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.

Figure 1. AG and UnAG protect C2C12 myotubes from dexamethasone-induced atrophy without induction of protein synthesis or hypertrophy.

(A) Myotube diameters were measured after 24-hour treatment in differentiation medium (DM) with 10 nM AG, 10 nM UnAG, and/or 1 μM dexamethasone (DEXA). In every experiment, 10 ng/ml IGF-1 was used as positive control for antiatrophic/hypertrophic activity. (B and C) Atrogin-1 and MuRF1 expression analysis upon dexamethasone treatment with or without AG and UnAG. (D) Treatment with 100 nM wortmannin (W) or 20 ng/ml rapamycin (R) reverted the antiatrophic activity of AG and UnAG on myotube diameter. Control myotubes in differentiation medium were treated with DMSO, a vehicle for both wortmannin and rapamycin. (E and F) Phosphorylation of Akt^S473 and FoxO3a^T32, detected by Western blotting, upon treatment for 20 minutes with 1 μM AG or UnAG. Shown are representative blots and quantification of 3 independent experiments. (G–I) IGF-1, but not AG and UnAG, induced protein synthesis, as determined by phosphorylation of S6K^T389 (G) or S6^S235/236 (H) and by incorporation of [³H]-leucine (I). (J) Effect of raptor and rictor silencing on protein levels, detected by Western blotting. (K) Silencing of rictor, but not of raptor, reverted the antiatrophic activity of AG and UnAG on the diameter of myotubes treated as in A. ^#P < 0.05, ^§P < 0.01 vs. DM control; *P < 0.01 vs. DEXA treatment.

Figure 2. AG and UnAG antiatrophic signaling is mediated by p38 and acts through a GPCR-dependent signaling pathway involving PI3Kβ.

(A) Phosphorylation of p38^T180/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 Akt^S473 was abolished upon treatment with 10 μM NF449, a Gα_s 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.

Figure 3. Myh6/Ghrl mice are protected from skeletal muscle atrophy induced by 48 hours of fasting.

(A–C) Effect of fasting on gastrocnemii. Mean percentage of gastrocnemius weight loss (A) and CSA reduction (B) of fasted Myh6/Ghrl (Tg) mice and WT littermates compared with fed animals. (C) Frequency distribution of gastrocnemii CSA of fasted Myh6/Ghrl and WT mice. (D–F) Effect of fasting on EDL muscles. Mean percentage of EDL muscle weight loss (D), CSA reduction (E), and CSA frequency distribution (F) of fasted Myh6/Ghrl and WT littermates. (G and H) Atrogin-1 and MuRF1 expression in gastrocnemii of fed and fasted Myh6/Ghrl mice and their WT littermates, determined by real-time RT-PCR. *P < 0.01 vs. WT. n = 7 (fed WT and Myh6/Ghrl); 5 (fasted WT); 6 (fasted Myh6/Ghrl); 3 (CSA loss and distribution, WT and Myh6/Ghrl).

Figure 4. Myh6/Ghrl mice are protected from denervation-induced skeletal muscle atrophy induced by sciatic nerve resection.

(A and B) Mean percentage of weight loss (A) and CSA reduction (B) of denervated gastrocnemius at 7 and 14 days after denervation, compared with the unperturbed side. (C and D) Frequency distribution of gastrocnemii CSA at 7 and 14 days after denervation in Myh6/Ghrl and WT mice. (E–H) CSA reduction and fiber area distribution of (E and F) EDL and (G and H) TA muscles at 7 days after denervation. (I and J) Atrogin-1 and MuRF1 expression, determined by real-time RT-PCR, in denervated gastrocnemii at 7 days after denervation, compared with the unperturbed side. **P < 0.01, *P < 0.05 vs. WT. n = 6 (WT); 5 (Myh6/Ghrl); 3 (CSA loss and distribution, WT and Myh6/Ghrl).

Figure 5. UnAG pharmacological treatment protects skeletal muscle from fasting- and denervation-induced atrophy in WT mice.

(A–C) Phosphorylation of Akt^S473, FoxO3a^T32, and p38^T180/Y182 in gastrocnemii of WT mice treated with 100 μg/kg UnAG or saline. At the indicated time points, gastrocnemii were removed and processed for Western blot analysis. Shown are representative blots and densitometric analysis of 3 independent experiments, normalized to untreated animals (not shown). (D–F) Mean percent weight loss (D), CSA reduction (E), and CSA frequency distribution (F) of gastrocnemii from fed or 48-hour fasted mice treated twice daily with 100 μg/kg UnAG or saline (n = 5 per group). Frequency distribution was measured in 3 mice per group. In D and E, percent reduction shown is between fasted and fed mice. (G–I) Mean percent weight loss (G), CSA reduction (H), and CSA frequency distribution (I) of gastrocnemii from mice treated with 100 μg/kg UnAG or saline twice daily for 7 days after sciatic nerve resection (n = 5 per group). Frequency distribution was measured in 3 mice per group. In G and H, percent reduction shown is between denervated gastrocnemii and gastrocnemii from the unperturbed side. *P < 0.05, **P < 0.01 vs. saline treatment.

Figure 6. AG and UnAG pharmacological treatment of Ghsr^–/– mice induces antiatrophic signaling and protects from fasting-induced skeletal muscle atrophy.

(A) Phosphorylation of Akt^S473 in gastrocnemii of Ghsr^–/– mice injected with 100 μg/kg AG or UnAG or with saline. 60 minutes after treatment, gastrocnemii were removed and processed for Western blot analysis. Shown are representative blots and densitometric analysis of 3 independent experiments. (B–D) Mean percentage weight loss (B), CSA reduction (C), and CSA frequency distribution (D) of gastrocnemii from fed or 48-hour fasted Ghsr^–/– mice injected s.c. twice daily with 100 μg/kg AG or UnAG or with saline (n = 5 per group). Frequency distribution was measured in 3 mice per group. In B and C, percent reduction is between fasted and fed mice. *P < 0.05, **P < 0.01 vs. saline treatment.