Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids

IF 8.4 1区医学 Q1 ENDOCRINOLOGY & METABOLISM

Diabetologia Pub Date : 2024-10-23 DOI:10.1007/s00125-024-06293-3

Nunzio Guccio, Constanza Alcaino, Emily L. Miedzybrodzka, Marta Santos-Hernández, Christopher A. Smith, Adam Davison, Rula Bany Bakar, Richard G. Kay, Frank Reimann, Fiona M. Gribble

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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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

Graphical Abstract

Abstract Image

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人十二指肠器官组织分泌葡萄糖依赖性促胰岛素多肽的分子机制

目的/假设葡萄糖依赖性促胰岛素多肽（GIP）是由小肠近端肠道内分泌K细胞分泌的一种增量激素。本研究旨在从分子和细胞水平探索人类 K 细胞的功能。方法CRISPR-Cas9 同源定向修复技术被用于在人类十二指肠器官组织的 GIP 基因座上插入编码黄色荧光蛋白（Venus）或基于 Epac 的 cAMP 传感器（Epac-S-H187）的转基因。通过 FACS 对荧光标记的 K 细胞进行纯化，以进行 RNA-seq 和肽组分析。利用 GIP 报告器官组织进行 GIP 分泌测定、使用 Fura-2 对 Ca2+ 进行活细胞成像、使用 Epac-S-H187 对 cAMP 进行活细胞成像，并进行基本的电生理特性分析。结果人十二指肠 K 细胞的核糖核酸序列分析表明，富集了多种参与营养感应的 G 蛋白偶联受体，包括 FFAR1、GPBAR1、GPR119、CASR 和 GPR142。葡萄糖诱导动作电位发射和细胞膜 Ca2+ 升高，并导致 GIP 分泌增加 1.8 倍，而钠葡萄糖协同转运体 1/2（SGLT1/2）阻断剂索他利氟嗪可抑制 GIP 分泌。激活长链脂肪酸受体游离脂肪酸受体 1（FFAR1）可诱导 GIP 分泌增加 2.7 倍，而色氨酸和苯丙氨酸可分别刺激分泌增加 2.8 倍和 2.1 倍。虽然 CASR 基因敲除会减弱细胞内 Ca2+ 的反应，但需要 CASR/GPR142 双基因敲除才能降低 GIP 对芳香族氨基酸的分泌反应。钙感受体（CASR）和G蛋白偶联受体142（GPR142）都有助于蛋白质刺激的GIP分泌。该模型将进一步用于确定调节糖尿病和肥胖症患者原生 GIP 分泌的潜在靶点。

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来源期刊

Diabetologia 医学-内分泌学与代谢

CiteScore

18.10

自引率

2.40%

发文量

193

审稿时长

1 months

期刊介绍： Diabetologia, the authoritative journal dedicated to diabetes research, holds high visibility through society membership, libraries, and social media. As the official journal of the European Association for the Study of Diabetes, it is ranked in the top quartile of the 2019 JCR Impact Factors in the Endocrinology & Metabolism category. The journal boasts dedicated and expert editorial teams committed to supporting authors throughout the peer review process.