Molecular Mechanisms Underlying Glucose-dependent Insulinotropic Polypeptide Secretion in Human Duodenal Organoids

Overview

Journal Diabetologia

Specialty Endocrinology

Date 2024 Oct 23

PMID 39441374

Authors

Nunzio Guccio

Constanza Alcaino

Emily L Miedzybrodzka

Marta Santos-Hernandez

Christopher A Smith

Adam Davison

Rula Bany Bakar

Richard G Kay

Frank Reimann

Fiona M Gribble

Affiliations

Soon will be listed here.

Abstract

Aims/hypothesis: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels.

Methods: CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing.

Results: RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids.

Conclusions/interpretation: The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.

Citing Articles

Single-cell transcriptomics of human organoid-derived enteroendocrine cell populations from the small intestine.

Smith C, Lu V, Bany Bakar R, Miedzybrodzka E, Davison A, Goldspink D J Physiol. 2024; .

PMID: 39639676 PMC: 7617304. DOI: 10.1113/JP287463.

Incretin hormones and obesity.

Alcaino C, Reimann F, Gribble F J Physiol. 2024; .

PMID: 39576749 PMC: 7617301. DOI: 10.1113/JP286293.

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