VVE was supplied by Korea Hanlim Co. (Seoul, Korea) and prepared as follows. After collecting ripe grapes, the seeds were separated from the pulp and shade-dried. Then the seeds were crushed into a powder, and the dried, powdered, seed material (1 kg) was extracted with 300 mL distilled water and 200 mL acetone. Then the extract was filtered and concentrated to a volume of 100 to 150 mL. Chloroform was added and the filtrates were pooled and evaporated below 50℃ in a vacuum drier to yield a final preparation of 3.5 g.

Male 4-week-old C57BL/6J mice were purchased from Charles River Laboratories (Tokyo, Japan) and used as reported previously [10]. Throughout the study, the mice were kept in a pathogen-free animal facility with a 12/12-hour light/dark cycle. The temperature and humidity in the room were maintained at 23℃ ± 1℃ and 53% ± 2%, respectively. The animal care protocol and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School.

For the first week (prestudy), the mice were provided with a pellet diet (Research Diets, New Brunswick, NJ, USA) for circumstantial adaptation. They were randomized into four groups: normal diet (ND), HD, HD with 100 mg/kg VVE (VVE100), and HD with 250 mg/kg VVE (VVE250). For the 12 weeks of the study, mice were fed an experimental AIN-93 modified diet (Table 1). VVE was dissolved in drinking water and administered orally at a regular time once per day. The ND and HD groups without VVE were allowed free access to the same amount of distilled water. Body weight, blood glucose, and food intake were measured at the outset and every following week, and blood glucose levels were measured via tail vein samples using Precision Xtra Plus (Abbott Laboratories, MediSense Products, Bedford, MA, USA). Serum triglyceride and cholesterol levels were measured using a commercial kit (Asan Pharmaceutical Co., Seoul, Korea). After 12 weeks of VVE treatment, an oral glucose tolerance test was performed as follows. After 12 hours of fasting, an oral glucose challenge with 50% glucose fluid (1.0 g/kg) was performed and blood samples (5 µL) were collected from the tail at 0, 30 minutes, and 1, 2, and 3 hours. On the final day of the experiments, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and a 3 mm punch skin biopsy was performed on the dorsum of the foot for quantification of innervation.

For immunohistochemical analysis of frozen microtome sections, skin tissues were fixed with periodate-lysine-paraformaldehyde (2% paraformaldehyde, 0.075 M lysine, 0.05 M phosphate buffer, pH 7.4, 0.01 M sodium m-periodate) solution for 24 hours. After thoroughly rinsing in phosphate-buffered saline (PBS) containing 20% glycerol and 0.1 M phosphate buffer for 48 hours at 4℃, tissues were cryoprotected with Tissue-Tec OCT compound (Miles, Elkhart, IN, USA). Sections 40 µm thick were cut perpendicular to the dermis on a sliding microtome (Leica CM 1510, Leica Microsystems AG, Wetzlar, Germany) and immersed in PBS for 15 minutes at room temperature. Then the samples were transferred to microcentrifuge tubes containing Dako Protein Block Serum-Free (Dako, Carpinteria, CA, USA) supplemented with 3% goat serum as blocking buffer. After blocking for 30 minutes on a shaker table at room temperature, sections were washed twice for 10 minutes with PBS and incubated with primary antibody against rabbit protein-gene-product 9.5 (Biogenesis, Poole, UK) at a dilution of 1:100 at 4℃ overnight. The antibodies were diluted in antibody diluent (Dako) supplemented with 1% goat serum. After thorough washing, secondary antibody (goat antirabbit IgG-FITC 1:200, Vector, Peterborough, UK) was loaded for 1 hour at room temperature in a dark room. After washing with PBS as before, sections were placed on slides and mounted with fluorescent mounting medium (Dako). Images were collected and analyzed using a Carl Zeiss Axioskop2 plus microscope (Carl Zeiss, Goettingen, Germany) and Axiovision 5.1 software. All biopsy slides were coded and mixed with a set of normal slides, and examiners were blinded to the code information.

Data are presented as means ± SD. One-way ANOVA with a Duncan's post hoc test was used to compare experimental groups. Differences were considered statistically significant if the p value was less than 0.05. Statistical analysis was performed using the SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA).

Mice were fed experimental diets (Table 1). Body weight changes according to energy intake were significantly greater in the HD diet groups. However, the weight change in the HD with VVE groups was less than in the HD alone group (Table 2). Serum lipid levels were also significantly different according to diet and administration of VVE (Table 3). However, VVE did not significantly inf luence weight change or lipid lowering, according to ANCOVA results (p > 0.05). Random blood glucose level was significantly increased in the HD group compared to the ND, but there was no significant difference between HD and HD + VVE, regardless of dose (Fig. 1A). Oral glucose challenge tests were carried out after 12 hours of fasting (Fig. 1B). We observed differences in blood glucose levels between the HD groups and the ND group (p < 0.05) but the trend towards VVE-dependent differences was not statistically significant.

The mean value of IENF density (number/mm) was 40.95 ± 5.40 in the ND mice and 28.70 ± 6.37 in the HD mice (p < 0.05). When VVE was administered, the mean value of IENF density in the HD + VVE mice was significantly higher: HD with VVE 100 mg/kg (41.14 ± 1.12) and HD with VVE 250 mg/kg (48.98 ± 7.01; p < 0.05) (Fig. 1C). The morphological findings of IENF in each group are depicted in Fig. 1D, showing an increased and less degenerated pattern of peripheral nerve fibers in the VVE treatment groups.