Introduction

The liver exhibits a remarkable capacity for complete regeneration following partial hepatectomy (PHx). In murine models, routinely used for investigating liver regeneration1, restoration of the tissue after ~ 35% PHx is achieved within a timeframe of fewer than seven days. This rapid proliferation surpasses even the robust growth observed in fast-proliferating tissues such as tumors. However, unlike tumors, the liver ceases to grow upon reaching its original mass, determined by cell number2,3,4. Liver regeneration involves in its initial phase a stage referred to as compensatory cellular hypertrophy (CCH), which results in rapid liver expansion after PHx due to increased hepatocyte size, followed by the phase of robust proliferation5,6,7. Not surprisingly, regeneration of liver tissue after PHx involves altered metabolism to support this highly dynamic process by engaging in particular anabolic pathways8,9,10,11. Both cell growth to a larger size and proliferation are dependent on mitochondrial function12,13.

The de novo pyrimidine synthesis pathway is important for cell proliferation to provide nucleic acid precursors. Not much is known about its role in liver regeneration. We have recently found that cancer cells with dysfunctional mitochondria import these organelles with their DNA payload via horizontal mitochondrial transport14,[15](https://www.nature.com/articles/s41467-025-65451-2#ref-CR15 “Dong, L. F. et al. Mitochondria on the move: Horizontal mitochondrial transfer in disease and health. J. Cell Biol. 222, https://doi.org/10.1083/jcb.202211044

(2023).“), in order to restore mitochondrial respiration that is critically linked to de novo pyrimidine synthesis, enabling transition through the S-phase of the cell cycle[15](https://www.nature.com/articles/s41467-025-65451-2#ref-CR15 “Dong, L. F. et al. Mitochondria on the move: Horizontal mitochondrial transfer in disease and health. J. Cell Biol. 222, https://doi.org/10.1083/jcb.202211044

(2023).“). It can be estimated that approximately half a billion cells need to form in the mouse liver within the span of < 7 days following ~35% PHx. Consequently, we postulated that such robust cell growth necessitates the engagement of de novo pyrimidine synthesis for rapid transition through the cell cycle.

We show here the critical role of de novo pyrimidine synthesis in liver regeneration linked to rapid metabolic remodeling, whereby detoxification of ammonia in the liver is suppressed in favor of the use of this toxic product to support anabolic pathways epitomized by the de novo pyrimidine pathway.

Results

Inhibition of dihydroorotate dehydrogenase suppresses liver regeneration

Our recent research showed that de novo pyrimidine synthesis (see scheme in Fig. 1a) is of importance for tumor formation that cancer cells devoid of mitochondrial DNA (mtDNA) import mitochondria with DNA from the surrounding stroma to restore respiration needed to drive conversion of dihydroorotate (DHO) to orotate. This is catalyzed by dihydroorotate dehydrogenase (DHODH), an enzyme coupled to the mitochondrial electron redox chain16. Since it takes 5–7 days to fully regenerate liver after removal of the left lateral lobe (Fig. 1b), which equates to about ~ 35% PHx17, we anticipated that generation of the high number of cells requires a switch from homeostasis to metabolic pathways that support rapid cell proliferation required for fast transition via the cell cycle, supported by de novo pyrimidine synthesis. It has been shown that inhibition of DHODH, using the specific inhibitor BAY-2402234 that suppresses activity of the enzyme with an IC50 of 3–5 nM18, results in suppression of tumor growth19. We therefore tested the effect of the inhibitor on liver regeneration following ~ 35% PHx (Fig. 1a, b). Figure 1c shows that the loss in body weight after PHx of about 1 g (assuming mouse weight of ~ 20 g) was followed by recovery to near that of control mice within 5 days. On the other hand, BAY-2402234 given to mice post-PHx by daily gavage (2 or 5 mg/kg) resulted in a complete halt in body weight increase. This is further corroborated by the liver to body weight ratio (LBWR), where the full restoration of liver in mice at 7 days is blocked by the DHODH inhibitor (Fig. 1d). Of note, treated mice exhibited similar liver status parameters compared to untreated mice 3–14 days after ~ 35% PHx (Supplementary Fig. 1) and had a 100% survival rate (Supplementary Fig. 2), suggesting that treatment with BAY-2402234 following 35% PHx is well tolerated and does not cause toxicity18.

Fig. 1: Liver regeneration and hepatocyte proliferation are suppressed by a DHODH inhibitor.

a Scheme of the de novo pyrimidine pathway illustrating the precursor substrates and respective enzymes to make the final product uridine-5-monophosphate (UMP), which can be inhibited by BAY-2402234 (BAY) specifically targeting DHODH in the mitochondria. b Mouse liver subjected to partial hepatectomy (PHx) showing removal of the left lateral lobe (~35% of the whole liver) with full regeneration of the remaining liver occurring within 7 days. Relative body weight (c) and liver/bodyweight ratio (LBWR, %) (d) of control mice and mice subjected to ~ 35% PHx with or without daily gavage of BAY. Assessment of proliferation using western blot (WB) of MCM2, PCNA, and pHH3 (e), and using the EdU assay (f) in control and ~35% PHx mice with or without daily gavage of BAY at the indicated timepoints. g A section of regenerating liver was imaged for E-cadherin (red) to demarcate the periportal region surrounding the portal vein (PV), GS (green) to stain the pericentral cells surrounding the central vein (CV), and EdU-positive cells (white) showing spatial localization of proliferating cells encompassing periportal to pericentral distribution within the liver (inset). For panel (c), multiple t tests using the Holm-Sidak method were used (control n = 4, PHx n = 8, PHx + 2 mg/kg BAY n = 4, PHx + 5 mg/kg BAY n = 4; *p-*values for control vs. PHx at days 1, 2, and 3 post-PHx are 0.0003, 0.0025, and 0.0122 respectively; *p-*values for PHx vs. PHx+2 mg/kg BAY at days 2, 3, 4, 5, 6 and 7 post-PHx are 0.0362, 0.0075, 0.0057, 0.0009, 0.0003, and 0.0061, respectively; *p-*values for PHx vs. PHx + 5 mg/kg BAY at days 2, 3, 4, 5, 6 and 7 post-PHx are 0.0370, 0.0370, 0.0085, 0.0264, 0.0040, and 0.0018, respectively). For panels (d and f), unpaired t test was used (d: control n = 15, day 0 post-PHx n = 4, day 3 post-PHx n = 15, day 3 post-PHx + 2 mg/kg BAY n = 10, day 7 post-PHx n = 15, day 7 post-PHx + 2 mg/kg BAY n = 4; *p-*values for control vs. post-PHx at days 0, 3, 3 + 2 mg/kg BAY, and 7 + 2 mg/kg BAY are < 0.0001, 0.0067, < 0.0001, and 0.0002, respectively; *p-*values for post-PHx day 3 vs. day 3 + 2 mg/kg BAY and post-PHx day 7 vs. day 7 + 2 mg/kg BAY are 0.0036 and 0.0035, respectively; (f) control n = 11, day 3 post-PHx n = 9, day 3 post-PHx + 2 mg/kg BAY n = 10, day 7 post-PHx n = 11, day 7 post-PHx + 2 mg/kg BAY n = 4; p-values for control vs. post-PHx days 3, 3 + 2 mg/kg BAY, 7, 7 + 2 mg/kg BAY are < 0.0001, 0.0007, < 0.0001, and < 0.0001, respectively; *p-*values for post-PHx day 3 vs. post-PHx day 3 + 2 mg/kg BAY, day 7, day 7 + 2 mg/kg BAY are < 0.0001; *p-*values for post-PHx day 3 + 2 mg/kg BAY vs. post-PHx day 7 and day 7 + 2 mg/kg BAY are 0.0017 and 0.0038, respectively). Images presented are representative of at least three independent biological experiments. Data are expressed as mean ± S.D. The symbols ‘*/+’, ‘**/++’, ‘***/+++’, and ‘****/++++’ denote statistical significance at p < 0 .05, p < 0.01, p < 0.001, and p < 0.0001, respectively; ns, not significant. Source data are provided as a Source Data File.

To test whether the DHODH inhibitor specifically blocks cell proliferation in liver tissue following PHx, we probed for known proliferation markers such as minichromosome maintenance complex component 2 (MCM2), proliferating cell nuclear antigen (PCNA), and phospho-histone H3 (pHH3), and performed an EdU (5-ethynyl-2’-deoxyuridine) assay20. The results demonstrate robust proliferation in regenerating liver peaking on day 3 after PHx, with slower proliferation on day 7 (Fig. 1e, f), and tissue distribution of proliferation encompassing whole liver parenchyma from periportal to pericentral localizations (Fig. 1g and Supplementary Fig. 3a). Treatment with BAY-2402234 considerably suppressed proliferation, as shown in Fig. 1e, f and Supplementary Fig. 3b.

De novo pyrimidine pathway is required for liver regeneration

We next investigated whether BAY-2402234 suppresses de novo pyrimidine synthesis (see scheme in Fig. 1a) in the context of liver regeneration. We first assessed the effect of the inhibitor on DHODH-dependent respiration (Fig. 2a) and DHODH activity (Fig. 2b), and found that suppression of both parameters considerably lowered the orotate-to-DHO ratio (Fig. 2c).

Fig. 2: Liver regeneration requires de novo pyrimidine synthesis.

Assessment of DHODH-dependent respiration (a), DHODH activity (b), and orotate-to-DHO ratio (c) in control mice and mice subjected to ~ 35% PHx with or without daily gavage of 2 mg/kg BAY-2402234 (BAY). LC-MS data showing levels of de novo pyrimidine metabolites glutamine (Gln), carbamoyl aspartate (CA), dihydroorotate (DHO), uridine monophosphate (UMP), their MALDI-TOF representative images with intensity quantification (d), and their MALDI-TOF spatial localization including glutamate (Glu) and aspartate (Asp) (e) demarcated by pericentral (C) GS staining (green), and periportal (P) taurocholic acid (TCA) accumulation in liver of control mice and mice subjected to ~ 35% PHx with or without daily gavage of 2 mg/kg BAY. MALDI-TOF images were acquired with the 9AA matrix and measured in the negative mode. Images are displayed with TIC normalization, 99% quantile hotspot removal, linear interpolation and weak denoising level. Peak intensities are rescaled to the full color map. For panels (a–d), unpaired t test was used, and data are expressed as mean ± S.D. (a: all groups have n = 4; control vs. day 3 post-PHx + 2 mg/kg BAY p < 0.0001; control vs. day 7 post-PHx + 2 mg/kg BAY p = 0.0003; day 3 post-PHx vs. day 3 post-PHx + 2 mg/kg BAY p < 0.0001; day 7 post-PHx vs. day 7 post-PHx + 2 mg/kg BAY p = 0.0028; b: all groups have n = 4; control vs. day 3 post-PHx + 2 mg/kg BAY p < 0.0001; control vs. day 7 post-PHx + 2 mg/kg BAY p < 0.0001; day 3 post-PHx vs. day 3 post-PHx + 2 mg/kg BAY p < 0.0001; day 7 post-PHx vs. day 7 post-PHx + 2 mg/kg BAY p = 0.0003; c: control, post-PHx days 3, 7 and 14 have n = 4; day 3 post-PHx + 2 mg/kg BAY has n = 5; day 3 post-PHx + 2 mg/kg BAY vs. control p = 0.0042; day 3 post-PHx + 2 mg/kg BAY vs. day 3 post-PHx p = 0.0034; day 3 post-PHx + 2 mg/kg BAY vs. day 7 post-PHx p = 0.0418; day 3 post-PHx + 2 mg/kg BAY vs. day 14 post-PHx p = 0.0319; d, Gln: n = 5 for all groups; control vs. day 3 post-PHx p = 0.0004; control vs. day 7 post-PHx p = 0.0476; d, CA: n = 4 for all groups; control vs. day 7 post-PHx p = 0.0004; control vs. day 3 post-PHx + 2 mg/kg BAY p = 0.0044; d, DHO: n = 4 for all groups; control vs. day 7 post-PHx p = 0.013; control vs. day 3 post-PHx + 2 mg/kg BAY p = 0.0011; d, UMP: n = 4 for all groups; control vs. day 3 post-PHx p = 0.0035; boxplot in d: midline depicts median value; the lower and upper hinges correspond to the first and third quartiles, representing the 25th and 75th percentiles respectively; the upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge; the lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge). The symbols ‘*/+’, ‘**/++’, ‘***/+++’, and ‘****/++++’ denote statistical significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Source data are provided as a Source Data File.

To extend our studies further, we applied LC-MS and MALDI imaging techniques to determine levels of de novo pyrimidine pathway intermediates. Figure 2d shows the level of glutamine (Gln), carbamoyl aspartate (CA), DHO and uridine monophosphate (UMP) in control liver and in liver on days 3, 7 and 14 post-PHx, as well as on day 3 after PHx in mice treated with BAY-2402234. LC-MS data show progressive increase in liver levels of Gln, CA and DHO from day 3 to day 7 post-PHx, followed by a reduction in levels similar to control on day 14 post-PHx. Interestingly, MALDI imaging analyses show high levels of aspartate (Asp), carbamoyl phosphate (CP), CA and DHO (also shown by LC-MS), precursors of orotate formed from DHO by DHODH, coupled with low levels of UMP and uridine diphosphate (UDP) on day 3 PHx mice treated with the DHODH inhibitor (Fig. 2d and Supplementary Fig. 4). Spatial mapping by combined MALDI imaging and immunostaining localized Gln pericentrally and glutamate (Glu) periportally in control and day 3 post-PHx with and without BAY-2402234 treatment (Fig. 2e). Notably, the high levels of orotate precursors observed in BAY-2402234-treated liver 3 days post-PHx exhibited differential localization. While CA and DHO were distributed across the whole liver parenchyma, aspartate (Asp) was mainly periportal, with its distribution similar in extent to Glu (Fig. 2e). We conclude from data in Figs. 1, 2, and Supplementary Fig. 4 that de novo pyrimidine synthesis is required for the proliferation of liver cells, and therefore for liver regeneration. In addition, as Asp and Glu are linked to the urea cycle, Asp accumulation periportally closely mirrors Glu spatial distribution upon BAY-2402234 treatment 3 days post-PHx, suggesting possible involvement of the urea cycle in fulfilling this requirement for de novo pyrimidine synthesis.

Urea cycle activity is lower in regenerating liver

A major liver function is detoxification of ammonia to urea. Since loss of hepatic tissue, e.g., due to resection, theoretically reduces its capacity to process ammonia, we queried the possibility of metabolic remodeling. Here, we hypothesize that the toxic metabolite ammonia, rather than being ‘wasted’ in the form of urea formed via the urea cycle (Fig. 3a), is utilized to support the increased demand for metabolic precursors necessary for anabolic pathway(s) of rapidly regenerating liver. To investigate this, we determined the state of the urea cycle during liver regeneration by treating mice with 15NH4Cl, given by intraperitoneal injection, and analyzing liver tissue by LC-MS to assess the fate of unlabeled and labeled ammonium chloride in the context of the urea cycle. Interestingly, urea cycle activity, assessed as the ratio of citrulline and CA (Fig. 3b), and as a function of ornithine transcarbamoylase (OTC) activity (ratio of citrulline and ornithine) (Fig. 3c), is lower following PHx from day 3–7, with a tendency to normalize to control mouse liver levels on day 14 post-PHx. Importantly, this effect on the urea cycle activity is mirrored by the levels of urea in the circulation (Fig. 3d and Supplementary Fig. 5).

Fig. 3: Hepatectomy is followed by low activity of the urea cycle.

a Scheme of the urea cycle showing the classical ‘entry’ of ammonia into the pathway as carbamoyl phosphate (CP), catalyzed by CPS1 and its eventual conversion into urea. Assessment of the urea cycle activity presented as a ratio of citrulline and carbamoyl aspartate (CA) (b), and as a ratio of citrulline and ornithine (OTC activity) (c), and the corresponding urea levels in the blood (d), analyzed in the liver or blood of control mice and mice subjected to ~ 35% PHx. e,** f** Evaluation of the m + 0 and m + 1 metabolites of the urea cycle following injection of 15NH4Cl in control mice and mice subjected to ~ 35% PHx. g Isotope ratio reported as fraction enrichment (%) of all possible nitrogen isotope variations. For panels (b–f), unpaired t test was used and data are expressed as mean ± S.D. (b: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0431; control vs. day 7 post-PHx p = 0.0374; c: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0140; control vs. day 7 post-PHx p = 0.0234; d: all groups have n = 5; control vs. day 3 post-PHx p = 0.0191; control vs. day 7 post-PHx p = 0.0173; e, Citrulline: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0072; control vs. day 7 post-PHx p = 0.0072; e, Arginosuccinate: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0025; control vs. day 7 post-PHx p = 0.0397; e, Arginine: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 7 post-PHx p = 0.0250; control vs. day 14 post-PHx p = 0.0057; e, Ornithine: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0060; control vs. day 14 post-PHx p = 0.0425; f, Citrulline: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0251; control vs. day 7 post-PHx p = 0.0083; f, Arginosuccinate: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0357; f, Arginine: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; f, Ornithine: control, day 14 post-PHx n = 4; day 3 post-PHx, day 7 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0292). The symbols ‘*’and ‘**’ denote statistical significance at p < 0.05 and p < 0.01, respectively. Source data are provided as a Source Data File.

Lower urea cycle activity, and as a consequence, lower blood urea levels, is further corroborated by LC-MS data showing lower citrulline and arginosuccinate concurrent with increased ornithine levels on days 3 and 7 post-PHx, and restoration to control levels at day 14 post-PHx (Fig. 3e), thereby confirming lower activity of the urea cycle in ammonia detoxification on days 3 and 7 post-PHx, and normalization at the time of completed liver regeneration. Of note, similar metabolic tendencies were observed for m + 1 isotopologues of metabolites (Fig. 3f), suggesting that the fate of injected ammonium chloride resembles metabolic processing of the unlabeled and presumably endogenous ammonia in the liver. Isotope fraction enrichment analysis confirms the metabolic processing of labeled ammonia via the urea cycle, and interestingly, demonstrated a more upstream enrichment of new intermediates made from the administered ammonia (Fig. 3g).

Ammonia that is not detoxified by urea cycle supports liver regeneration

The fact that the urea cycle activity is suppressed after PHx coupled with a tendency to a more upstream processing of new intermediates indicate that ammonia, which is detoxified via the urea cycle under homeostasis, can be redirected from upstream of the pathway towards anabolic reactions to support liver regeneration. This intriguing scenario, where a toxic metabolite is repurposed ‘in time of need’ to generate additional substrate to support efficient generation of pyrimidines by the de novo pathway, was further investigated. Figure 4a shows schematically the metabolite fluxes involving the urea cycle and de novo pyrimidine synthesis, highlighting diversion of the ammonia flow into de novo pyrimidine synthesis during liver regeneration. In this scheme, the entry points of ammonia into the pathway are (i) direct amination of Glu to Gln, catalyzed by glutamine synthetase (GS); (ii) reductive amination of α-ketoglutarate (α-KG) to Glu catalyzed by glutamate dehydrogenase (GDH), which can further incorporate ammonia by its amination to Gln, or be incorporated into the pathway as Asp via transamination catalyzed by glutamic-oxaloacetic transaminase 1 (GOT1); and (iii) incorporation into CP catalyzed by carbamoyl phosphate synthetase 1 (CPS1) that, along with Asp, can give rise to CA via the second reaction of the trifunctional enzyme CAD. Expression of these enzymes, including those of the de novo pathway is similar in regenerating liver 3 to 14 days post-PHx including liver treated with BAY-2402234 for 3 days post-PHx compared to control (Supplementary Fig. 6), suggesting that their levels are maintained post-PHx even after treatment with BAY-2402234 at the indicated timepoints.

In accordance with the scheme, we evaluated the regenerating liver from 15NH4Cl-injected mice for the presence of metabolites described, including their m + 1 and m + 2 isotopologues to track the flow of ammonia. Figure 4b shows the overall trend, whereby metabolites of de novo pyrimidine synthesis, Gln, DHO, orotate and UMP increase on days 3 and 7 post-PHx before normalizing to control levels on day 14, highlighting the need for de novo pyrimidines as the liver regenerates. A similar trend was observed for Asp, Glu and CA, suggesting the incorporation of ammonia into regenerating liver pyrimidine precursors via GDH and GOT1, and CPS1 in the urea pathway. Furthermore, the metabolic tendencies for the m + 1 and m + 2 isotopologues were similar to the unlabeled metabolites, pointing to direct incorporation of ammonia into the precursors shown in Fig. 4a. In addition, the accumulation of high levels of unlabeled and m + 1 and m + 2 DHO and CA coupled with low levels of UMP upon treatment with BAY-2402234 on day 3 provides further evidence for incorporation of ammonia into the de novo pyrimidine pathway. Analysis of the isotope fraction enrichment reveals that as much as ~50% of the new de novo pyrimidine intermediates Gln, CA, DHO and orotate stemmed from the administered ammonia (Fig. 4c). These results show that during liver regeneration, ammonia is diverted from urea cycle detoxification towards anabolic processes to form pyrimidines via the de novo pathway, thus allowing for fast cell proliferation to facilitate liver regeneration following PHx.

Liver regeneration involves zoning of metabolic pathways

On the premise that liver function is zonal21, where the urea cycle, dependent on CPS1 to ‘scavenge’ ammonia, is largely confined periportally, and Gln synthesis by GS strictly pericentrally, we propose that the entry points for ammonia incorporation to support de novo pyrimidine synthesis, and thus cell proliferation within regenerating liver, will be zonal as well (Fig. 5a; cf. Figure 1g and Supplementary Fig. 3a). This is further supported by our initial findings where Glu, an upstream urea cycle metabolite, localized periportally and Gln pericentrally (cf. Figure 2e). We mapped liver zones spanning periportal to pericentral localizations using specific functional markers glutaminase-2 (GLS2), GS, and CPS1 (Fig. 5b). While GLS2 and CPS1 co-localize periportally (Fig. 5b), CPS1 expression extends well beyond the GLS2 + periportal regions further into the parenchyma but is mutually exclusive with pericentral GS (Fig. 5c). This suggests that CPS1-mediated ammonia scavenging encompasses the majority of the hepatic zones, bounded only by the GS+ zone where it is not expressed. By resolving the tissue metabolites using MALDI imaging and mapping them against the functional liver zones described, we show de novo pyrimidine pathway metabolic flux along the defined functional zones of the liver tissue (Fig. 5d). This analysis, in tandem with the LC-MS and MALDI imaging data in Fig. 2d, e, and Fig. 4b shows accumulation of high levels of the orotate precursors, CA and DHO, in day 3 PHx mice treated with the DHODH inhibitor, but with spatial and fluxomic resolution. Of note, taurocholic acid (TCA) accumulates periportally, signifying precise mapping (Fig. 5d). This establishes applicability of the MALDI imaging method to functional spatial analysis of metabolic flux within liver tissue.

Fig. 5: Liver regeneration involves zoning of metabolic pathways.

a Scheme of functional liver zonation into glutaminase-2 positive (GLS2 + ) periportal (P), carbamoyl phosphate synthase positive (CPS1 + ) periportal (P) extending towards but mutually exclusive to glutamine synthetase positive (GS + ) pericentral (C) regions depicting how ammonia is ‘scavenged’ and incorporated into the de novo pyrimidine pathway spatially within a regenerating liver as it traverses the respective zones towards the direction of blood flow as indicated. The respective zones described are imaged in the mouse liver (b), showing periportal (P) to pericentral (C) staining using the functional markers GLS2 and GS, respectively, with the endothelium lining the blood vessels positive for CD31, and with the GS+ zone mutually exclusive with the CPS1 + zone (c). d Combined analyses using imaging to depict liver functional zones (upper panel) in tandem with MALDI-TOF analysis (lower panels) on the same zones (depicted by yellow line in imaging panels) using liver of control mice and mice subjected to ~ 35% PHx with or without daily gavage of 2 mg/kg BAY-2402234, showing metabolic fluxes of de novo pyrimidine metabolites from periportal (P) to pericentral (C) zones (arrows). Horizontal bars on lower panels represent metabolic flux set relative to control levels. Taurocholic acid (TCA) and GS staining were used to demarcate periportal and pericentral zones, respectively. e Similar combined analyses using imaging to identify liver functional zones as previously described using specific markers (upper panel) in combination with MALDI-FTICR (lower panels) in liver taken from 15NH4Cl-injected control mice and from mice subjected to ~ 35% PHx with and without daily gavage of 2 mg/kg BAY-2402234 showing distribution of m + 0 and m + 1 isotopologues of de novo pyrimidine precursor metabolites that can incorporate ammonia within the functional liver zones. Using MALDI-FTICR data on the functional zones identified through imaging (depicted by white arrow from P to C on upper panel of e), the periportal (P) to pericentral (C) m + 0 and m + 1 metabolic fluxes of isotopologues of pyrimidine precursors that can potentially incorporate ammonia, specifically Gln and Glu (f), CA, Asp and Glu (g; arrows point to the ‘P’ zone), and DHO and its upstream substrates Gln and CA (h; arrows point to the ‘C’ zone), are shown. MALDI-TOF and MALDI-FTICR images were acquired with the 9AA and DAN matrices measured in the negative mode, respectively. Images are displayed with TIC normalization (MALDI-TOF) or RMS normalization (MALDI-FTICR), 99% quantile hotspot removal, linear interpolation and weak denoising level. Peak intensities rescaled to full color map. Source data are provided as a Source Data File.

We applied this analysis to regenerating liver tissue following injection of 15NH4Cl and tracked the metabolic fluxes of labeled ammonia incorporation as it traverses the functional liver zones (Supplementary Fig. 7). Figure 5e illustrates the metabolic map of m + 0 and m + 1 isotopologues of pyrimidine precursors, zonally from periportal to pericentral in BAY-2402234-treated vs. untreated day 3 PHx and control livers. Detailed analyses show that higher levels of Glu and Gln in all PHx liver samples compared to controls are zonally distributed; whereas Glu is high periportally, Gln is high pericentrally (Fig. 5f). Interestingly, while Glu and Asp are higher in day 3 PHx liver compared to the control, treatment with the DHODH inhibitor shows accumulation of high levels of CA, coupled with combined reduction of Glu and Asp in the periportal zone (Fig. 5g), suggesting that Glu is transaminated to Asp and incorporated with CP to form CA within these zones. Notably, high levels of DHO accumulation are maintained along all liver zones in day 3 PHx liver treated with BAY-2402234 (Fig. 5h), indicating the availability of CA periportally and Gln pericentrally as DHO precursors, to facilitate de novo pyrimidine synthesis and, ultimately, supporting proliferation in the respective zones. Furthermore, the metabolic flux along the functional liver zones of m + 0 and m + 1 isotopologues are almost identical (Fig. 5e–h), demonstrating the incorporation of labeled ammonia into the respective de novo pyrimidine precursors spatially as they traverse the liver zones, as shown in Fig. 5a.

We applied a similar approach to regenerating liver after a more drastic resection by combined removal of left lateral and medial lobes, which equates to about ~ 60% PHx17 (Fig. 6), and noted certain differences. The loss of body weight by about 2 g (assuming mouse weight of ~20 g), tantamount to the amount of tissue loss at ~ 60% PHx17, did not fully recover to that of control mice at day 7 post-PHx (Fig. 6a), and is further reflected by the LBWR (Fig. 6b), suggesting an initial delay in regeneration possibly to make up for the bigger tissue loss. Although ~ 60% PHx mice treated daily with BAY-2402234 at 2 mg/kg have significantly reduced body weights and LBWR, the difference to untreated PHx mice only became significant from day 5 to day 7 post-PHx (Fig. 6a, b). EdU labeling revealed that despite an overall higher proliferative capacity (Figs. 1f, 6c), a significant proliferative difference can still be distinguished between treated and untreated mice on day 3 post-PHx (Fig. 6c) that is not reflected by body-weight and LBWR, suggesting effective attenuation of proliferation on day 3 post-PHx by BAY-2402234 when given at 2 mg/kg. Increasing the dose to 5 mg/kg however, while non-toxic to mice18 (Supplementary Fig. 2), resulted to 67% mortality 2 days post-PHx (Supplementary Fig. 2), indicating that further suppression of DHODH by administering higher doses of BAY-2402234 at ~ 60% hepatic tissue loss increased mortality. Likewise, expression of the functional markers GLS2, CPS1 and GS showed very similar localization effectively mapping liver zonation as described where proliferation by EdU labelling spanned across periportal to pericentral zones (Fig. 6d). Similarly, BAY-2402234 treatment effectively attenuated DHODH as reflected by lower respiration (Fig. 6e), lower activity (Fig. 6f), and lower orotate to DHO ratio (Fig. 6g). More importantly, lower urea cycle activity is again evident following ~ 60% PHx from day 3 to day 7, as reflected by lower ratios of citrulline and CA (Fig. 6h), citrulline and ornithine (Fig. 6i), and blood urea levels (Fig. 6j).

Fig. 6: Regenerating liver after ~ 60% partial hepatectomy has attenuated urea cycle with exacerbated pericentral ammonium use for de novo pyrimidine synthesis.

Relative body weight (a), liver/body weight ratio (LBWR, %) (b), and assessment of proliferation by EdU assay (c) in control mice and mice subjected to ~ 60% PHx, with or without daily gavage of 2 mg/kg of BAY-2402234 (BAY) and sham-operated mice as indicated. d Localization of EdU+ cells within the functional liver zones imaged by GLS2+ periportal (P) and CPS1 + zones extending towards a mutually exclusive GS+ pericentral zone (C). Assessments of DHODH-dependent respiration (e), DHODH activity (f), orotate-to-DHO ratio (g), urea cycle activity as a ratio of citrulline and carbamoyl aspartate (CA) (h), as a ratio of citrulline and ornithine (OTC activity) (i), and as blood urea levels (j), in control mice and mice subjected to ~ 60% PHx with or without daily gavage of 2 mg/kg BAY. k Combined analyses by liver functional zone imaging as described using specific markers (upper panel) in combination with MALDI-FTICR images (lower panels) of liver taken from 15NH4Cl-injected control mice and from mice subjected to ~ 60% PHx with and without daily gavage of 2 mg/kg BAY showing distribution of m + 0 and m + 1 isotopologues of de novo pyrimidine precursor metabolites that can incorporate ammonia within the functional liver zones. Using MALDI imaging data on the functional zones identified through imaging (depicted by white arrow from P to C on upper panel of k), the periportal (P) to pericentral (C) m + 0 and m + 1 metabolic fluxes of isotopologues of pyrimidine precursors that can potentially incorporate ammonia, specifically Gln and Glu (l), CA, Asp and Glu (m; arrows point to the ‘C’ zone), and DHO and its upstream substrates Gln and CA (n; arrows point to the ‘C’ zone), are shown. MALDI-FTICR images were acquired with the DAN matrix and measured in the negative mode. Images are displayed with RMS normalization, 99% quantile hotspot removal, linear interpolation and weak denoising level. Peak intensities rescaled to full colormap. For panel (a) multiple t tests using the Holm-Sidak method were used (control n = 5, control + BAY n = 4, sham n = 8, sham + BAY n = 8, PHx n = 10, PHx + BAY n = 13; control vs. day 1 sham p = 0.0196; control vs. day 1 sham+BAY p = 0.0205; *p-*values for control vs. PHx at days 1, 2, 3, 4, 5, 6 and 7 post-PHx are 0.000002, 0.00000005, 0.0001, 0.0004, 0.0010, 0.0002, and 0.0010, respectively; p-values for control vs. PHx + BAY at days 1, 2, 3, 4, 5, 6 and 7 post-PHx are 0.000000001, 0.0000009, 0.0000003, 0.00002, 0.00002, 0.000009, and 0.00001, respectively; *p-*values for PHx vs. PHx+BAY at days 5 and 7 post-PHx are 0.0278 and 0.0025, respectively). For panels (b, c), and (e–j), unpaired t test was used (b: control n = 4, days 3 and 7 ± BAY n = 3; *p-*values for control vs. post-PHx at days 3, 3 + BAY, 7, 7 + BAY are < 0.0001, 0.0001, 0.0001, and < 0.0001, respectively; day 7 post-PHx vs. day 7 post-PHx+BAY p = 0.0005; c: control n = 5, day 3 post-PHx ±BAY n = 10, day 7 post-PHx ± BAY n = 5; p-values for control vs. post-PHx days 3, 3 + BAY, 7, 7 + BAY are < 0.0001, < 0.0001, 0.0475, and 0.0002, respectively; day 3 post-PHx vs. day 3 post-PHx+BAY p = 0.0407; e: control n = 4, day 3 post-PHx n = 3, day 3 post-PHx+BAY n = 4, day 7 post-PHx ±BAY n = 3; control vs. day 3 post-PHx+BAY p = <0.0001; control vs. day 7 post-PHx+BAY p = 0.0005; day 3 post-PHx vs. day 3 post-PHx+BAY p < 0.0001; day 7 post-PHx vs. day 7 post-PHx + BAY p = 0.0014; f: control n = 3, day 3 post-PHx n = 3, day 3 post-PHx + BAY n = 4, day 7 post-PHx ± BAY n = 4; control vs. day 3 and 7 post-PHx+BAY p < 0.0001; day 3 post-PHx vs. day 3 post-PHx+BAY p = 0.0002; day 7 post-PHx vs. day 7 post-PHx + BAY p = 0.0123; g: all groups n = 3; p- values of day 3 post-PHx + BAY vs. post-PHx days 3,7, and 14 are 0.0037, 0.0462 and 0.0140, respectively; h: control n = 4; days 3, 7, and 14 post-PHx n = 5; control vs. day 3 post-PHx p = 0.0013; i: all groups n = 5; control vs. day 3 post-PHx p = 0.0408; control vs. day 7 post-PHx p = 0.0390; j: all groups n = 4; control vs. day 3 post-PHx p = 0.0001; control vs. day 3 post-PHx p = 0.0001; control vs. day 7 post-PHx p = 0.0019). Data are expressed as mean ± S.D. The symbols ‘*/+’, ‘**/++’, ‘***/+++’, and ‘****/++++’ denote statistical significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively; ns, not significant. Source data are provided as a Source Data File.

When injected with 15NH4Cl, labeled and unlabeled ammonia incorporated into de novo pyrimidine precursors similarly to ~ 35% PHx but accumulation of precursors, particularly Asp, Glu, CA, DHO, and orotate peaked earlier on day 3 post-PHx before tapering off on days 7 and 14 post-PHx (Fig. 4b and Supplementary Fig. 8a), suggesting earlier accumulation of precursors to support the higher proliferative capacity demonstrated by EdU labelling (Figs. 1f, 6c). In addition, we observed a 2-fold higher DHO accumulation upon treatment with BAY-2402234 on day 3 post-PHx (Fig. 4b and Supplementary Fig. 8a), suggesting that higher levels of precursors are being synthesized after ~ 60% PHx catering to increased proliferative capacity, and probably due to higher levels of ammonia processing as a consequence of greater tissue loss, as suggested by the isotope fraction enrichment data where up to ~75% of new CA and orotate stemmed from the labeled ammonia compared to only ~ 50% in ~ 35% PHx (Fig. 4c and Supplementary Fig. 8b). Interestingly, despite the same treatment with BAY-2402234 at 2 mg/kg for 3 days post-PHx, orotate accumulated 2-fold higher after ~ 60% PHx (Fig. 4b and Supplementary Fig. 8a), indicating ‘attenuation’ rather than ‘suppression’ of DHODH at the indicated dose after ~ 60% PHx, and hence, the significantly higher proliferation after ~ 60% PHx compared to ~35% PHx by EdU labelling in day 3 treated mice (Figs. 1f, 6c). Increasing the dose to 5 mg/kg, however, led to higher mortality (Supplementary Fig. 2).

When analyzed for the metabolic flux to show incorporation of ammonia into de novo pyrimidine precursors as it traverses the functional liver zones from periportal to pericentral 3 days post-PHx (Fig. 6k and Supplementary Fig. 9), incorporation into high levels of Glu and Gln showed periportal and pericentral accumulation, respectively (Fig. 6k, 6l). The high levels of Asp mirrored Glu with reducing trend from periportal to pericentral, indicative of the more periportal urea cycle activity incorporating ammonia into Glu, then transaminating to Asp (Fig. 6k, m). Interestingly, treatment with BAY-2402234 led to near depletion of Gln levels not observed in ~ 35% PHx (Figs. 5e, f, 6k, l), whose flux mirrors a concurrent higher level of the orotate precursors Asp, CA, and DHO peaking at the highest pericentrally (Fig. 6k, m, n). This suggests that under conditions of greater hepatic tissue loss (~ 60% PHx), a more active pericentral incorporation of ammonia into de novo pyrimidine precursors occurs. The almost iden