Role of Akt/GSK-3beta/beta-catenin transduction pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-treated rats

Abstract

Decrease of muscle IGF-I plays a critical role in muscle atrophy caused by glucocorticoids (GCs) because IGF-I gene electrotransfer prevents muscle atrophy caused by GCs. The goal of the present study was to identify the intracellular mediators responsible for the IGF-I anti-atrophic action in GC-induced muscle atrophy. We first assessed the IGF-I transduction pathway alterations caused by GC administration and their reversibility by local IGF-I overexpression performed by electrotransfer. Muscle atrophy induced by dexamethasone (dexa) administration occurred with a decrease in Akt (-53%; P<0.01) phosphorylation together with a decrease in beta-catenin protein levels (-40%; P<0.001). Prevention of atrophy by IGF-I was associated with restoration of Akt phosphorylation and beta-catenin levels. We then investigated whether muscle overexpression of these intracellular mediators could mimic the IGF-I anti-atrophic effects. Overexpression of a constitutively active form of Akt induced a marked fiber hypertrophy in dexa-treated animals (+175% of cross-sectional area; P<0.001) and prevented dexa-induced atrophy. This hypertrophy was associated with an increase in phosphorylated GSK-3beta (+17%; P<0.05) and in beta-catenin content (+35%; P<0.05). Furthermore, overexpression of a dominant-negative GSK-3beta or a stable form of beta-catenin increased fiber cross-sectional area by, respectively, 23% (P<0.001) and 29% (P<0.001) in dexa-treated rats, preventing completely the atrophic effect of GC. In conclusion, this work indicates that Akt, GSK-3beta, and beta-catenin probably contribute together to the IGF-I anti-atrophic effect in GC-induced muscle atrophy.

Figure 1

Decrease of Akt and p70S6K phosphorylation and β-catenin levels by dexa administration is reversed by local IGF-I overexpression. Tibial anterior muscles were injected and electroporated with a total of 100 μg pM1 plasmid at a concentration of 1 μg/μl (white column), whereas contralateral tibial anterior muscles were injected and electroporated with 100 μg pM1-hIGF-I plasmid at a concentration of 1 μg/μl (black column). Inset, Representative Western blot of phosphorylation forms (top) and total forms (bottom). Seven days after dexa administration, Akt phosphorylation (A), p70S6K phosphorylation (B), and β-catenin (D) were decreased without any changes in GSK3-β phosphorylation (C). IGF-I overexpression prevented these transduction pathway alterations observed in dexa-treated rats. Results are expressed as mean ± sem. ○, P < 0.05; ○○, P < 0.01; ○○○, P < 0.001 vs. ad libitum. *, P < 0.05; **, P < 0.01 vs. contralateral muscle.

Figure 2

caAkt muscle overexpression induces a marked fiber hypertrophy and prevents dexa-induced muscle atrophy. Overexpression of caAkt was detected by Western blot (A) and immunohistochemistry (B, top) in TA muscles electroporated with pCMV5-(m/p)Akt plasmid. caAkt overexpression induced a marked muscle fiber hypertrophy (black column) compared with surrounding UT muscle fibers (white column) (B, bottom). Results are expressed as mean ± sem. ***, P < 0.001 vs. UT muscles fibers of the same muscle. ○○○, P < 0.001 vs. ad libitum UT muscles fibers. ††, P < 0.01 vs. pair-fed caAkt transfected muscles fibers. •, P < 0.05 vs. ad libitum caAkt transfected muscles fibers. C, The proportion of large fibers in the three groups was larger in the caAkt DNA transfected muscle fibers (black column) compared with surrounding UT muscle fibers (white column). Statistical analysis was performed using the χ² Pearson test. ***, P < 0.001 for the three groups. A total of 1991 positive and 8280 UT muscle fibers were analyzed for measurements of fiber size.

Figure 3

caAkt overexpression stimulates p70S6K and GSK-3β phosphorylation and β-catenin levels in the muscle of dexa-treated rats. Prevention of dexa-induced muscle atrophy by caAkt overexpression was associated with a significant increase of phosphorylated p70S6K (A), GSK-3β (B), and total β-catenin levels (C), indicating that these molecules potentially contribute to the hypertrophic effects of Akt overexpression. Inset, Representative Western blot of phosphorylation forms (top) and total forms (bottom).*, < 0.05; **, P < 0.01 vs. contralateral muscle.

Figure 4

Dominant-negative (dn)GSK-3β overexpression induces a modest muscle fiber hypertrophy and prevents dexa-induced muscle atrophy. Overexpression of dnGSK-3β was detected by Western blot (A) and immunohistochemistry (B, top) in TA muscles electroporated with pcDNA3-dnGSK-3β plasmid. dnGSK-3β overexpression induced a modest muscle fiber hypertrophy (black column) compared with surrounding UT muscle fibers (white column) (B, bottom). Results are expressed as mean ± sem. **, P < 0.01; ***, P < 0.001 vs. UT muscles fibers of the same muscle. ○○, P < 0.01 vs. ad libitum UT muscles fibers. C, The proportion of large fibers in the three was greater in the dnGSK-3β DNA transfected muscle fibers (black column) compared with surrounding UT muscle fibers (white column). Statistical analysis was performed using the χ² Pearson test. ***, P < 0.001 for the three groups. A total of 6,354 positive and 16,431 UT muscle fibers were analyzed for measurements of fiber size.

Figure 5

ΔNβ-catenin overexpression prevents muscle atrophy in dexa-treated rats. Overexpression of ΔNβ-catenin was detected by Western blot (A) and immunohistochemistry (B, top) in TA muscles electroporated with pM1-ΔNβ-catenin plasmid. ΔNβ-catenin overexpression increases muscle fiber CSA (black column) compared with surrounding UT (white column) (B, bottom). Results are expressed as mean ± sem. ***, P < 0.001 vs. UT muscles fibers of the same muscle. ○, P < 0.05; ○○○, P < 0.001 vs. ad libitum UT muscles fibers. ‡, P < 0.05 vs. pair-fed UT muscles fibers. •, P < 0.05 vs. ad libitum ΔNβ-catenin transfected muscles fibers. C, The proportion of large fibers in the three groups was greater in the ΔNβ-catenin DNA transfected muscle fibers (black column) compared with surrounding UT muscle fibers (white column). Statistical analysis was performed using the χ² Pearson test. ***, P < 0.001 for the three groups. A total of 15,312 positive and 31,759 UT muscle fibers were analyzed for measurements of fiber size.