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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

The negative effects of tumor necrosis factor (TNF)-α on L6 myotubes are counteracted by ceramide-synthesis inhibition. (A) Myotube area was measured after 3 days in the presence or absence of 15 ng/ml TNF-α, with the addition of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l 3-0-methylsphingomyelin (OMS), or with myriocin plus either GW4869 or OMS. Shown are the mean ± SE of 5 to 9 experiments, with 10 fields considered for each condition. ***Different from all the other conditions: P ≤ 0.001. (B) ELISA quantification of the MHC content of myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The means ± SE of three measurements expressed as a percentage of control values are shown. ***Different from the other conditions: P ≤ 0.005. (C) Western blot analysis of myosin light chains (MLC) 1 and 3 from myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The diagram shows the quantification of MLC1 and MLC3 levels, after normalization by tubulin levels. Shown are the mean ± SE of three determinations. **Different from control and from TNF-α: P ≤ 0.002; *different from control: P ≤ 0.05. (D) Creatine kinase activity of myotubes treated or not by TNF-α, in the presence of 100 nmol/l myriocin, or 10 μmol/l GW4869, or 1 μmol/l OMS, or both myriocin and GW4869 or OMS. Shown are the mean ± SE of three determinations made in triplicate. ***Different from all the other conditions: P ≤ 0.001.
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Figure 2: The negative effects of tumor necrosis factor (TNF)-α on L6 myotubes are counteracted by ceramide-synthesis inhibition. (A) Myotube area was measured after 3 days in the presence or absence of 15 ng/ml TNF-α, with the addition of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l 3-0-methylsphingomyelin (OMS), or with myriocin plus either GW4869 or OMS. Shown are the mean ± SE of 5 to 9 experiments, with 10 fields considered for each condition. ***Different from all the other conditions: P ≤ 0.001. (B) ELISA quantification of the MHC content of myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The means ± SE of three measurements expressed as a percentage of control values are shown. ***Different from the other conditions: P ≤ 0.005. (C) Western blot analysis of myosin light chains (MLC) 1 and 3 from myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The diagram shows the quantification of MLC1 and MLC3 levels, after normalization by tubulin levels. Shown are the mean ± SE of three determinations. **Different from control and from TNF-α: P ≤ 0.002; *different from control: P ≤ 0.05. (D) Creatine kinase activity of myotubes treated or not by TNF-α, in the presence of 100 nmol/l myriocin, or 10 μmol/l GW4869, or 1 μmol/l OMS, or both myriocin and GW4869 or OMS. Shown are the mean ± SE of three determinations made in triplicate. ***Different from all the other conditions: P ≤ 0.001.

Mentions: To confirm the role of ceramide formation in the atrophic response to TNF-α, inhibitors of ceramide synthesis were added to the culture medium simultaneously with TNF-α. Ceramide can be formed by two different pathways (under the action of sphingomyelinases or through de novo synthesis), and TNF-α is known to activate both pathways [2,3]. Thus, two types of agents were used: myriocin, an inhibitor targeting de novo synthesis by selectively inhibiting the first step of the pathway catalyzed by serine-palmitoyl-CoA transferase [21], and GW4869 [22] and 3-O-methylsphingomyelin (OMS) [23], two inhibitors of sphingomyelinases. Myriocin was able to protect L6 myotubes from the TNF-α-induced decrease in surface. GW4869 and OMS were also able to counteract the TNF-α atrophic effect in L6 myotubes, suggesting that ceramide formed by either of the pathways mediates the atrophic effect of TNF-α (Figure 2a). The inhibitors had little influence on myotube surface in the absence of TNF-α, although there was a weak positive effect for GW4869 and OMS. No additive effects of the inhibitors of the two different ceramide-synthesis pathways were seen (Figure 2a).


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)

The negative effects of tumor necrosis factor (TNF)-α on L6 myotubes are counteracted by ceramide-synthesis inhibition. (A) Myotube area was measured after 3 days in the presence or absence of 15 ng/ml TNF-α, with the addition of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l 3-0-methylsphingomyelin (OMS), or with myriocin plus either GW4869 or OMS. Shown are the mean ± SE of 5 to 9 experiments, with 10 fields considered for each condition. ***Different from all the other conditions: P ≤ 0.001. (B) ELISA quantification of the MHC content of myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The means ± SE of three measurements expressed as a percentage of control values are shown. ***Different from the other conditions: P ≤ 0.005. (C) Western blot analysis of myosin light chains (MLC) 1 and 3 from myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The diagram shows the quantification of MLC1 and MLC3 levels, after normalization by tubulin levels. Shown are the mean ± SE of three determinations. **Different from control and from TNF-α: P ≤ 0.002; *different from control: P ≤ 0.05. (D) Creatine kinase activity of myotubes treated or not by TNF-α, in the presence of 100 nmol/l myriocin, or 10 μmol/l GW4869, or 1 μmol/l OMS, or both myriocin and GW4869 or OMS. Shown are the mean ± SE of three determinations made in triplicate. ***Different from all the other conditions: P ≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3344678&req=5

Figure 2: The negative effects of tumor necrosis factor (TNF)-α on L6 myotubes are counteracted by ceramide-synthesis inhibition. (A) Myotube area was measured after 3 days in the presence or absence of 15 ng/ml TNF-α, with the addition of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l 3-0-methylsphingomyelin (OMS), or with myriocin plus either GW4869 or OMS. Shown are the mean ± SE of 5 to 9 experiments, with 10 fields considered for each condition. ***Different from all the other conditions: P ≤ 0.001. (B) ELISA quantification of the MHC content of myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The means ± SE of three measurements expressed as a percentage of control values are shown. ***Different from the other conditions: P ≤ 0.005. (C) Western blot analysis of myosin light chains (MLC) 1 and 3 from myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The diagram shows the quantification of MLC1 and MLC3 levels, after normalization by tubulin levels. Shown are the mean ± SE of three determinations. **Different from control and from TNF-α: P ≤ 0.002; *different from control: P ≤ 0.05. (D) Creatine kinase activity of myotubes treated or not by TNF-α, in the presence of 100 nmol/l myriocin, or 10 μmol/l GW4869, or 1 μmol/l OMS, or both myriocin and GW4869 or OMS. Shown are the mean ± SE of three determinations made in triplicate. ***Different from all the other conditions: P ≤ 0.001.
Mentions: To confirm the role of ceramide formation in the atrophic response to TNF-α, inhibitors of ceramide synthesis were added to the culture medium simultaneously with TNF-α. Ceramide can be formed by two different pathways (under the action of sphingomyelinases or through de novo synthesis), and TNF-α is known to activate both pathways [2,3]. Thus, two types of agents were used: myriocin, an inhibitor targeting de novo synthesis by selectively inhibiting the first step of the pathway catalyzed by serine-palmitoyl-CoA transferase [21], and GW4869 [22] and 3-O-methylsphingomyelin (OMS) [23], two inhibitors of sphingomyelinases. Myriocin was able to protect L6 myotubes from the TNF-α-induced decrease in surface. GW4869 and OMS were also able to counteract the TNF-α atrophic effect in L6 myotubes, suggesting that ceramide formed by either of the pathways mediates the atrophic effect of TNF-α (Figure 2a). The inhibitors had little influence on myotube surface in the absence of TNF-α, although there was a weak positive effect for GW4869 and OMS. No additive effects of the inhibitors of the two different ceramide-synthesis pathways were seen (Figure 2a).

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