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Journal of Parenteral and Enteral Nutrition
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Original Communications

Comparative Effects of Glucagon-Like Peptide-2 (GLP-2), Growth Hormone (GH), and Keratinocyte Growth Factor (KGF) on Markers of Gut Adaptation After Massive Small Bowel Resection in Rats

Naohiro Washizawa, MD*,{dagger}, Li H. Gu, MD{dagger}, Liang Gu, BS{dagger}, Kyle P. Openo, MPH{dagger}, Dean P. Jones, PhD{dagger},{ddagger} and Thomas R. Ziegler, MD{dagger},{ddagger}

From the * Department of Surgery, Toho University School of Medicine, Tokyo, Japan; and the{dagger} Department of Medicine and{ddagger} Center for Clinical and Molecular Nutrition, Emory University School of Medicine, Atlanta, Georgia

Correspondence: Thomas R. Ziegler, MD, Suite GG-23, General Clinical Research Center, Emory University Hospital, 1364 Clifton Rd., Atlanta, GA 30322. Electronic mail may be sent to tzieg01{at}emory.edu.

Background: Administration of specific growth factors exert gut-trophic effects in animal models of massive small bowel resection (SBR); however, little comparative data are available. Our aim was to compare effects of a human glucagon-like peptide-2 (GLP-2) analog, recombinant growth hormone (GH) and recombinant keratinocyte growth factor (KGF) on jejunal, ileal, and colonic growth and functional indices after 80% SBR in rats. Methods: Thirty-seven male rats underwent small bowel transection (sham operation) with s.c. saline administration (control; Tx-S; n = 7) or 80% midjejuno-ileal resection (Rx) and treatment with either s.c. saline (Rx-S, n = 7), GLP-2 at 0.2 mg/kg/d (Rx-GLP-2; n = 8), GH at 3.0 mg/kg/d (Rx-GH; n = 8), or KGF at 3.0 mg/kg/d (Rx-KGF; n = 7) for 7 days. All groups were pair-fed to intake of Rx-S rats. Gut mucosal cell growth indices (wet weight, DNA and protein content, villus height, crypt depth, and total mucosal height) were measured. Expression of the cytoprotective trefoil peptide TFF3 was determined by Western blot. Gut mucosal concentrations of the tripeptide glutathione (L-glutamyl-L-cysteinyl-glycine) and glutathione disulfide (GSSG) were measured by high-performance liquid chromatography and the glutathione/GSSG ratio calculated. Results: SBR increased adaptive growth indices in jejunal, ileal, and colonic mucosa. GLP-2 treatment increased jejunal villus height and jejunal total mucosal height compared with effects of resection alone or resection with GH or KGF treatment. Both GH and KGF modestly increased colonic crypt depth after SBR. SBR did not affect small bowel or colonic goblet cell number or TFF3 expression; however, goblet cell number and TFF3 expression in both small bowel and colon were markedly up-regulated by KGF treatment and unaffected by GLP-2 and GH. SBR oxidized the ileal and colonic mucosal glutathione/GSSG redox pools. GLP-2 treatment after SBR increased the glutathione/GSSG ratio in jejunum, whereas KGF had an intermediate effect. In addition, GLP-2 (but not GH or KGF) prevented the SBR-induced oxidation of the glutathione/GSSG pools in both ileum and colon. Conclusions: GLP-2 exerts superior trophic effects on jejunal growth and also improves mucosal glutathione redox status throughout the bowel after massive SBR in rats. Both GH and KGF increase colonic mucosal growth in this model. KGF alone potently increases gut mucosal goblet cell number and expression of the cytoprotective trefoil peptide TFF3. The differential effects of GLP-2, GH and KGF administration in this model of short bowel syndrome suggest that individual therapy with these growth factors may not be an adequate strategy to maximally improve adaptive gut mucosal growth and cytoprotection after massive small intestinal resection. Future research should address the use of these agents in combination in short bowel syndrome.

Crohn's disease, intestinal ischemia, trauma, and other conditions may require massive or repeated small bowel resection (SBR), leading to short bowel syndrome, a major cause of intestinal failure.1,2 In animal models of massive SBR (eg, mouse, rat, rabbit, pig), the residual intestinal mucosa undergoes a dynamic growth response, a process termed intestinal adaptation.1,3–9 Depending on the location and degree of bowel resection, these models demonstrate variously increased small bowel crypt cell proliferation and a resultant increase in crypt depth and villus height over time, and, in some studies, modest colonic mucosal growth responses.10–12 Only limited data are available on intestinal adaptation from humans with short bowel syndrome; these studies suggest that the robust adaptive growth response observed in animal models may not occur.13,14 Current medical management for short bowel syndrome includes dietary modifications, oral rehydration solutions, antidiarrheal medications, and, in severe cases, parenteral nutrition (PN). Unfortunately, conventional PN does not have gut-trophic effects and does not reverse malabsorption in short bowel syndrome patients.1,2 Therefore, administration of recombinant growth factors is being actively studied as a method to improve gut mucosal growth or nutrient transport function in human short bowel syndrome.15–17

Several growth factors individually increase small bowel growth or nutrient transport function in animal models of short bowel syndrome, including glucagon-like peptide-2 (GLP-2), growth hormone (GH), keratinocyte growth factor (KGF), epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-1).7,8,18–38 In addition, emerging data demonstrate that both GH and GLP-2 variously improve intestinal macronutrient or electrolyte absorption in humans with short bowel syndrome.15–17,39,40 Further, GLP-2, GH, and KGF individually exert cytoprotective effects on small bowel and colonic mucosa in various models of mucosal inflammation.1,41–43 We previously showed that GH increased microvillus height (but not villus height or crypt depth) and up-regulated l-amino acid transport in small bowel mucosa of rabbits after massive SBR. This trophic effect on the microvillus compartment and stimulation of small bowel amino acid transport was additively increased by the combination of EGF and GH.28,29 Otherwise, little data are available comparing individual or additive effects of growth factors on gut mucosal indices in models of short bowel syndrome.31,36

Small intestinal and colonic goblet cells secrete mucins and TFF3, a member of the trefoil factor family formerly known as intestinal trefoil factor.44–47 Mucins and TFF3 are major constituents of the mucus layer that plays a key role in intestinal barrier function.46,47 Trefoil peptides play an important role in gastrointestinal cytoprotection and mucosal restitution after injury.46,47 Short bowel syndrome is associated with abnormal gut barrier function1,2; however, only limited data from 1 study are available on regulation of gut goblet cell number and TFF3 mRNA in a short bowel syndrome model.48 Of interest, KGF has particularly trophic effects on intestinal goblet cell expression in vivo.30,41,44,49

The tripeptide glutathione (L-glutamyl-L-cysteinyl-glycine) plays a key cytoprotective role in detoxification of cellular free radicals, toxins, and carcinogens in the intestine and other tissues. Cellular glutathione redox status appears to influence cellular proliferative and apoptotic responses1,50–52; thus, it is possible that local mucosal glutathione may mediate intestinal adaptive growth responses in short bowel syndrome. We showed that KGF prevents the decrease in small bowel and colonic glutathione and the glutathione/glutathione disulfide (GSSG) ratio in malnourished rats.53 However, whether gut mucosal glutathione is altered in adapting gut or regulated by growth factors after SBR is unknown. The purpose of this study was to compare effects of GLP-2, GH, and KGF on rat intestinal growth indices, goblet cell number, TFF3 expression, and glutathione redox after massive small intestinal resection.


   MATERIALS AND METHODS
Top
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 
Animals
Thirty-seven male Sprague-Dawley rats (170–210 g) were acclimated to the laboratory for 2 to 3 days in individual cages with ad libitum access to water and standard pelleted rat food (Laboratory Rodent Chow 5001; PMI Feeds Inc, St Louis, MO). Animals were housed in the Emory University Animal Care Facility. All procedures were approved by the Institutional Animal Care and Use Committee of Emory University.

Operative Procedures
Food was removed for 16 hours before laparotomy and intestinal surgery the following morning, as previously described.8 All operative procedures were performed under anesthesia with a mixture of i.p. ketamine (100 mg/mL) and xylazine (20 mg/mL), administered at doses of 0.1 to 0.15 mL/100 g body weight. The abdomen was opened by a midline incision and the ligament of Treitz and ileal-cecal junction identified and marked. In SBR rats, enterectomy was performed by removing approximately 80% of the small intestine, leaving approximately 10 cm of the terminal ileum and 10 cm of the proximal jejunum, which were anastomosed.8 Saline (0.9% sodium chloride; 10 mL) was given intraperitoneally for fluid resuscitation and the abdominal wall closed. Control rats (n = 7) underwent jejunal transection and anastomosis without bowel resection at a point 10 cm distal to the ligament of Treitz (sham operation; Tx-S). After recovery, rats were transferred back to individual cages and given water ad libitum overnight.

Treatment Regimens
The following morning and for the rest of the experiment, animals were pair-fed standard rat chow and given water ad libitum. Weight-matched rats were randomly divided into 5 study groups: control animals received twice-daily subcutaneous (s.c.) saline injections (9:00 AM and 5:00 PM). The 30 rats who had undergone 80% SBR received either twice-daily s.c. saline injections as SBR controls (Rx-S; N = 7) or s.c. injections of 1 of 3 growth factors. One group of rats (n = 8) received injections of a synthetic GLP-2 analog to human GLP-2 (0.1 mg/kg twice daily; Rx-GLP-2). The GLP-2 analog was synthesized with glycine substituted for alanine at position 2 to confer resistance to degradation by the enzyme dipeptidyl peptidase IV (American Peptide Company Inc, Sunnyvale, CA).54,55 Another group of animals (n = 8) received injections of recombinant human GH (Serostim; Serono Laboratories, Inc, Norwell, MA) at a dose of 1.5 mg/kg twice daily (Rx-GH). The fifth group of rats (n = 7) received s.c injections of recombinant human KGF (Amgen Inc, Thousand Oaks, CA) at a dose of 1.5 mg/kg twice daily (Rx-KGF). The average amount of daily food consumption in the Rx-S group was measured, and all other groups were fed this amount daily.

Body Weight and Tissue Isolation
The body weights of the rats were determined preoperatively after the acclimation period and daily during treatment. After the 7 days of saline or growth factor treatment, animals were anesthetized as above, and a laparotomy was performed by midline incision. This time point for studies on the gut adaptive response was chosen according to our previous studies in this model.7,8 Also, maximal adaptive growth of small bowel with rat mid-SBRs increases as the percentage of small bowel resection increases and occurs between 7 and 14 days after SBR in rats.3,4 The small intestine and colon were removed sequentially from the peritoneal cavity, and the lumen was flushed with ice-cold saline to clear intestinal contents. The small bowel and colon were individually suspended from a ring-stand with a constant distal weight and intestinal segments excised. Full-thickness 1- and 2-cm sections were obtained from proximal jejunum, distal ileum, and proximal colon (immediately distal to the cecum) for determination of TFF3 protein content and DNA and protein content, respectively. Mucosa was obtained from 4-cm segments of proximal jejunum, distal ileum, and proximal colon for glutathione redox studies. These segments were longitudinally cut and mucosa obtained by gentle scraping with a glass slide. All samples were weighed, immediately frozen in liquid nitrogen, and stored at –80°C. A defined 1-cm section from each intestinal segment was also fixed in 4% paraformaldehyde and paraffin-embedded for morphological analysis. Animals were killed by exsanguination after tissue removal.

Intestinal DNA and Protein Content
Full-thickness gut segments were homogenized in ice-cold buffer (50 mmol/L phosphate-buffered saline [PBS], 2 mol/L NaCl, 2 mmol/L EDTA), for 30 seconds using a Polytron (Brinkman Instruments Co, Westbury, NY) at a setting of 4, then immediately sonicated for 10 seconds. The DNA content per centimeter was determined by the fluorometric method56 and the protein content per centimeter by the Bradford method,57 with dye reagent from Bio-Rad Laboratories (Hercules, CA) and rabbit {gamma}-globulin as the protein standard.

Gut Mucosal Growth Indices
Paraffin-embedded sections of jejunum, ileum, and colon were stained with hematoxylin and eosin. Crypt depth and villus height were measured with a Leitz light microscope-video system (Panasonic 888 professional video system) by 2 investigators blinded to study group and the data averaged. A total of 15 well-oriented crypts and villi from each small bowel segment and 15 colonic crypts from each colon segment were measured per animal and averaged. Total mucosal height was calculated as the sum of crypt depth and villus height measurements in jejunal and ileal segments.

Gut Mucosal Goblet Cell Number
Goblet cells were identified by classical morphology in the small bowel and colonic sections in all animals.44 Total goblet cell number was determined from the crypt base to the luminal surface of 10 to 15 well-oriented crypt-villus units in each small bowel section and in 10 to 15 well-oriented colonic crypt units from the crypt base to the luminal surface. The average number of goblet cells per villus-crypt unit counted in small bowel segments and crypt units counted in colonic segments was calculated for each animal and group means were determined.

TFF3 Expression by Western Immunoblotting
A rabbit antirat polyclonal TFF3 antibody raised against a 21-residue synthetic peptide from the predicted C-terminal sequence of rat TFF3 was used in Western immunoblotting (kindly provided by Dr. Daniel Podolsky, Massachusetts General Hospital).46 Briefly, full-thickness small bowel and colonic sections were suspended in ice-cold lysis buffer (PBS, sodium deoxycholate, sodium dodecyl sulfate, NP-40, EDTA, chymotrypsin, thermolysin, pronase, papain, and pancreas extract). Samples were homogenized, centrifuged, and supernatants stored at –70°C. Sixty µg of solubilized protein/sample were resolved by SDS-PAGE in 16.5% Tris-Tricine gels (Bio-Rad Inc) and proteins transferred to Hybond ECL nitrocellulose membranes (Amersham, Arlington Heights, IL). Membranes were blocked for 60 minutes at 22°C with 5% dry milk, 0.1% BSA, 1X PBS and 0.05% Tween-20, and incubated with TFF3 antibody (1:500) in blocking solution. Bound antibody was detected with antirabbit horseradish peroxidase-conjugated secondary antibody (antirabbit IgG; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and chemiluminescence detection system reagents (Amersham). TFF3 protein was detected as an approximately 6.5-kDa band in rat jejunum, ileum, and colon, as we have previously described.44 Protein bands were quantified by densitometry (Computing Densitometer Model 300A; Molecular Dynamics).

Gut Mucosal GSSG Determination
After isolation, the jejunal, ileal, and colonic mucosal samples were immediately placed in a solution containing 5 g perchloric acid/L, 0.2 mol boric acid/L and 5 µmol {gamma}-glutamyl-glutamate/L for analysis by high-performance liquid chromatography (HPLC), as previously described.53 Precipitated tissue proteins were separated from the acid-soluble supernatant by microcentrifugation, and the protein pellet resuspended in 1 mol/L NaOH. Protein concentrations were measured using the Bradford method with rabbit {gamma}-globulin as the protein standard (Bio-Rad Laboratories). The acid-soluble supernatant was stored at –70°C for 2 to 4 weeks until thiol analysis, in which thiols were derivatized with dansyl chloride.53 For HPLC analysis, the dansyl-derivatized compounds, including glutathione and GSSG, were separated on a 3-aminopropyl column (5 mm; 4.6 mm x 25 cm; Custom LC, Houston, TX) using a Waters HPLC and autosampler system (Waters, Milford, MA) with fluorescence detection using bandpass filters (305–395 nm excitation, 510–650 nm emission; Gilson Medical Electronics, Middletown, WI). Quantitation of glutathione and GSSG was based on integration relative to the internal standard {gamma}-glutamyl-glutamate and expressed as nmol/mg protein. The glutathione/GSSG ratio was calculated as an index of mucosal glutathione pool redox state.53

Statistical Analysis
One-factor analysis of variance (ANOVA) was used to detect significant intergroup differences, in which case the 5 study groups were compared post hoc using the Fisher's protected least-significant difference test (Statview; Abacus Concepts, Berkeley, CA). p Values < .05 were considered statistically significant in all analyses. Data are expressed as mean ± SE.


   RESULTS
Top
MATERIALS AND METHODS
 RESULTS
DISCUSSION
 
Food Intake and Body Weight
Mean daily food intake in the 5 experimental groups was similar: TX-S, 10.8 ± 0.2; RX-S, 10.7 ± 0.2; RX-GLP-2, 11.1 ± 0.3; RX-GH, 10.6 ± 0.5; and RX-KGF, 9.7 ± 0.3 g/d (NS). The average preoperative body weight was similar in all study groups (not shown). The change in body weight from baseline until the end of study was similar in the non–KGF-treated groups (TX-S, +5 ± 6 g; RX-S +1 ± 5 g; RX-GLP-2, +4 ± 5 g and RX-GH, +10 ± 6 g; NS). The change in body weight in the RX-KGF group was –19 ± 6g(p < .05 vs the other 4 study groups).

Intestinal Wet Weight and DNA and Protein Contents
An adaptive increase in full-thickness jejunal and ileal growth indexes occurred with SBR, as expected (Table I). Colonic adaptation after SBR was evidenced by increased wet weight (+11%; p < .05), DNA content (+25%; p < .05) and protein content (+24%; NS) in the RX-S vs TX-S animals (Table I).


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  		TABLE I Effects of massive small bowel resection and GLP-2 analog, GH, or KGF on intestinal growth indexes

 

Differential and tissue-specific responses to the 3 growth factors were noted. In jejunum, GLP-2, GH, and KGF did not further increase wet weight compared with SBR alone (Table I). However, animals given GLP-2 demonstrated a significant 82% increase in jejunal DNA content compared with the TX-S group. GLP-2 also significantly increased DNA content in jejunum compared with the RX-S and RX-KGF groups. Both GH and KGF significantly increased jejunal protein content compared with control TX-S values.

GLP-2, GH, and KGF each increased ileal wet weight compared with resection alone (Table I). None of these agents further increased ileal DNA or protein content vs SBR alone, but the highest values occurred in the RX-GLP-2 group. In colon, GLP-2 and GH treatment did not alter wet weight, DNA content or protein content compared with bowel resection alone. KGF increased colonic wet weight vs TX-S, RX-S and RX-GLP-2 and decreased colonic DNA content compared with the RX-S, RX-GLP-2 and RX-GH groups (Table I).

Intestinal Morphology
Differential and tissue-specific mucosal growth responses occurred after 80% SBR and with GLP-2, GH, and KGF treatment in this model. SBR significantly increased jejunal and ileal villus height compared with control animals (by 17% and 18%, respectively; Fig. 1). In jejunum, treatment with GLP-2 (but not GH or KGF) further increased this adaptive growth response. None of the growth factors increased ileal villus height over effects of SBR alone (Fig. 1). There was a strong trend for both jejunal and ileal crypt depth to increase after SBR compared with TX-S rats (by 24% and 36%, respectively); however, this response was not statistically significant and was unaffected by treatment with any of the growth factors (Fig. 2). In contrast, colonic crypt depth was significantly increased vs transected control animals with GLP-2 (+21%), GH (+27%) and KGF (+29%) treatment after SBR (Fig. 2). Colonic crypt depth after SBR was also significantly increased by GH and KGF compared with the SBR control group (Fig. 2).


Figure 1
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  		FIG. 1. Adaptive increase in jejunal and ileal villus height after SBR ± growth factors and stimulation of this response in jejunal mucosa with GLP-2 treatment. Values are means ± SE. See text and legend of Figure 1 for details of operations, treatment regimens, morphologic assessment, and animal number/group. Symbols indicate p < .05 by 1-factor ANOVA with post hoc Fisher's test: {dagger}vs TX-S; *vs all other groups.

 

Figure 2
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  		FIG. 2. Crypt depth in jejunal, ileal, and colonic mucosa after SBR ± growth factors and stimulation of this response in colon with GLP-2, GH, and KGF treatment. Values are means ± SE. See text and legend of Figure 1 for details of operations, treatment regimens, morphologic assessment, and animal number/group. Symbols indicate p < .05 by 1-factor ANOVA with post hoc Fisher's test: {dagger}vs TX-S; vs RX-S.

 

In jejunum, total mucosal height was significantly increased with SBR alone, was not further increased by GH or KGF treatment, but was significantly increased by GLP-2 (Fig. 3). In ileum, total mucosal height was significantly increased by SBR but was unaffected by treatment with GLP-2, GH, or KGF.


Figure 3
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  		FIG. 3. Adaptive increase in jejunal and ileal total mucosal height after SBR and stimulation in jejunum by GLP-2 treatment. Values are means ± SE. See text and legend of Figure 1 for details of operations, treatment regimens, morphologic assessment, and animal number/group. Symbols indicate p < .05 by 1-factor ANOVA with post hoc Fisher's test: {dagger}vs TX-S; *vs all other groups.

 

Gut Mucosal Goblet Cell Number and TFF3 Protein Expression
The number of goblet cells in jejunal, ileal, and colonic mucosa was not altered by SBR alone or by GLP-2 or GH treatment after SBR. However, KGF administration markedly increased goblet cell number in all intestinal segments (Fig. 4). Expression of the cyto-protective goblet cell product TFF3 was not altered by SBR alone or by GLP-2 or GH treatment after SBR (Fig. 5). However, KGF treatment markedly increased TFF3 expression in jejunum (3.5-fold), ileum (50% increase), and especially colon (4.5-fold; Fig. 5 and Table II).


Figure 4
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  		FIG. 4. Small bowel and colonic goblet cell number. KGF treatment markedly increases the number of mucosal goblet cells in jejunum (A), ileum (B) and colon (C) after SBR. Values are means ± SE. See text and legend of Figure 1 for details of operations, treatment regimens, determination of crypt-villus goblet cell number, and animal number/group. The symbol indicates p < .05 by 1-factor ANOVA with post hoc Fisher's test: *vs all other groups.

 

Figure 5
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  		FIG. 5. KGF treatment markedly increases expression of TFF3 in jejunum after SBR. Values are means ± SE of densitometry units. See text and legend of Figure 1 for details of operations, treatment regimens, immunoblotting methods for TFF3 protein expression, and animal number/group. A representative autoradiogram showing the 6.5-kDa TFF3 band is shown above the quantitative histograms. The symbol indicates p < .05 by 1-factor ANOVA with post hoc Fisher's test: *vs all other groups.

 

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  		TABLE II Effects of massive small bowel resection and GLP-2 analog, GH, or KGF on TFF3 expression in ileum and colon

 

Gut Mucosal Glutathione and GSSG Concentrations
In jejunum, neither SBR nor administration of GLP-2, GH, or KGF after SBR altered mucosal concentrations of glutathione or GSSG, with the exception of an increase in GSSG in the RX-GH group (Table III). However, the glutathione/GSSG ratio, an index of the glutathione pool redox state,51 was significantly increased in the animals given GLP-2 after SBR compared with the TX-S, RX-S and RX-GH groups (Fig. 6A). In ileal mucosa by contrast, a significant 26% decrease in glutathione concentration occurred with SBR. The decrease in ileal glutathione after SBR was unaffected by GH or KGF but was modestly inhibited by GLP-2 (Table III). Ileal mucosal GSSG concentrations were unaffected by resection or growth factor treatment. The ileal glutathione/GSSG ratio was also significantly decreased by SBR alone (25% decrease) and was unaffected by GH or KGF treatment. However, the resection-induced decrease in the ileal glutathione/GSSG ratio was prevented GLP-2 treatment (Fig. 6B). In colon, neither SBR nor the 3 specific growth factors altered glutathione or GSSG concentrations (Table III). However, the glutathione/GSSG redox ratio was significantly decreased in resected rats compared with the transected controls (Fig. 6). This oxidative response to SBR was not altered by administration of GH or KGF but was prevented by administration of GLP-2 (Fig. 6C).


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  		TABLE III Effects of massive small bowel resection and GLP-2 analog, GH, or KGF on glutathione redox indexes

 

Figure 6
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  		FIG. 6. Effects of small bowel resection on gut mucosal glutathione/GSSG pool redox state: improvement in the glutathione/GSSG ratio with GLP-2 administration in small bowel and colon. Values are means ± SE in jejunum (A), ileum (B), and colon (C). See text and legend of Table I for abbreviations, details of operations, treatment regimens, HPLC methods for determination of gut mucosal glutathione and glutathione disulfide (GSSG) concentrations, and animal number/group. Symbols indicate p < .05 by 1-factor ANOVA with post hoc Fisher's test: {dagger}vs TX-S; VS RX-S; {ddagger}VS RX-GH.

 


   DISCUSSION
Top
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 
In this study, we report the comparative effects of GLP-2, GH, and KGF on intestinal adaptation in a rat model of massive SBR. Receptors for each of these growth factors are present throughout the intestine,42,58,59 and each agent has been shown to exert trophic and cytoprotective effects in small bowel and colonic mucosa in a variety of animal models, including short bowel syndrome, PN use and gut mucosal inflammation, and by chemical agents.19,20,22,24,26–33,36,41,42,60,61 damage induced We chose to compare these agents as each is currently being studied in the clinical setting as methods to enhance intestinal growth, repair, or absorptive function.15–17,39,40 GLP-2 has activity only on intestinal epithelial cells, and we used a GLP-2 analog resistant to degradation by DPP-IV that has more potent gut-trophic effects than native GLP-2 in intact animals.42,54 GH is a somatic growth factor with activity on a wide range of cell and tissue types. Several studies now suggest that recombinant GH increases gut nutrient absorption and lean body mass in human short bowel syndrome,15,40 and short-term use of this agent has been recently approved by the United States Food and Drug Administration for use in short bowel syndrome patients. KGF is a member of the fibroblast growth factor family which specifically stimulates growth and differentiation of epithelial cells in the gastrointestinal tract and other tissues, such as lung and skin.41,44,49 The cytoprotective effects of KGF on the gastrointestinal tract are currently being investigated in patients receiving chemotherapy/irradiation and in inflammatory bowel disease.

We found differential effects of GLP-2, GH, and KGF on postoperative body weight, an indicator of overall somatic growth. Animals in all SBR groups lost approximately 18 g body weight during the first 24 hours, reflecting loss of intestinal tissue, the catabolic effects of operation, and decreased food intake. Compared with resected-saline treated rats, animals receiving both GLP-2 and GH began to regain body weight increase at an earlier time point (3 vs 5 days), although group mean daily body weights were not significantly different. In contrast, body weight of KGF-treated rats remained at the postoperative level and did not increase over time. This differential change in body weight cannot be explained by differences in food intake, which was similar in all groups. The pattern for an earlier increase in body weight with GLP-2 and GH may reflect a whole-body anabolic effect, as seen in human short bowel syndrome with both GLP-2 and GH treatment.15,16,39,40 We did not determine gut nutrient absorption in the current study, but it is possible that GLP-2 and GH enhanced nutrient absorption relative to KGF. In human and animal models of short bowel syndrome, transport/absorption of various nutrients increased with administration of either GLP-2 or GH.28,29,33,36,37,42 KGF stimulates undifferentiated colonic cells to differentiate into goblet cells.44,47 Comparative studies on the effects of GLP-2, GH, and KGF on intestinal stem cell differentiation, nutrient transport/absorption, and body composition would be of interest.

Our 80% midjejuno-ileal resection model induced the expected adaptive increase in small bowel mucosal growth indices. We also observed a significant increase in colonic wet weight and DNA content and the tendency for colonic protein content to increase in the RX-S vs TX-S animals, confirming a colonic growth response to massive SBR. Additional studies to evaluate colonic growth and functional responses to massive resection of small bowel, including rates of colonocyte proliferation and apoptosis, microvillus height, expression of nutrient transporters and growth factors, and fluid/nutrient transport function would be of interest.

Full-thickness DNA and protein content may reflect cellularity changes in 1 or more layers of the intestine (eg, the mucosal, submucosal or muscularis propria [circular and longitudinal smooth muscle] layers). Full-thickness wet weight reflects both cellular mass and cellular and extracellular water content. All 3 growth factors increased ileal full-thickness wet weight after SBR compared with resected-saline treated rats but had no effect on jejunal wet weight. In colon, KGF increased wet weight, whereas GLP-2 and GH had no effect. We did not directly measure cell proliferation rates; however, DNA content is a commonly used indirect index of cell proliferation and protein content is an indirect index of total cell mass.2,7,8,54 Taken together, our DNA and protein content data suggest that that GLP-2, GH, and KGF affect full-thickness rat intestine in a differential and site-specific manner after SBR. It is possible that these changes reflect differential effects of these agents on specific cell types (eg, KGF alone increased goblet cell number) or on the mucosal vs the muscularis layers, as has been observed when IGF-1 and GH treatment was compared in PN-treated rats.62 GLP-2, GH, and KGF, given for 7 days, exerted differential and modest effects on specific gut mucosal growth parameters (villus height, crypt depth, total mucosal height) in a site-specific manner. The specific trophic effect on jejunal mucosa with the GLP-2 analog is consistent with the work of Scott et al.33 The GLP-2 analog also significantly improved D-xylose absorption in the SBR rats,33 consistent with studies showing improved gut nutrient transport with GLP-2 treatment in intact animals.42,61 In the only other study of GLP-2 in an animal short bowel syndrome model, Hirotani et al27 found that daily parenteral infusion of native GLP-2 for 7 days significantly increased jejunal mucosal wet weight, DNA, and protein content vs controls. This more restricted augmentation of gut growth with GLP-2 in bowel-resected animals differs from the generalized gut mucosal growth induced in small bowel and colon of intact rodents given either native GLP-2 or the more potent GLP-2 analog.42,54,60 In intact animals, GLP-2 increases crypt cell proliferation, crypt depth, and villus height throughout small bowel, enhances crypt cell proliferation and crypt depth in colon; GLP-2 also has antiapoptotic effects and improves gut barrier function.42,54,60,63 The reasons for the apparent differential gut mucosal growth response to GLP-2 between SBR and intact animal models is speculative but may relate to the lack of an as-yet-unidentified secondary small bowel–derived mediator of GLP-2 action that is missing in SBR animals. In addition, gut growth responses may already be maximally stimulated as a consequence of the SBR alone in some bowel segments. Levels of circulating (or local mucosal) GLP-2 or other putative mediators, such as IGF-1, in response to resection of different lengths of small bowel or specific segments would shed light on these questions.

Several studies of GH administration using a variety of dosing schedules in different animal models of short bowel syndrome have shown inconsistent effects on small intestinal growth; some studies demonstrate various trophic effects,19,20,22,24,28,35,38 whereas others showed no stimulation of small bowel growth with GH.5,36,64 In addition, a dissociation between mucosal growth responses and nutrient transport functions has been observed in some studies.29,36 Benhamou and colleagues19 showed that rats given human GH at approximately 0.8 mg/kg every other day for 28 days after 80% SBR demonstrate a 3-fold increase in residual jejunal-ileal length and a 20% increase in jejunal villus height and muscularis thickness vs resected controls. In a piglet model of 80% SBR, these investigators showed that GH increased residual small bowel length, but only in animals after SBR and not in unresected control animals.20 Gomez de Segura et al24 showed that human GH (1.0 mg/kg/d) given for 7 days significantly increased the rate of ileal mucosal cell proliferation and ileal villus height after either 90% SBR or 75% proximal colonic resection in rats. In contrast, other studies in rat models of massive SBR did not demonstrate trophic effects of GH in small bowel.5,36,64 In a rabbit model of 70% SBR, we showed that intramuscular administration of GH significantly increased small bowel brush border membrane transport of glutamine and leucine without altering villus height or crypt depth; however, GH significantly increased microvillus height in residual jejunum (10%) and ileum (20%) in this model.28,29 Sigalet and Martin36 showed that IV administration of human GH significantly increased glucose transport across ileal mucosa, without affecting ileal villus height or total mucosal height. Eizaguirre et al22 demonstrated that s.c. human GH (1.0 mg/kg/d) induced a 25% increase in the rate of colonic mucosal cell proliferation and in colonic crypt depth in rats subjected to 80% SBR + cecal resection and maintained on PN and GH for 10 days vs resected controls. Additional dose-finding and time-course studies on effects of GH on colonic growth indices, small bowel microvillus height, and gut mucosal nutrient transport functions in models of short bowel syndrome would be of interest. We do not know whether GH, GLP-2 analog, or KGF given for longer than 7 days would result in similar differential adaptive growth responses. Scott et al33 showed that the GLP-2 analog in a similar rat model of short bowel syndrome exerted similar trophic effects on jejunum and no effects on ileal growth at 6 and 21 days of treatment, respectively.

In the current study, KGF did not enhance adaptive growth in small bowel but significantly increased colonic crypt depth when determined 7 days after SBR. This is consistent with our earlier studies showing marked trophic effects of KGF on colonic mucosa in fasted/refed rats.49 Only 2 previous studies of KGF in short bowel syndrome have been published, and neither evaluated colonic responses. Using an identical dose of KGF as in the current study, we studied rats in the early adaptive phase 3 days after 75% SBR.30 KGF significantly increased wet weight, crypt depth, and total mucosal height in duodenum, jejunum, and ileum in this model (15% to 30% increase).30 In the recent study by Yang et al,37 jejunal endpoints were evaluated in mice after 55% SBR, with or without treatment with IV KGF (5.0 mg/kg/d) for 7 days. KGF significantly increased jejunal cell proliferation (30% increase), villus height (16% increase), and crypt depth (19% increase) compared with SBR control mice.37

Gut mucosal goblet cells play an important cytoprotective and gut barrier defense function, but effects of SBR and growth factor administration on expression of these cells or their secretory products have been very little studied.44,48 In our study, neither SBR alone nor the administration of GLP-2 or GH after SBR affected goblet cell number in small bowel or colon. In contrast, KGF administration markedly increased goblet cell number throughout the bowel (residual jejunum > residual ileum > colon).

In a study by Rubin et al,48 goblet cell number was significantly increased in residual ileum 7 days after 70% SBR in rats vs sham-operated controls; at 48 hours after resection, significant down-regulation of a novel goblet cell complementary DNA homologous to human and rat mucins occurred, but no change in ileal TFF3 mRNA was observed. Trefoil peptides (TFF1, TFF2 and TFF3) are a family of small molecules abundantly expressed by mucus-secreting cells of mammals, mainly in the gastrointestinal tract and play an important role in both gut barrier function and mucosal restitution after injury.46,47 Our data show that SBR alone or GH and GLP-2 treatment for 7 days after SBR does not alter TFF3 expression at the protein level. In the only other study investigating GH effects on trefoil peptide expression, transgenic mice overexpressing GH demonstrated an increase in TFF3 mRNA expression in colon during experimental colitis.43 In our earlier studies in intact rat and mouse models of fasting/refeeding and inflammatory bowel disease, KGF markedly increased small bowel and colonic goblet cell number and TFF3 mRNA and protein expression.41,44 KGF also up-regulated TFF2 in duodenum and jejunum in fasted or fasted/refed rats.44 Concomitant with the KGF-specific increase in goblet cell number throughout the bowel, we here confirm that KGF quantitatively increases intestinal TFF3 protein expression after SBR (colon > jejunum and ileum) (Fig. 5). Additional studies of KGF effects on growth of specific gut mucosal cell types, trefoil peptide expression and nutrient absorption and barrier functions in short bowel syndrome are indicated to further define effects of this agent in this clinical model.

As a major body antioxidant, glutathione is in equilibrium with GSSG and plays a key cytoprotective role in the detoxification of cellular free radicals and toxins.51 Glutathione is synthesized by gut mucosal cells, can be derived from diet, and may enter the lumen in bile or by direct secretion by mucosal cells.51 Glutathione appears to be required for normal intestinal function, in part, by protecting epithelial cell membranes from damage by electrophiles and fatty acid hydroperoxides and by maintaining luminal redox and mucus fluidity.50,51 In vitro studies in a variety of cell lines also suggest that intracellular glutathione may positively regulate cell proliferation.51,52 In intact mice, inhibition of glutathione synthesis by buthionine sulfoximine markedly decreases jejunal glutathione levels in association with villus atrophy and epithelial cell damage.65 These effects are prevented by oral glutathione, which increases intracellular free glutathione levels.65 In addition, gut mucosal damage, organ dysfunction, bacterial translocation, and increased mortality occur when glutathione is chemically depleted.1

We show that 80% SBR alone induces differential site-specific effects on the glutathione redox pool in the residual bowel (Fig. 6 and Table III). Jejunal mucosal glutathione, GSSG and the glutathione/GSSG ratio were maintained after SBR. In contrast, in ileal mucosa, SBR shifted the glutathione redox potential to a more oxidized state, as determined by the decrease in glutathione concentration and the glutathione/GSSG ratio. In colon, the glutathione/GSSG ratio was also significantly decreased with SBR, but this appeared to be caused by an increase in mucosal GSSG (Table III). We previously showed that KGF increases duodenal, ileal and colonic mucosal glutathione concentrations and the glutathione/GSSG ratio in fasted/refed rats.53 In that model, mucosal glutathione was positively correlated with ileal and colonic crypt depth.53 In contrast, in the current study KGF (or GH) did not alter small bowel or colonic glutathione, GSSG, or the glutathione/GSSG ratio after SBR. Thus, the gut glutathione response to KGF appears to be different in intact fasted/refed vs rats studied 7 days after SBR, but the mechanisms for this differential response in the 2 models are unclear.

Our study shows that administration of GLP-2 shifts the glutathione/GSSG pool to a more reducing state in jejunal, ileal, and colonic mucosa after SBR. Gut mucosal growth responses after SBR alone or with GLP-2, GH, or KGF treatment in ileum and colon did not correlate with local mucosal glutathione concentrations. The GLP-2-induced trophic effects in jejunum were associated with the most robust increase in the local glutathione/GSSG pool compared with responses in ileum and colon (Fig. 6). Thus, although a critical role for glutathione redox as a general mediator of intestinal adaptive growth responses in this animal model appears unlikely, it is possible that local glutathione may play a role in trophic effects of GLP-2.

In conclusion, GLP-2, GH, and KGF induce markedly different effects on gut mucosal growth responses, goblet cell number, TFF3 expression, and glutathione redox when determined 7 days after 80% jejuno-ileal resection in rats. Additional studies to define underlying mechanisms of action for these clinically relevant growth factors will be important, as these remain largely unknown. The differential effects we observed with GLP-2, GH, and KGF administration in this model of experimental short bowel syndrome suggest that combination therapy with these agents may be a strategy to maximally improve gut mucosal cytoprotection and adaptive gut growth in intestinal failure induced by massive resection of the bowel.


  
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MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 
This research was supported in part by grants from the National Institutes of Health R01 ES11195 (DPJ) and R01 DK55850 (TRZ) and support from the Department of Surgery, Toho University School of Medicine, Tokyo, Japan (NW).

Received for publication January 8, 2004. Accepted for publication June 18, 2004.

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Journal of Parenteral and Enteral Nutrition, Vol. 28, No. 6, 399-409 (2004)
DOI: 10.1177/0148607104028006399
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L. Wang, Y. Tang, D. C. Rubin, and M. S. Levin
Chronically administered retinoic acid has trophic effects in the rat small intestine and promotes adaptation in a resection model of short bowel syndrome
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1559 - G1569.
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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. Tian, N. Washizawa, L. H. Gu, M. S. Levin, L. Wang, D. C. Rubin, S. Mwangi, S. Srinivasan, Y. Gao, D. P. Jones, et al.
Stimulation of colonic mucosal growth associated with oxidized redox status in rats
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1081 - R1091.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. A. Helmrath, J. J. Fong, C. M. Dekaney, and S. J. Henning
Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice
Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G215 - G222.
[Abstract] [Full Text] [PDF]

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 JPEN J Parenter Enteral NutrHome page
M. E. Evans, J. Tian, L. H. Gu, D. P. Jones, and T. R. Ziegler
Dietary Supplementation With Orotate and Uracil Increases Adaptive Growth of Jejunal Mucosa After Massive Small Bowel Resection in Rats
JPEN J Parenter Enteral Nutr, September 1, 2005; 29(5): 315 - 321.
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