The protective role of D-glucose against 1-methyl-4-phenylpyridinium ion (MPP+): induced mitochondrial dysfunction in C6 astroglial cells

Abstract

Impaired mitochondrial function in glial and neuronal cells in the substantia nigra is one of the most likely causes of Parkinson's disease. In this study, we investigated the protective role of glucose on early key events associated with MPP(+)-induced changes in rat C6 astroglial cells. Studies were carried out to examine alterations in mitochondrial respiratory status, membrane potential, glutathione levels, and cell cycle phase inhibition at 48 h in 2 and 10 mM glucose in media. The results obtained suggest that MPP(+) caused significant cell death in 2 mM glucose with LC(50) 0.14 +/- 0.005 mM, while 10 mM glucose showed highly significant protection against MPP(+) toxicity with LC(50) 0.835 +/- 0.03 mM. This protection was not observed with cocaine, demonstrating its compound specificity. MPP(+) in 2 mM glucose decreased significantly mitochondrial respiration, membrane potential and glutathione levels in a dose dependent manner, while 10 mM glucose significantly restored them. MPP(+) in 2 mM glucose arrested the cells at G0/G1 and G2/M phases, demonstrating its dual inhibitory effects. However, in 10 mM glucose, MPP(+) caused G0/G1 arrest only. In summary, the results suggest that loss of cell viability in 2 mM glucose group with MPP(+) treatments was due to mitochondrial dysfunction caused by multilevel mechanism, involving significant decrease in mitochondrial respiration, membrane potential, glutathione levels, and dual arrest of cell phases, while 10 mM glucose rescued astroglial cells from MPP(+) toxicity by significant maintenance of these factors.

Fig. 1

Growth curves of glial cells. Cells with starting density of 5 × 10³ per well in 96-well plates (n = 4, and each value is the average absorbance of 6 wells, so final n at each time point = 24) with complete DMEM containing 2 or 10 mM glucose and 10% FBS were incubated for designated time points. The cells were stained with crystal violet and the absorbance at 540 nm was read in a plate reader. The absorbance values at any time point were so close to each other that error bars were not seen clearly on the plot. * Significant when compared to the respective zero hour controls (P<0.001). # There was no significant difference between the 2 and 10 mM glucose (P>0.05) groups

Fig. 2

a Concentration-effect curve. Glial cells were treated with MPP⁺ (0.1, 0.2 and 0.3 mM) in 2 mM glucose for 48 h. The absorbance for viability was measured, and a graph was plotted between log of MPP⁺ concentrations and the mean absorbance values by non-linear regression analysis method. b Effect of MPP⁺ on glial cell viability in 2 and 10 mM glucose after 48 h. * MPP⁺ treatments had significant effect when compared to the respective controls. # MPP⁺ treatments had significant effect on cell viability when 2 and 10 mM glucose groups were compared. Values represent the average of three independent studies (P<0.001, n = 16). c Lack of 10 mM glucose protection in glial cells treated with cocaine after 24 h. * Treatments had significant effects when compared to the respective controls (P<0.05, n = 12). # There was no significant difference between 2 and 10 mM glucose groups (P>0.05, n = 12)

Fig. 3

Effect of MPP⁺ on mitochondrial respiratory status (MRS) in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP⁺ for 48 h. Values represent the average of two independent studies (n = 6). * MPP⁺ had significant effect on MRS in comparison to the control containing 2 or 10 mM glucose (P<0.05, n = 6 One-way ANOVA, Bonferroni’s multiple comparison test). # Significant difference between MPP⁺ treatment groups with 2 and 10 mM glucose (P<0.001, n = 6, One-way ANOVA, Bonferroni’s multiple comparison test)

Fig. 4

Effect of MPP⁺ on mitochondrial membrane potential in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP⁺ for 48 h. Values represent the average of two independent studies (n = 8). * Significant effect of MPP⁺ in comparison to control groups containing 2 or 10 mM glucose (P<0.05, n = 8, One-way ANOVA, Bonferroni’s multiple comparison test). # Significant difference between MPP⁺ treatment groups with 2 and 10 mM glucose in media (P<0.001, n = 8, One-way ANOVA, Bonferroni’s multiple comparison test)

Fig. 5

Effect of MPP⁺ on GSH levels in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP⁺ for 48 h. Values represent the average of two independent studies (n = 12). * Significant in comparison to control containing 2 or 10 mM glucose in media (P<0.05, n = 12 One-way ANOVA, Bonferroni’s multiple comparison test). # Significant difference between MPP⁺ treatment groups with 2 and 10 mM glucose in media (P<0.001, n = 12, One-way ANOVA, Bonferroni’s multiple comparison test)

Fig. 6

Effect of MPP⁺ on glial cell cycle. Cells were grown in 100 × 15 mm culture dishes in DMEM with 2 mM (a) or 10 mM (b) glucose containing 10% FBS, and were treated with various concentrations of MPP⁺ for 48 h. Cells were stained by propidium iodide staining solution for 1 h in dark and analyzed by flow cytometry. Data were presented as mean ± SEM (n = 2, * P<0.05, significant in comparison to control, One-way ANOVA, Bonferroni’s multiple comparison test)