Animals

Experiments were performed in male C57Bl/6 mice aged 3, 12 and 14.2±1.2 months15 purchased from Harlan (Blackthorn, UK). Mice were housed in same-sex cages in groups of 4–6 (56 × 38 × 18 cm3, North Kent Plastics, Kent, UK) and individually identified by an ear notch. Mice were housed at 20±2 °C under a 12 h light/12 h dark photoperiod with lights on at 0700 hours. The diet used was standard rodent pelleted chow (CRM (P); Special Diets Services, Witham, UK) for AL-fed mice and the same diet, but as smaller pellets, was offered to DR mice. The smaller pellet size reduced competition for food. DR mice were offered 60% of AL intake (calculated based on average food intake in 90 control AL mice between 5 and 12 months of age) as one ration at 0930 hours daily. Half of the animals were subjected to DR, while the other half, matched for body mass, food intake and age, served as AL controls. Additionally, control, young mice were killed at 3 months of age. DR was introduced at 3 months of age and lasted for 9–12 months. At the age of 12 months, some mice from the AL and DR groups had their dietary regime changed AL to DR or DR to AL for 3 months. All mice were killed at the time points mentioned above and at the end of the experiment. All work complied with the guiding principles for the care and use of laboratory animals and was licensed by the UK Home Office (PPL60/3864).

A variety of tissues were collected. Tissues were frozen in optimal cutting temperature compound or OCT media for cryosections, snap-frozen in liquid nitrogen for biochemistry and fixed in 10% formalin for 24 h before processing and paraffin embedding. Cryosectioning was performed at 10 μm intervals and paraffin-embedded tissues were cut at 3 μm intervals. Haematoxylin–eosin (H&E)-stained mouse liver sections were graded for steatosis by a single expert liver pathologist (DT) who was not aware of the genotype/treatment.

Alb-Xpg transgenic mice, with a liver-specific Xpg gene inactivation, were generated and genotyped as previously described29. (We used organs produced in the previous study but generated additional mice under the same conditions to increase group size.) Xpgf/− Alb-Cre+ mice (in a C57BL6J/FVB F1 hybrid background; referred to as Alb-Cre) are heterozygous for Xpg in their entire body, except for the hepatocytes in the liver, which are homozygous for Xpg after Cre excision of the floxed allele. Littermates, with and without Cre-recombinase expression (Xpgf/+ Alb-Cre+ and Xpgf/− Alb-Cre− respectively), were used as controls (referred to as wt). Mice were maintained in a controlled environment (20–22 °C, 12 h light; 12 h dark cycle) and were housed in individual ventilated cages under specific pathogen-free conditions. All animals had AL access to water and standard mouse food (CRM pellets, SDS BP Nutrition Ltd.; gross energy content 4.39 kcal g−1 dry mass, digestible energy 3.2 kcal g−1). At 6 (control: 6 male, Xpg: 6 male, 1 female) and 12 months (control: 4 male, 2 female and Xpg: 3 male, 3 female) of age, mice were killed for tissue collection. Tissues were snap-frozen in liquid nitrogen, embedded in TissueTek and sliced in 10 μm thick cryosections or fixed overnight in 10% phosphate-buffered formalin, paraffin-embedded, sectioned at 3 μm and mounted on Superfrost Plus glass slides. Oil Red O and H&E images were generated using the NanoZoomer Digital slide scanner with the NDP view software (Hamamatsu Photonics, Japan).

A new stock of INK-ATTAC transgenic mice was generated and genotyped as previously described10. Mice were house at 2–5 mice per cage in a 12 h light/12 h dark cycle at 24 °C with free access to food (standard mouse diet, Lab Diet 5053, St Louis, MO, USA) and water in a pathogen-free facility. AP20187 (10 mg kg−1) was administered to 24-month-old mice by intraperitoneal injection every 3 days, for 3 months. For senolytic treatment, vehicle or D (5 mg kg−1) and Q (10 mg kg−1) in combination were administered by oral gavage once per month for 3 months. For dietary intervention studies, INK-ATTAC mice were housed 2–5 per cage, at 22±0.5 °C on a 12–12-h day–night cycle and provided food and water AL. Mice were randomly assigned into the chow diet or HF diet group. HF food was purchased from Research Diets (cat no #D12492, 60% of calories in this diet are from fat). Mice were injected intraperitoneally with AP20187 (10 mg kg−1) or vehicle for 3 days every 2 weeks for 10 weeks. All mice were killed at the age of 15 months (6 male HF (3 vehicle, 3 AP), 9 female HF (4 vehicle, 5 AP), 8 male control (4 vehicle, 4 AP) and 5 female control (2 vehicle, 3 AP)).

Db/db mice homozygotic males and females were purchased from Jackson Laboratory (Bar Harbor, ME, stock number: 000642). Mixed gender cohort consisting of 13 male db/db, 10 female db/db, 8 male db/+ and 8 female db/+ was first time treated at the age of 4 months. In total, four treatments (D (5 mg kg−1) and Q (50 mg kg−1) or vehicle (60% Phosal, 10% ethanol and 30% PEG-400) were administered for 5 consecutive days biweekly via oral gavage.). Animals were killed at the age of 6 months.

Ethical approval was granted by the LERC Newcastle University, UK Dutch Ethical Committee at Erasmus MC (permit # 139-12-18) and the IACUC at Mayo Clinic (Protocols A26713, A40312). The work was licensed by the UK Home Office (PPL 60/3864) and complied with the guiding principles for the care and use of laboratory animals.

Mouse adult fibroblasts

Ear clippings were transported and stored (not longer than 1 h) in DMEM on ice. Punches were washed three times with serum-free media, finely cut and incubated for 2–3 h at 37 °C in 2 mg ml−1 collagenase A in DMEM. A single-cell suspension was obtained by repeated pipetting and passing through a 24-G fine needle. Cells were centrifuged for 10 min at 1,000 r.p.m. and cultured in Advanced D-MEM/F-12 (DMEM, Invitrogen) plus 10% FCS (Sigma) in 3% O 2 5% CO 2 . Each cell strain was derived from a separate donor. MAFs were seeded and allowed to grow for 24 h and then X-ray irradiated with 5 or 10 Gy using a PXI X-Rad 225 (RPS Services Ltd) to induce cellular senescence. Alternatively, MAFs were treated with 100 nM of complex I inhibitor rotenone, which was replaced daily. Following 10 days of treatment, induction of senescent markers was observed.

Hepatocytes

Hepatocytes were isolated from the livers of wild-type mice by digestion with collagenase from Clostridium histolyticum (Sigma) and then filtered through a 70-μm cell strainer. Cells were collected by centrifugation (500 r.p.m. for 3 min), washed three times in Krebs–Ringer buffer (Sigma) and re-suspended in Williams medium E with 10% serum (WME Gibco) and plated onto collagen-coated plates (type I collagen, BD Biosciences). After 4 h, medium was removed and cells were cultured in fresh 10% or 0.5% Williams medium E. Hepatocytes were incubated at 37 °C and 3% oxygen overnight and were exposed the next day to 10 Gy irradiation in order to induce senescence. Following 10 Gy X-ray irradiation, hepatocytes acquire a morphology characteristic of senescence and SA-β-Gal activity after 6 days. Monitoring cell numbers revealed that a small percentage of hepatocytes experienced cell death after irradiation; however, most of the cells survived and acquired a senescent-like phenotype. Non-irradiated controls were analysed 1–2 days following isolation, at the same time as irradiation took place for the irradiated cells (this was necessary to prevent overgrowth of other cell types, which are present in very low numbers).

Subjects and histological examination of liver biopsies

Nine individuals with biopsy-proven NAFLD were evaluated. Demographic data are shown in Table 1. Liver biopsy was performed under radiological guidance. Liver tissue cores of mean length 16.3 mm, length range 9–30 mm were fixed in 10% neutral formalin and embedded in paraffin for histological examination. Tissue sections were stained with H&E and with Sirius Red Fast Green for visualizing collagen. Liver biopsies were reviewed by a single expert liver pathologist (DT) who was not aware of the clinical or immunohistochemical data. Histological diagnosis was based on currently accepted histopathological criteria for NAFLD/NASH47. The grade of steatosis (0–3), disease activity, including semi-quantification of lobular inflammation and hepatocyte ballooning, and stage of fibrosis (0–4) were assessed according to Kleiner et al.48 and Bedossa et al.49. The NAFLD activity score (range 0–8: sum of grade of steatosis, lobular inflammation 0–3 and hepatocyte ballooning 0–2)48 and activity according to SAF (range 0–4: sum of lobular inflammation 0–2 and hepatocyte ballooning 0–2)49 were calculated for each biopsy.

Oil Red O

Preparation of Oil Red O (Sigma-Aldrich, #O1391) working solution and staining of slides was performed according to Mehlem et al.50 and the manufacturer’s instructions. Briefly, Oil Red O working solution was prepared from stock solution mixed 3:2 with water and incubated at 4 °C for 10 min. Solution was filtered through 0.45-μm filters and applied on OCT-embedded liver sections for 5 min. Slides were washed twice in water, 15 min each wash, and mounted in vectashield mounting media. For representative images, sections were counterstained with haematoxylin. Samples were imaged within 6 h. Surface of lipid droplets was quantified using the ImageJ software by measuring area occupied by red pixels.

Nile red

In all, 2 μl of Nile red solution (Nile red (Sigma N3013) 150 μg ml−1 in acetone) were added to 1 ml 80% glycerol. Frozen OCT-embedded liver 10-μm sections were air dried for 30 min. MAFs were washed briefly with PBS and fixed for 10 min with 2% paraformaldehyde dissolved in PBS. DAPI solution was added for 10 min and afterwards sections were washed with PBS for 5 min. Some sections were stained with ActinGreen 488 (ThermoFisher, 1 drop in 0.5 ml PBS) for 30 min and washed with PBS for 3 × 5 min. In all, 20–30 μl of Nile red/glycerol were directly added to each section, mounted on a glass microscope slide and covered with a cover slip. Images were taken immediately after mounting.

BODIPY 493/503 staining

MAFs were washed briefly with PBS and fixed for 10 min with 2% paraformaldehyde dissolved in PBS. Cells were permeabilized with PBG for 30 min and incubated for 10 min with 4 ml ml−1 of BODIPY. Cells were washed with PBS for 3 × 5 min, stained with DAPI solution and mounted.

Histochemistry and immunofluorescence (IF)

Paraffin sections were deparaffinized with Histoclear and ethanol, and antigen was retrieved by incubation in 0.01 M citrate buffer (pH 6.0) at 95 °C for 10 min. Slides were incubated in 0.9% H 2 O 2 for 30 min and afterwards placed in blocking buffer (normal goat serum 1:60 in PBS/BSA, #S-1000; Vector Laboratories) for 30–60 min at room temperature (RT). Livers were further blocked with Avidin/Biotin (Vector Laboratories, no. SP-2001) for 15 min each. MAFs were washed briefly with PBS and fixed for 10 min with 2% paraformaldehyde dissolved in PBS. Cells were permeabilized for 45 min with PBG (0.5% BSA, 0.2% Fish Gelatine, 0.5% Triton X-100 in PBS). Primary antibodies were applied overnight at 4 °C. Slides were washed three times with PBS and incubated for 30 min with secondary antibody (no. PK-6101; Vector Lab). Antibodies were detected using a rabbit peroxidase ABC Kit (no. PK-6101; Vector Lab) according to the manufacturer’s instructions. Substrate was developed using NovaRed (no. SK-4800; Vector Lab) or 3′3′-diaminobenzidine (no. SK4100, Vector Lab). Sections were counterstained with haematoxylin. For IF, sections were treated as before, and after the secondary antibody incubation, Fluorescein Avidin DCS (1:500 in PBS, no. A-2011, Vector Lab) was applied for 20 min. For IF on MAFs, Alexa Fluor secondary antibody (1:2,000; Molecular Probes) was applied for 30 min at RT. Sections or cells were stained with DAPI for 5–10 min and mounted in vectashield mounting media.

p21 immunohistochemistry was performed on formalin-fixed sections using rat anti-p21 antibody (clone HUGO 291H, Abcam, UK) and the ImPRESS Rat immunodetection system (MP-7444, Vector laboratories, Country) using 3′3′-diaminobenzidine (Dako, UK) as chromagen followed by counterstaining with haematoxylin. Sections were then dehydrated and coverslipped. Ten blinded consecutive non-overlapping fields were acquired at × 200 magnification and quantified as previously described51.

Antibodies and dilutions

The nature, source and dilution of all antibodies used in the study are listed in Table 2.

Table 2 Nature, source and dilution of all antibodies used in the study. Full size table

RNA in situ hybridization

RNA-ISH was performed after RNAscope protocol from Advanced Cell Diagnostics Inc. (ACD). Paraffin sections were deparaffinized with Histoclear, rehydrated in graded ethanol (EtOH) and H 2 O 2 was applied for 10 min at RT followed by two washes in H 2 O. Sections were placed in hot retrieval reagent and heated for 30 min. After washes in H 2 O and 100% EtOH, sections were air dried. Sections were treated with protease plus for 30 min at 40 °C, washed with H 2 O and incubated with target probe (p16, eGFP) for 2 h at 40 °C. Afterwards, slides were washed with H 2 O followed by incubation with AMP1 (30 min at 40 °C) and next washed with wash buffer (WB) and AMP2 (15 min at 40 °C), WB and AMP3 (30 min at 40 °C), WB and AMP4 (15 min at 40 °C), WB and AMP5 (30 min at RT) and WB and, finally, AMP6 (15 min at RT). Finally, RNAscope 2.5 HD Reagent kit-RED was used for chromogenic labelling. After counterstaining with haematoxylin, sections were mounted and coverslipped.

Telomere and centromere fluorescent ISH

After γ-H2A.X IF, slides were washed three times in PBS, crosslinked with 4% paraformaldehyde for 20 min and dehydrated in graded ethanol. Sections were denatured for 10 min at 80 °C in hybridization buffer (70% formamide (Sigma UK), 25 mM MgCl 2 , 0.1 M Tris (pH 7.2), 5% blocking reagent (Roche, Germany)) containing 2.5 μg ml−1 Cy-3-labelled telomere-specific (CCCTAA) or FAM-labelled, CENPB-specific (centromere) (ATTCGTTGGAAACGGGA) peptide nucleic acid probe (Panagene), followed by hybridization for 2 h at RT in the dark. Slides were washed twice with 70% formamide in 2 × SSC for 15 min, followed by washes in 2 × SSC and PBS for 10 min. Sections were incubated with DAPI, mounted and imaged. In-depth Z-stacking was used (a minimum of 40 optical slices with × 63 objective) followed by Huygens (SVI) deconvolution. Relative telomere length was measured by telomere intensity per nucleus in one z plane.

Number of TAF per cell was assessed by quantification of partially or fully overlapping (in the same optical slice) signals from telomere probe and γ-H2A.X in slice-by-slice analysis. Number of decondensed centromeres was assessed by quantification of decondensed/elongated centromeres.

SA‐β‐Gal activity

For SA-β‐Gal activity, cells were fixed with 2% paraformaldehyde for 5 min, washed and incubated at 37 °C with fresh SA-β-Gal solution: 1 mg of 5-bromo-4-chloro-3-indolyl P3-D-galactoside (X-Gal) per ml (stock=20 mg of dimethylformamide per ml)/40 mM citric acid/sodium phosphate, pH 5.5/5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl2. Staining was evident after 24 h. Cells were washed and stained with DAPI for 10 min, washed and mounted. For SA-β‐Gal activity on the liver, 5-μm frozen sections were fixed with 0.5% glutaraldehyde for 15 min, washed with PBS and were incubated in SA-β-Gal staining solution for 18 h at 37 °C. Washed after incubation and counterstained with haematoxylin. Sections were dehydrated and mounted and 10–15 random fields were imaged per sections. Senescent hepatocytes were counted as a percentage of all hepatocytes per field.

Karyomegaly

In order to quantify the frequency of karyomegalic nuclei in the mouse liver precisely, nuclear staining was performed. Preparation of samples was performed as described by Wang et al.52. Briefly, OCT-embedded liver sections were washed three times with PBS and mounted in DAPI-containing mounting media and imaged. In-depth Z-stacking was used (a minimum of 40 optical slices with × 63 objective). Analysis of karyomegaly was performed using the ImageJ software. Karyomegaly was assessed using maximum-Z projections with a threshold of 127 μm2 of the nucleus area for cells to be considered karyomegalic.

Measurements of cellular bioenergetics

Cellular oxygen consumption rates were measured in a Seahorse XF24 Analyzer using unbuffered media (DMEM (Sigma, D-5030) supplemented with 5 mM D-glucose (Sigma), 2% L-Glutamate, 3% calf serum), and the relative changes in oxygen consumption rates after the addition of palmitate (100 μM) and etomoxir (4 μM) were calculated. Mitochondrial function in MAF was also determined in a Seahorse XF24 Analyzer (Agilent Technologies) by permeabilizing the cells using XF Plasma Membrane Permeabilizer (Agilent Technologies) according to the manufacturer’s instruction using complex I-linked substrate, pyruvate (10 mM)/Malate (1 mM). The state 3 was achieved by addition of 4 mM ADP and the state 4 by oligomycin (1 μg ml−1).

RNA-Seq

Strand-specific paired-end libraries for RNA-Seq were generated from DNAse-treated total RNA using Ribozero and ScriptSeq systems (Epicentre/Illumina) and run on an Illumina 2500 sequencer to obtain 100 base paired-end reads. Low quality reads were filtered out by Kraken53. The resulting filtered reads were mapped to the mouse genome version mm10 using Tophat54. Mapped reads were counted with htseq-count55 and read counts were normalized using deseq2 (ref. 56). In order to capture genes with the same expression pattern as the TAF and Oil Red O staining, said values were inserted into the normalized expression data set and then clustered with Biolayout express57 using a 0.7 minimum Pearson correlation and a 95 correlation value. Clustering was conducted using MCL implementation of Markov Cluster Algorithm58 using an inflation coefficient of 2.2 and a preinflation coefficient of 3.0. The cluster of genes with included TAF and oil lipid data were extracted and analysed for GO over-representation using the PANTHER database59.

Statistical analysis

Normal distribution and equal variance were assessed using the statistical software from Sigma Plot vs11.0. We conducted one‐way analysis of variance, two‐tailed t‐test and linear and nonlinear regression analysis tests using Sigma Plot v11.0 and GraphPad Prism 7.

Ethics

Approval was obtained from ‘Newcastle and North Tyneside Research Ethics Committee’ for the use of anonymized patient samples (approval reference: REC 06/Q0905/150).

Data availability

RNA-Seq data have been deposited in arrayexpress/ENA under accession code E-MTAB-5645. All data generated or analysed during this study are available within the paper and its Supplementary Information files and from the corresponding author on request.