6, e) and phosphorylated 4EBP1 (p-4EBP1) at S65 (b), phosphorylated p70S6K1 (p-p70S6K1) at T389 (d) and phosphorylated rpS6 (p-rpS6) at S235/236 (f) in 6-days denervated atrophic anterior tibial muscle (Den) compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated anterior tibial muscle sample was used as a reference sample and included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. *p < 0.05, **p < 0.01.

Figure 6

Akt mRNA expression. Akt1 and Akt2 mRNA expression in 6-days denervated atrophic hind-limb muscles (pooled muscle samples containing anterior tibial, extensor digitorum longus, soleus and gastrocnemius muscles) compared to innervated controls and expressed as fold change. Mean values ± standard error of the mean. *p < 0.05, **p < 0.01. The dotted line represents a fold change of 1, equal to no difference in expression between denervated and innervated muscles.