Article

Diabetologia

, Volume 50, Issue 8, pp 1732-1742

First online:

Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle

  • R. CazzolliAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical Research
  • , T. W. MitchellAffiliated withSchool of Health Sciences, University of Wollongong
  • , J. G. BurchfieldAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical Research
  • , D. J. PedersenAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical Research
  • , N. TurnerAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical ResearchSchool of Health Sciences, University of Wollongong
  • , T. J. BidenAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical Research
  • , C. Schmitz-PeifferAffiliated withDiabetes and Obesity Program, Garvan Institute of Medical Research Email author 

Abstract

Aims/hypothesis

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.

Materials and 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 ɛ, 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 ɛ 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/interpretation

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.

Keywords

Diacylglycerol Fatty acid Insulin resistance IRS-1 L6 muscle cells Phosphatidic acid Protein kinase C Skeletal muscle