Beneficial effects of exendin-4 on experimental polyneuropathy in diabetic mice

Abstract

Objective: The therapeutic potential of exendin-4, an agonist of the glucagon-like peptide-1 receptor (GLP-1R), on diabetic polyneuropathy (DPN) in streptozotocin (STZ)-induced diabetic mice was investigated.

Research design and methods: The presence of the GLP-1R in lumbar dorsal root ganglion (DRG) was evaluated by immunohistochemical analyses. DRG neurons were dissected from C57BL6/J mice and cultured with or without Schwann cell-conditioned media in the presence or absence of GLP-1 (7-37) or exendin-4. Then neurite outgrowth was determined. In animal-model experiments, mice were made diabetic by STZ administration, and after 12 weeks of diabetes, exendin-4 (10 nmol/kg) was intraperitoneally administered once daily for 4 weeks. Peripheral nerve function was determined by the current perception threshold and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood flow (SNBF) and intraepidermal nerve fiber densities (IENFDs) also were evaluated.

Results: The expression of the GLP-1R in DRG neurons was confirmed. GLP-1 (7-37) and exendin-4 significantly promoted neurite outgrowth of DRG neurons. Both GLP-1R agonists accelerated the impaired neurite outgrowth of DRG neurons cultured with Schwann cell-conditioned media that mimicked the diabetic condition. At the doses used, exendin-4 had no effect on blood glucose or HbA(1c) levels. Hypoalgesia and delayed MNCV and SNCV in diabetic mice were improved by exendin-4 without affecting the reduced SNBF. The decreased IENFDs in sole skins of diabetic mice were ameliorated by exendin-4.

Conclusions: Our findings indicate that exendin-4 ameliorates the severity of DPN, which may be achieved by its direct actions on DRG neurons and their axons.

FIG. 1.

Expression of GLP-1R in DRGs and sciatic nerves. A, C, D, and F: Immunohistochemically, GLP-1R (green) in DRG was detected with anti–GLP-1R antibody (sc-66911). Both DRG neurons, indicated by NF70 antibody (red) (E and F), and satellite glia cells, indicated by S100b antibody (red) (B and C), expressed GLP-1R. C, F, and G: Nuclei (blue) were stained with diaminido phenyl indol. A–F: White arrowheads indicate satellite glia cells. White arrows indicate neurons. G: GLP-1R protein (red) was not detected in DRG neurons of glp1r^−/− mice. H: The transcript levels of glp1r in DRGs and sciatic nerves of diabetic mice were not significantly different from those of normal mice (DRG: normal mice [N] [n = 6], 1 ± 0.54, threshold cycle value [C_t] of glp1r 34.8 ± 2.3, C_t of 18S rRNA 11.2 ± 1.0 and diabetic mice [DM] [n = 7], 0.65 ± 0.23, C_t of glp1r 34.0 ± 1.5, C_t of 18S rRNA 11.0 ± 1.4, P = 0.544; sciatic nerves: normal mice [n = 4], 1 ± 0.34, C_t of glp1r 33.6 ± 1.6, C_t of 18S rRNA 14.7 ± 1.1 and diabetic mice [n = 5], 1.95 ± 1.29, C_t of glp1r 34.5 ± 1.3, C_t of 18S rRNA 12.5 ± 2.8, P = 0.606). I: There were no significant differences in the protein levels of GLP-1R evaluated by Western blotting analyses between diabetic and normal mice (DRG: normal mice [n = 4], 1 ± 0.09 and diabetic mice [n = 4], 1.02 ± 0.20, P = 0.875; sciatic nerves: normal mice [n = 4], 1 ± 0.17 and diabetic mice [n = 4], 1.09 ± 1.35, P = 0.438). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Neurite outgrowth of DRG neurons by GLP-1 (7–37) and exendin-4 (Ex4). Representative fluorescence micrograph of DRG neurons cultured in the absence (A) or presence (B) of GLP-1 (7–37) (GLP-1) (10 nmol/L). GLP-1 (7–37) (10 nmol/L) or exendin-4 (0.1, 1, 10, and 100 nmol/L) increased the total neurite length (C) and joint number (D) of neurites. Results are means ± SD. CON, control medium. *P < 0.05 vs. control medium (n = 10–20). Control medium, joint number 23.5 ± 17.2 per cell, total length 833 ± 462 µm per cell, 10 nmol/L GLP-1 (7–37); joint number 54.8 ± 58.3, total length 2,786 ± 2,976, 0.1 nmol/L exendin-4; joint number 19.81 ± 29.59, total length 1,056.3 ± 904.5, 1 nmol/L exendin-4; joint number 32.23 ± 29.35, total length 761.9 ± 414.7, 10 nmol/L exendin-4; joint number 78.1 ± 58.4, total length 2,035 ± 1,162, 100 nmol/L exendin-4; and joint number 63.1 ± 33.8, total length 2,329 ± 1,104. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Neurite outgrowth of DRG neurons in IMS media with or without exendin-4 (Ex4). Total length (A) and joint number (B) of DRG neurons cultured in IMS media were measured. Decreased total length and joint number of DRG neurites cultured in HG were ameliorated by Ex4 in a dose-dependent fashion. Results are means ± SD. ●, Neurite cultured in HG-IMS media; ○, neurite cultured in NG-IMS media. NG-IMS media were obtained from IMS cultured in F-12 media with 5.5 mmol/L d-glucose; HG-IMS media were obtained from IMS cultured in F-12 media with 30 mmol/L d-glucose. **P < 0.005 vs. NG-IMS media without exendin-4; ††P < 0.005 vs. HG-IMS media without exendin-4. n = 10–20. Total length: NG 1,635 ± 1,014 μm per cell and HG 684 ± 410; joint number: NG 79.2 ± 52.5 per cell and HG 24.2 ± 23.8. Total length: HG with 10 nmol/L exendin-4 1,278 ± 457 and HG with 100 nmol/L exendin-4 2,045 ± 1,029. Joint number: HG with 10 nmol/L exendin-4 32.3 ± 12.0 and HG with 100 nmol/L exendin-4 55.1 ± 28.8. Total length: NG with 10 nmol/L exendin-4 1,839 ± 915 and NG with 100 nmol/L exendin-4 1,614 ± 821. Joint number: NG with 10 nmol/L exendin-4 72.7 ± 36.3 and NG with 100 nmol/L exendin-4 49.3 ± 22.3.

FIG. 4.

Evaluation of sensory nerve functions. Measurements of CPTs at 2,000 (A), 250 (B), and 5 (C) Hz by a neurometer were performed before and at the end of exendin-4 (Ex4) administration. CPTs for all pulses were significantly increased in the diabetic group (DM), and these deficits were significantly prevented by exendin-4. N, normal mice; S, saline. Results are means ± SD. #P < 0.05 vs. pretreatment normal mice; *P < 0.05 vs. saline-treated normal mice; †P < 0.05 vs. saline-treated diabetic mice. Saline-treated normal mice, n = 8; exendin-4–treated normal mice, n = 8; saline-treated diabetic mice, n = 9; exendin-4–treated diabetic mice, n = 9.

FIG. 5.

NCVs. MNCV (A) and SNCV (B) were measured before and after the treatment with exendin-4 (Ex4). DM, diabetic mice; N, normal mice; S, saline. Before the treatment: MNCV for normal mice 45.2 ± 2.7 m/s and diabetic mice 37.5 ± 8.2, P = 0.0341; SNCV for normal mice 33.0 ± 4.6 and diabetic mice 28.6 ± 7.2, P = 0.0489. After the treatment: MNCV for saline-treated diabetic mice 34.5 ± 8.1 and exendin-4–treated diabetic mice 43.3 ± 7.9, P = 0.0289; SNCV for saline-treated diabetic mice 28.6 ± 5.6 and exendin-4–treated diabetic mice 38.0 ± 8.5, P = 0.0201. Results are means ± SD. #P < 0.05 vs. pretreatment normal mice. *P < 0.05 vs. saline-treated normal mice. †P < 0.05 vs. saline-treated diabetic mice. Saline-treated normal mice, n = 7; exendin-4–treated normal mice, n = 8; saline-treated diabetic mice, n = 8; exendin-4–treated diabetic mice, n = 8.

FIG. 6.

IENFDs after the treatment with exendin-4 (Ex4). A: IENFs indicated by white arrowheads were detected by immunofluorescence assay with anti–PGP 9.5 antibody (red). B: Quantification of the density revealed a significant decrement in untreated diabetic mice and the significant amelioration by exendin-4 treatment. DM, diabetic mice; N, normal mice; S, saline. Bar: 20 μm. Saline-treated normal mice 38.5 ± 3.9, saline-treated diabetic mice 27.7 ± 1.9, exendin-4–treated normal mice 42.1 ± 2.4, and exendin-4–treated diabetic mice 40.2 ± 3.1 fibers per mm. Results are means ± SD. #P < 0.05 vs. pretreatment normal mice. *P < 0.05 vs. saline-treated normal mice. †P < 0.05 vs. saline-treated diabetic mice. Saline-treated normal mice, n = 4; exendin-4–treated normal mice, n = 3; saline-treated diabetic mice, n = 4; exendin-4–treated diabetic mice, n = 3. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

SNBF treated with or without exendin-4 (Ex4) in normal (N) or diabetic (DM) mice. S, saline. Saline-treated normal mice 19.4 ± 2.4, saline-treated diabetic mice 14.4 ± 3.6, and exendin-4–treated diabetic mice 15.2 ± 5.2 mL/min/100 g. Results are means ± SD. *P < 0.05 vs. saline-treated normal mice. Saline-treated normal mice, n = 9; exendin-4–treated normal mice, n = 8; saline-treated diabetic mice, n = 8; exendin-4–treated diabetic mice, n = 9.