{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/290641"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/290641","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Characterisation of L-cell secretory mechanisms and colonic enteroendocrine cell subpopulations","abstract":"Enteroendocrine cells (EECs) are chemosensitive cells of the gastrointestinal epithelium that exert a wide range of physiological effects via production and secretion of hormones in response to ingested nutrients, bacterial metabolites and systemic signals. Glucagon-like peptide-1 (GLP-1) is one such hormone secreted from so-called L-cells found in both the small and large intestines. GLP-1 exerts an anorexigenic effect and together with glucose- dependent insulinotropic polypeptide (GIP), restores postprandial normoglycaemia through the incretin effect. These effects are exploited by GLP-1 analogues in the treatment of type 2 diabetes. GLP-1 may also contribute to weight-loss and remission of type 2 diabetes following bariatric surgery which increases postprandial GLP-1 excursions. Here we investigated stimulus secretion coupling in L-cells. A novel 2D culture system from murine small intestinal organoids was established as an in vitro model. This was used to characterise synergistic stimulation of GLP-1 secretion in response to concomitant stimulation by bile acids through the Gs-protein coupled receptor GPBAR1 and free fatty acids through the Gq-coupled receptor FFAR1. Roughly half of colonic, but not small intestinal, L-cells co-produce the orexigenic peptide insulin-like peptide 5 (INSL5). This hitherto poorly examined subpopulation of L-cells was characterised through transcriptomic analysis, intracellular calcium imaging (using a novel GCaMP6F-based transgenic mouse model), LC/MS peptide quantification and 3D super resolution microscopy (3D-SIM). Based on the observed prevalent co-storage of GLP-1 and INSL5 in secretory vesicles and similar secretory responses of both hormones to a range of different stimuli strengths (including short chain fatty acids, angiotensin II and arginine vasopressin (AVP)) it was concluded that GLP-1 and INSL5 are co-secreted, rather than being selectively recruited by different stimuli. To further characterise the diversity of colonic EECs, single cell RNA-sequencing (scRNA-seq) was performed on cells isolated from mice with a pan-EEC fluorescent marker (NeuroD1- Cre:Rosa26-EYFP). This illustrated that INSL5-producing L-cells form one of two transcriptomically distinct subpopulations of L-cells in the murine colon, with the other distinguished by expression of neurotensin (Nts). Another major EEC subpopulation, enterochromaffin (EC) cells could be split into three groups, mechanosensitive and pro- inflammatory EC cells distinguished by Piezo2 and Tac1 expression, respectively and a third Sct-expressing group. Immunofluorescent labelling and RT-qPCR analysis revealed that the Nts-expressing and Insl5-expressing L-cell subpopulations are proximally and distally enriched in the murine colon, respectively. In primary cultures, angiotensin II and AVP stimulated INSL5, GLP-1 and PYY but not NTS secretion, correlating with selective expression profiles of the cognate receptors in the L-cell subpopulations. In summary, the work presented suggests that different L-cell subpopulations exist that respond to different stimuli, but that hormones co-expressed in individual L-cells are co- released upon stimulation. Differences in receptor expression between these subpopulations and other EEC-populations might be exploitable for selective hormone recruitment for the therapy of diabetes, obesity and other diseases.","abstract_html":"Enteroendocrine cells (EECs) are chemosensitive cells of the gastrointestinal epithelium that exert a wide range of physiological effects via production and secretion of hormones in response to ingested nutrients, bacterial metabolites and systemic signals. Glucagon-like peptide-1 (GLP-1) is one such hormone secreted from so-called L-cells found in both the small and large intestines. GLP-1 exerts an anorexigenic effect and together with glucose- dependent insulinotropic polypeptide (GIP), restores postprandial normoglycaemia through the incretin effect. These effects are exploited by GLP-1 analogues in the treatment of type 2 diabetes. GLP-1 may also contribute to weight-loss and remission of type 2 diabetes following bariatric surgery which increases postprandial GLP-1 excursions. Here we investigated stimulus secretion coupling in L-cells. A novel 2D culture system from murine small intestinal organoids was established as an in vitro model. This was used to characterise synergistic stimulation of GLP-1 secretion in response to concomitant stimulation by bile acids through the Gs-protein coupled receptor GPBAR1 and free fatty acids through the Gq-coupled receptor FFAR1. Roughly half of colonic, but not small intestinal, L-cells co-produce the orexigenic peptide insulin-like peptide 5 (INSL5). This hitherto poorly examined subpopulation of L-cells was characterised through transcriptomic analysis, intracellular calcium imaging (using a novel GCaMP6F-based transgenic mouse model), LC/MS peptide quantification and 3D super resolution microscopy (3D-SIM). Based on the observed prevalent co-storage of GLP-1 and INSL5 in secretory vesicles and similar secretory responses of both hormones to a range of different stimuli strengths (including short chain fatty acids, angiotensin II and arginine vasopressin (AVP)) it was concluded that GLP-1 and INSL5 are co-secreted, rather than being selectively recruited by different stimuli. To further characterise the diversity of colonic EECs, single cell RNA-sequencing (scRNA-seq) was performed on cells isolated from mice with a pan-EEC fluorescent marker (NeuroD1- Cre:Rosa26-EYFP). This illustrated that INSL5-producing L-cells form one of two transcriptomically distinct subpopulations of L-cells in the murine colon, with the other distinguished by expression of neurotensin (Nts). Another major EEC subpopulation, enterochromaffin (EC) cells could be split into three groups, mechanosensitive and pro- inflammatory EC cells distinguished by Piezo2 and Tac1 expression, respectively and a third Sct-expressing group. Immunofluorescent labelling and RT-qPCR analysis revealed that the Nts-expressing and Insl5-expressing L-cell subpopulations are proximally and distally enriched in the murine colon, respectively. In primary cultures, angiotensin II and AVP stimulated INSL5, GLP-1 and PYY but not NTS secretion, correlating with selective expression profiles of the cognate receptors in the L-cell subpopulations. In summary, the work presented suggests that different L-cell subpopulations exist that respond to different stimuli, but that hormones co-expressed in individual L-cells are co- released upon stimulation. Differences in receptor expression between these subpopulations and other EEC-populations might be exploitable for selective hormone recruitment for the therapy of diabetes, obesity and other diseases.","abstract_has_math":false,"creators":["Billing, Lawrence"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Gribble, Fiona","Reimann, Frank"],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-10-26","date_published":"2019-10-26","updated_at":"2026-07-22T22:23:59Z","subjects":["INSL5","GLP-1","Enteroendocrine cells","Intestinal Organoids","scRNA-seq","L-cells","Gut peptides","Diabetes","Obesity","Colon","Small intestine","Neurotensin"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c601b553-8327-4f2d-9ffc-5971b74b610b/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000230336766","0000000242322898","0000000193996377"],"render_values":[{"text":"0000-0002-3033-6766","href":"https://orcid.org/0000-0002-3033-6766","code":true},{"text":"0000-0002-4232-2898","href":"https://orcid.org/0000-0002-4232-2898","code":true},{"text":"0000-0001-9399-6377","href":"https://orcid.org/0000-0001-9399-6377","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.37850","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Gribble, Fiona","Reimann, Frank"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Funded through the Wellcome Trust doctoral training program in metabolic and cardiovascular disease."]},{"key":"dc:creator","label":"Author","values":["Billing, Lawrence"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000230336766","0000000242322898","0000000193996377"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2019-10-26"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/290641"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["INSL5","GLP-1","Enteroendocrine cells","Intestinal Organoids","scRNA-seq","L-cells","Gut peptides","Diabetes","Obesity","Colon","Small intestine","Neurotensin"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c601b553-8327-4f2d-9ffc-5971b74b610b/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.37850"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ddffab2f-971e-4387-996a-b8a2ab44b71b/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Enteroendocrine cells (EECs) are chemosensitive cells of the gastrointestinal epithelium that exert a wide range of physiological effects via production and secretion of hormones in response to ingested nutrients, bacterial metabolites and systemic signals. Glucagon-like peptide-1 (GLP-1) is one such hormone secreted from so-called L-cells found in both the small and large intestines. GLP-1 exerts an anorexigenic effect and together with glucose- dependent insulinotropic polypeptide (GIP), restores postprandial normoglycaemia through the incretin effect. These effects are exploited by GLP-1 analogues in the treatment of type 2 diabetes. GLP-1 may also contribute to weight-loss and remission of type 2 diabetes following bariatric surgery which increases postprandial GLP-1 excursions. Here we investigated stimulus secretion coupling in L-cells. A novel 2D culture system from murine small intestinal organoids was established as an in vitro model. This was used to characterise synergistic stimulation of GLP-1 secretion in response to concomitant stimulation by bile acids through the Gs-protein coupled receptor GPBAR1 and free fatty acids through the Gq-coupled receptor FFAR1. Roughly half of colonic, but not small intestinal, L-cells co-produce the orexigenic peptide insulin-like peptide 5 (INSL5). This hitherto poorly examined subpopulation of L-cells was characterised through transcriptomic analysis, intracellular calcium imaging (using a novel GCaMP6F-based transgenic mouse model), LC/MS peptide quantification and 3D super resolution microscopy (3D-SIM). Based on the observed prevalent co-storage of GLP-1 and INSL5 in secretory vesicles and similar secretory responses of both hormones to a range of different stimuli strengths (including short chain fatty acids, angiotensin II and arginine vasopressin (AVP)) it was concluded that GLP-1 and INSL5 are co-secreted, rather than being selectively recruited by different stimuli. To further characterise the diversity of colonic EECs, single cell RNA-sequencing (scRNA-seq) was performed on cells isolated from mice with a pan-EEC fluorescent marker (NeuroD1- Cre:Rosa26-EYFP). This illustrated that INSL5-producing L-cells form one of two transcriptomically distinct subpopulations of L-cells in the murine colon, with the other distinguished by expression of neurotensin (Nts). Another major EEC subpopulation, enterochromaffin (EC) cells could be split into three groups, mechanosensitive and pro- inflammatory EC cells distinguished by Piezo2 and Tac1 expression, respectively and a third Sct-expressing group. Immunofluorescent labelling and RT-qPCR analysis revealed that the Nts-expressing and Insl5-expressing L-cell subpopulations are proximally and distally enriched in the murine colon, respectively. In primary cultures, angiotensin II and AVP stimulated INSL5, GLP-1 and PYY but not NTS secretion, correlating with selective expression profiles of the cognate receptors in the L-cell subpopulations. In summary, the work presented suggests that different L-cell subpopulations exist that respond to different stimuli, but that hormones co-expressed in individual L-cells are co- released upon stimulation. Differences in receptor expression between these subpopulations and other EEC-populations might be exploitable for selective hormone recruitment for the therapy of diabetes, obesity and other diseases."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["5c5143fc0f38efb7ca7b80e1724eaabf","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Characterisation of L-cell secretory mechanisms and colonic enteroendocrine cell subpopulations"]}]}],"canonical_facts":{"dc:contributor.advisor":["Gribble, Fiona","Reimann, Frank"],"dc:contributor.sponsor":["Funded through the Wellcome Trust doctoral training program in metabolic and cardiovascular disease."],"dc:creator":["Billing, Lawrence"],"dc:creator.authoridentifier":["0000000230336766","0000000242322898","0000000193996377"],"dc:date.issued":["2019-10-26"],"dc:description.abstract":["Enteroendocrine cells (EECs) are chemosensitive cells of the gastrointestinal epithelium that exert a wide range of physiological effects via production and secretion of hormones in response to ingested nutrients, bacterial metabolites and systemic signals. Glucagon-like peptide-1 (GLP-1) is one such hormone secreted from so-called L-cells found in both the small and large intestines. GLP-1 exerts an anorexigenic effect and together with glucose- dependent insulinotropic polypeptide (GIP), restores postprandial normoglycaemia through the incretin effect. These effects are exploited by GLP-1 analogues in the treatment of type 2 diabetes. GLP-1 may also contribute to weight-loss and remission of type 2 diabetes following bariatric surgery which increases postprandial GLP-1 excursions. Here we investigated stimulus secretion coupling in L-cells. A novel 2D culture system from murine small intestinal organoids was established as an in vitro model. This was used to characterise synergistic stimulation of GLP-1 secretion in response to concomitant stimulation by bile acids through the Gs-protein coupled receptor GPBAR1 and free fatty acids through the Gq-coupled receptor FFAR1. Roughly half of colonic, but not small intestinal, L-cells co-produce the orexigenic peptide insulin-like peptide 5 (INSL5). This hitherto poorly examined subpopulation of L-cells was characterised through transcriptomic analysis, intracellular calcium imaging (using a novel GCaMP6F-based transgenic mouse model), LC/MS peptide quantification and 3D super resolution microscopy (3D-SIM). Based on the observed prevalent co-storage of GLP-1 and INSL5 in secretory vesicles and similar secretory responses of both hormones to a range of different stimuli strengths (including short chain fatty acids, angiotensin II and arginine vasopressin (AVP)) it was concluded that GLP-1 and INSL5 are co-secreted, rather than being selectively recruited by different stimuli. To further characterise the diversity of colonic EECs, single cell RNA-sequencing (scRNA-seq) was performed on cells isolated from mice with a pan-EEC fluorescent marker (NeuroD1- Cre:Rosa26-EYFP). This illustrated that INSL5-producing L-cells form one of two transcriptomically distinct subpopulations of L-cells in the murine colon, with the other distinguished by expression of neurotensin (Nts). Another major EEC subpopulation, enterochromaffin (EC) cells could be split into three groups, mechanosensitive and pro- inflammatory EC cells distinguished by Piezo2 and Tac1 expression, respectively and a third Sct-expressing group. Immunofluorescent labelling and RT-qPCR analysis revealed that the Nts-expressing and Insl5-expressing L-cell subpopulations are proximally and distally enriched in the murine colon, respectively. In primary cultures, angiotensin II and AVP stimulated INSL5, GLP-1 and PYY but not NTS secretion, correlating with selective expression profiles of the cognate receptors in the L-cell subpopulations. In summary, the work presented suggests that different L-cell subpopulations exist that respond to different stimuli, but that hormones co-expressed in individual L-cells are co- released upon stimulation. Differences in receptor expression between these subpopulations and other EEC-populations might be exploitable for selective hormone recruitment for the therapy of diabetes, obesity and other diseases."],"dc:format.checksum.md5":["5c5143fc0f38efb7ca7b80e1724eaabf","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["10.17863/CAM.37850"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ddffab2f-971e-4387-996a-b8a2ab44b71b/download"],"dc:language":["en"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/290641"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c601b553-8327-4f2d-9ffc-5971b74b610b/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["INSL5","GLP-1","Enteroendocrine cells","Intestinal Organoids","scRNA-seq","L-cells","Gut peptides","Diabetes","Obesity","Colon","Small intestine","Neurotensin"],"dc:title":["Characterisation of L-cell secretory mechanisms and colonic enteroendocrine cell subpopulations"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:59Z"}