Tumor-influenced amino acid transport activities in zonal-enriched hepatocyte populations

Alexandra M. Easson1, Timothy M. Pawlik1, Craig P. Fischer1, Jennifer L. Conroy1, Dennis Sgroi2, Wiley W. Souba1, and Barrie P. Bode1

1 Surgical Oncology Research Laboratories, Department of Surgery, and 2 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cancer influences hepatic amino acid metabolism in the host. To further investigate this relationship, the effects of an implanted fibrosarcoma on specific amino acid transport activities were measured in periportal (PP)- and perivenous (PV)-enriched rat hepatocyte populations. Na+-dependent glutamate transport rates were eightfold higher in PV than in PP preparations but were relatively unaffected during tumor growth. System N-mediated glutamine uptake was 75% higher in PV than in PP preparations and was stimulated up to twofold in both regions by tumor burdens of 9 ± 4% of carcass weight compared with hepatocytes from pair-fed control animals. Excessive tumor burdens (26 ± 7%) resulted in hypophagia, loss of PV-enriched system N activities, and reduced transporter stimulation. Conversely, saturable arginine uptake was enhanced fourfold in PP preparations and was induced twofold only after excessive tumor burden. These data suggest that hepatic amino acid transporters are differentially influenced by cancer in a spatial and temporal manner, and they represent the first report of reciprocal zonal enrichment of system N and saturable arginine uptake in the mammalian liver.

digitonin-collagenase; cancer; liver; glutamine; arginine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CANCER IS KNOWN TO INFLUENCE the nitrogen economy of the host, with a net efflux of amino acids such as glutamine from host tissues to tumors, where they are rapidly utilized (37). The methylcholanthrene (MCA)-induced fibrosarcoma is a transplantable tumor model in rats and has been used extensively by our group (10, 13, 15, 19, 41, 51) and others (7, 40, 47, 48) to study metabolic alterations in the host with cancer. It is a rapidly growing tumor that rarely metastasizes but is locally aggressive. Previous work with this model has shown that the liver switches from an organ of net glutamine balance to one of net release during tumor growth (10, 51). Surprisingly, concentrative (Na+-dependent) plasma membrane glutamine transport via system N is coordinately stimulated in the livers of tumor-bearing rats (TBR) by a mechanism that may partially involve the autocrine production of tumor necrosis factor-alpha by hepatocytes (21, 29). Liver arginine transport is also stimulated by tumor growth via an increased maximal velocity at the plasma membrane level (16) by a mechanism that may also involve tumor necrosis factor-alpha (29). These effects are tumor dependent, inasmuch as resection results in normalization of transport rates for both amino acids, albeit with different temporal kinetics (15).

The functional heterogeneity in glutamine metabolism along the liver acinus has been well established, with glutaminase (GAL) and urea cycle enzymes in the periportal (PP) hepatocytes and glutamine synthase (GS) expression restricted to only 5-7% of terminal perivenous (PV) hepatocytes (26). This "intercellular glutamine cycle" underlies the ability of this organ to serve in regulating glutamine balance and ensures the efficient detoxification of ammonia. Flux through GAL can be regulated by system N-mediated glutamine uptake (28, 34), especially when hepatic metabolism is accelerated, such as during catabolic states. Likewise, the slow transport of arginine across the hepatocyte plasma membrane has been well established (55) and is now known to be a result of the liver-specific expression of the CAT-2A gene (11, 35). The expression of this relatively inefficient transporter isoform has been proposed to protect circulating arginine from the marked hepatic arginase activity associated with the urea cycle (55). Thus both of these transporters play important roles in liver nitrogen metabolism. Furthermore, arginine and glutamine have garnered interest for potential use in immunomodulatory therapies for patients (1), so the regulation of their uptake by the liver during cancer is particularly of interest.

Most of the work regarding tumor-induced changes in hepatic amino acid transport has been performed in plasma membrane vesicles isolated from whole liver homogenates from animals with clinically relevant or supraphysiological tumor burdens. Therefore, the studies presented here were designed to comprehensively discriminate between the effects of tumor burden and food intake on these transport activities at the cellular level. We also chose to measure glutamine and arginine transport rates in hepatocytes from different positions in the liver acinus, inasmuch as this more refined examination may also offer initial clues into the disposition of each during tumor growth. These studies provide the first report of zone-dependent transport activities in the tumor-influenced liver, and the results indicate that glutamine and arginine transport rates are reciprocally enhanced along the acinus and are differentially enhanced by tumor growth.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animal tumor model. Adult male Fischer 344 rats (125-150 g; Charles River Laboratories, Wilmington, MA) were used in all studies, which received approval from the Massachusetts General Hospital Animal Care and Use Committee, according to the Guide for the Care and Use of Laboratory Animals. The animals were housed in individual wire cages under standard conditions (12:12-h light/dark cycles and ad libitum access to standard chow and water). After 1 wk of acclimatization, rats were randomly chosen to undergo bilateral subcutaneous flank implantation of 3-mm3 MCA-induced fibrosarcomas (TBR), as described previously (13), or a sham implantation procedure (control) under anesthesia. Control rats were pair fed to matched TBR chow consumption to control for tumor-induced anorexia. At specific times after tumor implantation, hepatocytes were isolated by the technique described below.

Tumor-induced anorexia. Food intake was measured daily, and tumor mass (in cm3) was determined as described by Morrison (38) every 48 h to day 27. To distinguish between the effects of duration and degree of tumor burden on the characteristic anorexia that develops in these animals, a separate group of 24 rats were implanted with one, two, or four MCA tumors on the same day (6 rats/group), and food intake was monitored daily thereafter.

Isolation of hepatocytes. To minimize the acute influence of prandial status on experimental results, all animals were subjected to an overnight fast before surgery on the following morning. Hepatocytes were isolated from the livers of animals by a modification (20) of the two-step collagenase perfusion technique of Seglen (49). For cell preparations enriched in hepatocytes from the PV or periportal (PP) acinar zones, the livers were first perfused antegrade or retrograde, respectively, with a 0.5% (wt/vol) digitonin solution, as originally described (32, 45). Briefly, after laparotomy the portal vein and inferior vena cava were cannulated and suture ligated with 16- and 14-gauge Teflon angiocatheters, respectively. The liver was rapidly cleared of blood by perfusion with calcium-free MEM for suspension cultures (S-MEM) at a flow rate of 20 ml/min. Thereafter, a 5 mg/ml digitonin solution in S-MEM was perfused antegrade at a flow rate of 4.5 ml/min for 25-40 s or retrograde at 5.5 ml/min for 40-60 s until the expected zonal destruction pattern was visible on the liver surface. Then, 75 ml of S-MEM was perfused at 15 ml/min in the opposite direction, and digestion was carried out with a 0.35 mg/ml collagenase solution in 100 ml of S-MEM supplemented with 0.5 mM CaCl2. For hepatocyte preparations from the entire liver (i.e., unenriched for PP or PV cells), the digitonin step was omitted. After enzymatic digestion, the liver was removed and gently agitated in cold S-MEM to release the hepatocytes, and the suspension was passed over eight-ply gauze and a metal cell strainer. The cell suspension was centrifuged three times at 50 g for 2 min at 4°C, and the pellet was resuspended in a 50% (vol/vol) Percoll solution and centrifuged at 800 g for 10 min to remove debris, nonviable cells, and any residual nonparenchymal cells (3).

The resulting cell pellet was resuspended in S-MEM and counted on a hemocytometer, and the viability was confirmed to be >= 85% by trypan blue exclusion. Hepatocytes were plated in 24-well culture plates (Costar, Cambridge, MA) previously coated with type 1 rat tail collagen (Collaborative Biomedical Products, Bedford, MA) in William's E media (Life Technologies, Grand Island, NY) at a density of 1.4 × 105 cells/cm2. Hepatocytes were maintained in a humidified atmosphere of 5% CO2-95% air in an incubator at 37°C for 3-4 h to allow the cells to adhere to the plates before transport assays.

Liver histology. Livers were perfused exactly as outlined above but were removed before the collagenase perfusion step. Slices were obtained from the left median lobe and placed in 4% neutral-buffered formalin. Formalin-fixed samples were embedded in paraffin, mounted on glass slides, stained with hematoxylin and eosin, and photographed.

RNA isolation and Northern blot procedure. A portion of the final hepatocyte suspension (107 cells) was removed from each preparation, centrifuged, and resuspended in Tri-Solv reagent (Biotecx, Houston, TX). Total cellular RNA was isolated by the one-step acid-phenol guanidinium procedure followed by an additional acid-phenol, phenol-chloroform-isoamyl alcohol, chloroform extraction and ethanol precipitation in the presence of sodium acetate. Equal amounts of total RNA (20 µg), as determined spectrophotometrically and by ethidium bromide staining, were fractionated by electrophoresis through denaturing 1% agarose gels containing 0.2 M formaldehyde, transferred to nylon membranes by capillary action, and ultraviolet cross-linked to the membrane.

The cDNAs utilized in this study to generate radiolabeled probes for Northern blot assays were rat phosphoenolpyruvate carboxykinase (PEPCK; Pst I, 2.67-kb fragment in pBR322, pPCK-10; American Type Culture Collection, Manassas, VA), rat liver GAL (EcoR 1, 0.6- and 1.4-kb fragments in pBluescript SK II; kindly provided by Dr. Malcolm Watford), rat GS (1.6-kb Pst I fragment in pBR322, pGSRK-1; American Type Culture Collection), and mouse beta -actin [Xho I/EcoR 1, 1.5-kb fragment in pBluescript SK M13(-); Stratagene, La Jolla, CA]. The cDNA inserts containing primarily coding sequence were excised from the plasmid with appropriate restriction enzymes, separated on agarose gels, excised, eluted, and used as templates to generate probes labeled with deoxy-[alpha -32P]CTP (NEN, Boston, MA) with a random primer labeling kit (Megaprime; Amersham, Arlington Heights, IL) according to the manufacturer's protocol. Hybridization with radiolabeled probe was performed overnight at 65°C in 5× sodium chloride-sodium phosphate-EDTA (SSPE) with 7.5× Denhardt's reagent, 0.5% SDS, and 0.1 mg/ml sheared herring sperm DNA after the membrane was blocked for 2 h under the same conditions. Blots were washed at 55°C three times each for 10 min in decreasing concentrations of SSPE and increasing concentrations of SDS until 0.1× SSPE and 1.0% SDS was reached.

Autoradiographic detection of the hybridization was achieved by exposure to X-ray film at -80°C. The hybridized probe was stripped off the membrane by boiling in 0.1% SDS, and the blots were reutilized for the Northern blot analyses of other genes. Quantitation of the hybridized mRNA bands was performed by laser densitometry (Molecular Dynamics, Sunnyvale, CA). The densitometric values for PP (PEPCK and GAL) and PV (GS) mRNA "markers" were normalized to those for beta -actin in the same sample.

Arginine transporter expression. Expression of cationic amino acid transporter genes was examined by Northern blot analysis using probes specific for CAT-2, CAT-2A, and CAT-1 (35). The CAT-1 cDNA [EcoR I/Xho I, 6.5-kb fragment in pBluescript KS(-)] was kindly provided by Dr. Maria Hatzoglou (2). CAT-2 and CAT-2A were examined with synthetic 58-base oligodeoxynucleotides complementary to the unique alternatively spliced mRNA region of each of the following transporter isoforms (30): CAT-2 (accession no. M62838) 1196-1253 and CAT-2A (accession no. L03290) 1100-1157. Northern blot analysis with the CAT-1 cDNA was carried out as described above; the CAT-2/2A oligodeoxynucleotides were end labeled with T4 polynucleotide kinase (Promega, Madison, WI) and [gamma -32P]ATP (NEN) and hybridized with Ultrahyb (Ambion, Austin, TX) according to the manufacturer's instructions. All subsequent steps were carried out as described above.

Amino acid transport. The transport of radiolabeled amino acids (all from Amersham) by hepatocyte monolayers was carried out by the cluster-tray method (22) with minor modifications as previously described (13, 19, 20). After the cells were washed twice with 2 ml/well of warm (37°C) Na+-free Krebs-Ringer phosphate buffer (choline-KRP, in which NaCl and Na2HPO4 were replaced with the corresponding choline salts) to remove extracellular amino acids and Na+, the transport assay was initiated by transferring 0.25 ml of warm (37°C) transport mixtures into each well. The composition of the transport mixtures allowed well-defined hepatic amino acid transport activities to be measured, and the mixtures were composed of choline-KRP or Na+-KRP plus specifically defined quantities of unlabeled and 3H-labeled amino acids as outlined below. Initial-rate transport assays were terminated after 30 s for glutamine and after 1 min in all other cases by three rapid washes with 2 ml/well of ice-cold PBS solution, and the plates were allowed to dry overnight.

Cells were lysed with 0.2 ml/well of 0.2 N NaOH + 0.2% SDS and incubated on a rotating platform for 1 h. One-half (0.1 ml) of the cell extract was neutralized with 10 µl of 2 N HCl and subjected to scintillation spectrophotometry (Top-Count; Packard Instruments, Meriden, CT). Protein content was determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL) to which transport velocities were normalized and expressed as picomoles of amino acid transported per milligram of protein per unit time. Values obtained in choline-KRP were subtracted from those obtained in Na+-KRP to yield Na+-dependent values.

Statistical analysis. Data were transformed into logarithmic form to normalize variation between groups of animals and then analyzed by ANOVA or a paired two-tailed Student's t-test where appropriate using the computer program Statview Student (Abacus Concepts, Berkeley, CA). Values are means ± SE from multiple preparations (quadruplicate samples per animal) and were considered significant when P < 0.050.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Tumor burden and food intake. TBR and matched control animals had similar carcass (tumor-excised) weights (198 ± 15 and 192 ± 18 g for controls and TBR, respectively) in the feeding study, suggesting that pair feeding was effective. Daily food intake decreased by 20-25% (on average by 4-5 g/day) only after the tumor burden reached 15-20% of carcass weight, consistent with earlier observations (10). Inasmuch as some tumors took longer to grow than others, the possibility was considered that the duration, rather than the magnitude, of tumor burden elicited hypophagia. However, when tested directly with multiple tumor implantations per animal, it was determined that the size of the tumor burden influenced food intake more than the duration of the tumor burden. Decline in average daily food intake was first observable when tumor burden exceeded 15-20%, which occurred in the tetra-implanted TBR at day 17 postimplantation, and at day 24 in the mono- and di-implanted TBR. On the basis of these results, the experimental groups were divided into small (<15%) and large (>15%) TBR to distinguish between tumor-specific and nutritional effects. Mean percent tumor burden and tumor weight were 9 ± 4% and 17 ± 8 g in the small and 26 ± 7% and 48 ± 12 g in the large TBR, respectively.

Zonal-enriched hepatocyte populations. The selective destruction of PP or PV hepatocytes via ante- or retrograde digitonin perfusion, respectively, before collagenase digestion is a well-established technique (32, 44) for the isolation of cells enriched in populations from "zones 1 and 2" or "zones 2 and 3" (24), as defined in Rappaport's classic functional model of the liver acinus (46). Figure 1 shows histologically the typical 20-30% PP or pericentral destruction patterns obtained after perfusion with 0.5% digitonin. The zonal enrichment of the cell preparations was confirmed using well-established specific mRNA markers (Fig. 2A): GS for PV cells and GAL and PEPCK for PP cells (54). When normalized to the hybridization intensity of beta -actin mRNA, GS was enhanced 13-fold in PV hepatocyte preparations compared with PP preparations in the large and small TB groups. This marked enrichment is a function of the restricted expression of GS mRNA in only 5-7% of the hepatocytes surrounding the terminal hepatic venule, which are more completely destroyed with digitonin. Conversely, GAL mRNA was enriched 2.1 (large TB)- to 3.5-fold (small TB) in PP compared with PV preparations, confirming the enrichment of each population (Fig. 2B). Consistent with previous observations (54), PEPCK mRNA was slightly less enriched in PP preparations than GAL (2.6-fold in small TB), but in the large TB group the PP enrichment disappeared. The reason for this observation is unclear but may be associated with progressively increased gluconeogenesis rates in the TBR (6, 39, 47). Nonetheless, the enrichments of PP and PV hepatocyte populations in this study were confirmed and consistent with previous reports. Finally, on the basis of the blots in Fig. 2A and other Northern analyses performed on unenriched hepatocyte preparations (not shown), there were no consistent tumor-dependent changes in GAL or GS mRNA abundance, suggesting that altered flux through these pathways does not involve changes in cognate mRNA levels (10).


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Fig. 1.   Histological appearance of rat liver after perfusion in the absence or presence of 0.5% (wt/vol) digitonin. Rat livers were perfused in the absence (A) or presence of 5 mg/ml digitonin in the antegrade (B) or retrograde (C) direction before hepatocyte isolation. Destruction of periportal (PP, zone 1; B) and perivenous (PV, zone 3; C) hepatocytes is readily apparent. Original magnification, ×79.



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Fig. 2.   Assessment of zonal enrichment in hepatocyte preparations. A: Northern blot analysis was performed with probes specific for liver glutaminase (GAL; 2.6-kb mRNA), phosphoenolpyruvate carboxykinase (PEPCK; 2.8-kb mRNA), glutamine synthase (GS; 3.1- and 1.7-kb mRNA), or beta -actin. Total RNA was obtained from PP or PV hepatocyte preparations from pair-fed sham-implanted (S) or tumor-bearing (T) animals and size fractionated on denaturing agarose gels. Each adjacent RNA sample is from matched pairs of animals in the small or large tumor burden group. The ethidium bromide-stained gel for each blot is shown to demonstrate equal loading per lane. B: laser densitometry quantification of the Northern blot exposures in A. Intensities of the signals for the specific probes were normalized to those of beta -actin in each of the samples. Values are means ± SD for each of the PP and PV groups (n = 4). *P < 0.050 vs. PV. C: Na+-dependent glutamate transport rates in PP, unenriched (no digitonin), or PV hepatocyte preparations from sham-implanted (control) or tumor-bearing rats (TBR). Transport of 50 µM L-glutamate was carried out as described in MATERIALS AND METHODS, and the resulting rates are means ± SE of 4 separate determinations from >= 10 animals. *P < 0.010 vs. PP.

Tumor burden and hepatic amino acid transport. Consistent with previous studies (5, 27), PV cells displayed accelerated Na+-dependent glutamate transport rates compared with PP hepatocytes [59 ± 5 and 9 ± 2 pmol · mg protein-1 · min-1 in PV and PP, respectively, in controls (P < 0.001) and 84 ± 20 and 11 ± 3 pmol · mg protein-1 · min-1 in PV and PP, respectively, in TBR (P < 0.001)]. The difference between Na+-dependent glutamate transport rates in control and tumor-influenced hepatocytes failed to reach statistical significance. These data (Fig. 2C) further confirm the zonal enrichments of the hepatocyte isolations.

The Na+-dependent uptake of glutamine in the tumor-influenced hepatocyte was also examined. Under certain conditions, system A has been reported to contribute to glutamine transport in hepatocytes (20, 25). To determine whether such a component exists or is induced by tumor growth in a position-dependent manner along the acinus, the system A-specific substrate alpha -(methylamino)-isobutyric acid was included at a concentration of 5 mM in the glutamine transport assay. As shown in Fig. 3, there was no evidence of a significant system A contribution to hepatic glutamine uptake in either region over the entire range of tumor burdens. This indicated that tumor-dependent glutamine transport stimulation was attributable to enhanced system N activity and is consistent with data reported previously in studies with plasma membrane vesicles from whole liver (29, 41).


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Fig. 3.   Assessment of system A contributions to Na+-dependent glutamine transport in PP and PV hepatocyte preparations from normal and tumor-bearing rats. Hepatocytes were isolated from tumor-bearing rats or their pair-fed controls and placed in culture for 3 h before transport measurement. Na+-dependent uptake of 50 µM L-glutamine was measured in the absence or presence of the system A-specific substrate alpha -(methylamino)-isobutyric acid (MeAIB) at 5 mM. Values are means ± SE of 4 separate determinations from 12 animals. *P < 0.010 vs. control. Data represent tumor burdens spanning the entire range used in the study (5-35%).

More detailed analysis of the hepatic response to tumor growth revealed some surprising and interesting results. Tumor burdens of 9 ± 4% elicited a 2.2-fold increase in system N activity in unenriched (no digitonin) hepatocyte preparations (P < 0.010) compared with those from pair-fed controls (Table 1). A separate study with tumors ranging from 0.004 to 9% of carcass weight indicated that tumor-dependent effects on glutamine uptake required a >= 4% burden. In contrast, large tumor burdens (26 ± 7%) collectively resulted in only a 26% induction of glutamine uptake, an effect that did not achieve statistical significance. These data indicated that cancer-dependent stimulation of system N activity occurs relatively early and wanes with excessive tumor burden. This effect appeared to be attributable in part to an 18% decrease in large TBR glutamine uptake (not significant) coupled with a 1.5-fold increase in large TBR control system N rates compared with small TBR controls (P < 0.010). Enhanced control system N activity may in turn result from reduced caloric intake during pair feeding (20).

                              
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Table 1.   System N and CAT-2A activity in PV, PP, and unenriched hepatocyte preparations

When these studies were extended to zonal-enriched hepatocyte populations, another surprising observation was made. System N activities were enhanced by an average of 75% in PV- vs. PP-enriched hepatocyte preparations from control and TBR early in tumor growth (P < 0.010; Table 1). Likewise, Na+-dependent glutamine transport rates were increased 60-70% in PP and PV tumor-influenced hepatocytes compared with controls (P < 0.050; Table 1), indicating that system N is equally induced by tumor burden in both regions. During excessive tumor burden (26 ± 7%), enrichment of system N activity in PV preparations was no longer evident in TBR (Table 1) because of a significant drop in PV transport rates (P < 0.050), an effect that partially contributes to decreased tumor-dependent activation in these animals. Control PV-to-PP system N activity ratios also decreased, but not as profoundly. Although all the values did not achieve statistical significance, this loss of PV system N enrichment was collectively attributable to increased PP activities and decreased PV activities in both groups of animals (Table 1). Thus prolonged tumor burden and its associated nutritional effects shift the emphasis on concentrative glutamine uptake from PV (zones 2 and 3) to PP (zones 1 and 2) regions of the liver.

Saturable arginine transport in the liver is mediated largely by a low-affinity Na+-independent transporter (55) that has been shown to be encoded by CAT-2A (11). Indeed, the activity was considerably lower than that for glutamine in control and TBR unenriched hepatocyte preparations (Table 1). In these hepatocyte preparations, arginine uptake was enhanced by 33% (not significant) and 67% (P < 0.050) in the small and large TBR groups, respectively. This observation was consistent with the graded induction of arginine uptake in liver plasma membrane vesicles with progressive tumor growth reported earlier (16). Contrary to system N activity, saturable arginine transport rates were enhanced fourfold in PP- vs. PV-enriched hepatocytes (P < 0.010) from small TBR groups and pair-fed controls. It is unclear why arginine transport rates in unenriched hepatocyte preparations failed to display intermediate values between those of PV and PP preparations, similar to that seen for glutamine (Table 1). This PP-enriched activity vanished in large TBR pair-fed controls, suggesting that the 20-25% decrease in caloric intake in these animals may elicit a metabolic/hormonal-dependent inhibition of augmented PP arginine uptake. Similarly, heightened PP arginine transport rates were attenuated by 33% in large TBR hepatocytes relative to small TBR, whereas the activities in PV preparations were enhanced by 33% compared with similar preparations from small TBR (Table 1). On the basis of the data, it appears that the presence of a tumor counteracts the nutritional effects in large TBR and preserves a portion of the PP-enriched arginine activity.

To our knowledge, this represents the first report of enhanced arginine uptake in zone 1 of the liver, so we sought to determine whether CAT-2A mRNA was correspondingly enhanced in this region. The Northern blot analysis shown in Fig. 4 suggests, however, that there is no obvious PP-enriched distribution of the CAT-2A mRNA (6.7 kb) in the control or tumor-influenced liver acinus, nor is there any apparent tumor influence on CAT-2A mRNA levels. Therefore, heightened arginine uptake in the PP preparations does not correspond to changes in CAT-2A mRNA, as originally hypothesized. Alternatively, we considered the possibility that another CAT isoform may underlie the fourfold-enhanced activity in PP preparations, but Northern blot analysis with CAT-1 and CAT-2 probes did not yield any detectable hybridizations (data not shown).


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Fig. 4.   Examination of CAT-2A expression in PP- and PV-enriched hepatocytes. Northern blot analysis of CAT-2A mRNA expression in PP- and PV-enriched hepatocytes isolated from sham-implanted controls (S) or tumor-bearing animals (T) was carried out using an end-labeled CAT-2A isoform-specific oligodeoxynucleotide. The ethidium bromide-stained size-fractionated total RNA gel demonstrates equal loading per lane. A single apparent 6.7-kb mRNA was detected and was quantified by laser densitometry (B). No tumor- or zone-dependent differences in CAT-2A mRNA were observed.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MCA fibrosarcoma model of cancer has been used extensively to study host-tumor metabolic relationships. It is now well established that tumor growth induces muscle proteolysis and amino acid efflux (9) as well as accelerated hepatic metabolism, such as protein synthesis and gluconeogenesis (47, 52). In support of heightened liver amino acid metabolism during tumor growth, the activities of several amino acid transport activities are coordinately stimulated as measured in plasma membrane vesicles isolated from whole liver homogenates (12, 15, 16, 29, 41). Initially, it was proposed that these activities increase in proportion to the tumor burden (12, 16). More recent reports in isolated hepatocytes, however, yielded preliminary indications that this may not be the case for glutamine (13, 19), although the problem was not specifically addressed in those reports. Many studies with this tumor model have been performed when the burdens exceeded 20% of carcass weight (12, 18), causing some to question its physiological relevance. The studies presented here were therefore undertaken to address these problems and comprehensively examine the effects of progressive tumor burden on specific amino acid transport activities in hepatocytes from different acinar zones. One of the reasons we chose to utilize hepatocytes instead of plasma membrane vesicles was the fact that they represent a more comprehensive assessment of tumor-influenced physiology. Factors that collectively contribute to measured transport rates, such as transmembrane electrochemical potentials and intracellular amino acid levels, are retained in hepatocytes but are lost during subcellular fractionation. Inasmuch as plasma membrane vesicles offer unequivocal assessments of changes only at the plasma membrane level, the two systems are complementary. Lastly, we chose to focus on the hepatic transport of glutamine and arginine, inasmuch as these two amino acids have been proposed for use in immunomodulatory nutritional therapies for cancer patients (1), and each plays an integral role in liver nitrogen metabolism.

Enriched system N activity (Table 1) in PV hepatocytes (zones 2 and 3) was an unexpected finding, because no precedent for this observation exists. An earlier study on amino acid transport activities in PP and PV hepatocytes reported no difference in Na+-dependent histidine (another system N substrate) transport rates between both zones (5). However, several differences in methodologies between our studies might provide reasons for this discrepancy. For example, in the present study, viable hepatocytes were separated from nonviable cells, nonparenchymal cells, and debris by Percoll gradient centrifugation, as introduced by Bilir and colleagues (3) for the digitonin-collagenase technique. On the basis of recent studies from our group that indicated differences in feeding habits between TBR and pair-fed controls (14), all animals were subjected to an overnight fast before hepatocyte isolation to eliminate influences from prandial status. In contrast, Burger and colleagues (5) utilized animals fed ad libitum and measured histidine uptake for 2 min, a time frame that may not assess initial-rate system N activity. Whether these collective differences sufficiently account for the discrepant results between our two studies remains to be determined.

Although this is the first report of PV-enriched system N activity, its significance was initially unclear and unexpected in the context of PP-enhanced glutaminase and urea synthesis (45, 54). A potential explanation for this observation was provided during the final submission of this manuscript when the gene encoding system N activity was isolated from rat (8) and mouse (23) cDNA libraries. On the basis of immunohistochemical studies in one of those reports (23), system N transporter expression was shown to be enriched in the PV regions of the liver acinus, supporting the results in our study (Table 1). In the other work (8), system N was shown to mediate the bidirectional transport of glutamine under physiological conditions. Its unique mechanism utilizes the countertransport of H+ and the transmembrane glutamine gradient to drive glutamine efflux and the Na+ gradient to drive uptake. Thus enhanced PV system N activity may be required for efficient glutamine release. Other possible reasons for enhanced PV system N activity were considered as well. Given the pronounced metabolism of glutamine in zone 1, a more robust transport activity may be required to support glutamine-dependent metabolism in zones 2 and 3 in the face of gradually diminishing concentrations of this amino acid along the acinus. This model is based on several independent observations. The affinity of system N for glutamine (Michaelis-Menten constant = 0.6-1 mM) correlates with the physiological levels of this amino acid in the blood. System N transport rates are therefore directly dictated by plasma glutamine levels. Also, studies by Low and colleagues (34) established the quantitative importance of system N activity in regulating intracellular hepatic glutamine metabolism. Therefore, enhanced system N activity may be necessary in zones 2 and 3 to support glutamine-dependent processes such as PV-enriched glutamate dehydrogenase flux (36) at levels equivalent to zone 1 metabolism.

The data in Table 1 show that system N activity is equally stimulated in both regions of the acinus by 9 ± 4% tumor burden, suggesting that this relative distribution of glutamine uptake is maintained and enhanced during clinically relevant tumor growth. This may represent a response to the diminished circulating arterial glutamine levels associated with the growth of this tumor (10, 41) coupled with an increased glutamine demand for gluconeogenesis and protein synthesis early in tumor growth (39, 53). During the course of these studies, it was determined that a 4% tumor burden was the minimum requirement for stimulation of system N activity, and previous studies in plasma membrane vesicles indicated that this effect is attributable to an increase in maximum transporter velocity (29). Only after supraphysiological tumor burden (26 ± 7%) and associated caloric restriction does this zonal arrangement cease to function (Table 1).

The pan-zonal stimulation of system N activity during cancer must be placed in the context of earlier in vivo work that served as the impetus for the present investigation. Those studies showed that a tumor burden of 6-9% caused the liver to switch to net glutamine output (51), whereas subsequent work showed that this response was secondary to a 35% decrease in GAL and a 43% increase in GS activities (10). A third study implicated transport in the switch to glutamine output, where it was demonstrated that a 7% tumor burden elicited a 2.7-fold increase in Na+-independent glutamine transport [system n (42)] and a 24% increase in system N as measured in plasma membrane vesicles (41). The net result is an enhanced ability to release glutamine from hepatocytes, which appeared to be a facilitative (Na+-independent) process (17). Those studies must be reevaluated in light of the recent report that system N mediates glutamine uptake and efflux, however (8). Our results indicate that early tumor burden stimulates system N activity in PP and PV hepatocyte populations (Table 1), which makes sense if the function of the liver is to provide glutamine during early tumor growth (51). Under these conditions, enhanced glutamine transport and metabolism in PP hepatocytes would be offset by enhanced GS activity (10) and system N-mediated efflux in the PV region. Given the recent data on system N transport mechanisms (8), modulation of the transmembrane glutamine and proton gradients would serve a regulatory role in determining the net efflux rate of glutamine from the liver. This concept is supported by previous observations (10, 12) that the liver exhibits increased glutamine content and output during early tumor burden (when system N is enhanced in both regions) but decreased glutamine content when the liver switches to net consumption in animals with large (>25%) tumor burdens [when the system N gradient disappears (Table 1)]. We speculate that the upregulation of system N in both zones in response to physiologically relevant tumor burden supports hepatic and extrahepatic demands for glutamine and allows the host to survive. Whereas data from whole liver support this paradigm, there are no data on the glutamine content of PP or PV hepatocytes in response to tumor burden, so substantiation of this proposed model awaits further work.

The other significant finding from the present study was the marked saturable arginine transport rates in PP hepatocytes relative to PV or unenriched preparations (Table 1). In contrast to the reciprocal arrangement for glutamine, arginine uptake rates were not stimulated in a statistically significant manner during clinically relevant tumor growth. It is unclear why arginine uptake appears to be markedly accelerated in zone 1. One possibility is that it may help drive the urea cycle in the context of high cytoplasmic glutamine in the extreme PP region (as discussed above), which in turn has been shown to inhibit arginine formation from citrulline in some cells (50). Recent work on this relationship in hepatocytes, however, has shown that glutamine actually enhances argininosuccinate synthase expression and activity through its effects on cell volume (43). A precedent for extracellular arginine driving hepatic ureagenesis was provided in earlier studies with patients afflicted with inborn errors in the urea cycle (4). The hepatic transport of arginine has been ascribed to CAT-2A (11, 35, 55), and our studies confirm the marked expression of this gene in rat hepatocytes (Fig. 4). However, the relative abundance of CAT-2A mRNA does not correlate with enhanced arginine transport activity in PP cells. If CAT-2A is responsible, then significant post-mRNA mechanisms such as enhanced translation or plasma membrane trafficking rates of the protein must underlie this observation. A similar situation exists for the GLUT-1 glucose transporter in the liver, where there are no detectable zonal differences in its mRNA, but only the terminal PV hepatocytes express the protein in the plasma membrane (3).

It is possible that an arginine transporter other than CAT-2A is responsible for the marked PP activity. Previous work from our laboratory in isolated liver plasma membrane vesicles showed that arginine uptake was mediated by high- and low-affinity transporters, both of which were stimulated by tumor burden (29). Clearly, the low-affinity transporter was CAT-2A, but the identity of the high-affinity carrier remains elusive. Northern blot analysis with CAT-1- and CAT-2-specific probes failed to yield detectable signals in the present study, a finding that is consistent with other reports that showed CAT-1 mRNA to be detectable in the liver only if it is induced to regenerate or if animals are injected with specific hormones (33). The possibility exists that CAT-1 may be responsible for enhanced PP arginine transport, but if it is only expressed in the first one or two hepatocytes surrounding the portal inflow, its mRNA may not be detectable in Northern blot analysis of total RNA. This hypothesis is supported by lack of an arginine transport "gradient" [i.e., no intermediate transport velocity value for unenriched hepatocyte preparations (Table 1)] and previous work based on its role as a viral receptor showing that CAT-1 is transiently expressed only around the portal triads during liver regeneration (56). It is unclear whether excessive tumor burden brings the liver to a state of regeneration, but our data also indicate little tumor-specific induction of arginine transport during physiologically relevant tumor burden (Table 1). It is also unclear why the PP-enriched arginine transport activity disappears only in hepatocytes from pair-fed control animals for the large tumor burden group. Such a response would have to involve the 20-25% decrease in caloric intake (the only variable) in these control animals, while the presence of the tumor somehow preserves PP arginine transport activity under the same conditions in the matched TBR. Clearly, the identity of the PP arginine transporter and its regulation by caloric restriction require further investigation.

In summary, the results presented here represent the first report of reciprocal zonal-enriched glutamine and arginine transport activities and demonstrate the differential effects of clinically relevant tumor growth on each. Although the stated objectives of reporting these effects at the cellular level were met, many new questions were raised based on the findings. This study will therefore serve as the basis for future investigations into the role and regulation of specific amino acid transporters along the liver acinus during cancer and the significance of tumor-influenced hepatic physiology to the survival of the host.


    ACKNOWLEDGEMENTS

We acknowledge and greatly appreciate the assistance of Dr. David Schoenfeld (Harvard Clinical Research Center) with the statistical analyses in this study. We are grateful to Dr. Jorge Gumucio for helpful discussions regarding technical aspects of the digitonin-collagenase perfusion technique.


    FOOTNOTES

This study was supported by National Cancer Institute Grant CA-57690 to W. W. Souba and by National Institute of Diabetes and Digestive and Kidney Diseases Harvard Clinical Nutrition Research Center Grant 1-P30-DK-40561 to B. P. Bode.

Address for reprint requests and other correspondence: B. P. Bode, Dept. of Biology, St. Louis University, 3507 Laclede Ave., St. Louis, MO 63103-2010 (E-mail: bodebp{at}slu.edu).

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.

Received 22 December 1999; accepted in final form 22 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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