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
{"title":"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","authors":"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","doi":"10.1007/s00125-024-06293-3","DOIUrl":null,"url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Aims/hypothesis</h3><p>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.</p><h3 data-test=\"abstract-sub-heading\">Methods</h3><p>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<sup>2+</sup> using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes <i>GPR142</i> and <i>CASR</i> were knocked out to evaluate roles in amino acid sensing.</p><h3 data-test=\"abstract-sub-heading\">Results</h3><p>RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including <i>FFAR1</i>, <i>GPBAR1</i>, <i>GPR119</i>, <i>CASR</i> and <i>GPR142</i>. Glucose induced action potential firing and cytosolic Ca<sup>2+</sup> 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 <i>CASR</i> knockout blunted intracellular Ca<sup>2+</sup> responses, a <i>CASR</i>/<i>GPR142</i> double knockout was needed to reduce GIP secretory responses to aromatic amino acids.</p><h3 data-test=\"abstract-sub-heading\">Conclusions/interpretation</h3><p>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.</p><h3 data-test=\"abstract-sub-heading\">Graphical Abstract</h3>\n","PeriodicalId":11164,"journal":{"name":"Diabetologia","volume":"1 1","pages":""},"PeriodicalIF":8.4000,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids\",\"authors\":\"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\",\"doi\":\"10.1007/s00125-024-06293-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<h3 data-test=\\\"abstract-sub-heading\\\">Aims/hypothesis</h3><p>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.</p><h3 data-test=\\\"abstract-sub-heading\\\">Methods</h3><p>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<sup>2+</sup> using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes <i>GPR142</i> and <i>CASR</i> were knocked out to evaluate roles in amino acid sensing.</p><h3 data-test=\\\"abstract-sub-heading\\\">Results</h3><p>RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including <i>FFAR1</i>, <i>GPBAR1</i>, <i>GPR119</i>, <i>CASR</i> and <i>GPR142</i>. Glucose induced action potential firing and cytosolic Ca<sup>2+</sup> 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 <i>CASR</i> knockout blunted intracellular Ca<sup>2+</sup> responses, a <i>CASR</i>/<i>GPR142</i> double knockout was needed to reduce GIP secretory responses to aromatic amino acids.</p><h3 data-test=\\\"abstract-sub-heading\\\">Conclusions/interpretation</h3><p>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.</p><h3 data-test=\\\"abstract-sub-heading\\\">Graphical Abstract</h3>\\n\",\"PeriodicalId\":11164,\"journal\":{\"name\":\"Diabetologia\",\"volume\":\"1 1\",\"pages\":\"\"},\"PeriodicalIF\":8.4000,\"publicationDate\":\"2024-10-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Diabetologia\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s00125-024-06293-3\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ENDOCRINOLOGY & METABOLISM\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Diabetologia","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s00125-024-06293-3","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids
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.
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.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
