Insulin-like growth factor I-mediated skeletal muscle hypertrophy is characterized by increased mTOR-p70S6K signaling without increased Akt phosphorylation

Abstract

Background: Insulin-like growth factor I (IGF-I) is an anabolic hormone that is known to induce skeletal muscle hypertrophy. However, the signaling pathways mediating IGF-I's hypertrophic effect in vivo are unknown.

Method: The phosphorylation of 46 proteins was investigated by Kinetworks proteomic analysis in the gastrocnemius muscle of transgenic mice overexpressing IGF-I myosin light chain/muscle specific IGF-I (MLC/mlgf-I) and wild-type littermates.

Results: In the hypertrophic muscle of MLC/mlgf-I mice, we observed increased phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1; 53% increase), the mammalian target of rapamycin (mTOR; 112% increase), and p70 S6 kinase (p70S6K) (254% increase) but no significant change in Akt phosphorylation (4% decrease). Furthermore, we found reduced phosphorylation of MAP kinase kinase 1 and 2 (MEK1/2) (60% decrease) and of mitogen-activated protein kinase kinases 3 and 6 (MKK3/6) (50% decrease) in muscle from transgenic mice, suggesting that the hypertrophic and mitogenic effects of IGF-I are mediated via distinct signaling pathways in skeletal muscle and that inhibition of the mitogen-activated protein (MAP) kinase pathway may be required for the IGF-I-induced hypertrophic effect. Single-fiber analysis revealed a trend toward a higher percentage of the fast twitch fibers (IIb and IIx) in the transgenic mice.

Conclusion: Persistent overexpression of IGF-I in mice skeletal muscle results in hypertrophy, which is likely mediated via the mTOR/p70S6K pathway, potentially via an Akt-independent signaling pathway.

Figure 1

Kinetworks KPSS 4.0 phosphoprotein analyses of wild-type (A) and transgenic (B) mouse gastrocnemius muscle. The identities of protein targets are indicated by arrows and numbers. Tg = MLC/mIgf-I transgenic mice.

Figure 2

Validation of phosphoprotein screen by Western blot. Phosphorylation levels of selected protein kinases in the gastrocnemius muscle from transgenic MLC/mIgf-I mice (T) and wild-type mice (W) were analyzed by Western blot.

Figure 3

Single-fiber analysis of mouse myosin heavy chain. A pictorial representation of the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for the MLC/mIgf-I mouse (A) and wild-type mouse (B) demonstrating the different pure and hybrid isoforms that are expressed for the MLC/mIgf-I and wild-type mice, respectively.