Evaluation of the mrna and Protein Expressions of Nutritional Biomarkers in the Gastrointestinal Mucosa of Patients with Small Intestinal Disorders

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1 ORIGINAL ARTICLE Evaluation of the mrna and Protein Expressions of Nutritional Biomarkers in the Gastrointestinal Mucosa of Patients with Small Intestinal Disorders Masanao Nakamura 1, Yoshiki Hirooka 2, Osamu Watanabe 1, Takeshi Yamamura 2, Kohei Funasaka 1, Eizaburo Ohno 1, Ryoji Miyahara 1, Hiroki Kawashima 1, Yoshie Shimoyama 3 and Hidemi Goto 1 Abstract Objective The objectives of this study were to investigate the mrna and protein expression of biomarkers related to absorption in the small intestinal mucosa of humans and determine the relationships between small intestinal diseases and nutrition. Methods The study subjects consisted of patients scheduled to undergo double-balloon endoscopy (DBE) or total colonoscopy for suspected gastrointestinal disorder in a clinical practice. Biopsies were taken from apparently normal mucosa in the visible areas of 6 parts of the intestines from the duodenum to the colon. The mrna expression of specific biomarkers (SGLT1, SGLT5, GIP, GLP, LAT1, LAT2, and NPC1L1) in the mucosa was compared among three patient groups: Inflammation, Tumor, and Control. Results Sixty-six patients participated in this study. Both routes of DBE were performed in 20 patients, in whom biopsy samples were obtained from the mucosa for all sections. There were no remarkable differences in the mrna expression levels among the 3 groups. However, SGLT1, GIP, GLP, and NPC1L1 exhibited specific distribution patterns. The expression levels of GIP and NPC1L1 were highest in the upper jejunum, but were extremely low in the terminal ileum and colon. A comparison of the mrna expression profile in each intestinal section revealed that the SGLT1 mrna expression in the Tumor group and the GIP mrna expression in the Inflammation group were significantly higher than the corresponding levels in the Control group in the upper jejunum. Conclusion The gastrointestinal mucosa of patients with small bowel diseases can maintain proper nutrient absorption, except in the upper jejunum. Key words: double-balloon endoscopy, nutrition, small bowel mucosa, mrna, protein (Intern Med 55: , 2016) () Introduction The small intestinal mucosa is the first location for the absorption of nutritional agents, namely, carbohydrates, proteins, and fat, and it plays an important role in the maintenance of nutritional homeostasis in humans (1). The surface of this mucosa harbors precise mechanisms to absorb various agents needed to maintain homeostasis in the human body. Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid hormone secreted by intestinal K cells in the mucosa, predominantly in the duodenum and proximal jejunum, in response to the luminal presence of nutrients (2). Niemann-Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process (3). NPC1L1 localizes to Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Japan, Department of Endoscopy, Nagoya University Hospital, Japan and Department of Pathology and Clinical Laboratories, Nagoya University Hospital, Japan Received for publication November 3, 2015; Accepted for publication November 29, 2015 Correspondence to Dr. Masanao Nakamura, makamura@med.nagoya-u.ac.jp 2145

2 the brush border membrane of absorptive enterocytes in the small intestine. The intestinal expression of NPC1L1 is downregulated by high cholesterol diets (4). Animal studies have demonstrated the location and distribution of such biomarkers in the gastrointestinal (GI) mucosa, which differ among animals (4, 5). Despite the current increase in interest in the role of nutrition in health and disease and the recognition, for over 20 years, that malabsorption syndrome is complicated by small bowel disorder, the relationships between small bowel mucosa and absorption remain poorly defined. Few human translational research studies have been performed. Much of this lack of clarity stems from the difficulties in agreeing upon a consensus concerning the diagnosis of these conditions: there are no gold standards or commonly available methodologies. Surgical specimens from the GI tract have provided some mechanistic information for specific local areas, however, there are no such data for the entire GI tract. Treatment protocols have been similarly supported by few evidence-based studies, and the most common elemental diets and supplemental agents are predominantly driven by custom rather than clinical trials. Nevertheless, clinical therapeutics targeting transporters on the surface of the small intestinal mucosa are available; the first such inhibitor targets dipeptidyl peptidase-4 (6, 7). With the advent of small intestinal endoscopy, we can now investigate the small intestinal mucosa and tissue by obtaining biopsies at multiple locations (8, 9). In patients with a small intestinal disorder, the small intestinal mucosa potentially loses the ability to perform certain functions (10). The aims of this study were to investigate the mrna and protein expressions of biomarkers related to nutrition in the GI mucosa and assess the relationships between small intestinal disease and nutrition. Materials and Methods Key inclusion criteria included patients who were years of age and scheduled to undergo total colonoscopy or double-balloon endoscopy (DBE), which accesses at least part of the small intestine in regular clinical practice. DBE was generally performed as previously described (11, 12). Key exclusion criteria included the following: (1) patients in whom it was difficult to reach the small intestine by endoscopy, (2) patients who had a poor general condition, namely, severe anemia, hypoalbuminemia, or fasting for more than one week, (3) patients from whom it was difficult to obtain informed consent, and (4) patients whom the physician judged to be ineligible. To analyze the distribution of biomarkers, the GI tract was divided into 6 parts (third portion of the duodenum, upper jejunum, deep jejunum, deep ileum, terminal ileum, and ascending colon). During colonoscopy, biopsies were obtained from the terminal ileum and the ascending colon. During anal route DBE, biopsies were obtained from the deep ileum in addition to the previously mentioned sections. During oral route DBE, biopsies were obtained from the third portion of the duodenum, upper jejunum, and deep jejunum. Two biopsy specimens were taken from normal mucosa in each section during endoscopy. One of the two specimens was soaked in an RNA stabilization solution for a mrna analysis. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction. RNA was transcribed to cdna by reverse transcription with a specific primer. The quantitative mrna expression of biomarkers, including transporters, was analyzed by realtime PCR (TaqMan method: StepOne, Applied Biosystems). GAPDH served as the endogenous control. We selected one sample that positively expressed each biomarker as the reference and evaluated the relative expression of each biomarker using the comparative Ct method. The other specimen was preserved in formalin for immunohistochemical staining. The specimen was fixed immediately in 4% paraformaldehyde in 0.1 M phosphate-buffered saline and embedded in paraffin, and 5 μm thick sections were mounted onto glass slides. A histological analysis of the biopsy samples was performed using Hematoxylin and Eosin (H&E)-stained slices from paraffin sections prior to immunohistochemical staining. These samples were confirmed to be normal mucosa according to the histological results. The details of the immunohistochemical staining have been described previously (13). The analyzed biomarkers included SGLT1 (glucose transporter), SGLT5 (fructose transporter), LAT1 (amino acid transporter), LAT2 (amino acid transporter), GIP, GLP (glucagon-like peptide), and NPC1L1. According to their final diagnosis, the enrolled patients were divided into the following 3 groups: Control, Inflammation, and Tumor. The Control group included patients in whom no gastrointestinal disorder was identified in the initial endoscopic procedure. When small bowel stenosis was found during DBE, balloon dilation was performed. In addition, biopsy specimens were obtained in front of and behind the stenosis, and the mrna expression levels were compared between these two locations. The primary endpoint of this study was the comparison of the longitudinal distribution of each biomarker in the GI tract among the 3 groups. Secondary endpoints included comparisons of the biomarker mrna expression in the each GI section among the 3 groups and of the distribution of the biomarker protein expression in the GI mucosa. This study was approved by the Ethics Committee of Nagoya University Graduate School of Medicine in This protocol was registered with the UMIN-CTR (UMIN ). The registered study focused on irritable bowel syndrome, but included the experimental study with regard to small bowel mucosa as a subanalysis. Written informed consent was obtained from each subject after explaining the purpose and possible complications of the study. Statistical analysis The statistical software package SPSS for Windows (SPSS, Chicago, USA) was used to analyze all data. The Mann-Whitney U test was used for comparisons of the relative mrna expression in each part according to the disease 2146

3 Figure 1. Number of endoscopies performed in the enrolled patients. type. In all the analyses, a p value of less than 0.05 was considered to be statistically significant. Results After providing their informed consent, 66 patients were enrolled in this study. There were 44 men, and the mean age of all the patients was 57±20 years. There were 25 patients in the Inflammation group, 25 in the Tumor group, and 16 in the Control group. The Inflammation group included 11 patients with Crohn s disease, 5 with ulcerative colitis, and 3 with intestinal tuberculosis as well as 6 patients classified as other. The Tumor group included 13 patients with colonic polyps, 3 with follicular lymphoma, 3 with Peutz-Jeghers syndrome and 2 with gastrointestinal stromal tumors as well as 4 patients classified as other. In total, 86 procedures were performed, and 452 biopsy specimens were obtained from 226 biopsy target sites (Fig. 1). H&E staining demonstrated that each biopsy sample included the epithelium, crypt and parts of the lamina propria. Longitudinal distribution of each biomarker in the gastrointestinal mucosa Fig. 1 shows that 20 patients underwent both routes of DBE and biopsy specimens were obtained from all 6 parts in these patients. This group included 8 Inflammation patients, 4 Tumor patients, and 8 Control patients. Quantitative analyses of the mrna expression of each biomarker were performed (Fig. 2). There were no remarkable differences among the 3 groups in the levels of any marker. The expression levels of GIP and NPC1L1 were the highest in the upper jejunum, but were extremely low in the terminal ileum and colon. The SGLT1 expression was similar to that of GIP and NPC1L1, but it was expressed in the terminal ileum. The GLP expression levels increased progressing toward the ileum, but it was not expressed in the colon. LAT1, LAT2, and SGLT5 did not show any specific expression patterns. Differences in the mrna expression profile in each GI section by the disease type Fig. 3 shows the number of obtained biopsy specimens according to the type of small bowel disorder. The Inflammation, Tumor, and Control groups did not have equal numbers of samples because this was an exploratory study. The mrna expression of each biomarker in each GI section was compared among the 3 groups (Table 1). The SGLT1 mrna expression in the Tumor group and the GIP mrna expression in the Inflammation group were significantly higher than the corresponding levels in the Control group in the upper jejunum (p=0.0161, ). However, none of the other mrna expression levels were significantly different among the 3 groups. Biomarkers in front of and behind the small intestinal stenosis Small bowel stenosis was detected in 4 lesions from 3 patients with established Crohn s disease. Balloon dilation was performed for all of these lesions. Biopsy specimens were subsequently obtained (Fig. 4). In 3 of the lesions, the GIP mrna expression in front of the stenosis was extremely high compared with that behind the stenosis. However, the mrna expression levels of the other biomarkers were not significantly different (Table 2). Distribution of biomarkers in the small bowel mucosa evidenced by immunohistochemical staining We conducted immunohistochemical staining to investigate the distribution of the biomarker protein expression in the GI mucosa. Fig. 5 shows positive immunohistochemical staining for the biomarker expression in the small bowel mucosa. We regarded brownish zones, areas, or dots to be positive for the protein expression. GIP-, GLP-, and NPC1L1-positive cells were mainly observed in the crypts. The expression of the other biomarkers, SGLT1, SGLT5, 2147

4 a b c d e f g Figure 2. The mrna expression of biomarkers in the GI mucosa. a: SGLT1: The expression level did not show any specific expression pattern. b: SGLT5: The expression level did not show any specific expression pattern. c: GIP: The expression level was the highest in the upper jejunum. d: GLP: The expression level increased progressing toward the ileum, but it was not expressed in the colon. e: LAT1: The expression level did not show any specific expression pattern. f: LAT2: The expression level did not show any specific expression pattern. g: NPC1L1: The expression level was the highest in the upper jejunum. LAT1, and LAT2, was spread uniformly in the villous epithelium and crypt. There were no positive cells in the lamina propria mucosa on any slides. 2148

5 Figure 3. The number of obtained biopsy specimens according to the type of small bowel disorder. Discussion This study is the first to report the distribution of nutritional biomarkers in the entire small intestinal mucosa in humans using endoscopic biopsy specimens. In addition, the biomarker distribution was not significantly different in patients with inflammatory disease or tumors compared with those in the Control group (Fig. 2). Therefore, it was hypothesized that the normal mucosa of the small intestine in patients with inflammatory disease or polyps has the same ability to obtain nutrition as that from healthy humans. The distribution of the SGLT1, GIP, GLP, and NPC1L1 expressions showed specific patterns. SGLT1 is a primary glucose transporter that is located on the surface of the jejunal mucosa, as demonstrated by the histopathology. Glucose can be absorbed in the wide area of the jejunum. GIP belongs to a class of molecules referred to as incretins. It is currently believed that the function of GIP is to induce insulin secretion, which is stimulated primarily by the hyperosmolarity of glucose in the duodenum (14). Therefore, it is reasonable that the peak GIP mrna expression was found in the jejunum. We previously investigated the relationships between the pancreas and the small intestine by evaluating endoscopic and histopathological findings of the proximal small intestine in patients with pancreatic diseases (13). We identified a close relationship between pancreatic juice and villous atrophy. Pancreatic juice affects the function of GIP, NPC1L1, and SGLT1, which are expressed mainly in the jejunum, rather than the function of GLP. SGLT1 was highly expressed in L cells from the small intestine, consistent with its known role in epithelial glucose absorption. Reimann et al. determined via immunofluorescence staining that SGLT1 localized to the apical membrane of ileal enterocytes and L cells, and we found the same result through immunohistochemical staining (15). GLP release is triggered by the ingestion of carbohydrates, fats, and protein and is believed to reflect, at least in part, the direct sensing of luminal nutrients by the apical processes of L cells. GLP release is also induced by SGLT1 secretion (16). We demonstrated that the expression of SGLT1 was highest in the jejunum and that the GLP expression was maximal in the ileum. These findings are reasonable given our current understanding of glucose absorption. GLP consists of GLP-1 and GLP-2, which are produced by intestinal endocrine L cells and co-secreted upon nutrient ingestion. GLP was generally believed to be produced by L cells in the lower intestine and colon (17-19); however, we demonstrated that both GLP-1 and GLP-2 were present throughout the small bowel, although there were quantitative differences among regions. Using the mrna expression and immunohistochemical staining analyses, we showed that human NPC1L1 is predominantly expressed in the jejunal mucosa and is not expressed in the terminal ileum or the ascending colon. Selective cholesterol absorption inhibitors, such as ezetimibe, inhibit the uptake and absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. These inhibitors directly act on NPC1L1 on the surface of the small intestinal mucosa. According to our analysis, we suggest that cholesterol cannot be absorbed in the distal ileum. Therefore, it is hypothesized that the jejunum is more responsible for the absorption of sugar groups and lipids than the ileum. The SGLT5, LAT1, and LAT2 expressions were observed at all parts of the GI tract without any particular expression pattern of these biomarkers. Immunohistochemical staining showed that nearly all epithelial cells were positive for these three biomarkers. Therefore, they are potential systemic factors. In this study, normal mucosae from patients with sus- 2149

6 Table 1. Relative Expression Level of mrna in Each Part according to Diseases. Control Tumor Inflammation p value Duodenum (n) SGLT1 2.70± ± ±0.67 n.s. SGLT5 0.14± ± ±0.03 n.s. LAT1 1.2± ± ±0.11 n.s. LAT2 1.07± ± ±0.17 n.s. GIP ± ± ±177.5 n.s. GLP 0.1± ± ±0.04 n.s. NPC1L ± ± ±35.69 n.s. Upper jejunum (n) SGLT1 2.90± ±0.76 * 3.25±0.517 * (vs.control) SGLT5 0.33± ±0.06 * 0.16±0.06 * (vs.control) LAT1 1.09± ± ±0.08 n.s. LAT2 0.92± ± ±0.18 n.s. GIP ± ± ±154.91* * (vs.control) GLP 0.28± ± ±0.1 n.s. NPC1L ± ± ±83.84 n.s. Deep jejunum (n) SGLT1 1.94± ± ±0.41 n.s. SGLT5 0.36± ± ±0.07 n.s. LAT1 0.96± ± ±0.11 n.s. LAT2 1.14± ± ±0.23 n.s. GIP ± ± ± n.s. GLP 1.06± ± ±0.18 n.s. NPC1L ± ± ±96.01 n.s. Deep ileum (n) SGLT1 2.66± ± ±0.27 n.s. SGLT5 0.11± ± ±0.02 n.s. LAT1 0.71± ± ±0.09 n.s. LAT2 1.08± ± ±0.19 n.s. GIP 91.73± ± ±30.45 n.s. GLP 1.49± ± ±0.41 n.s. NPC1L ± ± ±37.15 n.s. Termunal ileum (n) SGLT1 1.29± ± ±0.22 n.s. SGLT5 0.1± ±0.07 * 0.07±0.03 * (vs. Inflammation) LAT1 1.07± ± ±0.13 n.s. LAT2 1.78± ± ±0.24 n.s. GIP 2.62± ± ±2.71 n.s. GLP 7.64± ± ±2.09 n.s. NPC1L1 0.17± ± ±0.04 n.s. Ascending colon (n) SGLT1 0.06± ± ±0.01 n.s. SGLT5 0.17± ± ±0.1 n.s. LAT1 0.43± ± ±0.11 n.s. LAT2 1.04± ± ±0.3 n.s. GIP 0.01± ± ±0.02 n.s. GLP 0.17± ± ±0.04 n.s. NPC1L1 0.11± ± ±0.03 n.s. pected or established small intestinal disorders were analyzed. These normal mucosa samples were confirmed by histology. There were no differences in most comparisons among the 3 groups, except for in the upper jejunum, where the SGLT1 expression in the Tumor group and the GIP expression in the Inflammation group were significantly 2150

7 Figure 4. Balloon dilation and biopsies performed at locations of small intestinal stenosis in patients with Crohn s disease. Figure 5. Immunohistochemical staining for biomarkers in the small intestinal mucosa. Immunohistochemical staining showed GIP-, GLP-, and NPC1L1-positive cells mainly in the crypts. The expression patterns of the other biomarkers, SGLT1, SGLT5, LAT1, and LAT2, was spread uniformly in the villous epithelium and crypt. Table 2. Comparisons of the Relative Expression of mrna between Pre- and Post-small Bowel Stenosis in Crohn s Disease. Case 1 Case 2 Case 3 Case 4 (Deep jejunum) (Deep ileum) (Deep ileum) (Deep ileum) Pre Post Pre Post Pre Post Pre Post SGLT SGLT LAT LAT GIP GLP NPC1L higher. The upper jejunum may be easily damaged from a nutritional perspective; it is the most important section for nutritional absorption and/or malabsorption, even if the mucosa is normal. Small bowel stenosis may affect the absorption of nutritional agents because the nutritional supply will be insufficient at the anal end. Bacterial overgrowth may develop in front of the stenosis, and these bacteria consume energy from ingested food. In 3 of the 4 stenotic lesions, the GIP expression decreased only on the anal side of the stenosis. In addition, the differences in the GIP mrna expression were marked between the jejunum and the ileum in the distribution analysis. GIP may be a candidate starvation marker of the GI tract because the other biomarkers did not show differences in this region. There are some limitations associated with this study. Patients with small bowel disorders were divided into only 3 groups, however, these groups were not simple. For in- 2151

8 stance, patients with ulcerative colitis or Crohn s disease were placed in the same group, although clinically these patients exhibit many differences. Comparisons of the normal mucosa in the 3 groups did not reveal any differences, however, future investigations of biomarkers within the lesions in patients in these 3 groups or with certain diseases are necessary. Although this study focused on nutritional markers in the small intestinal mucosa and revealed their structure and distribution, further studies of their function should also be conducted. If such biomarkers cannot function normally, then the absorption of nutritional agents will be insufficient. In conclusion, this study demonstrated the distribution of nutrition-related biomarkers on the surface of GI mucosa. Even in patients with small bowel diseases, the GI mucosa may have the ability to maintain nutrient absorption, except in the upper jejunum. The authors state that they have no Conflict of Interest (COI). Acknowledgement The authors wish to thank Mr. Kuniyoshi Kito and Ms. Akina Oishi for their valuable technical support of this study. Financial Support This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology Japan. A part of this study was presented at the Digestive Disease Week Conference in the US (Washington, May 2015). References 1. Nisim AA, Allins AD. Enteral nutrition support. Nutrition 21: , Deacon CF, Ahrén B. Physiology of incretins in health and disease. Rev Diabet Stud 8: , Davis HR Jr, Altmann SW. Niemann-Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter. Biochim Biophys Acta 1791: , Damholt AB, Kofod H, Buchan A. Immunocytochemical evidence for a paracrine interaction between GIP and GLP-1-producing cells in canine small intestine. Cell Tissue Res 298: , Gilor C, Gilor S, Graves TK, et al. Distribution of K and L cells in the feline intestinal tract. Domest Anim Endocrinol 45: 49-54, Phan BA, Dayspring TD, Toth PP. Ezetimibe therapy: mechanism of action and clinical update. Vasc Health Risk Manag 8: , Karagiannis T, Paschos P, Paletas K, Matthews DR, Tsapas A. Dipeptidyl peptidase-4 inhibitors for treatment of type 2 diabetes mellitus in the clinical setting: systematic review and metaanalysis. BMJ 344: e1369, Yamamoto H, Kita H, Sunada K, et al. Clinical outcomes of double-balloon endoscopy for the diagnosis and treatment of small-intestinal diseases. Clin Gastroenterol Hepatol 2: , Rhee NA, Vilmann P, Hassan H, et al. The use of double-balloon enteroscopy in retrieving mucosal biopsies from the entire human gastrointestinal tract. Scand J Gastroenterol 49: , Takenaka H, Ohmiya N, Hirooka Y, et al. Endoscopic and imaging findings in protein-losing enteropathy. J Clin Gastroenterol 46: , Nakamura M, Niwa Y, Ohmiya N, et al. Preliminary comparison of capsule endoscopy and double-balloon enteroscopy in patients with suspected small-bowel bleeding. Endoscopy 38: 59-66, Nakamura M, Ohmiya N, Shirai O, et al. Route selection for double-balloon endoscopy, based on capsule transit time, in obscure gastrointestinal bleeding. J Gastroenterol 45: , Nakamura Y, Itoh A, Kawashima H, et al. Investigation of morphological and functional changes in the small intestine with pancreatic disease. Pancreas 44: , Thorens B. Glucagon-like peptide-1 and control of insulin secretion. Diabete Metab 21: , Reimann F, Habib AM, Tolhurst G, Parker HE, Rogers GJ, Gribble FM. Glucose sensing in L cells: a primary cell study. Cell Metab 8: , Gorboulev V, Schürmann A, Vallon V, et al. Na + -D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion. Diabetes 61: , Topstad D, Martin G, Sigalet D. Systemic GLP-2 levels do not limit adaptation after distal intestinal resection. J Pediatr Surg 36: , Jeppesen PB, Hartmann B, Thulesen J, et al. Elevated plasma glucagon-like peptide 1 and 2 concentrations in ileum resected short bowel patients with a preserved colon. Gut 47: , Jeppesen PB, Hartmann B, Hansen BS, Thulesen J, Holst JJ, Mortensen PB. Impaired meal stimulated glucagon-like peptide 2 response in ileal resected short bowel patients with intestinal failure. Gut 45: , The Japanese Society of Internal Medicine