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Department of Automation, Tsinghua University, Beijing 100084, ChinaInstitute for Brain and Cognitive Sciences, Tsinghua University, Beijing 100084, China
‡ These authors are co-senior authors and contributed equally.
Jing Li
Correspondence
Corresponding authors. Address: Department of Gastroenterology and Hepatology, Tongji Hospital, School of Medicine, Tongji University, 389 Xincun Road, Shanghai 200092, China; Tel.: +86-21-66111075, fax: +86-21-56050502.
‡ These authors are co-senior authors and contributed equally.
Changqing Yang
Correspondence
Corresponding authors. Address: Department of Gastroenterology and Hepatology, Tongji Hospital, School of Medicine, Tongji University, 389 Xincun Road, Shanghai 200092, China; Tel.: +86-21-66111075, fax: +86-21-56050502.
Intrahepatic Mas expression is upregulated in patients with DILI, and mice with APAP-induced liver injury.
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Mas1-/- mice are vulnerable to APAP-induced hepatotoxicity.
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Mas activation shows protective effects in hepatocytes by enhancing lipophagy and FAO, which are dependent on the AKT-FOXO1 axis.
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Mas arises as a novel therapeutic target for patients with APAP-induced liver injury.
Background & Aims
Acetaminophen (APAP) is the most common cause of drug-induced liver injury (DILI); however, treatment options are limited. Mas is a G protein-coupled receptor whose role in APAP-induced hepatotoxicity has not yet been examined.
Methods
Intrahepatic Mas expression was determined in both human and mouse DILI models. Mas1-/-, AlbcreMas1f/f, Ppara-/-, Mas1-/-Ppara-/- and wild-type mice were challenged with APAP for the in vivo analyses of Mas-AKT-FOXO1 axis-dependent lipophagy and fatty acid oxidation (FAO), using pharmacological compounds and genetic tools. Liver samples were collected for RNA-sequencing, proteomics, metabolomics, lipidomics, and metabolic flux analysis. Live-imaging of liver and histological, biochemical, and molecular studies were performed to evaluate APAP-induced hepatotoxicity in mice. Primary hepatocytes and hepatic cell lines were exposed to APAP for in vitro analysis.
Results
Intrahepatic Mas expression was significantly upregulated in human and mouse DILI models. Mice with systemic, liver-specific, or hepatocyte-specific Mas1 deficiency were vulnerable to APAP-induced hepatotoxicity. They exhibited substantially impaired lipophagy and downstream FAO, which was accompanied by the activation of AKT and suppression of FOXO1. In addition, the prophylactic activation of Mas conferred strong protection against APAP challenge in mice, with remarkably enhanced lipophagy and FAO dependent on the AKT-FOXO1 axis. Moreover, the protective effects of AVE0991 were substantially diminished by the inhibition of either lipophagy or FAO.
Conclusions
The activation of Mas on hepatocytes enhanced AKT-FOXO1-dependent lipophagy and downstream FAO, protecting mice from APAP-induced hepatotoxicity and indicating that hepatocyte-specific Mas might be a novel therapeutic target for DILI.
Impact and implications
Mas signalling arises as a novel therapeutic target for patients with APAP-induced liver injury. The Mas-AKT/FOXO1-fatty acid degradation pathway could be critical for the development of treatment strategies for APAP overdose. When Mas signalling is targeted, the extent of liver injury should be considered at the time of administration. These findings obtained from APAP-challenged mice still need to be confirmed in a clinical context.
The renin–angiotensin system (RAS), consists primarily of an enzymatic cascade in which angiotensinogen is converted to angiotensin (Ang) I and subsequently to Ang II by the actions of renin- and angiotensin-converting enzyme, respectively.1 Besides Ang II, other Ang peptides such as Ang-(1–7) may also have important biological activities. Ang-(1–7) is derived from Ang I and Ang II and activates the G protein-coupled receptor called Mas, which is widely expressed.2 The RAS, most well-known for being a potent regulator of blood pressure and hydromineral balance, is also involved in multiple physiological actions, including the regulation of key metabolic pathways, particularly related with glucose homeostasis and fatty acid metabolism.
Drug-induced liver injury (DILI) is a global primary health concern because it is the most common cause of acute liver failure (ALF) and the most frequently cited reason for drug withdrawal from the market.
Unfortunately, pharmacologic options are limited, providing an avenue for the discovery of new therapeutic targets. Acetaminophen (APAP) overdose, the leading cause of drug-induced ALF, is characterised by central lobular necrosis and substantially elevated transaminase levels. Owing to the availability of mouse models which are highly consistent with human pathophysiology, APAP is the most widely studied hepatotoxic drug. APAP toxicity occurs primarily due to its reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which rapidly depletes glutathione (GSH), impairs mitochondrial function, and induces nuclear DNA damage. Hepatocyte death subsequently activates Kupffer cells and induces hepatic infiltration of peripheral immune cells, further aggravating liver injury.
Mas specifically binds to angiotensin-(1-7), the major effector molecule of the non-classic renin-angiotensin system, which has recently been characterised as anti-inflammatory and anti-fibrotic. Mas is widely expressed in human and murine tissues and is especially enriched in monocyte-macrophages and the nervous system.
However, the role of Mas in DILI remains to be elucidated.
Macroautophagy is a process in which cells engulf and degrade damaged organelles and cytoplasmic macromolecules, providing ATP and substrates for subsequent cell metabolism. It is a recycling system widely present in eukaryotic cells and is usually upregulated under cellular stress. Lipophagy is a specific type of autophagy that selectively recognises lipid droplets (LDs) and efficiently integrates them into autophagosomes to degrade triglycerides (TGs) and release free fatty acids (FFAs),
providing substrates for fatty acid oxidation (FAO). In APAP-induced hepatotoxicity, enhanced autophagy is reportedly protective, as it reduces the production of reactive oxygen species by removing damaged mitochondria and APAP-protein adducts.
Notably, Mas is involved in both autophagy and lipid metabolism. The activation of Mas induces autophagy by inhibiting the PI3K-Akt-mTOR signalling pathway
Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.
Angiotensin-(1-7), the product of ACE2 ameliorates NAFLD by acting through its receptor Mas to regulate hepatic mitochondrial function and glycolipid metabolism.
This study has presented solid in vivo evidence of the protective role of activated hepatocyte Mas signalling in APAP overdose, through enhanced lipophagy and FAO and therefore suggests a novel therapeutic strategy for treating drug-induced ALF.
Materials and methods
Study design
To investigate whether and how Mas modulates APAP-induced hepatotoxicity, both in vivo and in vitro experiments were carried out in mice with systemic, liver-specific or hepatocyte-specific deficiency of Mas1, and in hepatocytes after APAP challenge, using pharmacological compounds and genetic tools. Detailed information on in vivo administration of small molecule agonists and inhibitors is provided in Table S2.
For further details regarding the materials and methods used, please refer to the CTAT table and supplementary information.
Results
Intrahepatic Mas expression is upregulated in human and mouse DILI
There was a significant upregulation of intrahepatic Mas in humans with DILI compared to healthy controls (Fig. 1A). Furthermore, multiplex immunohistochemistry (mIHC) revealed a wide expression pattern of Mas in both parenchymal (hepatocytes) and non-parenchymal cells, including monocytes, macrophages, neutrophils, hepatic stellate cells, and endothelial cells (Fig. 1B). Both protein (Fig. 1C) and mRNA (Fig. 1D) expression of intrahepatic Mas was found to be significantly upregulated in mice after challenge with different doses of APAP. The wide expression pattern of Mas was confirmed in multiple mouse tissues and cell types (Fig. 1E and Fig. S1A). Additionally, a remarkable upregulation of Mas mRNA was documented in both human and mouse hepatic cell lines after APAP challenge (Fig. S1B,C). Together, these findings suggest the possible involvement of Mas in DILI.
Fig. 1Intrahepatic expression of Mas in human and mouse drug-induced hepatotoxicity.
(A) Representative immunohistochemical staining and quantification of Mas in liver sections obtained from HC and DILI groups (both, n = 10). (B) Representative staining of Mas on hepatocytes (HNF4α+), HSCs (α-SMA+), endothelial cells (CD31+), macrophages (CD68+CD14-), neutrophils (CD16+CD68-CD14-) and monocytes (CD16+CD68+CD14+) by mIHC in human liver sections from HC and DILI groups. (C) Representative histological staining and quantification of Mas in liver sections from WT mice treated with vehicle or APAP (all, n = 4). (D) Intrahepatic mRNA expression of Mas1 in mice treated with vehicle or APAP (both, n = 4). (E) Representative staining of Mas on hepatocytes (HNF4α+), HSCs (α-SMA+, or GFAP+), endothelial cells (CD31+), macrophages (F4/80+), neutrophils (Ly6G+) and monocytes (CCR2+) by mIHC in liver sections from WT mice treated with vehicle or APAP. ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, One-way ANOVA with Tukey’s test). APAP, acetaminophen; DILI, drug-induced liver injury; HC, healthy control; WT, wild type.
Systemic Mas1 deficiency exacerbates APAP-induced hepatotoxicity in mice
To explore the role of Mas in vivo, the disease phenotype was compared between Mas1-/- and wild-type (WT) mice treated with APAP (300 mg/kg, 24 h). Interestingly, Mas1-/- mice showed substantial intolerance to APAP, as reflected by histological assessments of hepatocellular death and inflammatory infiltrates (Fig. 2A) and serum alanine aminotransferase levels (Fig. 2B). Consistently, when challenged with a lethal dose of APAP, Mas1-/- mice had a much lower survival rate (Fig. 2C). In addition, the dose- and time-dependent manner of APAP-induced hepatotoxicity was documented in Mas1-/- mice (Fig. S1F,H). A similar intolerance to APAP was observed in Mas1-/- mice under the fed state (Fig. S1G). Western blot (WB) revealed significantly higher levels of protein expression of markers for inflammation (IL-1β), mitochondrial stress (p-JNK), and cell death (cleaved-caspase3, Bax) in Mas1-/--APAP mouse livers (Fig. 2D). In transcriptomics, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analysis revealed substantially higher expression of genes enriched in the apoptosis, necrosis, ferroptosis, and inflammation pathways in the livers of Mas1-/- mice (Fig. 2E). Regarding the critical role of necroptosis in APAP-induced hepatotoxicity, as described,
the involvement of Mas signalling in necroptosis was also explored; however, insignificant findings were obtained (data not shown). Since APAP is metabolised into NAPQI by the cytochrome P450 isozyme CYP2E1 and further detoxified by GSH in the liver, the comparable levels of intrahepatic CYP2E1 and GSH between Mas1-/--APAP and WT-APAP mice (Fig. S1I) suggest that Mas is not involved in APAP metabolism. In APAP overdose, extensive necrosis results in sterile inflammation (SI) and recruitment of inflammatory cells. Here, intrahepatic neutrophils and macrophages in alive mice were monitored using digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT) intuitively (Fig. 2F). See Videos S1 and S2 for the hour-long details on immune cell behaviours, presented by time-lapse rendering volumes and quantitative comparisons. Both substantially enhanced activity (speed) and increased number of neutrophils were documented in the Mas1-/--APAP and WT-APAP groups.
Mice were challenged with APAP for 24 h (n = 6 per group). (A) Representative gross liver photographs, and intrahepatic staining with quantification (below) of H&E (necrotic areas circled with white lines), TUNEL (apoptosis), F4/80 (macrophages), and MPO (neutrophils). Arrows indicate positive staining. Scale bar: 100 μm. (B) Serum ALT. (C) Survival curves of WT and Mas1-/- mice treated with a lethal dose of APAP (650 mg/kg) (both, n = 12). (D) Representative immunoblots with quantification (below). (E) Heat maps show the intrahepatic expression of members of apoptosis, necrosis, ferroptosis, and inflammation pathways (n = 3, RNA-seq). (F) Maximum intensity projections of neutrophils (Ly6G) and macrophages (F4/80) in the vessels (WGA) of living mouse livers by DAOSLIMIT. Representative intravital images were captured at 3 and 22 h after APAP administration. The average migration speed of neutrophils, and temporal traces of their number during 3-4 h are shown. Scale bar: 50 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, One-way ANOVA, Log-rank test, and Mann-Whitney U test). ALT, alaninetransaminase; APAP, acetaminophen; WT, wild type.
Systemic Mas1 deficiency impairs lipophagy and FAO in APAP-challenged mice
Transcriptomics, proteomics, and metabolomics were used to explore the intrahepatic Mas signalling pathways in APAP overdose. The autophagy (FOXO, PI3K-Akt) and fatty acid degradation (PPAR, peroxisome) pathways were identified as the two most promising pathways, as reflected by KEGG analysis of differentially expressed mRNA and protein pairs (Fig. 3A), mRNAs (Fig. 3B), and metabolites (Fig. 3C). Heat maps (KEGG, transcriptomics) revealed downregulation of both the fatty acid degradation and autophagy pathways and upregulation of the lipid droplet pathway in the Mas1-/--APAP vs. WT-APAP groups (Fig. 3D). Consistently, gene ontology term analysis (transcriptomics) documented downregulation of fatty acid metabolic processes and peroxisomes and upregulation of inflammatory responses and neutrophil degranulation in Mas1-/--APAP vs. WT-APAP groups (Fig. S2A,B). Gene set-enrichment analysis also demonstrated the activation of the apoptosis and inflammation pathways and the suppression of fatty acid degradation and the autophagy and PPAR signalling pathways in Mas1-/--APAP vs. WT-APAP groups (Fig. S3A). Systemic Mas1 deficiency was suspected to impair autophagy and fatty acid degradation through the AKT-FOXO1 axis, in APAP overdose. To verify this assumption, we performed a series of analyses to determine the variations in Mas1-/--APAP livers compared to WT-APAP livers. First, by analysing the protein expression of corresponding markers, WB data suggested impaired autophagy (decreased LC3-II, ATG7, and LAMP1 with accumulated P62), lipolysis (decreased ATGL), and FAO (decreased ACOX1 and CPT1A), along with enhanced AKT (increased, activated, or phosphorylated AKT) and suppressed FOXO1 (decreased total and activated/acetylated FOXO1) signalling pathways in Mas1-/--APAP mice (Fig. 3E). Second, transmission electron microscopy (TEM) detected a significant decrease in the number of autophagic vacuoles (AVs) in Mas1-/--APAP mice (Fig. 3F,G). Third, immunofluorescence revealed a significant accumulation of LDs (BODIPY-positive dots) along with reduced lipophagy (BODIPY/LAMP1 co-localisation) and lipolysis (BODIPY/ATGL co-localisation) in Mas1-/--APAP mice (Fig. 3H-J). Fourth, a significant elevation in TG levels in Mas1-/--APAP mice was observed on biochemical assessment (Fig. 3K) and targeted lipidomics (Fig. S3B). Fifth, by determining the mRNA expression of genes involved in autophagy, FAO, lipolysis, and LD formation, qPCR analysis indicated impaired autophagy and FAO in Mas1-/--APAP mice (Fig. S3C). Taken together, the intolerance to APAP challenge exhibited by Mas1-/- mice may originate from insufficient lipophagy and FAO, possibly modulated by the AKT and FOXO1 signalling pathways.
Fig. 3Systemic Mas1 deficiency impairs lipophagy and FAO in APAP-induced mouse hepatotoxicity.
Mice livers were assessed by RNA-seq (n = 3), proteomics (n = 3) and metabolomics (n = 6). (A) The Spearman correlation pairs (p <0.05) generated using proteomics and RNA-seq data were rendered for further cluster analysis. KEGG pathway-enrichment analysis of differentially expressed pairs in WT-APAP and Mas1-/--APAP groups. The top 10 pathways (Fisher’s exact test) are presented. (B, C) KEGG pathway-enrichment analysis of differentially expressed genes (B, RNA-seq) and metabolites (C, metabolomics) in WT-APAP and Mas1-/--APAP groups. (D) Heat maps show the intrahepatic expression of members in fatty acid degradation, autophagy and lipid droplet formation pathways (RNA-seq). (E) Representative immunoblots with quantification. (F, G) Representative liver electron micrographs (F) with quantification (G) of AVs (black arrows). Scale bar: 2 μm. (H-J) Representative co-staining (H) of intrahepatic BODIPY/LAMP1 and BODIPY/ATGL. Nuclei were stained with DAPI. Scale bar: 10 μm. The quantification of LDs (BODIPY-positive dots) (I) and co-localisation (J) was carried out in at least 12 high-power fields (n = 4 per group). (K) Liver TG (n = 6). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; n.s., non-significant (Student’s t test, One-way ANOVA, Mann-Whitney U test). APAP, acetaminophen; TG, triglyceride; WT, wild type.
Prophylactic activation of Mas alleviates APAP-induced hepatotoxicity in mice by enhancing lipophagy and FAO
Mas was studied using AVE0991, a Mas activator. First, pre-administration of AVE0991 exerted incredible protective effects against APAP overdose, as evidenced by both histological and biochemical assessments (Fig. 4A,B). Moreover, AVE0991 substantially improved the survival rate of WT-APAP mice (Fig. 4C). In addition, AVE0991 significantly attenuated intrahepatic inflammation, cell death, and mitochondrial stress, as indicated by WB analysis (Fig. 4D). Furthermore, DAOSLIMIT demonstrated a negative effect of AVE0991 on liver-infiltrating neutrophils in APAP-treated mice (Fig. 4E). The dynamic behaviours of neutrophils with and without AVE0991 were compared in situ over 1 h (Videos S2-3). Unfortunately, due to the limitations of DAOSLIMIT, covering a narrow field of view that includes only a few monocytes, a conclusion could not be reached regarding monocytes (Fig. S4, Video S4). Additionally, AVE0991 exerted a dose-dependent prophylactic protective effect against APAP challenge in WT mice over different periods (Fig. S5A,C). AVE0991 exhibited similar effects in WT-APAP mice in the fed state (Fig. S5B). In this study, the specificity of AVE0991 for Mas was confirmed in Mas1-/--APAP mice (Fig. S5D). In addition, in the k-means clusters generated using transcriptomes, there was a substantially opposite trend in the gene expression profile between Mas1-/--APAP vs. WT-APAP and WT-APAP+AVE0991 vs. WT-APAP, indicating that Mas is the potential target of AVE0991 in APAP exposure (Fig. S4E). Subsequently, we focused on intrahepatic lipophagy and FAO to explore the mechanisms underlying the effects of AVE0991. The data demonstrated that AVE0991 significantly upregulated autophagy (TEM, Fig. 4F) and lipophagy (immunofluorescence, Fig. 4G) in WT-APAP mice, accompanied by the protein expression of markers of these processes (WB, Fig. 4H), suggesting rescued autophagy, lipolysis, and FAO, along with suppressed AKT and enhanced FOXO1 signalling. In transcriptomics, KEGG analysis showed enhanced fatty acid degradation and autophagy pathways and suppressed pathways of LD formation in WT-APAP+AVE0991 compared to WT-APAP mice (Fig. 4I). In metabolomics, AVE0991 administration was found to substantially increase the levels of monoacylglycerol (a neutral lipid product of triacylglycerol [TAG]), and phosphatidylethanolamine (PE), a phospholipid closely associated with autophagy (Fig. 4J). Targeted lipidomics was further applied to quantify intrahepatic TAG, DAG, FFA, and PE contents (Fig. 4K,L). These data collectively confirmed our speculation that AVE0991 may enhance the degradation of TAG to release FFAs for subsequent FAO. In the KEGG analysis of proteomics, the PPAR signalling pathway (the main regulator of key enzymes involved in FAO) was also identified among the top 20 upregulated pathways in WT-APAP+AVE0991 vs. WT-APAP mice (Fig. S5F), indicating the role of FAO in the AVE0991 mechanism. Consistently, in WT-APAP+AVE0991 vs. WT-APAP mouse livers, biochemical assessments detected decreased TG concentrations (Fig. S5G), while qPCR analysis documented mRNA expression of genes suggesting enhanced autophagy and FAO and suppressed formation of LDs (Fig. S5H). In this study, the potential crosstalk between PE and autophagy was preliminarily confirmed in Mas1-/--APAP mice using ethanolamine (the precursor of exogenous PE) (Fig. S5I-K). Notably, to obtain more direct evidence of FAO modulation induced by AVE0991, the metabolic flux of fatty acids was determined by the conversion of [U–13C]-palmitate to 13C-labelled acylcarnitine and acetyl-CoA (Fig. 4M). The data showed that AVE0991 significantly increased the in vivo turnover of fatty acids, as reflected by the significantly enhanced intrahepatic abundance of 13C-labelled palmitoyl-carnitine (C16), myristoyl-carnitine (C14), lauroyl-carnitine (C12), and acetyl-CoA (Fig. 4N–P). In summary, the prophylactic activation of Mas protects mice from APAP overdose by enhancing lipophagy and FAO.
Fig. 4Prophylactic activation of Mas induces lipophagy and FAO to ameliorate APAP-induced mouse hepatotoxicity.
Mice were pre-treated with or without AVE0991 before APAP challenge (n = 6 per group). (A) Representative liver photographs and intrahepatic staining with quantification of H&E, TUNEL, F4/80, and MPO. Scale bar: 100 μm. (B) Serum ALT. (C) Survival curves of WT mice treated with APAP (650 mg/kg) in the absence or presence of AVE0991 (n = 12). (D, H) Representative immunoblots with quantification. (E) Representative images by DAOSLIMIT with quantification of the neutrophil number and migration speed. Scale bar: 50 μm. (F) Representative liver electron micrographs and quantification of AVs. Scale bar: 2 μm. (G) Representative co-staining and quantification of BODIPY/LAMP1. Scale bar: 10 μm. (I) Heat maps show expression of members in fatty acid degradation, autophagy and lipid droplet pathways (RNA-seq). (J) Heat maps generated using metabolomics show levels of corresponding metabolites. (K, L) Heat maps generated using targeted lipidomics (n = 5) show levels of metabolites (K), and quantification (L). (M) Schematic diagram of U–13C-palmitate tracer administration. (N) Heatmap of metabolic flux depicting ratios of detected FAO and carnitine shuttle pathway-related metabolites. (O, P) The intrahepatic abundance of 13C-labelled (O) palmitoyl-carnitine (C16), myristoyl-carnitine (C14), lauroyl-carnitine (C12) and (P) acetyl-CoA assessed using LC/MS. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; n.s., non-significant (Student’s t test, Mann-Whitney U test, Log-rank test). ALT, alaninetransaminase; APAP, acetaminophen; AV, autophagic vacuole; DAG, Diacylglycerol; FFA, free fatty acid; MAG, monoglycerol; PE, phosphatidyl ethanolamine; TAG, triglyceride; TCA, tricarboxylic acid; TEM, transmission electron microscope; WT, wild type.
Lipophagy and FAO are critical players downstream of the prophylactic modulation of Mas in APAP-induced hepatotoxicity in mice
To determine the roles of lipophagy and FAO in Mas signalling after APAP challenge, we documented the beneficial roles of autophagy and FAO in WT-APAP mice by in vivo administration of rapamycin (RAPA, a classic autophagy inducer), chloroquine (CQ, an autophagy antagonist) (Fig. S6A), fenofibrate (a PPARα agonist), and lanifibranor (a pan-PPAR agonist) (Fig. S6B). Next, we enhanced both processes to determine whether they could reverse the disease phenotype of Mas1-/--APAP mice. Notably, the administration of RAPA, fenofibrate, and lanifibranor markedly alleviated APAP-induced hepatotoxicity (Fig. 5A,B). In addition, the pre-administration of etomoxir (an antagonist of FAO) significantly exacerbated liver injury in WT-APAP+AVE0991 mice (Fig. S6C,D). In Mas1-/--APAP mice, RAPA administration substantially enhanced intrahepatic lipophagy and FAO (as reflected by the upregulated mRNA (Fig. S7A) and protein (Fig. 5C) expression of the indicated markers of autophagy, lipolysis, and FAO and the increased AV number under TEM (Fig. 5D)), leading to decreased TGs (Fig. 5B) and LDs (Fig. 5E and Fig. S7A). Intriguingly, the substantially enhanced lipolysis and FAO (mRNA and protein expression of the indicated markers shown in Fig. 5F and Fig. S7B) induced by either fenofibrate or lanifibranor administration in Mas1-/--APAP mice was accompanied by a significant increase in AV number (Fig. 5D), suggesting a mutual dependence between the autophagy and FAO pathways.
Fig. 5Lipophagy and FAO are critical players downstream of the prophylactic modulation of Mas in APAP-induced mouse hepatotoxicity.
In A-G, mice were pre-treated with or without RAPA, fenofibrate or lanifibranor (n = 6 per group). (A, I) Representative liver photographs, and intrahepatic staining with quantification of H&E, TUNEL, and MPO. Scale bar: 100 μm. (B, J) Serum ALT and liver TG. (C, F) Representative immunoblots with quantification. (D) Representative liver electron micrographs with quantification of AVs. Scale bar: 2 μm. (E) Representative co-staining of BODIPY/LAMP1 with quantification of LDs. Scale bar: 10 μm. (G, L) Representative intrahepatic staining with quantification of H&E and TUNEL, and serum ALT. Scale bar: 100 μm. (H) Schematic diagram of AVE0991, CQ, and fenofibrate administration. (K) Representative co-staining and quantification of intrahepatic BODIPY/LAMP1. Scale bar: 10 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, One-way ANOVA, Mann-Whitney U test). ALT, alaninetransaminase; APAP, acetaminophen; AV, autophagic vacuole; CQ, chloroquine; RAPA, rapamycin; TG, triglyceride; TEM, transmission electron microscope; WT, wild type.
Since PPARα is the nuclear receptor that controls the expression of genes encoding peroxisomal and mitochondrial FAO enzymes, Mas1-/-Ppara-/- mice were generated to explore the upstream and downstream relationship between lipophagy and FAO in Mas signalling. Of note, Mas1-/-Ppara-/-vs. Mas1-/- mice showed a substantial resistance to the protective effect of RAPA (Fig 5G and Fig. S7C-E), suggesting that FAO acts downstream of lipophagy. Furthermore, AVE0991, CQ, and fenofibrate were administered to WT-APAP mice, as indicated (Fig. 5H); we found that, on APAP challenge, CQ substantially diminished the protective effects of AVE0991 and that subsequent fenofibrate administration substantially reversed the deleterious effects of CQ (Fig. 5I,J and Fig. S7F), indicating that autophagy is upstream of FAO. Consistently, the intrahepatic TG content (Fig. 5J) changed according to lipophagy activity (Fig. 5K) and the expression of markers of lipolysis, LDs, and FAO (Fig. S7F,G). In addition, Ppara-/- mice showed substantial resistance to the protective effects of AVE0991 against APAP overdose (Fig. 5L and Fig. S7H-J). In summary, in Mas signalling, lipophagy produces fatty acids as substrates to enhance downstream FAO.
AKT and FOXO1 are involved downstream of the prophylactic modulation of Mas in APAP-induced hepatotoxicity in mice
Recent studies have highlighted the role of FOXO1 in enhanced autophagy,
Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.
In this study, we identified FOXO1 and PI3K-AKT as the significant differentially expressed signalling pathways in Mas1-/--APAP and WT-APAP mice. In addition, enhanced AKT and suppressed FOXO1 were documented in Mas1-/--APAP mice, whereas WT-APAP+AVE0991 mice exhibited the opposite trend. The roles of AKT and FOXO1 were determined using perifosine (an inhibitor of p-AKT), AS1842856 (an inhibitor of FOXO1), and sirtinol (an inhibitor of deacetylation enzymes), and the intrahepatic processes of autophagy, lipolysis, and FAO were mainly evaluated by mRNA and protein expression of the indicated markers. In Mas1-/--APAP mice, perifosine substantially alleviated hepatotoxicity and TG accumulation, with increased total FOXO1 protein expression and enhanced autophagy, lipolysis, and FAO (Fig. 6A-C). In WT-APAP mice, AS1842856 substantially diminished the effects of AVE0991 and suppressed autophagy, lipolysis, and FAO (Fig. 6D-F). In contrast, sirtinol substantially reversed the disease phenotype of Mas1-/--APAP mice with increased protein levels of activated FOXO1 and enhanced autophagy, lipolysis, and FAO (Fig. 6G-I). In addition, enhanced lipophagy by sirtinol was confirmed by the co-localisation of BODIPY/LAMP1 (Fig. 6J). Therefore, after APAP challenge, Mas may modulate lipophagy and FAO through the negatively regulated AKT and FOXO1 signalling pathways.
Fig. 6AKT and FOXO1 are involved downstream of the prophylactic modulation of Mas in APAP-induced mouse hepatotoxicity.
Mice were pre-treated with or without perifosine (A-C), AS1842856 (D-F), or sirtinol (G-J) (n = 6 per group). (A, D, G) Representative intrahepatic staining with quantification of H&E, TUNEL and MPO. Serum ALT and liver TG. Scale bar: 100 μm. (B, E, H) Representative immunoblots with quantification. (C, F, I) Hepatic mRNA expression. (J) Representative co-staining and quantification of intrahepatic BODIPY/LAMP1. Scale bar: 10 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, Mann-Whitney U test).; ALT, alaninetransaminase; APAP, acetaminophen; H&E, hematoxylin-eosin; TG, triglyceride.
Intrahepatic and hepatocellular Mas1 deficiency exacerbate APAP-induced hepatotoxicity in mice
The roles of intrahepatic and hepatocellular Mas in APAP overdose were determined using adeno-associated virus 2/8-carrying short-hairpin (sh)Mas1 (Fig. S8A,B) and hepatocyte-specific Mas1-knockout (AlbcreMas1f/f) mice. We found that both WT-shMas1 (Fig. 7A-E) and AlbcreMas1f/f (Fig. 7F-I) mice exhibited a substantial intolerance to APAP challenge, as reflected by the aggravated hepatotoxicity accompanied by intrahepatic mRNA and protein expression of indicated markers, suggesting impaired autophagy, lipolysis, and FAO. Consistently, activated AKT and suppressed FOXO1 signalling pathways were also documented in WT-Sh-Mas1-APAP and AlbcreMas1f/f-APAP mice. In addition to the protein levels of ac-FOXO1, the activation of FOXO1 was evaluated by its nuclear translocation from the cytoplasm (Fig. S9A,B).
Fig. 7Intrahepatic and hepatocellular Mas deficiency exacerbate APAP-induced mouse hepatotoxicity.
WT mice were injected with AAV2/8 carrying shMas1 or shCtrl (n = 4 per group, A-E). Mas1f/f and AlbcreMas1f/f mice were used (n = 3 per group, F–I). AVE0991 was administrated prophylactically in A-I, while therapeutically in J-L. (A) Representative liver photographs and intrahepatic staining with quantification of H&E, TUNEL, and MPO. Scale bar: 100 μm. (B) Serum ALT and liver TG. (C, G) Hepatic mRNA expression. (D, H, K) Representative immunoblots with quantification. (E) Representative co-staining of intrahepatic BODIPY/LAMP1 and quantification of LDs. Scale bar: 10 μm. (F) Representative intrahepatic staining with quantification of H&E and TUNEL, and serum ALT. Scale bar: 100 μm. (I, L) Representative co-staining and quantification of intrahepatic BODIPY/LAMP1. Scale bar: 10 μm. (J) Representative intrahepatic staining with quantification of H&E (upper), and serum ALT (lower). Scale bar: 100 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, One-way ANOVA, Mann-Whitney U test). ALT, alaninetransaminase; APAP, acetaminophen; H&E, hematoxylin-eosin; Sh, short-hairpin; TG, triglyceride.
The therapeutic role of Mas activation in APAP-induced hepatotoxicity in mice
Considering the clinical significance of therapeutic administration in APAP overdose, AVE0991 was administered at different time points after APAP challenge. At 2 h post-APAP, AVE0991 still exerted protective effects against APAP overdose, as reflected by the substantially improved disease phenotype (Fig. 7J) and survival rate of WT-APAP mice (Fig. S9C). Consistently, administration of AVE0991 substantially restored impaired autophagy and fatty acid degradation (lipophagy, lipolysis, and FAO) in these mice, as reflected by WB and fluorescent co-staining (Fig. 7K,L). However, 24 to 48 h post-APAP, AVE0991 administration failed to show protection in WT-APAP mice (Fig. S9D,E). As illustrated by mIHC (Fig. S9F), AVE0991 (24 h post-APAP) exhibited insignificant effects on liver repair regardless of inflammatory infiltration, angiogenesis, and cell proliferation.
Mas modulates lipophagy and FAO in APAP-challenged human and mouse hepatocytes via AKT- and FOXO1-dependent pathways
The roles of AKT and FOXO1 were determined in vitro using small-interfering RNA (siRNA), siAkt (Fig. 8A-I) and siFoxo1 (Fig. 8J-P), to knock down the expression of corresponding genes in Mas signalling in mouse hepatocytes. Using the RFP-GFP-LC3 reporter, autophagic influx was evaluated by puncta formation, representing autophagosomes (yellow) and autolysosomes (red) (Fig. 8B,C,K). Based on the key steps of mitochondrial FAO shown in Fig. 8E, FAO activity was also evaluated by fatty acid turnover ([U–13C]-palmitate as the tracer) (Fig. 8F,G,M,N), fatty acid (FL-C16) trafficking to the mitochondria (Fig. 8H,O), and oxygen consumption rates (an indicator of mitochondrial respiratory capacity) (Fig. 8I,P). In primary hepatocytes obtained from Mas1-/- mice, siAkt significantly induced the protein expression of indicated markers, suggesting enhanced autophagy, lipolysis, and FAO, with FOXO1 signalling pathway activation during APAP challenge (Fig. 8A). Meanwhile, siAkt significantly increased autophagic influx, lipophagy (co-localisation of BODIPY/LAMP1), and FAO activity. Conversely, in primary hepatocytes obtained from WT mice, siFoxo1 administration substantially diminished the enhanced lipophagy and FAO induced by AVE0991 during APAP challenge (Fig. 8J-P). Consistently, AKT-FOXO1-dependent lipophagy and FAO in Mas signalling were also demonstrated in L02 (a human hepatic cell line) using human siAkt and siFoxo1 (Figs 8Q-V and S10). Additionally, the mechanisms of Mas were documented in AML12 (a mouse hepatic cell line) using pharmacological compounds (perifosine, AS1842856, sirtinol) (Fig. S11 and 12). In summary, in APAP challenge, Mas activation enhances lipophagy and FAO in hepatocytes by suppressing the AKT signalling pathway and activating the FOXO1 signalling pathway.
Fig. 8Mas modulates lipophagy and FAO in APAP-challenged human and mouse hepatocytes via AKT- and FOXO1-dependent pathways.
Primary mouse hepatocytes from Mas1-/- (A-I) or WT (J-P) mice, and human L02 cells (Q-V) were transfected with specific siRNAs (siAkt, or siFoxo1), or negative control (siNC) for 24 h. AVE0991 or A779 was administrated before APAP challenge. (A, J, Q, T) Representative immunoblots and quantification. (B) Schematic diagram of autophagic flux tracked by the mRFP-GFP-LC3 reporter. (C, K) Representative images and quantification of autophagic flux (n = 8 per group). Scale bar: 5 μm. (D, L) Representative co-staining and quantification of BODIPY/LAMP1 (n = 8 per group). Scale bar: 5 μm. (E) Schematic diagram of mitochondrial FAO. (F, M) Heatmap of metabolic flux depicting ratios of detected FAO and carnitine shuttle pathway-related metabolites. (G, N) The abundance of 13C-labelled acyl-carnitine and acyl-CoA. (H, O, R, U) Representative co-staining and quantification of FL-C16/Mito. Scale bar: 5 μm n = 8 per group. (I, P, S, V) Basal and maximal OCR (n = 3-4 per group). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (Student’s t test, Mann-Whitney U test). APAP, acetaminophen; FA, fatty acid; Mito, mitochondria; OCR, oxygen consumption rate; si, small-interfering; TCA, tricarboxylic acid; WT, wild type.
Due to the high associated morbidity and mortality, DILI is a significant health burden worldwide. Unfortunately, pharmacological treatment options are limited. Activation of Mas reportedly alleviates non-alcoholic steatosis, inflammation, and fibrosis in mouse livers
To our knowledge, this is the first study to demonstrate the beneficial role of Mas activation in APAP overdose. First, substantial upregulation of Mas expression after APAP exposure was observed in human and mouse hepatic tissues and cells. In addition, the increased expression of Mas was accompanied by suppression of the AKT signalling pathways and activation of the FOXO1 signalling pathway (Fig. S1D,E), both of which are downstream effectors of Mas signalling, as previously reported.
Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.
Thus, APAP enhanced the expression and activation of the Mas receptor in mice. Based on the acute nature of APAP overdose and the effects of Mas activation documented in WT-APAP mice, APAP-induced Mas expression may be a protective feedback mechanism; however, it was not sufficient to protect against hepatotoxicity. Further, mIHC staining illustrated that Mas was especially enriched in monocytes/macrophages and neutrophils, compared to hepatocytes. From this perspective, the enhanced liver infiltration of Mas-expressing cell populations could also contribute greatly to increased Mas expression. For the wide expression pattern of Mas, it is critical to investigate Mas signalling based on cell types. In this study, using different genetic techniques, we generated mice with systemic, liver-specific, and hepatocyte-specific Mas1 deficiency and demonstrated that the protective effects of Mas activation in APAP overdose were dependent on enhanced lipophagy and FAO in hepatocytes.
In APAP-induced hepatotoxicity, recent data have suggested the protective roles of lipophagy and FAO,
both of which are important fatty acid degradation pathways. Lipophagy facilitates the phagocytosis of LDs and subsequent degradation of TGs, producing fatty acids as substrates for subsequent FAO. A major strength of the present study is the diverse methodology applied to explore the interactions between lipophagy and FAO. The methods used were as follows: 1) expression of indicated markers of autophagy, lipolysis, and FAO were determined; 2) metabolites of neutral lipids and phospholipids were evaluated using both metabolomics and targeted lipidomics; 3) subcellular localisations of lipophagy (BODIPY/LAMP1), lipolysis (BODIPY/ATGL), and FAO (FL-C16/Mito) were illustrated by immunofluorescent co-staining; 4) metabolic flux of fatty acids was determined using the [U–13C]-palmitate tracer; 5) autophagic flux was determined using the RFP-GFP-LC3 reporter; and 6) FAO activity was quantified by oxygen consumption rates. Moreover, under different modulations of Mas signalling, the ‘gain- or loss-of function’ experiments of autophagy and FAO were performed using pharmacological compounds (RAPA, or CQ for autophagy; fenofibrate, lanifibranor, or etomoxir for FAO) and transgenic mice (Mas1-/-, Pparα-/-, Mas1-/-Pparα-/-). All the above experiments consistently demonstrated the protective roles of Mas-enhanced lipophagy and FAO in the context of APAP overdose.
FOXO1, a transcription factor negatively regulated by AKT, combines with Atg7 to enhance autophagic flux.
Melatonin protects mouse granulosa cells against oxidative damage by inhibiting FOXO1-mediated autophagy: implication of an antioxidation-independent mechanism.
In the context of APAP overdose, the substantial downregulation of p-AKT and upregulation of FOXO1/ac-FOXO1 protein expression was preliminarily presented as a protective feedback mechanism against hepatotoxicity. Furthermore, by utilising both pharmacological compounds and genetic tools, we consistently documented the significance of the AKT-FOXO1 axis in lipophagy and FAO modulated by Mas signalling. Moreover, the negative regulation and upstream-downstream relationships between AKT and FOXO1 were confirmed in Mas signalling in human and mouse hepatocytes. These findings could greatly improve our understanding of the molecular mechanisms involved in Mas-mediated fatty acid metabolism.
N-acetyl cysteine is an effective antidote against APAP overdose that acts by replenishing GSH; however, delayed administration may lead to treatment failure with liver transplantation as the last resort. Therefore, there is an unmet clinical need for novel therapeutic approaches. In the present study, the associations of Mas with lipophagy and FAO, and the AKT-FOXO1 axis were investigated based on its prophylactic modulation. Nevertheless, to evaluate the therapeutic potential of Mas activation, both pre- and post-administration of AVE0991 were applied in APAP-treated mice. It turned out that pre-administered AVE0991 exerted full protection against APAP challenge for different time periods. However, post-administered AVE0991 exerted insignificant effects at 24 and 48 h (the data were encouraging at 2 h). Hepatocellular necrosis in response to APAP exposure results in the release of damage-associated molecular patterns (DAMPs) by dying hepatocytes, which activate the innate immune system to trigger SI with neutrophils as the first recruited cell type.
Although SI is critical for liver repair, excessive SI can exacerbate liver injury. Another strength of the present study is the application of DAOSLIMIT
to dynamically observe SI in live mice challenged with APAP, which revealed the substantial downregulation of SI by pre-administered AVE0991. However, as illustrated by mIHC, post-administration of AVE0991 at 24 h barely influenced intrahepatic inflammation, angiogenesis, and proliferation, suggesting its insignificant role in liver repair and recovery. Combined with our understanding of the enhancement of lipophagy and FAO by activated hepatocyte-specific Mas signalling, it is speculated that AVE0991, when pre-administered, is most likely involved in the regulation of DAMP release from injured hepatocytes, thereby confining the extent of inflammation to a certain range to avoid excessive hepatocyte necrosis. Thus, when post-administered, AVE0991 exhibited diverse therapeutic effects depending on the level of DAMPs that had already been released and the normal hepatocyte reserve at the time of administration. Accordingly, human APAP overdose follows a pattern in which hepatic damage becomes apparent at 24-48 h, with hepatocyte death peaking at approximately 72 h.
Additionally, in APAP-treated mice, we observed that intrahepatic Mas1 mRNA expression began to decline after reaching its peak at 24 h (data not shown), also indicating a diminished role of Mas activation after that time point. Certainly, as a therapeutic target for DILI, the time frame of Mas activation still requires thorough investigation in both mouse models and patients. Nevertheless, these findings provide new clues to treating patients with APAP overdose by Mas activation, both prophylactically and therapeutically.
In summary, we have presented data in mice and humans demonstrating that activation of hepatocyte-specific Mas signalling alleviates APAP-induced hepatotoxicity through the enhancement of lipophagy and FAO, both of which are dependent on the suppression of the AKT signalling pathway and activation of the FOXO1 signalling pathway. However, the therapeutic benefits of Mas activation in APAP overdose need to be tested in large animal models of DILI before advancing to clinical trials.
This study was supported by grants from National Natural Science Foundation of China (No.818201008006 to Changqing Yang), Shanghai Municipal Key Specialty Project (No. SHSLCZDZK06801 to Changqing Yang), Clinical research plan of SHDC (No. SHDC2020CR2030B to Changqing Yang) and Shanghai Municipal Science and Technology Commission Original Exploration Project (No. 21ZR1481600 to Jing Li).
Conflict of interest
The authors declare no conflict of interest.
Please refer to the accompanying ICMJE disclosure forms for further details.
Authors’ contributions
Conception and design: Shuai Chen, Jing Li and Changqing Yang. Acquisition of data: Shuai Chen, Zhi Lu, Haoyu Jia, Bo Yang, Chun Liu, Yuxin Yang, Shuo Zhang, Zhijing Wang, Liu Yang, Shanshan Li. Analysis and interpretation of data: Shuai Chen, Zhi Lu and Yuxin Yang. Drafting of the article: Shuai Chen, Zhi Lu, Jing Li. Administrative, technical, or material support: Chun Liu, Zhi Lu, Yuxin Yang. Study supervision: Changqing Yang and Jing Li.
Data availability statement
The data that support the findings of this study are available from the corresponding authors upon request.
Acknowledgements
We would like to thank Prof. Qionghai Dai and Dr. Jiamin Wu from Institute of Brain and Cognitive Sciences of Tsinghua University for their assistance and technical support in intravital fluorescence imaging. We thank OE Biotech (Shanghai, China) for providing RNA-seq, Proteomics analysis and Metabolomics analysis, Yunna Zheng and Lei Luo for their assistance in data analysis.
Supplementary data
The following are the supplementary data to this article:
Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.
Angiotensin-(1-7), the product of ACE2 ameliorates NAFLD by acting through its receptor Mas to regulate hepatic mitochondrial function and glycolipid metabolism.
Melatonin protects mouse granulosa cells against oxidative damage by inhibiting FOXO1-mediated autophagy: implication of an antioxidation-independent mechanism.
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