Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice
Paolo E. Porporato, Nicoletta Filigheddu, Simone Reano, Michele Ferrara, Elia Angelino, Viola F. Gnocchi, Flavia Prodam, Giulia Ronchi, Sharmila Fagoonee, Michele Fornaro, Federica Chianale, Gianluca Baldanzi, Nicola Surico, Fabiola Sinigaglia, Isabelle Perroteau, Roy G. Smith, Yuxiang Sun, Stefano Geuna, Andrea Graziani
Paolo E. Porporato, Nicoletta Filigheddu, Simone Reano, Michele Ferrara, Elia Angelino, Viola F. Gnocchi, Flavia Prodam, Giulia Ronchi, Sharmila Fagoonee, Michele Fornaro, Federica Chianale, Gianluca Baldanzi, Nicola Surico, Fabiola Sinigaglia, Isabelle Perroteau, Roy G. Smith, Yuxiang Sun, Stefano Geuna, Andrea Graziani
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Research Article Muscle biology

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice

Abstract

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a–independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a–independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a–mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

Authors

Paolo E. Porporato, Nicoletta Filigheddu, Simone Reano, Michele Ferrara, Elia Angelino, Viola F. Gnocchi, Flavia Prodam, Giulia Ronchi, Sharmila Fagoonee, Michele Fornaro, Federica Chianale, Gianluca Baldanzi, Nicola Surico, Fabiola Sinigaglia, Isabelle Perroteau, Roy G. Smith, Yuxiang Sun, Stefano Geuna, Andrea Graziani

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Figure 2

AG and UnAG antiatrophic signaling is mediated by p38 and acts through a GPCR-dependent signaling pathway involving PI3Kβ.

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AG and UnAG antiatrophic signaling is mediated by p38 and acts through a...
(A) Phosphorylation of p38T180/Y182, detected by Western blotting, after 20-minute treatment with 1 μM AG or UnAG. Shown are representative blots and quantification of 3 independent experiments. (B) Treatment with the p38 inhibitor SB203580 (5 μM) reverted the antiatrophic activity of AG and UnAG on myotube diameter upon treatment with dexamethasone. (C and D) Atrogin-1 and MuRF1 expression analysis upon dexamethasone treatment with or without AG and UnAG in the presence or absence of 5 μM SB203580. (E) AG and UnAG phosphorylation of AktS473 was abolished upon treatment with 10 μM NF449, a Gαs subunit–selective G protein antagonist. Shown are representative blots and quantification of 3 independent experiments. (F) Treatment with 10 μM NF449 reverted the antiatrophic activity of AG and UnAG on myotube diameter upon dexamethasone treatment. (G) Treatment with 25 nM PIK-75, an inhibitor of PI3Kα, abolished the antiatrophic effect of IGF-1 on myotube diameter upon dexamethasone treatment, without affecting AG and UnAG activity. The antiatrophic effect was abrogated by treatment with 200 nM TGX-221, an inhibitor of PI3Kβ. (H) Atrogin-1 expression analysis upon dexamethasone treatment with AG, UnAG, and IGF-1 in the presence or absence of 200 nM TGX-221. In experiments with SB203580, NF449, PIK-75, and TGX-221, control myotubes in differentiation medium were treated with DMSO, a vehicle for all these compounds. #P < 0.05, §P < 0.01 vs. DM control; *P < 0.05, **P < 0.01 vs. DEXA treatment.