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

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 linoleatederived 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.

Protein Kinase C Function in Muscle, Liver, and β-Cells and Its Therapeutic Implications for Type 2 Diabetes

“…PKC activation is relatively specific to DAG species composed of unsaturated fatty acid side chains (47). Thus, we have demonstrated that the elevation in DAG observed in muscle cells treated with the saturated fatty acid palmitate does not greatly stimulate PKC translocation, while the relatively minor DAG changes caused by unsaturated fatty acids can do so (48,49). Because both saturated and unsaturated fatty acids cause insulin resistance, this supports the contention that saturated fatty acids do not act principally via the DAG/PKC pathway, but through other mechanisms including ceramide-dependent signaling (50 -53).…”

“…2], and a cross-talk mechanism involving sequestration of Akt by PKC in caveolae has also been proposed to contribute to the disruption of insulin signaling caused by saturated fatty acids [52].) The integral link between specific fatty acids, DAG, and PKC activation has been confirmed in studies addressing the effects of DAG kinase (DGK) isoforms, which lower intracellular DAG levels and reverse the activation of PKC, but these have also highlighted the complexity of DAG signaling (49,54,55). On the one hand, a reduction in DGK␦ activity potentially contributes to hyperglycemia-induced activation of PKC␦ and the resultant inhibition of insulin signaling in skeletal muscle from subjects with type 2 diabetes (55).…”

“…On the one hand, a reduction in DGK␦ activity potentially contributes to hyperglycemia-induced activation of PKC␦ and the resultant inhibition of insulin signaling in skeletal muscle from subjects with type 2 diabetes (55). On the other hand, overexpression of DGKε in muscle cells, while reducing the activation of several PKCs as expected because of chronic exposure to unsaturated fatty acid, in fact compounds defects in insulin signaling (49). Such discrepancies are perhaps explained by the different substrate specificities, regulation, and cellular localization of the DGK isoforms (56) involved.…”

“…In addition, and taken together with the failure of dominantnegative nPKC mutants to overcome insulin resistance in cultured myotubes (48), it can be argued that the diminished insulin action arising from lipid oversupply is not always simply explained by the accompanying DAG accumulation and PKC activation. Indeed, other lipid intermediates such as dilinoleoyl-phosphatidic acid (49) have been recently implicated as mediating insulin resistance induced by unsaturated fatty acids via pathways independent of PKC. This makes it essential to define precisely the situations where PKC does play a role and to quantify its contribution using in vivo models.…”

Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice

“…Cholesterol and cholesteryl ester were determined with a cholesterol assay kit (Calbiochem, San Diego, CA, USA). Diacylglycerol content was assessed by thin-layer chromatography (TLC), using a method adapted from Nakamura and Handa [14,15]. NEFA were determined in plasma samples from 1 week chow or fat-fed WT and Prkce −/− mice using the NEFA C kit from Wako Diagnostics (Richmond, VA, USA).…”