Expansion of intestinal progenitors and putative stem cells (pISC) occurs early and transiently following ileo-cecal resection (ICR). The mechanism controlling this process is not defined. We hypothesized that glucagon-like peptide-2 (GLP-2) would augment jejunal pISC expansion only when administered to mice immediately after ICR. Since recent reports demonstrated increases in intestinal insulin-like growth factor (IGF)-I following GLP-2 administration, we further hypothesized that increased intestinal IGF-I expression would correlate with pISC expansion following ICR. To assess this, GLP-2 or vehicle was administered to mice either immediately after resection (early) or before tissue harvest 6 wk following ICR (late). Histological analysis quantified proliferation and intestinal morphometrics. Serum levels of GLP-2 were measured by ELISA and jejunal IGF-I mRNA by qRT-PCR. Expansion of jejunal pISC was assessed by fluorescent-activated cell sorting of side population cells, immunohistochemistry for phosphorylated β-catenin at serine 552 (a pISC marker), percent of crypt fission, and total numbers of crypts per jejunal circumference. We found that early but not late GLP-2 treatment after ICR significantly augmented pISC expansion. Increases in jejunal IGF-I mRNA correlated temporally with early pISC expansion and effects of GLP-2. Early GLP-2 increased crypt fission and accelerated adaptive increases in crypt number and intestinal caliber. GLP-2 increased proliferation and intestinal morphometrics in all groups. This study shows that, in mice, GLP-2 promotes jejunal pISC expansion only in the period immediately following ICR. This is associated with increased IGF-I and accelerated adaptive increases in mucosal mass. These data provide clinical rationale relevant to the optimal timing of GLP-2 in patients with intestinal failure.
- intestinal adaptation
glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide secreted by intestinal L cells in the ileum and colon, which influences multiple aspects of intestinal homeostasis (9). In normal physiological states, GLP-2 inhibits gastrointestinal (GI) motility and gastric acid secretion, stimulates nutrient absorption, and reduces intestinal epithelial permeability (8). In adult total parenteral nutrition (TPN)-fed pigs, administration of GLP-2 increased intestinal blood flow (13) and decreased apoptosis (3, 30), yet, in a neonatal piglet model of short bowel syndrome, GLP-2 did not result in clinical improvement (28). This is in contrast to numerous rodent experiments where GLP-2 has been shown to augment the adaptive response following small bowel resection by increasing villus height, crypt depth, proliferation, and decreasing apoptosis (20, 24, 32). In humans as well as rodents, early morphological effects are observed with GLP-2 treatment, but these effects are not sustained following discontinuation of therapy (16).
Clinical findings in humans after bowel resection typically demonstrate increased intestinal caliber without changes in crypt/villus morphology (38). Following significant intestinal loss, TPN is often required to support both nutrition and fluid requirements. Although many of these patients are able to fully transition off TPN over the first one to two years following intestinal loss, limited treatment options are available to improve adaptive growth in those patients who remain dependent on TPN. Because of the potent mitogenic effects of GLP-2, there is considerable interest in its potential as a therapy for patients with chronic intestinal failure. Initial clinical studies evaluating Teduglutide, a GLP-2 analog resistant to degradation by dipeptidyl peptidase IV, have demonstrated improved intestinal wet weight and absorption in short bowel patients (18). To date, this represents the only hormonally induced adaptive intestinal growth (as evaluated by intestinal biopsies) in patients with chronic intestinal failure. Unfortunately, the effects of GLP-2 have not been clinically dramatic or sustained following discontinuation of the peptide (16). The limited clinical efficacy of GLP-2 may be due to the timing of GLP-2 therapy because most patients receive the growth factor many years after developing intestinal failure when humoral factors are less effective (16, 17).
Recent studies in our laboratory provided evidence that expansion of intestinal progenitors and putative stem cells (pISC) occurs early following ileo-cecal resection (ICR) (6). Resection-induced pISC expansion precedes increases in crypt fission and the production of new crypts, leading to sustained increases in the caliber of the remnant intestine (6). In our ICR model, sustained increases in intestinal caliber are not accompanied by maintained increases in proliferation, crypt depth, or villus height. This suggests that early pISC expansion, rather than maintained proliferation, is integral for adaptation following intestinal loss (6). The role of GLP-2 on pISC during the adaptive response following intestinal resection is not defined.
The GLP-2 receptor (GLP-2R) is predominantly expressed by enteroendocrine cells (37), enteric neurons (1, 26), and subepithelial myofibroblasts (27) throughout the GI tract, with highest expression in the jejunum (25). The lack of GLP-2R expression within enterocytes suggests that a secondary mechanism of action mediates the enterotrophic response to GLP-2 (8). Recently, administration of GLP-2 to fasting mice was shown to acutely increase secretion of intestinal insulin-like growth factor-I (IGF-I) from intestinal myofibroblasts in vitro and in vivo (10, 11). IGF-I-null animals did not demonstrate a trophic response to GLP-2, suggesting a key role of intestinal IGF-I mediation of the action of GLP-2 (10). Previous resection models have demonstrated that increases in IGF-I and IGF-I receptor mRNA occur in remnant bowel in rat following ICR (23, 39) and that administration of IGF-I further augmented adaptive growth (5).
The present study tested the hypotheses that exogenous GLP-2 would promote pISC expansion, resulting in increases in crypt number and bowel diameter if given during the early period of ISC expansion after ICR, and that this would correlate with enhanced local IGF-I expression. Since pISC expansion following resection occurs during a very limited window of time in our ICR model, we reasoned that therapy designed to augment this response may be effective only during a limited period, and GLP-2 would be ineffective or less effective when given at later times. Our findings suggest that GLP-2 indeed augments expansion of pISC only when given early after resection, despite increasing crypt proliferation, villus height, and crypt depth when given early or late after ICR.
MATERIALS AND METHODS
Our prior studies revealed that expansion of pISC occurs 3–5 days after ICR, leading to peaks in crypt fission at 7 days and increased intestinal circumference at 6 wks, a time when pISC expansion and crypt fission have returned to baseline (6). We have previously shown that sham-operated animals do not expand intestinal stem cells (6). Therefore, in our present study, to allow for comparison to most published GLP-2 studies, we used unoperated control mice given GLP-2 (100 μg/kg twice daily ip, human 126–158; California Peptide Research, Napa, CA) or vehicle (0.1% albumin in 0.45% NaCl) for either 4 or 7 days (Group A). These mice were compared with animals given GLP-2 or vehicle immediately after ICR and then killed at 4 days (Group B) or 7 days (Group C). Group D mice underwent ICR but did not receive GLP-2 or vehicle until the fifth week postoperatively and were studied after either 4 or 7 days of treatment (Fig. 1). There were at least five animals included in every time point, treatment group, and assay.
Animals and Operative Procedure
The University of North Carolina Institutional Animal Care and Use Committee approved the protocol for this study. Adult male C57BL/6J mice (10–12 wks, 25–30 g) were obtained from Jackson Laboratories (Bar Harbor, ME). Two days before the start of the experiment, all animals were switched to a liquid diet (Micro-Stabilized Rodent Liquid Diet; TestDiet, Richmond, IN). Mice were housed five per cage, and all mice within an individual cage were assigned to the same treatment group. Resection of the entire ileum and cecum was performed as previously described (6).
All groups received bromodeoxyuridine (BrdU, 120 mg/kg) 90 min before euthanasia except animals allocated to fluorescent-activated cell sorting (FACS) analysis of a side population (SP) of cells enriched for pISC on the basis of efflux of Hoechst 33342 (BrdU may quench Hoechst 33342 fluorescence) (7). Blood was obtained via cardiac puncture, serum separated, and stored at −80°C. The anastomosis and 1 cm proximal was discarded in all ICR animals. The 13 cm proximal to the ileo-cecal junction was discarded in unoperated controls. In all animals, the adjacent proximal segments of jejunum were dissected for histology and RNA. The first 1 cm was fixed in 10% unbuffered zinc formalin (Fisher Scientific, Kalamazoo, MI) for sectioning, and the second 1-cm segment of jejunum was snap frozen in liquid nitrogen for RNA or protein extraction and stored at −80°C. In mice designated for FACS sorting of SP cells, a single 5-cm segment was collected starting 1 cm proximal to the anastomosis in operated mice or 13 cm from the ileo-cecal valve in unoperated mice.
Histology and Analysis of Intestinal Morphometrics
Hematoxylin- and eosin-stained histological sections were analyzed for intestinal morphometry in a blinded manner with the use of digital images acquired with an AxioImager microscope. Crypt depth and villus height were measured in at least 10 well-oriented, full-length crypt-villus units and averaged for each sample. Proliferative index was assessed by calculating the ratio of BrdU-positive cells to total cells within 10 intact crypts. Submucosal circumference was measured with the use of AxioVision 4.6 as previously described (6). Percent crypt fission was scored in at least 100 crypts per mouse. A crypt undergoing fission was defined as a bifurcating crypt with a fissure creating two flask-shaped bases with a shared single crypt-villus junction. The number of crypts per 500 μm was counted in 10 well-oriented fields per animal, and the total number of crypts per submucosal circumference was calculated. Immunohistochemistry to identify phosphorylated β-catenin at serine 552 (β-cat p-ser552) was performed utilizing the antibody and methods provided by Dr. Linheng Li (15). Intestinal crypts were scored for the number of epithelial cells positive for β-cat p-ser552 per crypt.
RNA Isolation and Quantitative RT-PCR
Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and DNAase treated using the Turbo DNA-free kit (Ambion, Austin, TX) following manufacturer instructions. Quantitative real-time PCR was performed in triplicate using Applied Biosystems 7500 PCR system and TaqMan one-step RT-PCR Master Mix Reagents Kit (Applied Biosystems, Foster City, CA). Primer and probe sets for IGF-I (Mm00439559_m1) and β-actin (Mm00607939_sl) were purchased from Applied Biosystems. Data were analyzed using the ΔΔCt method with normalization to β-actin mRNA as the constitutive mRNA (31). Mean β-actin mRNA did not differ significantly between any groups evaluated. Pooled RNA from jejunum of five untreated adult C57BL/6J mice was used as the reference standard.
GLP-2 was quantified using 75 μl of serum and run in duplicate without dilution (Alpco Diagnostics, Salem, NH).
Single mucosal cell suspensions were prepared from 5 cm of jejunum as described previously (6). The percentage of CD45-negative SP cells was measured using the Hoechst 33342 staining method reported by Dekaney et al. (7).
All data are presented as means ± SE. Means ± SE for particular combinations of treatment group or time points were compared by two-way ANOVA with interactions. Contrasts of parameter estimates between categories of interest were computed, and P values were generated. This analysis allowed for post hoc testing for main effects of time (early vs. late), treatment (GLP-2 or vehicle), and operative procedure (unoperated vs. ICR), as well as interaction between these variables. P values < 0.05 were considered statistically significant.
Survival and Weight Gain
All mice were active and healthy throughout the study. There were no differences in survival (>90%) between groups. Similar weight gain was observed in unoperated control or ICR groups of mice receiving GLP-2 or vehicle.
Proliferation and Intestinal Morphometrics
Increases in the proliferative index occurred in all operative groups receiving GLP-2 compared with vehicle-treated controls (Fig. 2A, P < 0.001). Similarly, all groups demonstrated increases in villus height with GLP-2 treatment (Fig. 2B, P < 0.01). At 7 days and 6 wks following ICR, crypt depth was increased in response to GLP-2 treatment (Fig. 2C, P < 0.05).
Endogenous GLP-2 After ICR ± Exogenous GLP-2
Despite the loss of the ileum and proximal colon, we observed significant increases in endogenous serum GLP-2 in mice 4 days after ICR (4.2 ± 0.3 ng/ml) compared with unoperated controls (1.4 ± 0.3 ng/ml, P < 0.05), as recently reported following a proximal resection in rats (20, 24). Administration of GLP-2 for 4 days after ICR further augmented serum GLP-2 levels compared with ICR + vehicle (ICR + GLP-2: 7.4 ± 0.80 ng/ml, P < 0.05).
Measures Of Expansion of pISC Following ICR and GLP-2 Treatment
To investigate the influence of GLP-2 on intestinal stem cells, we used several parameters to measure pISC expansion.
Previous work from our laboratory has shown the CD45-negative SP to be a heterogenous epithelial fraction enriched with stem cells (7, 14) and a reasonable surrogate for pISC expansion (6). ICR resulted in an approximately sevenfold increase in the percent SP compared with nonoperated controls by 4 days following ICR (Fig. 3A, P < 0.001). Administration of GLP-2 for 4 days after ICR significantly augmented this response (P < 0.001) resulting in an ∼13-fold increase in the percent SP compared with unoperated control mice. GLP-2 did not affect the percent SP in unoperated mice (Group A) or when given at 5 wks after ICR (Group D, Fig. 3A).
Phosphorylated nuclear β-catenin at serine552.
β-catenin (p-ser552) is a recently described pISC marker reported by He et al. (15). In their model, a specific nuclear-localized form of β-catenin (p-ser552) colabeled with Musashi-1 in pISC and fissioning crypts (15). In our study, the number of nuclear β-catenin (p-ser552)-positive cells was significantly increased at 4 and 7 days after ICR compared with unoperated mice (Fig. 3B, P < 0.001) but was back to baseline by 6 wks after ICR. Administration of GLP-2 for 4 days after ICR significantly augmented this increase (P < 0.001), but this effect was transient and not sustained in mice given GLP-2 for 7 days after ICR. GLP-2 had no effect on the number of β-catenin (p-ser552)-positive cells per crypt in nonoperated mice (Group A) or those receiving GLP-2 at a late time point after ICR (Group D). β-catenin (p-ser552) cells were often found at the common wall of fissioning crypts (Fig. 3, D and E).
Crypt fission, number of crypts per circumference, and submucosal circumference.
Crypt fission is considered a primary mechanism by which pISC expansion increases crypt number and mucosal mass (21, 35). As previously described (6), significant increases in the percent crypt fission first occurred around 7 days following ICR in vehicle-treated animals (P < 0.01) and were back to baseline 6 wks after resection (Fig. 4A). Administration of GLP-2 significantly increased the percent of fissioning crypts only when given for 7 days following ICR (Group C, P < 0.001) and not when given to unoperated controls or 5 wks after ICR. Because increases in crypt fission should result in a greater total number of intestinal crypts, we determined the number of crypts per circumference (Fig. 4B). Consistent with our previous study (6), in vehicle-treated mice after ICR, significant increases in total crypt number were not observed until 6 wks following resection (P < 0.001). However, treatment with GLP-2 for 7 days immediately following ICR markedly increased the total number of crypts per circumference compared with vehicle-treated mice (Group C, P < 0.001), and this response was nearly equivalent to the increased crypt numbers observed in vehicle-treated mice at 6 wks after ICR. GLP-2 did not affect the total number of crypts in unoperated controls or when given 5 wks after ICR. Following resection, treatment with GLP-2 for 7 days dramatically increased the submucosal circumference compared with vehicle-treated controls (Fig. 4C, P < 0.001). In fact, as can be seen in Fig. 5, GLP-2 given for 7 days following ICR resulted in an increase in circumference equivalent to that normally observed at 6 wks after ICR (6). GLP-2 treatment did not affect intestinal circumference in unoperated controls or when given at 5 wks after ICR (Fig. 4C).
Intestinal IGF-I After ICR ± GLP-2
Given recent reports demonstrating that intestinal effects of GLP-2 are mediated by local IGF-I expression (10), we determined jejunal IGF-I mRNA following ICR. As shown in Fig. 6, a significant increase in jejunal IGF-I mRNA was observed 4 days after ICR. IGF-I mRNA levels remained elevated at 7 days but returned to baseline by 6 wks. Early administration of GLP-2 after resection significantly augmented ICR-induced intestinal IGF-I mRNA expression only at 4 days (Fig. 6, P < 0.01). Despite continued administration of GLP-2 for 7 days, IGF-I expression was not enhanced at this time compared with ICR + vehicle.
Increased proliferation, crypt depth, and villus height after bowel resection have been widely reported as early adaptive growth responses in a number of animal models, including our recently developed ICR model (6). GLP-2, a trophic growth factor presently under investigation in clinical trials in patients with intestinal failure, is known to enhance these adaptive responses in a number of animal models of resection (20, 24, 32). Our prior studies in the mouse ICR model provided evidence that early expansion of pISC and subsequent crypt fission contributes to sustained long-term adaptive growth manifest by dramatic increases in crypt number and intestinal caliber (6). This model provides a reproducible time course of intestinal stem cell expansion.
In this study, we tested whether the efficacy of GLP-2, a therapy typically administered months to years after surgery to patients with intestinal failure, differed when given immediately after ICR or later when the adaptive growth response has already occurred. Using two independent measures of pISC expansion, percent SP cells and β-catenin (p-ser552) staining, we demonstrated that GLP-2 enhanced ICR-induced SP cell expansion only when given immediately after ICR and not at later times. Furthermore, GLP-2 enhanced crypt fission only when given early after ICR. This was associated with a dramatically accelerated adaptive growth response with similar increases in mucosal circumference, crypt number, and intestinal caliber achieved within 7 days of GLP-2 given early after ICR as occurs within 6 wk after ICR in the absence of GLP-2 therapy. Interestingly, GLP-2 increased villus height, crypt depth, and proliferative index when given at either early or late time points following ICR. This indicates that there may be two different mechanisms of GLP-2 action in this study (Fig. 7). The effect of GLP-2 on pISC expansion, crypt fission, and total crypt number is limited to a period immediately following ICR and may potentially accelerate the adaptive process when given during this window. However, mitogenic effects of GLP-2 occur independently of resection or timing of treatment. This results in an increase in mucosal surface area as measured by increases in villus height, crypt depth, and proliferation. However, as highlighted by Jeppesen et al. (16), these changes will likely not persist when therapy is discontinued. Therefore, although GLP-2 treatment increases mucosal surface area regardless of when it is given, the early effects on stem cells and crypt fission may promote increased efficacy.
Intestinal stem cells are thought to be long-lived cells that continually provide epithelial progeny. Therapies that promote expansion of these cells may therefore result in sustained benefit after intestinal resection (4). Administration of Teduglutide (a GLP-2 analog) before radiation has previously been shown to increase the number of cells expressing the pISC marker Musashi-1 and the percentage of surviving crypts, supporting the ability of GLP-2 to promote pISC survival (2). Our previous work (6) using three independent assays of pISCs (SP sorting, BrdU label retention, and Musashi-1 staining) showed that all increased proportionately after ICR and led to the conclusion that the percent CD45-negative SP is a useful index of pISC number. In the present work, we chose to confirm the SP data with the β-catenin (p-ser552) staining because the latter (as opposed to other phosphorylated forms of β-catenin) seems to be convincingly confined to the stem cell zone (15). Both of these independent measures of ISC were augmented by GLP-2 only when administered immediately after ICR and not when GLP-2 was given to unoperated mice or at late time points after ICR. This, together with accelerated increases in crypt number and intestinal circumference, strongly supports a concept that early rather than late GLP-2 has the most dramatic and potentially sustainable effects on adaptive growth.
Presently, GLP-2 trials are underway in human patients with chronic intestinal failure. Given the extended period of time that has passed from their intestinal loss, our data suggest that GLP-2 would have no effect on the pISC in these patients. Although transient increases in crypt proliferation and villus height occur with GLP-2 treatment at late times after resection-induced intestinal loss, the inability of GLP-2 to expand ISC and crypt numbers likely compromises maximal and sustained intestinal adaptation and growth. Further clinical evaluation of early GLP-2 administration and its effects on ISC expansion and adaptive growth as a strategy to prevent intestinal failure after resection are warranted.
The observation that maintained weight gain and ultimate increases in mucosal circumference occurred in all mice following ICR regardless of GLP-2 treatment suggests that endogenous mechanisms alone are sufficient to promote a full adaptive response in our ICR model and may limit our ability to detect an enhanced magnitude of adaptive response with GLP-2. Furthermore, our mice were not pair fed because it is our experience that survival is unacceptably poor when housed individually following ICR. Thus the lack of difference in weight gain could have masked improved nutrient assimilation in GLP-2-treated animals if the latter had reduced food intake. However, we believe this is unlikely because a follow-up study showed no differences in liquid diet consumption with GLP-2 treatment 7 days following ICR (vehicle-treated: 11.5 ml/mouse per day vs. GLP-2-treated: 11.0 ml/mouse per day). The same study showed no significant effect of GLP-2 treatment on intestinal length (vehicle-treated mice: 17.9 ± 2.0 cm; GLP-2-treated mice: 16.8 ± 1.2 cm).
Our data in animals given GLP-2 for 7 days immediately after ICR clearly demonstrate that GLP-2 accelerates the long-term adaptive response since the increased mucosal circumference after just 7 days of early GLP-2 treatment was similar to the maximum increase of circumference attained in vehicle-treated animals 6 wks after ICR. Although increased intestinal circumference may be associated with stasis and bacterial overgrowth, this was not observed in our model during the present or previous study (6). Moreover, clinical bowel dilation is associated with human intestinal failure and is a prerequisite to any surgical lengthening procedure. Therefore, the ability of GLP-2 to expand pISC and accelerate adaptive growth is very clinically relevant because it could limit the duration of TPN required following intestinal loss. Additional studies to define trophic strategies that can expand pISC at late time points after intestinal resection will be of great interest.
Our data demonstrate that levels of jejunal IGF-I mRNA expression peak sevenfold at 4 days following ICR and are further augmented to 13-fold by early administration of exogenous GLP-2. These increases correlate temporally with increases in the percent SP and crypt fission and subsequent accelerated increases in bowel caliber, suggesting that GLP-2-induced ISC expansion may potentially be mediated via IGF-I. In the intestine, available evidence suggests that IGF-I is synthesized primarily by mesenchymal cells, myofibroblasts, fibroblasts, or smooth muscle and is thought to act in a paracrine fashion to regulate trophic responses of epithelial cells through the epithelial IGF-I receptor (IGF-IR) (34). Local increases in intestinal IGF-I expression and receptor are protective in models of IBD, irradiation, and adaptation following ICR (23, 39). Our present studies were limited to IGF-I mRNA because of previous work supporting the observation that IGF-I mRNA is a valid measure of IGF synthesis and that assays at the protein level can be complicated by rapid IGF-I secretion or interactions with IGF binding proteins (IGFBPs) (22). Recent evidence suggests that surgical resection is also associated with changes in IGFBPs, particularly IGFBP3, which is decreased, IGFBP4, which is increased, and IGFBP5, which is IGF regulated (12, 29, 39). On the basis of these data, it will be of interest to assess IGFBPs in the ICR model in future studies. Our present findings that GLP-2 induces IGF-I mRNA early after ICR are consistent with recent reports that GLP-2 induces IGF-I synthesis in intestinal myofibroblasts and that trophic effects of GLP-2 are dependent on intact IGF-I genes (10). In our study, the GLP-2-induced increases in IGF-I expression occurred only during a brief window of time at 4 days after ICR, correlating with pISC expansion, and were not sustained or further enhanced by 7 days of GLP-2. This narrow window of time for GLP-2-induced IGF-I expression and pISC expansion suggests that insight into mechanisms that could further drive resection or GLP-2-induced increases in local IGF-I may point to novel therapies to promote reexpansion of ISC in patients with chronic intestinal failure.
In summary, we provide novel evidence that expansion of pISC is augmented only by early administration of GLP-2 after ICR. In contrast, proliferative and morphological effects are independent of resection or timing of administration. New therapies designed to augment early pISC expansion following resection may be clinically useful for patients at high risk of developing intestinal failure. Since humoral factors may have much less efficacy in patients with established short-bowel syndrome (36), further studies investigating novel therapies to expand pISC in this difficult patient population are needed (19).
This work was funded by the following National Institutes of Health grants: NIH K08 DK067395-02, NIH R03 DK078796-01 (to M. Helmrath), NIH RO1 DK069585 (to S. Henning), and NIH T32 GM08450-14 (A. Garrison).
We thank Dr. Linheng Li for generously providing the β-catenin (p-ser552) antibody. We also thank Kirk McNaughton of the UNC Cell and Molecular Physiology Histology facility for skill and expertise with the immunohistochemistry and Joseph Galanko, Ph.D for help with the statistical analysis. We sincerely appreciate the help of Dr. Doug Burrin for early advice regarding GLP-2 and Dr. Rob Shulman for critical review of the manuscript.
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