2.2 Experimental procedures
2.2.1 Animals
A total of 60 weaned and specific-pathogen-free SD rats (male, 6–8 weeks, 180–190 g) were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The animal experiments were performed according to internationally followed ethical standards and approved by the research ethics committee of School of Public Health, Sun Yat-Sen University (NO. 2019-001). Rats were kept on a 12 h light/dark cycle in a temperature-controlled room maintained at (24 ± 1) ◦C with a relative humidity of (50 ± 5) % and were maintained on a standard chow diet. Rats were habituated to the conditions for 1 week before modeling.
2.2.2 Induction of diabetes
With reference to the method widely used in an abundance of literatures[16–18], rats were fed on a high-fat diet for 8 weeks and then received a single low dose of streptozotocin (STZ, 30 mg/kg) injection to induce T2DM model. Specifically, rats were randomly allocated to the blank control group (N = 20) and the model group (N = 40) according to body mass. Rats in the blank control group had a free normal diet (ND, Guangdong Medical Laboratory Animal Center, Foshan, China; contains 58% carbohydrates, 18% protein, 4.5% fats, and 4% essential vitamins and trace elements, total calorie is 3.45 kcal/g). Rats in the model group were kept on a high-fat diet (HFD, Guangdong Medical Laboratory Animal Center, Foshan, China; contains 53.6% normal chow, 15% sucrose, 10% lard, 20% protein, 1.2% cholesterol, 0.2% sodium cholate, total calorie is 4 kcal/g). After 8 weeks of feeding, rats in the model group were intraperitoneally injected with 1%STZ dissolved in 0.1M citric acid buffer (PH = 4.2–4.5) at a single dose of 30 mg/kg, while the blank control rats received same amount of 0.1M citric acid buffer injection. It’s important to note that the whole procedure of preparation and injection of STZ solution needs to avoid light and the dissolved STZ solution should be used as soon as possible (within 15 min). After STZ administration, model rats continued to eat ad libitum with the high-fat diet in order to attain relatively stable blood glucose level, then 14 days after STZ injection, random blood glucose (RBG) was measured by portable glucometer (Beijing Yicheng biotechnology co. LTD, China) through tail vein, and rats whose RBG were higher than or equal to 16.7 mmol/L were considered as diabetic[19] and were further used in the following experiment.
2.2.3 CR procedure
During high-fat diet feeding, the total food intake of rats in each cage was recorded to calculate average daily food intake per rat which turned out to be 28.54 ± 4.78 g/day. Thus, 30% CR (20 g/day) was applied in the following experiment. Diabetes model was successfully induced in a total of 29 rats after STZ administration, then according to blood glucose level, these diabetic rats were randomly assigned to the model control group eating ad libitum with high-fat diet (HFD + AL + STZ, MCT group, n = 15) or the CR intervention group feeding on high-fat diet but with 30% CR regimen as we stated above (HFD + CR + STZ, CR group, n = 14). In the meanwhile, rats in the blank control group continued to have a free normal diet (ND + AL, BCT group, n = 20). The intervention period lasted for 20 weeks. Water intake in all rats was free during the whole experiment. The whole process of this experiment was indicated in Fig. 1.
Body mass and water intake were recorded every week, while RBG and fasting blood glucose (FBG) were measured every 2 or 4 weeks respectively. To assess insulin resistance, insulin tolerance test (ITT) was conducted at baseline before modeling, after modeling, at 10 and 20 weeks post-treatment. In addition, FBG and fasting insulin (FINS) were also detected at the above indicated time points through tail vein after a 12 h overnight fasting, and insulin level was measured by ELISA operating according to the manufacturer’s instructions. Then, homeostasis model assessment of insulin resistance (HOMA-IR) was calculated with the following equation: HOMA-IR = FBG × FINS / 22.5. Intraperitoneal glucose tolerance test (IPGTT) and glucose stimulated insulin secretion (GSIS) were accomplished after modeling (before intervention), 10 and 20 weeks after intervention to evaluate glucose tolerance and islets secretion.
At the time point of 10 weeks and 20 weeks after intervention, 6 to 10 rats in each group were anesthetized by 2% pentobarbital sodium (60 mg/kg) after an overnight fasting (12 h), and then blood, liver, pancreas, skeletal muscle and white adipose tissue (WAT, specifically epididymal fat pad) were collected rapidly. Blood samples were centrifuged at 4℃,3500 rpm for 15 min to collected the serum (stored at -80℃ till use). Lipid profile was measured in the central laboratory of the First Affiliated hospital of Sun Yat-sen University.
Figure 1. Flow chart of animal study design
2.2.4 IPGTT
Rats were intraperitoneally injected with 50% glucose solution (2 g/kg) after overnight fasting for 12 h. Blood glucose was measured before injection (0 min) and 15 min, 30 min, 60 min, 90 min and 120 min after injection through tail vein with portable glucometer. Six rats form each group were randomly chose for use.
2.2.5 ITT
Rats were intraperitoneally injected with Gansulin-R (0.5 IU/kg) after fasting for 6 h. Blood glucose was measured before injection (0 min) and 15 min, 30 min, 45 min, 60 min and 90 min after injection through tail vein with portable glucometer. Six rats form each group were randomly chose for use.
2.2.6 GSIS
Rats were intraperitoneally injected with 50% glucose solution (2 g/kg) after 12 hours of fasting at night. Blood samples were collected from the tail vein before injection (0 min) and 15 min, 30 min and 60 min after injection. The blood samples were centrifuged at 3500 rpm, 4 °C for 15 minutes. The separated serum was collected and the insulin level was detected by ELISA. Six rats form each group were randomly chose for use.
2.2.7 Glucose uptake in isolated soleus
At the end of the experiment, glucose uptake in isolated soleus was measured in 4 rats from each group. The effect of CR in glucose uptake was detected with reference to the method described in previous study with slightly modifications[20–22]. Generally, the isolated fresh soleus was immediately rinsed with Krebs-Ringer buffer, removed the muscle fascia, tendons and attached connective tissue as far as possible. Subsequently, the muscle tissue was placed into the brain slice mold (Reward, China) to cut it into thin slices of similar size with a blade, then divided into three parts (roughly the same weight) and put into 2 ml ep tubes separately. The muscle tissue was then incubated with 1.5 ml oxygenated glucose-free Krebs-Hensleit buffer (KRH), containing 0.1% bovine serum albumin (BSA) and 10 mU/ml insulin (Gansulin-R) at 37 °C, 300 rpm (shaking) for 60 min to deplete intercellular glucose. After that, the liquid was removed, 1.5 ml oxygenated KRH (containing 11.1 mM glucose, 0.1%BSA, and 40 mM mannitol) with 0, 10 or 20 mU/ml insulin was added in. In the meantime, another tube contains equal volume of oxygenated KRH but no muscle tissue served as blank control. All the four tubes were incubated at 37 °C, 300 rpm (shaking) for 30 min. A 1 mL aliquot was collected from each incubation tube before and after the incubation, and the glucose concentration was determined. Glucose uptake of muscle was calculated as the amount of glucose (mg) taken up per gram of muscle tissue using the following formula: Muscle glucose uptake = (GCb-GCa)/ muscle tissue weight, where GCb and GCa are glucose concentrations before and after incubation, respectively.
2.2.8 Histological assessment
Liver and pancreas tissues from 3 rats in each group were randomly selected for histological evaluation. After fixation in 10% formalin overnight at 4 °C, one portion of liver and pancreas tissue from each rat were embedded in paraffin blocks, sliced at 10 µm thickness, and stained with hematoxylin and eosin (H&E) for histological studies. Besides, another portion of them were fixed in 4% paraformaldehyde overnight at 4 °C and embedded in OCT for frozen section, sliced at 10 µm thickness, then stained with Oil Red O and counterstained with hematoxylin to determine hepatic and pancreatic lipid accumulation. Photomicrographs were taken with a digital camera (Nikon Eclipse Ci-L, Japan).
2.2.9 Immunohistochemistry
Pancreas tissues from 3 rats in each group were randomly selected for insulin immunohistochemistry. After fixation in 10% formalin overnight at 4 °C, pancreas tissue samples were embedded in paraffin blocks, sliced at 10 µm thickness. Antigen retrieval was carried out in EDTA buffer (pH 9.0) by heating to 99 °C for 15 min. Subsequently, deparaffinized sections were incubated with 3% H2O2 for 25 min at room temperature to block endogenous peroxidase activity and then rinsed three times with phosphate-buffered saline (PBS). After blocking with 3% BSA for 30 min at room temperature, each section was incubated with anti-insulin antibody (1:600, Servicebio, China) in a wet box at 4 °C overnight. Thereafter, the sections were rinsed three times with PBS, and then incubated with horseradish peroxidase-conjugated secondary antibody (1:200, Servicebio, China) for 50 min at room temperature. After washing with PBS for three times, the sections were stained with a 3,3′-diaminobenzidine solution and then counterstained with haematoxylin for 3 min. Photomicrographs were taken with a digital camera (Nikon Eclipse Ci-L, Japan).
2.2.10 Immunofluorescence
Skeletal muscle from 3 rats in each group were randomly selected for GLUT4 immunofluorescence experiment. Briefly, after fixation in 4% paraformaldehyde overnight at 4 °C, followed equilibrated muscle tissues in 20% sucrose overnight until the tissues settle to bottom at 4 °C, then transferred them into 30% sucrose overnight till they settle to bottom at 4 °C, and next they were embedded in OCT for frozen section, sliced at 10 µm thickness. Subsequently, frozen sections were rinsed gently with 4 °C PBS for three times, fixed with 4 °C 4% PFA for 30 min at room temperature and then washed three times with PBS again. After incubation with 0.2% Trition-X100 in PBS for 20 min, sections were blocked with 3%BSA for 30 min at room temperature and then incubated overnight with rabbit anti-GLUT4 antibody (1:250, Abcam, ab654) at 4 °C in a wet box. After washing with Tris-buffered saline containing 0.1% Tween 20, the sections were co-incubated with donkey secondary antibody conjugated with Alexa Fluor® 568 (1:400, Invitrogen, A-11011) for 3 h at room temperature. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, 1 µg/ml, Sigma-Aldrich, USA). All photomicrographs were taken with an inverted fluorescence microscope (Nikon Eclipse Ti-E, Japan).
2.2.11 Western blotting
Total protein extracts of skeletal muscle and epididymal fat pad tissues were obtained by homogenizing and lysing with RIPA buffer (Beyotime, China), and then centrifuging at 14000 g for 5 min at 4 °C. The protein concentration was measured with a BCA assay kit (Beyotime, China). An equal amount of protein (20 µg) was loaded in each lane. Proteins were separated using sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and electrically transferred to a polyvinylidene difluoride membrane (Millipore). After blocking the membrane with 5% skim milk, target proteins were immunodetected using specific antibodies. After incubation with the secondary antibody, bands were visualized using an ECL plus kit (ThermoFisher, USA) and exposed to autoradiographic films according to the manufacturer’s instruction. The intensities of bands were performed using Image J 1.52q software.