Glucagon

ABSTRACT. Background: Parenteral nutrition and the absence of luminal feeding result in impaired intestinal growth and differentiation of enterocytes. Glucagon-like peptide 2 (GLP-2) and epidermal growth factor (EGF) have each been shown to have trophic effects on the intestine, and thus have the potential to benefit patients fed parenterally, such as those with intestinal failure from short bowel syndrome. We report studies aimed to determine whether there may be synergistic effects of these 2 peptides. Methods: Rats were established on parenteral nutrition (PN) and infused for 6 days with GLP-2 (20 µg/d), EGF (20 µg/d), or GLP-2 + EGF (20 µg/d of each). These groups were compared with untreated PN-fed and orally-fed controls. Tissue was obtained from small intestine and colon to determine growth, proliferation, and representative gene expression. Results: Small intestinal weight was increased by 75%, 43%, and 116% in the GLP-2, EGF, and GLP-2 + EGF groups, respectively, compared with PN controls (all p

To ensure adequate enterai nutrition from dietary sources, it is essential that there is sufficient functioning intestine to digest and absorb luminal nutrients. Loss of intestinal tissue, such as after surgical resection for Crohn's disease or ischemia, produces considerable adaptation in the remaining bowel, but malabsorption results when there is insufficient mass of intestinal tissue. Intestinal failure from this short bowel syndrome requires specialized, intensive nutrition support, usually involving parenteral nutrition (PN).1 Interventions that increase intestinal adaptation, mucosal mass, and gene expression will be of great benefit to patients who otherwise would require PN, if these changes enable enterai nutrients to be more successfully absorbed.

A number of growth factors have been shown to be important in the control of growth, differentiation, and repair of the gastrointestinal tract.2 Epidermal growth factor (EGF) is a major factor in the control of gut homeostasis and it has been shown to be a potent mitogen in both animal and human studies.3-6 The mature form of EGF comprises 53 amino acids with 3 intrachain disulphide bridges and is released from salivary glands and the Brunner's glands of the duodenum.7 EGF binds to the epidermal growth factor receptor (EGFR), which has tyrosine kinase activity. Under normal circumstances, the EGFR is expressed on the basolateral side of the intestinal epithelium8 and also binds other members of the EGF family, including TGF-α and amphiregulin.

More recently, glucagon-like peptide 2 (GLP-2) has been identified as another important agent stimulating cellular proliferation and differentiation of the intestine.9 GLP-2 derives from posttranslational processing of proglucagon by prohormone convertases 1 in intestinal L-cells producing a 33 amino-acid peptide.10-14 A GLP-2 receptor has been identified15 which is expressed on enteroendocrine cells.16 In the mouse, enteric neurons are necessary for the effects of GLP-2 on intestinal proliferation.17 Activation of the GLP-receptor in the rat intestinal mucosa has been reported to produce downstream events involving the cAMP/protein kinase A-dependent pathway18 and the immediate early gene, PC4/TIS7.19 In human subjects with short bowel syndrome, GLP-2 therapy has been shown to improve nutrient absorption and nutrition status.20

Although the induction of intestinal epithelial proliferation by GLP-2 has been the subject of several previous studies,21-25 only 1 investigated whether there was any interaction between GLP-2 and other growth factors, including EGF.26 This study used low doses of EGF and found no effect of EGF alone (unlike other previous studies) or in combination with GLP-2. It is necessary to investigate other experimental situations to determine whether the effects of EGF and GLP-2 can be combined to give additional benefit. In the PNfed rat, these growth-promoting effects can be studied without the influence of luminal factors.

In addition to their mitogenic effects, administration of EGF or GLP-2 in the PN-fed rat model results in changes in expression of certain genes in the intestine. Previously, we demonstrated changes with GLP-2 in sucrase-isomaltase expression,23 SGLT-I is also increased,25 and recently we have shown that different amino acid and peptide transporters are variably affected.27 We have also looked at another representative gene, expressed in differentiated cells in the distal small intestine, ileal bile acid binding protein (IBABP, gene symbol FABP6), which is important for the absorption of bile salts. The IBABP promoter has consensus sites for certain transcription factors that influence intestinal gene expression,28 including CDX2 and HNF4, and we now report studies attempting to detect changes in expression of these with the trophic peptides.

We report studies where the principal aim was to establish whether GLP-2 and EGF, when given in combination, can have additive and synergistic effects on small intestinal growth and differentiation in rats fed by PN. Such changes, if they also occur in human clinical situations, will allow enhanced intestinal function and may aid recovery from intestinal failure.

MATERIALS AND METHODS

PN-fed Rat Model

Male Wistar rats received PN via the jugular vein as previously described.3,23,29 The refrigerated PN diet was infused continuously into the rats by a multichannel peristaltic pump, at a rate of 60 mL/rat/d, giving 1.8 g nitrogen, 6.0 g lipid, 8.5 g glucose, and 1680 kJ/kg per day.

For the main experiments, 3 treatment groups were established: GLP-2, 20 µg/d; EGF, 20 µg/d; and GLP-2 with EGF, 20 µg/d of each. The peptides were administered continuously with the IV feed at submaximal effective doses as established in our previous studies on GLP-2 and EGF alone.3'24 In addition, some data on gene expression were obtained from another group of animals treated with GLP-2 of 40 μg/d, described in our previous publication.23 Two control groups were used: PN fed and oral chow fed. On the sixth postoperative day, all the animals were killed. This occurred 2 hours after an intraperitoneal injection of 1 mg/kg vincristine sulfate (David Bull Laboratories, Warwick, UK). Terminal anesthesia was induced by sodium pentobarbital injection. The weights of the whole animal, stomach, small intestine, cecum, and colon were measured, together with intestinal lengths. Tissue was obtained from proximal, middle, and distal regions of both the small and large intestine, at points 10%, 50%, and 90% along their respective lengths. These were fixed and stored in 70% ethanol for microdissection and proliferative analysis. All procedures were approved by the Cancer Research UK and Imperial College Animal Ethics Committee and covered by the British Home Office Animal Procedures Act, 1986.

Histologie Analysis and Microdissection Technique

Tissues for morphometric analyses were embedded in wax and 4-µm transverse sections were cut, mounted, and stained with hematoxylin and eosin. Representative slides were obtained from the proximal small intestine or midcolon. Individual crypts and villi from the proximal small intestine and crypts from the midcolonic site were traced using a calibrated drawing tube. The tracings were then scanned and the area determined using the National Institutes of Health Image Program.

To determine cell proliferation, tissue was fixed in Carnoy's fluid for 2 hours and stored in 70% ethanol until processed. The specimens were rehydrated and hydrolyzed in IM hydrochloric acid for 10 minutes at 60°C; tissue was then placed in Scruffs reagent for at least 45 minutes. Microdissection was performed on small and large intestinal tissue and the arrested metaphases counted in 10 and 20 crypts respectively.

Gene Expression

Total RNA was prepared from snap frozen mucosal scrapes of the terminal ileum as previously described.23 Aliquots of approximately 25 μg were electrophoresed on agarose gels and blotted on to Hybond N membranes (Amersham Buckinghamshire, UK). Each blot also included a sample of RNA from a single control RNA preparation, which was used to standardize between blots. Representative genes were chosen for the small intestine. Blots were probed with 32Plabeled probes for IBABP, HNF-4, Cdx-2, and 18S ribosomal RNA. Hybridization signals were quantified by phosphorimaging (Molecular Dynamics) and adjusted for 18S ribosomal RNA content and the control sample. The rat Cdx-2, HNF-4, IBABP probe, and the 18S ribosomal RNA oligonucleotides were previously described.30,31

Statistical Analysis

Results are expressed as means ± SEM. Analysis of variance for multiple comparisons, t test, or regression analyses were used where appropriate.

RESULTS

The effects of GLP-2 and EGF on gastrointestinal segmental weights are shown in Figure 1. GLP-2 infusion almost doubled the weight of the small intestine compared with PN-fed controls (p

Cellular proliferation (metaphases per crypt) more than doubled with GLP-2 (20 µg/d) in all regions of the small intestine (p

Morphometric studies were performed in the proximal small intestine and midcolon; in both, crypt area was significantly increased when GLP-2 was combined with EGF, compared with both PN-control and the GLP-2 groups (Fig. 3). Villus area in proximal small intestine was also increased by GLP-2 + EGF. On its own, EGF produced no effects in proximal small intestine, but the combination of GLP-2 + EGF was significantly different from GLP-2 alone, indicating a synergistic effect on both villus and crypt areas (p

To attempt to gain more information about changes in gene expression in differentiated cells after GLP-2, Northern blots of distal ileal RNA from the different treatment groups were probed for IBABP, Cdx-2, HNF-4, and 18S ribosomal RNA (see Fig. 5 for representative blots). The phosphorimager data, presented in Table I, were variable and only partly corrected by standardizing for 18S ribosomal RNA. GLP-2 + EGF significantly increased IBABP expression 1.3-fold compared with PN controls (p

DISCUSSION

These data demonstrate that GLP-2 and EGF have additive effects increasing small intestinal weight, cellular proliferation, and morphometric measurements of villus and crypt area. The resultant hyperplasia was very dramatic; the measures of proliferation in the GLP-2 + EGF group were even greater than those in the orally-fed rats.

The mechanisms for the effects of GLP-2 and EGF on the intestine have been the subject of recent studies and, although our present study is largely descriptive, these merit review in the context of our findings. GLP-2 appears to bind to a G-protein-coupled receptor, transcripts of which are found with the largest concentration in the jejunum, although they are also found in the ileum and colon.15 Immunohistochemical localization shows that expression in rats and humans can be detected in a proportion of chromogranin-positive cells in the stomach/duodenum, small intestine, and colon.16 It seems likely that cAMP and protein kinase A activation are involved in the effects of GLP-2 on its target cells.18 However, other downstream molecules that may mediate the effects of GLP-2 have been the subject of further investigation, including certain immediate early genes.19 Other cellular sites of action may be implicated, as suggested by the report of the importance of enteric neuronal involvement in GLP-2 actions in the mouse.17 The mechanisms of the effects of EGF have been more thoroughly studied and are direct on the epithelial cells, where the EGFR has been demonstrated.8 A large number of postreceptor events have been described for EGF, involving activation of various protein kinase cascades. EGF actions can be blocked by tyrosine kinase inhibitors with benefit in gastrointestinal cancers.32 Changes induced by EGF include some that are also found with GLP-2, such as increased expression of the immediate early gene PC4/TIS7.19 The additive or synergistic effects of co-administration of GLP-2 and EGF found in the small intestine in the present study are in line with their effects on different pathways that converge and indicates that a combination of these agents may have a greater therapeutic benefit than giving either one alone.

A previous study26 investigating the potential synergy between GLP-2 and other agents, including EGF, conflicts with our results. No effect on small intestinal weight was found with EGF alone, and no increase in growth was seen when given in combination. These investigators used a dipeptidyl peptidase IV resistant GLP-2 analog with 1 or all of the following 4 growth factors: EGF, long insulin-like growth factor (IGF)-I, IGF-2, human growth hormone (GH). The combination of GLP-2 and either GH, IGF-I, or all growth factors together exhibited greater increases in histologic parameters of small intestinal growth than with GLP-2 alone. Surprisingly, EGF alone (1 µg twice daily, by subcutaneous injection) caused no increase in small intestinal growth and did not augment growth stimulated by GLP-2.26 The reason for this discrepancy is likely due to different doses or routes of administration, as both peptides were given subcutaneously. We have previously shown that many peptides are more potent when given IV.24,33 The animals in the Drucker paper were also given free access to food, unlike ours who received all their nutrition parenterally.

In the colon, this study demonstrated there were no significant effects of GLP-2, given at 20 µg/d, on cellular proliferation or tissue weight. However, at a higher dose of GLP-2 (40 µg/d), colonie cellular proliferation was significantly increased in our previous study,23 though no effect on colonie weight or crypt depth was seen. In contrast, EGF increased cellular proliferation by 2-3 fold in the proximal and distal colon, and this was reflected in the near doubling of colonie weight. In the midcolonic region, EGF gave a 50% increase in crypt area, but the change in cellular proliferation did not achieve significance. Combining EGF with GLP-2, however, resulted in a significant effect at this site. These regional differences result from the small numbers of measurements and the variability of metaphase determinations, rather than any significant additive effect of GLP-2 on the proliferation produced by EGF in the colon, unlike in the small intestine where both peptides alone are capable of producing significant increases.

Having previously shown that GLP-2 increased the expression of sucrase-isomaltase in jejunum, we wanted to perform initial studies of changes in representative genes found in differentiated cells in other segments of the small intestine. IBABP is only expressed in the ileum; GLP-2 and EGF in combination significantly increased transcript levels by 1.3-fold, an effect likely to represent increased differentiation of the ileal enterocytes. Furthermore, expression of the transcription factor HNF-4 was associated with IBABP expression in controls and GLP-2 treated animals. No important changes in Cdx2 transcript levels could be detected. Although the IBABP promoter can be affected by the farnesoid X receptor and is capable of binding Cdx2,28 the mechanisms regulating changes in IBABP expression with differentiation are not completely understood. These data suggest, however, that regulation by HNF-4 may be involved and it will be of interest to perform further experiments to determine these mechanisms, and those for upregulation of HNF-4.

The therapeutic potential of GLP-2 and EGF in clinical situations, including intestinal failure and short bowel syndrome, is becoming apparent. GLP-2 has been used successfully in initial trials in short bowel syndrome,20 and our study suggests that combining it with EGF, or other trophic factors, will yield greater clinically useful results. Furthermore, EGF has been used safely and apparently successfully in children with necrotizing enterocolitis and microvillus atrophy.4,5 Caution must be shown, however, in administering promitogenic factors via the systemic circulation because of the risks of progression of premalignant lesions in the gut or at distant sites.34,35 It is important that the risks and benefits of such an approach are carefully considered, and that the lowest therapeutic doses of peptides are used, taking advantage of potential synergistic effects between agents, where, as shown in the present paper, the combined benefit is greater than the sum of the individual responses.32

In conclusion, we describe that GLP-2 and EGF separately and together augment growth of the small intestine in the parenterally-fed rat, and many of their effects are additive or synergistic, particularly in proximal small intestine. The potential for therapeutic use of these peptide growth factors in parenteral feeding suggests the need for further, detailed studies into their mechanisms and effects in other models and clinical nutrition support.

ACKNOWLEDGMENTS

Dr Kitchen was supported by grants from the Digestive Disorders Foundation, the Dunhill Medical Trust, and an educational grant from Fresenius Kabi. Additional support for the work came from the Kati Jacobs Appeal.

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Paul A. Kitchen, MB, PhD, MRCP*[dagger]; Robert A. Goodlad, PhD, DSc[double dagger]; Anthony J. FitzGerald, MSc[double dagger][double dagger]; Nikki Mandir, MSc[double dagger]; Mohammed A. Ghatei, PhD, DSc¶; Stephen R. Bloom, DSc, FRCP¶; Jorge Berlanga-Acosta, PhD§; Raymond J. Playford, PhD, FRCPt; Alastair Forbes, MD, FRCP*; and Julian R.F. Walters, MB, FRCP[dagger]

From *St Mark's Hospital, Imperial College London, Harrow, United Kingdom; the [dagger]Gastroenterology section, Department of Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom; iHistopathology Unit, Cancer Research UK, London, United Kingdom; ¶ Department of Metabolic Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom; and ^Centre for Genetic Engineering and Biotechnology, Havana, Cuba

Received for publication September 10, 2004.

Accepted for publication March 11, 2005.

Correspondence: Dr. J.R.F. Walters, Gastroenterology section, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK. Electronic mail may be sent to julian.walters@imperial.ac.uk.

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