Streptozotocin (STZ) was obtained from Sigma (St. Louis, MO, USA). Blood glucose test strip and glucometer were delivered from Roche (Changsha, Hunan, China). Primary antibodies anti-phosphor-Ser 9 GSK3β (ab131097), GSK3β (ab32391) and β-actin (ab8226) were purchased from Abcam (Cambridge, UK). Anti-phosphor-Tyr607 PI3K (#17366), PI3K (#4228), phosphor-Ser308 AKT (#4060), and AKT (#9272) were from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Pra-C was purchased from Shanghai PureOne Biotechnology (Shanghai, China) with 98% of purity. All chemicals and reagents used were of standard biochemical quality.
Animals And Establishment Of Type 2 Diabetic Model
Male C57BL/6 mice aged 5–7 weeks were got from the Experimental Animal Center of the Air Force Military Medical University (Xi’an, China). Mice were fed under standard laboratory conditions (12 h light/12 h dark, temperature 22–26 °C, air humidity 55–60%) with water and mice chow ad libitum. The experimental procedures were approved by the Institutional Animal Care and Use Committee of The Fourth Military Medical University, and are complied with the Protection of Animals and Principles of laboratory animal care" (NIH publication No. 86 − 23, revised 1985). The number of animals used and their suffering were greatly minimized. To induce T2DM model, mice got HFD (60% kcal fat, Ke’ao Biotech, Xi’an, China) feeding for 4 weeks, a single dose of STZ (60 mg/kg, dissolved in 0.1 M citrate buffer) was intraperitoneally injected after 12 h fasting, while other control mice were given regular food and same volume of 0.1 M citrate buffer injection. Model mice then continuously fed with HFD for 1 week and got blood glucose test by single tail tip pricks. HFD fed mice with fasting blood glucose levels over 13 mM were considered the successful establishment of T2DM. Blood glucose levels were measured every 2 weeks. All mice were allowed to acclimate for 24 h of laboratory environment before any room change.
After establishment of T2DM model induction, mice were randomly divided into 2 groups, T2DM and Pra-C treatment, and were continuously fed with HFD. Pra-C was dissolved in 0.9% saline and administrated intraperitoneally (i.p., 3 mg/kg/d) at 9 a.m. once a day for 4 weeks from 5th week, while T2DM group mice got equal volume of saline. Pra-C dose was selected base on previous study . Control mice were fed with conventional chow. Behavior tests were performed 3 weeks after treatment. Fasting blood glucose and body weight were detected before final sacrifice.
New Objection Recognition (NOR) Test
NOR test was performed in a sound-proof polyvinyl chloride box (25 × 25 cm bottom, 25 cm height) with a digital camera on the roof. On Day 1, single mouse was placed in the box to acclimate the new environment for 10 min. On Day 2, mouse was put into box with two cylinders (5 cm in diameter, 5 cm height) fixed in symmetrical corner of the box (training trial). On Day 3, every group was equally divided into two groups randomly. For the new object recognition, one cylinder was replaced with a cone (5 cm in diameter, 5 cm height) (testing trial). For the object-place recognition test, the former cylinders were fixed in parallel corners with one in the former location as the training trial and the other in a different location. In both training and testing trials, mice freely explored for 10 min, and time spent exploring each object or location was recorded and scored by software (Jiliang, Shanghai, China). After each single experiment, the box and objects were wiped with 75% ethanol. The proportion of exploration time on new object or new object-location was defined as the “Descrimination index” expressed by the ration of (TN-TF)/(TN + TF) (TN: time spent on exploring the new object or location; TF: time spent on exploring the familiar object or location).
Morris Water Maze (MWM) Test
MWM test was conducted in a circular tank (120 cm in diameter and 50 cm deep) with white wall decorated by different bold marks in four directions. An escape platform (10 cm in the diameter) was erected in the midway position between the center and wall of the tank. Before experiment, opaque water (24 ± 1 °C, with unharmed white coating) was filled in the tank to a height 1.0 cm above the platform. Mice were put into water and trained for four trials (different starting positions) per day with 10 min inter-trial intervals for four consecutive days. During each trial, mice were allowed to explore until they found the hidden platform and held on the platform for 20 s before returning to a holding cage. Mice failed to find the platform in 60 s were guided to the platform by a wooden stick. Probe test was performed 24 h after the final training trial, mice were put into the same height water filled tank with undersurface hidden platform for 90 s and their swim path was recorded.
Western Blot Analysis
Western blot analysis was conducted as described previously . After behavior tests and blood glucose test, mice were anesthetized with urethane (1.5 g/kg) and then rapidly decapitated. Brain was immediately placed on ice; hippocampus and hypothalamus were removed, respectively collected in 1.5 mL tubes and stored at -80 ℃ until further analysis. Tissues were homogenized by RIPA lysis buffer and separated by centrifugation at 13,000 rpm for 20 min at 4 °C, and supernatant was collected. All proteins were quantified by BCA protein assay Kit (Pierce Biotech, USA) and equal amounts (30 µg) of protein were subjected to western blot analysis. Samples were separated via 10% SDS-PAGE gel and electrotransferred onto PVDF membranes (Millipore, USA), which were blocked with primary antibodies at 4 °C overnight, washed 3 times with Tris-phosphate buffer containing 0.05% Tween 20 (TBST) for 10 min of each time and were further incubated with HRP-conjugated secondary antibodies. Bands were developed using enhanced chemiluminescence detection (Genshare Biological, China) and imaged by Tanon imaging system (Tanon 4200, China).
Field Electrophysiological Recording
Hippocampal slices from mice were prepared as previously described . In short, slices (300 µm) were cut in oxygenated solution (in mM): 250 Sucrose, 2.5 KCl, 0.5 CaCl2, 6 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, and 10 D-glucose. After cutting, the slices recovered at 34 °C in ACSF (in mM) as follows: 124 NaCl, 4.4 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 NaHCO3, and 10 D-glucose. After 10 min, the slices were placed in ACSF at room temperature for an additional 1–2 h, gassed with 95% O2-5% CO2. A commercial MED64 recording system (Panasonic Alpha-Med Sciences, Osaka, Japan) was used to record field excitatory postsynaptic potentials (fEPSPs), and the procedure was similar to previous study . Briefly, single hippocampal slice was placed in a probe (MED-P515A, 8 × 8 array; interpolar distance, 150 µm), positioned on the Schaffer collateral-CA1 pathway of the dorsal hippocampus, with oxygenated ACSF (30.0 °C, 1–2 ml/min) perfusion. After a recovery period of at least 1 h for the slices in the recording chamber, biphasic constant-current pulse stimulation (0.2 ms) was applied to the stimulation channel to evoke the fEPSPs in the channels closest to the stimulation site. Stable baseline responses were recorded for at least 40 min, L-LTP was induced by a strong theta burst stimulation (TBS, 5 bursts at 5 Hz, repeated 5 times at 10 s intervals, four pulses at 100 Hz for each burst) , then test stimulus was repeatedly given once per minute for at least 3 h. The fEPSPs were measured and analysed using Mobius software (Panasonic Alpha-Med Sciences, Tokyo, Japan).
All data were presented as mean ± SEM. The statistical significance of differences between groups were analyzed with One-way analysis of variance (ANOVA) followed by LSD and S-N-K(s) t-tests. In all cases, the criterion for statistical significance was p < 0.05.