Changes in Skeletal Muscle Protein Metabolism Signaling Induced by Glutamine Supplementation and Exercise

Abstract

Aim: To evaluate the effects of resistance exercise training (RET) and/or glutamine supplementation (GS) on signaling protein synthesis in adult rat skeletal muscles.

Methods: The following groups were studied: (1) control, no exercise (C); (2) exercise, hypertrophy resistance exercise training protocol (T); (3) no exercise, supplemented with glutamine (G); and (4) exercise and supplemented with glutamine (GT). The rats performed hypertrophic training, climbing a vertical ladder with a height of 1.1 m at an 80° incline relative to the horizontal with extra weights tied to their tails. The RET was performed three days a week for five weeks. Each training session consisted of six ladder climbs. The extra weight load was progressively increased for each animal during each training session. The G groups received daily L-glutamine by gavage (one g per kilogram of body weight per day) for five weeks. The C group received the same volume of water during the same period. The rats were euthanized, and the extensor digitorum longus (EDL) muscles from both hind limbs were removed and immediately weighed. Glutamine and glutamate concentrations were measured, and histological, signaling protein contents, and mRNA expression analyses were performed.

Results: Supplementation with free L-glutamine increased the glutamine concentration in the EDL muscle in the C group. The glutamate concentration was augmented in the EDL muscles from T rats. The EDL muscle mass did not change, but a significant rise was reported in the cross-sectional area (CSA) of the fibers in the three experimental groups. The levels of the phosphorylated proteins (pAkt/Akt, pp70S6K/p70S6K, p4E-BP1/4E-BP1, and pS6/S6 ratios) were significantly increased in EDL muscles of G rats, and the activation of p4E-BP1 was present in T rats. The fiber CSAs of the EDL muscles in T, G, and GT rats were increased compared to the C group. These changes were accompanied by a reduction in the 26 proteasome activity of EDL muscles from T rats.

Conclusion: Five weeks of GS and/or RET induced muscle hypertrophy, as indicated by the increased CSAs of the EDL muscle fibers. The increase in CSA was mediated via the upregulated phosphorylation of Akt, 4E-BP1, p70S6k, and S6 in G animals and 4E-BP1 in T animals. In the EDL muscles from T animals, a decrease in proteasome activity, favoring a further increase in the CSA of the muscle fibers, was reported.

Keywords: 26S proteasome; 4E-BP-1; hypertrophy; pS6; physical exercise; proteasome.

Figure 1

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the concentrations of glutamine (a) and glutamate (b) in the EDL muscle. (c) Glutamine/glutamate ratio in EDL muscle. The results are presented as the mean ± SEM (standard error of the mean) and were compared using a two-way ANOVA test and the Bonferroni post-test (n = 5 to 6 animals per group). * p = 0.0422 versus T (exercise, hypertrophy resistance training exercise protocol). ^# p = 0.0447 versus C (Control, no exercise). ^& p = 0.0286 versus T (exercise, hypertrophy resistance training exercise protocol).

Figure 2

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the expression of IGF-1 mRNA in the EDL muscle. The results are presented as the mean ± SEM (standard error of the mean) and were compared using a two-way ANOVA test and the Bonferroni post-test (n = 5 to 6 animals per group).* p < 0.05 vs. C. Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise, supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 3

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the levels of phosphorylated Akt-1 (a) and p70S6K (b) in the EDL muscle. Representative bands of the proteins are shown in (c). The results are presented as the mean ± SEM (standard error of the mean) and were compared using a two-way ANOVA and the Bonferroni post-test (n = 8 to 9 animals per group). * p < 0.05 vs. C. Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 4

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the levels of phosphorylated P-4E-BP1 (a) and pS6 (b) in the EDL muscle. Representative bands of the proteins are shown in (c). The results are presented as the mean ± SEM (standard error of the mean) and were compared using a two-way ANOVA and the Bonferroni post-test (n = 8 to 9 animals per group). * p < 0.05 vs. C. Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise, supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 5

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the correlation between muscle glutamine content and pAkt (n = 20), pp70s6k (n = 21), p4E-BP1 (n = 21) and pS6 (n = 21) levels in the EDL muscle of the animals. (A) Correlation between EDL muscle glutamine concentration and pAkt/Akt. (B) Correlation between EDL muscle glutamine concentration and ppP70S6k/pP70S6k. (C) Correlation between EDL muscle glutamine concentration and p4E-BP1/4E-BP1. (D) Correlation between EDL muscle glutamine concentration and pS6/S6.Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 6

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the cross-sectional area (CSA) of muscle fibers of the EDL. (a) CSA (μm²) Mean ± SEM (standard error of the mean), and (b) muscle fibers CSA images (20×) obtained via optical microscopy after hematoxylin and eosin staining (HE). One thousand fibers were analyzed per group. Significant differences were reported using 95% confidence interval, as described in Materials and Methods (n = 6 animals per group). Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 7

Effects of L-glutamine supplementation (daily doses of 1 g/kg body weight for five weeks) associated or not with resistance exercise training on the activity of the 26S proteasome in the EDL muscle. The results are presented as the mean ± SEM (standard error of the mean) and were compared using two-way ANOVA and the Bonferroni post-test. (n = 5 animals per group). * p < 0.05 vs. C. Control, no exercise (C); exercise, hypertrophy resistance exercise training protocol (T); no exercise supplemented with glutamine (G); and exercise and supplemented with glutamine (GT).

Figure 8

Summary of the findings. Control, no exercise (C); exercise, hypertrophy resistance training exercise protocol (T); no exercise supplemented with glutamine (G); and exercise and supplemented with glutamine (GT). The increase in muscle CSAs in the three experimental groups was mediated via upregulated phosphorylation of Akt, 4E-BP1, p70S6k, and S6 in G and 4E-BP1 in T rats. Proteasome activity was decreased in trained rats, favoring a further increase in the muscle fibers CSA. Abbreviations: CSA, cross-sectional area; IGF1, Insulin-like growth factor 1; phosphorylated-Akt, protein kinase B; phosphorylated-S6, ribosomal protein S6; phosphorylated-4E-BP1, eukaryotic initiation factor 4E-binding protein 1.