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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

Effects of tumor necrosis factor (TNF)-α on protein metabolism in L6 myotubes are prevented by ceramide-synthesis inhibition. (A) Myotubes were treated for 12 hours with TNF-α, in the presence or absence of 100 nmol/l myriocin, or 1 μmol/l OMS. The rate of protein synthesis was measured by adding [3H]-tyrosine to the culture medium, and counting the radioactivity present in trichloroacetic acid (TCA) precipitates of the cells, in relation to the total protein content. The results are the mean ± SE of three determinations. ++Different from control: P = 0.001; *different from TNF-α alone: P≤ 0.05. (B) Proteolysis was measured in L6 myotubes labeled with [3H]-tyrosine for 48 h, and treated for 12 hours with TNF-α in the presence or absence of 100 nmol/l myriocin, 1 μmol/l OMS, or both. TCA-soluble radioactivity released in the medium was measured and related to the total incorporated radioactivity. The results are the mean ± SE of three determinations. *Different from TNF-α alone: P < 0.05. (C) The mRNA levels of Atrogin-1 and LC3b were measured by reverse transcription quantitative PCR in myotubes treated or not with TNF-α in the presence of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l OMS, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 5 measurements in duplicate. **Different from control without drug: P = 0.01; +++different from TNF-α alone: P≤ 0.001, +P < 0.05. (D) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. Phospho- Ser253 Foxo3 in cell extracts was analyzed by western blotting. Results were normalized by total Foxo3 protein amounts. *Different from all other values: P < 0.02.
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Figure 5: Effects of tumor necrosis factor (TNF)-α on protein metabolism in L6 myotubes are prevented by ceramide-synthesis inhibition. (A) Myotubes were treated for 12 hours with TNF-α, in the presence or absence of 100 nmol/l myriocin, or 1 μmol/l OMS. The rate of protein synthesis was measured by adding [3H]-tyrosine to the culture medium, and counting the radioactivity present in trichloroacetic acid (TCA) precipitates of the cells, in relation to the total protein content. The results are the mean ± SE of three determinations. ++Different from control: P = 0.001; *different from TNF-α alone: P≤ 0.05. (B) Proteolysis was measured in L6 myotubes labeled with [3H]-tyrosine for 48 h, and treated for 12 hours with TNF-α in the presence or absence of 100 nmol/l myriocin, 1 μmol/l OMS, or both. TCA-soluble radioactivity released in the medium was measured and related to the total incorporated radioactivity. The results are the mean ± SE of three determinations. *Different from TNF-α alone: P < 0.05. (C) The mRNA levels of Atrogin-1 and LC3b were measured by reverse transcription quantitative PCR in myotubes treated or not with TNF-α in the presence of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l OMS, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 5 measurements in duplicate. **Different from control without drug: P = 0.01; +++different from TNF-α alone: P≤ 0.001, +P < 0.05. (D) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. Phospho- Ser253 Foxo3 in cell extracts was analyzed by western blotting. Results were normalized by total Foxo3 protein amounts. *Different from all other values: P < 0.02.

Mentions: Protein synthesis rate was evaluated in L6 myotubes by measuring the incorporation of [3H]-tyrosine into neosynthesized proteins. The marked decrease in protein synthesis induced by a 12-hour TNF-α treatment was significantly counteracted by the addition of either myriocin or OMS (Figure 5a). Proteolysis was also quantified in [3H]-tyrosine labeled L6 myotubes, by measuring the release of trichloroacetic acid-soluble radioactivity. In the presence of TNF-α for 12 hours, both myriocin and OMS were able to decrease proteolysis significantly (Figure 5b). These results strongly suggest that ceramide formation negatively influences protein synthesis, whereas it activates proteolysis.


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)

Effects of tumor necrosis factor (TNF)-α on protein metabolism in L6 myotubes are prevented by ceramide-synthesis inhibition. (A) Myotubes were treated for 12 hours with TNF-α, in the presence or absence of 100 nmol/l myriocin, or 1 μmol/l OMS. The rate of protein synthesis was measured by adding [3H]-tyrosine to the culture medium, and counting the radioactivity present in trichloroacetic acid (TCA) precipitates of the cells, in relation to the total protein content. The results are the mean ± SE of three determinations. ++Different from control: P = 0.001; *different from TNF-α alone: P≤ 0.05. (B) Proteolysis was measured in L6 myotubes labeled with [3H]-tyrosine for 48 h, and treated for 12 hours with TNF-α in the presence or absence of 100 nmol/l myriocin, 1 μmol/l OMS, or both. TCA-soluble radioactivity released in the medium was measured and related to the total incorporated radioactivity. The results are the mean ± SE of three determinations. *Different from TNF-α alone: P < 0.05. (C) The mRNA levels of Atrogin-1 and LC3b were measured by reverse transcription quantitative PCR in myotubes treated or not with TNF-α in the presence of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l OMS, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 5 measurements in duplicate. **Different from control without drug: P = 0.01; +++different from TNF-α alone: P≤ 0.001, +P < 0.05. (D) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. Phospho- Ser253 Foxo3 in cell extracts was analyzed by western blotting. Results were normalized by total Foxo3 protein amounts. *Different from all other values: P < 0.02.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3344678&req=5

Figure 5: Effects of tumor necrosis factor (TNF)-α on protein metabolism in L6 myotubes are prevented by ceramide-synthesis inhibition. (A) Myotubes were treated for 12 hours with TNF-α, in the presence or absence of 100 nmol/l myriocin, or 1 μmol/l OMS. The rate of protein synthesis was measured by adding [3H]-tyrosine to the culture medium, and counting the radioactivity present in trichloroacetic acid (TCA) precipitates of the cells, in relation to the total protein content. The results are the mean ± SE of three determinations. ++Different from control: P = 0.001; *different from TNF-α alone: P≤ 0.05. (B) Proteolysis was measured in L6 myotubes labeled with [3H]-tyrosine for 48 h, and treated for 12 hours with TNF-α in the presence or absence of 100 nmol/l myriocin, 1 μmol/l OMS, or both. TCA-soluble radioactivity released in the medium was measured and related to the total incorporated radioactivity. The results are the mean ± SE of three determinations. *Different from TNF-α alone: P < 0.05. (C) The mRNA levels of Atrogin-1 and LC3b were measured by reverse transcription quantitative PCR in myotubes treated or not with TNF-α in the presence of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l OMS, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 5 measurements in duplicate. **Different from control without drug: P = 0.01; +++different from TNF-α alone: P≤ 0.001, +P < 0.05. (D) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. Phospho- Ser253 Foxo3 in cell extracts was analyzed by western blotting. Results were normalized by total Foxo3 protein amounts. *Different from all other values: P < 0.02.
Mentions: Protein synthesis rate was evaluated in L6 myotubes by measuring the incorporation of [3H]-tyrosine into neosynthesized proteins. The marked decrease in protein synthesis induced by a 12-hour TNF-α treatment was significantly counteracted by the addition of either myriocin or OMS (Figure 5a). Proteolysis was also quantified in [3H]-tyrosine labeled L6 myotubes, by measuring the release of trichloroacetic acid-soluble radioactivity. In the presence of TNF-α for 12 hours, both myriocin and OMS were able to decrease proteolysis significantly (Figure 5b). These results strongly suggest that ceramide formation negatively influences protein synthesis, whereas it activates proteolysis.

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