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Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment

Journal Article


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Abstract


  • AIMS/HYPOTHESIS: An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

    Methods CerS isoforms CerS1, CerS2, CerS4, CerS5 and

    CerS6 were overexpressed in L6 myotubes using adenovirus,

    and cells were treated with palmitate and stimulated with

    insulin. Alternatively, CerS isoforms were knocked down

    using siRNAs. Sphingolipids were examined by mass spectrometry

    and tracer incorporation. Phosphorylation of IRS1

    and Akt was measured by immunoblotting, while glucose

    disposal was assessed by measuring GLUT4 translocation

    and the incorporation of [14C]glucose into glycogen.

    Results Palmitate treatment increased the levels of several

    ceramides but reduced the levels of sphingomyelins, while

    insulin had no effect. The fatty acid also inhibited insulinstimulated

    Akt phosphorylation and glycogen synthesis.

    Overexpression of CerS isoforms increased specific

    ceramides. Unexpectedly, the overexpression of CerS1 and

    CerS6 promoted insulin action, while no isoform had inhibitory

    effects. CerS6 knockdown had effects reciprocal to those

    of CerS6 overexpression.

    Conclusions/interpretation Palmitate may increase intracellular

    ceramide levels through sphingomyelin hydrolysis as well

    as de novo synthesis, but no particular species were implicated

    in the generation of insulin resistance. The modulation of

    ceramides through an alteration of CerS expression does not

    affect the action of insulin in the same way as ceramide

    generation by palmitate treatment. Conversely, certain

    isoforms promote insulin action, indicating the importance

    of ceramides in cell function.

    METHODS: CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen.

    RESULTS: Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

    CONCLUSIONS/INTERPRETATION: Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.

UOW Authors


  •   Frangioudakis, Georgia (external author)
  •   Diakanastasis, Barbara (external author)
  •   Liao, Bing-Qing M. (external author)
  •   Saville, Jennifer T. (external author)
  •   Hoffman, Nolan J. (external author)
  •   Mitchell, Todd
  •   Schmitz-Peiffer, Carsten (external author)

Publication Date


  • 2013

Citation


  • Frangioudakis, G., Diakanastasis, B., Liao, B. M., Saville, J. T., Hoffman, N. J., Mitchell, T. W. & Schmitz-Peiffer, C. (2013). Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment. Diabetologia, 56 (12), 2697-2701.

Scopus Eid


  • 2-s2.0-84888014316

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2214&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1196

Number Of Pages


  • 4

Start Page


  • 2697

End Page


  • 2701

Volume


  • 56

Issue


  • 12

Abstract


  • AIMS/HYPOTHESIS: An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

    Methods CerS isoforms CerS1, CerS2, CerS4, CerS5 and

    CerS6 were overexpressed in L6 myotubes using adenovirus,

    and cells were treated with palmitate and stimulated with

    insulin. Alternatively, CerS isoforms were knocked down

    using siRNAs. Sphingolipids were examined by mass spectrometry

    and tracer incorporation. Phosphorylation of IRS1

    and Akt was measured by immunoblotting, while glucose

    disposal was assessed by measuring GLUT4 translocation

    and the incorporation of [14C]glucose into glycogen.

    Results Palmitate treatment increased the levels of several

    ceramides but reduced the levels of sphingomyelins, while

    insulin had no effect. The fatty acid also inhibited insulinstimulated

    Akt phosphorylation and glycogen synthesis.

    Overexpression of CerS isoforms increased specific

    ceramides. Unexpectedly, the overexpression of CerS1 and

    CerS6 promoted insulin action, while no isoform had inhibitory

    effects. CerS6 knockdown had effects reciprocal to those

    of CerS6 overexpression.

    Conclusions/interpretation Palmitate may increase intracellular

    ceramide levels through sphingomyelin hydrolysis as well

    as de novo synthesis, but no particular species were implicated

    in the generation of insulin resistance. The modulation of

    ceramides through an alteration of CerS expression does not

    affect the action of insulin in the same way as ceramide

    generation by palmitate treatment. Conversely, certain

    isoforms promote insulin action, indicating the importance

    of ceramides in cell function.

    METHODS: CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen.

    RESULTS: Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

    CONCLUSIONS/INTERPRETATION: Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.

UOW Authors


  •   Frangioudakis, Georgia (external author)
  •   Diakanastasis, Barbara (external author)
  •   Liao, Bing-Qing M. (external author)
  •   Saville, Jennifer T. (external author)
  •   Hoffman, Nolan J. (external author)
  •   Mitchell, Todd
  •   Schmitz-Peiffer, Carsten (external author)

Publication Date


  • 2013

Citation


  • Frangioudakis, G., Diakanastasis, B., Liao, B. M., Saville, J. T., Hoffman, N. J., Mitchell, T. W. & Schmitz-Peiffer, C. (2013). Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment. Diabetologia, 56 (12), 2697-2701.

Scopus Eid


  • 2-s2.0-84888014316

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2214&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1196

Number Of Pages


  • 4

Start Page


  • 2697

End Page


  • 2701

Volume


  • 56

Issue


  • 12