Animal experiments

Animal care and experiments were conducted in accordance with the guidelines of the Animal Care Committee of Zhejiang University. Male C57BL/6 mice aged 8–10 weeks were purchased from the Model Animal Research Center of Nanjing University for the investigations. The mice were divided into three groups based on age: young group (2–4 months), middle-aged group (9–12 months), and old group (24–26 months). The livers were collected from mice of different ages.

To selectively inactivate the IGF2 gene in the liver, CRISPR-Cas9 technology was used to insert loxP sites on flanking exons 2 of the IGF2 gene in fertilized mouse eggs to generate IGF2 floxed (IGF2f/f) mice (CKOCMS190327JN1 + CKOCMS190327JN2-B, Cyagen Biosciences). IGF2f/f mice were crossbred with Albumin-cre (Alb-cre) mice (Strain #:003574, The Jackson Laboratory) to generate IGF2f/+;Alb-Cre (IGF2f/+Cre) mice. Then, IGF2f/+Cre mice were crossbred with IGF2f/f mice to generate liver-specific IGF2-deficient mice, termed as IGF2f/f; Alb-Cre (IGF2f/fCre). The IGF2f/f littermates were used as controls (IGF2f/f). Genotyping for the IGF2 floxed allele, and Alb-Cre gene was performed by PCR and genome sequencing using DNA extracted from tails. All mice used in the study were bred on a C57BL/6 background. Mice were maintained under standard conditions of 22 ± 2 °C, 50% to 60% relative humidity, and a 12-h light–dark diurnal cycle (lights on at 6: 00 A.M.), and had free access to water and food. High doses of d-galactose (d-gal) are widely used to establish mouse aging models and to explore the mechanisms underlying liver aging (Azman et al. 2021). 8-week-old male mice (25 ± 2 g) were randomly assigned into four groups (n = 5–7) according to genotype and whether they were administrated with d-gal: IGF2f/f, IGF2f/fCre, IGF2f/f + d-gal, and IGF2f/fCre +  d-gal. The d-gal model group received intraperitoneal injections of 120 mg/kg/day of d-gal (59-23-4, Sigma-Aldrich) at a concentration of 12 g/L for 8 weeks, whereas the mice in the saline control group received equivalent volumes of physiological saline. Mice were sacrificed at the end of the experiments, and the livers of the mice were collected.

Glucose and insulin tolerance tests

For the intraperitoneal glucose tolerance testing (IPGTT), mice were fasted for 16 h and then were given glucose (2 g/kg body weight). For the insulin tolerance test (ITT), mice received an intraperitoneal injection of insulin (1 IU/kg body weight) after 4 h of fasting. The blood glucose level from the tail vein was measured at 0 (baseline), 15, 30, 60, and 120 min; glucose levels were immediately measured using One Touch Ultra glucose strips (LifeScan).

Enzyme-linked immunosorbent assay

The serum insulin levels were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using a wide range mouse insulin immunoassay kit (MS300, EZassay) following the manufacturer’s instructions.

Assessment of liver function and histology

Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an Alanine aminotransferase Assay Kit (C009-2-1, Nanjing JianCheng) and an Aspartate aminotransferase Assay Kit (C010-2-1, Nanjing JianCheng) according to the manufacturer’s protocol, respectively. The liver tissue samples were preserved in 4% paraformaldehyde, followed by embedding in paraffin. Sections of 4 μm thickness were then obtained and subjected to hematoxylin and eosin (H&E) staining for histological analysis.

RNA-Seq and analysis

Global transcriptome profiling was performed by RNA-Seq using liver tissues of IGF2f/f +  d-gal and IGF2f/fCre + d-gal mice (n = 3). RNA was extracted, sequenced, and analyzed by a custom service provided by Novogene using an Illumina NovaSeq 6000. Bioinformatics analysis was performed using the OmicStudio tools at https://www.omicstudio.cn/tool. The cut-off for differential expression threshold was based on |log2fold change (log2FC) |> 1 and P-value < 0.05.

Cell culture

The immortalized normal mouse hepatocyte cell line (AML12) was purchased from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture medium (GNM12500, Genomcell.bio), containing 10% fetal bovine serum (S-FBS-EU-015,SERANA), 100 U/mL Penicillin–Streptomycin (BL505A, Biosharp), 1% Insulin–Transferrin–Selenium (C0345, Beyotime), and 40 ng/mL dexamethasone (D4902, Sigma-Aldrich) at 37 ℃ in 5% CO2. For induction of cellular senescence, cells were exposed to 20 g/L d-gal for 48 h or exposed to 100 µM hydrogen peroxide (H2O2) for 2 h and cultured for 2 days in normal media. To elevate nicotinamide adenine dinucleotide (NAD+) levels, nicotinamide riboside (NR) (1341-23-7, Zhengzhou Acme Chemical Co. Ltd) at a dosage of 1 mM was supplemented to the culture medium for 48 h before measurement.

Cells infection and transfection

For infection and transfection, AML12 cells were seeded in 6-well plates (105 cells/mL) and cultured overnight (30–40% confluency). The specific short hairpin RNA (shRNA) against IGF2 (sh-IGF2) and its corresponding negative control (sh-NC), as well as their lentiviral vector construction, were acquired from Genechem (Shanghai, China). Cells were infected with lentivirus with a multiplicity of infection (MOI) of 10 and incubated with 6 μg/mL polybrene for 12 h to increases the efficiency of infection. Then, the supernatant was replaced by the growth medium. When the cells reached 80% confluency, 3 μg/mL puromycin was added to the culture medium to eliminate uninfected cells. When stably transfected cells appeared, the concentration of puromycin in the culture medium was reduced to 1 ug/mL for continuous screening of infected cells. To knock down CEBPB, small interfering RNAs (siRNAs) were used. Cells were transiently transfected with si-CEBPB or si-NC (TSINGKE, Beijing, China) via Lipo 8000 (C0533, Beyotime) according to the manufacturer’s protocol. Cells were harvested after transfection for 48 h. The sequences of shRNA and siRNA are listed in Additional file 1: Table S1.

RNA isolation and real-time quantitative PCR

Total RNA was isolated from AML12 cells and mouse liver tissues using AG RNAex Pro reagent (AG21102, Accurate Biotechnology) and reverse transcribed using an Evo M-MLV RT Premix kit (AG11706, Accurate Biotechnology) according to the manufacturer’s protocols. Real-time quantitative PCR (RT-qPCR) was performed utilizing the SYBR Green Premix Pro Taq HS qPCR kit (AG11701, Accurate Biotechnology) on a LightCycler 480 PCR System (Roche, Basel, Switzerland). The relative mRNA expression levels were calculated using the 2−ΔΔCT method and normalized to GAPDH mRNA as the internal control. The various primer sets used in this study are shown in Additional file 1: Table S2.

Western blotting

Total protein was extracted from AML12 cells and mouse liver tissues using RIPA lysis (FD009, FdBio Science) containing protease and phosphatase inhibitors, and protein concentrations were measured via BCA Protein Assay Reagent (FD2001, FdBio Science). Equal amounts of proteins were denatured and subjected to 10% or 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride membrane, and blocked with 5% non-fat milk for 1 h at room temperature. After blocking, the membranes were incubated overnight with primary antibodies at 4 °C. Primary antibodies were diluted at 1:1000 and included IGF2 (ab9574, Abcam), P53 (10442-1-AP, Proteintech), P21 (A11454, Abclonal), P16 (A0262, Abclonal), CEBPB (sc-7962, Santa Cruz Biotechnology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), p38 (8690S, Cell Signaling Technology), p-p38 (4511S, Cell Signaling Technology). After washing, the membranes were incubated with a secondary anti-mouse (FDM007, FdBio Science; diluted at 1:5000) or anti-rabbit antibody (FDR007, FdBio Science; diluted at 1:5000) for 1 h at room temperature, and protein signals were visualized using an enhanced chemiluminescence kit (FD8030, FdBio Science).

Senescence-associated-β-galactosidase assay

The senescence-associated-β-galactosidase (SA-β-gal) assay was performed using a SA-β-gal Staining Kit (C0602, Beyotime) according to the manufacturer’s instructions. In brief, frozen liver sections and AML12 cells were prepared, washed, fixed, and stained with the β-galactosidase staining solution in a 37 °C dry incubator (without CO2). The SA-β-gal-positive cells exhibited a blue color, and photos were taken with an optical microscope (Olympus, Tokyo, Japan).

Immunofluorescence

After de-waxing and rehydration, liver sections were antigen retrieved in Tris/EDTA buffer (pH 9.0) for 20 min. AML12 cells seeded on coverslips were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 15 min. After blocking the liver sections and cells with 5% bovine serum albumin (FD0030, FdBio Science) for 1 h, they were incubated with primary antibodies against F4/80 (13-4801-82, Invitrogen; diluted at 1:100) and γ-H2AX (bs-2560R, Bioss; diluted at 1:200) overnight at 4 °C. After removing the primary antibody by washing, they were incubated with DyLight 488 AffiniPure Goat Anti-Mouse IgG (FD0150, FdBio Science; diluted at 1:500) or DyLight 594 AffiniPure Goat Anti-Rabbit IgG (FD0129, FdBio Science; diluted at 1:500) for 1 h at room temperature, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Observation and photo-taking were conducted using a fluorescence microscope (Olympus, Tokyo, Japan), and fluorescence intensity was measured using Image-J software.

Cell viability assay

Cell viability was determined by the cell counting kit-8 assay (CCK-8; C0038, Beyotime). AML12 cells were seeded at a density of 0.5 × 104 cells per well in 96-well plates. After cell treatment, the culture supernatant was removed, and the cells were incubated in a culture medium containing CCK8 for 2 h at 37 °C in the dark. The absorbance at 450 nm was measured by a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Cell cycle analysis

The cell cycle and apoptosis were analyzed using commercially available kits (C1052, Beyotime) according to the manufacturer’s instructions. Briefly, AML12 cells were harvested, fixed with 70% ethanol overnight at 4 °C, and incubated with propidium iodide staining solution in the dark for 30 min. The cell cycle analysis was performed using a flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using ModFit LT 4.1 software.

Measurement of oxygen consumption rate

Oxygen consumption rates of AML12 cells were examined using the XF Mito Stress Test Kit (103015-100, Agilent) on an XFe96 Extracellular Flux Analyzer following the manufacturer’s protocols. One day before the assay, 8000 cells per well were seeded in XF 96-wells microplates, and a sensor cartridge was hydrated in XF Calibrant at 37 °C in a CO2-free incubator overnight. On the day of the assay, the cells were incubated in 180 mL of XF assay medium (XF Base Medium supplemented with 1 mM pyruvate, 2 mM l-glutamine, and 10 mM glucose adjusted to pH 7.4) at 37 °C in a non-CO2 incubator for 1 h before initiation of measurements. During the incubation period, 10 × stocks of drugs in XF media were successively loaded into the injection ports in the XFe 96 sensor cartridge, and the final concentrations of drugs after injections were: 1.0 μM Oligomycin, 1.0 μM Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 μM Rotenone/antimycin A (Rot/AA). The plates were assayed using an XFe96 analyzer to measure the oxygen consumption rate (OCR) over time. This was done by sequentially adding different drugs and monitoring the OCR response. The OCR of cells was normalized to cell number, and datasets were analyzed by Wave software (Agilent).

Measurement of ROS production

Reactive oxygen species (ROS) production was identified using a ROS assay kit (S0033S, Beyotime) according to the manufacturer’s instructions. Briefly, AML12 cells were washed with PBS and incubated with 10 μM 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFH-DA) probes without light for 30 min at 37 °C. The cells were washed twice with PBS, resuspended in PBS, and subjected to flow cytometric analysis (BD Biosciences, San Jose, CA, USA).

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was detected using the JC-1 assay kit (M8650, Solarbio) following the manufacturer’s instructions. AML12 cells were stained live in a growth medium with the fluorescent probe JC-1 (1:200) at 37 °C for 20 min. After washing, the cells were resuspended in PBS and subjected to flow cytometric analysis (BD Biosciences, San Jose, CA, USA).

Mitochondrial ROS

The mitochondrial ROS was detected using the MitoSOX™ Red mitochondrial superoxide indicator (M36008, Invitrogen). AML12 cells cultured in 12-well plates were incubated with 5 μM MitoSOX™ reagent working solution for 10 min at 37 °C, protected from light. Cells were washed with PBS three times, and the images were captured using an fluorescence microscope (ZEISS, Oberkochen, Germany).

Transmission electron microscopy

AML12 cells were fixed with 2.5% glutaraldehyde in PBS buffer for 2 h at room temperature, followed by overnight fixation at 4 °C. The cells were then post-fixed in 1% osmic acid at 37 °C for 1 h and stained with aqueous 2% uranyl acetate for 30 min. After dehydration with an ascending gradual series of ethanol (50%, 70%, 90%, and 100%) for 15 min each, the samples were transferred to absolute acetone for 20 min. Cells were further penetrated with a 1:1 mixture of absolute acetone and embedding agent for 2 h and then embedded in a pure embedding agent. The samples were cut into approximately 100-nm-thick ultrathin sections with a Leica UC7 ultramicrotome and examined with a transmission electron microscope (Tecnai T10, Holland).

Measurement of NAD +/NADH ratios

NAD+ and NADH were quantified using a commercial kit (N6035, UElandy). 1 × 106 cells were treated with 200 μL of NAD+ /NADH extraction buffer. To detect the total amounts of NAD+ and NADH, 20 μL of extracted samples were transferred into 96-well plates. To detect NADH, 20 μL of extracted samples were heated to 60 °C for 30 min and transferred into 96-well plates. Then, the samples were mixed with enzyme mix, NADH developer and NAD buffer, and OD450 was measured. Standard curves (0–200 pmol) were generated for quantification.

Statistical analysis

All experiments were repeated in triplicate. Statistical analysis was performed using GraphPad Prism 8 software. Continuous data were expressed as means ± SEM. Comparisons between the two groups were made using unpaired student t-tests. Differences between more than two independent groups were analyzed using one-way ANOVA. A P-value < 0.05 was statistically significant.