Untargeted Metabolomics Analysis of ABCC6-Deficient Mice

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Untargeted metabolomics analysis of ABCC6-deficient mice discloses an altered metabolic liver profile Mie Rostved Rasmussen, Kirstine Lykke Nielsen, Mia Roest Laursen, Camilla Bak Nielsen, Pia Svendsen, Henrik Dimke, Erik Ilsø Christensen, Mogens Johannsen, and Søren Kragh Moestrup J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00669 • Publication Date (Web): 19 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Journal of Proteome Research

Untargeted metabolomics analysis of ABCC6deficient mice discloses an altered metabolic liver profile

‡,

Mie R. Rasmussen1 Kirstine L. Nielsen2

‡

, Mia R. Laursen2, Camilla B. Nielsen2, Pia Svendsen3,

Henrik Dimke,3,4, Erik I. Christensen1, Mogens Johannsen2*, Søren K. Moestrup1,3,5*

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2

Department of Biomedicine, Aarhus University, Aarhus, Denmark

Department of Forensic Medicine, Section for Forensic Chemistry, Aarhus University, Aarhus, Denmark

Departments of 3Cancer and Inflammation and 4Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 5

Department of Clinical Biochemistry, Pharmacology, Odense University Hospital, Odense, Denmark

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ACS Paragon Plus Environment


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KEYWORDS ABCC6; pseudoxanthoma elasticum (PXE); metabolomics; UPLC-HR-TOF-MS; N-acetylated amino acids; acyl glycine

ABSTRACT

Loss-of-function

mutations

in

the

transmembrane

ABCC6

transport

protein

cause

pseudoxanthoma elasticum - an ectopic, metabolic mineralization disorder affecting the skin, eye and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation.

Untargeted metabolomics using liquid chromatography mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5) that is involved in acetylation reactions were accumulated in the liver. None of the identified metabolites seem to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.

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INTRODUCTION Mutations in the gene encoding the ATP-binding cassette (ABC) protein ABCC6 cause pseudoxanthoma elasticum (PXE)1-4. The disorder manifests in ectopic mineralization of elastinrich tissues such as the skin, arterial vessels and Bruch’s membrane of the retina. Similar mineralization manifestations are observed in mice with targeted disruption of the Abcc6 gene5-6. ABCC6 is capable of transporting organic anions in an ATP-dependent manner as demonstrated in an in vitro vesicular transport assay7-8. Yet, physiologically relevant substrates for this transporter have not been identified. Vitamin K and adenosine have been tested as potential candidates, but no experimental evidence for these compounds as ABCC6 substrates exists9-13. Recent studies reported ABCC6 to affect cellular nucleoside triphosphate release in vitro14-15. An untargeted metabolomics study demonstrated that culture medium from ABCC6expressing human embryonic kidney 293 (HEK293) cells contained increased levels of the nucleosides inosine and guanosine, nucleoside phosphates and nucleotide sugars compared to media from control cells14-15. However, an in vitro vesicular transport assay showed that nucleoside triphosphates were not transported by the ABCC6 protein14. Nevertheless, the study provides interesting, novel PXE pathomechanistic information with respect to the role of pyrophosphate (PPi), which is released from nucleoside triphosphates and acts as an important factor to prevent ectopic mineralization. However, the exact role of the ABCC6 protein in this mechanism remains unknown14-16. The ABCC6 protein is expressed predominantly in the liver. Although, the subcellular localization of ABCC6 in the liver has been a matter of debate, the predominant view is that the protein localizes to basolateral membrane domains of human and mouse hepatocytes6,17-18. Recently, this view was challenged by Martin et al., who reported that ABCC6 is associated with 3



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hepatic mitochondria-associated membranes – sites of dynamic contacts between mitochondria and the endoplasmatic reticulum19. This statement was based on fractionation of homogenized tissue from mice by ultracentrifugation. Subsequent immunoblotting showed enrichment of ABCC6 in mitochondrial subcellular fractions. These observations were retorted in a research commentary by Pomozi and coworkers who employed immunofluorescent labeling of mouse liver tissue sections and mouse primary hepatocytes to reconfirm a basolateral localization of the protein20. A basolateral subcellular localization is in accordance with a presumed functional role of ABCC6 in transport of cytosolic compounds from the liver to the blood. Surprisingly, PXE-affected tissues are devoid of ABCC6 protein expression. PXE is thus characterized as a metabolic disorder21. This view finds supporting evidence in several experimental studies of a PXE mouse model with targeted disruption of the Abcc6 gene, and it is suggested that the ectopic mineralization phenotype associated with PXE may be caused by absence of an unknown anti-mineralizing compound normally exported from the liver to the blood by ABCC622-23. Alternatively, mineralizing perturbations may be secondary effects of intoxication of the liver resulting from hepatic accumulation of the ABCC6 substrate (or several substrates)24. This substrate accumulation in the liver may elicit changes in the regulation of hepatic genes encoding proteins that directly or indirectly have pro-mineralizing effects in the blood. This has not been demonstrated in liver tissue, but knockdown of the ABCC6 gene in human HepG2 cells was shown to cause downregulation of genes encoding the antimineralization proteins NT5E, Fetuin A and Osteopontin and upregulation of genes encoding mineralization-promoting proteins such as TNAP25. The physiological substrate transported by ABCC6 is yet unknown. To investigate if the presence of ABCC6 has a measurable impact on the profile of metabolic substances in the liver 4


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cytosol, untargeted metabolomics was employed. Specifically, liver extracts of Abcc6-/- null mice that represent a PXE animal model were characterized using ultra performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS).

MATERIALS AND METHODS Animals Abcc6 gene knockout mice, that were generated on a C57BL/6 background5, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) (strain name: B6.129S1-Abcc6tm1Jfk/J5) and mated to C57BL/6 mice (Taconic, Ry, Denmark) to generate a heterozygous generation. The offspring obtained from intercrosses of this heterozygous generation were genotyped (GENterprise Genomics, Mainz, Germany), and mice homozygous for the Abcc6 mutant allele were designated Abcc6 gene knockout (KO) mice, whereas mice without genetically modified Abcc6 alleles were used as wild-type controls (WT). The liver study included seven KO mice and seven WT mice of which all were 7-9-week-old males. Mice were killed by cervical dislocation and the liver was removed post-mortem. The organ was crushed manually using a mortar. Three samples per animal were collected, put on dry ice and stored at -80˚C. Plasma was obtained from three KO and three WT male mice. Mice were anaesthetized using isoflurane before retro-orbital blood collection into heparin-coated tubes. All mice were killed by cervical dislocation immediately following blood sampling. Plasma samples were prepared by centrifugation at 4000 RPM for 15 min at 4°C and frozen at -20˚C until extraction and analysis. Urine was obtained from six KO and two WT male mice as outlined below. The mice were housed in a conventional animal facility. Animal housing and handling were in compliance with Danish regulations.

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Metabolic cage experiments KO and WT mice were housed individually in metabolic cages in a temperature-controlled environment with an average ambient temperature of 24.5°C degrees and humidity of 53%. Mice were maintained on a standard rodent diet (Altromin #1324) supplemented with 3.3% agar and water (food to water was 1:1.6). During the entire course of the experiment, animals were allowed free access to food and water. After an acclimatization period of 2 days, urine was collected over a 24-hour period, frozen, and maintained at -80°C prior to metabolomics analysis. Metabolic cage experiments were conducted in accordance with Danish law under the animal experimental permit #2014-15-0201-00043.

ABCC6 antibodies The rat monoclonal antibodies M6II-24 and M6II-68 (MON9805 and MON9806, Sanbio Nordic Biosite, Copenhagen, Denmark) both detect the same intracellular epitope of the mouse ABCC6 protein. The goat polyclonal antibody S-20 (sc-5787, Santa Cruz Biotechnology - AH Diagnostics, Aarhus, Denmark) detects the extracellular N-terminus of the mouse ABCC6 protein.

Western blotting Mouse liver tissue was homogenized in radio-immunoprecipitation assay buffer (SigmaAldrich, Broendby, Denmark) supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Hvidovre, Denmark) by a rotor stator homogenizer. The homogenate was centrifuged twice at 10,000 g for 10 min at 4˚C. The protein concentration of the supernatant was determined by a bicinchroninic acid assay (Pierce - Fischer Scientific, Roskilde, Denmark). 6


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Mouse liver homogenate (50 µg) was subjected to SDS-PAGE essentially as described19. To maintain proteins in a reduced state, samples were added NuPAGE Sample Reducing Agent (Invitrogen, Naerum, Denmark) and the running buffer was supplemented with NuPAGE Antioxidant (Invitrogen). Mouse ABCC6 protein was detected by incubation with the monoclonal anti-mouse ABCC6 antibodies M6II-24 and M6II-48 (1:20 dilution) for 16 h followed by incubation with an anti-rat alkaline phosphatase-conjugated antibody (Abcam, Cambridge, UK) for 2 h. Mouse ABCC6 protein was also detected by the polyclonal anti-mouse S-20 antibody (1:100 dilution) for 16 h followed by incubation with an anti-goat alkaline phosphatase-conjugated antibody (Sigma-Aldrich) for 2 h. Protein bands were developed by the nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate system (Roche). GAPDH protein was detected by a monoclonal anti-GAPDH peroxidase-conjugated antibody (SigmaAldrich) and protein bands were developed by an enhanced chemiluminescence western blotting substrate (Pierce) using a FUJI FLA3000 Gel Doc (Fujifilm, Düsseldorf, Germany). Blots that were incubated with secondary antibody only were included as controls.

Electron microscopy Livers from KO and WT mice were fixed by retrograde perfusion through the aorta with 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. The tissue was trimmed into small blocks, further fixed by immersion for 1 h in the same fixative, infiltrated with 2.3 M sucrose for 30 min and finally frozen in liquid nitrogen. 70-90 nm tissue cryosections were obtained at 100˚C with a Leica EM FC6 cryoultramicrotome (Leica Microsystems, Ballerup, Denmark) as previously described26.

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For EM immunolabeling, tissue sections were incubated with the primary rat monoclonal antibody M6II-24 (Sanbio) (1:10 dilution) at 4˚C overnight followed by incubation at room temperature for 1 h with 10 nm gold particles coupled to anti-rat IgG (BioCell, Cardiff, UK). The cryosections were embedded in methylcellulose containing 0.3% uranyl acetate and studied in a FEI CM100 electron microscope (FEI). Controls were prepared by incubation with secondary antibodies alone or with non-specific IgG.

Extraction of metabolites from liver tissue samples Extraction of metabolites from liver tissue was performed essentially as previously described27. Tissue homogenization was achieved in 1 mL ice-cold methanol/water (1:1 v/v) in a TissueLyser (Qiagen, Copenhagen, Denmark) (25 Hz, 5 min) using a stainless steel bead. Following centrifugation at 15,700 × g for 5 min at 4˚C, supernatants were collected and transferred to Eppendorf tubes. The extracts were evaporated to dryness and stored at -80˚C. Prior to analysis, samples were reconstituted in 800 µL 0.1% formic acid in water. Samples were centrifuged to remove particles, and a quality control (QC) sample was prepared by pooling equal volumes of all samples.

Extraction of metabolites from plasma A volume of 100 µL of each sample was aliquoted into separate glass tubes, added 300 µL icecold methanol and mixed for protein precipitation. The tubes were kept at 4˚C for 10 min and then centrifuged at 2876 × g at 4˚C for 10 min. Sample supernatants were transferred to clean glass tubes and evaporated under a stream of N2. The precipitates were added 300 µL 8


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methanol/water (4:1 v/v), mixed and centrifuged at 2876 × g at 4˚C for 10 min. The supernatants were transferred to the corresponding glass tubes as before and evaporated to dryness. The dried extracts were reconstituted in 50 µL 0.1% formic acid in water and a QC-sample was prepared by pooling equal volumes of all samples.

Preparation of urine samples Prior to analysis a volume of 100 µL of each sample was aliquoted into separate glass tubes and added 100 µL of 5% acetonitrile and 0.1% formic acid in water. The mixture was centrifuged at 2876 × g at 4˚C for 30 min, supernatants were collected, and QC-samples were prepared by pooling equal volumes of all samples. Urinary creatinine concentrations were determined by colorimetry using an ABX Pentra Creatinine 120 CP kit (Horiba ABX SAS, Montpellier, France) according to the instructions provided by the manufacturer.

UPLC-HR-TOF-MS analysis All samples were analyzed on a UPLC-HR-TOF-MS instrument using an ACQUITY I-Class UPLC system (Waters Corporation, Milford, MA, USA) coupled to a Bruker maXis Impact QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in negative (-) and positive (+) electrospray ionization (ESI) modes. Chromatographic separation of liver, plasma, and urine samples was performed within 21 min using gradient elution on an ACQUITY UPLC HSS T3 C18 column (2.1 mm x 100 mm, 1.8 µm). Mobile phase A was 0.1% formic acid in water and mobile phase B was methanol/acetonitrile (1:1 v/v) with 0.1% formic acid. The column temperature was 50°C. The gradient was kept at 0% B for 2 min, linearly increased to 40% B within 6 min, linearly increased 9



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to 60% B within 6.5 min, linearly increased to 88% B within 11 min, linearly increased to 100% B within 11.5 min, and kept at 100% B until 18 min. The column was returned to 0% B within 19.5 min and kept at 0% B until 21 min for column equilibration. The flow rate was 0.4 mL/min and the injection volume was 10 µL (ESI-) and 4 µL (ESI+) for liver samples, 5 µL for plasma samples (ESI-), and 2 µL for urine samples (ESI- and ESI+). The sample temperature was 6°C. Calibration was performed in the beginning of each run by sodium formate acetate in ESI+ mode and sodium formate in ESI- mode. Samples were randomly injected. QC samples were injected in the beginning to condition the column and in between samples for quality assessment of instrument performance. A mass range of 50-1000 m/z and a sampling rate of 4 Hz were used. The capillary voltage was 4000 V in ESI+ mode and 2500 V in ESI- mode. The nebulizing gas pressure was 1.2 bar and the drying gas flow and temperature were 8.0 L/min and 220°C, respectively, in both ESI modes.

Data preprocessing The raw UPLC-HR-TOF-MS data were converted to mzXML files by CompassXport 3.0.6. (Bruker Daltonics, Bremen, Germany). The mzXML files were processed with XCMS28 in R (version 3.1.2, http://www.R-project.org). The initial scans containing calibration masses and dead volume (18 sec) were removed from all spectra. Subsequently, the centWave algorithm29 was used for peak picking with a resolution of 10 ppm (ESI+) or 12 ppm (ESI-) and a signal-tonoise ratio of 6. Retention time correction was conducted using the Obiwarp algorithm30. Gap filling was conducted in order to recover missing signals from raw data. Features should be present in at least 50% of all samples within one sample group to be included in further analysis. Isotopes, ion source fragments, and adducts were annotated using CAMERA31. 10


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To account for differences in liver tissue weight between samples, the intensity of each individual feature in a sample was normalized to the total intensity of the chromatograms and log-transformed prior to statistical analysis. Urine samples were normalized to the total intensity or creatinine concentrations, and plasma samples were normalized to the total intensity.

Statistical analysis The liver samples were analysed using multivariate analysis in SIMCA version 13 (Umetrics, Umeå, Sweden). Pareto scaling was applied before principal component analysis (PCA) and orthogonal projections to latent structures discriminative analysis (OPLS-DA). PCA served as an exploratory visualization of the data, whereas OPLS-DA was used to locate differences between KO and WT mice. The parameters Q2 and R2X(R2Y) (the X matrix was the metabolite features, and the Y matrix was the KO/WT groups) were used to evaluate the performance of the OPLSDA model. Q2 was obtained by seven-fold cross-validation and explains the predictability of the model, whereas R2 explains how well the model fit the data. The predictability of the OPLS-DA model was further validated by an independent test set; the data set was randomly divided into training and test sets containing 67% and 33% of the samples, respectively. Features with variable importance parameters (VIPs) > 1 were considered largely important for class separation. To further evaluate significant differences, a Welch’s two-sample t test with equal or unequal variance based on an F-test was employed. A P-value < 0.05 was considered significant. In order to correct for multiple comparisons, false discovery rate (FDR) q values were calculated and reported.

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Metabolite identification Features, that were significantly increased or decreased, were searched against an in-house database and/or in METLIN (https://metlin.scripps.edu), the human metabolome database (http://www.hmdb.ca), and MetFrag (http://msbi.ipb-halle.de/MetFrag) for tentative compound identification. Confirmation of identity was performed by matching the m/z-values, fragments, and retention time values to the available authentic standards.

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RESULTS Immuno-electron microscopy of ABCC6 in mouse liver To unambiguously visualize the subcellular localization of ABCC6, livers of KO and WT mice were inspected using immuno-electron microscopy (immuno-EM) with the three anti-mouse ABCC6 antibodies that were used by Martin et al.19 and Pomozi et al.20. Furthermore, the specificity of the employed antibodies was investigated. The anti-mouse ABCC6 antibodies M6II-24, M6II-68 and S-20 detect a ~160 kDa protein corresponding to the expected size of the ABCC6 protein in liver homogenate of WT mice, whereas this protein is absent in samples from ABCC6-deficient KO mice (Figure 1). The M6II-68 and S-20 antibodies do however also detect other proteins in liver homogenates, whereas the M6II-24 antibody exhibits specificity for the mouse ABCC6 protein. Immuno-gold labeling using the M6II-24 antibody clearly detects ABCC6 in basolateral (perisinusoidal) membranes of hepatocytes of WT mice (Figure 2A). No immuno-gold labeling in intracellular compartments or organelles was detected in the hepatocytes of WT mice (Figure 2A). Moreover, liver cryosections of KO mice were devoid of ABCC6-specific labeling (Figure 2B). In conclusion, we confirmed a basolateral localization of ABCC6, which indicate that a substrate from the liver cytosol will be secreted in plasma.

UPLC-HR-TOF-MS analysis of metabolites in mouse liver To investigate the importance of functional ABCC6 on the profile of metabolites in the liver cytosol, liver extracts of KO male mice and WT male mice were analyzed by UPLC-HR-TOFMS. The PCA scores plot of ESI- data (Figure 3) showed that KO mice cluster slightly to the right, whereas the WT mice expressed larger variation. This variation also seemed to affect the OPLS13



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DA model showing a rather low predictability with Q2(cum) = 0.347. The R2Y(cum) = 0.992 was much higher than Q2 and the low R2X(cum) = 0.463 could additionally indicate some model overfitting. The sensitivity (true KO) and specificity (true WT) from the test set validation was 67% and 83%, respectively. The metabolic identity of up- and downregulated features in KO mice was investigated as it was expected that accumulating metabolites in the liver of KO mice may directly or indirectly be related to ABCC6 protein function. Despite of the rather low predictability of the OPLS-DA model, features with VIP-values > 1 together with P-values < 0.05 were used to select features for identification. More features were significantly upregulated than downregulated in KO mice (Figure 4). Among 16 identified metabolites (Table 1), 14 were verified by an authentic standard. N-αacetylcitrulline was verified by its fragments and standards of L-citrulline and N-α-acetylarginine. The metabolite 3-dehydro-D-glucose 6-phosphate was verified by its fragments and the standard of D-glucose 6-phosphate. Various unknowns (Table S-1) could not be identified; however, several of the metabolites were found to contain a phosphate group based on fragments with positive m/z-values of 78.9591 (O3P) and 96.9696 (H2O4P). The data in ESI+ mode contained many multiple charged species considered to be protein/peptides. Thus, this data set was primarily used to verify the findings found in ESI- mode showing similar fold changes (Table 2). The findings from the liver samples were additionally searched for in plasma and urine samples from KO and WT mice. No significant differences in the plasma levels of the detected metabolites between KO and WT mice were evident (Table 2). However, the compounds N-

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acetylmethionine and 4-pyridoxic acid were found to be significantly downregulated in urine of KO mice compared to WT (Table 2).

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DISCUSSION AND FUTURE PERSPECTIVES At present, the precise mechanism by which ABCC6 prevents deleterious, aberrant calcification in healthy individuals remains to be fully resolved and therefore it is the matter of intense investigation. In this context, it is of key importance to know the precise subcellular ABCC6 localization, which has been a matter of debate. In the present study, immuno-EM ultrastructurally visualized ABCC6 in basolateral (perisinusoidal) membranes of mouse livers. No reactivity was detected in KO mouse livers, which validates the specificity of our findings. No specific intracellular detection of the ABCC6 protein was evident in our study. ABCC6 has also been reported to be expressed in basolateral membranes in the kidney32. We were however not able to detect any significant staining by immunohistochemistry in mouse kidney using the same antibodies as used for detection of the ABCC6 protein in mouse liver (Rasmussen et al., data not shown). The present characterization of liver extracts of KO mice with an Abcc6-/- genotype by untargeted metabolomics points to changes in the metabolic profile in the liver of these mice compared to control mice. Interestingly, data implies that ABCC6-deficiency causes hepatic accumulation of substances as features were primarily elevated in liver extracts of KO mice compared to control samples – although only small increases were detected. However, none of the identified liver metabolites were found to be significantly affected in plasma. On the other hand, accumulation of N-acetylmethionine and 4-pyridoxic acid in the liver was connected to decreased excretion in urine of KO mice. Whether this accumulation of metabolites in liver is a direct or indirect effect of an absent transport function of ABCC6 needs further investigations. Furthermore, we do not know how concentrations of ABCC6 substrates in liver and urine are linked. It is possible that an ABCC6 substrate from hepatocytes is excreted to plasma and 16


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eliminated by active or passive excretion in the urine. Conversely, one could also envision a compound generated in the liver and reclaimed by the kidney, with ABCC6 acting as a secretory transporter in the basolateral proximal tubular membrane. This is the first metabolomics study of the liver in ABCC6-deficient animals. A metabolomics study has previously been conducted in cultured dermal fibroblasts from PXE patients33. However, the liver is the absolute main tissue for ABCC6 expression34 and there is therefore no reason to believe that the same metabolic changes are present in the liver and the fibroblasts. The expression level of ABCC6 in liver has been shown to correlate with extrahepatic calcification35 and it is a prevailing hypothesis that causative changes in the liver account for the disease in other organs21-24,

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. Instead, an inverse metabolic footprint might be seen in a PXE-affected liver

versus peripheral tissues such as skin and vessels, if one or several ABCC6 substrates accumulate in the liver and therefore do not reach their target tissues. By comparison of the metabolomics data in the cultured human skin fibroblasts study33 and the present study of mouse liver, we noticed that elevated levels of pantothenic acid in KO mouse liver could be associated with decreased levels in human PXE fibroblasts, although no alterations in panthothenic acid in the blood could be detected from the present study. Further functional studies may investigate pantothenic acid as a potential substrate for ABCC6 although the present literature does not link this vitamin to extrahepatic mineralization. Pantothenic acid is ingested from food and may to some extent pass through the liver. It is an essential precursor for coenzyme A, which is important for acylation reactions. From a clinical perspective pantothenic acid has been shown to have wound-healing effects on the skin37. Additionally, Kuzaj et al. reported xanthine to be reduced in PXE fibroblasts33, which could be in connection to a decreased level of xanthosine and AMP found in the liver of KO mice in the 17



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present study. This could indicate a perturbation of purine metabolism, which might be a response of mitochondria to oxidative stress38. Alterations in purine metabolism could perhaps also be linked to reduced plasma PPi levels previously shown in ABCC6-deficient mice14. However, no differences in AMP or xanthosine levels between KO and WT mice could be detected in plasma. Succinyladenosine was furthermore increased in the liver samples of KO mice. Succinyladenosine is the product of dephosphorylation of adenylosuccinic acid, which is an intermediate in the conversion of IMP into AMP39; additionally adding to the low levels of liver AMP. Along with the increase in pantothenic acid important for Coenzyme A production and acetylations, several N-acetylated amino acids were found to be upregulated in livers of ABCC6 KO mice, whereas other related amino acids; glutamine, glutamate, and 3-sulfinoalanine were all downregulated in the liver. N-acetylated amino acids may originate from the catabolism of intracellular N-terminally acetylated proteins. Acetylation serves to protect intracellular proteins from proteolysis. During protein catabolism, acylpeptide hydrolase releases the N-acetylated amino acid that is deacetylated by aminoacylase-1 or -2 making the amino acid available for new protein synthesis40. It has been shown that N-acetylmethionine and N-acetylglutamate are some of the best substrates for aminoacylase-141. These amino acids are also the most abundant residues at the N-termini of acetylated proteins41. N-acetylglutamate is furthermore an essential cofactor of carbamoyl phosphate synthase 1 in the urea cycle, in which the identified glutamine, glutamate, and N-acetylcitrulline are involved. Increased levels of N-acetylglutamate and especially N-acetylcitrulline could thus suggest an effect on the urea cycle, when ABCC6 transport function is abrogated. These increases may additionally divert carbamoyl phosphate from the urea cycle into pyrimidine synthesis leading to 18


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accumulation of intermediates in this pathway42, which includes uridine that was found to be upregulated, although not entirely significant (P = 0.074), in the liver of KO mice. The compound 4-pyridoxic acid is a catabolic product of vitamin B6 and is usually excreted in urine. In KO mice, it was found to be up-regulated in the liver, which is the site for vitamin B6 metabolism. This could suggest the ABCC6 protein to be involved in transport of vitamins and/or their metabolites. Pyridoxal 5’-phosphate, the metabolically active form of vitamin B6, is essential for metabolism of amino acids including methionine. Previous studies have investigated whether ABCC6 is involved in transport of vitamin K or derivatives important for the carboxylation of glutamate to form gla-residues in for instance matrix Gla protein involved in mineralization. However, no studies so far have established such a role of ABCC69-12. Neither did any of our data point in that direction. The peptides Val-Phe and Ile-Pro-Ile (Diprotin A) which were upregulated in the ABCC6 KO mice might be breakdown products that may have physiological cell signaling effects. Diprotin A is highly expressed in the liver and is known to be an inhibitor of dipeptidyl aminopeptidase IV, whereas any signaling effects of Val-Phe is unknown. Downregulation of 2-hydroxy-3-methylbutyric acid in the liver of KO mice could furthermore indicate changes in fatty acid and/or amino acid metabolism, as it originates primarily from ketogenesis and the metabolism of valine, leucine and isoleucine. Neither the presence nor function of 3-dehydro-D-glucose 6-phosphate could be established. The analysis of the vesicular content from an in vitro transport assay by UPLC-HR-TOF-MS a so-called transportomics approach - has proved to be a useful tool to identify ABC transporter substrates43-44. This approach was tested on ABCC6-expressing vesicles incubated with and without ATP and with cell culture media from ABCC6-expressing cells compared to vesicles 19



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with no ABCC6 protein. It was, however, not possible to find compounds that accumulated in ABCC6 vesicles in an ATP-dependent manner. However, potential candidate ABCC6 substrates found in the liver samples could be tested for ATP-dependent transport by ABCC6-expressing membrane vesicles using an approach similar to the transportomics setup14, 45. In conclusion, the present metabolomics study implies that ABCC6 protein expression affects liver metabolism as suggested from the fact that ABCC6 absence induces an altered metabolic profile reflected in small changed levels of metabolites in liver tissue. Thus, the data set is in agreement with the hypothesis of PXE being a metabolic disease originating in the liver21. However, how the suggested ABCC6 transport function is linked to an altered metabolic profile disclosed in the present study warrants further investigations.

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FIGURES

Figure 1. Specificity of anti-mouse ABCC6 antibodies by western blotting of liver from a WT (Abcc6+/+) mouse and a KO (Abcc6-/-) mouse. Mouse liver homogenate was probed with the rat monoclonal anti-mouse ABCC6 antibodies M6II-24 and M6II-68 and the goat polyclonal anti-mouse ABCC6 S-20 antibody. A ~160 kDa protein is detected in the lysate of WT mice, and is absent in the lysate of ABCC6-deficient mice. M6II-68 and S-20 do however also detect other proteins, and are thus not perfectly specific for the mouse ABCC6 protein. The membrane was reprobed with an anti-GAPDH antibody to ensure that equal amounts of protein were present in all lanes (lower insert). The blots are representative of western blots of livers from three WT mice and three KO mice.

21 ACS Paragon Plus Environment


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Figure 2. Immuno-electron microscopy images using the anti-mouse ABCC6 antibody M6II24. (A) Gold particles are located over the basolateral (perisinusoidal) membranes of hepatocytes of WT mice (arrows and insert), whereas no significant intracellular labeling is detected (two examples of WT mouse cryosections are shown). Neither is labeling seen in the apical membrane that faces the bile canaliculi. (B) No labeling is evident in the liver of KO mice. BC: bile canaliculi (apical membrane), M: mitochondria, PS: perisinusoidal space, S: sinusoide. (C) Schematic representation of hepatocytes. The dashed box outlines the subcellular area visualized by electron microscopy. Images are representative of the electronmicroscopic scanning of immuno-gold-stained sections from two WT mice and two KO mice. 22 ACS Paragon Plus Environment

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Additional controls were prepared by incubation of liver sections of WT mice with secondary antibodies alone or with non-specific IgG (data not shown).



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KO QC

15

WT 10 5 0 -5 -10 -15 -20 -30

-20

-10

0 tPS[1]

10

20

Figure 3. PCA scores plot to visualize differences between KO and WT liver samples. The ellipse represents Hotelling’s T2 (95%). KO (n = 16), WT (n = 19), quality controls (QC, n = 10). A slight drift of the QC samples can be visualized caused by the liver samples accumulating dirt on the column.


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A

B 3.5 3 2.5

-log10(P-value)

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2 1.5 1 0.5 0 -2

-1.5

-1

-0.5

0

0.5

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log2 (fold change KO/WT)

Figure 4. More features are significantly increased than decreased in liver extracts of KO mice. (A) Volcano plot of all detected features. The horizontal line represents the level of significance (P-value 0.05), whereas the vertical line represents no fold-change. The features in the upper right quadrant defined by the two lines represent features that are significantly upregulated in the liver of KO mice. The features in the upper left quadrant represent compounds that are significantly downregulated in liver samples of KO mice. More features are significantly upregulated (188 features) than downregulated (115 features) in KO mice compared to WT mice. (B) Histogram of fold-changes of significantly elevated (>1) and downregulated (

Untargeted Metabolomics Analysis of ABCC6-Deficient Mice

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