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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:14-18, January 2005

Biochemical and Molecular Actions of Nutrients

Growth Hormone and Epidermal Growth Factor Upregulate Specific Sodium-Dependent Glutamine Uptake Systems in Human Intestinal C2BBe1 Cells1,2

Edward C. Ray3, Nelly E. Avissar3,4, Rabih Salloum and Harry C. Sax

Department of Surgery, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642

4To whom correspondence should be addressed. E-mail: nelly_avissar{at}urmc.rochester.edu.


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Glutamine (Gln) is one of the major oxidative fuels of the enterocyte and enters from the lumen via Na+-dependent transport mechanisms. When given parenterally, growth hormone (GH) + epidermal growth factor (EGF) increase apical Gln uptake after massive enterectomy in rabbits. Although both receptors are basolateral, GH and EGF are present in luminal contents. We hypothesized that short-term luminal growth factor exposure to enterocytes increases apical Gln uptake by selective upregulation of systems A, B0,+, or ASC+B0. A monolayer of C2BBe1 cells was exposed for 10 or 60 min to GH (500 µg/L), EGF (100 µg/L), both, or neither. Initial uptake of [3H]Gln (50 µmol/L) was measured in the presence of Na+ or choline. The contributions of systems A, B0,+, and ASC+B0 were determined by competitive inhibition with arginine and/or {alpha}-(methylamino)butyric acid. Gln uptake was linear for up to 8 min. Na+-independent transport was negligible. Under control conditions the relative contributions of systems A, B0,+, and ASC+B0 were 0, 19 ± 6, and 80 ± 4%, respectively. GH alone had no effect on Gln transport. After 10 min of EGF exposure, Na+-dependent Gln uptake increased by 50% (P < 0.001) with no change in individual transport systems. Combined EGF and GH for 60 min increased Gln transport by system B0,+ nearly 250% (P < 0.001) and system A from undetectable levels to 16% of total transport (P < 0.01). Thus, short-term luminal exposure to EGF+GH increases Na+-dependent Gln transport mainly by upregulating system B0+.

KEY WORDS: • enterocytes • amino acid transport systems A, ASC, B0, B0,+ • glutamine • epidermal growth factor • growth hormone

Short-bowel syndrome (SBS)5 is a devastating condition resulting from massive enterectomy (1,2). Increasing luminal nutrient transport is crucial to enhance adaptation. Therapeutic modalities, including numerous growth factors and cytokines in combination with nutrients, have been used to enhance the adaptive process (3–11). Our previous work in a rabbit model of SBS showed that long-term parenteral epidermal growth factor (EGF) plus growth hormone (GH) enhanced Na+-dependent amino acid uptake in vivo (12,13). In this study, we discuss the short-term in vitro effects of apically applied EGF and/or GH. We focused on glutamine (Gln) because it is one of the major oxidative fuels for the enterocyte (2,14). In addition, Gln has clinical applicability through its trophic and cytoprotective effects on the small bowel (15)

Gln is primarily transported into the cell from the gut lumen by Na+-dependent neutral amino acid transporters. Of these, the main contributors are the broad-spectrum system B0 and the transport system for neutral amino acids (ASC) with minor contributions by system B0,+ and possibly system A (16–22). Proteins that confer these system activities in heterologous expression systems have been recently cloned (20–23). Na+-dependent neutral amino acid transporters belonging to the ASC family (ATB0/ASCT2) and B0 (B0AT1) correspond to system ASC and B0, respectively, and are broad-spectrum carriers responsible for the majority of Gln movement across the intestinal brush border. System B0,+ (ATB0,+) may be important because of its ability to transport both neutral and basic amino acids including arginine (Arg) and its analogs. The ubiquitous system A (ATA2) is widely regulated in several cell types, but is mainly a basolateral transporter. This system transports primarily short-chain and N-methylated amino acids such as {alpha}-(methylamino)butyric acid (MeAIB) (23–25).

A subclone of Caco-2 cells, C2BBe1 is known for its enhanced small-intestine-like brush border protein expression (26). In culture, these cells form a tight monolayer with apical/basolateral polarity (27) and have been useful for studying the effects of growth factors and cytokines on intestinal cells in vitro (28–32).

The mechanisms by which EGF and GH affect transport have not been well defined (9,33–35). While some of these effects may be due to increased expression of intestinal transport systems during adaptation (30), there may also be nontranslational effects leading to activation and membrane insertion of preformed transporters.

Our objective was to characterize the short-term effects of apically administered EGF and/or GH on apical Gln transport, similar to the effects of feeding. We hypothesized that short-term provision of luminal growth factors increases overall Gln uptake and alters the relative contribution of the different transporters.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Reagents and chemicals. Reagents and chemicals of analytic purity were purchased from Sigma Chemical. Radiolabeled L-[3H]Gln was purchased from Amersham Pharmacia Biotech. EGF was from Upstate Biotechnologies USA and GH (Nutropin) was a gift from Genentech.

    Cell culture. C2BBe1 [American Type Culture Collection No. CRL-2102], a subclone of Caco-2 cells with enhanced brush border expression, was grown as described before (36). Subconfluent cells (passages 10 to 15) were split and subcultured in 24-well culture plates at a density of 105 cells/well and grown until 3 d postconfluence (37). Cells were serum starved 24 h prior to experiments. Cultures were treated for 10 or 60 min with GH (500 µg/L), EGF (100 µg/L), both, or serum-free medium (vehicle, untreated control).

    Transport experiments. Transport experiments were performed in modified cluster trays (29). Cells were washed twice with 37°C choline uptake buffer (each liter contained 145 mmol choline chloride, 3 mmol K2HPO4, 1 mmol CaCl2, 1 mmol MgCl2, 10 mmol Hepes-Tris, 5 mmol glucose, pH 7.4). Warm (37°C) uptake buffer containing [3H]Gln (50 µmol/L, 2 mCi/L) and 145 mmol/L of either sodium chloride or choline chloride (with or without an excess of competitive inhibitors) was added simultaneously to each well. After 2–8 min, the cells were washed 3 times each with ice-cold choline uptake. The cells in each well were solubilized with 2 g/L SDS with 0.2 mol/L NaOH. Half of each cell homogenate was resuspended in Cytoscint scintillation cocktail (ICN Biomedicals) and radioactivity was measured using liquid scintillation spectrometry (LS 8000, Beckman Instruments). Protein concentration determination was performed on the remaining homogenate using bicinchoninic acid protein assay reagent kit (Pierce Biochemicals) according to the manufacturer’s instructions. Absorbance at 545 nm was measured using a Benchmark plate reader (Bio-Rad). Bovine serum albumin served as standard. Uptake activity was expressed as pmol Gln · min–1 · mg protein–1 or as a percentage of the measured control uptake activity.

Na+-dependent uptake was defined as uptake in the presence of Na+ minus uptake in the presence of choline. The contribution of systems A, B0,+, and ASC+B0 to the net Na+-dependent uptake was determined by comparing the total Na+-dependent uptake of Gln to that in the presence of the competitive inhibitors MeAIB (5 mmol/L, transported only by system A) and Arg (5 mmol/L, transported mainly by system B0,+), individually and together. The residual uptake in the presence of both inhibitors represented systems ASC+B0.

    Statistical analysis. Results are reported as means ± SEM. Data were analyzed by one-way ANOVA (and post hoc Bonferroni multiple comparisons test where appropriate) on the GraphPad Prism statistical software package. Differences were considered significant at P < 0.05. A two-way ANOVA with treatment group and time as variables (and post hoc Bonferroni multiple comparisons test) was also performed. Each value represents the mean of 4 experiments with 6 duplicate measurements per experiment.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Na+-dependent Gln uptake time-course. Gln uptake was linear up to 8 min, as was the calculated transport of each major Gln transporter (data not shown). The 2-min time point was used for all subsequent studies because it was well within the linear portion of the uptake curve.

    Total and individual systems Gln uptake. Na+-independent Gln uptake accounted for 10% of total transport (data not shown). Total and individual system Na+-dependent uptakes of control monolayers were determined (Table 1). The relative contributions of system A, B0,+, and ASC+B0 were ~0, 20, and 80%, respectively. Systems ASC+B0 were therefore the main apical Gln transporters under control conditions. Compared to uptake measured at 10 min, there was a 50% decrease in the total and system-specific uptake after 60 min of incubation (P < 0.0001). This might be due to a longer incubation period after exposure to air during medium change (38).


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  		TABLE 1 Total and system-specific Na+-dependent glutamine uptake after 10 and 60 min of exposure of C2BBe1 cells to uptake buffer1

 
    Total and individual systems Gln uptake with GH and/or EGF treatment. After 10 min of EGF exposure, total Na+-dependent Gln uptake increased by 50% (P < 0.001) (Fig. 1). Ten minutes of growth factor treatment did not change the contributions of the individual systems to the total Gln transport (data not shown).



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  		FIGURE 1 Total Na+-dependent Gln uptake in C2BBe1 cells after treatment with vehicle (control) or with EGF or GH alone or in combination (EGF+GH) for 10 min. Uptake of Gln was measured for 2 min. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.001.

 
After 60 min of exposure, the EGF effect disappeared (Fig. 2a). EGF or GH alone did not increase total or system-specific Gln transport (Fig. 2a–c and data not shown). However, total Gln transport was increased 50% with combined EGF and GH. This increase was due to a near 250% increase in system B0,+ activity (P < 0.001) (Fig. 2b). Systems ASC+B0 activities were reduced by half with combined EGF and GH treatment (Fig. 2c). System A contribution to total uptake increased from 0 to 16% (P < 0.01) (data not shown). The most important effect on uptake was the addition of growth factors; this was confirmed by two-way ANOVA with post hoc Bonferroni test. The interaction between the effect of growth factors and time of exposure was significant (P < 0.04).



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  		FIGURE 2 Total (a), system B0,+ (b), and systems ASC+B0 (c) Na+-dependent Gln uptake in C2BBe1 cells after treatment with vehicle (control) and with EGF and GH alone or in combination (EGF+GH) for 60 min. Uptake of Gln was measured for 2 min. Values are means ± SEM, n = 4. Means without a common letter differ, P < 0.001.

 
Ten minutes of combined EGF+GH treatment did not change the relative contributions of each system to the total uptake (Fig. 3). EGF+GH treatment for 60 min increased the contribution of system A to detectable levels and changed the distribution of the other systems such that system B0,+ became the major system instead ASC+B0 (Fig. 3).



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  		FIGURE 3 Relative contribution of Na+-dependent transport systems A, B0,+, and ASC+B0 (ASC/B0) to Gln uptake in C2BBe1 cells following combined epidermal growth factor and growth hormone treatments for 10 and 60 min. 100% = Values are percentage of total Na+-dependent Gln uptake with GH + EGF treatment for 10 or 60 min expressed as means ± SEM, n = 4. For each system, means without a common letter differ, P < 0.0001.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Apical Gln transport in C2BBe1 cells, like in the parental cell line, Caco-2, is mainly Na+-dependent (18,31,32). Caco-2 cells do not express B0,+ activity, whereas in C2BBe1 cells, similar to human intestine (unpublished results), B0,+ contributes 20% to the total activity (18,31,32). This finding supports the use of C2BBe1 cells as a model for the small intestine. System A is not involved in apical Gln uptake (see Results) (18,23,31,32). The detection of system A activity in response to growth factor treatment could reflect directing of low amounts of the transporter from intracellular pools to the apical membrane.

After 60 min of combined EGF and GH treatment, system B0,+ was upregulated 250% (Fig. 2c) and systems ASC+B0 activity was reduced to a half of its baseline level. Short-term upregulation of system B0,+ might be advantageous under stressful conditions. System B0,+ has a broader substrate specificity, importing a broad range of neutral and basic amino acids in their L and D configurations. In addition, it has a much higher concentrative capacity, because it is driven by 2 Na+ and 1 Cl–, rather than by 1 Na+ (39). Since the completion of these studies, reports of B0AT1 cloning and characterization were published (20–22). Our experiments were not designed to differentiate between B0AT1 and ATB0/ASCT2 although tryptophan is a potential competitive inhibitor of B0AT1. There are systems other than B0,+ with Gln transport activity inhibited by Arg (y+LAT and ATA3), but these are basolateral transporters. System y+L exchanges preloaded intracellular basic amino acids with Gln. For the low-affinity ATA3, Gln is not the preferred substrate. If these systems are present in C2BBe1 cells, their contribution to apical Arg-inhibited Gln transport might be either absent or minimal (40,41).

Although we were able to detect ATB0 and ATA2 messages in C2BBe1 cells, we were not able to detect an ATB0,+ message under the same conditions that revealed massive ATB0,+ message expression in lung tissue (data not shown). Therefore, the protein responsible for B0,+ activity in C2BBe1 cells could be different from the classical characterized ATB0,+ protein.

Very-short-term EGF exposure (10 min) transiently enhanced total Gln uptake, although it is difficult to tell which system(s) is responsible. Whatever transient effect EGF has on uptake with only 10 min of exposure, the presence of GH leads to a disappearance of this effect. The synergy between GH and EGF is not manifested until after the 10-min treatment time point. In a preadipocyte cell line, 3T3-F442A, GH inhibits binding of EGF to EGFR after 10 min of incubation, with a gradual return to baseline by 60 min (42). If a similar mechanism occurs in our model, this may explain the differential effect of EGF on transport in the presence of GH over the first 60 min. In contrast to short-term effects of EGF, 48 h of EGF treatment increased Caco-2 ATB0 activity due to de novo ATB0 synthesis (30).

Although the majority of EGF receptor is on the basolateral membrane, the main sources of EGF are luminal secretions, and luminal EGF enhances adaptation (43–47). In Caco-2 cells, C2BBe1 cells, primary cancer cells, and adapting gut there are small amounts of functional EGF receptor on apical membrane (28,48,49) (and unpublished results). GH is found in milk and apical GH has been shown to enhance oligopeptide transporter 1 activity in Caco-2 cells (50,51). At present, it is not definitively known whether the effects of GH and EGF on C2BBe1 monolayers are via apical receptors or whether the effects are elicited by some paracellular leak in the monolayer and activation of basolateral receptors.

The mechanisms responsible for the increased system A and B0,+ activities and the decrease in ASC+B0 activity after short-term growth factor treatment are not known. Translational effects as well as trafficking of premade transporters from intracellular pools to the brush border surface (52) or the activation of transporters already present in the apical membrane (53) may be involved. The synergy between GH and EGF in increasing the specific transporters might be due to a cross talk between the 2 receptor signal transduction pathways. For example, GH can induce EGFR phosphorylation through the Jak/STAT pathway (54). We are investigating these interactions in a Transwell culture model.

The major component of apical Gln transport in both C2BBe1 cells and human jejunum is system B0+ASC with a small contribution of system B0,+ and minimal contribution of system A. System B0,+ is the main Na+-dependent apical Gln transporter upregulated by EGF+GH. C2BBe1 cells in culture serve as a model for intestinal epithelium’s ability to rapidly augment its absorptive surface transport capacity. Understanding the role of cytokines, hormones, and growth factors in mediating these processes offers potential for developing new treatments for SBS and other malabsorptive conditions.


   FOOTNOTES
 
1 Presented at the American College of Surgeons Forum, New Orleans, LA, October 2001 [Ray, E. C., Avissar, N. E., Salloum, R. & Sax, H. C. (2001) Growth hormone and epidermal growth factor alter the relative contributions of intestinal glutamine transport systems. Surg. Forum 52: 5–6]. Back

2 Supported by NIH Grant R01-DK47989-09. Back

3 Equal contribution by first and second authors. Back

5 Abbreviations used: Arg, arginine; ASC, Na+-dependent amino acid transport system for neutral amino acids; ATB0/ASCT2, Na+-dependent neutral amino acid transporter belonging to the ASC family; B0AT1, Na+-dependent broad-spectrum neutral amino acid transporter B0; EGF, epidermal growth factor; GH, growth hormone; Gln, glutamine; MeAIB, {alpha}-(methylamino)butyric acid; SBS, short-bowel syndrome. Back

Manuscript received 9 June 2004. Initial review completed 13 July 2004. Revision accepted 19 October 2004.


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