Deregulation of transcription factors controlling intestinal epithelial cell differentiation; a predisposing factor for reduced enteroendocrine cell number in morbidly obese individuals

Abstract

Morbidly obese patients exhibit impaired secretion of gut hormones that may contribute to the development of obesity. After bariatric surgery there is a dramatic increase in gut hormone release. In this study, gastric and duodenal tissues were endoscopically collected from lean, and morbidly obese subjects before and 3 months after laparoscopic sleeve gastrectomy (LSG). Tissue morphology, abundance of chromogranin A, gut hormones, α-defensin, mucin 2, Na⁺/glucose co-transporter 1 (SGLT1) and transcription factors, Hes1, HATH1, NeuroD1, and Ngn3, were determined. In obese patients, the total number of enteroendocrine cells (EEC) and EECs containing gut hormones were significantly reduced in the stomach and duodenum, compared to lean, and returned to normality post-LSG. No changes in villus height/crypt depth were observed. A significant increase in mucin 2 and SGLT1 expression was detected in the obese duodenum. Expression levels of transcription factors required for differentiation of absorptive and secretory cell lineages were altered. We propose that in obesity, there is deregulation in differentiation of intestinal epithelial cell lineages that may influence the levels of released gut hormones. Post-LSG cellular differentiation profile is restored. An understanding of molecular mechanisms controlling epithelial cell differentiation in the obese intestine assists in the development of non-invasive therapeutic strategies.

Figure 1

Expression of chromogranin A, CCK and ghrelin proteins in human duodenal biopsies. Profile of the enteroendocrine cell marker, chromogranin A (A, ChA) and the gut hormones CCK (B) and ghrelin (C), was determined in the duodenal biopsies of lean (■), obese () and post-operative obese, (□) by immunohistochemistry. Bar charts (on the right) show number of cells counted expressing ChA or gut hormones. Statistical significance was determined by a One-way ANOVA with differences between means identified using a Holm-Sidak multiple comparison post-test where, *p < 0.05, **p < 0.01 and ***p < 0.001. Scale bars are either 100 or 50 µm. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (Dapi).

Figure 2

Expression profile of chromogranin A mRNA in duodenal biopsies. Relative mRNA abundance in lean (■) and obese () duodenum for ChA (enteroendocrine cell marker) determined by qPCR. Values are means ± SEM, normalised to RNA polymerase IIA (POLR2A) m RNA expression, n = 4. Statistical significance was determined by a Student’s t-test where *p < 0.05.

Figure 3

PYY, GLP-1 and GLP-2 protein expression in human duodenal biopsies. Profile of gut hormones, PYY (A), GLP-1 (B) and GLP-2 (C) was determined in the duodenal biopsies of lean (■), obese () and post-operative obese, (□) by immunohistochemistry. Bar charts (on the right) show number of cells counted expressing the gut hormones. Statistical significance was determined by a One-way ANOVA with differences between means identified using a Holm-Sidak multiple comparison post-test test where *p < 0.05, ***p < 0.001. Scale bars are either 100, 50 or 25 µm. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (Dapi).

Figure 4

Expression profile of defensin5 and mucin-2 in duodenal biopsies. (A) Relative mRNA abundance in lean (■) and obese () duodenum for DEFA5 (Paneth cell marker) and MUC2 (goblet cell marker) determined by qPCR. Values are means ± SEM, normalised to RNA polymerase IIA (POLR2A) m RNA expression, n = 4. (B) Profile of defensin 5 (DEFA5; red) and mucin 2 (MUC2; green) determined in lean (■), obese () and post-operative obese, (□) duodenum by immunohistochemistry. Bar charts (on the right) show total number of cells counted expressing either DEFA5 or MUC2. Statistical significance was determined by a Student’s t-test (A) or One-way ANOVA with differences between means identified using Tukey’s multiple comparison post-test (B) where **p < 0.01; ***p < 0.001. Scale bars are 25 µm. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (Dapi).

Figure 5

SGLT1, Hes1, NeuroD1, Ngn3, and HATH1 protein expression (western blot) in duodenal biopsies of lean, obese and post-operative. Protein contents of BBMV (20 µg protein), or cell lysates (100 µg protein) separated from duodenal biopsies were subjected to western blot analysis using antibodies to SGLT1 (A), transcription factors: Hes1 (B), NeuroD1 (C), Ngn3 (D), HATH1 (E) and β-actin; the latter was used as a loading control. Upper panel shows abundance of SGLT1, the transcription factor and β-actin proteins determined by western blots and lower panel depicts the densitometric analysis of western blots normalising protein abundance to that of β–actin: in lean (L) (■), obese (Ob) () and post-operative obese, (POb) (□) individuals. Results are shown as mean ± SEM; n = 7–10. Statistically significant results determined using a One-way ANOVA with differences between means identified using a Holm-Sidak multiple comparison post-test where *P < 0.05 and ***P < 0.001. For clarity, boxed western blot images are displayed. Full length blots are shown in Supplementary Figure S4.