The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 μg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2′,3′-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.
The small intestine is an important organ that serves as a vital site for nutrient digestion and absorption. Accordingly, it represents the main site of exposure to toxic food contaminants and therapeutic drugs. Once inside the enterocytes, many of these compounds are metabolized and become substrates of apical transporters that efflux them back to the intestinal lumen. Glutathione transferase (GST) and UDP-glucuronosyltransferase are major biotransformation systems involved in metabolism of a large variety of food contaminants. Further excretion of their anionic derivatives is mediated by a primary-active ATP-dependent pump, identified as multidrug resistance-associated protein 2 (Mrp2 or Abcc2) (Keppler et al., 1997; Mottino et al., 2000). Metabolic and transport systems share the same gradient of expression along the intestine, and their coordinated action, particularly at the proximal enterocyte, limits the absorption of xenobiotics, thus acting as a chemical barrier (Dietrich et al., 2003).
Increased activity of these systems is particularly necessary under conditions of increased food intake. This is the case in postpartum lactating rats, mainly at the latter stage of lactation (14–21 days after delivery), when food intake is increased 2- to 4-fold over control females (Cripps and Williams, 1975). Lactating rats increase their intestinal capacity to secrete conjugated xenobiotics (Mottino et al., 2001) and reabsorb bile salts (Mottino et al., 2002), which was associated with an increased expression of the respective transporters, Mrp2 and the ileal apical sodium-dependent bile acid transporter. These modifications would suggest an adaptive response to the increased food consumption imposed by the increased energy demands present in lactation caused by milk production. This adaptation implies a higher exposure to potentially toxic food contaminants. These changes probably are mediated by hormonal factors, with prolactin being a major candidate. However, whereas this hormone is able to induce the expression of basolateral hepatic transporters involved in bile salt uptake (Liu et al., 1995), other studies failed to demonstrate any effect on the expression of the intestinal apical transporters Mrp2 and Abst (Mottino et al., 2001, 2002).
An alternative candidate, whose plasma levels increase in lactation, is glucagon-like peptide 2 (GLP-2) (Jacobs et al., 1981). GLP-2 is a biologically active peptide secreted by the L-type enteroendocrine cells of the intestinal epithelium with trophic properties directed to the gut, affecting mucosal morphology, function, and integrity (Drucker, 2002). GLP-2 represents an important regulator of intestinal function under both physiological and pathological conditions, although its mechanism of action remains unclear. GLP-2 acts through a seven-transmembrane spanning G protein-coupled receptor (GLP-2R) classified as a member of the glucagon receptor family; GLP-2R expression is highest at the proximal small intestine and decreases distally (Munroe et al., 1999). This distribution coincides with the preferential localization of Mrp2 and conjugating enzymes. The cellular localization of GLP-2R is not yet completely defined and seems to depend on the species studied. GLP-2R was detected in intestinal enteroendocrine cells in humans (Yusta et al., 2000), in enteric neurons in mice (Bjerknes and Cheng, 2001), and in subepithelial myofibroblasts in humans, rats, and mice (Ørskov et al., 2005). The presence of GLP-2R in the enterocyte has not been demonstrated, and consequently, it remains unclear whether the intestinotrophic effects of GLP2 are caused by a direct effect on enterocytes or are mediated by secondary factors, which makes the mechanism of GLP-2 action in vivo especially complex. The downstream mediators and mechanistic pathways underlying GLP-2 action also remain ill-defined because, in part, of a lack of cell models expressing the endogenous receptor. However, the activation of transfected GLP-2R results in an increase in intracellular cAMP, activation of cAMP-dependent protein kinase A (PKA), and increases in cAMP-response element- and activator protein-1 (AP-1)-dependent transcription (Munroe et al., 1999; Yusta et al., 1999; Boushey et al., 2001).
Until now, no study has evaluated the effects of GLP-2 on intestinal Mrp2 chemical barrier function. Such information would contribute to understanding the physiology of adaptive changes occurring in intestine during lactation. It could also be of clinical relevance, because GLP-2 is considered as a novel therapeutic tool in several situations of intestinal injury (Arthur et al., 2004; Wallis et al., 2007). Therefore, we examined the effect of administration of GLP-2 on expression and activity of Mrp2 and GST in the rat and the potential mediation of cAMP in GLP-2 action. We found that GLP-2 was able to induce both systems at protein and mRNA levels, and regulation of expression of Mrp2 probably was mediated by cAMP.
Materials and Methods
Phenylmethylsulfonyl fluoride, pepstatin A, 3-isobutyl-1-methylxanthine, 1-chloro-2,4-dinitrobenzene (CDNB), 2′, 3′-dideoxyadenosine (DDA), dithiothreitol (DTT), and dihydrochloride hydrate (H89) were obtained from Sigma-Aldrich (St. Louis, MO). Rat GLP-2 was obtained from American Peptide Co., Inc. (Sunnyvale, CA). 2-Methylbutane was obtained from Acros Organics (Fairlawn, NJ). All other chemicals and reagents used were commercial products of analytical-grade purity.
Animals and Treatment
Adult female Wistar rats (200–230 g), 90 days old, were used. Animals had free access to food and water and received humane care as outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The rats were randomly divided in two experimental groups. GLP-2-treated rats (GLP-2 group) were administered GLP-2 dissolved in sterile phosphate-buffered saline (PBS) (12 μg/100 g b.wt.) by subcutaneous injection every 12 h for 5 consecutive days. Control rats (control group) received subcutaneous injections of the vehicle (PBS) according to the same schedule described for GLP-2.
In addition, we evaluated the effect of coadministration of DDA, a specific inhibitor of adenylyl cyclase, with GLP-2 on intestinal Mrp2 expression. In the DDA group, rats received intraperitoneal injections of DDA dissolved in 0.9% sterile saline (15 μg/100 g b.wt.) every 12 h for 5 consecutive days (Wang et al., 2001). In the DDA+GLP-2 group, rats received injections of DDA, as in the DDA group, 10 min before every administration of GLP-2, as in the GLP-2 group. As a control group, rats received injections of both vehicles, saline intraperitoneally and PBS subcutaneously every 12 h for 5 consecutive days.
Specimen Collection, Morphometric Analysis, and Cell Isolation
Unless otherwise stated, animals were anesthetized with urethane (1000 mg/kg b.wt. i.p.) and sacrificed 18 h after the last GLP-2 injection. For collection of the proximal jejunum used in this study, the first 15 cm starting from the pyloric valve and corresponding to the duodenum were excluded, and the following 30 cm were taken as the proximal jejunum. This segment was carefully rinsed with ice-cold saline, dried with filter paper, and weighed. For Western blot studies, the jejunum was immediately opened lengthwise, the mucus layer was carefully removed, and the mucosa was obtained by scraping, weighed, and used for brush border membrane (BBM) or cytosol preparations. For Mrp2 transport studies, 3-cm segments were isolated from the proximal jejunum and carefully rinsed with ice-cold saline and immediately used in the everted sac preparation. For confocal microscopy analysis of Mrp2 localization, small rings were cut from this same region of intestine, gently frozen in liquid nitrogen-cooled 2-methylbutane, and kept at −70°C until use in slice preparation.
For real-time PCR studies, animals were anesthetized and sacrificed 15 h after the last GLP-2 injection, and total RNA was prepared from the proximal jejunum by using TRIzol reagent (Invitrogen, Carlsbad, CA), following the manufacturer's protocol.
For detection of cAMP in mucosal homogenates, animals were anesthetized and sacrificed 2 h after the last GLP-2 injection. The mucosal tissue from proximal jejunum was obtained as described above, and an aliquot of 50 mg was immediately frozen and kept in liquid nitrogen until use in cAMP determination.
Measurement of the Villus Height.
To evaluate the effect of GLP-2 on the villus height, jejunum sections from GLP-2 and control groups were prepared with a Zeiss Microm HM500 microtome cryostat (Carl Zeiss Inc., Thornwood, NY) and stained with hematoxylin and eosin for examination by light microscopy. The morphometric analysis of villus height was performed by using Image J 1.34m software (available at http://rsb.info.nih.gov/ij) and measuring the distance between the top and the base of the villus.
Isolation of Enterocytes.
The isolated enterocyte model was used to evaluate the effect of GLP-2 on villus regional-dependent expression of Mrp2 and villin and production of intracellular cAMP. Enterocytes were isolated by the standard low-temperature method as described by Flint et al. (1991) with some modifications. This method ensures preservation of the structural and biochemical integrity of the cells. Because Mrp2 is expressed mainly in mature enterocytes located at the villus tip, we first separated this region (fraction T) from the middle portion (fraction M) of the villus. In brief, the proximal jejunum was removed and immediately washed with ice-cold Hanks' balanced salt solution. It was everted, cut into small pieces, and stirred on ice for 5 min in oxygenated Hanks' balanced salt solution containing 0.5 mM DTT. The intestinal sheets were then transferred into oxygenated calcium-chelating buffer (27 mM Na-citrate, 5 mM Na2HPO4, 96 mM NaCl, 8 mM KH2PO4, 1.5 mM KCl, 20 mM d-sorbitol, 20 mM sucrose, 2 mM glutamine, 1.5 mM DTT, pH 7.40, 4°C) and stirred on ice for 10 min, obtaining a first fraction of villus cells, fraction T. The residual tissue pieces were then transferred to new cold chelating buffer and stirred on ice for 15 min to obtain fraction M. The isolated enterocytes from both extractions were collected by centrifugation (1000g, 6 min at 4°C) and washed twice with PBS. All solutions contained 100 μM 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) and protease inhibitors (0.1 μM phenylmethylsulfonyl fluoride, 35 nM pepstatin A). The efficiency of the fractionating procedure was monitored by examination of the residual tissue stained with hematoxylin-eosin, which confirmed that only upper and middle portions of the villus were removed during the procedure. The two cell subpopulations were characterized by determining functional properties associated with differentiated epithelial cells. Alkaline phosphatase and sucrase-isomaltase activities were assayed as described previously by Walsh et al. (2003) and Heinonen and Lahti (1981), respectively. These enzymes show higher activities in the upper villus zone than in the rest of the villus.
For assessment of Mrp2 and villin expression, BBMs were prepared from isolated cells immediately after their isolation. Cellular cAMP was also determined immediately after cell isolation.
To evaluate the presence of endocrine cells in the fractions of isolated epithelial cells, we performed RT-PCR for chromogranin A, an established marker of endocrine cells. Aliquots of the first-strand reaction were used as templates for PCR using Taq polymerase (Promega, Madison, WI). Primer pairs used for PCR of rat chromogranin A were based on the sequences reported previously by Walsh et al. (2003): sense, 5′-GAGGGTCCTCTCCATCCTTC-3′; antisense, 5′-CGCCTTCTCCTCTTTCTCCT-3′. β-Actin was used as an internal control in RT-PCR experiments, and primer pairs were: sense, 5′-CAACCTTCTTGCAGCTCCTC-3′; antisense, 5′-TTCTGACCCATACCCACCAT-3′. Amplification of cDNA for both chromogranin A and actin was performed at an annealing temperature of 58°C for 26 cycles, resulting in the generation of 550- and 210-bp products, respectively. Analysis of the PCR products was performed by agarose gel electrophoresis followed by visualization by ethidium bromide staining.
Evaluation of Mrp2 and GST Expression and Activity
Western Blot Studies.
BBMs were prepared from mucosa samples or isolated enterocytes as described by Mottino et al. (2000). Cytosolic fractions were obtained from intestinal mucosa by ultracentrifugation methodology (Catania et al., 2000). Protein concentration was measured by using bovine serum albumin as standard (Lowry et al., 1951). Aliquots of the BBM and cytosol preparations were kept on ice and used the same day in Western blot studies of Mrp2, villin, and GST as described previously (Mottino et al., 2000; Arias et al., 2009). Apical Mrp2 and villin were detected in BBMs using a mouse monoclonal antibody to human MRP2 (M2 III-6; Alexis Laboratories, San Diego, CA) and human villin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The expression of the major GST classes present in intestine were evaluated by using goat antisera against rat α and μ GSTs (GS9 and GS23, respectively; Oxford Biomedical Research, Oxford, MI) and rabbit antiserum against human π GST (Immunotech, Marseille, France). Equal loading and transfer of proteins were checked by Ponceau S staining of the membranes. The immunoreactive bands were quantified with Gel-Pro Analyzer software (MediaCybernetics, Inc., Bethesda, MD).
For in situ immunodetection of Mrp2, intestinal rings from jejunum were sectioned (thickness, 5 μm) and fixed as described previously (Arias et al., 2009). Mrp2 and the tight junction protein zonula occludens 1 (ZO-1) were detected with the respective antibodies, and the cell nuclei were detected with 4,6-diamino-2-phenylindole blue fluorescence as described previously (Arias et al., 2009). All confocal studies were performed with a Nikon (Tokyo, Japan) C1 Plus microscope. To ensure comparable staining and image capture performance for the different groups belonging to the same experimental protocol, intestinal slices were prepared on the same day, mounted on the same glass slide, and subjected to the staining procedure and microscopy analysis simultaneously.
Real-Time Polymerase Chain Reaction Studies.
cDNA was produced by using the SuperScript Preamplification System for first-strand cDNA synthesis according to the manufacturer's instructions (Invitrogen). Real-time quantitative PCR was performed on cDNA samples using a Stratagene (La Jolla, CA) Mx3000P. Sequences of primer pairs and conditions for Mrp2, GST, and 18S were designed to optimally detect the respective mRNAs. All sequences and conditions are summarized in Table 1. Quantification of the target cDNAs in all samples was normalized to 18S ribosomal RNA (Cttarget − Ct18S = ΔCt) and the difference in expression for the target cDNA in the treated group was expressed relative to the amount in the control group (ΔCttreated − ΔCtcontrol = ΔΔCt). Fold changes in target gene expression were determined by taking 2 to the power of this number (2ΔΔCt).
To characterize the effect of GLP-2 on intestinal Mrp2 efflux activity, the in vitro model of everted sacs was chosen. Three-centimeter segments from proximal jejunum were everted and incubated in the presence of 100 μM CDNB in the mucosal compartment as described previously (Mottino et al., 2001), for 0, 10, 20, 30, and 60 min. After diffusion of CDNB into the enterocyte, and further endogenous conjugation with glutathione, the product dinitrophenyl-S-glutathione (DNP-SG) was detected by high-performance liquid chromatography in the same mucosal compartment (Mottino et al., 2001).
The glutathione-conjugating activity toward CDNB was assayed in the cytosol from the proximal jejunum as described previously (Catania et al., 2000).
Evaluation of cAMP Formation
The effect of GLP-2 on intestinal cAMP formation was examined in both mucosal homogenates from proximal jejunum and tip and middle villus enterocyte subpopulations (T and M fractions, respectively). For detection of cAMP in homogenates, 50-mg aliquots of frozen homogenate were pulverized manually by using a mortar and diluted with PBS to approximately 10 mg of protein per ml. For detection of cAMP in isolated cells, cell suspensions were diluted with PBS to approximately 5000 cells/30 μl. cAMP levels were detected by using an enzyme immunoassay system kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK), following the manufacturer's instructions. The cAMP values in the different treatment groups are expressed relative to the control group, which was defined as 100%.
Data are presented as mean ± S.D. All statistical analysis was performed by using Student's t test except for DDA studies in which we performed one-way analysis of variance, followed by Bonferroni's test. Values of p < 0.05 were considered statistically significant.
Effect of GLP-2 on Intestinal Mass and Villus Height.
The hypertrophic effect of GLP-2 on the intestinal mucosa was confirmed at both the macroscopic and microscopic levels in the proximal jejunum, where Mrp2 and GST expression and activity were evaluated. We found an increase in the mass of the proximal 30 cm of jejunum in response to GLP-2 administration (5.95 ± 0.25 and 4.92 ± 0.42 g for GLP-2 and control groups, respectively; n = 4; p < 0.05). Morphometric analysis revealed that jejunal villus height was significantly increased by 25% in GLP-2-treated rats (Fig. 1).
Effect of GLP-2 on Mrp2 Expression and Activity.
Figure 2 A shows that Mrp2 expression, detected in BBM, was increased in the GLP-2 group by 60% with respect to control. The effect of GLP-2 on Mrp2 localization at the villus was analyzed in situ in the proximal jejunum by confocal immunofluorescence microscopy. Mrp2 was labeled with red fluorescence and the tight junction ZO-1 was labeled with green fluorescence, and the cell nucleus of enterocytes is shown in blue. Figure 2B shows that detection of Mrp2 was restricted to the luminal surface of the enterocyte, mostly outside of the region delimited by ZO-1 in both control and GLP-2 groups, indicating that GLP-2 treatment did not affect normal localization of this transporter. To establish whether up-regulation of Mrp2 protein expression resulted from increased expression of its mRNA, we performed real-time PCR studies 15 h after the last injection of GLP-2. This time point was selected on the basis of a previous study reporting maximal increases of intestinal Mrp2 mRNA level 15 h after the last injection with an Mrp2 inducer (Ruiz et al., 2009). Mrp2 mRNA expression in GLP-2-treated rats increased by 111% (n = 4; p < 0.05) with respect to the control group, suggesting transcriptional regulation.
To characterize the relationship between Mrp2 expression and its transport activity, we evaluated the secretion rate of DNP-SG, a typical substrate for Mrp2, by using the model of everted intestinal sacs. In this model, CDNB diffuses passively from the intestinal lumen into the enterocyte and its metabolite, DNP-SG, is effluxed back to the mucosal compartment via Mrp2. Figure 2C shows that the excretion of DNP-SG was substantially increased by GLP-2 treatment. At the 60-min period, detection of DNP-SG on the mucosal side was 1.6-fold higher in the GLP-2 group compared with control, agreeing well with the increased expression of Mrp2 determined by Western blotting.
Effect of GLP-2 on GST Expression and Activity.
Although transport mediated by Mrp2 is considered the rate-limiting step in the overall disposition of CDNB by the rat enterocyte (Mottino et al., 2001), conjugation with glutathione, either spontaneous or enzymatic, is a prerequisite for CDNB to become an Mrp2 substrate. To test whether GLP-2 might affect the efficiency of conversion of CDNB to DNP-SG, as tested in the everted intestinal sac model, we evaluated the activity and expression of the major GST classes present in rat intestine. Figure 3 A shows a 64% induction of the α class of GST in the GLP-2 group with respect to control, whereas the μ and π classes remained unchanged. We also assessed GSTYa2 mRNA levels, the only isoform belonging to the α class detected in rat intestine (Catania et al., 2000) by real-time PCR 15 h after the last injection of GLP-2. The result showed a significant 125% increase in GLP-2-treated rats (n = 4; p < 0.05) with respect to control, suggesting a transcriptional regulation. Increased expression of GSTα was consistent with GLP-2 induction of cytosolic GST activity by 52% (Fig. 3B) over control animals.
Relationship between Mrp2 Induction and cAMP Formation.
Participation of cAMP, a major mediator of GLP-2 actions, was further investigated. The content of this cyclic nucleotide was measured in tissue homogenates from jejunal mucosa prepared 2 h after the last injection with GLP-2, in addition to detection of expression of Mrp2 by Western blotting in BBM prepared from the same tissues. The 2-h period was found to be optimal for detection of increased synthesis of cAMP in rat hypothalamus in response to central administration with corticotrophin-releasing hormone (Wang et al., 2001). GLP-2 treatment significantly increased cAMP levels 161% (Fig. 4 A) over that in control rats and was accompanied by a 72% induction of Mrp2 protein (Fig. 4B). A direct association between increased production of cAMP and induction of Mrp2 expression by GLP-2 was demonstrated by administering the adenylyl cyclase inhibitor DDA. Indeed, Fig. 4B shows that DDA, administered 10 min before GLP-2, prevented both the increase in cAMP formation (Fig. 4A) and Mrp2 expression by GLP-2.
In an attempt to establish whether the action of GLP-2 on Mrp2, involving increased production of cAMP, was restricted to a specific region in the villus, we further evaluated the action of GLP-2 in two different cell subpopulations. A first characterization of T versus M cell subpopulations was performed by assessment of enzymatic activities linked to differentiated enterocytes, which are expected to be more abundant at the villus tip. The study confirmed that the T fraction was indeed more enriched in both alkaline phosphatase and sucrase-isomaltase than the M fraction (25% increases for both enzyme activities; Table 2). Other authors reported increases of 40% for alkaline phosphatase and 30% for sucrase-isomaltase activities under similar, although not identical, fractionation conditions (Weiser, 1973; Flint et al., 1991). Table 2 also shows that GLP-2 treatment did not affect enzyme marker enrichment in any fraction. As a second characterization, we evaluated the presence of cell types different from enterocytes through detection of chromogranin A, an enteroendocrine cell marker. We found that the mRNA transcript of chromogranin A was not detected in the T subpopulation, whereas it was present in the M fraction of the villus (Fig. 5). We then evaluated the potential differential response of both cell fractions to GLP-2 administration. We found that both T and M subpopulations isolated from rats treated with GLP-2 showed a significant increase in cAMP levels (182 and 121%, respectively) with respect to the control group (Fig. 6 A). Mrp2 expression was also significantly increased by GLP-2 treatment in both the T (136%) and M (68%) fractions with respect to control rats (p < 0.01; Fig. 6B), the response being significantly higher in T versus M fractions (p < 0.05). In contrast to Mrp2, expression of villin, a constitutive component of the BBM also associated with differentiated enterocytes, was not affected by GLP-2 in either fraction (Fig. 6B).
Conjugating enzymes and Mrp2, acting together, may affect bioavailability of therapeutic drugs and restrict absorption of food contaminants, thus providing a defense against toxic injury (Dietrich et al., 2003). Food intake is significantly increased 2- to 4-fold in lactating rats, together with a significant increase in the size and surface of the small intestine (Cripps and Williams, 1975; Mottino et al., 2001). We have shown that Mrp2 expression and activity were also increased in these animals, suggesting an adaptive mechanism for dealing with increased food consumption, and potential food contaminants. Maximal increases were observed at the mid and late stages of lactation, coinciding with maximal increases in food intake and growth of the intestine (Mottino et al., 2001). Prolactin and GLP-2 serum levels are increased during lactation (Jacobs et al., 1981; Liu et al., 1990), suggesting a potential role in regulating expression of intestinal Mrp2, although prolactin was found not to be involved (Mottino et al., 2001). We demonstrate here for the first time a role for GLP-2 in regulating expression and activity of jejunal Mrp2 and show that activation of adenylyl cyclase is a likely mediator. Thus, GLP-2 may be a candidate to explain up-regulation of intestinal Mrp2 in lactating rats. It is noteworthy that the trophic action of GLP-2 is known to principally affect the proximal small intestine, where Mrp2 exhibits its maximal expression, whereas the expression of P-glycoprotein, present mainly at the distal small intestine, was shown not to be affected by the hormone (Brubaker et al., 1997).
GLP-2 is produced by specialized, L-type endocrine cells, mainly at the distal small intestine (Ørskov et al., 1986). GLP-2 is implicated in the regulation of intestinal growth and absorptive function under physiological conditions such as development (Drucker, 2002) or during lactation (Jacobs et al., 1981). Promotion of the increase of intestinal size and weight is associated at least in part with increased villus height (Tsai et al., 1997). As a result, the mucosal surface is expected to be significantly increased, consistent with adaptation to the increased energy demands and consequently the need for absorption of nutrients. In addition, GLP-2 stimulates hexose transport through significant increases in both hexose and sodium/glucose cotransporter-1 transport activity and increases the activity of specific digestive enzymes, such as duodenal maltase, sucrase, lactase, glutamyl transpeptidase, and dipeptidyl-peptidase IV (Cheeseman and Tsang, 1996; Brubaker et al., 1997). We here demonstrated that GLP-2 was able to induce the expression of jejunal Mrp2 and the specific isoform of GST, GSTYa2. Increases in mRNA levels suggest a selective regulation of the respective genes, rather than a mere stimulation of overall protein synthesis in the growing tissue.
The trophic action of GLP-2 is also relevant in experimental pathological states in the rat. For example, Scott et al. (1998) found that a potent protease-resistant analog of GLP-2 increased the adaptive morphologic response to massive intestinal resection. In addition, this hormone analog was able to restore absorptive functions decreased by resection, as detected by using d-xylose. Likewise, coinfusion of GLP-2 and parenteral intravenous nutrition prevents the mucosal hypoplasia that occurs in the absence of enteral nutrition (Chance et al., 1997). It is noteworthy that endogenous GLP-2 plasma levels were found to be increased in response to small intestinal resection, also indicating an important adaptive role during recovery from surgery (Ljungmann et al., 2001). Likewise, plasma levels of GLP-2 also increased in response to ileal-jejunal transposition surgery in rats, in association with a pronounced growth of the transposed segment (Thulesen et al., 2001). All of these findings have led to the postulate that GLP-2 may be of therapeutic value in a variety of intestinal dysfunctional conditions. Data from preclinical studies in models of inflammatory bowel disease (Yazbeck et al., 2009) and pilot studies in humans (Wallis et al., 2007) support this assumption. We currently provide evidence favoring an additional role for GLP-2, providing protection to the growing/regenerating intestinal tissue against chemical injury. Indeed, selective restriction to absorption of common substrates of GST and Mrp2 is demonstrated from the experiments in everted sacs, where CDNB was added to the mucosal side and the resulting metabolite, DNP-SG, was more efficiently recovered in the same side in GLP-2-treated versus control rats. Because Mrp2 is probably the rate-limiting step in the overall disposition of CDNB by the rat enterocyte (Mottino et al., 2001), induction of expression of this transporter rather than of GST is primarily responsible for the increased excretion of DNP-SG from the cell to the luminal compartment. Our finding agrees well with a previous report by Benjamin et al. (2000) demonstrating that administration of GLP-2 of human origin, or alternatively a synthetic protease-resistant analog, to normal mice, results in increased membrane barrier function of the intestinal epithelium by reducing transcellular and paracellular movement of small compounds and macromolecules. Epithelial permeability is increased in inflammatory bowel disease and celiac disease because of alterations in transcellular and paracellular routes, respectively, whereas simultaneous alterations in both routes are detected in food allergies (Bjarnason et al., 1995). Based on these findings, Benjamin et al. (2000) proposed administration of GLP-2 as a beneficial therapeutic strategy for restoring the barrier function, thus limiting intestinal uptake of allergens and toxins.
GLP-2 acts through the G protein-coupled receptor GLP-2R, whose expression is tissue-specific, with the highest levels in the jejunum. However, this receptor was found not to be present in enterocytes (Ørskov et al., 2005), the main target cells of GLP-2 actions, and therefore, it is not clear whether the effects of GLP-2 on these cells are direct or indirect. Walsh et al. (2003) studied the GLP-2 signaling pathway in cells isolated from rat intestinal mucosa expressing mRNA transcripts for the GLP-2R, as well as for chromogranin A and β-tubulin III, markers for enteroendocrine and neural cells, respectively. They found increased cAMP production in response to a degradation-resistant analog of GLP-2. In addition, GLP-2 treatment of isolated mucosal cells increased [3H]thymidine incorporation, and this was prevented by inhibition of the PKA pathway. These results provided the first evidence that activation of rat GLP-2R is linked to activation of a cAMP/PKA-dependent, growth-promoting pathway in vitro. Likewise, activation of GLP-2R signaling in heterologous cells expressing a transfected rat or human GLP-2R leads to activation of adenylyl cyclase, increased intracellular cAMP, followed by activation of PKA and increased transcription of cAMP-response element- and AP-1-dependent genes (Munroe et al., 1999; Yusta et al., 1999; Boushey et al., 2001). In this study, we performed experiments to determine whether Mrp2 regulation by GLP-2 was also mediated by cAMP. The studies performed in vivo using the adenylyl cyclase inhibitor DDA unambiguously demonstrated cAMP participation. The downstream pathways after adenylyl cyclase activation and targeting Mrp2 gene are unknown, but could involve the above-described transcription factors. In support of this possibility, it is known that the Mrp2 gene promoter region contains a nucleotide sequence capable of interacting with AP-1 and regulating its expression (Kauffmann and Schrenk, 1998; Haimeur et al., 2004). Mrp2 presents a gradient along the villus, with the highest expression at the tip, coincident with maximal cell differentiation (Mottino et al., 2000). We further explored whether induction of Mrp2 by GLP-2 was restricted to this same region or extended to other regions down the villus and its potential association with the presence of cell types other than enterocytes. Endocrine cells, for example, were found to express the GLP-2R in rodents, humans, and pigs and respond to hormone stimulation by modulating other cell functions through a paracrine communication (Yusta et al., 2000; Guan et al., 2006). We were able to prepare two different subpopulations of mucosal cells exhibiting a differential proportion of enterocytes versus endocrine cells. We found that both subpopulations were able to respond significantly to GLP-2 by increasing Mrp2 expression. In spite of the fact that the effect was more visible in the T subpopulation, the induction observed in the middle region of the villus, where Mrp2 is constitutively less expressed, suggests an increased area of protection exerted by Mrp2. This is of particular relevance considering that the trophic action of GLP-2 involved increases in the surface of the intestine caused by villus growth. It is noteworthy that we previously observed that lactating rats also exhibit an extension in the area of the villus expressing Mrp2 as detected by immunohistochemistry together with a pronounced increase in villus height (Mottino et al., 2001). We also observed that cAMP was increased in response to GLP-2 in both cell subpopulations. It is not possible to be conclusive on whether the action of GLP-2 was exerted directly on the enterocyte or through a different cell type, such as endocrine cells. However, the finding that the T fraction, where chromogranin A was not detected, exhibited a similar increase in cAMP production in response to GLP-2 as the M fraction, suggests such a possibility. Studies demonstrating uptake of a radioactive analog of GLP-2 by rat enterocytes (Thulesen et al., 2000) indicate that a direct interaction between these cells and the hormone is possible. Whether this interaction involves an alternative GLP-2R subtype, not yet identified, or other members of the glucagon receptor family, is not known. This hypothesis and the alternative possibility that GLP-2 acts primarily on a different cell type both are depicted in Fig. 7 . A schematic representation of the action of GLP-2 on expression of GST and Mrp2, and mediation by adenylyl cyclase, together with the coordinated action of both systems to efficiently metabolize CDNB and excrete DNP-SG from the enterocyte, are also depicted.
In conclusion, we demonstrated that GLP-2 positively modulates the expression and activity of Mrp2 and GSTα in rats, and activation of adenylyl cyclase is involved in Mrp2 induction. These findings are of pathophysiologial relevance and indicate a role for GLP-2 in the prevention of cell toxicity under conditions of intestinal damage or as a complement to its trophic action during development, lactation, or tissue regeneration.
This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica [Proyecto de Investigación Científica y Tecnológica Grant 05-26306], Consejo Nacional de Investigaciones Científicas y Técnicas [Proyecto de Investigación Plurianual Grant 6442], and Universidad Nacional de Rosario [Proyecto de Investigación y Desarrollo Grant BIO-112].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- glucagon-like peptide 2
- GLP-2 receptor
- glutathione transferase
- multidrug resistance-associated protein 2
- protein kinase A
- activator protein-1
- brush border membrane
- zonula occludens 1
- real-time polymerase chain reaction
- phosphate-buffered saline
- Received June 3, 2010.
- Accepted August 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics