Glucocorticoids and 11β-HSD1 are major regulators of intramyocellular protein metabolism

Abstract

The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, 'Cushing's syndrome', create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11β-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use of selective 11β-HSD1 inhibitors to ameliorate muscle-wasting effects associated with glucocorticoid excess.

Keywords: 11β-HSD1; Cushing’s syndrome; glucocorticoid; protein metabolism.

Figure 1

Treatment of C2C12 myotubes with corticosterone (CORT: 62.5–1000nM, 24h) decreased [³H]tyrosine incorporation into cellular proteins (protein synthesis). (A) Paralleled by increased TCA-soluble radioactivity released into media from cells pre-labeled with [³H]tyrosine (protein degradation). (B) Data analyzed using one-way ANOVA. C2C12 myotube area was decreased following treatment with CORT (250nM, 24h) and 11-dehydrocorticosterone (11DHC, 250nM, 24h). (C) The selective 11β-HSD1 inhibitor, PF-877423 (PF: 2.5μM, 24h), blocked the effects of 11DHC. Myotube area quantified from images using ImageJ software and analyzed using two-way ANOVA (C). Data expressed as mean±s.e.m. of n=6 experiments. (**P<0.01, ***P<0.001 vs ctrl, ^$$$P<0.001 vs 11DHC).

Figure 2

[³H]tyrosine incorporation into cellular proteins in C2C12 myotubes (protein synthesis) was decreased by both corticosterone (CORT: 250nM, 24h) and 11-dehydrocorticosterone (11DHC: 250nM, 24h). The selective 11β-HSD1 inhibitor, PF-877423 (PF: 2.5μM, 24h), blocked the effects of 11DHC (A). Both CORT (250nM, 24h) and 11DHC (250nM, 24h) decreased IGF1 mRNA expression in C2C12 myotubes (B), but Eif2b1 (C), 4E-BP1 (D), mTOR (E), Eif6 (F), and Ep300 (G) mRNA expression was unchanged. Serine-2448 phosphorylation of mTOR and threonine-37/46 phosphorylation of 4E-BP1 were decreased by both CORT (250nM, 24h) and 11DHC (250nM, 24h) in C2C12 myotubes, without effecting total protein levels. The effects of 11DHC on IGF1 expression, p-mTOR and p-4E-BP1 were reversed by PF-877423 (PF: 2.5μM, 24h) (B and H). Data expressed as mean ±s.e.m. of n=6 experiments, and analyzed using two-way ANOVAs. Western blot analyses were carried out on at least three different preps. (*P<0.05, **P<0.01, ***P<0.001 vs ctrl, ^$P<0.05, ^$$P<0.01 vs 11DHC).

Figure 3

TCA-soluble radioactivity released into the culture media from C2C12 myotubes pre-labeled with [³H]tyrosine was increased by both corticosterone (CORT: 250nM, 24h) and 11-dehydrocorticosterone (11DHC: 250nM, 24h). The selective 11β-HSD1 inhibitor, PF-877423 (PF: 2.5μM, 24h), blocked the effects of 11DHC (A). Both CORT (250nM, 24h) and 11DHC (250nM, 24h) increased atrogin-1 (B), MuRF1 (C), Mstn (D), and FOXO3a (E) mRNA expression in C2C12 myotubes. Total protein levels of MuRF1 were also increased by both CORT and 11DHC (F). The effects of 11DHC on atrogin-1, MuRF1, Mstn, and FOXO3a mRNA expression as well as MuRF1 total protein levels was reversed by PF-877423 (PF: 2.5μM, 24h) (B, C, D, E and F). Data expressed as mean ±s.e.m. of n=6 experiments, and analyzed using two-way ANOVAs. Western blot analyses were carried out on at least three different preps. (*P<0.05, **P<0.01 vs ctrl, ^$$P<0.01 vs 11DHC).

Figure 4

C2C12 myoblast proliferation was decreased by corticosterone (CORT: 100–500nM, 48h), but not 11-dehydrocorticosterone (11DHC: 100–500nM, 48h), in a concentration-dependent manner (A). C2C12 myoblasts treated with CORT (100–500nM, 24h) had deceased mRNA expression of IGF1 (B) and Myf5 (C), while Mstn expression was increased (D), whereas CORT was without effect on CASP3 (E) and CASP7 (F) expression. Low oxoreductase activity of 11β-HSD1 (conversion of inactive 11DHC to active CORT) was detected in C2C12 myoblasts, with activity increasing across myocyte differentiation (G). Data expressed as mean ±s.e.m. of n=6 experiments, and analyzed using one-way ANOVAs. (*P<0.05, **P 0.01, ***P 0.001 vs ctrl).

Figure 5

Treatment of differentiated human primary myotubes with cortisone (250nM, 24h) decreased [³H]tyrosine incorporation into cellular proteins (protein synthesis) (A), paralleled by increased TCA-soluble radioactivity released into the culture media from cells pre-labeled with [³H]tyrosine (protein degradation) (B). The selective 11β-HSD1 inhibitor, PF-877423 (PF: 2.5μM, 24h), blocked the effects of cortisone on both protein synthesis and protein degradation. Proliferation of human primary myoblasts was decreased by cortisol (100–500nM, 48h) in a concentration-dependent manner, but not cortisone (100–500nM, 48h), (C). Data expressed as mean±s.e.m. of n=6 experiments, and analyzed using one- or two-way ANOVAs. (*P<0.05, **P<0.01, ***P<0.001 vs ctrl, ^$P<0.05 vs cortisone).