Enhanced Ghrelin Levels and Hypothalamic Orexigenic AgRP and NPY Neuropeptide Expression in Models of Jejuno-Colonic Short Bowel Syndrome

Abstract

Short bowel syndrome (SBS) patients developing hyperphagia have a better outcome. Gastrointestinal endocrine adaptations help to improve intestinal functions and food behaviour. We investigated neuroendocrine adaptations in SBS patients and rat models with jejuno-ileal (IR-JI) or jejuno-colonic (IR-JC) anastomosis with and without parenteral nutrition. Circulating levels of ghrelin, PYY, GLP-1, and GLP-2 were determined in SBS rat models and patients. Levels of mRNA for proglucagon, PYY and for hypothalamic neuropeptides were quantified by qRT-PCR in SBS rat models. Histology and immunostaining for Ki67, GLP-1 and PYY were performed in SBS rats. IR-JC rats, but not IR-JI, exhibited significantly higher crypt depths and number of Ki67-positive cells than sham. Fasting and/or postprandial plasma ghrelin and PYY concentrations were higher, or tend to be higher, in IR-JC rats and SBS-JC patients than in controls. Proglucagon and Pyy mRNA levels were significantly enhanced in IR-JC rats. Levels of mRNA coding hypothalamic orexigenic NPY and AgRP peptides were significantly higher in IR-JC than in sham rats. We demonstrate an increase of plasma ghrelin concentrations, major changes in hypothalamic neuropeptides levels and greater induction of PYY in SBS-JC rats and patients suggesting that jejuno-colonic continuity creates a peculiar environment promoting further gut-brain adaptations.

Figure 1. Body weight loss, plasma leptin levels and histological adaptation of the remaining colon mucosa after intestinal resection in rats.

(A) Schematic representation of the surgeries and colonic tissue collections at day 7 post-surgery. Length of resected or remnant part of intestinal segments are not proportional. (B) Comparative time-dependent body weight loss represented as percent of the preoperative weight and (C) total calorie intake (oral intake and parenteral nutrition) in IR jejuno-ileal (IR-JI), IR jejuno-colonic (IR-JC), IR jejuno-colonic PN (IR-JC PN) and sham-operated (Sham) rats during 7 days after surgery. Black box corresponds to the period of the postoperative care. (D,E) Plasma levels of leptin (D) and albumin (E) in the same groups. Data are represented as mean ± SEM of n = 6 for sham, n = 4 to 6 for IR jejuno-ileal, n = 10 for IR jejuno-colonic, n = 5 to 6 for IR jejuno-colonic with PN. *P < 0.05, **P < 0.01, vs sham-operated rats based on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons.

Figure 2. histological adaptation of the remaining colon mucosa after intestinal resection in rats.

(A) Representative photomicrographs of colon mucosa of Sham, IR jejuno-ileal (JI), IR jejuno-colonic (JC) and IR jejuno-colonic with PN (JC-PN) rats immunostained with an anti-Ki67 antibody. (B) Measurement of colon mucosa crypt depth in μm (at least 5 crypts analyzed by rat). (C) Quantification of Ki67 positive cells per crypt (at least 5 crypts analyzed by rat), (D) density of Ki67 positive cells expressed as number of Ki67 positive cells per 100 μm of crypt. (E,F) Representative photomicrographs of colon mucosa of sham and IR-JC rats stained with Periodic Acid Schiff (PAS) (E) and Representative photomicrographs of colon mucosa of sham and IR-JC rats immunostained with anti-Muc2 antibody (F) with quantification of Muc2 positive cells per area in mm². Data are represented as mean ± SEM of n = 6 for sham, n = 4 to 6 for IR jejuno-ileal, n = 10 for IR jejuno-colonic, n = 5 to 6 for for IR jejuno-colonic with PN. *P < 0.05, **P < 0.01, vs sham-operated rats based on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons. Scale bar: 50 μm.

Figure 3. Increased levels of plasma gut hormones after intestinal resection in rats.

Fasting plasma levels of ghrelin (A), GLP-1 (B) GLP-2 (C) and PYY (D) in IR jejuno-ileal (JI), IR jejuno-colonic (JC), IR jejuno-colonic with PN (JC-PN) and sham-operated (Sham) overnight fasted rats 7 days after surgery. Data are represented as mean ± SEM of n = 6 for sham, n = 4 or not available (NA) for IR-JI, n = 4 to 10 for IR JC, n = 4 to 6 or not available (NA) for IR-JC PN. *P < 0.05, **P < 0.01, vs sham-operated rats based on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons.

Figure 4. Unchanged number of PYY and GLP-1 positive cells but enhanced levels of proglucagon and Pyy mRNA after intestinal resection in rats.

(A,B) Representative photomicrographs of GLP-1 (A) and PYY (B) immunostaining on colon mucosa sections from sham-operated and IR-JC rats (cytoplasm of GLP-1 or PYY cells are stained in brown), scale bar:50 μm. (C,D) Quantification of GLP-1 (C) and PYY (D) positive cells per area in mm². Data are represented as mean ± SEM of n = 4 for sham, n = 4 or not available (NA) for IR jejuno-ileal (JI), n = 5 for IR jejuno-colonic (JC), n = 6 for for IR jejuno-colonic with PN (JC-PN). (E–F) Colonic mucosa mRNA levels of proglucagon (Gcg) (E) and Pyy (F) normalized to L19, 7 days after surgery. Data are represented as mean ± SEM of n = 6 for sham, n = 5 for IR-JI, n = 9 for IR-JC, n = 6 for IR-JC with PN. *P < 0.05, **P < 0.01, ***P < 0.001 vs sham-operated rats and ^#P < 0.05 ^##P < 0.01 vs IR-JC based on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons.

Figure 5. Expression of mRNA coding for hypothalamic orexigenic (AgRP, NPY) and anorexigenic (POMC, CART) neuropeptides after intestinal resection in rats.

Expression of (A) Agrp, (B) Npy, (C) Pomc and (D) Cart mRNA normalized to Hprt mRNA in hypothalamus in IR jejuno-ileal (JI), IR jejuno-colonic (JC), IR jejuno-colonic with PN (JC-PN) and sham-operated (Sham) rats 7 days after surgery. Data are represented as mean ± SEM of n = 6 for sham, n = 5 for IR-JI, n = 10 for IR-JC, n = 6 for for IR-JC with PN.*P < 0.05, ***P < 0.001 vs sham-operated rats based on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons.

Figure 6. Increased fasting and post-prandial levels of plasma active Ghrelin, GLP-1, GLP2 and PYY in SBS patients.

Plasma concentrations and secretory response to a calibrated meal (750 kcal) in SBS jejuno-ileal (JI) and SBS jejuno-colonic (JC) patients compared to healthy subjects (C) of: (A,B) active ghrelin (pg/mL) before (T0) and after (T30, T90) the meal (A) and ghrelin secretory response expressed as the total area under the curve (AUC) (B) GLP-1 (pg/mL) before (T0) and after (T30, T90) the meal (C) and GLP-1 secretory response expressed as the incremental area under the curve (iAUC) (D); (E,F) GLP-2 (pg/mL) before (T0) and after (T30, T90) the meal (E) and secretory response expressed as the iAUC (F PYY (pg/mL) before (T0) and after (T30, T90) the meal (G), and secretory response expressed as the iAUC (H). All values are expressed as the mean ± S.E.M of n = 5 for healthy subjects, n = 5 for jejuno-ileal SBS patients and n = 4 for jejuno-colonic SBS patients. *P < 0.05, **P < 0.01, ***P < 0.001 vs healthy subjects and ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs jejuno-ileal SBS patients based for A, C, E, G, on Bonferroni’s multiple comparisons test and for B, D, F, H on non-parametric Kruskal-Wallis test followed by Dunn’s adjusted multiple comparisons.