What Is Known/What Is New
What Is Known
- Structural adaptation includes intestinal lengthening and mucosal hyperplasia.
- Human milk contains factors important for gut growth, including insulin-like growth factor-1 and epidermal growth factor. These may benefit intestinal adaptation.
- Milk factor globule epidermal growth factor-8 is a major proteins in human milk not yet studied for adaptation.
What Is New
- Post massive intestinal resection in piglets, the ileum is the main site of intestinal lengthening.
- Milk fat globule epidermal growth factor-8 treatment increases lengthening of the ileum after resection.
- Growth of the ileum with milk fat globule epidermal growth factor-8 treatment was associated with increased gene expression for I insulin-like growth factor-1 and epidermal growth factor-1 in the ileum.
Human milk has essential biological functions for neonatal gastrointestinal and immune function and development (1). In this context, the benefits of human milk are particularly significant for preterm infants, where exclusive use of mothers’ own milk and/or donor human milk have been associated with reduction in the risk of necrotizing enterocolitis (NEC) and sepsis (1,2). Preterm infants are at increased risk of intestinal failure secondary to short bowel syndrome (SBS) from causes such as NEC, gastroschisis and congenital intestinal atresia's. In the setting of pediatric SBS, use of human milk over formula can decrease the duration of parenteral nutrition (PN) dependency (3). This is further supported by animal research using neonatal models of SBS showing enhanced adaptation with use of colostrum (4).
After lactose, fat is the second largest component of mammalian milk and a key nutrient supporting the growth of neonates. Given the hydrophobic nature of lipids, this nutrient is contained within milk fat globules formed and secreted in the mammary ducts (5). The milk fat globule (MFG) is composed of a triglyceride core, surrounded by a cellular membrane of phospholipids, cholesterol, protein and glycoproteins (6). This outer membrane contains many of the bioactive protein factors of mammalian milk that support immune and gastrointestinal function and development (6).
To date, eight major and over a hundred minor MFG proteins have been identified (6). The majority appear to have roles in immune function and protection (5,6). Milk fat globule epidermal growth factor-8 (MFG-E8), formerly lactadherin, is one of those major MFG proteins in human milk (6). Importantly, this protein is protected from gastric degradation in preterm infants (5,7). The structure of MFG-E8 has been conserved across many mammalian species, including human and bovine, suggestive of an important role in mammalian development (8).
Notable functions that have been associated with MFG-E8 include protection against rotavirus gastroenteritis in neonates, reduction in intestinal inflammation and enhanced tissue repair in animal models (9) and, more recently, a potential role in fat absorption (10,11). Furthermore, MFG-E8 has genetic homology with epidermal growth factor (EGF) and shares an identical binding domain within the EGF receptor (Egfr) (12). In the context of SBS, EGF has been of therapeutic interest to our group and to others as it appears to enhance intestinal structural adaptation to surgical resection (13,14). Given this background, the aim of this proof of principle pilot study was to determine whether recombinant human MFG-E8 treatment of neonatal piglets with SBS improves fat absorption and/or enhances structural adaptation.
Animals and Surgery
Animal studies were conducted in compliance with the Canadian Council on Animal Care guidelines and approved by the University of Alberta Animal Policy & Welfare Committee. Neonatal Landrace-Large White cross male piglets underwent general anesthesia for jugular venous catheterization, laparotomy, intestinal length measures, short bowel surgery and insertion of a gastrostomy (G) tube (13). Piglets were allocated to the following surgical groups: 75% mid-intestinal resection (leaving equal lengths of remnant jejunum and ileum) with jejunoileal anastomosis (JI group) or 75% distal-intestinal resection (including all of the ileum and proximal 5 cm of colon) with jejunocolic anastomosis (JC group).
Piglets were randomized to receive either saline (control) or 5 mg/kg MFG-E8, delivered daily via G tube at equal volumes. The MFG-E8 dose was extrapolated from studies in rodents (from an effective dose of 50 μg/kg for fat absorption) using allometric scaling (15). Recombinant human MFG-E8 was supplied in kind by the manufacturer (Empire Biotechnologies, Inc., California, USA).
Postoperatively, piglets were maintained in metabolic cages with a 12-hour light/dark cycle and provided pain relief and antibiotic prophylaxis for catheter sepsis as previously described (13).
Immediately postoperatively, piglets received PN advanced to meet 100% of daily caloric intake by day 2, when they commenced enteral nutrition (EN) at 40% of daily caloric requirements via G-tube. PN and EN were delivered continuously. Target PN nutrient intakes (100%) were: 1100 kJ/kg/day, 37% of parenteral energy from carbohydrate and 36% from fat (13). Target enteral fat delivery was 4 g/kg per day.
Enteral Fat Absorption
Fecal effluent was collected for 48 hours, beginning on study day 5, into drainable ostomy appliances (Two-Piece Pouch System; Hollister, Aurora, Ontario, Canada). Data were included only when fecal collections were complete without leakages. Samples were freeze-dried and fat was subsequently extracted by petroleum ether distillation (13). Enteral fat absorption was calculated by subtracting the average fecal fat per pig (g/kg per day) from the total amount of enteral lipid infused (g/kg per day) to determine the average fat absorption (g/kg per day) and is expressed as percentage fat absorption.
Tissue Collection and Morphology
On study day 7, piglets were anesthetized and underwent terminal laparotomy where final intestinal lengths were measured. The small intestine from the ligament of Treitz to ileocecal valve (JI) or jejunocolic anastomosis (JC) was removed, emptied of fecal matter and weighed. In JI piglets, the length of jejunum was measured proximal to the surgical anastomosis and the length of ileum was measured distal to the anastomosis. Mucosal scrapings of 20 cm segments of jejunum (immediately distal to ligament of Treitz) and ileum (immediately proximal to ileocecal valve) were obtained and weighed, then corrected for length and expressed as g/cm. Cross-sectional jejunal (JI and JC) and ileal (JI) segments were preserved in 10% buffered formaldehyde for histology. Villus height and crypt depth were measured on H&E-stained jejunal and ileal cross-sections (Nikon Eclipse 80i; Nikon, Tokyo, Japan) by a certified veterinary pathologist blinded to treatment. Ten well-oriented villi and crypts were measured on 2 to 3 different cross sections per piglet.
Real-Time Reverse Transcriptase-Quantitative Polymerase Chain Reaction
Jejunal and ileal mucosa was isolated by scraping of rapidly thawed intestinal segments that had been stored at −80 °C, and the remaining tissue identified as ‘muscle’, although this included any residual submucosa and serosa. Expression of genes related to intestinal growth, nutrient absorption, tight junction function, mucosal proliferation and apoptosis were assessed by extraction of total RNA (RNeasy Plus mini kit with Qiashredder; QIAGEN Inc., Germantown, MD) followed by reverse-transcription (5X All-in-One RT MasterMix; Applied Biological Materials Inc., Richmond, BC, Canada) and real-time-semi-quantitative PCR, with TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA) using primers as listed in Supplemental Digital Content, Table 1, https://links.lww.com/MPG/B872). Relative messenger RNA (mRNA) expression was quantified using the Livak method with 18S ribosomal RNA as the internal control, as validated (13,16).
Results are expressed as mean ± SEM per experimental group. Data were analyzed by 2-way ANOVA (main effects and interaction for surgical anatomy and treatment), followed by Tukey post-hoc analysis. Significance was set at P less than 0.05.
Piglet characteristics are shown Table 1. All piglets were healthy on trial, without presumed sepsis. No differences in body weight (P = 0.06) or weight gain (P = 0.78) were observed with either surgery or treatment.
Percentage total fat absorption was increased in JI compared with all JC piglets by 15% (corrected model P = 0.02; anatomy P = 0.004) (Table 1). There was no effect of MFG-E8 treatment (P = 0.44); JI piglets had on average 85% fat absorption without treatment and 84% with treatment, JC piglets had on average 67% fat absorption without treatment and 73% with treatment.
Gross Morphology and Histopathology
Intestinal lengthening was observed only in JI piglets, mainly in the ileal segment (Table 1). Total length increase over remnant surgical length in JI piglets was +44% and in JC piglets was -2% (P < 0.001). In JI piglets, on average the jejunal segment lengthened 29% without MFG-E8 treatment and 24% with treatment (P = 0.75). In contrast, the ileal segment lengthened 49% without treatment and this was increased to 71% with treatment (P = 0.02). Findings are summarized in Figure 1.
Overall small bowel weight was greater in JI compared to JC piglets, by 48% (P < 0.001) (see Table 1), with no treatment effect. Jejunal scraping weight was not different between the 2 surgical groups (P = 0.09). In JI piglets, ileal scraping weight was not different with treatment (P = 0.30). No surgical group or treatment differences in villus height (P = 0.98) or crypt depth (P = 0.33) were observed (Table 1).
Gene Expression: Mucosa
Given the above findings in JI piglets, we evaluated relative gene expression for mucosal transcripts involved in pre- and post-natal intestinal growth and function in order to evaluate further both site-specific (jejunum vs ileum) and treatment differences (Fig. 2). Overall, in ileum as compared with jejunum, there was a significant increase in expression of insulin like growth factor-1 (Igf-1) by 83% (P = 0.004). There was also a site × treatment interaction and Igf-1 expression increased in ileum with MFG-E8 treatment as compared with jejunum (P = 0.02). Overall, expression of Egf was increased 3.5 fold in ileum compared with jejunum (P < 0.001) and expression of Egfr was increased 60% (P = 0.02). There was a site × treatment interaction and Egfr expression increased in ileum with MFG-E8 as compared with jejunum (P = 0.04). No changes or differences in mucosal expression of Gcg, the prohormone for the intestinal growth factor glucagon-like peptide, or in that of the GLP-2 receptor Glp2r were detected.
There were no differences in mucosal gene expression for transcripts relevant for fat absorption (Cd36, Fatp4), carbohydrate absorption (Slca1, Slca2), proliferation (Mki67), general transcriptional regulation (Cdx2) or tight junction proteins (Cldn5, Cldn7), except for Claudin-2 (Cldn2), the expression of which was increased in the ileum compared with jejunum (P = 0.03), without a treatment effect (Supplemental Digital Content, Figure 1, https://links.lww.com/MPG/B872).
Gene Expression Muscle
Similarly, relative gene expression, jejunal and ileal muscle, for transcripts involved in pre- and post-natal intestinal growth and function were evaluated in JI piglet's (Fig. 3). In the ileum as compared with the jejunum, there was a significant increase in expression of Glp2r by 29% (P = 0.01), and there was a site-specific treatment interaction for Glp2r expression increased in ileum as compared with jejunum (P = 0.05). No changes or differences in other growth factors or their receptors were observed.
In this pilot study, treatment of short bowel neonatal piglets with recombinant bovine MFG-E8 resulted in site-specific trophic effects in the remnant small intestine. Notably, in piglets with retained ileum treatment resulted in increased linear growth of the ileal segment in association with increased mucosal expression of the intestinal growth regulators Igf1 and Egfr. No treatment effects were observed in the absence of remnant ileum.
Neonatal piglets are recognized as a suitable model for neonatal gastrointestinal development (17). Creating neonatal piglet models of surgical short bowel that are anatomically distinct has allowed us to demonstrate that loss of the ileum is associated with marked reduction in potential for adaptation after surgical resection, with increased PN dependency postsurgical resection (18,19). This was confirmed in the current study, wherein increased intestinal length and intestinal weight were greater in the JI model compared with the JC model. We had not previously determined that the site of intestinal lengthening postresection in the JI is actually in the ileum, rather than jejunum. In this study, the treatment effect with MFG-E8 was only seen in the ileum in the JI piglets.
We are not aware of ileal growth predominance in SBS—with and without treatments— having been reported in other animal studies. Few studies have focused on intestinal lengthening; most focus has been on mucosal hyperplasia. In rodents with JI anatomy, lengthening has been shown to be most prominent in the ileum (20), whereas in rodents with JC anatomy, mucosal hyperplasia is significantly restricted, whereas length changes were not reported (21). Sigalet et al (22) characterized changes in intestinal weight, diameter and length in juvenile pigs over 16 weeks in a JI model similar to our own. In this model, mucosal hyperplasia occurred in the ileum more than jejunum. Notably, the greatest contribution to increased absorptive surface area was intestinal diameter and length. Similar to our own findings in pigs, Thompson et al (23), demonstrated in dogs that intestinal lengthening is more likely given a proximal versus a distal resection. Interestingly, in that model, 5 cm of ileum was retained and was associated with much more potential for intestinal growth than we observe in piglets without remnant ileum.
JI models have been studied that show site-specific treatment effects with other trophic treatments. Growth hormone has been shown to increase equally ileal and jejunal lengthening in SBS piglets (24). In rodents, IGF treatment has been shown to increase small bowel length proximally (25). EGF treatment increases intestinal length in rodents, site not specified; however, mucosal hyperplasia occurred only in the ileum (26). In contrast, in our SBS piglet models, we have shown that GLP-2 treatment does not increase intestinal growth in piglets with retained ileum over and above normal potential, but it does when the ileum is resected and can be further enhanced with EGF treatment (13). It is, therefore, clear that site-specific effects need to be considered. A one-size fits-all trophic strategy appears unlikely to benefit all neonates with SBS, given the diversity of surgical anatomy in this population. Further, this speaks to the likelihood that trophic factors might be combined to optimize site-specific effects. Given the potential costs of these therapies, further research to better understand efficacy according to remnant anatomy is warranted.
In the current study, we did not observe any differences in mucosal adaptation, although we have previously shown increased mucosal hyperplasia in the JI compared with JC piglets (19). It is possible this reflected the small sample sizes. Regardless, treatment with MFG-E8 did not have any effect on mucosal adaptation in either model. This raises the possibility that, after surgical resection in neonates, intestinal linear growth and mucosal hyperplasia are distinct events, plausibly subject to different timing and regulation.
This is potentially also true for normal neonatal postnatal lengthening. To date, our understanding of intestinal growth in human neonates has been limited to fetal autopsy studies or measurements undertaken in surgical neonates (27–29). Most intestinal linear growth occurs prenatally, appearing to continue postnatally until 5 years of age (29). Prenatally, the process of elongation commences before villus formation, subject to epithelial and mesenchymal signalling (30). The trophic factors that drive this process perinatally include IGF-1 and EGF (31). Both are prominent in amniotic fluid as well as in maternal colostrum in humans and pigs (6,32). In this study, the expression of Ifg-1 and Egf and their receptors were increased in ileum over jejunum in JI piglets. Furthermore, the differences in growth of ileum with MFG-E8 treatment was plausibly mediated directly via Egfr, or indirectly through IGF-1 or EGF, given that treatment increased expression of both Igf1 and the Egfr in the ileum.
Linear intestinal growth must involve all layers of the intestine: mucosa, submucosa, muscle and serosa. Although research on intestinal adaptation has focused primarily on the mucosa, it is known that muscle adaptation occurs after small bowel resection and mechanical stress can promote linear intestinal growth (33). In the current study, we examined the molecular signalling relevant to this layer in the tissue below the scraped-off mucosa that contained submucosa, muscle and serosa. The only difference was for Glp2r, interesting given the likely role of GLP-2 in postnatal intestinal growth after enteral nutrition, and given the likely role for IGF-1 and EGF as secondary messengers for GLP-2 (34).
Treatment, however, did not significantly increase overall intestinal length or increase fat absorption in the present study. This and the small differences in overall length changes limits utility for SBS of this treatment as it seems unlikely this would benefit weaning from PN. Previous studies in mature rodents had suggested that MFG-E8 has a significant role in fat absorption, hence our interest in this treatment as a potential therapeutic strategy for SBS. In piglets without ileum, there was a small increase in fecal fat absorption (on average +6%), although this did not reach statistical significance, likely because of the small number of animals studied. Alternatively, there may be a species difference or there may be differential roles for MFG-E8 in neonatal versus mature mammals. This remains, however, speculative as, to date, few studies on the role of MFG-E8 in fat absorption outside of adult rodents have been conducted (10,11).
NEC, one of the most common surgical emergencies of preterm neonates that leads to SBS most commonly affects the ileum and right colon (35). There has been considerable interest in EGF as a potential therapy for NEC (36,37). Notably, 1 mechanism by which EGF has efficacy is by reducing ileal apoptosis, also a proposed mechanism for the anti-inflammatory effects of MFG-E8 in the gut (38). We speculate that the role of MFG-E8 should be investigated in animal models of NEC, given our findings in this pilot study of increased ileal Egfr expression (39).
Limitations of this study include the small sample sizes and the short duration of treatment, both of which may have been inadequate to see significant treatment effects. Furthermore, we acknowledge that length measurements are subject to variability that cannot be completely eliminated; however, the technique for measurement has been standardized in place in our laboratory for 15 years. All measurements are performed with a premeasured, 60 cm, 3-0 silk suture. The bowel is measured along the antimesenteric border of the bowel, taking care not to over stretch the bowel. Final measurements at terminal laparotomy are taken with the bowel in situ, while animals are alive (to maintain bowel tone), after lysis of adhesions. All measurements are performed by a single experienced technician (the same person for the past 15 years) on every piglet to ensure consistency but this individual was not done blinded to treatment.
Many bioactive factors of the MFG are likely to play a role in postnatal gut development and may be therapeutic targets for intestinal diseases of developing neonates. We studied MFG-E8, a major protein of the MFG shown to have a role in neonatal immune function and in intestinal repair and fat absorption in adults (6,9–11). We found that MFG-E8 had site-specific effects on the neonatal piglet intestine after surgical resection, increasing both the length of the remnant ileum and mucosal gene expression of Ifg1 and Egfr. This peptide may have a limited role for neonatal SBS, given lack of remnant ileum is the more common anatomy. This study, however, does provide new information about the potential role of MFG-E8 in neonatal gut development that may be relevant for intestinal diseases that specifically target the ileum, such as NEC.
The authors are grateful to Dr K. Austin and Ms J.A. Chalmers for technical assistance with the RT-qPCR studies. We acknowledge the important contribution of Charlane Gorsak, dedicated animal technician, for assistance with the anesthesia for all piglets.
1. Wagner CL, Taylor SN, Johnson D. Host factors in amniotic fluid and breast milk that contribute to gut maturation. Clin Rev Allergy Immunol
2. Schanler RJ, Atkinson SA. Effects of nutrients in human milk on the recipient premature infant. J Mammary Gland Biol Neoplasia
3. Andorsky DJ, Lund DP, Lillehei CW, et al. Nutritional and other postoperative management of neonates with short bowel
syndrome correlates with clinical outcomes. J Pediatr
4. Nagy ES, Paris MC, Taylor RG, et al. Colostrum protein concentrate enhances intestinal adaptation after massive small bowel resection in juvenile pigs. J Pediatr Gastroenterol Nutr
5. Hamosh M, Peterson JA, Henderson TR, et al. Protective function of human milk: the milk fat globule. Semin Perinatol
6. Chatterton DE, Nguyen DN, Bering SB, et al. Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns. Int J Biochem Cell Biol
7. Giuffrida MG, Cavaletto M, Lamberti C, et al. Proteolysis of milk fat globule membrane proteins in preterm milk: a transient phenomenon with a possible biological role? Int J Immunopathol Pharmacol
8. Khaldi N, Shields DC. Shift in the isoelectric-point of milk proteins as a consequence of adaptive divergence between the milks of mammalian species. Biol Direct
9. Bu HF, Zuo XL, Wang X, et al. Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J Clin Invest
10. Khalifeh-Soltani A, McKleroy W, Sakuma S, et al. Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids. Nat Med
11. Khalifeh-Soltani A, Gupta D, Ha A, et al. Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity. JCI Insight
12. Larocca D, Peterson JA, Urrea R, et al. A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains. Cancer Res
13. Lim DW, Diane A, Muto M, et al. Differential effects on intestinal adaptation following exogenous glucagon-like peptide 2 therapy with and without enteral nutrition in neonatal short bowel
syndrome [formula: see text]. JPEN J Parenter Enteral Nutr
14. Sham J, Martin G, Meddings JB, et al. Epidermal growth factor improves nutritional outcome in a rat model of short bowel
syndrome. J Pediatr Surg
15. Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm
16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods
17. Guilloteau P, Zabielski R, Hammon HM, et al. Nutritional programming of gastrointestinal tract development. Is the pig a good model for man? Nutr Res Rev
18. Suri M, Turner JM, Sigalet DL, et al. Exogenous glucagon-like peptide-2 improves outcomes of intestinal adaptation in a distal-intestinal resection neonatal piglet model of short bowel
syndrome. Pediatr Res
19. Turner JM, Wales PW, Nation PN, et al. Novel neonatal piglet models of surgical short bowel
syndrome with intestinal failure. J Pediatr Gastroenterol Nutr
20. Dou Y, Lu X, Zhao J, et al. Morphometric and biomechanical remodelling in the intestine after small bowel resection in the rat. Neurogastroenterol Motil
21. Appleton GV, Bristol JB, Williamson RC. Proximal enterectomy provides a stronger systemic stimulus to intestinal adaptation than distal enterectomy. Gut
1987; 28 Suppl:165168.
22. Sigalet DL, Lees GM, Aherne F, et al. The physiology of adaptation to small bowel resection in the pig: an integrated study of morphological and functional changes. J Pediatr Surg
23. Thompson JS, Quigley EM, Adrian TE. Factors affecting outcome following proximal and distal intestinal resection in the dog: an examination of the relative roles of mucosal adaptation, motility, luminal factors, and enteric peptides. Dig Dis Sci
24. Benhamou PH, Canarelli JP, Richard S, et al. Human recombinant growth hormone increases small bowel lengthening after massive small bowel resection in piglets. J Pediatr Surg
25. Lemmey AB, Ballard FJ, Martin AA, et al. Treatment with IGF-I peptides improves function of the remnant gut following small bowel resection in rats. Growth Factors
26. Chaet MS, Arya G, Ziegler MM, et al. Epidermal growth factor enhances intestinal adaptation after massive small bowel resection. J Pediatr Surg
27. FitzSimmons J, Chinn A. Shepard TH Normal length of the human fetal gastrointestinal tract. Pediatr Pathol
28. Marnerides A, Ghazi S, Sundberg A, et al. Development of fetal intestinal length during 2nd-trimester in normal and pathologic pregnancies. Pediatr Dev Pathol
29. Struijs MC, Diamond IR, de Silva N, et al. Establishing norms for intestinal length in children. J Pediatr Surg
30. Montgomery RK, Mulberg AE, Grand RJ. Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology
31. Kimble RM, Breier BH, Gluckman PD, et al. Enteral IGF-I enhances fetal growth and gastrointestinal development in oesophageal ligated fetal sheep. J Endocrinol
32. Dasgupta S, Arya S, Choudhary S, et al. Amniotic fluid: Source of trophic factors for the developing intestine. World J Gastrointest Pathophysiol
33. Park J, Puapong DP, Wu BM, et al. Enterogenesis by mechanical lengthening: morphology and function of the lengthened small intestine. J Pediatr Surg
34. Brubaker PL. Glucagon-like peptide-2 and the regulation of intestinal growth and function. Compr Physiol
35. Israel EJ. Neonatal necrotizing enterocolitis, a disease of the immature intestinal mucosal barrier. Acta Paediatr Suppl
36. Dvorak B, Halpern MD, Holubec H, et al. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol
37. Shin CE, Falcone RA Jr, Stuart L, et al. Diminished epidermal growth factor levels in infants with necrotizing enterocolitis. J Pediatr Surg
38. Clark JA, Lane RH, Maclennan NK, et al. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol
39. Fagbemi AO, Wright N, Lakhoo K, et al. Immunoreactive epidermal growth factor receptors are present in gastrointestinal epithelial cells of preterm infants with necrotising enterocolitis. Early Hum Dev