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. 2014:2014:674164.
doi: 10.1155/2014/674164. Epub 2014 Jun 15.

Insulin blocks glutamate-induced neurotoxicity in differentiated SH-SY5Y neuronal cells

Madhavan Nampoothiri  1 , Neetinkumar D Reddy  1 , Jessy John  1 , Nitesh Kumar  1 , Gopalan Kutty Nampurath  1 , Mallikarjuna Rao Chamallamudi  1
Affiliations

Insulin blocks glutamate-induced neurotoxicity in differentiated SH-SY5Y neuronal cells

Madhavan Nampoothiri et al. Behav Neurol. 2014.

Abstract

Insulin is a cytokine which promotes cell growth. Recently, a few published reports on insulin in different cell lines support the antiapoptotic effect of insulin. But the reports fail to explain the role of insulin in modulating glutamate-mediated neuronal cell death through excitotoxicity. Thus, we examined the neuroprotective effect of insulin on glutamate-induced toxicity on differentiated SH-SY5Y neuronal cells. Changes in cell viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) based assay, while apoptotic damage was detected by acridine orange/ethidium bromide and Hoechst staining. Intracellular reactive oxygen species (ROS) accumulation and morphological alterations were also measured. Treatment with glutamate induced apoptosis, elevated ROS levels and caused damage to neurons. Insulin was able to attenuate the glutamate-induced excitotoxic damage to neuronal cells.

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Figures

Figure 1
Figure 1
Effect of different concentrations of glutamate on cell viability in (a) undifferentiated SH-SY5Y cells and (b) differentiated SH-SY5Y cells. Values are expressed as mean ± SEM of three tests in triplicate. Statistical analysis was done by using one-way ANOVA followed by Tukey's multiple comparison test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to control group.
Figure 2
Figure 2
AO/EB staining in differentiated SH-SY5Y cells after treatment. (a) Control, (b) glutamate (20 mM), (c) glutamate (20 mM) + insulin (0.1 μM), and (d) glutamate (20 mM) + insulin (1 μM). White arrow: live cell; yellow arrow: apoptotic cell.
Figure 3
Figure 3
Hoechst staining in differentiated SH-SY5Y cells after treatment. (a) Control, (b) glutamate (20 mM), (c) glutamate (20 mM) + insulin (0.1 μM), and (d) glutamate (20 mM) + insulin (1 μM). White arrow: live cell; yellow arrow: apoptotic cell.
Figure 4
Figure 4
Effect of treatments on morphology of differentiated SH-SY5Y cells. (a) Control, (b) glutamate (20 mM), (c) glutamate + insulin (0.1 μM), and (d) glutamate + insulin (1 μM).
Figure 5
Figure 5
Effect of insulin pretreatment on the glutamate-induced ROS accumulation in differentiated SH-SY5Y cells. Values are expressed as mean ± SEM of three tests in triplicate. Statistical analysis was done by using one-way ANOVA followed by Tukey's multiple comparison test. ∗∗∗P < 0.001 compared to control group, # P < 0.05 compared to glutamate group, and ## P < 0.01 compared to glutamate group.
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