The experimental rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China), and housed in the laboratory animal centre of Ruijin hospital, which is affiliated to the medical college of Shanghai Jiao Tong University. The temperature was maintained between 22–24°C, and rats were subjected to a 12/12 h light/ dark cycle (lights on at 07:00 am) and provided food (Shanghai SLAC company, China) and water ad libitum. The animal study was approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China) and was conducted in accordance with the ethical principles governing animal welfare, rearing, and experimentation.
Dexamethasone treatment and experimental method
The basal body weight and fasting blood glucose (FBG) values of 35 male Wistar rats (250–300 g) were measured after one week of adaptive feeding. The experimental rats were randomly divided into 2 groups, 20 in the dexamethasone treatment group (SDM), and 15 in the control group (CTL). SDM rats were injected intraperitoneally with dexamethasone (Sigma–Aldrich, USA) (10 mg/kg) daily(27), and 5 were used for orbital blood collection, 5 for glucose tolerance tests, and 10 for PET/CT imaging. CTL rats were intraperitoneally injected with a corresponding dose of normal saline daily, and 5 were used for orbital blood collection, 5 for glucose tolerance tests, and 5 for PET/CT imaging. In the SDM and CTL groups, changes in body weight and FBG were monitored every other day. Orbital blood collection, glucose tolerance tests, and PET imaging were performed on days 0, 3, 7, 11, and 15 of the experimental time frame.
The basal body weight and FBG values of 10 male GK rats, spontaneous T2DM rats (100–150 g), were measured after one week of adaptive feeding with ordinary feed, and the first imaging was performed. The administration of the high-fat diet increased the disease process, and changes in body weight and blood glucose were monitored once a week, and small animal imaging was performed every 1-2 months.
FBG, serum insulin, and intraperitoneal glucose tolerance test
After 12 h fasting, blood was drawn from the tail tip of the rats, and their FBG was measured using a Bayan automatic blood glucose meter(28) (Contour TM TS, Bayer, Germany). Similarly, blood was collected from the orbits of the rats, under anesthesia(29). The blood was centrifuged at 2000 × g at 4°C for 20 min, before the supernatant was collected and stored at -80℃. Serum insulin was detected with a rat insulin ELISA kits (30) (Rat Insulin ELISA Kit, Crystal Chem, USA).
Intraperitoneal glucose tolerance test: after overnight fasting, the rats were given a glucose load (1 g/kg) by intraperitoneal injection of a 50% glucose solution. The blood glucose was measured at 0, 30, 60, 90, and 120 min following glucose loading. For statistical analyses, the blood glucose change curves were plotted and the area under the curve (AUC) calculated(31).
Micro PET/CT imaging
After 12 h overnight fasting, rats were anaesthetized with 3% pentobarbital sodium (Sigma-Aldrich, USA) (30 mg/kg) by intraperitoneal injection. The tail vein was injected with 18F-FDG at a dose of approximately 7.4 MBq. After 30 min, rats were fixed in the prone position on the center of a Micro PET/CT (Inveon MM Platform, Siemens Preclinical Solutions, Knoxville, Tennessee, USA) scan bed field of view (FOV), and scanned under anesthesia. The PET/CT equipment has a resolution of 1.5 mm, an aperture of 5.7 cm, and an axial FOV of 8.5 cm. The micro PET/CT equipment acquisition workstation was Inveon Acquisition Workplace (IAW) 220.127.116.11. A new workflow was established before data acquisition, including CT Acquisition, Reconstruction, PET Acquisition, PET Histogram, and PET Reconstruction. The static scan data was collected under the conditions of 80 kV voltage, 500 μA current, and 1100 ms exposure for 10 min, and then PET data was collected. The collected data was reconstructed with IAW software through attenuation correction, and the three-dimensional ordered subsets maximization algorithm (OSEM3D) was used to reconstruct the coronal, transverse, and sagittal tomographic images for analysis. The reconstructed images were obtained using Siemens Inveon Research Workplace (IRW) 3.0 to obtain 3D Regions of Interest (ROI). In this study, the skeletal muscle comprised the upper limb epitrochlearis muscle(32), and the upper right portion of the liver was utilized. Finally, the %ID/g max value of the ROI was obtained for quantitative analysis(33).
RNA extraction and cDNA synthesis from muscle were performed using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, China Da Lian), respectively, according to the manufacturers’ protocols. Total RNA (500 ng) was amplified on a StepOne Fast Real-Time PCR System (Thermo Fisher Scientific), using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, China Da Lian) for real-time PCR after cDNA synthesis. The standard curve for quantification was derived as per a modified version of a previously described method. Fold change of the gene expression was calculated by 2−ΔCt relative to the internal reference gene (glyceraldehyde3-phosphate dehydrogenase, GAPDH). The primer sequences used are as follows: GLUT4: forward 5′-GGGCTGTGAGTGAGTGCTTTC-3′, reverse 5′- CAGCGAGGCAAGGCTAGA-3′; insulin receptor substrate 1 (IRS-1): forward 5′-ATGTGGAAATGGCTCGGA-3′, reverse 5′-TAAGGCAGCAAAGGGTAGGC-3′; Phosphatidylinositol 3-kinase (PI3K)-p85α: forward 5′-TTAAACGCGAAGGCAACGA-3′, reverse 5′-CAGTCTCCTCCTGCTGTCGAT-3′;GAPDH: forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
After the rats were euthanized, the excised pancreatic tissue and epitrochlearis muscle of the upper limbs were immersed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 48 h. Tissues were washed in 70% ethanol, embedded in paraffin, and sectioned onto glass slides. The slides were dewaxed with xylene, dehydrated with ethanol, and washed with PBS. The slides were then incubated in a hydrogen peroxide blocking solution for 10 min at 18-25℃ to block endogenous peroxidase activity. Anti-masking/epitope retrieval of the antigen was performed by high pressure heating of 1 mMol Tris-EDTA (pH 9.0). Slides were incubated in a protein blocking solution (Sigma-Aldrich, USA), before the pancreatic tissue was tested for insulin (1:100, ab7842, Abcam, CA, USA) and glucagon (1:200, ab10988, Abcam, CA, USA). Skeletal muscle was tested for GLUT4 (1:500, ab654, Abcam, CA, USA), IRS-1 (1:100, ab52167, Abcam, CA, USA), and PI3Kp85α (1:100, ab182651, Abcam, CA, USA). After overnight incubation at 4°C, the slides were immunostained with secondary antibodies from EnVision reagent (HRP/rabbit and mouse, Dako, K5007 kit, Denmark). DAB was added and allowed to develop under microscope observation until brown staining was visible. Slides were counterstained with hematoxylin (Sigma-Aldrich, USA). Representative slides of insulin and glucagon were used to quantify the mass and islet area of islet β and α cells.
Results are presented as mean ± standard error of the mean (unless otherwise stated). Statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). *, statistical significance where P<0.05; **, statistical significance where P<0.01; ***, statistical significance where P<0.001; ****, statistical significance where P<0.0001.