Materials/Chemicals

The cell culture medium RPMI-1640, fetal bovine serum (FBS), penicillin-streptomycin solution (dual antibody; BL505A), and β-mercaptoethanol (M301574) were purchased from VivaCell (Shanghai, China), Biosharp (Hefei, China), and Aladdin (Shanghai, China). D-(+)-Glucose (≥ 99.5%), and ATP (D7378) were purchased from Beyotime (Shanghai, China). Irisin (HY-P70664) and ML385 (HY-100,523) were purchased from MCE (NJ, USA). Streptozotocin (STD) was purchased from Solarbio (Beijing, China). The DCFH-DA Reactive oxygen ROS fluorescent probe was purchased from Meilunbio® (Dalian, China). Carboxyl -H2DCFDA (C400) and Carboxyl -DCFDA were purchased from Invitrogen (USA).

ELISA kits for insulin (INS) (CSB-E05071 m), interleukin 18 (IL-18; CSB-E04609 m), and IL-1β (CSB-E08054 m) were purchased from CUSABIO (Wuhan, China). The Caspase 1 Activity Assay Kit (C1101), Glucagon ELISA Kit, and hematoxylin-eosin (HE) staining kit were purchased from Beyotime. The kits for detecting malondialdehyde (MDA; A003-4-1) and superoxide dismutase (SOD; A001-3-2) were purchased from Nanjing Jiancheng Bioengineering Research Institute (China). PrimeScript™ RT Kit was purchased from Qiagen (Germany).

Anti-GSDMD N-Terminal (DF13758), anti-GSDMD (20770-1-AP), anti-Nrf2 (GB113808), anti-FNDC5 (23995-1-AP), anti-β-actin (AB0035), anti-NLRP3 (DF7438), anti-ASC (DF6304), anti-Lamin B1 (12232-1-AP), anti-TXNIP (18243-1-AP), anti-TrX1 (AF7577), anti-DAPI (G1012), anti-Insulin (GB13121), anti-glucagon (GB11097), anti-caspase1 (af5418), anti-Cleaved-Caspase 1 (Asp296), and p20 (AF4005) were purchased from Proteintech (Wuhan, China), Abways (Beijing, China), Servicebio (Wuhan, China), and Affinity Biosciences (Jiangsu, China).

T2DM pancreatic β-cell model

The mouse islet β-cells Min6 (FH0390) were purchased from FuHeng (Shanghai, China). The complete growth medium for Min6 consisted of 89% RPMI-1640, 10% FBS, 1% dual antibody, and 0.05 mM β-mercaptoethanol. To induce the T2DM cell model, the Min6 cells were exposed to a high glucose (HG; 25 mmol/L) condition for 24 h. Then, ELISA was performed to evaluate the effectiveness of the modeling process.

Cell group

To investigate the effect of irisin, Min6 cells were divided into six groups, including the Control group, HG group, HG + PBS group, HG + irisin group, HG + irisin + ATP group, and HG + irisin + ML385 group. The cells in the Control group were cultured in a normal medium supplemented with PBS, the cells in the HG group were cultured in the HG medium supplemented with PBS, and the cells in the HG + irisin group were cultured in the HG medium supplemented with 100 ng/mL irisin. The processing time was 24 h. The cells in the ATP treatment group were additionally treated with 3 mM ATP for 45 min. The cells in the ML385 treatment group were treated with 2 μM ML385 for 30 min.

T2DM mouse model and collection of biological indicators

Animal experiments were conducted following protocols approved by the First People’s Hospital of Yunnan Province, the Affiliated Hospital of Kunming University of Science and Technology. Male C57BL/6J mice were purchased from Bei You Biotechnology. All mice were raised in metabolic cages at 22 °C, and a 12-h/12-h light-dark cycle was maintained.

The mice were divided into five groups at random (N = 6 mice/group): the Control group, Model group, Model + PBS group, Model + Irisin group, and Model + Irisin + ML385 group. A high-fat diet (HFD, 45% kcal, MS1607, Shanghai Maokang Biotechnology Co., Ltd., China) plus low-dose STD was used to construct the T2DM mouse model. When the mice were five weeks old, they were fed HFD consisting of 45 kcal% for an additional four weeks. The mice induced with insulin resistance were given a single intraperitoneal injection of 50 mg/kg STZ. The fasting blood glucose level (FBG) equal to or greater than 11.1 mmol/L was classified as diabetic [23]. The diabetic mice in the Model + Irisin group were administered a peritoneal injection of irisin (0.25 μg/kg/day) for 21 days. The diabetic mice in the Model + Irisin + ML385 group were administered a peritoneal injection of irisin (0.25 μg/kg/day) along with ML385 (30 mg/kg/day) for 21 days. In the control group, mice were given a basal diet and administered an intraperitoneal injection of an equal amount of 0.1 mmol/L sodium citrate buffer. For ML385 treatment, mice were intraperitoneally injected with ML385 (30 mg/kg/day) for three weeks. Blood samples were collected from the tail vein of the mice for further analysis.

The oral glucose tolerance test (OGTT) of the mice was performed at the end of the intervention. Briefly, after the mice were starved for 15 h and fed glucose via oral gavage (2 g/kg), their fasting plasma insulin (FINS) and FBG (at 0, 30, 60, and 120 min) levels were measured using ELISA kits. The levels were evaluated using the following equations: HOMA-β = 20×FINS/(FBG-3.5), HOMA-IR = FBG× FINS/22.5.

The mice were euthanized by carbon dioxide (CO2) asphyxiation. Their pancreas was isolated by collagenase digestion of the pancreas and snap-frozen at − 80 °C for subsequent assays. Some of the pancreatic tissues were fixed in 4% paraformaldehyde and subsequently processed to form paraffin samples. These samples were pre-cut to a thickness of 6 μm using a slicer (RM2016, Leica, Germany).

Western blotting analysis

The Min6 cells were washed with PBS to remove the culture medium, and then, they were lysed with a RIPA lysis buffer on ice for 8 min. The pancreatic tissues were initially snipped and then subjected to lytic ultrasound treatment. The protein concentration was assessed by adding BCA solution. The protein samples, which were denatured in advance, were electrophoretically separated on polyacrylamide gel. Following this, the proteins were electrically transferred onto a nitrocellulose membrane. The membranes were washed and blocked at room temperature for 1 h using non-fat powdered milk. They were incubated with primary antibodies overnight at 4 °C. The following day, they were incubated with secondary antibodies at room temperature for 1 h. Finally, color reaction was performed using the pre-mixed ECL luminescent substrate.

Caspase 1 activity

The cells were collected after incubation for 48 h. The enzyme activity of caspase 1 was measured using a Caspase 1 Activity Assay Kit following the manufacturer’s instructions. Briefly, the cells were mixed with lysate on ice to be cleaved for 15 min and then incubated with Ac-YVAD-pNA (2 mM) at 37 °C for 80 min. The OD value was recorded at 405 nm using a spectrophotometer (1510, Thermo Fisher).

ELISA

The cell supernatants were collected after incubation for 48 h. The level of insulin and the secretion of IL-1β and IL-18 in the cell supernatants or mouse serum samples were measured using an ELISA kit following the manufacturer’s instructions. Briefly, the diluted cell supernatant was introduced into the bottom of the well coated with monoclonal antibodies and left to incubate at 37 °C for 1 h. Subsequently, the enzyme-labeled solution was incubated for another 1 h. The color reaction was conducted for 10 min, and it was terminated using a stop solution. The OD value was recorded at 450 nm using a spectrophotometer (1510, Thermo Fisher).

Oxidative stress assays

The cells were collected and incubated with 5 μM DCFH-DA probe, which was diluted in serum-free medium, in a wet box for 30 min at 37 °C. The images were captured by the THUNDER Imager (Leica, Germany).

For pancreatic tissues, 100 μg of total protein was incubated at 37 °C with either oxidation-sensitive carboxy-H2DCFDA (C400) or oxidation-insensitive carboxy-DCFDA (C369); the final concentration of both was 25 μM. The measurements were recorded at 0 and 30 min using a fluorescent enzyme-labeled apparatus with excitation at 485 nm and emission at 530 nm. The data were expressed as multiples of the C400/C369 ratio. The cell lysate or mouse serum samples were used to determine the MDA and SOD levels using specific reagent kits, and the OD values were read at 530 nm and 450 nm, respectively.

Immunofluorescence staining

The sections of cells and pancreatic tissues first underwent antigen repair with EDTA (pH 9.0) for 27 min, and then, they were sealed in goat serum for 30 min. The sections were blocked and added with diluted antibodies, and subsequently, incubated in a wet box at 4 °C overnight. The following day, the corresponding fluorescently labeled secondary antibodies were added respectively and incubated for another 50 min in the wet box. DAPI was used to re-stain the nucleus. The fluorescence intensity of GSDMD (Green) was measured to assess the level of pyroptosis. The fluorescence intensity of insulin and glucagon was measured to assess pancreatic function. The distribution of insulin (red) and glucagon (green) was observed by fluorescence microscopy. The β-cells are the source of insulin, and α cells are the source of glucagon. Immunohistochemical double-labeling of caspase-1 and islet β-cells was performed.

Histological staining

The sections of the pancreas were stained using the H&E staining kit. Briefly, the samples were incubated with hematoxylin for 4 min, followed by the application of eosin solution for 30 s. The images were captured using an inverted microscope (CKX3-SLP, Olympus).

Statistical analysis

The data were analyzed using GraphPad Prism version 8.0. All experiments were performed with at least three replicates. The outcomes were presented as the mean ± SD, and the statistical differences were assessed by Student’s t-test or ANOVA. Image J was used for the semi-quantitative analysis. All results were considered to be statistically significant at P < 0.05.