Characterization of the scavenger cell proteome in mouse and rat liver

Distribution of ammonium metabolism-related proteins in rodent livers

The distribution of GS, GLS2, CPS1, GLT1 and RhBG protein in liver sections was investigated by immunofluorescence analyses (Figure 1). GLS2⁺ hepatocytes were confined to the periportal zone, GS⁺ scavenger cells surrounded the central vein and both subpopulations were clearly demarked by a mid-zone constituted by GLS2⁻/GS⁻ hepatocytes in rodent liver slices. CPS1 was found in GLS2⁺ and in hepatocytes of the transitional zone but not in GS⁺ scavenger cells. As shown in Figure 1, GS strongly colocalized with RhBG as well as with GLT1 in scavenger cells from both, rat and mice. These data suggest that the localization of the ammonium metabolism-related proteins GLS2, CPS1 and GS is similar in mouse and rat livers. Furthermore, the clear and specific labeling of perivenous scavenger cells by the antibodies directed against GLT1 and GS confirmed their suitability for isolating perivenous scavenger cells from the liver.

Figure 1:

Metabolic zonation of the liver.

Immunofluorescence analyses of glutamine synthetase (GS), glutaminase 2 (GLS2), carbamoylphosphate synthetase 1 (CPS1), glutamate/aspartate transporter II (GLT1) and ammonium transporter Rh type B (RhBG) protein in (A) mouse and (B) rat liver sections. Cell nuclei were counterstained with Hoechst 34580.

Characterization of the cellular proteome of scavenger cells from rat and mouse livers

For the characterization of the proteome of GS-expressing scavenger cells in rat and mouse livers, GS⁺ (perivenous) cells and GS⁻ (periportal and midzonal) hepatocytes were purified by means of FACS sorting (Supplementary Figure 1). Protein lysates of the separated cells were then subjected to mass spectrometry in order to identify individual protein profiles of GS⁺ scavenger hepatocytes (GS⁺ HCs) and GS⁻ hepatocytes (GS⁻ HCs). As illustrated in Figure 2, a total of 1717 and of 1263 proteins was quantified in isolated mouse or rat hepatocytes, respectively. Among the 1717 quantified proteins in mouse, 1503 (87.5%) proteins showed comparable abundances in both, GS⁺ and GS⁻ hepatocytes. In GS⁺ hepatocytes, 130 (7.6%) proteins showed a significantly higher abundance compared to GS⁻ hepatocytes (q-value < 0.05), whereas 84 (4.9%) proteins were higher abundant in GS⁻ HCs (Figure 2A and B). As expected, scavenger cell markers such as GS, GLT1 and ornithine aminotransferase (OAT), were detected in higher abundances in GS⁺ hepatocytes (Figure 2C, left panel). Moreover, proteins characteristic for periportal hepatocytes such as mitochondrial ornithine carbamoyltransferase (OTC), argininosuccinate synthase (ASS1) and phosphoenolpyruvate carboxykinase (PCK1) were only barely detected in this cell population (Figure 2C, left panel). Interestingly, heat shock protein 25 (HSP25), basal transcription factor 3 (BTF3) and RNAse 4 were higher abundant in GS⁺ HCs compared to GS⁻ mouse hepatocytes. Further proteins showing higher abundances in GS⁺ scavenger hepatocytes, are shown in Figure 2B and Table 1.

Figure 2:

Proteome analysis of GS expressing scavenger hepatocytes and GS negative hepatocytes in mouse and rat.

Hepatocyte subpopulations were isolated from mouse and rat livers and analyzed by mass spectrometry as described in materials and methods (n = 4, respectively). (A) Pie charts illustrating the number of proteins differentially abundant in GS⁺ or GS⁻ hepatocytes (HCs) from mice (left panel) or rat (right panel). (B) Volcano plots illustrating differentially abundant proteins in scavenger cells (GS⁺ hepatocytes) compared to periportal hepatocytes (GS⁻ hepatocytes) in mice (left panel) or rat (right panel). Proteins significantly higher abundant in GS⁺ hepatocytes are labeled in green and proteins higher abundant in GS⁻ hepatocytes are represented in red. The fold change represents the difference of the means of the respective log₂ LFQ intensity values. (C) Bar chart indicating fold changes of selected proteins in GS⁺ scavenger cells (upper panel) or GS⁻ hepatocytes (lower panel) in mice (left) and rat (right), respectively.

In contrast to GS⁺ hepatocytes, GS⁻ hepatocytes showed significantly higher abundances of GLS2, PCK1 and urea cycle enzymes such as CPS1, OTC, ASS1 and arginase 1 (ARG1), whereas the amounts of GS⁺ scavenger cell markers were very low (Figure 2C, left panel).

Proteome profiles of GS⁺ and GS⁻ hepatocytes slightly differed between mouse and rat (Supplementary Figure 2). For instance, glutathione S-transferase alpha-5 (GSTA5) and scavenger receptor class B member 1 (SCARB1) were detected in higher abundances in rat but not in mouse GS⁺ hepatocytes (Figure 2C, right panel and Supplementary Figure 2).

Protein network and gene ontology (GO) biological terms analyses revealed an enrichment of proteins related to 115 and 173 biological processes in mouse and rat GS⁺ scavenger cells compared to GS⁻ hepatocytes, respectively (Supplementary Figure 3 and Supplementary Table 2). Some of these categories such as “xenobiotic metabolic process”, “response to drug” and “glutathione metabolic process” are well established in perivenous hepatocytes (Jungermann 1988).

To further validate the mass spectrometry data, we performed immunofluorescence analyses on mouse liver sections which allow for the investigation of both, the spatial protein distribution as well as the heterogeneity of protein expression within hepatocyte subpopulations on liver sections. As depicted in Figure 3A, heat shock protein 25 (HSP25) and basic transcription factor 3 (BTF3) immunofluorescence was high in the majority of GS⁺ scavenger cells. However, in a subset of GS⁺ hepatocytes and in GS⁻ cells, the BTF3 and HSP25 immunofluorescence intensities were very weak or only barely detectable. Roughly 68.1% of the GS⁺ hepatocytes showed high staining intensities of HSP25, while 56.6% of GS⁺ hepatocytes were strongly positive for BTF3.

Figure 3:

Validation of proteome analysis by immunofluorescence analysis in mouse liver sections.

(A) Analyses of glutamine synthetase (GS), heat shock protein 25 (HSP25) and basic transcription factor 3 (BTF3) by immunofluorescence and fluorescence microscopy. Cell nuclei were counterstained with Hoechst 34580. (B) Proportions of scavenger cells showing high or low levels of HSP25 (left) or BTF3 (right), respectively.

These data show that protein expression differs among GS⁺ hepatocytes which is indicative for the existence of scavenger cell subpopulations.

Characterization of the cellular proteome of periportal hepatocytes in mouse liver

In following experiments, we aimed to characterize the proteome of periportal GLS2⁺ hepatocytes in relation to GS⁺ hepatocytes in mouse liver. Due to antibody incompatibilities, scavenger cells were labeled in these experiments not with antibodies directed against GS, but with antibodies detecting an extracellular epitope of GLT1⁺. Periportal hepatocytes were labeled with antibodies directed against E-cadherin and GLS2. As shown by immunofluorescence analysis on liver sections, virtually all E-cadherin expressing periportal hepatocytes coexpressed GLS2 (Figure 4A) suggesting the suitability of E-cadherin as an epitope for the purification of GLS2⁺ periportal hepatocytes. Moreover, in isolated primary mouse hepatocytes, a strong colocalization of GLS2 and E-cadherin was observed, while GLT1⁺ hepatocytes did not coexpress E-cadherin (Figure 4A, lower panel and Supplementary Figure 4). Next, GLT1⁺ and E-cadherin⁺ hepatocytes were isolated by FACS sorting (Supplementary Figure 5) and their proteome profile was analyzed by mass spectrometry and bioinformatic analyses (Figure 4B and C). As shown in Figure 4D, GS was present in higher amounts in the fraction of GLT1⁺ hepatocytes but only barely detectable in the fraction of E-cadherin expressing hepatocytes. Of note, abundances of perivenous scavenger cell marker such as GS and GLT1 were lower in GLT1⁺ sorted perivenous hepatocytes compared to GS⁺-sorted perivenous hepatocytes. This may be explained by the different experimental set-up (lower signal intensity of GLT-staining) or by a lower purity of the GLT1⁺ and E-Cad⁺ sorted cells due to technical limitations. Conversely, GLS2 was only weakly detectable in the fraction of GLT1⁺ hepatocytes but highly abundant in the fraction of E-cadherin⁺ hepatocytes (Figure 4D). These data indicate a strong enrichment of GS-expressing perivenous scavenger hepatocytes and GLS2-expressing periportal hepatocytes in FACS sorted fractions of GLT1⁺ and E-cadherin⁺ hepatocytes, respectively.

Figure 4:

Proteome analysis of GLT1⁺ scavenger hepatocytes and E-cadherin⁺ periportal hepatocytes in mouse. E-cadherin⁺ and GLT1⁺ hepatocytes were isolated from mouse livers and analyzed by mass spectrometry as described in Materials and methods (n = 3). (A) Immunofluorescence analysis of E-cadherin (E-Cad), glutamate/aspartate transporter II (GLT1) and glutaminase 2 (GLS2) in mouse liver sections or in isolated mouse hepatocytes. Cell nuclei were counterstained with Hoechst 34580. (B) Pie charts illustrating the number of proteins differentially abundant in mice in GLT1⁺ or E-Cad⁺ hepatocytes (HCs). (C) Volcano plots illustrating differentially abundant proteins in GLT1-expressing scavenger cells (GLT1⁺ HCs) compared to periportal E-cadherin⁺ hepatocytes (E-Cad⁺ HCs) from mice. Proteins significantly higher abundant in GLT1⁺ hepatocytes are labeled in green and proteins with higher abundances in periportal E-Cad⁺ hepatocytes are represented in red. The fold change represents the difference of the means of the respective log₂ LFQ intensity values. (D) Bar chart indicating fold changes of selected proteins in GLT1⁺ scavenger cells (upper panel) or E-Cad⁺ periportal hepatocytes (lower panel) in mice. (E) Venn diagram indicating the overlap of proteins higher abundant in perivenous (GS⁺ and GLT1⁺) hepatocytes (left panel) or in GS⁻ and E-Cad⁺ hepatocytes (right panel).

As shown by proteome analysis the abundances of 77 proteins were lower and of 48 proteins were higher in E-cadherin⁺ hepatocytes when compared to GLT1⁺ hepatocytes. Besides proteins well-known to be expressed in periportal hepatocytes, the proteome analysis revealed a number of proteins which have not yet been recognized to be highly abundant in E-cadherin⁺ hepatocytes compared to GLT1⁺ scavenger cells. Among these proteins were mitochondrial glycine dehydrogenase (GLDC), sideroflexin-1 or mitochondrial aldehyde dehydrogenase X (Table 2). Protein network and GO biological terms analyses revealed an enrichment of proteins related to 107 biological processes in mouse E-Cad⁺ hepatocytes compared to GLT1⁺ hepatocytes, respectively (Supplementary Figure 3 and Supplementary Table 2). Proteins that were enriched in E-Cad⁺ hepatocytes relate to biological processes already established in these cells such as “organonitrogen compound metabolic process” or “urea cycle” (Supplementary Figure 3 and Supplementary Table 2).

The results of the two independent proteome analyses partially overlapped (Figure 4E). For instance, 48 proteins were concordantly more abundant in both, GS⁺ hepatocytes (compared to GS⁻ hepatocytes) and GLT1⁺ hepatocytes (when compared to E-cadherin⁺ hepatocytes). Moreover, 25 proteins were identified as being higher abundant in GS⁻ periportal hepatocytes (compared to GS⁺ hepatocytes) as well as in E-cadherin⁺ hepatocytes (compared to GLT1⁺ hepatocytes) (Figure 4E). Moreover, the comparison of enriched GO terms revealed that 69 biological process were enriched in both, GS⁺ and GLT1⁺ hepatocytes in mouse (Supplementary Figure 6). The comparison of GS⁻ hepatocytes and E-Cad⁺ periportal hepatocytes showed that 80 terms were likewise enriched in these hepatocytes (Supplementary Figure 6). Interestingly, 103 additional biological processes were found enriched only in GS⁻ hepatocytes, which may be dedicated to the fact that the GS⁻ subpopulation comprises periportal (GLS2⁺) as well as midzonal hepatocytes.

The differences of the proteome and GO analyses resulting from the two different approaches may be explained by the fact that the GS⁺ perivenous hepatocytes were compared to the periportal plus mid-zone hepatocytes, while perivenous GLT1⁺ hepatocytes were directly compared to periportal E-cadherin⁺ cells.

Effects of dietary protein load on levels and distribution of key proteins involved in ammonium metabolism in mouse liver

With the aim to investigate whether levels and the distribution of proteins involved in ammonia metabolism change in response to the dietary nitrogen load, mice were fed with standard chow or with low or high protein diet for two weeks (Supplementary Figure 7). While protein levels of the scavenger cell marker GS remained unchanged, levels of RhBG were elevated in mice fed with high protein diet compared to low protein or standard diet (Figure 5A). HSP25 protein levels were substantially decreased in low protein fed mice and tended to be increased in livers of animals receiving a high protein diet (Figure 5A).

Figure 5:

Effects of dietary protein content on the expression of RhBG, GS, HSP25, GLS2, GLT1 and CPS1 in mouse liver.

C57BL/6J mice at the age of eight-weeks were fed ad libitum with control, low protein or high protein diet for 14 days as described in Materials and methods (n = 3). Animals were sacrificed, livers were dissected and snap-frozen in liquid nitrogen. (A) Immunofluorescence analyses of glutamine synthetase (GS) and glutaminase 2 (GLS2), carbamoylphosphate synthetase 1 (CPS1), ammonium transporter Rh type B (RhBG), heat shock protein 25 (HSP25) or glutamate transporter (GLT1). Cell nuclei were counterstained with Hoechst 34580. (B) Western Blot analyses of GS and GLS2 protein lysates prepared from livers of mice fed with diets with different protein content. Data represent the average ± standard error of the mean of three animals per condition (n = 3). *Statistically significantly different. (C) Relative quantification of GLS2 positive hepatocytes in livers from mice fed with low or high protein diet. Liver sections were stained with phalloidin-TRITC (red) to identify individual cells and for GLS2 (green) as indicated. The number of GLS2 positive and GLS2 negative hepatocytes was evaluated for three animals per condition in 5–10 distinct positions in each liver (left panel). Bar chart indicates percentage of GLS2 positive hepatocytes in mouse liver (right panel).

In periportal hepatocytes, GLS2 protein levels were elevated after high protein diet (Figure 5, Supplementary Figure 8). In order to quantify GLS2⁺ hepatocytes after high protein diet, individual hepatocytes were discriminated on liver slices by staining polymerized actin filaments with phalloidin-TRITC and GLS2⁺ hepatocytes were counted. Here we found, that the amount of GLS2⁺ hepatocytes was significantly increased in mice fed with a high protein diet (59.2 ± 1.5%) compared to those fed with a low protein diet (50.9 ± 2.4%) (Figure 5C). This was reflected by an enlargement of the area comprising GLS2⁺ HCs.

These data show that the dietary protein intake can not only alter the expression of proteins related to ammonium metabolism, as already shown decades ago for urea cycle enzymes by Schimke (Schimke 1962), but also their subacinar distribution in mouse liver. This especially holds for GLS2, which provides ammonium ions for urea synthesis. Thus, the increase of the GLS2⁺ zone at the expense of the GLS2⁻ and GS⁻ mid-zone suggests that more hepatocytes become recruited for urea synthesis in response to high protein diet.

Animals and treatments

All animal experiments were reviewed and approved by the appropriate authorities and were performed in accordance with the German animal protection law (North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection, reference number Az. 81-02.04.2016.A289). In order to study the effect of protein load on the liver zonation, eight-weeks old male C57BL/6J mice (n = 3) were fed ad libitum with either low protein diet (9 kJ% protein, Ssniff, Soest, Germany), high protein diet (49 kJ% protein, Ssniff) or standard chow (26 kJ% protein, Ssniff) for two weeks. All animals had free access to water and food. Detailed compositions of all diets are given in the supplement (Supplementary Figure 7). At the end of the treatment, mouse livers were briefly perfused with physiological saline to remove residual blood, dissected, shock frozen and analyzed by Western blot and immunofluorescence analyses.

Hepatocyte isolation, staining and sorting

Hepatocytes were isolated from eight-weeks old C57BL/6J mice using a two-step collagenase perfusion method. Briefly, livers were perfused via the portal vein with Ca²⁺-free HEPES buffer (HANKS buffer) for 3 min, followed by perfusion with HANKS buffer supplemented with 5 mM CaCl₂ and 0.4 mg/ml type II collagenase for 15 min at 42 °C. After digestion with collagenase, the liver was dissected and carefully disrupted with forceps in Krebs-Henseleit-buffer. The resulting cell suspension was filtrated through a filter with a diameter of 70 μm. The isolated single hepatocytes were immediately fixed with 4% formaldehyde for 10 min at room temperature (RT) and permeabilized with 0.3% (v/v) Tween-20 in PBS. Thereafter, hepatocytes were incubated in blocking reagent (3% bovine serum albumin (BSA) in PBS) for 30 min. Cells were stained for the intracellular marker glutamine synthetase (GS) using the mouse anti-glutamine synthetase antibody (#610518, BD Bioscience, Heidelberg, Germany) at a concentration of 1:100 in blocking reagent for 1 h at RT. For the labeling of extracellular markers anti-glutamate transporter 1 (#ab41621, Abcam, Cambridge, UK) or E-cadherin (#610182, BD Biosciences), isolated hepatocytes were treated with blocking solution 3% BSA (15 min) without prior fixation and permeabilization. Cells were then stained with fluorophore-conjugated secondary antibodies (1:100 1 h, RT) and cell nuclei were counterstained with Hoechst 34580 (1:5000, Thermo Fisher Scientific, Schwerte, Germany). Hepatocytes were separated and collected by using FACSAria III (BD Biosciences). A 2.0 neutral density filter in front of the forward scatter detector was used to decrease the FSC signal. The sorting of primary hepatocytes was performed using a 100 µm Nozzle. The collected Hoechst⁺-labeled hepatocytes were divided in GS positive (GS⁺) and GS negative (GS⁻) hepatocytes or GLT1 (GLT1⁺) and E-cadherin (E-Cad⁺) positive hepatocytes, respectively. For proteome analysis, 500,000 cells of each fraction were collected (n = 3–4).

Mass spectrometric analysis of GS positive and negative cells

In order to characterize the similarities and differences between different hepatocyte populations, quantitative mass spectrometry was conducted essentially as described (Grube et al. 2018). Briefly, cell lysates were prepared and analyzed from three to four independent replicates per group. Five µg of protein per sample were prepared by in-gel digestion with trypsin after reduction and alkylation with iodoacetamide. Finally, 500 ng of resulting peptides were separated using a 2 h gradient via an Ultimate 3000 rapid separation liquid chromatograph system on C18 material. Separated peptides were sprayed by an electrospray ionization nano source directly into a Q Exactive plus mass spectrometer operated in data-dependent positive mode. First, survey scans were recorded with a resolution of 70,000 (or 140,000 for the comparison of GLT1⁺ and E-Cad⁺ cells) and subsequently up to ten 2- and 3-fold charged precursors were selected by the quadrupole (2 m/z isolation window), fragmented by higher-energy collisional dissociation and analyzed at a resolution of 17,500. For the analysis of GS⁺ and GS⁻ hepatocytes, spectra were further processed for peptide and protein identification as well as precursor spectra-based quantification with MaxQuant version 1.6.1.0 (Max Planck Institute for Biochemistry, Planegg, Germany) using standard parameters if not stated otherwise. For the three different experiments, different versions of MaxQuant and protein datasets downloaded from the UniProt knowledgebase were used: MaxQuant 1.6.1.0 and 52548 Mus musculus sequences (UP000000589, 18th January 2018) for the comparison of mouse GS⁺ and GS⁻ cells, MaxQuant 1.6.3.4 and 29951 Rattus norvegicus sequences (UP000002494, 10th April 2019) for the comparison of rat GS⁺ and GS⁻ cells and MaxQuant 1.6.10.43 and 55192 Mus musculus sequences (UP000000589, 7th June 2019) for the comparison of mouse GLT1⁺ and E-Cad⁺ cells. Carbamidomethylation at cysteines was considered as fixed and protein N-terminal acetylation as well as methionine oxidation as variable modifications. The ‘match between runs’ function was enabled as well as label-free quantification, peptides and proteins were accepted at a false discovery rate of 1%. Quantitative data (LFQ intensities) was further processed with Perseus version 1.6.1.1 (Max Planck Institute for Biochemistry, Planegg, Germany) for GS⁺ and GS⁻ hepatocytes or with Perseus 1.6.6.0 (Max Planck Institute for Biochemistry) for GLT1⁺ and E-Cad⁺ hepatocytes. Here, only proteins were considered showing at least two different peptides and three valid intensity values in at least one group. To reveal differences between the groups, the significant analysis of microarrays method (S ₀ = 0.8 for GS⁺ and GS⁻ hepatocytes or S ₀ = 0.6 for GLT1⁺ and E-Cad⁺ hepatocytes, FDR = 5%; (Tusher et al. 2001)) based on Student’s t-tests was applied to log₂-transformed values. This method includes permutations of repeated measurements to estimate the false discovery rate and, therefore, accounts for repeated measurements in this case several hundred/thousands of proteins. Before analysis, missing values were filled in with values drawn from a down-shifted normal distribution (width 0.3, downshift 1.8 standard deviations). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2019) partner repository (dataset ID: PXD023561).

Proteins showing significant differences in the aforementioned comparisons were separately analyzed for higher and lower abundant proteins by STRING v11 (Szklarczyk et al. 2019). Here, a network analysis was carried out for categorical enrichment and network analysis. Only high confidence interactions were considered from textmining, experiments, databases and co-expression. Respective proteins have been further analyzed by STRING for enriched gene ontology biological process terms. Only terms were reported which were associated with a Benjamini-Hochberg corrected p-value < 0.05.

Western blot

For Western blot analysis, liver tissue was homogenized using a pistil and thoroughly lysed in ice-cold lysis buffer (10 mM Tris pH 7.4, 1% (v/v) Triton X-100, 0.5% (v/v) NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 0.2 mM PMSF). Cell debris was removed from soluble proteins by centrifugation at 20,000 g at 4 °C for 10 min. Protein concentrations of liver homogenates were determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany). Equal amounts of protein mixed with gel loading buffer were utilized for polyacrylamide gel electrophoresis on 10–15% SDS polyacrylamide gels as described before (Görg et al. 2019; Qvartskhava et al. 2015). After electrophoresis, proteins were transferred on nitrocellulose membranes by semi-dry blotting technique. Membranes were incubated in 5% BSA in Tris-buffered saline containing 0.01% (v/v) Tween-20 (TBST) for 30 min. Subsequently, membranes were incubated with anti-GAPDH antibody (1:5000, #H86504M, Biodesign International, Saco, USA) for 1 h at RT, while incubation with antibodies directed against GS (1:1000, #610518, BD Bioscience), GLS2 (1:1000, #ab93434, Abcam) were conducted at 4 °C RT overnight. Membranes were then washed three times with TBST and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies at a concentration of 1:10,000, for 1 h at RT (HRP-coupled goat-anti mouse antibody [#170-6516, Bio-Rad Laboratories]; HRP-coupled goat-anti rabbit antibody [#P0448, Dako, Biozol, Echig, Germany]; HRP-coupled mouse-anti rabbit antibody [#sc2357, Santa Cruz, Heidelberg, Germany]). Unbound antibodies were removed by washing the blots three times with TBST. HRP activity was detected using ECL Western Blotting Substrate (Promega, Walldorf, Germany) and images were acquired using ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories). Densitometric analysis was performed using Image Lab software (Bio-Rad Laboratories). Relative protein levels of a given protein were quantified by analyzing the raw signal intensities from the digitally acquired pictures and normalizing to GAPDH levels for each sample.

Immunofluorescence staining

Cryosections (7 µm) of mouse and rat livers were prepared using the Leica Cryostat CM1950 (Leica Biosystems, Wetzlar, Germany). Sections were fixed with ice-cold 100% methanol for 10 min and washed three times with PBS. Sections were blocked with 5% BSA in PBS for 30 min and incubated with primary antibody solutions at 4 °C overnight. Anti-GS (#610518, BD Bioscience) and anti-RhBG (#ab106801, Abcam) antibodies were used at a concentration of 1:500, while anti-GLS2 (#ab93434, Abcam), anti-CPS1 (#ab3682, Abcam), anti-GLT1 (#ab41621, Abcam), anti-E-Cad (#610182, BD Biosciences), anti-HSP25 (#ADI-SPA-801-F, Enzo Life Sciences, Lörrach, Germany), and anti-BTF3 (#ab203517, Abcam) were used at 1:200 in blocking buffer. Following three washing steps with PBS, sections were incubated with fluorochrome-coupled secondary antibodies (1:500) and Hoechst 34580 (1:20,000) for counterstaining of the nuclei for 1 h at RT. Sections were mounted with Fluoromount-G (Thermo Fisher Scientific) and immunofluorescence analysis was performed using the epifluorescence microscope Observer.Z1 (ZEISS, Oberkochen, Germany) or confocal laser scanning microscopy (LSM510 and LSM880, ZEISS).

Quantification of GLS2⁺ and GLS2⁻ hepatocytes in the liver

GLS2⁺ and GLS2⁻ hepatocytes were quantified in fixed liver slices costained with anti-GLS2 antibodies anti-GLS2 (#ab93434, 1:200, Abcam) and phalloidin-TRITC (#P1951, 1:200, Sigma-Aldrich). Per liver slice 5–10 immunofluorescence pictures were acquired using an epifluorescence microscope (dx25, ObserverZ.1, ZEISS). Individual hepatocytes were discriminated by membraneous f-actin staining. Numbers of hepatocytes along the acinus were determined in each picture straight from the centers of the portal to the central vein. The number of GLS2⁺ hepatocytes surrounding one portal vein was determined in triplicates, averaged and given relative to the total number of hepatocytes.

Statistical analysis

Data are presented as means ± standard error of the mean (SEM). Statistical differences between the groups were determined using Student’s t-test or one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test (GraphPad Prism V5.01, La Jolla, USA) as appropriate. A p-value ≤ 0.05 was considered significant.