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

De Larichaudy J, Zufferli A, Serra F, Isidori AM, Naro F, Dessalle K, Desgeorges M, Piraud M, Cheillan D, Vidal H, Lefai E, Némoz G - Skelet Muscle (2012)

Bottom Line: In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis.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.Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lyon University, INSERM U1060, CarMeN Laboratory, University Lyon-1, Institut National de la Recherche Agronomique UMR1235, INSA-Lyon, F-69600 Oullins, France. georges.nemoz@insa-lyon.fr.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: Ceramide 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.

No MeSH data available.


Related in: MedlinePlus

S1P interferes with the trophic effects of tumor necrosis factor (TNF)-α in L6 myotubes. (A) Myotube area was measured after 3 days of treatment with or without 15 ng/ml TNF-α, in the presence of 1 μmol/l exogenous S1P or 1 μmol/l inhibitor of the S1P receptor FTY720. Shown are the mean ± SE of 10 fields. +++Different from control without drug: P < 0.0001; **different from TNF-α alone, P = 0.004. (B) Creatine kinase activity of myotubes treated for 3 days with TNF-α, without or with 1 μmol/l exogenous S1P, 1 μmol/l FTY720, or 10 μmol/l of the sphingosine kinase inhibitor dihydrosphingosine (DHS). Results are shown as the mean ± SE of 3 to 7 experiments performed in triplicate. +++Different from control: P < 0.003; **different from TNF-α alone: P < 0.01, *P < 0.05. (C) Myotube area was measured after 3 days of treatment with 5 μmol/l C6 ceramide, without or with 1 μmol/l S1P or 10 μmol/l of the sphingosine kinase inhibitor dimethylsphingosine (DMS). Shown are the mean ± SE of 10 fields. ***Different from C6 ceramide alone: P < 0.005.
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Figure 4: S1P interferes with the trophic effects of tumor necrosis factor (TNF)-α in L6 myotubes. (A) Myotube area was measured after 3 days of treatment with or without 15 ng/ml TNF-α, in the presence of 1 μmol/l exogenous S1P or 1 μmol/l inhibitor of the S1P receptor FTY720. Shown are the mean ± SE of 10 fields. +++Different from control without drug: P < 0.0001; **different from TNF-α alone, P = 0.004. (B) Creatine kinase activity of myotubes treated for 3 days with TNF-α, without or with 1 μmol/l exogenous S1P, 1 μmol/l FTY720, or 10 μmol/l of the sphingosine kinase inhibitor dihydrosphingosine (DHS). Results are shown as the mean ± SE of 3 to 7 experiments performed in triplicate. +++Different from control: P < 0.003; **different from TNF-α alone: P < 0.01, *P < 0.05. (C) Myotube area was measured after 3 days of treatment with 5 μmol/l C6 ceramide, without or with 1 μmol/l S1P or 10 μmol/l of the sphingosine kinase inhibitor dimethylsphingosine (DMS). Shown are the mean ± SE of 10 fields. ***Different from C6 ceramide alone: P < 0.005.

Mentions: Because ceramide can be rapidly metabolized in the cell, and potentially converted into the bioactive mediator S1P through the sequential action of ceramidases and sphingosine kinases [5], we evaluated the effects of S1P on myotubes. In L6 myotubes, exogenous S1P in the presence of TNF-α had a positive effect, on myotube surface and on CK activity (Figure 4a, b), suggesting that ceramide metabolization into S1P can induce effects opposite to that of ceramide itself. This antagonistic action was also supported by the observation that S1P also decreased the atrophic effects of ceramide (Figure 4c). Conversely, inhibition of S1P biosynthesis by the addition of the sphingosine kinase inhibitors D-L-threo-dihydro-sphingosine (DHS) and N,N-dimethylsphingosine (DMS) [24] increased the effects of TNF-α and ceramide on myotube surface or CK activity, supporting the assumption that S1P at least partly antagonizes the effects of ceramide (Figure 4b, c). S1P can be secreted and is known to activate a set of specific membrane surface receptors, of which S1P1, S1P2, and S1P3 are expressed in muscle cells [25]. The effects of FTY720, a compound functionally acting as an inhibitor of S1P receptors [26], were thus evaluated. FTY720 itself had negative effects on myotubes, consistent with a positive influence of S1P, but FTY720 did not amplify the negative effects of TNF-α (Figure 4a, b).


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

De Larichaudy J, Zufferli A, Serra F, Isidori AM, Naro F, Dessalle K, Desgeorges M, Piraud M, Cheillan D, Vidal H, Lefai E, Némoz G - Skelet Muscle (2012)

S1P interferes with the trophic effects of tumor necrosis factor (TNF)-α in L6 myotubes. (A) Myotube area was measured after 3 days of treatment with or without 15 ng/ml TNF-α, in the presence of 1 μmol/l exogenous S1P or 1 μmol/l inhibitor of the S1P receptor FTY720. Shown are the mean ± SE of 10 fields. +++Different from control without drug: P < 0.0001; **different from TNF-α alone, P = 0.004. (B) Creatine kinase activity of myotubes treated for 3 days with TNF-α, without or with 1 μmol/l exogenous S1P, 1 μmol/l FTY720, or 10 μmol/l of the sphingosine kinase inhibitor dihydrosphingosine (DHS). Results are shown as the mean ± SE of 3 to 7 experiments performed in triplicate. +++Different from control: P < 0.003; **different from TNF-α alone: P < 0.01, *P < 0.05. (C) Myotube area was measured after 3 days of treatment with 5 μmol/l C6 ceramide, without or with 1 μmol/l S1P or 10 μmol/l of the sphingosine kinase inhibitor dimethylsphingosine (DMS). Shown are the mean ± SE of 10 fields. ***Different from C6 ceramide alone: P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: S1P interferes with the trophic effects of tumor necrosis factor (TNF)-α in L6 myotubes. (A) Myotube area was measured after 3 days of treatment with or without 15 ng/ml TNF-α, in the presence of 1 μmol/l exogenous S1P or 1 μmol/l inhibitor of the S1P receptor FTY720. Shown are the mean ± SE of 10 fields. +++Different from control without drug: P < 0.0001; **different from TNF-α alone, P = 0.004. (B) Creatine kinase activity of myotubes treated for 3 days with TNF-α, without or with 1 μmol/l exogenous S1P, 1 μmol/l FTY720, or 10 μmol/l of the sphingosine kinase inhibitor dihydrosphingosine (DHS). Results are shown as the mean ± SE of 3 to 7 experiments performed in triplicate. +++Different from control: P < 0.003; **different from TNF-α alone: P < 0.01, *P < 0.05. (C) Myotube area was measured after 3 days of treatment with 5 μmol/l C6 ceramide, without or with 1 μmol/l S1P or 10 μmol/l of the sphingosine kinase inhibitor dimethylsphingosine (DMS). Shown are the mean ± SE of 10 fields. ***Different from C6 ceramide alone: P < 0.005.
Mentions: Because ceramide can be rapidly metabolized in the cell, and potentially converted into the bioactive mediator S1P through the sequential action of ceramidases and sphingosine kinases [5], we evaluated the effects of S1P on myotubes. In L6 myotubes, exogenous S1P in the presence of TNF-α had a positive effect, on myotube surface and on CK activity (Figure 4a, b), suggesting that ceramide metabolization into S1P can induce effects opposite to that of ceramide itself. This antagonistic action was also supported by the observation that S1P also decreased the atrophic effects of ceramide (Figure 4c). Conversely, inhibition of S1P biosynthesis by the addition of the sphingosine kinase inhibitors D-L-threo-dihydro-sphingosine (DHS) and N,N-dimethylsphingosine (DMS) [24] increased the effects of TNF-α and ceramide on myotube surface or CK activity, supporting the assumption that S1P at least partly antagonizes the effects of ceramide (Figure 4b, c). S1P can be secreted and is known to activate a set of specific membrane surface receptors, of which S1P1, S1P2, and S1P3 are expressed in muscle cells [25]. The effects of FTY720, a compound functionally acting as an inhibitor of S1P receptors [26], were thus evaluated. FTY720 itself had negative effects on myotubes, consistent with a positive influence of S1P, but FTY720 did not amplify the negative effects of TNF-α (Figure 4a, b).

Bottom Line: In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis.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.Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lyon University, INSERM U1060, CarMeN Laboratory, University Lyon-1, Institut National de la Recherche Agronomique UMR1235, INSA-Lyon, F-69600 Oullins, France. georges.nemoz@insa-lyon.fr.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: Ceramide 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.

No MeSH data available.


Related in: MedlinePlus