p53 and TIGAR promote redox control to protect against metabolic dysfunction-associated steatohepatitis

Abstract

Background & Aims

TP53 is a potent tumour suppressor that coordinates diverse stress response programmes, ranging from pro-survival activities to cell death. p53 is also engaged during tissue damage and repair, including within the liver. Metabolic dysfunction-associated steatohepatitis (MASH) is a major driver of hepatocellular carcinoma, but our understanding of the molecular determinants of MASH remains incomplete. Here, we investigate p53 activity throughout MASH development, with implications for disease prevention.

Methods

This study utilises non-invasive imaging of p53 activity and liver-specific p53 deletion within the context of mouse models of diet and genetically induced MASH. Histopathological analyses are employed to monitor differential disease progression. Molecular mechanisms are assessed within an in vitro obesogenic system utilising western blotting and flow cytometry. Human relevance is examined through transcriptomic analyses of patients with MASH.

Results

Using a p53 reporter mouse, we report early and sustained activation of hepatic p53 in response to a high-fat and high-sugar diet (p <0.05 at 100 days in males, p <0.001 at 200 days in females). Liver-specific loss of p53 accelerates progression of benign fatty liver disease to MASH, which is characterised by high levels of reactive oxygen species, extensive fibrosis, and chronic inflammation (all p <0.0001, n = 13 per high-fat high-sugar group). Our findings indicate that p53 induces the antioxidant gene TP53-induced glycolysis and apoptosis regulator (TIGAR) in vivo and in vitro. We show that loss of TIGAR exacerbates lipid peroxidation during MASH development in vivo (p <0.001) and that TIGAR is engaged in human MASH (p <0.001).

Conclusions

Our work demonstrates an important role for the p53-TIGAR axis in protecting against MASH and implicates redox control as a barrier against disease progression that is therapeutically targetable.

Impact and implications

p53 is of intense interest as a potent tumour suppressor and compounds targeting the pathway have been developed and trialled as anti-cancer therapies. Our findings suggest that early activation of p53 is similarly protective against metabolic dysfunction-associated steatohepatitis and that redox control is an important mediator of this protection. Further studies evaluating the efficacy of proactive activation of p53 in the liver to prevent metabolic dysfunction-associated steatohepatitis, or administration of targeted antioxidants to augment p53 redox protection, could provide new treatment approaches for a condition with few approved therapies.

Graphical abstract

Highlights

• •Loss of hepatic Trp53 promotes progression from MASLD to MASH with elevated lipid peroxidation, fibrosis, and chronic inflammation.
• •p53, in part through TIGAR, supports hepatic redox control in response to high-fat and high-sugar conditions.
• •The antioxidant N-acetyl cysteine normalises elevated lipid peroxidation levels present in TIGAR-deficient obese mice.

Introduction

Liver cancer (specifically hepatocellular carcinoma [HCC]) is a common and lethal disease with limited treatment options and increasing prevalence.^1,2 HCC arising from metabolic dysfunction-associated steatohepatitis (MASH, formerly called non-alcoholic steatohepatitis [NASH]³) represents the fastest growing fraction of HCC mortality globally and has become the leading cause of HCC in some Western countries, replacing viral hepatitis.^2,4,5 Liver disease progression from benign metabolic dysfunction-associated steatotic liver disease (MASLD, formerly called non-alcoholic fatty liver disease [NAFLD]³) to diet-induced MASH is strongly linked with the consumption of a high-fat and high-sugar diet as well as with features of metabolic syndrome (MetS), including obesity, insulin resistance, hypertension, and hyperlipidaemia.[6], [7], [8], [9] Of concern, MASH has especially high prevalence in patients with co-morbid type 2 diabetes mellitus (T2DM). Disease progression is particularly rapid in this cohort, and the development of HCC is more common.¹⁰

Characteristics of MASH include chronic inflammation, fibrosis, and high levels of reactive oxygen species (ROS), which overlap with hallmarks of cancer.^8,9,[11], [12], [13] This connection is reinforced by data from patients with MASH, where cancer-promoting and tumour-suppressive pathways are active, including the classic tumour suppressor protein TP53 (p53).[14], [15], [16], [17], [18], [19], [20], [21] Consistent with these observations, p53 has also been implicated in the aetiology of MetS more broadly. The p53 proline 72 to arginine (P72R) polymorphism, for example, has been reported to promote metabolic dysfunction in a humanised mouse model²² and is associated with increased T2DM sequelae in humans.²³

The p53 transcription factor coordinates a diverse cellular stress response to balance adaptation and cell survival against senescence and cell death.[24], [25], [26], [27], [28] As in many tissues, both unrestrained activation of p53 and the absence of p53 in the liver can be lethal.^29,30 Outside of these severe cases, however, context is an important mediator of p53 activity in the liver. On the one hand, undue p53 pathway activation has been shown to impede liver regeneration, promote fibrosis, and support HCC development following chronic liver damage.[31], [32], [33], [34], [35] p53-mediated apoptosis has also been reported to promote MASH in a nutritional stress-induced methionine and choline-deficient diet model.³⁶ Even so, p53 can also exert clear hepatoprotective activity. During damage-induced liver regeneration, for example, p53 has been shown to limit inflammation and fibrosis, protect against fatty acid-induced apoptosis, act to maintain mitotic fidelity, and promote detoxification of damaging lipid peroxides.[37], [38], [39], [40], [41], [42]

Redox control is an important aspect of p53 function more broadly and is commonly coordinated by downstream antioxidant effector proteins including TP53-inducible glycolysis and apoptosis regulator (TIGAR), amongst many others.²⁵ TIGAR is a fructose-2,6-bisphosphatase that acts in this capacity to limit accumulation of fructose-2,6-bisphosphate, a potent allosteric activator of phosphofructokinase-1.^43,44 Consequently, TIGAR activation reduces glycolytic flux and promotes the generation of NADPH in support of redox robustness.[43], [44], [45] Functionally, TIGAR has been shown to support intestinal regeneration and promote tumorigenesis, in part by enhancing the detoxification of peroxidised lipids and keeping ROS levels low.⁴⁶

Our work demonstrates that p53-mediated redox control protects against the development of MASH in vivo. We have further identified TIGAR as an important mediator of protective p53 activity in this context and provide evidence that antioxidant therapy can ameliorate features of MASH.

Materials and methods

Mice

Procedures involving mice were performed under Home Office licence numbers 70/8645, 70/8468, PP6345023 and PP1389725. Experiments were conducted in accordance with the Animals (Scientific Procedures) Act 1986 and the EU Directive 2010 and were sanctioned by Local Ethical Review Process (University of Glasgow) and The Francis Crick Institute.

p53^FL/FL (Trp53^tm1Brn), Albumin-Cre (Speer6-ps1^{Tg(Alb-cre)21Mgn}), PG13-iRFP (Hprt^{Tm1(p53RE-iRFP [pg13])Bea}), and Tigar^-/- mice were described previously[46], [47], [48], [49] and were fully backcrossed to C57BL6/J (N10). Lep^ob(B6.Cg-Lep^ob/J) and Lepr^db (BKS.Cg-Dock7^m+/+Lepr^dbJ) heterozygous mice were purchased from Charles River (Strain codes: 606 and 607). Albumin-Cre; Trp53^FL/FL compound mice were previously described.⁴¹ Lep^ob/ob; Albumin-Cre; Trp53^FL/FL mice were generated by interbreeding Lep^ob/WT and Albumin-Cre; Trp53^FL/FL mice. Lepr^db/db; Tigar^-/- mice were generated by interbreeding Lepr^db/WT and Tigar^WT/- mice.

For further information, see supplementary methods.

Long-term diet experiments

Male 65–75-day old mice were shifted onto either an obesogenic high-fat and high-sugar (HFHS) diet or remained on normal mouse chow (control diet) for up to 1 year. HFHS diet (AIN-76A, 58R3) was purchased from TestDiet and contained 59% energy from fat, 15% energy from protein, 25% energy from carbohydrates, was rich in sucrose, and did not contain cholesterol. The control normal mouse chow (DS801752G10R, Special Diet Services/SDS) contained 9% energy from fat, 21% energy from protein, 70% energy from carbohydrates and reduced sucrose.

For further information, see supplementary methods.

In vivo imaging

Throughout long-term diet experiments, cohort mice were imaged longitudinally on a Pearl Impulse Small Animal Imaging System (LI-COR) as previously described.⁵⁰ For further information, see supplementary methods.

Histology and immunohistochemistry

Staining for H&E, p21, Picrosirius red (PSR) and Oil red O (ORO) were performed as previously described.⁴¹ F4/80 (ab6640, Abcam) and malondialdehyde (MDA) (ab243066, Abcam) immunohistochemical (IHC) staining were performed on a Leica Bond Rx autostainer as previously described for p21.⁴¹

For further information, see supplementary methods.

Staining quantification

All sections except for those from Oil red O-stained slides were scanned at 20x magnification using a Leica Aperio AT2 slide scanner (Leica Microsystems, UK) prior to subsequent analysis. Staining quantification was undertaken utilising QuPath software (v0.5.1).⁵¹ Analyses were conducted on a random order of samples blinded to the genotype and treatment regimen of a given sample until the summation of results.

For further information, see supplementary methods.

Cell culture

HepG2 (HB-8065) cells were obtained from ATCC and Hep53.4 cells (Cellosaurus CVCL_5765) were obtained from Cytion (Product number 400200). HepG2 TP53 knockout (p53 KO) cells were generated through CRISPR-mediated targeting of TP53 as previously described.⁵² Stock flasks were maintained in DMEM (low glucose) (Gibco cat# 11880036) supplemented with 2 mM L-Glutamine (Gibco cat# 25030032), penicillin/streptomycin (Gibco cat# 15070063), and 5% FBS (Merck cat# F7524). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂.

For further information, see supplementary methods.

Transfection with siRNA

Studies utilising small-interfering RNA (siRNA) knockdown were performed as previously described⁵² using ON-TARGETplus SMARTpool constructs (Horizon Discovery Biosciences). For further information, see supplementary methods.

In vitro HFHS medium formulation

To create the HFHS medium formulation, a concentrated lipid mixture stock solution of fatty acids conjugated to fatty acid-free BSA (10% solution in PBS) (Merck cat# A1595) was created as previously described.⁵³ The final concentrations of conjugated fatty acids used in the HFHS medium were: 150 μM palmitate (Merck cat# P5585), 150 μM stearic acid (Thermo Scientific cat# A12244.06), 25 μM linoleic acid (Merck cat# L9530), 2.8 μM linolenic acid (Thermo Scientific cat# 215040050), 0.14 μM oleic acid (Thermo Scientific cat# 270290050) and 2 μM arachidonic acid (Sigma, cat# 10931). These amounts were chosen to broadly match the lipid distribution found in the murine HFHS diet. HFHS medium was further supplemented with 20 mM D-Glucose (Merck cat# G7528) and 25 mM D-Fructose (Thermo Scientific cat# A17718.30).

For further information, see supplementary methods.

Flow cytometry

Cells were labelled with CellROX Deep Red reagent (7.5 μM, Thermo Fisher Scientific cat# C10422) and BODIPY 493/503 (5 μM, Thermo Fisher Scientific cat# D3922). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Merck cat# D9542) was added to a final concentration of 1 μg/ml to each sample and was used to identify viable cells for analysis on a MACSQuant Analyzer 10 (Miltenyi Biotec). FlowJo 10.10.0 (BD Biosciences) was utilised for downstream analysis.

For further information, see supplementary methods.

Western blotting

Western blotting was performed as previously described.⁵² For further information, see supplementary methods.

Analysis of publicly available transcriptomics datasets

Human and mouse RNA-seq datasets were accessed through the NCBI Gene Expression Omnibus (GEO) portal via GEO accession numbers: GSE135251, GSE130970, and GSE35961.^16,54,55 Analysis of TIGAR/Tigar gene expression was completed using the built-in NCBI GEO2R analysis platform and sample groups were defined based on included information from each respective submission. In each of the three datasets, TIGAR/Tigar expression was significantly differentially expressed according to the p values included in the data associated with each GEO submission. Further analyses were completed using GraphPad Prism.

Data plotting and statistical analysis

Data were plotted using GraphPad Prism 10 (GraphPad) and presented as mean with min/max floating bars and individual data points (Figs 1 and S1) or mean +/- SEM with individual data points (Fig. 2 onwards). Statistical tests were chosen based on standard tests utilised in the field and are specified within each figure legend. Underlying assumptions for these tests were assumed to be met although not explicitly examined. Comparisons examining two independent variables, such as diet and Trp53 status, utilised two-way ANOVA analyses with Holm-Sidak’s multiple comparisons testing and multiplicity-adjusted p values. Where one independent variable was examined, one-way ANOVA analyses or an unpaired two-tailed t-test with Welch’s correction was performed based on the number of groups. Survival curve analysis was conducted using Log-rank (Mantel-Cox) testing. p values are denoted as follows: n.s., not significant, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Figures were prepared using Adobe Illustrator 2024 (Adobe).

Fig. 1.

Liver p53 is engaged in response to a high-fat and high-sugar diet.

(A,B) Male P53rep mice were given an obesogenic HFHS diet at 65-75 days of age or put onto a comparable purified low-fat control diet (chow) and imaged after the indicated days. Representative images (A) and P53rep signal quantification (B) normalised to the initial liver signal identified per mouse. n = 12 chow and n = 13 HFHS mice at 0 days, 50 days, 100 days; n = 7 chow and n = 10 HFHS at 150 days; n = 7 chow and n = 7 HFHS at 200 days, 250 days, 300 days. Data presented as mean +/- range with individual data points and analysed using a two-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. (C,D) Representative images (C) and quantifications (D) of female mice on same diet and imaging regimen as in (A,B). n = 9 chow and n = 8 HFHS mice for 0 days, 50 days, 100 days; n = 6 chow and n = 6 HFHS for 150 days, n = 6 chow and n = 5 HFHS at 200 days; and n = 6 chow and n = 4 HFHS for 250 days and 300 days. Data presented and analysed as (B). (E,F) Representative images (E) and quantifications (F) of ex vivo P53rep iRFP signal in tissues after 100 days on diet. Organ layout and LUT intensity profile as shown. P53rep intensity normalised to kidney signal in each mouse. n = 1 male and n = 3 female mice in the chow group and n = 3 male and n = 2 female mice in the HFHS group. Data presented as mean +/- SEM and analysed using two-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. (G) Quantification of IHC staining for CDKN1A/P21 in P53rep mice after 100 days on either HFHS or control (chow) diet. N-numbers and data presentation as in (E,F). Data analysed using an unpaired t-test with Welch’s correction.

n.s., not significant, ∗p <0.05, ∗∗p <0.01, ∗∗∗∗p <0.0001. HFHS, high-fat and high-sugar; IHC, immunohistochemical; P/S, pancreas/spleen.

Fig. 2.

Liver-specific loss of p53 accelerates diet-induced MASH.

(A) Representative liver H&E images and staining for p21, lipid content (ORO), lipid peroxidation (MDA), fibrosis (PSR), and macrophages (F4/80) in male Albumin-Cre+; Trp53 WT (Trp53^WT) and Albumin-Cre+; Trp53^FL/FL (Trp53^livΔ) mice after 1 year on control chow diet or obesogenic HFHS diet. ORO images are representative of n = 4 Trp53^WT chow-fed mice, n = 3 Trp53^livΔ chow-fed mice and n = 3 HFHS-fed mice per genotype; scale bars 40 μm. For the remainder, representative of n = 9 Trp53^WT chow-fed mice, n = 8 Trp53^livΔ chow-fed mice and n = 13 HFHS-fed mice per genotype. Scale bars 20 μm. Arrows denote p21-positive hepatocytes or F4/80-positive macrophage-engulfed hepatocytes (lipogranulomas). (B-E) Quantification of p21-positive hepatocytes per field (B), ORO stain area (C), MDA-positive stain area (D), PSR stain area (E), and F4/80-positive lipogranulomas per field (F) in mice from (A). WT: Trp53^WT, FL: Trp53^livΔ. N-numbers as in (A). Data presented as mean +/- SEM and analysed using two-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. n.s., not significant, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. HFHS, high-fat and high-sugar; MDA, malondialdehyde; ORO, Oil red O; PSR, Picrosirius red; ROS, reactive oxygen species; WT, wild-type.

Results

Liver p53 is engaged in response to a high-fat and high-sugar diet

In humans, increased expression of either TP53 or the p53 target gene CDKN1A/p21 (p21) is correlated with MASH, liver fibrosis, and T2DM.^20,21 We have previously utilised a p53 reporter mouse to non-invasively monitor the p53 pathway after total body gamma-irradiation and during hepatotoxin-mediated liver regeneration.⁴⁹ In p53 reporter mice, active p53 induces expression of the near-infrared fluorescent protein iRFP713 (iRFP) that is linked to the synthetic PG13 p53-responsive promoter. The resulting iRFP fluorescence can be monitored non-invasively and longitudinally (Fig. S1A).⁴⁹

To explore the dynamics of p53 activity during diet-induced liver disease in greater detail, male and female p53 reporter mice were shifted onto either a purified HFHS diet⁵⁶ or a comparable purified low-fat control diet, both of which are suitable for imaging. Mice were maintained on either diet for a period of up to 300 days and regularly monitored for weight gain and iRFP expression from the p53 reporter (Figs. 1A-D and S1B-D). As expected, both male and female mice exhibited weight gain on the HFHS diet relative to mice on the control diet (chow) within the first 50 days (Fig. S1C and D). Consistent with reports linking p53 activity with MASH,^20,21,36 we observed a robust signal from the p53 reporter in both male and female mice by the end of our study (Figs. 1A–D and S1B). Interestingly, there was also significant and sustained activation of the p53 reporter within male mice beginning much earlier, starting at 100 days on the HFHS diet and increasing thereafter (Fig. 1A,B). At this timepoint, although HFHS-fed mice of both sexes had gained significant weight, they did not exhibit changes in oral glucose tolerance (Fig. S1E and F), suggesting that diet-induced MetS was still in its early stages.

Although it is possible to differentiate the spatial distribution of iRFP expression on whole-body scans, for example between liver- and gut region-specific signals, organ overlap within a region of interest can sometimes obfuscate the source of the iRFP signal within the two-dimensional images. Ex vivo analysis of organs is unambiguous, however, and confirmed that p53 induction was restricted to the liver in HFHS-fed mice sampled after 100 days (Fig. 1E,F). This was validated by IHC staining for p21, confirming diet-dependent induction of the p53 pathway at this time point (Figs. 1G and S1G,H).

Together, these findings reveal that p53 induction is an early and sustained feature of the hepatic response to chronic consumption of a HFHS diet. Although male and female mice both induce p53, its induction is particularly rapid and robust in male mice in vivo.

Liver-specific loss of p53 accelerates diet-induced MASH

Within the liver, p53 activity can be protective or harmful, determined in part by the extent and duration of underlying liver damage.³⁴ To explore potential functional roles for p53 activity during diet-induced liver disease, we created cohorts of mice harbouring liver-specific deletion of Trp53 (Albumin-Cre+; Trp53^FL/FL/Trp53^livΔ mice) and proceeded to characterise the long-term response to the HFHS diet within this model. Importantly, Trp53^livΔ mice retain wild-type p53 expression in all tissues except the liver. They are therefore not susceptible to developing cancer before approximately 2 years of age, as previously reported,⁴¹ and allow for long-term studies into the effects of p53 loss on liver biology. Based on our findings in Fig. 1, we focused on examining the response to the HFHS diet within male mice in these experiments to provide the best signal-to-noise resolution of potential aspects of p53 function.

In Trp53^livΔ male mice, we observed clear signs of MASH after a period of 1 year on the HFHS diet that remained largely absent in Albumin-Cre+; Trp53 wild-type (Trp53^WT) mice fed the same diet (Figs. 2A–F and S2A,B). Consistent with our observations in p53 reporter mice, we observed increased expression of p21 within HFHS-fed Trp53^WT mice that did not occur in Trp53^livΔ mice (Figs. 2A,B). Instead, HFHS-fed Trp53^livΔ mice exhibited histopathological features consistent with MASH, including abundant steatosis, hepatocyte ballooning, the accumulation of Mallory-Denk bodies, hepatic hypertrophy, and inflammatory infiltrates in the liver that were largely absent within HFHS-fed Trp53^WT mice (Figs. 2A and S2A). MASH within HFHS-fed Trp53^livΔ mice was further characterised by significantly increased IHC staining for MDA, a marker of lipid peroxidation, substantial staining for PSR, a marker of fibrosis, and a clear increase in the presence of hepatic lipogranulomas, consistent with increased chronic inflammation (Figs. 2A–F and S2B). This was not the case in HFHS-fed Trp53^WT mice, where features of MASLD such as lipid accumulation were evident to a similar extent (Figs. 2A,C), but with significantly less staining for MDA, PSR, or lipogranulomas observed. Based on these findings, we concluded that p53 activity can act to oppose MASLD to MASH progression.

Loss of liver p53 is lethal in the Lep^ob/ob leptin-deficient genetic model of obesity

Leptin-deficient Lep^ob/ob (ob/ob) mice develop progressive liver disease with features of MASLD from an early age that progresses to include evidence of MASH within 20-30 weeks as a consequence of chronic hyperphagia.^57,58 To examine whether p53 also exerts protective activity against liver disease within this context, we created cohorts of male and female ob/ob mice harbouring liver-specific deletion of Trp53 (ob/ob; Albumin-Cre+; Trp53^FL/FL/ob/ob; Trp53^livΔ) and proceeded to assess liver disease development within this model compared with Trp53 wild-type ob/ob mice (ob/ob; Albumin-Cre+/ob/ob; Trp53^WT).

Consistent with published reports,⁵⁷ we observed significant steatosis in our ob/ob; Trp53^WT mice but this did not appreciably progress to MASH within the study period (Fig. 3A). Unexpectedly, however, the loss of liver Trp53 was lethal in male and female ob/ob; Trp53^livΔ mice (Fig. S3A and B). In comparison to ob/ob; Trp53^WT mice, where all cohort mice survived to the study endpoint, a median survival of just 319 days was observed in ob/ob; Trp53^livΔ mice (Fig. S3A and B). Survival within this cohort appeared to be independent of sex, with both male and female mice exhibiting a median survival based on humane clinical endpoint of between 295 days of age (male) and 319 days (female). Histopathological analysis of endpoint samples confirmed a preponderance of liver tumours within ob/ob; Trp53^livΔ mice (n = 8/10 with evidence of liver tumours at necropsy with n = 5/10 confirmed to have significant tumour burden by histopathology), consistent with a strong liver-related phenotype.

Fig. 3.

Loss of liver p53 is lethal in leptin-deficient (Lep^ob/ob) obese mice. (A) Representative liver H&E images and staining for MDA, PSR, and F4/80 in Albumin-Cre+; Lep^ob/ob; Trp53 WT (ob/ob; Trp53^WT) (n = 14 total: n = 8 male and n = 6 female) and Albumin-Cre+; Lep^ob/ob; Trp53^FL/FL (ob/ob; Trp53^livΔ) (n = 10 total: n = 4 male and n = 6 female) mice. ob/ob; Trp53^livΔ mice were sampled when reaching endpoint due to clinical signs of moderate severity. Of these, n = 5/10 had liver tumours that were confirmed by histology and n = 5/10 had small tumours or lacked macroscopic tumour burden, as reflected in downstream analyses. ob/ob; Trp53^WT mice were all healthy at endpoint and sampled at timepoints consistent with the observed survival of ob/ob; Trp53^FL/FL mice, in a time range from 269-323 days of age. Macrophage-engulfed steatotic hepatocytes (lipogranulomas) depicted by arrows. Scale bars 20 μm. (B-E) Quantification of liver images from (A), including MDA-positive stain area (B), PSR stain area (C), F4/80-positive lipogranulomas per field (D), and total F4/80-positive staining (E) in mice from (A). WT: ob/ob; Trp53^WT, FL: ob/ob; Trp53^livΔ. N-numbers as in (A). Data presented as mean +/- SEM and analysed using an ordinary one-way ANOVA and Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. n.s., not significant, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. Liv, liver; MDA, malondialdehyde; PSR, Picrosirius red; Tum, tumour; WT, wild-type.

As with Trp53^livΔ HFHS-fed mice (Fig. 2), liver sections from ob/ob; Trp53^livΔ mice at endpoint were characterised by elevated IHC staining for MDA (Fig. 3A,B). Liver tumours from ob/ob; Trp53^livΔ mice also exhibited extensive fibrosis (Fig. 3A,C). Unlike in the HFHS diet model, however, ob/ob; Trp53^livΔ tumour mice had fewer lipogranulomas than age-matched ob/ob; Trp53^WT mice, although overall macrophage infiltration was significantly increased (Figs. 3A and 3D,E). These potentially related findings could be due to differences in macrophage response to MASLD/MASH in the ob/ob model compared with diet-induced disease, which would be in accordance with previous studies.⁵⁷ Collectively, these results suggest that, as in diet-induced liver disease, liver Trp53 acts to limit MASLD to MASH progression in the ob/ob leptin-deficient genetic model of obesity in vivo.

p53 engages TIGAR and supports redox control in response to nutrient excess

Comparison of our findings across diet-induced and genetic models of MASLD highlighted diminished redox control as one of the common connecting characteristics. Pursuing this thread further within human MASH, we found evidence for increased expression of TIGAR, a key mediator of p53 redox regulation, in publicly available transcriptomic data from patients with MASH characterised by elevated fibrosis score (F2–F4)⁵⁴ or a high NAFLD activity score (NAS 5-6)¹⁶ compared with less severe MASH, MASLD, or control tissue (Figs. 4A and S4A). Tigar expression was similarly elevated in MASLD/MASH compared with normal liver within a murine dataset comparing the effects of 8 weeks of a methionine and choline-deficient high-fat diet with normal chow (Fig. S4B).⁵⁵

Fig. 4.

p53 engages TIGAR and supports redox control in response to nutrient excess. (A) Analysis of TIGAR gene expression in the public transcriptomics dataset GSE135251 comparing a control group, patients with MASLD/MASH with low fibrosis scores (F0/F1), and patients with MASH with high fibrosis scores (F2–F4). n = 10 control, n = 85 F0/F1, and n = 121 F2–F4 samples. Data presented in transcripts per kilobase million (TPKM) and analysed using an ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values. (B,C) Western blot analysis (B) and quantification (C) of TIGAR, CDKN1A/p21, and MDM2 expression in TP53 WT and TP53-deficient HepG2 cells cultured in baseline medium supplemented with BSA or HFHS medium formulation for 48 h n = 8 independent experiments. COXIV used as loading control. TIGAR abundance represents sum of bands. Protein MW ladder (in kDa) as depicted. Data presented as mean +/- SEM with data points and analysed by two-way ANOVA with Holm-Sidak’s multiple comparisons test and multiplicity-adjusted p values.

(D,E) Western blot analysis (D) and quantification (E) of TIGAR, p21, and MDM2 expression in Trp53 WT Hep53.4 HCC cells as in (B,C). n = 7 independent experiments. Data presented and analysed as in (B,C). (F) Analysis of ROS (CellROX Deep Red) via flow cytometry in TP53 WT and TP53 KO HepG2 cell lines cultured in same media formulations as (B,C). n = 5 independent experiments. Data presented as MFI relative to WT BSA treatment with individual data points per experiment. Data analysed as in (C).

(G,H) Analysis of ROS (CellROX Deep Red) via flow cytometry in HepG2 (G) or Hep53.4 (H) cells treated with either non-targeting control (NT) or siRNA against TP53/Trp53 (p53) or TIGAR/Tigar (TIG) for 96 h and cultured as in (B/C). n = 7 independent experiments for (G). Data from n = 7 experiments for NT and Trp53 siRNA and n = 3 for Tigar siRNA for (H). Data presented and analysed as in (F). (I,J) Western blot analysis (I) and quantification (J) of TIGAR expression in liver lysates created from Trp53^WT (WT) and Trp53^livΔ (FL) mice after 1 year of HFHS diet or control (chow) (as in Fig. 2). n = 4 Trp53^WT chow-fed mice, n = 6 Trp53^WT HFHS-fed mice, n = 6 Trp53^livΔ chow-fed mice and n = 7 Trp53^livΔ HFHS-fed mice. ACTIN used as loading control. TIGAR abundance represents sum of bands. Protein MW ladder (in kDa) as depicted. Data presented and analysed as in (C), comparing TIGAR expression between chow and HFHS samples within each liver Trp53 group. (K) Representative H&E images and staining for MDA in female Lepr^db/db (db/db; Tigar^WT) and Lepr^db/db; Tigar^-/-(db/db; Tigar^-/-) mice at 100 days of age. Mice were either provided with normal water throughout or treated with NAC in the drinking water ad libitum from 42 days of age (+NAC). Representative of n = 6 Tigar WT; db/db and n = 10 Tigar^-/-; db/db mice on normal water and n = 8 NAC-treated mice per cohort. Scale bars 20 μm. (L) Quantification of MDA to assess lipid peroxidation in mice from (K). N-numbers as in (K). WT: db/db; Tigar^WT, KO: db/db; Tigar^-/-, CHOW: mice with normal drinking water, +NAC: mice treated with NAC drinking water. Data presented and analysed as in (B/C).

n.s., not significant, ∗p <0.05 ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. HFHS, high-fat and high-sugar; KO, knockout; MDA, malondialdehyde; MFI, median fluorescence intensity; MW, molecular weight; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; siRNA, small-interfering RNA; WT, wild-type.

To explore these observations further in vitro, we turned to human (HepG2) and murine (Hep53.4) HCC cell lines that maintain wild-type p53.^41,59 Treatment of either cell line, but not HepG2 cells with CRISPR-mediated knockout of TP53 (TP53 KO), with medium formulated to model the composition of our HFHS murine diet increased expression of the p53 target genes p21 and MDM2 as well as TIGAR (Fig. 4B-E). Functionally, although TP53 WT and TP53 KO HepG2 cells accumulated similar amounts of lipid after 48 h of growth within HFHS medium, HepG2 TP53 KO cells exhibited significantly increased levels of ROS that were not observed within HFHS-treated HepG2 TP53 WT cells (Figs. 4F and S4C). This response was also observed after siRNA-mediated depletion of TP53/Trp53 in HepG2 or Hep53.4 cells (Figs. 4G,H and S4D-I). Treatment with the antioxidant N-acetyl-cysteine (NAC) was sufficient to restore redox control to HFHS-treated TP53 KO HepG2 cells without altering lipid accumulation, consistent with p53-mediated redox control supporting this process (Fig. S4J and K). Conversely, siRNA-mediated knockdown of TIGAR/Tigar in both HepG2 and Hep53.4 cells phenocopied CRISPR-mediated TP53 deletion, resulting in significantly increased ROS in HFHS conditions without altering lipid accumulation (Figs. 4G,H and S4F,I).

To expand these findings in vivo, we first confirmed that Tigar expression was elevated in our 1-year HFHS-fed Trp53^WT mice compared with HFHS-fed Trp53^livΔ mice (Fig. S4L). Consistent with our in vitro findings, we also observed increased TIGAR levels in 1-year HFHS-fed Trp53^WT mice compared with chow-fed controls – induction that was absent in HFHS-fed Trp53^livΔ mice (Figs. 4I,J). Even so, a subset of both chow and HFHS-fed Trp53^livΔ mice also exhibited elevated levels of TIGAR (Figs. 4I,J), consistent with previous reports describing p53-independent TIGAR induction.^60,61

Next, we sought to validate the importance of Tigar expression itself in MASLD. Since the leptin-deficient genetic model of obesity yielded a rapid and potent example of repercussions for liver Trp53 loss (Fig. 3), we decided to examine the consequences of Tigar loss in a similar setting. Unfortunately, we were not able to readily generate ob/ob; Tigar-deficient double homozygous mice, likely owing to genetic linkage arising from the fact that Tigar and leptin (Lep) share murine chromosome 6. Instead, we created a comparable system using leptin receptor-deficient (Lepr^db/db (db/db)) mice harbouring whole-body deletion of Tigar (db/db; Tigar^-/- mice). Considering the already rapid lethality that we observed in ob/ob; Trp53^livΔ mice (Fig. S3A), we decided to focus on liver disease development within cohorts of female db/db; Tigar^-/- mice as the lifespan of male db/db mice is markedly decreased compared with female mice in most strains.^58,62

As in the ob/ob model, db/db; Tigar^-/- female mice exhibited histopathological features of MASLD including steatosis and hepatocyte ballooning at 100 days of age alongside significantly increased IHC staining for MDA – indicative of elevated lipid peroxidation that was absent in db/db; Tigar^WT mice (Fig. 4K,L). NAC supplementation in the drinking water of db/db; Tigar^-/- mice contributed to normalised histology and reduced IHC staining for MDA in these animals, suggesting that antioxidant supplementation could also prevent increased lipid peroxidation in vivo. Together, these findings suggest that Tigar is important for limiting liver disease progression within the db/db genetic model and that targeting ROS directly with an antioxidant can alleviate detrimental effects of Tigar loss in vivo.

Discussion

During the transition from MASLD to MASH, the liver undergoes progressive rewiring of hepatic metabolism, significant inflammation, development of tissue damage, and engagement of cancer-associated signalling programmes.^8,9 Many of these attributes are also important hallmarks of cancer.¹¹ With this overlap in mind, we have interrogated the activity of the tumour suppressor protein p53 throughout MASH development. Our findings identify p53-mediated redox control as an important protective feature of the hepatic response to high fat and high sugar in vivo, suggest that TIGAR contributes to ROS detoxification, and highlight that the p53-TIGAR axis may be similarly engaged in human disease.

Across experimental models, we have found that loss of hepatic p53 is associated with worsening liver disease and high levels of lipid peroxidation. In Trp53/TP53 wild-type murine and human HCC cell lines, we show that the antioxidant gene Tigar/TIGAR is induced after HFHS treatment and supports redox control. The same is true in vivo, although interestingly, we also observed p53-independent TIGAR induction within a subset of liver Trp53-deficient mice. This observation may reflect reliance on additional antioxidant programmes, such as the NRF2 pathway which can also engage TIGAR,⁶¹ as compensation for Trp53 loss.

In response to HFHS medium, loss of either p53 or TIGAR exacerbates redox stress, which can be rescued with NAC supplementation, confirming the role of TIGAR in supporting p53-mediated ROS control. Consistent with these observations, in the db/db genetic model of obesity, we show that Tigar-deficient mice exhibit evidence of enhanced liver disease and increased lipid peroxidation from an early age that can also be rescued by NAC supplementation. Our findings support the conclusion that TIGAR-mediated redox control, likely alongside additional functions of p53, helps to protect the liver against deleterious consequences of chronic consumption of a HFHS diet. This protection is lost in the absence of liver p53 or TIGAR but can be partially restored with antioxidant treatment.

Our results add nuance to reports in mouse models and human patients where p53 induction and/or increased p53 pathway activity are associated with the progression of MASLD to MASH.^20,21,36,63 We also observe greater p53 activity in advanced vs. early stages of diet-induced liver disease. Nevertheless, induction of p53 in our reporter mice is clearly a much earlier feature of adaptation to HFHS diet stress, at least in male mice, and occurs prior to evidence of, for example, glucose intolerance or the onset of MASH. This observation raises an important question as to what aspect of MASLD aetiology drives early p53 activation. This is an area where more research is warranted. Equally, considering the observed disparity in p53 timing, it would be interesting to examine whether the noted sex differences we observed in p53 response reflect different underlying male/female p53 biology or are a consequence of the well-documented differing kinetics observed for MASH development in females.[64], [65], [66] Nevertheless, our findings suggest a dynamic interaction may exist between diet-induced stress and p53 activity. In this model, modest activation of p53 early in MASLD development may be sufficient to coordinate redox protection, but this could eventually switch into a pro-disease progression paradigm later when p53 signalling increases above a critical threshold and p53-mediated apoptosis or senescence, for example, is achieved.³⁶ Such a model would be consistent with more general literature on the threshold mechanisms inherent in determining the p53 response.⁶⁷

Focusing on this point further, it is tempting to propose metabolic zonation or oxygen tension as potential mediators of the p53 response during MASLD to MASH progression. Diet-induced steatosis begins pericentrally, as we observed within our HFHS diet model and as is reported in human patients.⁶⁸ This region of the liver maintains low oxygen tension compared with periportal regions of the liver.⁶⁸ Even mild hypoxia is known to alter p53 activity, including changing the regulation of p53-mediated apoptosis and altering the p53 response to ROS.^69,70 One of the main features of MASLD progression to MASH is the overall expansion of steatosis from a zonally-restricted localisation to a global distribution, with lipid accumulation, inflammation, and fibrosis occurring across the liver lobule, including within more oxygenated areas of the liver where p53 activity is normalised. Future work could examine this possibility, for example by mapping the spatio-temporal distribution of p53 activation throughout MASLD progression to MASH.

Taken together, our results underscore the importance of p53 and TIGAR for protecting the liver against damage associated with diet-induced liver disease. They also suggest that antioxidant interventions may have efficacy in preventing some of the deleterious aspects of MASH. Interestingly, considering that MetS is a multi-organ condition, our findings also suggest that nuances of the p53 response to diet stress are disease-state specific and potentially not shared across all metabolic organs.

Abbreviations

HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; NAFLD, non-alcoholic fatty liver disease; TP53, tumour protein p53 (human); Trp53, transformation related protein 53 (mouse); HFHS, high-fat and high-sugar; IHC, immunohistochemical; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; NADPH, nicotinamide adenine dinucleotide phosphate; MDA, malondialdehyde; NAS, NAFLD activity score; MetS, metabolic syndrome; NAC, N-acetyl-cysteine; PSR, Picrosirius red; ROS, reactive oxygen species; siRNA, small-interfering RNA; T2DM, type 2 diabetes mellitus; TIGAR, TP53-induced glycolysis and apoptosis regulator.

Financial support

This work was supported by Tenovus Scotland [S22-05], the United Kingdom Medical Research Council [MR/X018512/1], and the Academy of Medical Sciences (AMS) Springboard scheme [SBF008\1034], which is joint-funded by the AMS, the Wellcome Trust, the Government Department of Business, Energy and Industrial Strategy (BEIS), the British Heart Foundation and Diabetes UK. Additional support was provided by The Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2073), the United Kingdom Medical Research Council (CC2073), and the Wellcome Trust (CC2073), and the Cancer Research UK Scotland Institute which receives its core funding from Cancer Research UK grant C596/A17196 and A31287. KB also receives support from Cancer Research UK (A29799), both KB and DMW receive support from the UK Medical Research Council (MC_PC_21042) and KHV received support from Cancer Research UK (C596/26855) and the European Research Council (ERC-2020-ADG PancObese 10102064).

Authors’ contributions

CRediT (Contributor Roles Taxonomy) author statement: CIW: Methodology, Investigation, Formal Analysis, Writing- Original Draft Preparation, Writing- Review & Editing; ECC: Methodology, Investigation, Formal Analysis, Writing-Review & Editing; DA, NC, LB, MH, VM, DMW: Investigation, Writing- Review & Editing; KB, KHV: Conceptualization, Supervision, Funding acquisition, Writing- Review & Editing; TJH: Conceptualization, Investigation, Formal Analysis, Writing- Original Draft Preparation, Writing- Review & Editing, Supervision, Project Administration, Funding Acquisition.

Data availability statement

All data needed to evaluate the conclusions in the paper are present either within the paper itself or the included Supplementary Materials. Underlying raw data from this study are available from the corresponding author upon reasonable request.

Conflict of interest

KHV is on the board of directors and shareholder of Bristol Myers Squibb and on the science advisory board (with stock options) of PMV Pharma, RAZE Therapeutics, Volastra Pharmaceuticals and Kovina Therapeutics. She is on the scientific advisory board of Ludwig Cancer Research and is a co-founder and shareholder of Faeth Therapeutics. She has been in receipt of research funding from Astex Pharmaceuticals and AstraZeneca and contributed to the CRUK Cancer Research Technology filing of patent application WO/2017/144877. The other authors declare no competing interests.

Please refer to the accompanying ICMJE disclosure forms for further details.

Supplementary data

The following are the Supplementary data to this article:

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