Neuroprotective effect of the glucagon-like peptide-1 receptor agonist, synthetic exendin-4, in streptozotocin-induced diabetic rats

Abstract

Background and purpose: Glucagon-like peptide-1 (GLP-1) receptors are widely expressed in neural tissues and diminish neuronal degeneration or induce neuronal differentiation. The aim of this study was to investigate the effect of the GLP-1 pathway on peripheral nerves in streptozotocin-induced diabetic rats.

Experimental approach: Diabetic and nondiabetic rats were treated with the GLP-1 receptor agonist, synthetic exendin-4 (i.p., 1 nmol·kg(-1)·day(-1)) or placebo for 24 weeks, and current perception threshold values, cAMP levels and nerve fibre size in the sciatic nerve were measured. We also investigated GLP-1 receptor expression, quantitative changes in PGP9.5-positive intraepidermal nerve fibres and cleaved caspase 3-stained Schwann cells by immunohistochemistry.

Key results: GLP-1 receptor expression was detected in the sciatic nerve and skin. After exendin-4 treatment, the increase seen in current perception threshold values at 2000 and 250 Hz in diabetic rats was reduced. Also, the decrease in myelinated fibre size or axon/fibre area ratio in the sciatic nerve and the loss of intraepidermal nerve fibre in the skin of diabetic rats were ameliorated. These responses were closely associated with the attenuation of Schwann cell apoptosis and improvement in the cAMP level in exendin-4-treated diabetic rats, compared with placebo-treated animals.

Conclusion and implications: Synthetic exendin-4 may prevent peripheral nerve degeneration induced by diabetes in an animal model, supporting the hypothesis that GLP-1 may be useful in peripheral neuropathy. The neuroprotection is probably attributable to GLP-1 receptor activation, antiapoptotic effects and restoration of cAMP content.

Figure 1

Changes in oral glucose tolerance test (OGTT) in nondiabetic and diabetic rats with or without exendin-4. Data are expressed as mean ± SEM for six to eight animals. NOR = vehicle-treated nondiabetic group; DM = vehicle-treated diabetic group; EXE-4 = treated with 1 nmol·kg⁻¹·day⁻¹ exendin-4. ##P < 0.01 vs. nondiabetic rats; *P < 0.05 versus vehicle treated diabetic rats.

Figure 2

Effects of exendin-4 treatment on current perception thresholds at frequencies of 2000 and 250 HZ in diabetic rats over 24 weeks. Data are expressed as mean ± SEM. NOR = vehicle-treated nondiabetic group; DM = vehicle-treated diabetic group; EXE-4 = treated with 1 nmol·kg⁻¹·day⁻¹ exendin-4. *P < 0.05 vs. vehicle-treated diabetic rats; ^$P < 0.05 week 24 versus week 0 in vehicle-treated diabetic group.

Figure 3

Morphology of sciatic nerve cross-sections stained with toluidine blue in nondiabetic (A and B) and diabetic rats (C and D) treated with (B and D) or without exendin-4 (A and C). Exendin-4 treatment prevented the reduction in calibre of myelinated fibres in diabetic rats. Bar = 20 µm.

Figure 4

(A) Representative microphotographs of PGP9.5-immunoreactive intraepidermal nerve fibres in the dorsum and toes of nondiabetic and diabetic rats with or without exendin-4. Skin 40-µm sections were collected in week 24 from six to eight rats per group. (B and C) Bar and line graphs of epidermal innervation in the dorsum (B) and toe (C) in weeks 0, 8, 16 and/or 24. NOR = vehicle-treated nondiabetic group; DM = vehicle-treated diabetic group; EXE-4 = treated with 1 nmol·kg⁻¹·day⁻¹ exendin-4. #P < 0.05 DM versus NOR; *P < 0.05 and **P < 0.01 DM + EXE-4 versus DM. Bar = 100 µm.

Figure 5

(A and C) Expression of GLP-1 receptors in sciatic nerve and skin in week 24. GLP-1 receptor was expressed predominantly in epidermis (arrowhead) and around skin hair follicles (arrow) of the skin. (B and D) No immunoreactivity was observed in the non-immune serum controls for sciatic nerve and skin. Sections of 10 µm were stained with rabbit anti-GLP-1 receptor or normal rabbit serum. Bar = 100 µm.

Figure 6

(A) Double-labelling fluorescence of sciatic nerves in nondiabetic and diabetic rats with or without exendin-4. Arrows indicate the nuclei of apoptotic Schwann cells that were dual-stained by anti-cleaved caspase 3 (green) and DAPI (blue-white). (B) Bar graph shows cleaved caspase 3-stained Schwann cells as a percentage of total number of DAPI-stained cells. NOR = vehicle treated nondiabetic group; DM = vehicle-treated diabetic group; EXE-4 = treated with 1 nmol·kg⁻¹·day⁻¹ exendin-4. #P < 0.05 DM versus NOR; *P < 0.05 DM + EXE-4 versus DM. Bar = 20 µm.

Figure 7

Effects of exendin-4 treatment on cAMP levels in sciatic nerves of nondiabetic and diabetic rats. Data are expressed as mean ± SEM. NOR = vehicle-treated nondiabetic group; DM = vehicle-treated diabetic group; EXE-4 = treated with 1 nmol·kg⁻¹·day⁻¹ exendin-4. #P < 0.05 DM versus NOR; *P < 0.05 DM + EXE-4 versus DM.