The COS was obtained via chitosan degradation using an enzyme–membrane coupling reactor system reported in previous studies with some modifications (Qin et al. 2018). Affinity (Cincinnati, OH, USA) provided the mouse monoclonal anti-p-eIF2α (Ser52), p-IRS1 (Ser307), and p-JNK (Thrl83/Tyrl85) antibodies. Cell Signaling Technology (Beverly, MA, USA) provided the anti-Bax, Bcl-2, BIP, CHOP, NF-κB (p-p65), p-AKT (Ser473), cleaved-PARP, cleaved-caspase-9, cleaved-caspase-3, and GAPDH mouse monoclonal antibodies. The qPCR reagents were purchased from Vazyme (Nanjing, China), while the Shanghai Jining Co., Ltd. (Shanghai, China) and Nanjing Jiancheng Bioengineering Institute (Nanjing, China) provided the mouse enzyme-linked immunosorbent assay (ELISA) kits. The commercial kits with standard protocols were acquired from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
The final COS product quantitation and characterization were performed using MALDI-TOF-MS (a 4700 Proteomics Analyzer; Applied Biosystems, Foster City, CA) and an HPLC system equipped with an ELSD detector (Shimadzu 20A; Shimadzu, Kyoto, Japan).
Animals and treatment
The Animal Care and Use Committee of Laboratory Animals provided research ethics approval. The specific-pathogen-free (SPF) grade C57BL/6 mice were provided by the Shanghai Jihui Experimental Animal Feeding Co., Ltd (Shanghai, China, Animal Certificate Number: 20170012004449). The mice were raised at the Chengqin Biotechnology (Shanghai) Co., Ltd (License number: SYXK (HU) 2019-0013).
The mice had free access to water and food and were divided into six groups (n = 8 per group). The standard diet (Research Diet D12450J) group (Control) was given a daily saline dose as solvent control. The remaining groups received a high-fat diet (HFD) (Research Diet D12492) containing 20% protein, 20% carbohydrate, and 60% fat. After an 8-week breeding period, the mice were inspected for glucose tolerance. Streptozotocin (STZ) dissolved in 0.1 mol/L sodium citrate–hydrochloric acid buffer (at pH = 4.5) was injected daily at a low 35 mg/kg dose over the next 5 days. The glucose levels in the blood of the mice were measured randomly after STZ injection at 3 days, 7 days, and 10 days using a glucometer. The model was established successfully when the blood sugar level exceeded 16.7 mmol/L.
Acarbose, metformin, and COS were dissolved in 0.9% NaCl, different doses of which were intragastrically administered to the intervention groups. The groups and dosages are shown in Fig. 1. All the mice were killed after 8 weeks and a fasting period of 18 h.
The insulin tolerance test (ITT) and oral glucose tolerance test (OGTT)
The ITT and OGTT were conducted at the end of the administration cycle. The mice were subjected to an overnight fasting period of 12 h, after which they were given a 2 g/kg glucose dose. The glucose levels in the blood samples obtained from the tail veins were evaluated at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min using a Roche glucometer (Germany). The ITT was performed after a one-week recovery period. The mice were fasted for 6 h, after which they received an injection of 1 unit of insulin per 1 kg into the peritoneum. The subsequently collected blood samples were used for blood glucose level assessment. The area under the curve (AUC) was measured to quantify the OGTT and ITT.
Serum and tissue collection
Clean 1.5 mL Eppendorf tubes were used to collect blood from the intra-orbital venous plexus. The blood samples were stored overnight at 4 °C and centrifuged at 106g for 15 min, after which the supernatant was collected using clean EP tubes and stored at − 80 °C. The livers, pancreas, epididymal fat, femoral skeletal muscles, hearts, spleens, and kidneys were dissected and weighed. The livers, fat, muscles, pancreas, small intestines, and spleens of each group were cryopreserved and fixed with paraformaldehyde. Additional samples were collected and stored at − 80 °C for subsequent detection.
Biochemical analysis of the serum and liver
Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the insulin and glycosylated hemoglobin levels in the serum of the mice. The homeostasis model assessment for IR (HOMA-IR) index was calculated using the following formula: fasting insulin value (μU/mL)/22.5 × fasting blood glucose value (mmol/L).
The total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels in the serum and livers of the mice were determined using commercial kits obtained from the Nanjing Institute of Bioengineering (Nanjing, China). The total protein was normalized according to the results and determined using a Bradford protein assay kit (Bio-Rad, Hercules, CA).
The glycogen content in the muscle and liver tissues was measured using glycogen determination kits acquired from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) as per the instructions of the manufacturer.
Intestinal glycosidase activity
Fresh duodenal content was collected immediately after dissection. This material was diluted at different proportions, homogenized mechanically using an ice-water bath, and centrifuged at 1300g for 10 min using a refrigerated centrifuge. The supernatant was subsequently collected, and the digestive enzyme activity was tested according to the instructions of the manufacturer.
The liver, muscle, small intestine, and pancreatic tissues obtained from each group of mice were fixed in a 10% paraformaldehyde solution, dehydrated with graded ethanol, and placed in paraffin. Then, 5-μm sections of the samples were stained with hematoxylin and eosin (HE), after which the histopathological changes were monitored using a vertical optical microscope (Nikon Eclipse CI, NIKON, Japan).
Immunohistochemical assessment of the mouse pancreas
The pancreatic paraffin sections were dewaxed and dehydrated. The tissue slices were incubated overnight with the primary IR (Servicebio, GB11334, 1:300) and glucagon resistance (Servicebio, GB13097, 1:100) antibodies at 4 °C. The tissue slices were then washed and incubated at room temperature for 1 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Servicebio, GB25303, 1:400) and Alexa Fluor Cy3-conjugated goat anti-mouse IgG (Servicebio, GB21303, 1:300). The slices were rewashed and stained with DAPI for 10 min at room temperature. A confocal fluorescence microscope (Ti-U, NIKON, Japan) was used to obtain images of the slices.
Real-time fluorescence quantitative PCR
The total RNA in the mouse tissues was acquired using an extraction RNA kit (Vazyme Biotech Co. Ltd., Nanjing, China) according to the instructions of the manufacturer, after which the absorbance was measured at 260/280 nm to determine the quantity and purity of the RNA. Then, cDNA was synthesized from the total RNA using a reverse transcription kit obtained from the Vazyme Biotech Co. Ltd. (Nanjing, China). Next, qPCR was performed using SYBR Premix Ex Taq (Vazyme Biotech Co. Ltd., Nanjing, China) according to the instructions of the manufacturer. The detected mRNA-to-GAPDH ratio was determined with GAPDH considered an internal control. Additional file 1: Table S1 provides the target gene primers.
The liver, muscle, and pancreatic samples were homogenized in a lysis buffer. A mixture of phosphate and protease inhibitors was then dissolved in the lysis buffer, after which the lysate protein concentrations were identified using a BCA protein detection kit. Then, 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used to separate an equal amount of protein, which was transferred to a polyvinylidene fluoride (PVDF) membrane obtained from GE Healthcare (Buckinghamshire, UK). This membrane was subjected to incubation for 1 h at room temperature in a blocking agent containing 5% skim milk powder or bovine albumin (BSA), after which it was further incubated at 4 °C with specific primary antibodies, such as GAPDH (1:3000 dilution), p-eIF2α (1:1000 dilution), p-JNK (1:1000 dilution), BIP (1:1000 dilution), p-Akt (1:1000 dilution), and p-IRS1 (1:1000 dilution). The membrane was then incubated at room temperature for 1 h with the secondary antibody, after which the blot was developed using a chemiluminescence (ECL) detection kit. Finally, the intensity of the bands was quantified via grayscale analysis.
Chem 3D software was used to construct the molecules, while the compound configuration was optimized via MM2 molecular mechanics. The three-dimensional protein structure is available in the RCSB Protein Data Bank (www.rcsb.org) for the proteins used in this study (PDB Codes: 3TOP, 2QMJ, 1GZO). Autodock Vina 1.1.2 was employed for semi-flexible docking, while Pymol and LigPlot were used for plotting.
GraphPad Prism 6 mapping software was used for plotting. The experimental data were expressed as mean ± standard deviation (SD). The differences between the groups were determined using one-way analysis of variance (ANOVA), Duncan’s test, and SPSS software, while p < 0.05 was deemed statistically significant.
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