title: Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle.
abstract: OBJECTIVE
  Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle.
  
  
  METHODS
  Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase epsilon, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry.
  
  
  RESULTS
  Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase epsilon reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells.
  
  
  CONCLUSIONS
  These data indicate that linoleate-derived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.
authors.affiliation: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Cazzolli
authors.forename: T W
authors.initials: TW
authors.lastname: Mitchell
authors.forename: J G
authors.initials: JG
authors.lastname: Burchfield
authors.forename: D J
authors.initials: DJ
authors.lastname: Pedersen
authors.forename: N
authors.initials: N
authors.lastname: Turner
authors.forename: T J
authors.initials: TJ
authors.lastname: Biden
authors.forename: C
authors.initials: C
authors.lastname: Schmitz-Peiffer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019852:Diacylglycerol Kinase / Q000378:metabolism; D015151:Immunoblotting; D055504:Insulin Receptor Substrate Proteins; D019787:Linoleic Acid / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D014443:Tyrosine / Q000378:metabolism
chemical_list: D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010712:Phosphatidic Acids; D010750:Phosphoproteins; D014443:Tyrosine; D019787:Linoleic Acid; D019869:Phosphatidylinositol 3-Kinases; D019852:Diacylglycerol Kinase