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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

Inhibitors of ceramide synthesis upregulate anti-atrophic signaling pathways. (A) Myotubes were treated for 3 days with or without tumor necrosis factor (TNF)-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869, or both. (Left panel) Phospholipase (PL)D1 mRNA levels were measured by reverse transcription quantitative PCR, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 6 measurements in duplicate. *Different from TNF-α alone: P = 0.05, **P < 0.001; different from control: P≤ 0.05. (Right panel) PLD1 protein in cell extracts was analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three experiments. **Different from control: P < 0.01; +different from TNF-α alone: P = 0.02. (B) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. (Left panel) S6K1 mRNA levels were measured by reverse transcription quantitative PCR and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of three measurements in duplicate. **Different from both control and TNF-α alone: P < 0.01. (Right panel) Total S6K1 protein and phospho-Thr389 S6K1 in cell extracts were analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three determinations. +Different from control: P < 0.05; ++P < 0.02; **different from both control and TNF-α alone: P < 0.02. (C) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869. Ph-Ser473 Akt was analyzed by western blotting. Results were normalized to the amount of (left panel) tubulin or (right panel) total Akt, and are the mean ± SE of 3 to 5 determinations. NS, not significantly different from control. ***Different from TNF-α: P < 0.001, **P < 0.01, *P < 0.05.
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Figure 6: Inhibitors of ceramide synthesis upregulate anti-atrophic signaling pathways. (A) Myotubes were treated for 3 days with or without tumor necrosis factor (TNF)-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869, or both. (Left panel) Phospholipase (PL)D1 mRNA levels were measured by reverse transcription quantitative PCR, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 6 measurements in duplicate. *Different from TNF-α alone: P = 0.05, **P < 0.001; different from control: P≤ 0.05. (Right panel) PLD1 protein in cell extracts was analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three experiments. **Different from control: P < 0.01; +different from TNF-α alone: P = 0.02. (B) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. (Left panel) S6K1 mRNA levels were measured by reverse transcription quantitative PCR and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of three measurements in duplicate. **Different from both control and TNF-α alone: P < 0.01. (Right panel) Total S6K1 protein and phospho-Thr389 S6K1 in cell extracts were analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three determinations. +Different from control: P < 0.05; ++P < 0.02; **different from both control and TNF-α alone: P < 0.02. (C) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869. Ph-Ser473 Akt was analyzed by western blotting. Results were normalized to the amount of (left panel) tubulin or (right panel) total Akt, and are the mean ± SE of 3 to 5 determinations. NS, not significantly different from control. ***Different from TNF-α: P < 0.001, **P < 0.01, *P < 0.05.

Mentions: Ceramide is known to be a downregulator of the expression of PLD, particularly the PLD1 isoform [9,31]. PLD is in turn an activator of mTOR kinase, a major regulator of protein synthesis and degradation [15], closely involved in muscle-tissue homeostasis [32]. We therefore assessed the influence of TNF-α and ceramide-synthesis inhibitors on the expression of PLD1 in L6 myotubes. Myriocin by itself had no influence on PLD1 mRNA, whereas GW4868 alone moderately increased the PLD1 mRNA level. TNF-α strongly repressed the expression of PLD1 mRNA, and ceramide-synthesis inhibitors rescued its expression, with myriocin resulting in partial, and GW4869 in total rescue. In fact, potentiation of the effects of the two inhibitors on PLD1 mRNA levels was seen (Figure 6a). These effects were confirmed at the protein level, with TNF-α inducing a marked decrease in PLD1 protein, which was rescued by either myriocin or GW4869 (Figure 6a). These results thus indicated that TNF-α lowers PLD1 expression in myotubes through the production of ceramide both by the de novo pathway and by sphingomyelinase activation.


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)

Inhibitors of ceramide synthesis upregulate anti-atrophic signaling pathways. (A) Myotubes were treated for 3 days with or without tumor necrosis factor (TNF)-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869, or both. (Left panel) Phospholipase (PL)D1 mRNA levels were measured by reverse transcription quantitative PCR, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 6 measurements in duplicate. *Different from TNF-α alone: P = 0.05, **P < 0.001; different from control: P≤ 0.05. (Right panel) PLD1 protein in cell extracts was analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three experiments. **Different from control: P < 0.01; +different from TNF-α alone: P = 0.02. (B) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. (Left panel) S6K1 mRNA levels were measured by reverse transcription quantitative PCR and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of three measurements in duplicate. **Different from both control and TNF-α alone: P < 0.01. (Right panel) Total S6K1 protein and phospho-Thr389 S6K1 in cell extracts were analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three determinations. +Different from control: P < 0.05; ++P < 0.02; **different from both control and TNF-α alone: P < 0.02. (C) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869. Ph-Ser473 Akt was analyzed by western blotting. Results were normalized to the amount of (left panel) tubulin or (right panel) total Akt, and are the mean ± SE of 3 to 5 determinations. NS, not significantly different from control. ***Different from TNF-α: P < 0.001, **P < 0.01, *P < 0.05.
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Figure 6: Inhibitors of ceramide synthesis upregulate anti-atrophic signaling pathways. (A) Myotubes were treated for 3 days with or without tumor necrosis factor (TNF)-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869, or both. (Left panel) Phospholipase (PL)D1 mRNA levels were measured by reverse transcription quantitative PCR, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 6 measurements in duplicate. *Different from TNF-α alone: P = 0.05, **P < 0.001; different from control: P≤ 0.05. (Right panel) PLD1 protein in cell extracts was analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three experiments. **Different from control: P < 0.01; +different from TNF-α alone: P = 0.02. (B) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. (Left panel) S6K1 mRNA levels were measured by reverse transcription quantitative PCR and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of three measurements in duplicate. **Different from both control and TNF-α alone: P < 0.01. (Right panel) Total S6K1 protein and phospho-Thr389 S6K1 in cell extracts were analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three determinations. +Different from control: P < 0.05; ++P < 0.02; **different from both control and TNF-α alone: P < 0.02. (C) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869. Ph-Ser473 Akt was analyzed by western blotting. Results were normalized to the amount of (left panel) tubulin or (right panel) total Akt, and are the mean ± SE of 3 to 5 determinations. NS, not significantly different from control. ***Different from TNF-α: P < 0.001, **P < 0.01, *P < 0.05.
Mentions: Ceramide is known to be a downregulator of the expression of PLD, particularly the PLD1 isoform [9,31]. PLD is in turn an activator of mTOR kinase, a major regulator of protein synthesis and degradation [15], closely involved in muscle-tissue homeostasis [32]. We therefore assessed the influence of TNF-α and ceramide-synthesis inhibitors on the expression of PLD1 in L6 myotubes. Myriocin by itself had no influence on PLD1 mRNA, whereas GW4868 alone moderately increased the PLD1 mRNA level. TNF-α strongly repressed the expression of PLD1 mRNA, and ceramide-synthesis inhibitors rescued its expression, with myriocin resulting in partial, and GW4869 in total rescue. In fact, potentiation of the effects of the two inhibitors on PLD1 mRNA levels was seen (Figure 6a). These effects were confirmed at the protein level, with TNF-α inducing a marked decrease in PLD1 protein, which was rescued by either myriocin or GW4869 (Figure 6a). These results thus indicated that TNF-α lowers PLD1 expression in myotubes through the production of ceramide both by the de novo pathway and by sphingomyelinase activation.

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