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Comparative Study
. 2007 Oct 8:8:84.
doi: 10.1186/1471-2202-8-84.

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

Tatiana P Storozhevykh  1 , Yana E Senilova, Nadezhda A Persiyantseva, Vsevolod G Pinelis, Igor A Pomytkin
Affiliations
Comparative Study

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

Tatiana P Storozhevykh et al. BMC Neurosci. .

Abstract

Background: Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons.

Results: Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN.

Conclusion: Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.

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Figures

Figure 1
Figure 1
Effect of N-acetylcysteine on Insulin-stimulated H2O2 production and the insulin receptor autophosphorylation in cerebellar granule neurons. A: CGN cultures were pre-incubated for 30 min in the absence or presence of N-acetylcysteine (5 mmol/l) in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L). H2O2 release from cultures for 1 min was measured as described in Materials and Methods. Results were normalized by cell density. Columns represent the means ± SD of H2O2 values obtained from five to nine cultures. Dotted line represents a detection limit of the assay (7 nmol/L). B: CGN cultures were pre-incubated for 30 min in the absence or presence of N-acetylcysteine (5 mmol/l) in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L) for 20 min. Autophosphorylation of insulin receptor was measured as described in Materials and Methods. In each experiment, amount of phosphorylated insulin receptor β-subunit (pYpY-IR) was normalized to total amount of insulin receptor β-subunit and expressed as a percentage of the response produced to 100 nmol/L insulin. Columns represent the means ± SD of pYpY-IR values obtained from four to nine culture dishes. *P < 0.05 vs. control.†P < 0.05 vs. insulin.
Figure 2
Figure 2
Effects of malonate and FCCP on insulin-stimulated H2O2 production and insulin receptor autophosphorylation in cerebellar granule neurons. A: CGN cultures were pre-incubated for 30 min in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L) in the absence or presence of malonate (2 mmol/L) or FCCP (0.5 μmol/L). H2O2 release from cultures for 1 min was measured as described in Materials and Methods. Results were normalized by cell density. Columns represent the means ± SD of H2O2 values obtained from five to nine cultures. Dotted line represents a detection limit of the assay (7 nmol/L). B: CGN cultures were pre-incubated for 30 min in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L) for 20 min. Malonate (2 mmol/l) or FCCP (0.5 μmol/L) were added to cultures 5 min before the insulin exposure. Autophosphorylation of insulin receptor was measured as described in Materials and Methods. In each experiment, amount of phosphorylated insulin receptor β-subunit (pYpY-IR) was normalized to total amount of insulin receptor β-subunit and expressed as a percentage of the response produced to 100 nmol/L insulin. Columns represent the means ± SD of pYpY-IR values obtained from four to nine culture dishes. *P < 0.05 vs. control.†P < 0.05 vs. insulin.
Figure 3
Figure 3
Effects of dicholine salt of succinic acid on insulin-stimulated insulin receptor autophosphorylation in cerebellar granule neurons. CGN cultures were pre-incubated for 30 min in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L), insulin (5 nmol/L), CS (50 μmol/L), or a combination of insulin (5 nmol/L) and CS (50 μmol/L) for 20 min. Autophosphorylation of insulin receptor was measured as described in Materials and Methods. In each experiment, amount of phosphorylated insulin receptor β-subunit (pYpY-IR) was normalized to total amount of insulin receptor β-subunit and expressed as a percentage of the response produced to 100 nmol/L insulin. Columns represent the means ± SD of pYpY-IR values obtained from four to nine culture dishes. *P < 0.05 vs. control.†P < 0.05 vs. insulin (100 nmol/L). #P < 0.05 vs. insulin (5 nmol/L).
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