Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats

<div><p>Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex’s action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.</p></div

Effect of BCAA and Dex administration on body weight, food intake and muscle mass in the GH-treated SDRs.

<p>A. Dex treatment for 5 days elicited a reduction in body weight gain in the GH-administered SDRs irrespective of BCAA administration. BCAA did not increase body weight gain. B. Food intake by the Dex-treated SDRs was decreased irrespective of BCAA administration. C. Dex reduced soleus muscle mass, and BCAA recovered this reduction. D. Dex reduced EDL muscle mass, but BCAA did not recover this reduction. *, <i>P</i> < 0.05 vs. control group.</p

Effect of BCAA and Dex administration on body weight, food intake and muscle mass in the SDRs.

<p>A. Treatment with Dex for 5 days elicited a reduction in body weight gain in the SDRs irrespective of BCAA administration. BCAA did not increase body weight gain. B, C, D. Neither Dex nor BCAA affected food intake, soleus or EDL muscle mass. *, <i>P</i> < 0.05 vs. control group.</p

Phosphorylations of 4E-BP1 and p70S6K in the soleus muscles of the GH-administered SDRs.

<p>A. Western blots for phospho (Thr37/46) and total 4E-BP1 and phospho (Thr389) and total p70S6K in the GH-administered SDRs. B. BCAA tended to increase the phosphorylation of 4E-BP1 in the GH-treated SDR muscles in the presence and absence of Dex. C. BCAA significantly increased the phosphorylation of p70S6K in the presence of Dex and showed a tendency to elevate the phosphorylation in the absence of Dex. ¶, <i>P</i> < 0.05 vs. Dex-treated group.</p

The CSAs of the muscle fibers in the soleus and EDL muscles of the SDRs treated with dexamethasone, BCAA or both.

<p>A. ATPase staining (pH 10.7) of soleus muscle fibers from the SDRs. In this staining, type 1 fibers are stained light, and type 2 fibers are dark. Scale bar: 100 μm. B. ATPase staining (pH 10.7) of EDL muscle fibers from the SDRs. C. BCAA did not increase the CSAs in the soleus muscles. Dex elicited a slight increment in the CSAs compared to the control. The administration of both Dex and BCAA resulted in a decrease in CSA compared to the Dex-treated SDRs. *, <i>P</i> < 0.05 vs. control group; ¶, <i>P</i> < 0.05 vs. Dex-treated group. D. BCAA did not increase but rather decreased the CSAs in the EDL muscles. Dex elicited a slight increment in CSA, and the administration of both Dex and BCAA resulted in a decrease in CSA compared to the control and Dex-treated SDRs, respectively. *, <i>P</i> < 0.05 vs. control group; ¶, <i>P</i> < 0.05 vs. Dex-treated group.</p

Phosphorylations of 4E-BP1 and p70S6K in soleus muscles of the SDR.

<p>A. Western blots for phospho (Thr37/46) and total 4E-BP1 and phospho (Thr389) and total p70S6K. B. BCAA did not increase the phosphorylation of 4E-BP1 in the SDR muscles in the presence or absence of Dex. C. BCAA did not increase the phosphorylation of p70S6K in the presence or absence of Dex.</p

Comparison of basal mRNA levels between the SDR and the GH-treated SDR muscles.

<p>A, B, D and F. MuRF1, FoxO1, FoxO4 and REDD2 mRNA levels in rectus femoris muscle were not different between the GH-treated SDRs and the control SDRs. C, E and G. FoxO3, REDD1 and IGF-I mRNA levels were higher in the GH-treated SDRs than in the control SDRs. H. GR mRNA level was lower in the GH-treated SDRs than in the control SDRs. *, <i>P</i> < 0.05 vs. control SDRs (A ~ H). I. In soleus (gray column) and EDL muscles (black column), GR mRNA levels were lower in GH-treated SDRs and SD rats than in SDRs. *, <i>P</i> < 0.05 vs. normal SD rats (wild); ¶, <i>P</i> < 0.05 vs. SDRs.</p

Effects of BCAA on the dexamethasone-induced protein degradation pathway and GR mRNA levels in the SDR muscles.

<p>A. Dex increased Bnip3 mRNA level, but BCAA did not influence the Dex-elevated Bnip3 mRNA level in the EDL muscles of SDRs. Dex caused a similar response in the soleus muscles, but it was not significant. B, C and D. Neither Dex nor BCAA exhibited a significant influence on atrogin-1, MuRF1 or REDD1 mRNA levels. E. Dex increased the REDD2 mRNA level. BCAA exhibited a tendency to suppress the overexpression of REDD2 induced by Dex, but this difference was not significant. F, G, H, I and J. Neither Dex nor BCAA exhibited a significant influence on FoxO1, FoxO3, FoxO4, myostatin or IGF-I mRNA levels. K. GR mRNA level was not different between soleus and EDL muscles. Black column: EDL muscles, gray column: soleus muscles. *, <i>P</i> < 0.05 vs. control group; +, <i>P</i> < 0.05 vs. BCAA-treated group.</p

Muscle fiber CSAs of the soleus and EDL muscles in the GH-administered SDRs treated with dexamethasone, BCAA or both.

<p>A. ATPase staining (pH 10.7) of the soleus muscle fibers in GH-treated SDRs. In this staining, type 1 fiber are stained light, and type 2 fibers are dark. Scale bar: 100 μm. B. ATPase staining (pH 10.7) of the EDL muscle fibers of the GH-treated SDRs. C. Dex elicited a decrease in the CSA of the soleus muscles, and BCAA significantly recovered this decrease in CSA in the Dex-treated SDRs when supplemented with GH. *, <i>P</i> < 0.05 vs. control group; ¶, <i>P</i> < 0.05 vs. Dex-treated group. D. Dex elicited a decrease in the CSAs of the EDL muscles in the GH-treated SDRs. BCAA partially but significantly recovered the decrease in CSA.</p

Contents of the mTORC1 components and the signaling molecules up- and downstream of mTORC1 in the soleus muscles of the SDR and SD rats.

<p>A. Western blots for mTOR, Raptor and GβL in the SDR and wild-type SD rat muscles. B. The level of mTOR protein in the SDR muscles was not different from that in the SD rats. Raptor and GβL were increased in the SDR muscles compared to the SD rat muscles. C. Western blots for PI3K, Akt, 4E-BP1 and p70S6K in the SDR and SD rat muscles. D. The contents of PI3K and Akt, which are molecules that are upstream of mTORC1, were not different between the SDR and SD rats. The p70S6K content in the SDR muscles was not different from that in the SD rat muscles. The 4E-BP1 protein level was greater in the SDRs than in the SD rat.</p