Insulin resistance states are associated with endothelial dysfunction, but the molecular mechanisms underlying this association are incompletely understood. Impairment of insulin stimulated endothelial NO production is proposed to be a key mechanism in this process. In cultured human aortic endothelial cell (HAEC) models, insulin stimulates an intracellular signalling cascade resulting in activating phosphorylation of Insulin Receptor Substrate-1 (IRS-1), Protein kinase B (PKB / Akt) and endothelial Nitric Oxide Synthase (eNOS), with resultant NO release. The pro-inflammatory adipokine Tumour Necrosis Factor Alpha (TNF-α) is over-expressed in models of insulin resistance, acting in humans to inhibit both insulin-stimulated glucose uptake and endothelial-dependant vasodilatation. Studies in bovine aortic endothelial cells have shown that preincubation with TNF-α inhibits insulin stimulated NO production, with concomitant inhibition of activating phosphorylation of IRS-1, PKB and eNOS but the precise molecular pathway that accounts for the inhibition seen is unclear. Accordingly, we studied the interaction between TNF-α and insulin-mediated NO production in HAECs.
We have demonstrated that pre-incubation with TNF-α inhibits insulin stimulated NO production. In contrast to bovine studies this was not associated with reduced activating phosphorylation of PKB (Ser473) or eNOS (Ser1177). A previously uncharacterised eNOS residue (Ser 617) is phosphorylated in response to insulin and appears to be unaltered by TNF-α preincubation. Phosphorylation of IRS-1 residue Ser 312 has been proposed as a key inhibitory signalling mechanism in insulin resistance in adipocytes and skeletal muscle, however in our model no change was demonstrable. TNF-α activates JNK and IKK, and these kinases may form the intracellular link between TNF-α and insulin signalling. We hypothesise that the reduction in NO production in this model is not solely due to impairment of the insulin signalling cascade. A potential mechanism of action currently being investigated is a reduction in NO bioavailability through quenching by superoxide, produced in response to TNF-α.