TNF-α- and tumor-induced skeletal muscle atrophy involves sphingolipid metabolism

Joffrey De Larichaudy,Filippo Serra,Etienne Lefai,Andrea M Isidori,Alessandra Zufferli,Monique Piraud,Kevin Dessalle,Marine Desgeorges,Georges Némoz,David Cheillan,Hubert Vidal,Fabio Naro

doi:10.1186/2044-5040-2-2

Abstract

BackgroundMuscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.ResultsWe addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.ConclusionsCeramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.

Highlights

Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality
Other parameters reflecting the functional status of the differentiated muscle cells were significantly reduced by tumor necrosis factor (TNF)-a treatment, such as the myosin heavy chain (MHC) content, as evaluated by ELISA (Figure 1b), and the creatine kinase (CK) activity (Figure 1c)
To assess whether ceramide accumulation could explain the atrophic effects of TNF-a, we investigated the effects of myotube treatment by exogenous ceramide

Summary

Introduction

Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Ceramide has been shown to inhibit myogenic differentiation [9], amino acid transport, mammalian target of rapamycin (mTOR) activity, and protein synthesis in myotubes [10] It can enhance pathways involved in proteolysis, such as the nuclear factor (NF)B pathway [11] and autophagy [12,13]. We hypothesized that the biosynthesis of sphingolipid mediators, ceramide, participates in the mechanisms leading to muscle loss associated with pathological states To test this assumption, we used differentiated L6 and C2C12 myotubes treated with TNF-a as in vitro models of muscle atrophy, and an in vivo mouse model of tumor-induced cachexia [14]. The protective action of the inhibitor of de novo sphingolipid synthesis myriocin [19], which we observed both in vitro and in vivo during tumor-induced cachexia, suggests that preventing ceramide accumulation could represent a promising strategy to preserve muscle mass against the atrophy associated with a number of chronic diseases

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