Animals and diet
Protocol approved by the Institutional Animal Care and Use Committee of Hanyang University was used for all animal experiments (HY-IACUC-11-059). Six week old male Wistar rats (Orient, Gyeonggi-do, Korea) were housed in individual ventilated in an air-conditioned room maintained at 22±2°C with a 12 h light–dark cycle. After one week of acclimatization, twenty rats were intraperitonially injected with STZ (70 mg/kg body weight in 0.05 M citrate buffer; Sigma Co, St. Louis, MO, USA) once, and then blood glucose concentration was measured to confirm the development of diabetes mellitus. After 3 days of STZ injection, twenty STZ-induced diabetic rats and five normal rats without STX injection were fed with an AIN-93M diet (Research Diets, New Brunswick, NJ, USA) and water ad libitum for 6 weeks, and the body weight and food intake were recorded every week.
Sorghum (Sorghum bicolor L. Moench cv. Hwanggeumchal-susu) was grown at the Department of Functional Crops, National Institute of Crop Science, Rural Development Administration, Milyang, Korea during the 2010 growing season. Voucher herbarium specimens were deposited with the reference number (KNICS-579) in the Herbarium of the Department of Functional Crops. The botanical identification was made by Dr. Ill-Min Chung of Konkuk University. Sorghum was finely ground using a Pin-type Mill (DK-201, Sejung Tech, Daegu, Korea) and extracted using 80% fermented ethanol at room temperature for 24 h while shaking (WiseCube WIS-RL010, Daihan Scientific Co., Ltd., Seoul, Korea). The extracts were centrifuged at 5,000 g for 30 min and the supernatants were filtered through Advantec 2 filter paper (Advantec Toyo Kaisha Ltd., Tokyo, Japan). The filtrates were evaporated using a rotary evaporator (Eyela N-1000, Tokyo Rikakikai Co., Tokyo, Japan) at 40°C, and then freeze-dried in vacuum (FDT-8612, OPERON, Kimpo, Korea). The dried extract was stored at −20°C.
During a six-week period of diet, normal control rats (NC) were orally administrated 0.1 mL of saline, and twenty STZ-induced diabetic rats were randomly divided into four groups that orally administrated either 0.1 mL of saline (DM) or 0.1 mL of saline with a 0.4 g/kg body weight of SE (DM-SE 0.4), 0.6 g/kg body weight of SE (DM-SE 0.6) or 0.7 mg/ kg body weight of glibenclamide (DM-G) using gavage (n = 5 per group).
Intraperitoneal glucose tolerance tests (IPGTT) were performed by intraperitoneal injection of 25% glucose (2 g/kg body weight) on the last day of the experiment after an overnight fast. Blood samples were collected from the tail vein at 30, 60, and 120 min after injection.
At the end of the experimental period, all rats were anesthetized with an intraperitoneal injection of tiletamine (25 mg/kg), zolazepam (25 mg/kg), and xylazine (10 mg/kg) after overnight fasting. Blood was collected into SST tubes (BD Vacutainer, Franklin Lakes, NJ, USA) and centrifuged at 3000 g for 15 min (HA 1000–3, Hanil Sciences Industrial CO. Ltd., Incheon, Korea). Organs and adipose tissues were harvested, rinsed with saline and then weighed. Serum and tissue samples were stored at −80°C.
Glucose concentration during IPGTT was determined with AccuCheck (Roche Diagnostics, Indianapolis, IN, USA). The values of the area under the glucose time curve (AUC) were calculated using the glucose levels at each time point during IPGTT. The serum levels of triglycerides, total- and HDL-cholesterol, glucose, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were determined using a commercially available kit (Asan Pharm., Hwaseong, Korea) with a spectrophotometer (DU 600, Beckman Coulter, Inc., Indianapolis, IN, USA). LDL-cholesterol concentration was calculated using the Friedwald formula. The serum insulin level was determined using an ultra-sensitive rat insulin enzyme linked immunosorbent assay kit (Crystal Chem, Downers Grove, IL, USA) with a microplate reader (iMark, Bio-Rad Laboratories, Hercules, CA, USA).
Liver and skeletal muscles were homogenized in a 0.8 ml ice-cold lysis buffer (20 mM HEPES, 0.25 M sucrose, 0.5 mM EDTA, 2 mM dithiothreitol, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1mM Na3VO4, pH 7.5). The homogenates were centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was centrifuged at 20,000 g for 1 h at 4°C to obtain cytosolic fraction. For the membrane fraction of skeletal muscle, the pellets were re-suspended in 250 μl of lysis buffer and 1% (v/v) triton X-100, incubated on ice for 30 min, and centrifuged at 200,000 g for 30 min at 4°C. The protein concentrations were determined using a Bradford assay with bovine serum albumin (Bio-Rad, Hercules, CA, USA) as the standard. Equal amounts of protein (30μg) from liver or skeletal muscle was separated on 8% SDS-PAGE and transferred to a polyvinylidine fluoride membrane (0.45 μm, Immobilon-P transfer membrane, Millipore, USA). After blocking, the membranes were incubated overnight with a primary antibody for PEPCK (1:1,000, Cell Signaling Technology, Beverly, MA, USA), p38 (1:1,000, Cell Signaling Technology, Beverly, MA, USA), phospho p-38 (1:500, Cell Signaling Technology, Beverly, MA, USA), AMPK (1:1,000, Cell Signaling Technology, Beverly, MA, USA), phospho AMPK (1:500, Cell Signaling Technology, Beverly, MA, USA), GLUT4 (1:2000, abCam, Cambridge, UK), Akt (1:1,000, Cell Signaling Technology, Beverly, MA, USA), or phospho-Akt (1:500, Cell Signaling Technology, Beverly, MA, USA) in Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk at 4°C. After washing in TBST, the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit or mouse IgG (1:5,000, Cell Signaling Technology, Beverly, MA, USA) for 1 h. The immunoreactive signals were developed using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Piscataway, NJ, USA) and exposed to Kodak film. The relative and normalized protein expression was calculated by β-action (1:1,000, BD Transduction Laboratories, NJ, USA).
All data are expressed as the mean ± standard error of the mean (SEM). Statistical differences among the groups were calculated by the analysis of variance (ANOVA) followed by Duncan’s multiple range test (SPSS 18.0 version., Chicago, IL, USA). Differences with p < 0.05 were considered significant.