We have previously shown that unsaturated fatty acids amplify platelet-derived-growth-factor (PDGF)-induced protein kinase C (PKC) activation in vascular smooth-muscle cells (VSMCs). Diacylglycerol-induced PKC activation is normally terminated by diacylglycerol kinases (DGKs). We thus hypothesized that fatty acids act by inhibiting a DGK. Fractionation of VSMC extracts demonstrated that the DGK α isoform was the major DGK activity present. PDGF markedly increased the DGK activity of cultured cells. An inhibitor selective for the DGK α isoform,R59949[3-{2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone], abolished the growth-factor-induced increase in DGK activity, but had little effect on basal activity. PDGF thus selectively activates DGKα. Epidermal growth factor and α-thrombin stimulated total DGK activity similarly to PDGF. Activation by epidermal growth factor was sensitive to R59949, again suggesting involvement of DGKα. However, the α-thrombin-induced activity was unaffected by this agent. Unsaturated fatty acids inhibited growth-factor-induced DGKα activation, but had no effect on basal activity. Fatty acids also amplified the PDGF-induced increase in cell diacylglycerol content. These results indicate that inhibition of DGKα contributes to fatty-acid-induced amplification of PKC activation. Increased levels of fatty acids in diabetes may thus contribute to chronic PKC activation associated with this disorder.

Abbreviations used: DAG, sn-1,2-diacylglycerol; DGK, diacylglycerol kinase; diC10, sn-1,2-didecanoylglycerol; EGF, epidermal growth factor; NEFA, non-esterified (‘free’) fatty acid; FGFb, fibroblast growth factor-2; MEM, (Earle's) minimal essential medium; PA, phosphatidic acid; PDGF, platelet-derived growth factor; PI, phosphoinositide; PKC, protein kinase C; R59949, 3-{2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone; VSMC, vascular smooth-muscle cell.

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