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

Abstract

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 Ca²⁺ concentrations [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺ 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, pS⁴⁷³), 44% less activation of mTOR (assessed by mTOR pS²⁴⁴⁸), and 38% less inhibition of glycogen synthase kinase β (GSK3β) (assessed by GSK3β pS⁹) 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.

Main text

An acute impairment in the biological response to insulin in the brain, known as central insulin resistance, was identified during stroke [1] and traumatic brain injury [2], for which excitotoxicity, which is caused by excessive glutamate release, is a key pathogenic factor [3].

The exact molecular link between insulin resistance and glutamate excitotoxicity remains unclear. Based on published data, an abnormal rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and decreased mitochondrial inner membrane potential (ΔΨ_m) are factors associated with the excitotoxic glutamate [4,5,6], which could potentially affect insulin signalling. The presence of Ca²⁺ (1 mM) has been shown to reduce the insulin-induced tyrosine phosphorylation of the insulin receptor (IR) in hippocampal synaptic preparations [7]. Glutamate has been shown to reduce the tyrosine phosphorylation of the IR when added after the prolonged insulin-mediated stimulation of hippocampal neuronal cultures [8]. Protonophore-induced decreases in ΔΨ_m have been shown to evoke concomitant decreases in the IR tyrosine phosphorylation in response to insulin, indicating that mitochondrial depolarisation is an independent causative factor for neuronal insulin resistance [9]. However, the effects of glutamate-evoked changes in [Ca²⁺]_i and ΔΨ_m on the insulin-induced activation of the IR/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK)3β pathways have never been studied. Here, we investigated whether excitotoxic glutamate affects the insulin-induced phosphorylation of IR/Akt/mTOR pathway components during significant mitochondrial depolarisations caused by the massive influx of Ca²⁺ into the cells.

To determine the times during which glutamate induces significant mitochondrial depolarisation, rat cortical neurons were exposed to 100 μM glutamate and changes in [Ca²⁺]_i, and ΔΨ_m were monitored for 30 min. Detailed methods for the preparation of primary cortical neuronal cultures and measuring [Ca²⁺]_i and ΔΨ_m are described in Additional file 1. As expected, glutamate evoked a rapid increase in [Ca²⁺]_i, followed by a decreased in ΔΨ_m in the cells (Fig. 1a and b, Additional file 2: Tables S3 and S4). After 30 min of glutamate exposure, the mean [Ca²⁺]_i level increased by 2.4-fold above baseline (F_148,8732 = 44.8, P < 0.0001), and the mean ΔΨ_m value significantly decreased by 1.6-fold below baseline (F_148,8732 = 182.8, P < 0.0001) (Fig. 1a–c, one-way analysis of variance [ANOVA] with repeated measures, followed by Tukey’s post hoc test). Therefore, the 30-min interval for glutamate exposure was selected for subsequent experiments.

Fig. 1

Excitotoxic glutamate inhibits IR/Akt/mTOR pathway

a Dynamics of [Ca²⁺]_i and b ΔΨ_m in single rat cortical neurons, loaded simultaneously with Fura-FF and Rh123 dyes and exposed to 100 μM glutamate. Grey lines represent sixty single neurons. Blue and red lines represent the respective means of [Ca²⁺]_i and ΔΨ_m, averaged across sixty individual neurons at every time point. c Fura-FF and Rh123 fluorescence 30 min after the onset of glutamate exposure, expressed as the fold increase over baseline (at − 5 min). Data are the mean ± SEM from sixty neurons. ****P < 0.0001 compared to respective baselines (one-way ANOVA with repeated measures, followed by Tukey’s post hoc test). d-g Levels of d IRβ pY^1150/1151, e Akt pS⁴⁷³, f mTOR pS²⁴⁴⁸, and g GSK3β pS⁹ in rat cortical neurons exposed to 0 nM (C) or 100 nM insulin for 15 min (I), 100 μM glutamate for 30 min g, or sequentially to 100 μM glutamate for 30 min and 100 nM insulin for 15 min (G + I). Bar graphs represent the levels of the phosphoproteins, normalised against respective total protein levels, in cell lysates and expressed as a percentage of levels in insulin-treated cells (group I). Each value represents the mean ± SEM from six independent cultures (cell populations obtained from twelve separate rats, two per culture). ^###P < 0.001, ^####P < 0.0001 compared with untreated control c; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with insulin i (one-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons). h Scheme illustrating the inhibitory effects of glutamate on the insulin-induced activation of the IR/Akt/mTOR pathway.

Next, we investigated the effects of glutamate on the insulin-induced activation of signalling molecules in the IR/Akt/mTOR and GSK3β pathways. Rat cortical neuronal cultures were sequentially exposed to either 100 μM excitotoxic glutamate concentration or no glutamate for 30 min, then stimulated with 100 nM insulin for 15 min and lysed. The levels of the tyrosine-phosphorylated IR β-subunit (IRβ pY^1150/1151), the serine-phosphorylated Ser/Thr kinase Akt (Akt pS⁴⁷³), the serine-phosphorylated mammalian target of rapamycin (mTOR pS²⁴⁴⁸), and the serine-phosphorylated glycogen synthase kinase 3β (GSK3β pS⁹) were measured in the cell lysates and normalised against the total levels of these proteins, as outlined in Additional file 1. A one-way ANOVA analysis revealed a significant difference between groups for IRβ pY^1150/1151 (Fig. 1d, F_3,20 = 23.62, P < 0.0001), Akt pS⁴⁷³ (Fig. 1e, F_3,20 = 388.6, P < 0.0001), mTOR pS²⁴⁴⁸ (Fig. 1f, F_3,20 = 19.69, P < 0.0001), and GSK3β pS⁹ (Fig. 1g, F_3,20 = 4.48, P = 0.0146). Tukey’s multiple comparisons test showed that insulin stimulation resulted in a significant increase in IRβ pY^1150/1151 (Fig. 1d, P < 0.0001), Akt pS⁴⁷³ (Fig. 1e, P < 0.0001), and mTOR pS²⁴⁴⁸ (Fig. 1f, P < 0.001) levels compared with their respective controls (Additional file 1: Tables S1 and S2). In the glutamate pre-treated neurons, insulin induced 48% less IR phosphorylation at pY^1150/1151 (Fig. 1d, P < 0.001), 72% less Akt phosphorylation at pS⁴⁷³ (Fig. 1e, P < 0.0001), 44% less mTOR phosphorylation at pS²⁴⁴⁸ (Fig. 1f, P < 0.0001), and 38% less GSK3β phosphorylation at pS⁹ (Fig. 1g, P < 0.01) compared with non-glutamate treated controls. Neuronal IR becomes fully active following autophosphorylation at Y^1150/1151 [10], which triggers downstream signalling via the Akt/mTOR and GSK3β pathways. The serine-phosphorylation of Akt at S⁴⁷³ [11] and mTOR at S²⁴⁴⁸ [12] are crucial for their activation, whereas the serine-phosphorylation of GSK3β at S⁹ [13] leads to its inhibition. Therefore, our results suggested that excitotoxic glutamate inhibits insulin-induced IR activation and the downstream IR/Akt/mTOR signalling pathway (Fig. 1h).

The primary finding of the present study was that excitotoxic glutamate inhibits the IR/Akt/mTOR pathway, resulting in the development of acute neuronal insulin resistance during periods of significant mitochondrial depolarisation caused by glutamate-evoked massive influxes of Ca²⁺. This rapid loss of neuronal insulin sensitivity appears to be one of the earliest pathological events associated with glutamate excitotoxicity. These results are in complete agreement with our previous findings that mitochondria control IR autophosphorylation in neurons and that mitochondrial depolarisation causes the loss of insulin sensitivity during the IR autophosphorylation stage [9, 14]. Recently we showed that pre-treatment with insulin prevents the glutamate-evoked increases in [Ca²⁺]_i and decreases in ΔΨ_m, protecting rat cortical neurons against excitotoxicity [15]. The glutamate effect and the protective effects of insulin were both completely abrogated by MK 801, an inhibitor of Ca²⁺ influx, via the N-methyl-D-aspartate (NMDA) receptor and the plasmalemmal Na⁺/Ca²⁺ exchanger operating in reverse mode [15]. Collectively, these findings suggested that the modulation of intracellular Ca²⁺ levels plays a critical role in negative cross-talk between insulin and glutamate signalling during excitotoxicity. Glutamate induces an increase in [Ca²⁺]_i and a decrease in ΔΨ_m, which inhibit IR activation. In turn, insulin prevents the glutamate-evoked rise in [Ca²⁺]_i and mitochondrial depolarisation, protecting against excitotoxicity.

In conclusion, this study showed that glutamate excitotoxicity is causative for central insulin resistance and may induce the acute loss of insulin signalling within minutes under mitochondrial depolarisation conditions. Therefore, the use of agents designed to prevent mitochondrial depolarisation may be a reasonable approach to the treatment of acute neuronal insulin resistance during excitotoxicity.