Glucagon-Like Peptide-1 Receptor Agonist Protects Dorsal Root Ganglion Neurons against Oxidative Insult

Abstract

Objective: Diabetic polyneuropathy (DPN) is one of the most prevalent diabetic complications. We previously demonstrated that exendin-4 (Ex4), a glucagon-like peptide-1 receptor agonist (GLP-1RA), has beneficial effects in animal models of DPN. We hypothesized that GLP-1 signaling would protect neurons of the peripheral nervous system from oxidative insult in DPN. Here, the therapeutic potential of GLP-1RAs on DPN was investigated in depth using the cellular oxidative insult model applied to the dorsal root ganglion (DRG) neuronal cell line.

Research design and methods: Immortalized DRG neuronal 50B11 cells were cultured with and without hydrogen peroxide in the presence or absence of Ex4 or GLP-1(7-37). Cytotoxicity and viability were determined using a lactate dehydrogenase assay and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt), respectively. Antioxidant enzyme activity was evaluated using a superoxide dismutase assay. Alteration of neuronal characteristics of 50B11 cells induced by GLP-1RAs was evaluated with immunocytochemistry utilizing antibodies for transient receptor potential vanilloid subfamily member 1, substance P, and calcitonin gene-related peptide. Cell proliferation and apoptosis were also examined by ethynyl deoxyuridine incorporation assay and APOPercentage dye, respectively. The neurite projection ratio induced by treatment with GLP-1RAs was counted. Intracellular activation of adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling was also quantified after treatment with GLP-1RAs.

Results: Neither Ex4 nor GLP-1(7-37) demonstrated cytotoxicity in the cells. An MTS assay revealed that GLP-1RAs amended impaired cell viability induced by oxidative insult in 50B11 cells. GLP-1RAs activated superoxide dismutase. GLP-1RAs induced no alteration of the distribution pattern in neuronal markers. Ex4 rescued the cells from oxidative insult-induced apoptosis. GLP-1RAs suppressed proliferation and promoted neurite projections. No GLP-1RAs induced an accumulation of cAMP.

Conclusions: Our findings indicate that GLP-1RAs have neuroprotective potential which is achieved by their direct actions on DRG neurons. Beneficial effects of GLP-1RAs on DPN could be related to these direct actions on DRG neurons.

Figure 1

Cell cytotoxicity of GLP-1 receptor agonists (GLP-1RAs) in dorsal root ganglion (DRG) neurons. Cytotoxicity was determined 24 hours after treatment with GLP-1RAs or forskolin using LDH assay. No significant difference was detected between neurons treated with GLP-1RAs or forskolin and those without treatment (control). Concentrations of GLP-1RAs; exendin-4: 0.1, 1, 10, and 100 nM; GLP-1: 1, 10 nM. Ctrl: control; Fskln: forskolin; error bar: standard deviation. n = 3 in each group.

Figure 2

Distribution of sensory neuronal markers in the dorsal root ganglion (DRG) neuron cell line treated with exendin-4. Pictures on the left side are neurons without any treatment. Pictures on the right side are neurons treated with 100 nM exendin-4 for 36 hours. TRPV1: red (a), substance P: green (b), CGRP: green: DAPI (c), scale 100 μm.

Figure 3

Cell viability in dorsal root ganglion (DRG) neurons treated with GLP-1 receptor agonists. Cell viability was quantified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS). Although hydrogen peroxide (H₂O₂) significantly decreased the cell viability of DRG neurons, GLP-1 receptor agonists, exendin-4 and GLP-1, and forskolin, an activator of adenylate cyclase, prevent the reduction of cell viability induced by H₂O₂. White bar: no supplementation of H₂O₂; hatched bar: 0.01 mM H₂O₂; filled bar: 0.1 mM H₂O₂; Ctrl: control; H₂O₂: hydrogen peroxide; ^# p < 0.01 versus control without H₂O₂; ^∗ p < 0.05 versus control with 0.1 mM H₂O₂; ^∗∗ p < 0.005 versus control with 0.1 mM H₂O₂; error bar: standard deviation. n = 3 in each group.

Figure 4

Superoxide dismutase- (SOD-) like activity in dorsal root ganglion (DRG) neurons treated with glucagon-like peptide-1 (GLP-1) receptor agonists. Oxidative insult induced by 30-minute treatment with 0.1 mM hydrogen peroxide increased SOD-like activity in the neurons supplemented with 10 nM GLP-1 or 100 nM exendin-4. ^∗ p < 0.001 versus no treated cell with H₂O₂. H₂O₂: hydrogen peroxide; Ctrl: control; GLP-1: cells supplemented with 10 nM GLP-1; Ex-4: cells supplemented with 100 nM exendin-4. Error bar means standard deviation. n = 6 or 7 in each group.

Figure 5

Apoptosis in dorsal root ganglion (DRG) neurons treated with exendin-4. Apoptosis induced by 3-hour treatment with 0.1 mM hydrogen peroxide was partially inhibited in the neurons supplemented with 100 nM exendin-4 or 10 μM forskolin. ^∗ p < 0.05 versus control; H₂O₂: hydrogen peroxide; PC: positive control of apoptosis; GLP-1: glucagon-like peptide-1; Ex-4: exendin-4. Error bar means standard deviation. n = 8 in each group.

Figure 6

Proliferation rate of dorsal root ganglion (DRG) neurons treated with GLP-1 receptor agonists. Proliferation rate assessed by EdU assay revealed that both GLP-1 receptor agonists, exendin-4 and GLP-1, suppressed proliferation of DRG neurons. Ctrl: control; Ex-4: cells supplemented with 100 nM exendin-4; GLP-1: cells supplemented with 10 nM GLP-1; ^∗ p < 0.05 versus control; error bar: standard deviation. n = 9 in each group.

Figure 7

Neurite outgrowth of dorsal root ganglion (DRG) neurons. The ratio of neurite-positive neurons increased in cells supplemented with GLP-1 receptor agonists, exendin-4 and GLP-1, as well as cells which were supplemented with forskolin. Ctrl: control; Ex-4: cells supplemented with 100 nM exendin-4; GLP-1: cells supplemented with 10 nM GLP-1; forskolin: cells supplemented with 10 nM forskolin; ^∗∗ p < 0.001 versus control; error bar: standard deviation. n = 9 or 15 in each group.

Figure 8

Intracellular cyclic adenylate monophosphate (cAMP) accumulation in neurons treated with GLP-1 receptor agonists. The cAMP accumulation was measured 20 minutes after exposure to 100 nM exendin-4, 10 nM GLP-1, or 10 μM forskolin. Both GLP-1 receptor agonists, exendin-4 and GLP-1, provoked no significant cAMP accumulation. Ex-4: cells supplemented with 100 nM exendin-4; ^∗∗ p < 0.001 versus control; error bar: standard deviation. n = 5 or 6 in each group.