Metformin Preserves Peripheral Nerve Damage with Comparable Effects to Alpha Lipoic Acid in Streptozotocin/High-Fat Diet Induced Diabetic Rats

Abstract

Background: Metformin is widely marketed medication for the treatment of diabetes, but its pharmacological effect on diabetic peripheral neuropathy remains unclear. In this study, the effect of metformin on peripheral nerves in diabetic rats was investigated using diverse neuronal parameters of nerve fibers.

Methods: Rats were assigned to one of four groups (n=7 to 10 per group): normal, diabetes mellitus (DM), DM+metformin (100 mg/kg), and DM+alpha lipoic acid (ALA, 100 mg/kg). DM was induced by streptozotocin/high-fat diet (STZ/HFD). After 12 weeks, the sensory thresholds to mechanical and heat stimuli were assessed. Repeated sensory tests, immunofluorescence microscopic comparison of peripheral nerves, and biochemical blood analysis were performed after 24 weeks.

Results: Both DM+metformin and DM+ALA groups showed similar trends to diverse sensory tests at 24 weeks compared to DM group although the degree of change were different according to the stimulated senses. There was no significant difference in the comparison of the intraepidermal nerve fiber density (IENFD) of peripheral nerves between the DM+metformin and DM+ALA groups (11.83±0.07 fibers/mm vs. 12.37±1.82 fibers/mm, respectively). Both groups showed preserved IENFD significantly compared with DM group (8.46±1.98 fibers/mm, P<0.05). Sciatic nerve morphology of the experimental animals showed a similar trend to the IENFD, with respect to axonal diameter, myelin sheath thickness, and myelinated fiber diameter.

Conclusion: Metformin has beneficial pharmacological effects on the preservation of peripheral nerves in diabetic rats and its effects are comparable to those of ALA.

Keywords: Diabetes mellitus; Metformin; Peripheral nerves; Thioctic acid; Vitamin B12.

Fig. 1. (A) Body weight, (B) blood glucose levels, and (C) glycosylated hemoglobin levels in the experimental groups. Body weight was increased gradually in all experimental groups, but diabetes mellitus (DM)+alpha lipoic acid (ALA) group showed less increased trend compared with other groups although significant differences were not present. Glucose levels were significant higher in DM groups than normal group; however, DM+metformin group showed lower glucose level significantly compared with other DM group. Data are presented as mean±standard deviation (n=7 to 10 per group). ^aP<0.05 vs. normal controls, ^bP<0.05 vs. DM (n=7 to 10 per group).

Fig. 2. Sensory test using (A) Von Frey filament, (B) hot plate, (C) tail flick, and (D) Randall-Selitto responses in the experimental groups. Mechanical allodynia, threshold to hot sense, and pressure response were more sensitive in diabetes mellitus (DM) group during experimental period and these responses were partly blunted by metformin or alpha lipoic acid (ALA) treatment although there were no significant differences between DM+metformin and DM+ALA groups. Data are presented as mean±standard deviation (n=7 to 10 per group). ^aP<0.05 vs. normal controls, ^bP<0.05 vs. DM (n=7 to 10 per group).

Fig. 3. Intraepidermal nerve fiber density (IENFD) according to the time passage (A) and metformin (MET) or alpha lipoic acid (ALA) treatment (B) in experimental groups and immunohistochemistry images (C) of the cutaneous small nerve fibers in experimental groups (a: normal; b: diabetes mellitus [DM]; c: DM+MET; d: DM+ALA). Arrows indicate immunostained small nerve fiber. Both DM+MET and DM+ALA groups showed less reduced IENFD irrespective of experimental period compared with DM group and significant differences were not present between DM+MET and DM+ALA groups. Data are presented as mean±standard deviation (n=7 to 10 per group). ^aP0.05 vs. normal controls, ^bP<0.05 vs. DM (n=7 to 10 per group).

Fig. 4. The comparison of sciatic nerves in the respect of (A) axon diameter, (B) myelin sheath thickness, and (C) myelinated fiber diameter of experimental groups. There were no significant differences in the three morphometric parameters between diabetes mellitus (DM)+metformin and DM+alpha lipoic acid (ALA) groups. Data are presented as mean±standard deviation (n=7 to 10 per group). ^aP0.05 vs. normal controls, ^bP<0.05 vs. DM (n=7 to 10 per group).

Fig. 5. Stained morphological findings of sciatic nerves (×1,000) using toluidine blue in the experimental groups. (A) Normal, (B) diabetes mellitus (DM), (C) DM+metformin (MET), (D) DM+alpha lipoic acid (ALA).

Fig. 6. Blood levels of (A) vitamin B12, (B) glutathione, and (C) tumor necrosis factor α (TNF-α) in the experimental groups at 24 weeks. Vitamin B12 and glutathione levels were not different among experimental groups; however, TNF-α was most high in diabetes mellitus (DM) group compared with other experimental groups and DM+metformin or DM+alpha lipoic acid (ALA) groups showed reduced trend significantly compared with DM group. Data are presented as mean±standard deviation (n=7 to 10 per group). ^aP0.05 vs. normal controls, ^bP<0.05 vs. DM (n=7 to 10 per group).