title: 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.
abstract: OBJECTIVE
  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 [(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
  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.
authors.affiliation: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia.
authors.forename: Georgia
authors.initials: G
authors.lastname: Frangioudakis
authors.forename: Barbara
authors.initials: B
authors.lastname: Diakanastasis
authors.forename: Bing-Qing M
authors.initials: BQ
authors.lastname: Liao
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Saville
authors.forename: Nolan J
authors.initials: NJ
authors.lastname: Hoffman
authors.forename: Todd W
authors.initials: TW
authors.lastname: Mitchell
authors.forename: Carsten
authors.initials: C
authors.lastname: Schmitz-Peiffer
mesh_terms: D002518:Ceramides / Q000096:biosynthesis / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis*; D018485:Muscle Fibers, Skeletal; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism
chemical_list: D002518:Ceramides; D007328:Insulin; D008565:Membrane Proteins; D010168:Palmitates; D020033:Protein Isoforms; D013107:Sphingolipids; D006003:Glycogen; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D005947:Glucose