Ileo-colonic delivery of conjugated bile acids improves glucose homeostasis via colonic GLP-1-producing enteroendocrine cells in human obesity and diabetes

Abstract

Background: The bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes.

Methods: We conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes.

Findings: Human colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine.

Interpretations: In humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876.

Funding: Research support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology.

Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1

Co-Expression of GLP-1 with BA pathway signalling molecules in human colon and in vitro NCI-H716 L-cell model. A) Double immunofluorescence staining of whole human colonic tissue zoomed to GLP-1 (green) positive EECs co-expressing TGR5 (pink), with channel and merged displays of staining. Red bars for all images indicate 10 µm. Differentiated NCI-H716 cells, incubated with 1 mM CBAS in control media or control media alone for 2 h increments over the course of 6 h. Media was replaced in-between time point. Resulting culture supernatants were assayed for protein levels of b) GLP-1 c) FGF19. Differentiated H716 cell mRNA expression of d) GCG and e) FGF19 after 24 h treatments with 1 mM CBAS, 1 mM CDCA, compared to control media expression. Data are presented as mean±SEM. Significance testing used the student t-test for protein secretion studies, and ANOVA for mRNA expression; *p<0·05, **p<0·01, ***p<0·001.

Fig. 2

Co-Expression of TGR5 in FACS-isolated human colonic EEC a) diagram and b) corresponding FACS-plots demonstrating a sequential flow-cytometric gating strategy to isolate GLP-1+ EEC from single cells suspensions of human colonic biopsies. c) Triple immunofluorescence staining of (from top) GLP-1+ FACS-isolated human enteroendocrine cells, GLP-1- FACS-isolated human colonic cells, and no primary antibody controls. Both sorted GLP-1 positive and negative cells contained populations of TGR5 (pink) compared to negative control. D) Quantification of TGR-5 positive cells in GLP-1+ and GLP-1- FACS-isolated human colonic cells. Red bar indicates 10 µm. Data are presented as mean±SEM. Significance testing used the student t-test; *p<0·05, **p<0·01, ***p<0·001.

Fig. 3

Basolateral application of taurodeoxycholic acid triggers stronger GLP-1 response compared to apical application in human and mouse colon. GLP-1 release was measured in the basolateral compartment following addition of taurodeoxycholic Acid (TDCA, 100 μM) to the apical (AP) or basolateral (BAS) compartments of colonic tissue sections of a) human and b) mouse mounted in Ussing chambers. Water was added for control experiments (CTR). Human: Adj p<0·05 AP vs CTR, p<0·0001 BAS vs CTR, p<0·001 AP vs BAS; Mouse: p<0·01 BAS vs CTR, p<0·01 AP vs BAS. c) Double immunofluorescence staining of whole human colonic tissue with GLP-1 (green) positive EEC and colonocytes producing ASBT (pink) with individual channel and merged displays of staining. Red bars for all images indicate 20 µm.

Fig. 4

Fasting serum Fibroblast Growth Factor (FGF) 19, 7aC4 and bile acids levels in patients with healthy weight, overweight and obesity based on BMI and type 2 diabetes. a) Fasting FGF19, c) serum fasting bile acids, and e) fasting 7aC4 in participants with normal weight, overweigh and obesity; b) Fasting FGF19, d) serum fasting bile acids, and f) fasting 7aC4 in participants with obesity with or without diabetes, Data are represented as median [IQR]. ANOVA*p<0·05.

Fig. 5

Effect of Ileocolonic delivery of conjugated bile acids (IC-CBAS) in glucose homeostasis and glucose regulation in patients with obesity and type 2 diabetes. (a,b) Glucose concentrations at baseline and after treatment with (a) placebo, and (b) with IC—CBAS (grey curve= at baseline; black curve= after treatment). Arrows at 0 and 240 min indicate meal ingestion. (c) delta fasting glucose, (d) delta AAB00-120 min and (e) delta AUC0-120 min of postprandial glucose, and (f) delta fasting fructosamine after treatment period. <GLP-1 concentrations at baseline and after treatment with g) placebo and h) IC—CBAS (i) delta GLP-1 AUC0-360 min, (j) delta AAB0-360 min c-peptide, (k) delta fasting insulin, and (l) delta AAB0-120 min insulin after treatment period. Data are presented as mean±SEM. Wilcoxon test treatment vs. placebo: #p<0·1. *p<0·05, **p<0·01.

Fig. 6

Effect of Ileocolonic delivery of conjugated bile acids (IC-CBAS) on bile acid pathways in patients with obesity and type 2 diabetes. (a) Total faecal bile acids (circles, baseline; and squares, treatment); (b) delta total fecal bile acids and (c) delta primary faecal bile acids; (d) delta total faecal bile acids by group (circles, placebo; and squares, IC-CBAS); (e) delta conjugated faecal bile acids, (f) delta postprandial serum bile acids, (g) delta C4, (h) delta fasting FGF19, (i) delta fasting total cholesterol, (j) delta fasting cLDL, (k) correlation between ∆ weight and ∆ faecal bile acids; (l) correlation between ∆ fructosamine with ∆ faecal bile acids after 1-month treatment with IC-CBAS, and (m). correlation between ∆ postprandial GLP-1 at 45 min with ∆ faecal bile acids after 1-month treatment with IC-CBAS Data are presented as mean±SEM. Wilcoxon, *p<0·05, ** p<0·01. ANCOVA, ^p<0·05, (Figs. a–j).