C57BL/KsJ-db/db and normal non-diabetic mice (C57BL/KsJ-db/+ or +/+) 7 weeks of age were supplied by Japan SLC (Japan). The mice were housed one animal per cage during the experiment in a room with controlled temperature (23 ± 2℃), humidity (55 ± 10%), and light (08:00 a.m.~20:00 p.m.). The animals were fed a commercial laboratory chow for mice (CJ Cheiljedang, Korea) and water ad libitum. The mice were divided into four groups according to the treatment received: the DB group (C57BL/KsJ-db/db mice, n = 12), ND group (C57BL/KsJ-db/+ or +/+ mice, n = 12), DB-SF group (C57BL/KsJ-db/db mice, n = 12), and ND-SF group (C57BL/KsJ-db/+ or +/+ mice, n = 12). The DB-SF and ND-SF groups were given a 20% SFH solution prepared in water for 6 weeks. The animals were sacrificed at 14 weeks of age. The mice were allowed to acclimate to SFH treatment for 1 week before the experiments by receiving increasing concentrations in drinking water. This study was conducted in accordance with the Institutional Animal Care and Use Committees of Hallym University (Korea).

The raw cocoons were purchased from Sindobiosilk (Korea). SFH was prepared from the cocoons of Bombyx mori (Chinese silkworm). Briefly, serine and other impurities were removed from raw cocoons (1 kg) with 5% Na₂CO₃. Silk fibroin was treated with 2 N HCl at 100℃ for 48 h and neutralized using a 2N NaOH solution. The silk fibroin solution was mixed with chymotrypsin (Sigma, USA) and allowed to undergo proteolysis [16]. Low-molecular weight silk fibroin was acquired through a desalting process using ion exchange chromatography as previously described [16]. Amino acid composition of the SFH made by mentioned above (Table 1) was determined with an amino acid analyzer (Pico-Tag System and Waters 486/600/626; Waters, USA).

Body weights of all mice were measured with an animal balance (Mettler Toledo, USA) before and every week during the experiments. Blood samples (9:00 a.m.) were collected from the infraorbital plexus of the mice while under ether anesthesia. The plasma was obtained by centrifugation and frozen until assayed for insulin contents. Thirty micro liter of whole blood was added to the test strip and blood glucose was measured with an enzymatic dry-chemistry method (Reflotron dry-chemistry analyzer; F. Hoffmann-La Roche, Switzerland). A radioimmunoassay (RIA) was used to measure plasma insulin concentrations using a commercial RIA kit (Linco Research, USA). Absorbance at 450 nm and 590 nm was measured in a microtiter plate reader (Spectramax L; Molecular Devices, USA) within 5 min.

After 6 weeks of SFH treatment, an intraperitoneal glucose tolerance test (IPGTT) was performed 3 days before the mice were sacrificed. Mice were fasted for 12 h and then 2 g/kg of a D-glucose (Sigma, USA) solution was administered intraperitoneally. Blood samples were obtained prior to, immediately after, and 30, 60, and 120 min after glucose injection. Plasma glucose and insulin levels were measured as described above. Total area under the curve (AUC) during the course of the IPGTT was calculated according to the trapezoid rule [13].

After 6 weeks of SFH treatment, whole pancreatic tissue was obtained from each mouse. Pancreatic tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4-µm sections with an automatic rotary microtome (2050 Supercut microtome; Reichert-Jung, Germany). The sections were incubated overnight with rabbit antibodies against pancreatic insulin (1 : 300) and glucagon (1 : 300) (Dako, Denmark) at 4℃. The sections were then incubated with peroxidase-conjugated secondary antibodies (Vectastain ABC; Vector Laboratories, USA) and developed with 3, 3'-diaminobenzidine.

Islet size and β cell mass were determined as previously described [2,6,20]. To measure islet size, a 40× magnification field was chosen at random. The islets in this field and those in the immediately adjacent fields were examined until 20 islets had been evaluated. Each of these 20 islets was evaluated to determine the total islet cross-sectional area. The percentage of the insulin-positive area (insulin immunoreactive area per µm² of pancreatic epithelium in the sections) was measured by computer-assisted stereological morphometry using an Olympus BH-2 microscope (Olympus, Japan) connected to a Hewlett-Packard (USA) computer with Image-Pro Plus software (Media Cybernetics, USA). Fibrotic tissue outside the pancreatic lobules was excluded from the measurements. Pancreatic β cell mass was calculated by multiplying the percentage of insulin-positive areas by the weight of the corresponding pancreatic tissues.

Results are presented as the mean ± SE. To make comparisons between two groups, an unpaired Student's t-test was used. For multiple comparisons, a one-way ANOVA was performed by GraphPad Prism 4 (GraphPad Software, USA). p-values less than 0.05 were considered statistically significant.

In order to determine whether SFH can decrease blood glucose levels in db/db mice, SFH was administered to the mice for 6 weeks via drinking water. High blood glucose concentrations (18.05 ± 3.59 mmol/L) of the db/db mice gradually decreased throughout the treatment period. After 6 weeks of SFH treatment, blood glucose levels (8.83 ± 3.35 mmol/L) of the db/db animals decreased to that of the normal mice (Fig. 1A). In contrast, blood glucose levels of untreated db/db mice gradually increased from 22.94 ± 2.92 mmol/L to 32.95 ± 6.59 mmol/L within 6 weeks. SFH did not have any hypoglycemic effect on the normal mice as expected.

Throughout the experimental period, SFH treatment did not induce changes of body weight in either normal or db/db mice (Fig. 1B), indicating that SFH treatment did not have a marked nutritional effect. Glycosylated hemoglobin (HbA1c) levels of the SFH-treated db/db mice were significantly lower compared to those of the untreated db/db mice (6.55 ± 0.76 vs. 10.11 ± 1.51%, p < 0.01), indicating that glucose levels in SFH-treated db/db mice were maintained at near normal levels throughout the treatment period (Fig. 1C). Taken together, these results suggest that SFH had an anti-diabetic effect on the db/db mice.

To verify whether SFH improves glucose tolerance, we performed an IPGTT on db/db mice that had been administered SFH for 6 weeks. Blood glucose levels of the SFH-treated db/db mice were significantly lower at 30, 60, and 120 min after glucose injection compared to the untreated db/db mice (Fig. 2A), suggesting that SFH improves glucose tolerance. Plasma insulin levels of SFH-treated db/db mice were higher at 30, 60, and 120 min after glucose injection compared to the untreated db/db mice (Fig. 2B). Results from the IPGTT (Fig. 2C) showed that the AUC for glucose levels in SFH-treated db/db mice was significantly less than that of untreated db/db mice (2,751.2 ± 756.7 vs. 4,538.2 ± 425.9 mmol/L × min, respectively; p < 0.01). In addition, the AUC for insulin levels of the SFH-treated db/db mice (Fig. 2C) was significantly larger than that of untreated db/db mice (609.2 ± 197.6 vs. 292.7 ± 2.65 pmol/L × min, respectively; p < 0.01), indicating that SFH improved pancreatic β cell function. Taken together, these results suggest that SFH improved pancreatic β cell function and glucose tolerance of the db/db mice.

Since SFH treatment had a dramatic anti-diabetic effect on db/db mice, we examined histological changes in the pancreas. Pancreatic islets of the control mice (ND) contained well-granulated β cells with normal insulin immunoreactivity and peripherally located α cells (Fig. 3A and D). In comparison, insulin immunoreactivity was weak and glucagon-immunoreactive cells were observed in the center of the islets from the pancreas of control db/db mice (Fig. 3B and E). Islets from SFH-treated db/db mice had intense insulin immunoreactivity and glucagon-immunoreactive cells were redistributed at the islet periphery (Fig. 3C and F).

In order to determine how SFH increased insulin production in the db/db mice, pancreatic β cell volume was measured by morphometric analysis. Results of the analyses showed that islet density and β cell mass in the SFH-treated db/db mice were significantly increased compared to untreated db/db mice (Table 2). In addition, α cell volume was significantly decreased in the SFH-treated db/db mice relative to the untreated db/db mice (Table 2). These immunohistochemical observations were closely correlated to our biochemical measurements demonstrating the anti-diabetic effects of SFH, suggesting that this compound helped maintain pancreatic β cell integrity.