Excitotoxic glutamate causes neuronal insulin resistance by inhibiting insulin receptor/Akt/mTOR pathway

AIM: An impaired biological response to insulin in the brain, known as central insulin resistance, was identified during stroke and traumatic brain injury, for which glutamate excitotoxicity is a common pathogenic factor. The exact molecular link between excitotoxicity and central insulin resistance remains unclear. To explore this issue, the present study aimed to investigate the effects of glutamate-evoked increases in intracellular free Ca(2+) concentrations [Ca(2+)](i) and mitochondrial depolarisations, two key factors associated with excitotoxicity, on the insulin-induced activation of the insulin receptor (IR) and components of the Akt/ mammalian target of rapamycin (mTOR) pathway in primary cultures of rat cortical neurons. METHODS: Changes in [Ca(2+)](i) and mitochondrial inner membrane potentials (ΔΨ(m)) were monitored in rat cultured cortical neurons, using the fluorescent indicators Fura-FF and Rhodamine 123, respectively. The levels of active, phosphorylated signalling molecules associated with the IR/Akt/mTOR pathway were measured with the multiplex fluorescent immunoassay. RESULTS: When significant mitochondrial depolarisations occurred due to glutamate-evoked massive influxes of Ca(2+) into the cells, insulin induced 48% less activation of the IR (assessed by IR tyrosine phosphorylation, pY(1150/1151)), 72% less activation of Akt (assessed by Akt serine phosphorylation, pS(473)), 44% less activation of mTOR (assessed by mTOR pS(2448)), and 38% less inhibition of glycogen synthase kinase β (GSK3β) (assessed by GSK3β pS(9)) compared with respective controls. These results suggested that excitotoxic glutamate inhibits signalling via the IR/Akt/mTOR pathway at multiple levels, including the IR, resulting in the development of acute neuronal insulin resistance within minutes, as an early pathological event associated with excitotoxicity.