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. 2015 Jun 8;10(6):e0128263.
doi: 10.1371/journal.pone.0128263. eCollection 2015.

Protective Effects of Clenbuterol Against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

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Protective Effects of Clenbuterol Against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

Daisuke Umeki et al. PLoS One. .
Free PMC article

Abstract

Background: Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition.

Methodology: We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB.

Results and conclusion: Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Changes of body weight, daily consumption of food and water, and daily intake of CB and energy.
(A) Body weight (BW: g) of CB was similar to the Control (P = NS vs. Control). On the other hand, BW of both the DEX and CB+DEX groups was significantly smaller than that in the Control (**P < 0.01 vs. Control in each case). (B-C) No significant difference in daily consumption of food (B) or water (C) was observed among the CB, DEX, CB+DEX, and the Control groups (P = NS vs. Control in each case).(D-E) No significant difference was observed in daily intake of CB per baseline BW between the CB and CB+DEX groups (D) or in energy intake among the CB, DEX, CB+DEX, and the Control groups (E) (P = NS vs. Control in each case). The values of BW, consumption of food, consumption of water, CB intake, and energy intake in the Control group were taken as 100% in each determination.
Fig 2
Fig 2. Masseter muscle hypertrophy after treatment with CB and/or DEX for 2 weeks.
(A) Masseter muscle mass weight of CB was significantly greater, and that of DEX was significantly smaller than that of the Control (**P < 0.01 vs. Control in each case). Importantly, the DEX-mediated inhibition was suppressed by co-treatment with CB (CB+DEX vs. DEX, ## P < 0.01). (B) Representative images of HE staining of masseter muscle of rats in the Control, CB, DEX, and CB+DEX groups. Scale bar: 100 μm. (C) Fiber diameter of CB was significantly greater and that of DEX was significantly smaller than that of the Control (**P < 0.01 vs. Control in each case). DEX-mediated decrease was suppressed by co-treatment with CB (CB+DEX vs. DEX, # P < 0.05). (D) CSA of CB was significantly greater (**P < 0.01) and that of DEX was significantly smaller (*P < 0.05) than that of the Control. DEX-mediated inhibition was suppressed by the co-treatment of CB (## P < 0.01 vs. DEX). Masseter mass, fiber diameter and CSA in the Control were taken as 100% in each determination.
Fig 3
Fig 3. Changes in composition of MHC isoforms in masseter muscle after treatment with CB and/or DEX for 2 weeks.
(A) Protein expression level of MHC-IIa in the CB group was significantly smaller than that of the Control (**P < 0.01), while those in the DEX and CB+DEX groups were similar to the Control (P = NS vs. Control in each case) (B) Protein expression level of MHC-IId/x in the CB group was similar to that of the Control (P = NS), while those of MHC-IId/x in the DEX and CB+DEX groups tended to be greater than that of the Control, though without significance (P = NS vs. Control in each case). (C) Protein expression level of MHC-IIb in the CB group was significantly greater and that in the DEX group was significantly smaller than that in the Control group (**P < 0.01 vs. Control in each case). The DEX-mediated decrease of MHC-IIb was suppressed by co-treatment with CB (# P < 0.05). (D) Representative silver staining of MHC isoforms in the masseter muscle. Expression of MHC-I was not observed. The density of the MHC-Neo band was too low to permit quantification. The average amount of each MHC isoform expression in the Control was taken as 100% in each determination.
Fig 4
Fig 4. Changes in expression of β2-AR and glucocorticoid receptor after treatment with CB and/or DEX for 2 weeks.
(A) Expression of β2-AR protein in masseter muscle of the CB, DEX, and CB+DEX groups was smaller than that in the Control (*P < 0.05, **P < 0.01 vs. Control), but the magnitude of the attenuation was similar among the three groups. (B) There was no change in expression of glucocorticoid receptor protein in the CB, DEX, and CB+DEX groups (P = NS vs. Control in each case). (C) Representative immunoblotting results for β2-AR and glucocorticoid receptor. The amount of expression in the Control was taken as 100% in each determination. GAPDH; glyceraldehyde 3-phosphate dehydrogenase
Fig 5
Fig 5. Effects of CB and DEX on IGF1 expression, myostatin expression, and Akt phosphorylation in masseter muscle after treatment with CB and/or DEX for 2 weeks.
(A) IGF1 expression in masseter muscle of the CB group was greater than that of the Control (**P < 0.01). Conversely, IGF1 expression of the DEX group was smaller than that of the Control (*P < 0.05). The DEX-mediated inhibition of IGF1 was suppressed by co-treatment with CB (CB+DEX vs. DEX, # P < 0.05) (B) Expression of myostatin protein was similar in all four groups (P = NS vs. Control in each case). (C) Phosphorylation of Akt on serine 473 in the CB, DEX, and CB+DEX groups was significantly greater than that in the Control (*P < 0.05, **P < 0.01 vs. Control). (D) Representative immunoblotting results for IGF1, myostatin, and phosphorylated Akt, and total Akt. The amount of expression or phosphorylation level in the Control was taken as 100% in each determination. p-Akt, phosphorylated Akt at serine 473; t-Akt, total Akt, GAPDH; glyceraldehyde 3- phosphate dehydrogenase
Fig 6
Fig 6. Effects of CB and DEX on phosphorylation of FOXO1, FOXO3a, and S6K1.
(A) Phosphorylation of FOXO1 at serine 259 in the CB, DEX, and CB+DEX groups was similarly and significantly greater than that in the Control (**P < 0.01 vs. Control in each case). (B) Phosphorylation of FOXO3a at serine 253 in CB, DEX, and CB+DEX was similarly and significantly greater than that in the Control (**P < 0.01 vs. Control in each case). (C) Phosphorylation of S6K1 on threonine 389 in the CB group was significantly greater than that in the Control (*P < 0.05), while that in the DEX group was significantly lower than that in the Control (*P < 0.05). Importantly, DEX-induced dephosphorylation at this site was significantly suppressed by co-treatment with CB (CB+DEX vs. DEX (# P < 0.05)). (D) Representative immunoblotting results for phosphorylated and total FOXO1,FOXO3a, and S6K1. The amount of phosphorylation in the Control was taken as 100% in each determination. p-FOXO1, phosphorylated FOXO1 at serine 259; t-FOXO1, total FOXO1; p-FOXO3a, phosphorylated FOXO3a at serine 253; t-FOXO3a, total FOXO3a, p-S6K1, phosphorylated S6K1 at threonine 389; total S6K1, t-S6K1.
Fig 7
Fig 7. Changes in expression of atrogin-1 and MuRF1 in masseter muscle after treatment with CB and/or DEX for 2 weeks.
(A and B) Expression levels of atrogin-1 mRNA (A) and MuRF1 mRNA (B) in the DEX group were greater than those of the Control (*P < 0.05, **P < 0.01 vs. Control in each case). DEX-induced upregulation of atrogin-1 mRNA expression, as well as MuRF1 mRNA expression, was unaffected by co-treatment with CB. The amount of mRNA expression in the Control was taken as 100% in each determination.
Fig 8
Fig 8. (A-C) Phosphorylation of NFATc1 on serine 259, phosphorylation of NFATc3 on serine 265, and expression of modulatory calcineurin-interacting protein 1 were similar in all four groups (P = NS vs. Control in each case).
(D) Representative immunoblotting results for phosphorylated and total NFATc1 and NFATc3, together with expression of modulatory calcineurin-interacting protein 1. The amount of phosphorylation (NFATc1 and NFATc3) or expression (modulatory calcineurin-interacting protein 1) in the Control was taken as 100% in each determination. p-NFATc1; phosphorylated NFATc1 at serine 259; t-NFATc1; total NFATc1; p-NFATc3, phosphorylated NFATc3 at serine 265, t-NFATc3; total NFATc3, GAPDH; glyceraldehyde 3- phosphate dehydrogenase
Fig 9
Fig 9. Schematic illustration of the proposed pathways involved in the protective effects of CB against DEX-induced muscle atrophy.
CB as well as DEX induces phosphorylation of Akt on serine 476. Activation of the Akt/mTOR pathway was increased by CB, but decreased by DEX. Activation of the Akt/FOXO pathway was similarly increased by CB and DEX, indicating that another pathway that is independent of Akt/FOXO pathway may account for the augmented expression of atrogin-1 and MuRF1 in DEX-treated group. Solid black lines represent findings in this study and solid grey lines represent findings reported previously [5,13,16,48]. IGF1; insulin growth factor 1, REDD1; regulated in development and DNA damage responses 1, PI3K; phosphoinositide 3-kinase, KLF15; Kruppe-like factor 15

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Grant support

This study was supported in part by grants from a Grant-in-Aid for Scientific Research on Innovative Areas (22136009: Dr. Okumura), and grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology [Drs. Umeki (26861803), Mototani (22791147), Suita (24790219), Fujita (25460296), Nakamura (23593052), Okumura (23591087)], Takeda Science Foundation (Dr. Okumura), Yokohama Foundation for Advancement of Medical Science (Dr. Okumura), Yokohama Academic Foundation (Dr. Ohnuki), Research Foundation for Community Medicine (Dr. Okumura), and Suzuken Memorial Foundation (14-014: Dr. Okumura).
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