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Nutrient-Gene Interactions

Increased Expression of Specific Intestinal Amino Acid and Peptide Transporter mRNA in Rats Fed by TPN Is Reversed by GLP-2¹

Alison Howard, Robert A. Goodlad^*, Julian R. F. Walters, Dianne Ford and Barry H. Hirst²

Institute for Cell and Molecular Biosciences, University of Newcastle Medical School, Newcastle upon Tyne, UK; ^* Cancer Research UK, Lincoln’s Inn Fields, London, UK; and Faculty of Medicine, Imperial College London, UK

²To whom correspondence should be addressed. E-mail: barry.hirst{at}ncl.ac.uk.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Intestinal function depends on the presence of luminal nutrients and is altered during starvation and refeeding. Amino acids are essential for enterocytes, but the luminal supply is compromised with changes in dietary intake. To test the hypothesis that during periods of restricted luminal nutrient availability mucosal cells undergo adaptations aimed toward preserving amino acid supply, the expression of amino acid and peptide transporter mRNAs was quantified in rats with no oral intake, whose nutritional status was maintained with total parenteral nutrition (TPN). The role of the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) was investigated in the adaptive responses. Rats were administered TPN with or without exogenous GLP-2. Amino acid and peptide transporter mRNAs in small intestine mucosa were measured by semiquantitative RT-PCR. Compared with orally fed rats, removal of luminal nutrition increased the expression of ASCT1, SAT2, and GLYT1 mRNAs in the duodenum and of ASCT2, EAAC1, NBAT, and PepT1 mRNAs in the ileum. CAT1, PAT1, and SN2 mRNA abundances were unaffected. GLP-2 reversed these effects. Three subgroups of transporters were identified by regional differences in response to TPN. This may reflect differing roles for substrates of transporters located apically and basally and along the proximal-distal axis of the intestine. The importance of maintaining amino acid supply for intestinal mucosal cells is illustrated.

KEY WORDS: • amino acid transport • parenteral nutrition • glucagon-like peptide-2

Amino acids are required by cells of the small intestinal mucosa for protein and nucleotide synthesis and for energy generation. Glutamine is the preferred fuel of enterocytes but other amino acids, particularly glutamate and aspartate, are also extensively catabolized (1). Glycine, cysteine, and glutamate are essential for the production of glutathione (2), and both threonine and glutamine are used at high levels in the synthesis and secretion of the secretory mucins (3,4). Consequently, maintenance of gut function requires a constant supply of amino acids. These may be synthesized de novo in the mucosal cells or obtained from luminal and arterial supplies by uptake at the apical and basolateral membranes, respectively (4). In humans and pigs, the proportion of absorbed dietary amino acids utilized by the intestinal mucosa is important, accounting for 30–40% of some essential amino acids and rising to almost 90% for glutamate (4). Less is known of the importance of basolateral amino acid uptake. However, radioactive tracer studies show that amino acids from both dietary and arterial sources are used in mucosal protein synthesis and energy generation and that, even in the fed state, there is uptake across both apical and basal membranes (4). Amino acids and small peptides are transported across intestinal enterocyte plasma membranes by a large number of transport systems with distinct molecular and functional characteristics (5). Activity and expression of intestinal amino acid transporters and the intestinal peptide transporter PepT1 are subject to regulation by substrate availability, including manipulations of dietary protein intake. However, the nature (whether increase or decrease) and magnitude of the effect varies with the amino acid and the region of intestine under study (6–11).

Starvation in humans and animals removes the major luminal supply of amino acids derived from the diet. Small amounts derived from protein secretion and cell loss into the intestine will still be found, particularly in the distal gut. In clinical situations, it is frequently necessary to limit oral intake, and this can have major effects on intestinal function. Parenteral nutrition is used in intestinal failure to maintain overall nutrition, but it is associated with intestinal atrophy and other alterations of mucosal function such as increased bacterial translocation. Increased knowledge of the molecular mechanisms underlying these changes will facilitate the development of parenteral formulae that avoid these negative effects.

To test the hypothesis that during periods of restricted luminal nutrient availability mucosal cells undergo adaptations aimed at preserving or maximizing amino acid supply, we investigated changes in amino acid and peptide transporter mRNA expression in a rat model of total parenteral nutrition (TPN). Unlike dietary restriction or starvation, TPN maintains the nutritional status of the animals and thus allows determination of the role of the mucosal cells and luminal signaling in controlling transporter expression without the confounding effects of malnutrition. We quantified mRNA expression for a series of 10 transporters for amino acids and peptides important for intestinal function. The transporters illustrate varying patterns of expression along the longitudinal axis of the intestine (vide infra) and varying localization to luminal and basolateral plasma membrane domains (Table 1). The expression of these transporters was determined in both proximal and distal small intestine of rats administered TPN and in orally fed control rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals. Male Wistar rats (Harlan UK) with a mean starting weight of 200 g were used. All procedures were approved by the Imperial Cancer Research Fund Animal Ethics Committee. Before surgery, all rats were given a combination anesthetic consisting of 0.1 mL Hypnorm i.m. (Janssen Animal Health) and 0.1 mL diazepam i.p. (Phoenix Pharmaceuticals). For TPN feeding, a silastic cannula was placed into the right jugular vein and connected to a fluid swivel joint (Linton Instruments) as previously described (18). The rats were housed individually in wire-bottomed cages with free access to water.

Four groups of rats, n = 6/group, were used. Groups 1–3 were established on TPN and administered the basic TPN diet for 7 d. The TPN diet³ was pumped into the rats by a multichannel peristaltic pump, at a rate of 60 mL/(rat · d), giving 1.8 g N, 6.0 g lipid, 8.5 g glucose, and 1047 kJ/(kg · d). As previously described (19), the TPN diet was formulated to be energetically equivalent to a daily intake of 20 g of standard rat diet,⁴ which was fed orally to the fourth control group of rats. Rats in group 2 were continuously co-infused with native GLP-2 [100 µg/(rat · d); synthesized and characterized as previously described (13)] and those in group 3 were administered GLP-2 [100 µg/(rat · d)] as a bolus through the external jugular line.

    Tissue preparation. At the completion of the study, the rats were anesthetized with pentobarbitone and killed by cardiac puncture. The small intestine was rapidly excised, washed with PBS, and the length measured. Mucosal scrapings were obtained from the duodenum and ileum (defined by percentage length), snap-frozen, and stored at –80°C for RNA extraction.

For determination of patterns of transporter expression along the proximal-distal axis of rat intestine, the small intestine was excised and divided lengthwise into 6 equal segments. The mucosal layer of each segment was harvested by scraping, snap-frozen, and stored at –80°C.

    RT-PCR. A series of 9 amino acid transporters and the peptide transporter, PepT1, were chosen for the study. The intestinal expression and functional activity of these transporters were demonstrated before this study (20–27). The selected group encompasses a wide spectrum of substrate specificities, with differential localization to apical and basolateral membrane domains (Table 1). The relative abundance of transporter mRNA was determined by semiquantitative RT-PCR. Total cellular RNA was extracted from the snap-frozen samples using the SV Total RNA extraction kit (Promega). Concentration of RNA was quantified by measuring absorbance at 260 nm, and RNA integrity was verified by electrophoresis on a 1% (wt:v) agarose formaldehyde gel stained with ethidium bromide. Reverse transcription was performed by incubating 1 µg total RNA, 500 ng random hexamers (Amersham Pharmacia), 0.5 mmol/L each of the 4 dNTPS, 1X reaction buffer, 20 U RNasin (Promega), and 100 U M-MLV Reverse Transcriptase (Promega) in a final volume of 20 µL at 42°C for 1 h.

The oligonucleotide primers (Table 2) used for the detection of the transporters were synthesized by the Molecular Biology Unit, University of Newcastle, Newcastle, UK. PCR was performed in a Hybaid PCR Express thermal cycler (ThermoHybaid). Semiquantitative PCR reactions were performed in a 25 µL total volume containing 2 µL template cDNA, 0.2 mmol/L dNTPs, 1X PCR buffer, 1.25 U TAq DNA polymerase (ThermoStart TAq, ABgene), and 18S rRNA primers and competimers (QuantumRNA 18S Internal Standards, Ambion). All PCR reactions used a hot-start procedure (incubation at 95°C for 15 min to activate the enzyme before beginning cycling), denaturation at 95°C for 30 s, extension at 72°C for 30 s, and a final incubation at 72°C for 10 min. PCR products were electrophoresed on 2% (wt:v) agarose/Tris-borate EDTA gels, stained with ethidium bromide, and analyzed on an AlphaInnotech Gel Document system. mRNA abundance was expressed as band intensity relative to 18S rRNA band intensity.

Statistical analysis.

Results are expressed as means ± SEM. Statistical comparisons were made using one-way ANOVA and post-tested using Bonferroni’s Multiple Comparison test (Prism Graph Pad) or by paired t test. Differences between means were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Distribution of transporter mRNA along the rat intestine. To determine the pattern of expression of each transporter along the proximal-distal axis of the intestine, semiquantitative RT-PCR was performed on RNA extracted from mucosa taken from segments of small intestine of orally fed control rats. Negative controls, in which reverse transcriptase was omitted from the reaction, yielded no product. NBAT and PepT1 were expressed at approximately equal levels in all small intestinal segments (Fig. 1). ASCT1, ASCT2, GLYT1, CAT1, and SAT2 demonstrated a pattern of increased expression along the small intestine, with the expression increasing by 20–80% from duodenum to ileum (Table 3). EAAC1 was detectable in the duodenum and proximal jejunum, but expression was at a very low, nonquantifiable level in contrast to the ileum in which EAAC1 levels were quantifiable (Fig. 1). SN2 was found at high levels in the first proximal segment of small intestine but, although detectable by RT-PCR, was at low levels in all other segments. PAT1 was expressed throughout the small intestine but expression in ileum was significantly lower than in all other segments and 50% less than in duodenum. The duodenum and ileum were selected for investigation of changes in response to TPN, reflecting the diversity of expression of amino acid and peptide transporters in the small intestine (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIGURE 1 Relative abundance of transporter mRNA along the proximal-distal axis of rat small intestine, measured by semiquantitative RT-PCR. mRNA abundance is expressed relative to segment 1, the duodenum, which is given an arbitrary value of 1. Results are expressed as the mean of 3 measurements ± SEM (from a single animal). Means without a common letter differ (P < 0.05, ANOVA with Bonferroni’s multiple comparison post-test). Where letters are not given, differences were not significant. 1–6: segments of small intestine from (1) duodenum to (6) ileum. *EAAC1 mRNA abundance could not be reliably quantified in proximal small intestine (values are shown relative to segment 3).

View larger version (20K): [in this window] [in a new window]   FIGURE 2 Relative mRNA abundance of amino acid/peptide transporters in duodenal mucosa of rats administered the control (C), TPN (T), TPN + GLP-2 infusion (T+Gi) or TPN + GLP-2 bolus (T+Gb) diet. mRNA abundance of orally fed, control rats was set at 100%. Values are means ± SEM, n = 6/diet. Means without a common letter differ, P < 0.05 (ANOVA with Bonferroni’s multiple comparison post-test). Where letters are not given means do not differ. *EAAC1 mRNA abundance could not be quantified in proximal small intestine.

    Effect of TPN on Transporter mRNA expression in distal small intestine. EAAC1, ASCT2, NBAT, and PepT1 mRNAs were greater than in controls in the distal small intestine of rats administered TPN (Fig. 3). PepT1 mRNA was increased by 173% (control: 0.95 ± 0.15, TPN 2.6 ± 0.25; P < 0.001), ASCT2 by107% (0.89 ± 0.07, 1.84 ± 0.17; P < 0.001), EAAC1 by 77% (1.43 ± 0.1, 2.53 ± 0.25; P < 0.005), and NBAT by 39% (0.64 ± 0.5, 0.89 ± 0.07; P < 0.01). The abundances of ASCT1, GLYT1, CAT1, PAT1, SAT2, and SN2 mRNAs in distal small intestine were not affected by TPN (Fig. 3).

View larger version (20K): [in this window] [in a new window]   FIGURE 3 Relative mRNA abundance of amino acid/peptide transporters in ileal mucosa of rats administered the control (C), TPN (T), TPN + GLP-2 infusion (T+Gi) or TPN + GLP-2 bolus (T+Gb) diet. mRNA abundance of orally fed, control rats was set at 100%. Values are means ± SEM, n = 6/diet. Means without a common letter differ, P < 0.05 (ANOVA with Bonferroni’s multiple comparison post-test).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Carrier-mediated transport of amino acids in intestinal mucosa serves 2 purposes, i.e., transfer of dietary protein and other components of the luminal milieu from the gut lumen to the circulatory system and supply of nutrients to the mucosal cells themselves. Mucosal cells require amino acids for energy, synthesis of proteins and nucleic acids, and for maintenance of intestinal defense systems, including glutathione and mucin production. During TPN, the small intestine is functionally redundant in terms of nutrient absorption; thus, minimal absorptive transport across enterocytes and downregulation of transport activities might be predicted. In contrast to this expectation, transporter mRNA expression was increased or unaltered during TPN. This may reflect the requirement to maintain mucosal cell amino acid supply during luminal starvation. Commensurate with this inference are earlier studies in humans demonstrating increased or maintained duodenal intramucosal content of 17 amino acids in volunteers after 10–14 d of TPN (28). Similarly, studies in rats and pigs showed that 6–14 d of parenteral nutrition affected the capacity of the intestine to absorb amino acids only marginally and for some amino acids and dipeptides, it had no effect at all (29,30). Burrin et al. (30) further demonstrated that the apparent decreased absorption of some essential amino acids was due to increased mucosal utilization. Together these results illustrate the absolute requirement of the gut mucosa for amino acids.

Expression of mRNA for all 9 amino acid transporters and the peptide transporter PepT1 was detected along the proximal-distal axis of the rat small intestine. With the exceptions of SN2 and PAT1, transporter mRNA expression was either uniform or demonstrated a slight increase along the proximal-distal axis. This pattern is in agreement with previous studies on a smaller subset of transporters in rat small intestine, PepT1, NBAT, and EAAC1 (20). SN2 mRNA was expressed at high levels in the first proximal part of the small intestine, compared with other regions. This may reflect a particular role for glutamine, a SN2 substrate, in this region. PAT1 expression was significantly lower in the distal ileum than in all other segments.

The regulatory effect of nutrient deprivation separates amino acid/peptide transporters into the following 3 groups: 1) upregulated in the proximal small intestine (ASCT1, SAT2 and, GLYT1), 2) upregulated in the distal small intestine (ASCT2, EAAC1, NBAT, and PepT1), and 3) unregulated in either segment (CAT1, PAT1, and SN2). Previous studies noted that the patterns (increase or decrease) and magnitude of the effect in response to a variety of dietary manipulations vary according to the individual amino acid/peptide transport system and position along the longitudinal intestinal axis (6). The basis for the division of transporters into 3 groups defined by their response to removal of luminal nutrients appears complex, although reflecting common regulatory elements/pathways. The groups are not characterized by common substrates, transport mechanism, or pattern of expression along the proximal-distal axis of the intestine. Villus/crypt localization also does not appear to be important. PepT1, NBAT, and EAAC1 are all upregulated in the distal small intestine by TPN. However, although PepT1 and NBAT are found throughout the length of the villus, EAAC1 is found only in crypts and cells at the villus base (20). GLYT1, upregulated in the proximal small intestine in rats, is found in crypts and all parts of the villus in the human small intestine (23). An important factor may be the localization of the transporters to different plasma membrane domains. Of those transporters for which a precise localization was demonstrated, PepT1, EAAC1, and NBAT are all located to the brush border membrane (20). In humans, GLYT1 is found at both the apical and basal surfaces, although basolateral expression is much greater than apical (23). Functional studies have placed system A and ASCT1 activity at the basal surface of intestinal cells (21,26), whereas ATB⁰, the rabbit and human homologue of ASCT2, is confined to apical membranes (22). These patterns of distribution coincide with the division of proximally and distally regulated transporters. We speculate that during TPN, the availability of luminal amino acids is particularly limited in the proximal small intestine, compared with the orally fed state. Thus basolateral transporters may be induced to feed the enterocytes. In contrast, in the distal small intestine during TPN, apical transporters may be induced to maximize recovery of nondietary, luminal amino acids. Although these suggestions are speculative, recent work demonstrated an apparent metabolic compartmentation in intestinal mucosa, in which the source, arterial or luminal, of an amino acid dictates its fate (4). Differential regulation of transporters on the basis of apical/basal location may indicate differing uses of amino acids by the regions of the intestine during dietary restriction.

The effects of parenteral nutrition may be mediated by a range of signals, including changes in the composition of the luminal milieu and thus substrate availability, and/or altered production of specific regulatory factors such as gastrointestinal hormones and growth factors. The intestinotrophic peptide GLP-2 is normally produced in the L-cells of the distal intestine in response to enteral nutrient intake. Circulating concentrations of GLP-2 in pigs are correlated with the percentage of nutrient intake provided orally, which is minimal, although not ablated, during TPN (31). GLP-2 is thought to mediate the effects of nutrients on gut growth and function and was shown to increase glycine and galactose absorption (32) and regulate expression of a number of intestinal genes including sucrase-isomaltase (16,17) and the sodium-dependent glucose transporter (33). When co-infused with parenteral diets, GLP-2 prevents the gut hypoplasia characteristic of TPN (12).

Compared with TPN-fed rats, GLP-2 decreased the expression of ASCT1, SAT2, and GLYT1 in both duodenum and ileum and of EAAC1, ASCT2, NBAT, PepT1, and PAT1 in ileum only. CAT1 and SN2 were decreased only by the GLP-2 bolus. With the exception of PAT1, the apparent division of the transporters into separate groups corresponded to that found for the effect of TPN compared with oral feeding. In a recent study, Guan et al. (34) demonstrated an increased uptake of some amino acids by the portal drained viscera after GLP-2 infusion in TPN-fed neonatal pigs. Although these results would at first appear to be inconsistent with those described here, the apparent disparity may be explained by differential effects of acute vs. chronic GLP-2 administration. Guan et al. (34) investigated the response to acute GLP-2 infusion (4 h) after 7 d of TPN. In contrast, our study examined the effects of chronic exposure (7 d) to GLP-2 throughout the full period of TPN. That the effects of chronic and acute GLP-2 treatment do indeed differ was recognized by Guan and co-workers (34) who showed that intestinal proteolysis is decreased after chronic but not acute GLP-2 treatment. A recent report of GLP-2 receptor desensitization after preexposure to GLP-2 in isolated rat mucosal cells may provide a mechanistic explanation (35). Preservation of cellular protein after chronic GLP-2 treatment may reduce the requirement for amino acid uptake, thus leading to the decrease in transporter mRNA expression observed in our study.

The role of GLP-2 in regulating amino acid and peptide transport under normal conditions has yet to be elucidated, and few studies pertain to this. In one study, exogenous GLP-2 given to orally fed rats increased glycine uptake in jejunal inverted-sleeve preparations (32), but work conducted in humans showed that ingestion of a protein meal did not stimulate release of bioactive GLP-2 (15). In the current study, we found that compared with oral feeding, and in contrast to the effect of TPN, GLP-2 given during TPN led to a decrease in specific transporter mRNA in specific regions along the small intestinal length. Plasma concentrations of GLP-2 were strikingly reduced in food-deprived rats (36) and in pigs administered TPN (31). In the current study, decreased plasma GLP-2 concentration during TPN may contribute to the observed increases in the expression of amino acid and peptide transporters.

Overall, the results of this work show that changes in transporter mRNA level are part of the mucosal response to removal of luminal nutrition and to exogenous GLP-2. Intestinal adaptation is a multilevel process, involving changes at both the transcriptional and translational levels as well as changes in the activity of functional proteins. Quantitation of changes in mRNA abundance, whether the result of altered gene transcription or mRNA stability, are important to understand the overall process of intestinal adaptation. Caution must be exercised in drawing conclusions on how changes in mRNA affect transporter function. However, a direct relation between mRNA levels and protein expression or functional activity was demonstrated for several transporters. In a study of PepT1 in rat small intestine, Ihara et al. (37) showed a 161% increase in jejunal mucosa PepT1 mRNA level after 10 d of TPN, which is comparable to the increase in ileal PepT1 mRNA reported here (173%). In that work, the increase in mRNA level was similar to changes in PepT1 protein density at the brush border membrane. Previously, we showed that the increased PepT1 mRNA level induced in Caco-2 cells by the dipeptide glycyl-glutamine is accompanied by similar increases in both PepT1 protein abundance and activity (38). In nonintestinal cell lines, mRNA and protein levels and functional activity were shown to be directly related for the transporters SAT2 (39) and CAT1 (40). These observations suggest that the changes in transporter mRNA identified in the current work may be accompanied by corresponding alterations in protein abundance. However further work requiring the development of specific antibodies to these rat proteins is warranted before firm conclusions can be made.

We conclude that the removal of luminal nutrition increases the expression of amino acid and peptide transporter mRNAs in small intestine. Three subgroups of transporters that may share common regulatory elements were identified on the basis of regional differences in responses to TPN and GLP-2. The identification of these subgroups illustrates the difficulties in discerning patterns of expression when classification is based on proximal-distal patterns of expression together with global functional descriptors, such as transport (41). GLP-2 downregulates transporter mRNA levels during parenteral nutrition; whether it plays an important role in regulating protein and amino acid uptake by enterocytes during normal, oral feeding patterns remains to be established. The investment of cellular resources in the production of new transporter mRNA during TPN, when energy sources are restricted, emphasizes the importance of a continuous supply of amino acids for maintenance of gut mucosal function.

	ACKNOWLEDGMENTS

 
The expert technical assistance of Maxine Geggie is gratefully acknowledged.

	FOOTNOTES

 
¹ Supported by Biotechnology and Biological Sciences Research Council (13/D09247) and Wellcome Trust (070194) grants.

³ 3 L of the TPN diet contained 2000 mL Vamin glucose (Pharmacia), 84mL Intralipid 20% (KabiVitrum), 133mL dextrose 50% (Clintec Nutrition), 8mL Vitilipid Infant (KabiVitrum), 1 vial Solvito (KabiVitrum), 19 mL Addiphos (KabiVitrum), 12 mL 10% calcium gluconate (Phoenix Pharmaceuticals), 6.7 mL of 30% sodium chloride solution (Martindale Pharmaceuticals), and 3.4 mL of 50% magnesium sulfate (Aurum Pharmaceuticals).

⁴ Rat and Mouse No. 1 Maintenance [(RM1) Special Diets Services Limited], containing 2.71% crude oil, 14.38% crude protein, 4.65% crude fiber, and giving 11.90 MJ/kg.

Manuscript received 5 April 2004. Initial review completed 6 May 2004. Revision accepted 2 August 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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