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Am J Physiol Gastrointest Liver Physiol 293: G75-G83, 2007. First published March 15, 2007; doi:10.1152/ajpgi.00245.2006
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LIVER AND BILIARY TRACT

Significant contribution of liver nonparenchymal cells to metabolism of ammonia and lactate and cocultivation augments the functions of a bioartificial liver

Geir I. Nedredal,1,2 Kjetil Elvevold,3 Lars M. Ytrebø,2,4 Ole-Martin Fuskevåg,5 Ingvild Pettersen,3 Kjell Bertheussen,6 Bodil Langbakk,7 Bård Smedsrød,3 and Arthur Revhaug1,2

1Department of Digestive Surgery, University Hospital of Northern Norway, Tromsø; 2Surgical Research Laboratory, Institute of Clinical Medicine, and 3Department of Experimental Pathology, University of Tromsø, Tromsø; 4Department of Anesthesia and Intensive Care, University Hospital of Northern Norway, Tromsø; 5Department of Pharmacology, University of Tromsø, Tromsø; and 6Department of Obstetrics and Gynecology and 7Department of Clinical Chemistry, University Hospital of Northern Norway, Tromsø, Norway

Submitted 5 June 2006 ; accepted in final form 13 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A bioartificial liver (BAL) will bridge patients with acute liver failure (ALF) to either spontaneous regeneration or liver transplantation. The nitrogen metabolism is important in ALF, and the metabolism of nonparenchymal liver cells (NPCs) is poorly understood. The scope of this study was to investigate whether cocultivation of hepatocytes with NPCs would augment the functions of a BAL (HN-BAL) compared with a BAL equipped with only hepatocytes (H-BAL). In addition, NPCs were similarly cultivated alone. The cells were cultivated for 8 days in simulated microgravity with serum-free growth medium. With NPCs, initial ammonia and lactate production were fivefold and over twofold higher compared with later time periods despite sufficient oxygen supply. Initial lactate production and glutamine consumption were threefold higher in HN-BAL than in H-BAL. With NPCs, initial glutamine consumption was two- to threefold higher compared with later time periods, whereas initial ornithine production and arginine consumption were over four- and eightfold higher compared with later time periods. In NPCs, the conversion of glutamine to glutamate and ammonia can be explained by the presence of glutaminase, as revealed by PCR analysis. Drug metabolism and clearance of aggregated gamma globulin, probes administered to test functions of hepatocytes and NPCs, respectively, were higher in HN-BAL than in H-BAL. In conclusion, NPCs produce ammonia by hydrolysis of amino acids and may contribute to the pathogenesis of ALF. High amounts of lactate are produced by NPCs under nonhypoxic conditions. Cocultivation augments differentiated functions such as drug metabolism and clearance of aggregated {gamma}-globulin.

liver sinusoidal endothelial cells; kidney-type glutaminase; acute liver failure; hyperammonemia; glutamine

THE LIVER IS THE MOST IMPORTANT ORGAN for ammonia removal in the body. Ammonia (i.e., the ammonia ion) is produced during the breakdown of amino acids and proteins and then incorporated by the hepatocytes into urea. Hyperammonemia is toxic and believed to be important in the development of cerebral edema and subsequent intracranial hypertension in acute liver failure (28). Therapeutic approaches to lower ammonia levels have included plasmapheresis, albumin-bound dialysis, and bioartificial liver (BAL) based on isolated hepatocytes (8).

Most studies including BAL have focused on the function of hepatocytes, ignoring the important liver function of clearing the blood circulation of waste macromolecules, functions carried out by nonparenchymal cells (NPCs) (10, 20). The few reports on implementation of NPCs in novel BAL systems have focused on the potential support of these cells for hepatocyte function; little attention has been paid to the metabolic contribution of NPCs themselves (12, 15). The nitrogen and energy metabolism of NPCs have been poorly studied, although there is a report indicating that glutamine is an important energy source for these cells (26). Kidney-type glutaminase is present in rat NPCs, strengthening the idea of conversion of glutamine to glutamate and ammonia by these cells (16), whereas in hepatocytes liver-type glutaminase is present (22).

During liver transplantation the liver becomes a net producer of lactate. A study showed that there is a correlation between the increased transhepatic lactate gradient and increased systemic lactate during clamping of the liver (29). Moreover, the lactic acidosis seen during transplantation for acute liver failure decreased after the donor liver was transplanted (19). In this study, the authors addressed the source for the hepatic lactate production.

The long-term cultivation of hepatocytes and stellate cells has been extensively studied, whereas the cultivation of liver sinusoidal endothelial cells (LSECs) has been studied only to a limited extent. However, in a recent study, our group has shown that expansion and specific differentiation patterns can be stimulated in cultured pig LSECs in a new synthetic serum-free growth medium, DM110/SS, for up to 20 days (9).

Cultivation of hepatocytes is associated with difficulties to maintain differentiated phenotypic function in long-term cultures, but several groups have cultivated primary hepatocytes in simulated microgravity and shown maintenance of metabolic functions (7, 31). Furthermore, reports indicate the stabilization of differentiated phenotypic functions of hepatocytes in coculture with other cell types (2, 5, 18).

Therefore, we hypothesized that a BAL equipped with NPCs and hepatocytes would enhance cell-cell interactions and preserve differentiated functions in a BAL using DM110/SS. Furthermore, the nitrogen and substrate metabolism in a BAL equipped with either hepatocytes or both hepatocytes and NPCs were investigated. Drug metabolism by hepatocytes and receptor-mediated endocytosis of NPCs was used as a specific functional marker to probe the differentiated functions of these cells in BAL.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

Chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Collagenase P was obtained from Roche (Oslo, Norway). Paracetamol was obtained from Norsk Medisinaldepot (Oslo, Norway). Immunoglobulin ({gamma}-globulin) was obtained from Pharmacia & Upjohn (Oslo, Norway). Aggregated {gamma}-globulin (AGG) was prepared as previously described (1). HPLC grade methanol was purchased from J. T. Baker (Deventer, The Netherlands), whereas isoamyl alcohol sodium hydroxide, sodium carbonate, and formic acid were supplied by Merck-Schuchardt (Hohenbrunn, Germany). N-7084 was a gift from H. Lundbeck (Nykobing, Denmark). n-Hexan was obtained from Riedel de Haën (Seelze, Germany). Water was purified on a Milli-Q purification system (Millipore, Bedford, MA). One liter of growth medium DM110/SS was composed of 6.0 g of DME powder (Bio-Whittaker), 6.8 g of MCDB 110 powder, 2.6 g of NaHCO3, and antibiotics (penicillin, streptomycin, and gentamycin) supplemented with 5% tissue synthetic serum (tSS) (4). tSS was obtained from Medi-Cult (Jyllinge, Denmark). Collagen-coated dextran microcarriers (Cytodex 3, diameter ~175 µm) obtained from Pharmacia Biotech (Uppsala, Sweden) were prepared according to the manufacturer and inoculated at a concentration of 4.0 x 10–3 g/ml.

Liver Cell Culture in Simulated Microgravity

The experimental protocol was approved by the local steering committee of the Norwegian Experimental Animal Board. All animals received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (Publication No. 86-23, Revised 1985). Liver cells from castrated male pigs weighing 7–8 kg (Sus scrofa domesticus) were isolated using a two-step collagenase perfusion technique and subsequent enrichment of NPCs as described previously (20). The viabilities of hepatocytes and NPCs were >95% as judged by the trypan blue exclusion test. Two sets of experiments were performed according to the experiment outline in Fig. 1.


Figure 1
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  		Fig. 1. An outline of the 2 sets of experiments. The first set of experiments consisted of bioartificial livers (BAL) randomly allocated into 2 groups: hepatocytes cultivated in BAL (H-BAL) and hepatocytes cocultivated with nonparenchymal cells (NPCs) in BAL (HN-BAL). Note that in HN-BAL, NPCs were added to hepatocytes after 5 h. Aggregated {gamma}-globulin (AGG) and drugs (imipramine, nortriptyline, caffeine, and paracetamol) were administered at 25, 73, and 144 h to test functions of NPCs and hepatocytes. The second set of experiments consisted of NPCs cultivated alone. NEM, nitrogen and energy metabolism; m1–m3, minutes 1–3.

 
Perfusion bioreactors with volumes of 250 and 500 ml with separate oxygenators were obtained from Synthecon (Houston, TX) (Fig. 2). The first set of experiments consisted of BAL randomly allocated into two groups: 2.0 x 109 hepatocytes cultivated in a 500-ml BAL (H-BAL) and 2.0 x 109 hepatocytes cocultivated with 1.0 x 109 NPCs in a 500-ml BAL (HN-BAL). The second set of experiments consisted of 1.0 x 109 NPCs cultivated alone in a 250-ml bioreactor.


Figure 2
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  		Fig. 2. Schematic drawing of BAL. The bioreactor was rotated horizontally around its axis by an electric motor simulating microgravity. The solid and dotted black lines indicate the flow of the growth medium. The growth medium was oxygenated in an in-line oxygenator consisting of silicone tubes in a coil with an electric fan at the end to increase the gas convection. Cells attached to microcarriers were maintained in a suspension by balancing their sedimentation-induced gravity with centrifugation caused by bioreactor rotation (30–40 rpm).

 
For both sets of experiments, fresh DM110/SS was replenished after 23, 71, and 143 h. The inner core of the bioreactor was covered with a nylon mesh (F. Burmeister, Oslo, Norway), with an average mesh diameter of 5 µm. Bioreactors, growth medium, and microcarriers were screened for pyrogenes by a limulus amebocyte lysate assay.

Biochemical Analysis

All samples were centrifuged at 1.900 g at +4°C immediately after sampling and frozen at –70°C for later analysis. AGG was measured with a human IgG-Fc ELISA quantitation kit, whereas albumin was measured with a pig albumin ELISA quantification kit, both from Bethyl Laboratories (Montgomery, TX). The kit for lactate measurement was obtained from Roche Diagnostics (Indianapolis, IN). The concentrations for glucose and ammonia were determined using an automated enzymatic method (Cobas Fara II; Roche, Basel, Switzerland). A kit for pyruvate (pyruvate-726) was obtained from Sigma-Aldrich. The amount of DNA was quantified by measuring mithramycin binding to double-stranded DNA, and the fluorescence was measured in a spectrofluorometer with emission set at 510 nm and excitation set at 420 nm (13). Samples for amino acids and urea were precipitated in 20% trichloroacetic acid, centrifuged, and thereafter analyzed as described previously (11). Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were analyzed using standard automated techniques at the Department of Clinical Chemistry, University Hospital of Northern Norway. The results were correlated to the amount of DNA, representing the numbers of cells.

RNA Isolation and RT-PCR

Total RNA was extracted from NPCs and hepatocytes using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. Samples were treated with DNase. cDNA was synthesized using 2.0 µg of total RNA that was reverse transcribed in a final volume of 50 µl with the SuperScript preamplification kit (Life Technologies, Gaithersburg, MD). Gene-specific PCR was performed in 50 µl of reaction mixture containing 2 µl of cDNA (from isolated RNA), 2.5 units of Taq DNA polymerase (New England BioLabs), 20 mM Tris·HCl (pH 8.3 at 25°C), 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, 0.2 mM deoxynucleotide triphosphate mix, 1 M of each primer, 0.2 mM deoxynucleotide triphosphate mix, and 0.2 µM of each primer. The amplified glutaminase fragment was generated from a nucleotide sequence with accession number AF490841 (21).

Glutaminase PCR and GAPDH PCR

The PCR mixture described above was used. The following primer pair was used for glutaminase: 5'-CTGGATGCTTCTGGTTTGGCCATT-3' (sense) and 5'-ACCCTACTCCAACAGTAAGTGCGT-3' (antisense), where a 334-bp fragment was expected. The following primer pair was used for GAPDH: 5'-GCAAATTCCACGGCACAGTCA-3' (sense) and 5'-TCACGCCACAGTTTCCCAGAG-3' (antisense), where a 433-bp fragment was expected. PCR was performed at 94°C for 5 min (first denaturation/hot start) and then at 94°C for 45 s (denaturation), 56°C (58°C for GAPDH) for 1 min (annealing), and 72°C for 1 min (extension) for 35 cycles with a 10-min final extension at 72°C.

PCR amplifications were performed in a PTC-200 Peltier thermal cycler (MJ Research, MA). PCR products were analyzed using agarose gel (1.5%) electrophoresis and photographed under UV light.

Oxygen Consumption Measurements

The "arterial" partial pressure of oxygen (19–21 kPa) and the pH (7.3–7.4) in the growth medium were kept constant by having a perfusion rate of 5 ml/min in BAL, whereas there were no perfusion required in NPCs because there was less oxygen consumption. The partial pressures of oxygen in "arterial" and "venous" samples (Fig. 2) were analyzed with a blood gas analyzer (Rapidlab 865; Chiron Diagnostics). The sampling was performed with a plastic syringe and immediately analyzed to avoid equilibration with atmospheric air. All experiments were performed in a humidified CO2 standard incubator with 95% air-5% CO2 at 37°C.

O2 consumption in BAL (nmol/min) = {Delta}arteriovenous PO2 x flow x 1.18 x 10–3, where {Delta}arteriovenous PO2 (mmHg) = arterial PO2 – venous PO2, flow was 5 ml/min, and 1.18 x 10–3 (µmol·ml–1·mmHg–1) was a constant derived from the Bunsen solubility coefficient for oxygen (0.03 µmol O2·ml–1·mmHg–1) and the volume of 1 µmol of oxygen (25.4 µl) at 37°C (32).

O2 consumption in NPCs cultivated alone (µmol/l) = PO2 x 1.18 x 10–3, where PO2 equals venous PO2, and there is no flow through the bioreactor. The values for consumption of oxygen by NPCs were corrected for the fall in oxygen concentration observed in the cell-free controls.

Drug Concentration Analysis

To assess the metabolic capacity of hepatocytes, we administered the following drugs at twice the therapeutic range for humans: nortriptyline [tests the activity of cytochrome P-450 (CYP) 1A2], imipramine (tests the activity of CYP2C19), caffeine (tests the phase II metabolic activity), and paracetamol (tests the activities of UDP-glucuronosyltransferase and CYP2E1). Paracetamol was detected by fluorescence polarization immunoassay (FPIA; TDFLX, Abbott Laboratories), whereas imipramine, nortriptyline, and caffeine were detected by a triple quadropole tandem mass spectrometer (Quattro Micro; Micromass, Manchester, UK) (27). For separation of the compounds, a high-pressure liquid chromatograph (2695 Alliance) separation system consisting of an autosampler, a binary pump, and a degasser was equipped with a Xterra MS C18 3.5-µm 2.1 x 20-mm IS column (Waters, Milford, MA). Stock standard solutions were prepared by dissolving imipramine, nortriptyline, and caffeine in methanol to obtain a concentration of 2 mM. These solutions were stored at –20°C. Growth medium standard samples were prepared by dilution of the stock solutions with drug-free plasma at the following concentrations: 20, 40, 80 160, 320, 640, 1,280, and 2,048 nM. The extraction procedure for the compounds has been described previously (27). For liquid chromatography and mass spectrometry, the mobile phase consisted of 50% methanol in 5 mM aqueous formic acid (isocratic) with a flow rate of 0.2 ml/min. Ionization was achieved by using electrospray in the positive ionization mode. Quantitative analysis was performed in the multiple reaction monitoring. The following transitions of protonated precursor ion -> product ion in multiple reaction monitoring were monitored: m/z 195 -> 138, m/z 264 -> 91, m/z 281 -> 86, and m/z 304 -> 91 for caffeine, nortriptyline, imipramine, and N-7084, respectively.

Statistics

We applied SPSS 11.0 software (Chicago, IL) for statistical analysis. In the first set of experiments, two-way (time and group) analysis of variance for repeated measurements was applied for continuous variables. An overall significance in analyses of variance for repeated measurements (F-test, P < 0.05) may be attributable to either the effect of time (PT) or the interaction for group and time (PGT). PGT < 0.05 denotes a significant difference between the groups dependent of time, whereas PG < 0.05 denotes a significant difference between the groups independent of time. Significant differences between the groups are given by either PGT or PG. If both PGT and PG are significant, only PGT is denoted since PGT is more robust than PG. PT < 0.05 denotes a significant change for both groups in time. Greenhouse-Geisser epsilon factor probability levels were used when Mauchly's test of sphericity was significant. In the second set of experiments, the statistical analyses were performed with paired t-tests between the first and last measurement in each period.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Nitrogen Metabolism

Ammonia and urea. The production of ammonia was significant in both groups (H-BAL and HN-BAL) in all periods with no differences between them (PT < 0.05) (Fig. 3). The production of urea, on the other hand, was over 1.5 times higher in H-BAL than in HN-BAL in periods 1 and 2 (PGT < 0.05), and in period 4 (PG < 0.05), whereas there was significant production in both groups in periods 1–4 (PT < 0.05). In NPCs, initial (period 1) ammonia production was fivefold higher compared with later time periods (Fig. 4), and the production was significant in periods 1–3 (P < 0.05), whereas urea was not produced.


Figure 3
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  		Fig. 3. Production of ammonia and urea in BAL. Results are presented as differences between the last and first measurement, divided by the number of hours each period lasted (means ± SD). Positive values denote production. Two-way ANOVA was performed for each of the parameters in each of the periods; PGT < 0.05 denotes a significant difference between the groups dependent on time, whereas PG < 0.05 denotes a significant difference between the groups independent of time, and PT < 0.05 denotes a significant change for both groups in time. The values were corrected for ammonia generated in the cell-free controls.

 

Figure 4
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  		Fig. 4. Glutamine, ammonia, and glutamate in NPCs. Results are presented as differences between the last and first measurement divided by the number of hours each period lasted (means ± SD). Positive values denote production, and negative values denote consumption. The first and last measurements in each period were compared. Paired t-tests were performed between the first and last measurement in each period, with indication of statistically significant differences by P < 0.05. The values were corrected for ammonia generated in the cell-free controls. There was no production of urea.

 
Amino acids. Glutamine consumption was threefold higher in HN-BAL than in H-BAL in period 1 (PGT < 0.05), the consumption was higher in HN-BAL than in H-BAL in period 2 (PGT < 0.05), and the consumption was significant in both groups in all four periods (PT < 0.05) (Fig. 5). Glutamate production was higher in HN-BAL than in H-BAL in period 1 (PGT < 0.05), whereas the production in period 2 was higher in H-BAL than in HN-BAL (PG < 0.05). In periods 1–3, there was significant production of glutamate in both groups (PT < 0.05). Ornithine production was higher in H-BAL than in HN-BAL in period 1 (PGT < 0.05), and there was significant production in both groups in periods 1–4 (PT < 0.05) (Table 1). In contrast, there was significant consumption of arginine in both groups with no differences between them in all four periods (PT < 0.05). In NPCs, glutamine was by far the most metabolized amino acid, and the consumption was two- to threefold higher in period 1 compared with later time periods (P < 0.05), whereas glutamate production was significant in periods 1 and 2 (P < 0.05) (Fig. 4). The ornithine production was fourfold higher in period 1 compared with later time periods (P < 0.05), whereas the arginine consumption was eightfold higher in period 1 compared with later time periods (P < 0.05) (Table 2).


Figure 5
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  		Fig. 5. Consumption of glutamine and production of glutamate in BAL. Results are presented as differences between the last and first measurement, divided by the number of hours each period lasted (means ± SD). Negative values denote consumption, and positive values denote production. Two-way ANOVA was performed for each of the parameters in each of the periods. The values were corrected for ammonia generated in the cell-free controls.

 

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  		Table 1. Amino acids, lactate, glucose, and pyruvate in bioartificial liver

 

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  		Table 2. Amino acids, lactate, pyruvate, glucose, LDH, and AST in nonparenchymal cells

 
Kidney-type glutaminase transcription in NPCs and hepatocytes. Since high amounts of ammonia were produced and glutamine consumed in our BAL, we examined the existence of kidney-type glutaminase in NPCs and hepatocytes by RT-PCR analysis. The product detected by gel electrophoresis indicates the existence of glutaminase in both NPCs and hepatocytes (Fig. 6). GAPDH was used as a positive control.


Figure 6
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  		Fig. 6. Expression analysis of kidney-type glutaminase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in NPCs and hepatocytes (Hep). The blot shows that RT-PCR products of the expected size were obtained. N, negative control sample.

 
Lactate Metabolism

Lactate released (or produced) into the medium was over threefold higher in HN-BAL than in H-BAL in period 1 (PGT < 0.05), and there was significant production in both groups in periods 1–4 (PT < 0.05) (Table 1). There was consumed pyruvate in both groups with no differences between them in period 1 (PT < 0.05). There was no significant production or consumption of glucose in either experimental set. In NPCs, the lactate production in period 1 was over twofold higher compared with periods 2 and 3, and the production was significant in periods 1–3 (P < 0.05) (Table 2). The consumption of pyruvate in NPCs was eightfold higher in period 1 compared with later time periods, and the consumption was only significant in period 1 (P < 0.05).

Oxygen Consumption

The consumption of oxygen was higher in HN-BAL than in H-BAL, indicating higher metabolic activities in HN-BAL (PGT < 0.05), and there was significant consumption of oxygen in both groups (PT < 0.05) (Fig. 7). In NPCs, the oxygen consumption was over threefold higher in period 1 compared with later time periods, and the consumption was significant in all four periods (P < 0.05) (Fig. 8). The partial pressure of oxygen was kept almost twice the physiological levels (18–22 kPa), ensuring that no hypoxia was present.


Figure 7
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  		Fig. 7. Oxygen "arteriovenous" flux in BAL. Positive values denote consumption. Results are presented as means ± SD. Two-way ANOVA was performed for the results in each of the periods.

 

Figure 8
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  		Fig. 8. Oxygen consumption by NPCs. The first and last measurements in each period were compared. Results are presented as differences between the first and second measurements in each period, divided by the number of hours each period lasted (means ± SD). Paired t-tests were performed between the first and last measurement in each period, with indication of statistically significant differences by P < 0.05. In period 1 the comparison was performed between the second and third measurements. The values were corrected for the fall in oxygen concentration observed in the cell-free controls.

 
Functional Properties of Hepatocytes and NPCs in BAL

The metabolism of both paracetamol, a test of the UDP-glucuronosyltransferase and CYP2E1 in hepatocytes, and nortriptyline, a test of CYP1A2 in hepatocytes, was higher in HN-BAL than in H-BAL in period 2 (PGT < 0.05 and PG < 0.05, respectively) but similar in both groups in periods 2–4 (PT < 0.05) (Table 3). The metabolism of caffeine, a test of the phase II metabolic activity in hepatocytes, was higher in HN-BAL than in H-BAL in periods 2 and 3 (PGT < 0.05) but similar in both groups in periods 2–4 (PT < 0.05). The metabolism of imipramine, a test of CYP2C19 capacity, was similar in both groups with no differences between them in periods 2–4 (PT < 0.05). The clearance of AGG, an NPC-specific function, was (as expected) higher in HN-BAL than in H-BAL in periods 2 and 3 (PG < 0.05) but similar in both groups in periods 2–4 (PT < 0.05). Albumin synthesis, a characteristic hepatocyte synthetic function, was higher in HN-BAL than in H-BAL in periods 1 and 2 (PGT < 0.05) but similar in periods 1–4 (PT < 0.05). Finally, LDH, a marker for the viability of the cells, was released in both groups with no differences between them in periods 1–3 (PT < 0.05) (Table 3), whereas in NPCs there were significant releases of both LDH and AST only in period 1 (P < 0.05) (Table 2).


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  		Table 3. Drug metabolism, clearance of aggregated gamma globulin, synthesis of albumin, and the release of LDH in BAL

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated for the first time that NPCs produce high amounts of ammonia. In addition, high amounts of lactate were produced under nonhypoxic conditions by NPCs. We also observed that the production of lactate was threefold higher in HN-BAL than in H-BAL, clearly indicating that NPCs are the main producer. Furthermore, the cocultivation of hepatocytes with NPCs in HN-BAL augments specific cell functions such as drug metabolism, clearance of AGG, and synthesis of albumin.

The most probable source of ammonia in both experimental sets was glutamine and arginine. Both were utilized in considerable amounts, and both have hydrolyzable amino groups that during hydrolysis will be released as ammonia into the growth medium. HN-BAL consumed threefold more glutamine than H-BAL, and NPCs cultivated alone consumed glutamine to an extent comparable to that of HN-BAL. These observations identify NPCs as the main contributors to ammonia production, and indeed, PCR analysis demonstrated the presence of kidney-type glutaminase in NPCs, as shown earlier in rat NPCs (16). Another substantial finding was the metabolism of ornithine. Ornithine, together with urea, is a split product of arginine and H2O in the urea cycle. It was produced by NPCs even when no coproduction of urea was observed. The source of ornithine produced in NPCs is not known. In addition, BAL with NPCs surprisingly produced less ornithine than BAL without NPCs. Whereas NPCs have beneficial effects on hepatocytic drug metabolism and albumin synthesis, NPCs seem to have a detrimental effect on the urea synthesis. This might be due to the higher ammonia concentration in HN-BAL that reached a peak of ~1 mM in the growth medium. Previous reports have actually shown reduced synthesis of urea when hepatocytes were cocultivated with NPCs in an ammonia concentration of 5 mM (15). Another study showed reduced ammonia removal and urea synthesis even at an ammonia concentration of 0.1 mM (14). A similar phenomenon was observed in chronic liver failure patients with elevated concentrations of ammonia in the blood (23). The capacity of liver slices from these patients to synthesize urea was decreased by 80%. The reduced ability to synthesize urea during hyperammonia conditions is consistent with our findings, since the hyperammonia in HN-BAL seems to reduce the urea synthesis capacity. Another possible explanation for the reduced urea synthesis in the HN-BAL could be the presence of activated Kupffer cells among the NPCs. Activated Kupffer cells produce reactive oxygen species that might affect urea synthesis (30).

There are two possible explanations for the high lactate production by the NPCs in our experiments. First, LSECs in vivo clear the blood of numerous physiological and foreign waste macromolecules (10, 25), and it has been reported that rat LSECs produce lactate and acetate as the final degradation products of the endocytosed waste macromolecules (24). Second, the pyruvate in the growth medium was consumed and most likely anaerobically converted to lactate. This is a plausible explanation, because NPCs have few mitochondria and thereby perform limited oxidative phosphorylation (6).

The consumption and production of metabolites and substrates were highest in both BAL groups and in NPCs cultivated alone in the first period. Likewise, the oxygen consumption was highest in the first period. Accordingly, a prerequisite for a BAL to function properly is adequate oxygen delivery, substrate availability, and stable pH. Lack of fulfillment of these parameters, especially initially, will result in loss of differentiated functions and a dysfunctional BAL. The cocultivation setting in this BAL stimulated differentiated functions of both the NPCs and hepatocytes. The drugs tested were metabolized faster when hepatocytes were cocultivated with NPCs than when cultivated alone, suggesting that NPCs stimulate the cytochrome activity of the hepatocytes. Cytochrome activity, as tested by the metabolism of lignocaine, has earlier been shown to be stimulated in a coculture of hepatocytes with biliary epithelial cells (3). Cocultivation in our study also enhanced the synthesis of albumin, which also has been reported by others (17).

In conclusion, we postulate a new source of the release of ammonia into the blood circulation: the production of ammonia by liver NPCs (Fig. 9). The produced ammonia may be released in two different ways: first, the kidney-type glutaminase present in NPCs catalyzes the conversion of glutamine to glutamate and ammonia; and, second, LSECs carry large amounts of lysosomal hydrolases with high degrading capacities (10) that degrade an array of soluble waste macromolecules and thus give rise to ammonia as a degradation product. Furthermore, high amounts of lactate are produced by NPCs despite sufficient oxygen supply.


Figure 9
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  		Fig. 9. Established and postulated novel pathways of production of ammonia and lactate by NPCs. The novel findings of ammonia and lactate production by NPCs under nonhypoxia are marked with dotted arrows. The established metabolism of glutamine by glutaminase in hepatocytes is also indicated. In the in vivo situation, where hepatocytes are separated from the underlying endothelium by the space of Disse, it would appear that there is a close collaboration between NPCs and hepatocytes. Ammonia and lactate produced by the NPCs travel across the space of Disse to the hepatocytes, where ammonia is converted to urea. Moreover, lactate from NPCs can readily be converted by the hepatocytes to glucose in the cytosol or energy equivalents in the mitochondrion. Urea and other molecules produced by hepatocytes probably enter the circulation by passing through the fenestrae of the liver sinusoidal endothelial cells. LDH, lactate dehydrogenase.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Norwegian Research Council (Oslo, Norway).


   ACKNOWLEDGMENTS
 
We are grateful to Hege Hagerup, Torfinn Solvang, Hanne Mæhre, and Wenche Bakkelund (University of Tromsø, Tromsø, Norway) for skillful technical assistance. The excellent analysis service provided by Ellinor Hareide and Dr. Pål Falkenberg is highly appreciated. We are grateful for the advice of Dr. Peter A. G. McCourt and the statistical advice of Dr. Tom Wilsgaard.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. I. Nedredal, Dept. of Digestive Surgery, Univ. Hospital of Northern Norway, 9038 Tromsø, Norway (e-mail: geir.ivar.nedredal{at}fagmed.uit.no)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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