Porcine liver sinusoidal endothelial cells contribute significantly to intrahepatic ammonia...

Porcine Liver Sinusoidal Endothelial Cells ContributeSignificantly to Intrahepatic Ammonia Metabolism

Geir I. Nedredal,1 Kjetil Elvevold,2 Lars M. Ytrebø,3 Ole-Martin Fuskevåg,4 Ingvild Pettersen,2 Peter A. G. McCourt,2

Kjell Bertheussen,5 Bård Smedsrød,2* and Arthur Revhaug1*

Ammonia metabolism in the liver has been largely credited to hepatocytes (HCs). We have shownthat liver nonparenchymal cells that include liver sinusoidal endothelial cells (LSECs) produceammonia. To address the limited knowledge regarding a role for LSECs in ammonia metabolism,we investigated the ammonia metabolism of isolated LSECs and HCs under three differentconditions: (1) bioreactors containing LSECs (LSEC-bioreactors), (2) bioreactors containingHCs (HC-bioreactors), and (3) separate bioreactors containing LSECs and HCs connected insequence (Seq-bioreactors). Our results showed that LSEC-bioreactors released six-fold moreammonia (22.2 nM/hour/106 cells) into the growth media than HC-bioreactors (3.3 nM/hour/106 cells) and Seq-bioreactors (3.8 nM/hour/106 cells). The glutamate released by LSEC-biore-actors (32.0 nM/hour/106 cells) was over four-fold larger than that released by HC-bioreactorsand Seq-bioreactors (<7 nM/hour/106 cells). LSEC-bioreactors and HC-bioreactors consumedlarge amounts of glutamine (>25 nM/hour/106 cells). Glutaminase is known for catalyzingglutamine into glutamate and ammonia. To determine if this mechanism may be responsible forthe large levels of glutamate and ammonia found in LSEC-bioreactors, immunolabeling ofglutaminase and messenger RNA expression were tested. Our results demonstrated that glutam-inase was present with colocalization of an LSEC-specific functional probe in lysosomes ofLSECs. Furthermore, using a nucleotide sequence specific for kidney-type glutaminase, reverse-transcription polymerase chain reaction revealed that this isoform of glutaminase was expressedin porcine LSECs. Conclusion: LSECs released large amounts of ammonia, perhaps due to thepresence of glutaminase in lysosomes. The ammonia and glutamate released by LSECs in Seq-bioreactors were used by hepatocytes, suggesting an intrahepatic collaboration between these twocell types. (HEPATOLOGY 2009;50:900-908.)

Since it was revealed that the liver plays a role innitrogen metabolism (ureagenesis), ammonia de-toxification, and amino acid metabolism,1 nitrogen

metabolism has been largely credited to hepatocytes(HCs).2 Later electron microscopical studies in the early1970s revealed the existence of liver sinusoidal endothelialcells (LSECs).3 Improved cultivating techniques and theadvent of specific and functional probes for LSECs inrecent decades have opened a new era for the study of thefunctions of LSECs.4,5 In a previous study, we showedthat cultured liver nonparenchymal cells (NPCs) that in-clude LSECs produce significant amounts of ammonia(ammonium-ions), most likely from glutamine.6 On asingle cell level, NPCs used four times more glutaminethan HCs. A plausible explanation for this difference wasreflected in a study that showed different expressions ofglutamine transporter systems in LSECs and HCs.7 Thesame study revealed that LSECs expressed kidney-typeglutaminase as opposed to liver-type glutaminase in HCs.The kidney-type glutaminase is probably also expressed inendothelial cells other than LSECs.8 The ammonia pro-duction by NPCs, may have implications for a better un-

Abbreviations: AST, aspartate aminotransferase; atmosO2, atmospheric oxygentensions; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; HC, hepatocyte; HC-bioreactor, bioreactor containing hepatocytes;KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; LSEC-bioreactor, biore-actor containing LSECs; NPC, nonparenchymal cell; OUR, oxygen uptake rate;physO2, physiological oxygen tensions; RT-PCR, reverse-transcription polymerasechain reaction; SC, stellate cell; Seq-bioreactor, sequentially connected LSEC-bio-reactor and HC-bioreactor.

From the 1Department of Digestive Surgery, University Hospital Northern Nor-way and Institute of Clinical Medicine, and the 2Department of Cell Biology andHistology, University of Tromsø, Tromsø,Norway; the 3Department of Anesthesiaand Intensive Care; the 4Department of Clinical Pharmacology; and the 5Depart-ment of Obstetrics and Gynecology, University Hospital Northern Norway.

Received September 23, 2008; accepted May 6, 2009.Supported by the Norwegian Research Council.*These authors contributed equally to this work.Address reprint requests to: Geir I. Nedredal, Division of Transplant Surgery,

Department of Surgery, Mayo Clinic, Rochester, MN 55902. E-mail:[email protected]; fax: 507-266-4893.

Copyright © 2009 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.23076Potential conflicts of interest: none.Additional Supporting Information may be found in the online version of this

article.

900

derstanding of the pathophysiology of hyperammonemiain relation to liver disease.9

Under normal conditions, the intrahepatic turnover ofglutamine is excessive, requiring both a rapid breakdownand a high capacity for resynthesis.10 This phenomenonprovides the basis for efficient and highly adaptive regu-lation of body nitrogen balance.2,9 In the periportal re-gion, glutamine is hydrolyzed to ammonia and glutamateto fuel the urea synthesis. Accordingly, the concentrationof ammonia declines during downstream passage throughthe sinusoids, and in the pericentral region a small popu-lation of the total HC population (5%-7%) scavenge re-sidual ammonia to resynthesize glutamine from ammoniaand glutamate.

Today, most in vitro studies on liver cells are performedin incubators with atmospheric oxygen tensions (at-mosO2) that result in oxygen tensions around 139 mmHg, far above the physiological oxygen tensions (physO2)being between 91 and 10 mm Hg (�10 mm Hg is hyp-oxia).11 The oxygen tensions within the liver lobules arebelieved to be between 70 mm Hg in the periportal regionand decrease to 25 mm Hg in the pericentral region of theliver acini.12 Cell types such as embryonic stem cells havebeen shown to grow better in incubators at physO2 thanat atmosO2.13 Primary rat LSECs cultured at physO2 sus-tained better survival and maintained their differentiatedfeatures better than LSECs cultured at atmosO2.14

In vivo, LSECs have a large capacity for clearing theblood of numerous physiological and foreign waste mac-romolecules by receptor-mediated endocytosis.4,15 The fi-nal degradation products of the waste macromolecules(hyaluronan and serglycin) are lactate and acetate.16,17

During anaerobic conditions, most mammalian cells re-lease lactate, mainly via glycolysis. NPCs produced largeamounts of lactate, indicating that they are highly meta-bolically active cells, even at atmosO2. NPCs favored ananaerobic oxidation of monosaccharides.6

In the present study, bioreactors containing porcineLSECs (LSEC-bioreactors), bioreactors containing HCs(HC-bioreactors), and separate bioreactors containingLSECs and HCs connected in sequence (Seq-bioreactors)were cultivated at physO2 (91 mm Hg) in simulated mi-crogravity that has been shown to maintain metabolicfunctions of HCs.18 We found that the LSEC-bioreactorsused approximately the same amount of glutamine as theHC-bioreactors, but the LSEC-bioreactors released six-fold and four-fold more ammonia and glutamate than theHC-bioreactors into the growth media, respectively. Im-munolabeling and reverse-transcription polymerase chainreaction (RT-PCR) confirmed the presence of the enzymeglutaminase in the lysosomes of the LSECs. Furthermore,LSEC-bioreactors released five-fold more lactate into the

growth media than HC-bioreactors. The ammonia, glu-tamate, and lactate released by LSECs were used by HCsin the Seq-bioreactors.

Materials and MethodsChemicals were obtained from Sigma-Aldrich (St.

Louis, MO) unless stated otherwise. Collagenase P wasobtained from Roche, Norway. One liter of growth me-dium, DM110/SS, was composed of 6.0 g DME powder(BioWhittaker, Walkersville, MD), 6.8 g MCDB 110-powder, 2.6 g NaHCO3 (Supporting Table 1), and anti-biotics (penicillin, streptomycin, and gentamycin)supplemented with a prototype version of 5% tissue syn-thetic serum.19 Formaldehyde-treated serum albumin(0.8 mg/mL) was incubated with the fluorochrome fluo-rescein isothiocyanate (FITC) in sodium carbonate buffer(0.5 mol/L [pH 9.5]) in a protein/dye weight ratio of 1:1at 4°C overnight. Unreacted fluorochrome was removedby way of gel filtration through a PD-10 column equili-brated and eluted with phosphate-buffered saline withoutCa2� or Mg2�. The final concentrations of the growthfactors in the growth media were for fibroblast growthfactor 20 ng/mL, insulin-like growth factor l 10 ng/mL(both from R&D Systems, Minneapolis, MN), and por-cine endothelial growth factor 1 ng/mL (a gift from L.Erickson, Guelph, Canada). Collagen-coated dextranmicrocarriers were obtained from Pharmacia Biotech(Sweden). The microcarriers were inoculated at a concen-tration of 4.0 mg/mL. Two mouse polyclonal antibodies(clones 2G6 and 2B10) against porcine macrophages werekindly provided by Dr. Angela Berndt (Jena, Germany).Monoclonal mouse anti-human desmin (clone D33) wasobtained from DAKO (Denmark). Polyclonal mouse an-ti-human glutaminase was obtained from Novus Biologi-cals (Littleton, CO). Monoclonal goat anti-mouseimmunoglobulin G, TRITC-conjugate, was obtainedfrom Zymed (San Francisco, CA).

Cell Isolation and Bioreactors. All animals receivedcare according to the criteria outlined in the Guide for theCare and Use of Laboratory Animals prepared by the Na-tional Academy of Sciences and published by the Na-tional Institutes of Health (publication 86-23, revised1985). LSECs and HCs were isolated from male pigs (Susscrofa domesticus) weighing 7-8 kg.20 Briefly, the liverswere perfused with a HEPES buffer to wash out bloodcells before perfusion with a HEPES buffer containingcollagenase. To pellet the HCs, the resulting cell suspen-sions were subjected to velocity centrifugation (50g). Theresulting supernatants were concentrated by centrifuga-tion (850g), thereafter enriched in a 21% Optiprep (Ny-comed, Oslo) density solution at 3,300g. The enrichedNPCs were subjected to elutriation centrifugation to iso-

HEPATOLOGY, Vol. 50, No. 3, 2009 NEDREDAL ET AL. 901

late LSECs. The viabilities of the cells were �95% asjudged by a trypan blue exclusion test. No pyrogenes weredetected on Limulus amebocyte lysate assay.

Perfusion bioreactors with volumes of 250 mL and 500mL with oxygenators were obtained from Synthecon(Houston, TX) (Fig. 1A). The 250-mL bioreactors wereinoculated with 6.9 � 108 � 8.2 � 107 LSECs (LSEC-bioreactors [n � 7]), and the 500-mL bioreactors wereinoculated with 2.0 � 109 � 2.9 � 106 HCs (HC-bio-reactors [n � 7]). Separate bioreactors containing 6.9 �108 � 8.2 � 107 LSECs and 2.0 � 109 � 2.9 � 106 HCswere connected in sequence (Seq-bioreactors [n � 7])(Fig. 1B). The inner cores of the bioreactors were coveredwith a nylon mesh with a diameter of 5 �m obtained fromF. Burmeister (Oslo, Norway). The nylon mesh was su-tured with polypropylene sutures (Ethicon, NJ). The flowrate was kept at 25 mL/minute with a peristaltic pump(Allmed, Sweden), and the rate was confirmed with aninline flow probe (Medi-Stim, Norway). Extra channels

were drilled in the inner cores of the bioreactors to ensuremore uniform oxygenation of the cells (dotted horizontallines in the outer part of the inner core). Fresh DM110/SSwas replenished between periods 1 and 2. The cell samplesfor DNA and protein measurements were sampled di-rectly from the cell compartments of the bioreactors. ThepH was between 7.3 and 7.4.

Assays. Lactate and glucose concentrations were mea-sured with a YSI-analyzer (Cobas, Switzerland). Albuminwas measured with a pig albumin enzyme-linked immu-nosorbent assay kit obtained from Bethyl Laboratories(Montgomery, TX). Pyruvate was measured with a Pyru-vate-726 kit (Sigma-Aldrich). Ammonia, amino acids,and urea were analyzed as described.21 Immediately aftersampling, the samples were precipitated in 20% trichlo-roacetic acid and centrifuged, and we then performedaffinity and ion exchange chromatography. The ammoniaconcentrations were confirmed with an automated enzy-matic method (Cobas Fara II, Roche, Switzerland). Pro-

Fig. 1. Illustration of the perfusion bioreac-tor and outline of the experiments. (A) Theperfusion bioreactor was rotated horizontallyaround its axis, thereby simulating microgravity.The cells were attached to microcarriers main-tained in a suspension by balancing their sed-imentation-induced gravity with centrifugationcaused by the rotation of the cell compartment(30-40 rpm). The nylon mesh (with an averagediameter of 5 �m) prevented the cells fromescaping the cell compartment. (B) The exper-iments consisted of bioreactors allocated intothree groups: (1) LSEC-bioreactors (n � 7), (2)HC-bioreactors (n � 7), and (3) Seq-bioreac-tors (n � 7).

902 NEDREDAL ET AL. HEPATOLOGY, September 2009

tein was assayed as described by Lowry et al.22 Lactatedehydrogenase, aspartate aminotransferase (AST), andalanine aminotransferase released into growth media wereanalyzed by standard automated techniques at the De-partment of Clinical Chemistry, University Hospital ofNorthern Norway. For the measurement of the intracel-lular contents of lactate dehydrogenase, AST, and alanineaminotransferase, the purified LSECs and HCs werethawed from �70°C to 22°C, centrifuged, washed inphosphate-buffered saline, and sonicated before analysis.DNA concentrations in the bioreactors were determinedby the binding of mithramycin to DNA.23 All biochemi-cal values were normalized to the cellular DNA content.

RNA Isolation and RT-PCR. The total RNA wasextracted from the different liver cell populations usingTRIZOL reagent (Invitrogen, Carlsbad, CA) accordingto the manufacturer’s protocol. Complementatry DNAwas synthesized using 2.0 �g total RNA that was reverse-transcribed to a final volume of 50 �L using theSuperScript Preamplification kit (Invitrogen). PCR am-plifications were performed in a PTC-200 Peltier Ther-mal Cycler (Bio-Rad, Hercules, CA). Primer pairs were:glutaminase, 5�-CTGGATGCTTCTGGTTTGGC-CATT-3� (sense) and 5�-ACCCTACTCCAACAGTA-AGTGCGT-3� (antisense) where a 334-bp fragment wasexpected; glyceraldehyde-3-phosphate dehydrogenase(GAPDH), 5�-GCAAATTCCACGGCACAGTCA-3�(sense) and 5�-TCACGCCACAGTTTCCCAGAG-3�(antisense) where a 433-bp fragment was expected. PCRproducts were analyzed by agarose gel (1.5%) electro-phoresis and photographed under ultraviolet light.

Immunolabeling. To determine the purity of the cul-tures of LSECs, the LSEC fraction after the centrifugalelutriation was seeded on fibronectin-coated glass cover-slips and labeled as described.20 The purity of the LSECcultures varied between 80% and 95%. The other celltypes in the LSEC cultures were stellate cells (SCs) (3%-11%) as judged by staining with anti-desmin antibody,Kupffer cells (KCs) (1%-7%) by staining with anti-por-cine macrophage antibodies, and HCs (1%-2%) asjudged by simple morphology.

To investigate the localization of glutaminase, we cul-tivated NPCs and HCs on fibronectin-coated glass cover-slips. The cells were incubated with a specific andfunctional probe for LSECs, FITC-labeled formalde-hyde-treated serum albumin (10 �g/mL), for 10 minutes.The cells were then fixed in cold methanol for 5 minutes.The fixed cells were incubated for 1 hour with polyclonalmouse anti-human glutaminase (1:100 dilution), andthereafter additional 1-hour incubation with TRITC-conjugated monoclonal goat anti-mouse immunoglobu-lin G (1:200 dilution). The examination of the specimens

was performed with an Axiophot photomicroscopeequipped with fluorescence optics (Zeiss Axiophot, Ger-many).

Oxygen Uptake Rate. The experiments were per-formed at physO2 in a humidified CO2 incubator withsupplemental O2 control (12% O2) (Galaxy R, RSBio-tech, UK). The partial pressures of oxygen were measuredby sampling growth media in capillary glass tubes takensimultaneously from the arterial and the venous ports ofthe bioreactors (Fig. 1A) and analyzed immediately in ablood-gas analyzer (Rapidlab 865, Chiron Diagnostics,UK).

Oxygen uptake rate (OUR) (nmol/minute) � arte-rial-venous pO2 � flow � 1.18 � 10�3, 1.18 � 10�3

�mol/mL/mmHg was a constant derived from the Bun-sen solubility coefficient for oxygen 37°C.24 The resultswere adjusted for the OUR measured in cell-free controls.

Statistics. We used the SPSS 11.0 statistical packageto establish significance between groups (SPSS, Chicago,IL). Two-way (time and group) analysis of variance forrepeated measurements was applied to test whether anystatistical significance existed between the three groups,followed by the LSD post hoc comparison test. An overallsignificance in analyses of variance for repeated measure-ments (F-test; P � 0.05) may be attributable to either theeffect of time (PT) or the interaction of group and time(PGT). PGT � 0.05 denoted a significant difference be-tween the groups dependent of time, whereas PG � 0.05denoted a significant difference between the groups inde-pendent of time. If both PGT and PG were significant, thenonly PGT was denoted, because PGT is more robust thanPG. PT � 0.05 denoted a significant change in time for thegroups.

ResultsAmmonia Metabolism

Ammonia and Urea. The ammonia released into thegrowth media by LSEC-bioreactors (22.2 � 5.1 nM/hour/106 cells) was six-fold larger than that of HC-biore-actors (3.3 � 1.8 nM/hour/106 cells) in period 1 (PGT �0.05) (Fig. 2A). In period 2, the ammonia release byLSEC-bioreactors was four-fold larger (17.0 � 2.8 nM/hour/106 cells) than that of LSEC-containing Seq-biore-actors (3.8 � 1.8 nM/hour/106 cells) (PGT � 0.05). Theurea production in the HC-bioreactors was 11.5 � 2.0nM/hour/106 cells, whereas there was no urea productionas expected in the LSEC-bioreactors in period 1 (Fig. 2B).There was a significant urea production in both the HC-bioreactors and the Seq-bioreactors in period 2: 14.3 �2.5 nM/hour/106 cells and 14.5 � 5.0 nM/hour/106

cells, respectively (PT � 0.05).


Glutamine and Glutamate. We attempted to iden-tify the source of the ammonia release. There was a largeremoval of glutamine both in LSEC-bioreactors (27.7 �11.8 nM/hour/106 cells) and HC-bioreactors (28.8 � 1.7nM/hour/106 cells) (PT � 0.05) (Fig. 2C). The largestremoval of glutamine in period 2 was by the LSEC-bio-reactors (25.1 � 2.3 nM/hour/106 cells) (PGT � 0.05),whereas the glutamine removal by HC-bioreactors andSeq-bioreactors was 8.8 � 2.8 nM/hour/106 cells and10.0 � 2.0 nM/hour/106 cells, respectively. The corre-sponding glutamate release by the LSEC-bioreactors inperiod 1 was 32.0 � 8.5 nM/hour/106 cells, which wereover four-fold larger than the HC-bioreactors (6.7 � 1.5nM/hour/106 cells) (Fig. 2D) (PGT � 0.05). Moreover,the LSEC-bioreactors released 26.1 � 1.1 nM/hour/106

cells glutamate in period 2 (PGT � 0.05), which was overnine-fold more than the HC-bioreactors (2.7 � 1.1 nM/hour/106 cells) and Seq-bioreactors (1.3 � 0.6 nM/hour/106 cells). For the net removal/release of amino acids,urea, and ammonia (Supporting Table 2). Cell-free con-trol was also performed to determine the rate of autohy-drolysis (Supporting Table 3).

Localization of Glutaminase in Isolated LiverCells. We then attempted to determine the mechanism

of ammonia release by LSECs. Kidney-type glutaminasemessenger RNA was present in all examined liver cells,including HCs, as revealed on RT-PCR analysis (Fig. 3).We next investigated the intracellular localization of glu-taminase by immunolabeling-cultivated NPCs (LSECs,SCs, and KCs). The LSECs showed strong immunolabel-ing of glutaminase, and anti-glutaminase colocalized withthe LSEC-specific functional probe, FITC-labeled form-aldehyde-treated serum albumin, that accumulated in ly-sosomes of LSECs (Fig. 4). There was weaker labeling of

Fig. 2. Ammonia metabolism in the bioreactors. The net release of (A) ammonia and (B) urea, (C) the net removal of glutamine, and (D) the netrelease of glutamate were presented as differences between the last and the first measurement divided by the number of hours each period lastedper 106 cells (mean � standard deviation). The positive values denoted net release into the growth media, and the negative values denoted netremoval of glutamine from the growth media. PGT � 0.05 denoted a significant difference between the groups dependent of time, whereas PT � 0.05denoted a significant change for the groups in time. The ammonia figures were corrected for ammonia generated in the cell-free controls. Urea wasbelow detectable limits in LSEC-bioreactors. White bars represent LSEC-bioreactors; black bars represent HC-bioreactors; gray bars representSeq-bioreactors.

Fig. 3. Expression analysis of kidney-type glutaminase and GAPDH inLSECs, SCs, KCs, and HCs. RT-PCR products detected by way of gelelectrophoresis of expected size were obtained. N was the negativecontrol sample, and GAPDH was used as a positive control. Note theweaker signal in the KC band due to the smaller amount of isolated KCs.


SCs as compared with LSECs. The labeling of HCs re-vealed punctuated cytoplasmic structures.

Lactate MetabolismAt physO2, the release of lactate in LSEC-bioreactors

(87.5 � 8.9 nM/hour/106 cells) was over five-fold largerthan for HC-bioreactors (15.9 � 1.9 nM/hour/106 cells)in period 1 (PGT � 0.05) (Fig. 5). There was also a largerelease of lactate by LSEC-bioreactors in period 2 (42.2 �6.3 nM/hour/106 cells) (PGT � 0.05), whereas there was arather small lactate release by LSEC-containing Seq-bio-reactors (7.4 � 1.7 nM/hour/106 cells). The large lactaterelease coincided with large glucose removal by LSEC-bioreactors (34.8 � 9.3 nM/hour/106 cells) as comparedwith HC-bioreactors (3.9 � 7.3 nM/hour/106 cells) inperiod 1 (PGT � 0.05) (Table 1). The glucose consump-tion was larger by the LSEC-bioreactors (28.3 � 5.7 nM/hour/106 cells) as compared with both the HC-bioreactors and the Seq-bioreactors in period 2 (PGT �0.05). In period 2, there was a three-fold larger pyruvateremoval by LSEC-bioreactors (28.2 � 10.0 nM/hour/106

cells) than for both the HC-bioreactors and the Seq-bio-reactors (7.7 � 1.7 nM/hour/106 cells and 5.2 � 1.5nM/hour/106 cells, respectively) (PGT � 0.05).

Functional Features of BioreactorsThe bioreactors maintained their cellular performance

throughout the periods: the results are summarized inTable 2. The oxygen uptake rate by LSEC-bioreactors was25-29 nmol/minute (2% of the HC-bioreactors and theSeq-bioreactors). The release of AST (4.3 � 0.5 mU/hour/106 cells) into the growth media in the LSEC-bio-

Fig. 4. Fluorescence micrographs of immunolabeled glutaminase incultured NPCs and HCs. (A) Red fluorescence was from immunolabeledanti-glutaminase antibody (large arrows) residing in the lysosomes ofLSECs. SCs revealed weak anti-glutaminase labeling (small arrows). (B)Green fluorescence was from endocytosed LSEC-specific probe (FITC-labeled modified albumin) (large arrows). Red and green fluorescencecolocalized in the lysosomes of LSECs. (C) Anti-glutaminase labeling wasevenly distributed in the cytosol of the HCs. (D) Negative control withoutanti-glutaminase antibody showed no labeling (scale bars, 20 �m).

Fig. 5. Net release of lactate. The results are presented as differencesbetween the last and the first measurement divided by the number ofhours each period lasted per 106 cells (mean � standard deviation).PGT � 0.05 denoted a significant difference between the groups depen-dent on time. White bars represent LSEC-bioreactors; black bars repre-sent HC-bioreactors; gray bar represents Seq-bioreactors.

Table 1. Net Release and Removal of Glucose, Pyruvate, andAmino Acids in Bioreactors

Period 1 Period 2

GlucoseLSEC-bioreactors �34.8 � 9.3* �28.3 � 5.7*HC-bioreactors �3.9 � 7.3 �4.9 � 8.3Seq-bioreactors NA �5.0 � 8.9

PyruvateLSEC-bioreactors �36.2 � 11.8‡ �28.2 � 10.0*HC-bioreactors �20.6 � 1.1‡ �7.7 � 1.7Seq-bioreactors NA �5.2 � 1.5‡

ArginineLSEC-bioreactors �20.4 � 9.5*,‡ �12.2 � 4.7*HC-bioreactors �10.8 � 1.4‡ �11.5 � 1.2*Seq-bioreactors NA �6.5 � 0.9‡

OrnithineLSEC-bioreactors 22.7 � 5.8†,‡ 5.6 � 2.1‡HC-bioreactors 10.9 � 0.6‡ 9.4 � 1.1*,‡Seq-bioreactors NA 5.5 � 0.6‡

Only the amino acids (out of the total 21 amino acids except for glutamine andglutamate) that showed a significant release or removal are presented. Results arepresented as the differences between the last and the first measurement dividedby the number of hours each period lasted per 106 cells (mean � standarddeviation). Positive values denoted net release into the growth media; negativevalues denoted net removal from the growth media.

Abbreviation: NA, not available.*Significant difference between the groups dependent on time (PGT � 0.05).†Significant difference between the groups independent of time (PG � 0.05).‡Significant change for the groups in time (PT � 0.05). The amino acid figures

were corrected for autohydrolysis based on cell-free control. Unit: nM/hour/106

cells.


reactors in period 1 was an unexpected finding. Wetherefore measured the intracellular content of AST byfreezing and then thawing isolated LSECs and HCs; theamount of AST was 49.1 � 13.1 mU/hour/106 cells and529.4 � 130.9 mU/hour/106 cells, respectively (data notshown).

Discussion

Ammonia Metabolism. Intrahepatic ammonia me-tabolism is largely credited to HCs. Since the discovery ofLSECs in the 1970s,3 no research regarding the role ofLSECs in ammonia metabolism has been conducted. Ourresults show that LSEC-bioreactors released six-fold andfour-fold more ammonia and glutamate, respectively,than HC-bioreactors. The LSEC-bioreactors used ap-proximately the same amount of glutamine as HC-biore-actors. In Seq-bioreactors, where LSEC-bioreactors andthe HC-bioreactors are connected in sequence, theamounts of ammonia and lactate were as low as the levelsobserved in HC-bioreactors. RT-PCR analysis revealedthe presence of kidney-type glutaminase in porcineLSECs. Immunolabeling revealed colocalization of glu-taminase with the specific LSEC-probe (FITC-labeledmodified albumin) in the lysosomes of LSECs; however,we do not know the catalytic activity of this enzyme in

these acidic vesicles. A study on metabolic acidosis andkidney-type glutaminase suggested that this enzyme has ahigh activity at a low pH,25 indicating that its lysosomallocalization may maintain its enzyme activity.

LSEC-bioreactors used approximately 29 nM glu-tamine/hour/106 cells, released 31 nM glutamate/hour/106 cells, and released 23 nM ammonia/hour/106 cells.This indicated that after hydrolysis of glutamine byLSEC-bioreactors, little or no further metabolism of am-monia and glutamate occurred by LSEC-bioreactors, asopposed to the HC-bioreactors and Seq-bioreactors.Only 3 nM ammonia/hour/106 cells were released byHC-bioreactors into the growth media together with 12nM urea/hour/106 cells. Because two ammonia moleculesare incorporated into one urea molecule, a total of 24 nMammonia/hour/106 cells can be explained stoichiometri-cally. Likewise, glutamate was most likely further metab-olized by the HCs, because only 8 nM glutamate/hour/106 cells was released by HC-bioreactors, indicating a truerelease of 24 nM glutamate/hour/106 cells. The low levelsof glutamate suggested a further breakdown of this aminoacid in HC-bioreactors.26 The removal of arginine byLSEC-bioreactors was 20.4 nM/hour/106 cells, whereasthe release of ornithine into the growth media was 22.7nM/hour/106 cells. Arginase hydrolyses arginine to ureaand ornithine in the urea cycle. Because there was no ureaproduction in the LSEC-bioreactors, the conversion ofarginine to ornithine must occur by an alternative en-zyme—namely, arginine:glycine aminotransferase.27 Thisenzymatic reaction uses glycine. The LSEC-bioreactorsshowed no net release (or production) of glycine. Glycine,however, is also a substrate for several other enzymes thatmay mask its release.

Glutaminase Activity in Endothelial Cells. Theonly previous study of kidney-type glutaminase did notreveal the activity of this enzyme in LSECs.7 However,Wu et al.8 and Leighton et al.28 reported maximal glutam-inase activities of 260 and 634 nmol/minute/mg proteinlysates in venular and pulmonary endothelial cells, respec-tively. These studies demonstrated the activity of kidney-type glutaminase, assuming that the kidney-type variantexisted in endothelial cells other than LSECs. Calculationof glutamine degradation in intact cells in period 1showed a cellular glutaminase activity of 10.9 and 0.5nmol/minute/mg in LSEC-bioreactors and HC-bioreac-tors, indicating that the activity was 21-fold larger forLSECs than HCs. This difference in glutaminase activitycan be explained by different Km values: the Km for thekidney-type variant was 2-5 mM glutamine, and the Km

for the liver-type variant was 8-40 mM,29 indicating thatLSECs contribute more than HCs to the degradation ofglutamine.

Table 2. Functional Features of Bioreactors

Period 1 Period 2

Oxygen uptake rateLSEC-bioreactors 29 � 17 25 � 16HC-bioreactors 1,246 � 103 1,227 � 222Seq-bioreactors NA 1,337 � 173

AlbuminLSEC-bioreactors BDL BDLHC-bioreactors 1.7 � 0.6 1.6 � 0.5Seq-bioreactors NA 1.7 � 0.5

ASTLSEC-bioreactors 4.3 � 0.5 1.8 � 0.9HC-bioreactors 4.9 � 0.8 1.9 � 0.5Seq-bioreactors NA 1.1 � 0.4

ALTLSEC-bioreactors 0.0 � 0.0 0.0 � 0.0HC-bioreactors 0.1 � 0.1 0.0 � 0.0Seq-bioreactors NA 0.0 � 0.0

LDHLSEC-bioreactors 3.7 � 1.4 1.5 � 0.8HC-bioreactors 4.6 � 1.2 1.7 � 0.6Seq-bioreactors NA 1.3 � 0.7

Results are presented as differences between the last and the first measure-ment in each period divided by the number of hours each period lasted per 106

cells (mean � standard deviation)—except for the oxygen uptake rate, which ispresented as nmol/minute (mean � standard deviation). Positive values denotednet release to the growth medium. ALT, AST, and LDH units: mU/hour/106 cells.Albumin units: ng/hour/106 HCs.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;BDL, below detectable limit; LDH, lactate dehydrogenase; NA, not available.


The NPCs of the liver occupy only 3% of the livervolume.30 In contrast, NPCs constitute approximately33% of the total number of liver cells; LSECs constituting21% of the number.31 The glutamine removal per cell wassimilar for LSECs and HCs in the Seq-bioreactors group,where the LSEC/HC number ratio was 1:3, resemblingthe in vivo ratio between these two cell types. Can this betaken to reflect the situation in the intact liver? Consider-ing the cellular architecture of the intact sinusoid, LSECsprovide a large and efficient interaction surface throughwhich HCs stay in contact with the blood. This will to alarge extent make up for the fact that the HCs constitutethree times as many cells as LSECs in the sinusoid.

Lactate Metabolism. The release of lactate by LSEC-bioreactors in the present study was substantial (87.5 nMlactate/hour/106 cells), and the removal of glucose was34.8 nM/hour/106 cells. The products of glycolysis aretwo pyruvate per glucose. The stoichiometric conversionof glucose to lactate yielded 69.6 nM lactate; therefore,approximately 18 nM lactate must come from a differentsource. One probable source is pyruvate, which underanaerobic conditions is known to be reduced to lactate invigorously contracting muscles or in erythrocytes. LSEC-bioreactors consumed 36 nM pyruvate/hour/106 cells atphysO2; half of this pyruvate was perhaps completely oxi-dated in the tricarboxylic acid cycle, whereas the otherhalf was reduced to lactate. Recently, we showed that theNPCs released large amounts of lactate with no glucoseuse at atmosO2.6 In the present study, we improved theculture conditions by adding the growth factors insulin-like growth factor 1, endothelial growth factor, and fibro-blast growth factor in a serum-free synthetic growthmedium.32 With this growth medium, the LSEC-biore-actors consumed 34.8 nM glucose/hour/106 cells atphysO2. Rat LSECs cultivated both at physO2 and at-mosO2, with the same growth medium but without thegrowth factors, released more than two-fold lactate atphysO2.14 The large release of lactate at physO2 was notdue to anaerobic oxidation of glucose, because the glucosein the growth media in this study was unaltered.

Functional Features of the Bioreactors. The biore-actors maintained their cellular performance throughoutthe periods (the OUR and the synthesis of albumin). TheOURs of the HC-bioreactors and Seq-bioreactors werenearly the same; the Seq-bioreactors had a slightly higherOUR, perhaps due to the presence of LSECs. There was asurprisingly large amount of AST in the medium of theLSEC-bioreactors, suggesting that this enzyme resides in-tracellularly in LSECs. This assumption was confirmedwhen the intracellular content of AST in LSECs was mea-sured. The AST amounts in LSEC-bioreactors cannot be

explained by HC contamination, because only 1%-2% ofHCs were found in LSEC-bioreactors.

In conclusion, we demonstrate that LSECs release sig-nificant amounts of ammonia that may be due to thepresence of kidney-type glutaminase in the lysosomes ofLSECs.

Acknowledgment: We express our gratitude to EllinorHareide for analytical service, Hege Hagerup for technicalassistance, and Tom Wilsgaard for statistical advice.

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Porcine liver sinusoidal endothelial cells contribute significantly to intrahepatic ammonia...

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