Obesity Surgery, 3, Case 31+323 Report Alkaline Phosphatase, Sucrase, and Insulin-like Growth Factor Receptors are Present in Atrophied Small Bowel 14 Years after Jejunoileal Bypass Ruth S. MacDonald, MD, FACS, Barbro PhD, RD; William H. Thornton, Jr, MS, MT; Boyd E. Terry, A.-L. Barrett, MT; Edward H. Adelstein, MD Departments of Food Science and Human Columbia, Missouri, USA. Nutrition, The authors obtained atrophied and hypertrophied small intestinal tissue from a patient undergoing jejunoileal (JI) bypass reversal. Tissue from both segments was examined for insulin, insulin-like growth factor-l (IGF-I) and IGF-II receptors, and alkaline phosphatase and sucrase. We were interested in the potential of the atrophied segment to respond to luminal stimulation once the food train was re-established. Within the atrophic segment, flow cytometric evaluation of the receptors revealed (expressed as percent positive staining cells): insulin, 17.1%; IGF-I, 33.6%; and IGF-II, 60.6%, while the immunoreactive sucrase was 87.7% and alkaline phosphatase was 88.6%. Actual sucrase activity (expressed as glucose generated) in this segment was 17.9 nglminlpg protein and alkaline phosphatase was 28.0 U/L/w protein as assessed by conventional methods. Receptor evaluation in the hypertrophic segment demonstrated 9.7% positive staining cells for insulin, 26.6% for IGF-I and 70.2% for IGF-II. Immunoreactive sucrase was 91.2% and alkaline phosphatase was 91.4%. Enzyme activity for sucrase was 10.4 nglminlpg protein and for alkaline phosphatase was 59.4 U/L/ag protein. This data suggests that even in atrophied bowel insulin and IGF receptors as well as sucrase and alkaline phosphatase enzymes are present and may assist in the rapid recovery of the atrophied portion following reversal of the JI bypass. Key words: growth factors. Jejunoileal bypass, intestinal enzymes, Presented in part at the 32nd Annual Meeting of the American College of Nutrition, Clearwater Beach, Florida, September 1991. Reprint requests to: Ruth S. MacDonald, PhD. Food Science and Human Nutrition, 122 Eckles Hall, University of Missouri, Columbia, MO 65211, USA. Tel: 314-882-4113; fax: 314-882-0596. This is a Missouri Agricultural Experiment Station paper no. 11, 784. 0 1993 Rapid Communications Surgery, and Pathology, University of Missouri, Introduction The blind loop formed following jejunoileal (JI) bypass becomes atrophic, due to the absence of lumenal stimulation and nutriture. This static environment contributes to many of the adverse side-effects of the surgical procedure, especially immunological response to bacterial toxins.’ Additionally, protein malnutrition induced by a short-gut effect and bypass enteropathy may ultimately result in the need for reoperation to restore continuity, and replacement with a gastric restrictive procedure (vertical banded gastroplasty). Rasmussenand coworkers’ reported arthralgia, vitamin B12 and iron deficiency among the primary complications in a long-term evaluation of JI bypass. Calcium and magnesiummalabsorption, osteoporosis, electrolyte imbalance, as well as renal complications have also been reported, including casesof interstitial nephritis caused by oxalate deposition3 and urinary calculi.’ Insulin and the insulin-like growth factors (IGF) are involved in tissue growth and differentiation,4,5 and participate in the differentiation of villus epithelial cells in the small intestine.6,7In the newborn IGF-II plays a key role in small intestinal growth,’ and this role may account for the rapid growth response of atrophic tissuefollowing JI bypassreoperation. Certainly, at the cellular level, insulin and IGF-I receptors are involved in regulating metabolism of nutrients.’ The performance of JI bypass to control the morbidly obese patient has fallen into disfavor of Oxford Ltd Obesity Surgery, 3, 1993 319 MacDonald et al. because of the serious side-effects. Many patients require reoperation to alleviate these side-effects, necessitating alternative means of weight control. Following JI bypass the bypassed segment of the small intestine will atrophy, especially the area removed from exposure to the food train. This atrophic segment is capable of absorbing nutrients upon JI bypass take-down, utilizing an adaptation period with an elemental diet followed by a more sophisticated diet; yet little is understood about the cellular mechanisms involved in this tissue recovery. We have examined tissue from both the atrophic and hypertrophic segments of the small intestine in a patient who had undergone JI bypass surgery 14 years prior and was currently undergoing take-down of the bypass, for insulin, IGF-I and IGF-II receptors, as well as alkaline phosphatase and sucrase. Histology Sections proximal to the original transection were obtained and labelled atrophied (from the areathat was bypassed) and hypertrophied (from the shortened, absorptive area). The atrophied sample was 2.5 cm in width when flat and the hypertrophied sample was 8 cm when flat. Sections from each sample were opened, pinned onto Styrofoam and placed into a 2.5% glutaraldehyde solution. Fixed sections were paraffin embedded and cut for routine histology or transferred to 1% osmium tetroxide for post fixation and electron microscopy. Sections were then embedded in Epon 812, thin sectioned and stained with lead citrate and uranyl acetate and viewed on a Phillips 300 transmission electron microscope. Additional sections were sent to pathology for routine processing. Flow Cytometry Materials and Methods Case Report CJT, a d&year-old female housewife, presented to one of us (BET) with recurring fatigue, carpopedal spasms, abdominal bloating with cramps, and migratory polyarthritis in fingers, wrists, elbows, knees and ankles.At age 29 shehad undergone JI bypass to treat morbid obesity, weighing 124.5 kg (height 1.71 m) with a Body Mass Index (BMI) of 43 kg/m’, losing to 70 kg (BMI 24 kg/m’) and then gradually regaining to 85 kg (BMI 29 kg/m’) over 13 years. Repeated hospitalization for electrolyte abnormalities precipitated the decision to reverse the JI bypass and to control weight by vertical banded gastroplasty. This patient was obese as a child and had bilateral hip pinning at age 13 for slipped capital femoral epiphysis. After JI bypass, cholecystectomy was necessary. She was maintained on potassium, calcium, magnesium, vitamins and a high protein diet. She noted chronic fatigue, some muscle weakness, and depression with limitation of activity. She denied tobacco and alcohol intake, but had lost senseof taste and smell. Jaundice and kidney stones were denied. Easy bruising was noted. Five to seven loose bowel movements were common each day. Episodes of bloating with cramps were frequent. Serum potassium was 3.5 mEq, but renal and liver function tests were within normal limits. The bypass was reversed, with the jejunal segments of 8 cm and 2.5 cm diameter reconstituted and vertical banded gastroplasty performed. Liver biopsy showed mild steatosis without fibrosis. 320 Obesity surgery, 3, 1993 Sections from the atrophied and hypertrophied samples were placed into hyaluronidase solution consisting of 10 mM glucose, 5.4 mM KCl, 120 mM NaCl, 2 mM Na,HPO,, 35 mM mannitol, 200 IU/L hyaluronidase and phenylmethylsulfonyl fluoride, in a procedure adapted from Ahrenstedt et a1.I’ In the laboratory sections were inverted onto 13 x 100 mm glasstubes and returned to the hyaluronidase solution at 37°C with agitation for 1 h. Additional mucosawas removed by scraping with a glassslide and placed into the hyaluronidase solution. Mucosal cells were mixed vigorously and concentrated by centrifugation. The cell pellet was washed twice in phosphate buffered saline.Cell counts were performed in a hemocytometer and cell sampleswere prepared for staining. Both the atrophied and hypertrophied sampleswere stained for insulin, IGF-I and IGF-II receptors. Insulin receptors were identified using fluorescent labelled insulin prepared by us.rr IGF-I receptor antibody (Upstate Biochemical Co., Lake Placid, NY) and IGF-II receptor antibody (a generous gift from Dr Richard MacDonald, University of Nebraska)were used in conjunction with a fluorescein-labelled second antibody. The cells were also incubated with antibodies to alkaline phosphatase (Accurate Antibody, Westbury, NY), and, sucrase (a generous gift from Dr Andrea Quaroni, Syracuse University) followed by a fluorescein-labelled second antibody. Appropriate controls were included. Cells were analyzed by flow cytometry using a Coulter EPICS 7.53 (Coulter Corporation, Hialeah, FL), assessinglog green fluorescence with a minimum of 5,000 cells analyzed. Results are expressed as percent positive staining cells compared to control cells. Enzymes and Growth Factor Receptors in Atrophied Bowel Alkaline Phosphatase Assays and Sucrase Enzyme Tissuehomogenates were assessedfor protein content using the BioRad micromethod (BioRad Chemical Division, Richmond, CA). Sections from the atrophied and hypertrophied samples in hyaluronidase buffer were scraped with a glass slide removing the mucosa. Mucosal sampleswere homogenized in a HEPESbuffer and frozen at -60°C until analysis. Alkaline phosphatase activity was analyzed on homogenate samplesby a commercially available kit. Samples were diluted with saline and absorbance measured at 405 nm. Sucrase activity was assessed using the method of Dahlqvist” measuring the amount of glucose generated. Glucose was assessedusing a commercially available kit, with absorbance measured at 450 nm. Table 1. Receptor expression hypertrophied and atrophied reversal of JI bypass Insulin Receptora IGF-I Receptor IGF-II Receptor Alkaline Phosphatase lmmunoreactive Enzymaticb Sucrase lmmunoreactive EnzymaticC and enzyme small bowel activity in following Hypertrophied Atrophied 9.7% 26.6% 70.2% 17.1% 33.6% 60.8% 91.4% 59.4 U/L 88.6% 28.0 U/L 91.2% 10.4 ng/min 87.7% 17.9 nglmin a Receptors and immunoreactive enzyme flow cytometry. Data expressed as percent compared to control cells. b Alkaline phosphatase enzyme activity protein. ‘Sucrase enzyme activity expressed erated/min/pg protein. data as analyzed positive staining expressed as ng as glucose by cells UILIpg gen- Results Histological Examination Sections from each area prepared for electron microscopy were relatively unremarkable. In the atrophic tissue, microvilli were intact with glycocalyx present. Villus epithelial cells were normal, and numerous goblet cells visible. Villi were notably hypertrophied in tissue from the absorptive segment, yet cellular morphology was similar to that of the atrophic segment. Goblet cells were present but appeared to be less in number than in the atrophic sections. Paneth cells were also visible in the hypertrophied sections, yet lacking in the atrophied sections. Microscopic evaluation from pathology revealed focal artifactual changes but no pathologic lesions. A liver biopsy performed at the time of surgery revealed mild steatosis with normal lobular architecture. There was slight pericentral venous fibrosis and a very slight increasein portal fibrosis. No cirrhosis was seen. for the hypertrophic segment. IGF-I receptor expression was more prominent in both segments, with the atrophic segment having 33.6% positive cells and 26.6% positive cells for the hypertrophied segment. Expression of IGF-II receptors was very prominent in both groups with greater expression in the hypertrophied segment (70.2% vs 60.8% in the atrophied segment). Evaluation of immunoreactive sucraseand alkaline phosphatase revealed similarly high levels in both segments(Table I), with 91.2% positive for sucrasein the hypertrophied segment and 87.7% positive in the atrophied segment. Percent positive alkaline phosphatase for the hypertrophied segment was 91.4% and 88.6% for the atrophied segment. Interestingly, actual enzyme activity within each of these segments was quite different as seenin Table 1. Enzyme Analysis Flow Cytometry Analysis of insulin and IGF receptors revealed similar expression from both the hypertrophied and atrophied segments as seen in Table 1. Insulin receptors demonstrated the smallestpercent positive cells when compared to control, with the atrophic segment expressing a greater percent positive, 17.1% vs 9.7% Alkaline phosphataseactivity within the hypertrophic tissue was 59.4 U/L/pg protein compared to 28.0 U/L/pg protein for the tissue obtained from the atrophied area. Sucraseactivity was low in both tissue segments; the hypertrophic homogenate generated 10.4 ng glucose/min/pg protein, while the atrophic homogenate generated 17.9 ng glucose/min/pg protein. Obesity Surgery, 3, 1993 321 MacDonald et al. Discussion References The cellular morphology of the atrophic segment was similar to that of the hypertrophic segment, microvilli and goblet cells were present and the villus epithelial cells appeared normal. The cells received nutrients and hormones via the circulatory system; however, normal lumenal stimulation by the food train was absent, and bacterial colonization may have affected cells within this atrophic segmenti The presence of insulin and IGF receptors on both segmentsis not surprising. These receptors are located primarily on the basolateral area of the cellsi4-i7 responding to circulating insulin, IGF-I and IGF-II. Certainly the IGFs play a role in intestinal growth and differentiation4,i8 and insulin as well may be involved.6 Receptor expression will change following surgical alteration of the small bowe116,i8with an increased insulin binding by 12 h following resection, and IGF-II receptors increasing immediately following surgery. Flow cytometric evaluation of receptor expression revealed little difference between the two segments. This most likely reflects a static environment both within the small intestine and with circulating levels of hormone, a result of adaptation considering bypass surgery was performed 14 years prior. Following reoperation one would speculate that receptor levels would change as the atrophic segment recovered. Perhaps the most interesting finding was the presence of alkaline phosphatase and sucrase in the atrophied segment.While there was decreasedalkaline phosphatase activity when compared to the hypertrophied segment, sucrase activity was actually somewhat higher when assessedusing the enzymatic assay. These enzymes were present at similarly high levels in both the atrophic and hypertrophic segments as demonstrated by immunoreactive evaluation using flow cytometry. The presence of these enzymes may assistin the recovery of the atrophic tissue following JI bypass reversal. Our observations demonstrate the presence of insulin and IGF receptors as well as alkaline phosphataseand sucrasein both the hypertrophic and atrophic segments of the small intestine of a patient who had undergone JI bypass 14 years ago. Additionally, the cellular morphology within the atrophic segment was unremarkable when compared to the hypertrophic segment. These findings suggest that the ability of the atrophied small bowel to adapt following reoperation, even in the absenceof lumenal nutrient exposure and stimulation, can be attributed to the capability of the cells to maintain normal morphology, growth factor receptors, and enzymes. 322 Obesify surgery, 3, 1993 1. Corrodi P, WidemanPA, SutterVL, et al. Bacterialflora of the smallbowel before and after bypassprocedure for morbid obesity.] lnfecDis 1978; 137: 1-5. 2. Rasmussen I, EnbladP, AroseniusKE.Jejunoileal bypass for morbid obesity. Report of a serieswith long-term results.Acfa Ckir Stand 1989; 155: 401-7. 3. Verani R, Nasir M, Foley R. Granulomatous interstitial nephritisafter a jejunoilealbypass:anultrastructuraland histochemicalstudy. Am ] Nepkrol 1989; 9: 51-5. 4. BallesterosM, Scott CD, Baxter RC. Developmental regulation of insulin-like growth factor-II/mannose 6-phosphatereceptormRNA in the rat. Biockem Biopkys Res Commun 1990; 172: 775-9. 5. Fraslon C, Bourbon JR. Comparisonof effects of epidermal and insulin-like growth factors, gastrin releasingpeptide and retinoic acid on fetal lung cell growth and maturation in vitro. Biockem Biopkys Acfa 1992; 1123: 65-75. 6. Buts J-P, De Keyser N, KolanowskiJ, ef al. Hormonal regulation of the rat smallintestine:responsiveness of villus and crypt cells to insulin during the suckling period and unresponsiveness after weaning.Pediaf Res 1990; 27: 161-4. 7. Park JHY, Vanderhoof JA, Blackwood D, et al. Characterization of type I and type II insulin-like growth factor receptors in an epithelial cell line. Endocrinology 1990; 126: 2998-3005. 8. SchofieldPN, Valerie TE. Regulationof humanIGF-II transcription in fetal and adult tissues.Development 1987; 101: 793-803. 9. Weiland M, Bahr F, Hohne M, et al. The signaling potential of the receptorsfor insulin and insulin-like growth factor I (IGF-I) in 3T3-Ll adipocytes: comparisonof glucosetransport activity, induction of oncogenec-fos, glucosetransportermRNA, and DNA synthesis.] Cell Pkysiol 1991; 149: 428-35. 10. Ahrenstedt0, KnutsonF,KnutsonL, ef al. Cell recovery during segmentalintestinalperfusionin healthy adult subjectsand patients with Crohn’sdisease.Gtrf 1991; 32:170-3. 11. Thornton WH Jr, MacDonald RS. Flow cytometric evaluation of insulin and insulin-like growth factor receptor expressionfollowing smallbowel resection. FASEB ] 1992; 6: A1841 (Abstract). Anal 12. Dahlqvist A. Assay of intestinaldisaccharidases. Ckem 1967; 22:99-107. 13. Leung F, Drenick EJ, Stanley TM. Intestinal bypass complications involving the excluded small bowel segment.Am 1 Gasfroenferol 1982; 77: 67-72. 14. Heinz-ErianP, KesslerU, Funk B, et al. Identification and in situ localization of the insulin-like growth Enzymes and Growth Factor Receptors in Atrophied factor-II/mannose-6-phosphate (IGF-II/M6P) receptor in the rat gastrointestinal tract: comparison with the IGF-I receptor. Endocrinology 1991; 129: 1769-78. 15. Young GP, Taranto TM, Jonas HA, et al. Insulin-like growth factors and the developing and mature rat small intestine: receptors and biological actions. Digestion 1990; 46 (suppl 2): 240-52. 16. MacDonald RS, Park JHY, Thornton WH Jr. Insulin, IGF-1 and IGF-2 receptors in rat small intestine following massive small bowel resection: analysis by binding, flow cytometry and immunohistochemistry. In press Dig Dis Sci 1993. Bowel Gingerich RL, Gilbert WR, Comens PG, et al. Identification and characterization of insulin receptors in basolateral membranes of dog intestinal mucosa. Diabetes 1989; 36: 1124-9. 18. MacDonald RS, Steel-Goodwin L, Smith RJ. Influence of dietary fiber on insulin receptors in rat intestinal mucosa. Ann Nufr Mefab 1991; 35: 328-38. 17. (Received 31 ]anuary 1993; accepted 14 April 1993) Obesity Surgery, 3, 1993 323