Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model

Abstract

Although diabetic polyneuropathy (DPN) is a frequent diabetic complication, no effective therapeutic approach has been established. Glucagon is a crucial hormone for glucose homeostasis but has pleiotropic effects, including neuroprotective effects in the central nervous system. However, the importance of glucagon in the peripheral nervous system (PNS) has not been clarified. Here, we hypothesized that glucagon might have a neuroprotective function in the PNS. The immortalized rat dorsal root ganglion (DRG) neuronal cell line 50B11 was treated with methylglyoxal (MG) to mimic an in vitro DPN model. The cells were cultured with or without glucagon or MG. Neurotoxicity, survival, apoptosis, neurite projection, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were examined. Glucagon had no cytotoxicity and rescued the cells from neurotoxicity. Cell survival was increased by glucagon. The ratio of apoptotic cells, which was increased by MG, was reduced by glucagon. Neurite outgrowth was accelerated in glucagon-treated cells. Cyclic AMP and PKA accumulated in the cells after glucagon stimulation. In conclusion, glucagon protected the DRG neuronal cells from MG-induced cellular stress. The cAMP/PKA pathway may have significant roles in those protective effects of glucagon. Glucagon may be a potential target for the treatment of DPN.

Keywords: 50B11; PKA; apoptosis; cAMP; diabetic polyneuropathy; glucagon; methylglyoxal; peripheral neuronal cell.

Figure 1

Cytotoxicity assay: (A) After 6 h of treatment with glucagon or forskolin, no significant cytotoxicity was identified. (B,C) Although 0.1 and 0.5 mmol/L methylglyoxal (MG) exhibited significant cytotoxicity in the cells, glucagon (1, 100 pmol/L) and forskolin (25 µmol/L) significantly attenuated cytotoxicity. # p < 0.001 versus control without MG; ** p < 0.005 versus control, * p < 0.05 versus control. Ctrl: control, Gcg: glucagon, MG: methylglyoxal, Fsk: forskolin, mM: mmol/L, pM: pmol/L.

Figure 2

Cell survival assay: (A–C) MG significantly reduced the survival of dorsal root ganglion (DRG) neuronal cells. Glucagon and forskolin increased the cell survival which was reduced by MG. # p < 0.05 versus control without MG, glucagon, or forskolin; ## p < 0.001 versus control without MG, glucagon, or forskolin; * p < 0.01 and ** p < 0.001 versus control. n = 3 in each group. Error bars: standard deviation. Ctrl: control, Gcg: glucagon, MG: methylglyoxal, Fsk: forskolin, mM: mmol/L, pM: pmol/L.

Figure 3

Apoptosis estimation: (A) Apoptotic cells were stained with purple dye. Many apoptotic cells were observed in the cells treated with 0.5 mM MG (control). However, the number of apoptotic cells was low in cells treated with glucagon or forskolin. Scale bar: 200 µm. (B) The percentage of apoptosis was significantly reduced in the cells treated with glucagon or forskolin. # p < 0.05 versus MG (−); * p < 0.05 versus MG treatment without glucagon or forskolin (control). n = 3 in each group. Error bars: standard deviation. Gcg: glucagon, MG: methylglyoxal, Fsk: forskolin, pM: pmol/L, mM: mmol/L.

Figure 4

Mitochondrial reactive oxygen species (ROS) measurement: (A) The fluorescence images of MitoSOX™ (red) showed mitochondrial ROS production of neuronal cells with or without glucagon or forskolin in the presence or absence of MG. Blue: DAPI (nuclei). Scale bar: 200 μm. (B) The fluorescence intensity quantified by ImageJ software. The production of ROS by MG was significantly reduced in the cells treated with glucagon or forskolin. # p < 0.05 versus MG (−); * p < 0.05 versus control with MG; n = 3 in each group. Error bars: standard deviation, Gcg: glucagon, MG: methylglyoxal, Fsk: forskolin, mM: mmol/L, pM: pmol/L.

Figure 5

Neurite outgrowth: (A) Light microscopic photography showed the neurite projection in DRG neuronal cells with or without glucagon and forskolin. Arrowheads indicate cells with neurite. Scale bar: 100 μm. (B) The percentage of neurite positive neurons was higher in the cells treated with glucagon and forskolin. ** p < 0.001 versus control; * p < 0.05 versus control; n = 3 in each condition; Error bars: standard deviation. Gcg: glucagon, MG: methylglyoxal, Fsk: forskolin, pM: pmol/L.

Figure 6

Cyclic adenosine monophosphate (cAMP) assay: Accumulation of cAMP was found in the cells treated with 1 pmol/L glucagon or 10 μmol/L forskolin. * p < 0.05 versus control, ** p < 0.001 versus control; Error bars: standard deviation. Gcg: glucagon, Fsk: forskolin.

Figure 7

Protein kinase A (PKA) detection: (A) The activity of PKA significantly increased in the cells treated with 1 or 100 pmol/L of glucagon. (B) When the cells were treated with glucagon in the presence of the PKA inhibitor H89, no significant increase in PKA was observed. * p < 0.05 versus control, ** p < 0.01 versus control, Error bars: standard deviation. Gcg: glucagon, Fsk: forskolin, pM: pmol/L.