516 UROCORTIN 3 EXPRESSION IN HUMAN ENTEROENDOCRINE CELLS AND CORRELATION WITH PLASMA GLP1 LEVELS IN HUMAN OBESITY

Abstract

Introduction Enteroendocrine cells (EECs) are hormone secretory cells sparsely distributed throughout the GI tract, and implicated in obesity. Intestinal L-type EECs secrete Glucagon-Like peptide 1(GLP-1) in response to a meal to promote satiety and maintain glucose homeostasis. Abnormal post prandial satiety (APS) is a phenotypic subset of obesity characterized by blunted post-prandial levels of GLP-1. Intestinal D-type EECs release somatostatin (SST) to inhibit GLP-1 release from L cells via the STT-receptor. In the pancreas, somatostatin release from delta cell is stimulated by pancreatic urocortin-3 from the beta cells. It is unknown whether urocortin-3 is expressed in intestinal EEC cells. Thus, we aimed to study the role of UCN3 in intestinal L, and D- type EECs, and its association with GLP-1 secretion in human obesity. Methods We studied a total of 34 participants, 17 healthy controls (age 38±13y, BMI 23±2kg/m2, 59% female), and 17 with obesity (age 39±9y, BMI 37±5kg/m2, 65% female). Obesity was sub-grouped into: Abnormal postprandial satiety (APS, low postprandial GLP-1, n=6) and normal post prandial satiety (NPS, normal postprandial GLP-1, n=11). Human colonic biopsies from participants were obtained by sigmoidoscopy and preserved for scRNA-seq (CryoStorCS10), gene expression (RNAlater), or microscopy studies (10%NBF). Single-cell transcriptomics was completed using the Chromium 10X Genomics platform followed by Illumina HiSeq4000 sequencing. Blood was collected from participants at fasting, and postprandial at 15, 45, and 90 minutes. Immunofluorescence (IF) confocal microscopy was used to detect protein expression of UCN3 in L-type EECs in the gut mucosa. UCN3 mRNA expression was determined by RT-qPCR. Blood GLP-1 levels were determined by ELISA. Results Our transcriptomic investigation of EECs in human obesity revealed expression of UCN3 in Chromogranin A-positive EECs, and GLP-1 positive L-type EECs, previously not known to be expressed in EECs (Fig1A). Protein expression of UCN3 in GLP-1 positive EEC was confirmed using immunofluorescence in human ileum and colon (Fig 1B). Colonic UCN3 mRNA expression was positively associated with BMI (r=0.43, p Conclusions These findings suggest that intestinal UCN3 is overexpressed in obesity, and further overexpressed in APS obesity phenotype. This study introduces a new pathophysiological underpinning of obesity where negative feedback mechanisms of SST-mediated GLP-1 inhibition are exaggerated, and may potentially promote blunted postprandial GLP-1 levels and decreased satiety in obesity. Download : Download high-res image (100KB) Download : Download full-size image Figure 1 . Expression of Urocortin 3 (UCN3) in human enteroendocrine cells (EECs). (A) Ratio of cell population clusters of the human colonic mucosa in either all EECs or just GCG-expressing L-Type EECs, expressing UCN3 of 10 patients (n=4 lean, n=6 obesityall). (B) Double immunoflurescent staining of the human intestinal mucosa demonstrating expression of UCN3 (Red) in a subset of GLP-1 (Green) L-type EECs. Red bar indicates 20µm. Download : Download high-res image (95KB) Download : Download full-size image Figure 2 . Urocortin 3 (UCN3) Associations with BMI and GLP-1 Release. (A) Association of colonic UCN3 mRNA expression (A) to BMI (kg/m2). Relative Human colonic mucosal mRNA expression of UCN3 comparing (B) lean and all-obesity; and (C) lean and obesity with and without APS. Association of intestinal UCN3 expression with (D) plasma concentrations of GLP-1 measured 45 and (C) resulting association of GLP-1 area under the curve (AUC). Significance testing used a linear regression for A,D, E, and a two-tailed student t-test compared between all groupings; # p