Animal study

The Ethics Committee on the Use and Care of Animals at Xi’an Jiaotong University approved the study protocol. The animals (male C57BL/6J mice, aged 7–8 weeks, weighing 18–22 g) received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. The mice were randomized into 2 groups, fed by a high-fat diet (HFD) or normal diet for 8 weeks. HFD (60% fat, 20% protein and 20% carbohydrates) purchased from Jiangsu Synergy Co., Ltd. Streptozotocin (STZ, 60 mg/kg body weight; Sigma-Aldrich, USA) was then intraperitoneally injected to HFD-fed mice (for 3 consecutive days, 60 mg/kg body weight) to further induce T2DM model. After following fasting for 12 h, animals with fasting blood glucose (FBG) levels ranging from 15 to 25 mmol/L measured by Ruidian Performa glucometer (Shenzheng Ruidian Technology Co., Ltd, Shenzheng, China). 3 days after STZ injection were considered diabetic. Subsequently, all T2DM mice and the normal mice were randomly divided into 3 subgroups as follows: (1) normal diet group (control group); (2) HFD diet model (T2DM model group) (3) liraglutide (Lira, Novo Alle, DK-2880 Bagsvaerd, Denmark) intervention group or ferrostatin-1 (Fer-1, MedChemExpress, Shanghai, China) intervention group. Lira (300 mg/kg/day) was administrated three times a week for 6 weeks. Fer-1 group was intraperitoneal injected with ferrostatin-1 at 1 mg/kg daily for 6 weeks. Meanwhile, the mice in the control group were received injection of equivalent volume of normal saline for 6 weeks. All animals were maintained under standardized conditions (consistent temperature 22 ± 1 °C, relative humidity 50 ± 10%, and 12 h light/dark cycle). Animals were provided with a corresponding diet and allowed free access to tap water. Body weight (BW) and FBG were measured weekly.

Glucose tolerance test (IPGTT and insulin tolerance test (ITT)

Mice were injected intraperitoneally with glucose (2 g/kg body weight) when the mice were fasted for 12 h at the 5th week during the drug administration. Blood samples were taken by tail prick at 0, 30, 60, and 120 min for the measurement of blood glucose levels using glucometer. Area under the curve (AUC) of IPTGG was calculated. ITT was carried out in the next day. The mice which were fasted 6 h were injected with insulin (0.75 U/kg body weight) (Jiangsu Wanbang Biochemistry Medicine Co. Ltd., Xuzhou, China). Glucose was measured at 0, 15, 30, 60 and 120 min after insulin injection. Area under the curve (AUC) of ITT was calculated.

Serum analyses

The serum level of total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to instructions of reagent kits (Nanjing jiancheng Bioengineering Institute, Nanjing, China).

Histopathologic examination

Liver tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. Embedded tissue was cut to 4-μm thickness which were then stained with hematoxylin–eosin (H&E) kit (Servicebio, Wuhan, China) for histological assessment. Oil-red O staining was conducted with frozen liver tissues using a commercially available Oil-Red O stain kit (Servicebio, Wuhan, China) according to the instruction. For immunohistochemistry (IHC), the deparaffinized sections were subjected to heat-mediated antigen retrieval and blocked in 3% H2O2 for 20 min. The sections were stained overnight with a primary anti-cleaved caspase 3 antibody (1:200; CST, #9664), anti-4HNE antibody (1:200, Abcam, ab46545) after blocking with bovine serum albumin for 1 h at room temperature. The sections were incubated with a secondary antibody (1:5000; HRP, Donkey Anti-Goat IgG, Proteintech, Wuhan, China) after washing with PBS for 15 min at room temperature. Then, the colour were developed with a 3,3′-diaminobenzidine tetrahydrochloride (DAB; Servicebio, Wuhan, China) after PBS wash, and then the nuclei were restrained with Harris hematoxylin for 3 min. After dehydrating and sealing, images were obtained using a microscope (BX51, Olympus, Tokyo, Japan).

RNA-sequencing

RNA-seq was performed on triplicate sample from liver of three group mice (Control, Model and Lira). The total RNA was extracted using Trizol (Ambion, USA). RNA quality was verified using Agilent 2100 Bio-analyzer (Agilent Technologies, Santa Clara, CA). RNA sequencing libraries were generated using Agilent High Sensitivity DNA Kit, and sequenced on an Illumina Novaseq 6000 platform. Sequencing was performed at Bioprofile Co. Ltd (Shanghai, China). The filtered reads were mapped to the mouse genome reference sequence (GRCm39.dna.toplevel.fa Ensembl release103) using HISAT2. Gene expression levels were calculated by the fragments per kilobase of transcript per million mapped reads (FPKM) values. Genes were considered differentially expressed when ∣log2 (fold change)∣ > 1.5 and P-value < 0.05.

Real-time PCR

Total RNA was extracted from liver of mouse using TRI Gen (GenStar, China), and reverse-transcribed with the ABscript II cDNA First Strand Synthesis Kit (ABclonal, China). Real-time PCR was conducted by using ABScript III RT Master Mix for qPCR (ABclonal, China). β-actin was used as an internal control and Relative expression of target mRNA was calculated based on the 2−ΔΔCt comparative method. The primer sequences used are listed in Table 1.

Table 1 Sequences of primers for real-time PCRCell culture studies

The human liver cancer cell lines HepG2, Hep3B and PLC cell lines obtained from the Cancer Research Institute, Xi’an Jiaotong University. HepG2, Hep3B and PLC cells were cultured in Dulbecco’s modified Eagle medium (Gibco, US) in a 37 ◦C incubator with an atmosphere of 5% CO2. All the medium supplemented with 10% fetal bovine serum (Excell Bio, China) and 1% penicillin–streptomycin (Solarbio, Beijing, China).

Cell viability assay

Cell viability was measured as described previously using the Cell Counting Kit-8 (CCK8) (Enogene, Nanjing, China). In brief, cells were seeded in 96-well plates at a density 4 × 103–8 × 103 cells per well and incubated for 24 h. Then, the cells were exposed to different treatment. Cells were treated with glucose (Solarbio, Beijing, China)., ferroptosis inducers, erastin (Selleckchem, Houston, USA), or RSL3 (Selleckchem, Houston, USA); Liraglutide; cell death inhibitors, including ferrostatin-1 (Selleckchem, Houston, USA), necrostatin-1 s (Selleckchem, Houston, USA), or Z-VAD-fmk (Selleckchem, Houston, USA); AMPK inhibitor, compound C (MedChemExpress, USA); After that, the medium in each well was replaced with 100 µl fresh medium containing 10 µl CCK8 reagent. After incubation for 1 h at 37 °C, 5% CO2 incubator. The absorbance at a wavelength of 450 nm was measured using a Multiskan Spectrum (Thermo Scientific, Waltham, MA, USA).

Measurement of intracellular ferrous iron level (Fe2+)

The intracellular Fe2+ level was determined using RhoNox-1 (HY-D1533, MCE). First, cells were cultured on sterile coverslips. Then, remove the coverslip from the medium and aspirate excess medium. Furthermore, add 100uL of working solution, gently shake it to completely covers the cells, and then incutate at 37℃ for 1 h. At last, wash twice with medium, 5 min each time and observe by fluorescence microscopy.

Western blotting

Western blotting to analyze protein expression was performed as previously described (Yi et al. 2022). Briefly, liver tissues and cell pellets were lysed using RIPA lysis buffer (Millipore) and the protein concentration was determined by a Pierce BCA kit (Thermo, Waltham, MA, USA). The primary antibodies and concentrations used for western blotting were: phospho-AMPKα (Thr172, 1:1000, 2535, Cell Signaling, Boston, USA), AMPKα (1:1000, 5832, Cell Signaling, Boston, USA), phospho-ACC (S79, 3661, 1:1000, Cell Signaling, Boston, USA), ACC (1:1000, 3662, Cell Signaling, Boston, USA), phospho-S6 (Ser240/244, 1:1000, 2215, Cell Signaling, Boston, USA), S6 (1:1000, 2217, Cell Signaling, Boston, USA), phospho-S6K (Thr229/389, 1:1000, 9202, Cell Signaling, Boston, USA), S6K (1:1000, 9205, Cell Signaling, Boston, USA), TFRC (1:1000, A5865, Abclonal, Wuhan, China), ACSL4 (sc-271800, 1:1000, Santa Cruz), SLC7A11 (12691S, 1:1000, Cell Signaling, Boston, USA), GPX4 (52455S, 1:1000, Cell Signaling, Boston, USA), beta actin (TA811000S, 1:2000, Origene), Tubulin (80762-1-RR, 1:10,000, Proteintech, Wuhan, China). The secondary antibodies used were: horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, 1:5000 dilution, Wuhan, China), horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology, 1:5000 dilution). Proteins were visualized with the ECL Western blotting substrate (32,109, ThermoScientific, USA).

Statistical analysis

Data are presented as means ± standard deviation (SD), with at least 3 independent biological replicates in each group. The difference between the two groups was tested by using unpaired Student’s t test with GraphPad Prism 8 (GraphPad Software, Inc.). One-way analysis of variance or two-way analysis of variance was used to determine the significance among three or more groups replicates. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, non-significant). The band intensity of western blotting and fluorescence intensity of Fe2+ are measured by using Image J. All samples from cells were collected from at least 3 independent biological replicates.