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Original Investigation | Association of VA Surgeons, SURGICAL CARE OF THE AGING POPULATION

Glucagonlike Peptide 2 Analogue Teduglutide:  Stimulation of Proliferation but Reduction of Differentiation in Human Caco-2 Intestinal Epithelial Cells

Lakshmi S. Chaturvedi, PhD1,2,3; Marc D. Basson, MD, PhD, MBA1,2
[+] Author Affiliations
1Department of Surgery, College of Human Medicine, Michigan State University, East Lansing
2Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan
3Department of Anesthesiology, Wayne State University, Detroit, Michigan
JAMA Surg. 2013;148(11):1037-1042. doi:10.1001/jamasurg.2013.3731.
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Importance  Short bowel syndrome occurs when a shortened intestine cannot absorb sufficient nutrients or fluids. Teduglutide is a recombinant analogue of human glucagonlike peptide 2 that reduces dependence on parenteral nutrition in patients with short bowel syndrome by promoting enterocytic proliferation, increasing the absorptive surface area. However, enterocyte function depends not only on the number of cells that are present but also on differentiated features that facilitate nutrient absorption and digestion.

Objective  To test the hypothesis that teduglutide impairs human intestinal epithelial differentiation.

Design and Setting  We investigated the effects of teduglutide in the modulation of proliferation and differentiation in human Caco-2 intestinal epithelial cells at a basic science laboratory. This was an in vitro study using Caco-2 cells, a human-derived intestinal epithelial cell line commonly used to model enterocytic biology.

Exposure  Cells were exposed to teduglutide or vehicle control.

Main Outcomes and Measures  We analyzed the cell cycle by bromodeoxyuridine incorporation or propidium iodide staining and flow cytometry and measured cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. We used quantitative reverse transcription–polymerase chain reaction to assay the expression of the enterocytic differentiation markers villin, sucrase-isomaltase, glucose transporter 2 (GLUT2), and dipeptidyl peptidase 4 (DPP-4), as well as that of the putative differentiation signals schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2). Villin promoter activity was measured by a luciferase-based assay.

Results  The MTS assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated controls at a maximal 500nM (n = 6, P < .05). Teduglutide increased bromodeoxyuridine-positive cells vs untreated controls by a mean (SD) of 19.4% (2.3%) vs 12.0% (0.8%) (n = 6, P < .05) and increased the S-phase fraction by flow cytometric analysis. Teduglutide reduced the mean (SD) expression of villin by 29% (6%), Cdx2 by 31% (10%), DPP-4 by 15% (6%), GLUT2 by 40% (11%), SLFN12 by 61% (14%), and sucrase-isomaltase by 28% (8%) (n = 6, P < .05 for all).

Conclusions and Relevance  Teduglutide increased Caco-2 proliferation but tended to inhibit intestinal epithelial differentiation. The effects of mitogenic stimulation with teduglutide in patients with short bowel syndrome might be greater if the more numerous teduglutide-treated cells could be stimulated toward a more fully differentiated phenotype.

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Figure 1.
Effect of Teduglutide on Human Caco-2 Intestinal Epithelial Cell Proliferation

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) dye assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated control cells at a maximal 500nM (n = 6, P < .05). A, Teduglutide (250-1000nM) significantly increased cell numbers compared with untreated control cells as measured by the MTS assay. B, Teduglutide (500nM) and human epidermal growth factor (EGF) (50 ng/mL), used as a positive control, significantly increased the proportion of bromodeoxyuridine (BrdU)–positive cells vs untreated control cell populations.aP < .05. Gray bar indicates treated cells. The plus sign and minus sign indicate whether the cells received or did not receive what is written on the side in the figure.

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Figure 2.
Effect of Teduglutide on the Human Caco-2 Intestinal Epithelial Cell Cycle

Teduglutide increased bromodeoxyuridine-positive cells vs untreated control cells by a mean (SD) of 19.4% (2.3%) vs 12.0% (0.8%) (n = 6, P < .05) and increased the S-phase fraction by flow cytometric analysis. Teduglutide (500nM) significantly increased the diploid (Dip) S-phase fraction compared with untreated control cells as measured by flow cytometry. A, Typical cell cycle distribution. B, Our results. PI-A indicates propidium iodide A.aP < .05. Black bar indicates treated cells. The plus sign and minus sign indicate whether the cells received or did not receive what is written on the side in the figure.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Effect of Teduglutide on the Expression of Differentiation Marker Transcripts in Human Caco-2 Intestinal Epithelial Cells

Teduglutide reduced the expression of villin, dipeptidyl peptidase 4 (DPP-4), sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) differentiation markers compared with untreated control cells as measured by quantitative reverse transcription–polymerase chain reaction (n = 6, P < .05 for all). mRNA indicates messenger RNA; rRNA, ribosomal RNA.aP < .05. The plus sign and minus sign indicate whether the cells received or did not receive what is written on the side in the figure.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.
Effect of Teduglutide on the Expression of 2 Intracellular Signal Proteins That May Influence Differentiation in Human Caco-2 Intestinal Epithelial Cells

Teduglutide reduced the expression of schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2) compared with untreated control cells as measured by quantitative reverse transcription–polymerase chain reaction (n = 6, P < .05 for both). mRNA indicates messenger RNA; rRNA, ribosomal RNA.aP < .05. The plus sign and minus sign indicate whether the cells received or did not receive what is written on the side in the figure.

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