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Article ; Online: Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells.

Psichas, Arianna / Tolhurst, Gwen / Brighton, Cheryl A / Gribble, Fiona M / Reimann, Frank

Journal of visualized experiments : JoVE

2017 , Issue 122

Abstract: The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of ...

Abstract	The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.
MeSH term(s)	Animals ; Bombesin/pharmacology ; Cell Culture Techniques/instrumentation ; Cell Culture Techniques/methods ; Enteroendocrine Cells/cytology ; Enteroendocrine Cells/drug effects ; Enteroendocrine Cells/metabolism ; Gastrointestinal Hormones/metabolism ; Glucagon-Like Peptide 1/metabolism ; Intestine, Small/cytology ; Mice, Inbred C57BL ; Mice, Transgenic ; Potassium Chloride/pharmacology
Chemical Substances	Gastrointestinal Hormones ; Potassium Chloride (660YQ98I10) ; Glucagon-Like Peptide 1 (89750-14-1) ; Bombesin (PX9AZU7QPK)
Language	English
Publishing date	2017-04-20
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/55687
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Intestinal sensing of nutrients.

Tolhurst, Gwen / Reimann, Frank / Gribble, Fiona M

Handbook of experimental pharmacology

2012 , Issue 209, Page(s) 309–335

Abstract: Ingestion of a meal triggers a range of physiological responses both within and outside the gut, and results in the remote modulation of appetite and glucose homeostasis. Luminal contents are sensed by specialised chemosensitive cells scattered ... ...

Abstract	Ingestion of a meal triggers a range of physiological responses both within and outside the gut, and results in the remote modulation of appetite and glucose homeostasis. Luminal contents are sensed by specialised chemosensitive cells scattered throughout the intestinal epithelium. These enteroendocrine and tuft cells make direct contact with the gut lumen and release a range of chemical mediators, which can either act in a paracrine fashion interacting with neighbouring cells and nerve endings or as classical circulating hormones. At the molecular level, the chemosensory machinery involves multiple and complex signalling pathways including activation of G-protein-coupled receptors and solute carrier transporters. This chapter will discuss our current knowledge of the molecular mechanisms underlying intestinal chemosensation with a particular focus on the relatively well-characterised nutrient-triggered secretion from the enteroendocrine system.
MeSH term(s)	Animals ; Appetite Regulation ; Chemoreceptor Cells/metabolism ; Dietary Carbohydrates/administration & dosage ; Dietary Carbohydrates/metabolism ; Dietary Fats/administration & dosage ; Dietary Fats/metabolism ; Dietary Proteins/administration & dosage ; Dietary Proteins/metabolism ; Eating ; Enteroendocrine Cells/metabolism ; Humans ; Intestinal Mucosa/metabolism ; Signal Transduction
Chemical Substances	Dietary Carbohydrates ; Dietary Fats ; Dietary Proteins
Language	English
Publishing date	2012-01-11
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ISSN	0171-2004
ISSN	0171-2004
DOI	10.1007/978-3-642-24716-3_14
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Article ; Online: G-protein-coupled receptors in intestinal chemosensation.

Reimann, Frank / Tolhurst, Gwen / Gribble, Fiona M

Cell metabolism

2012 Volume 15, Issue 4, Page(s) 421–431

Abstract: Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and ... ...

Abstract	Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and peripheral nutrient disposal. G-protein-coupled receptors responsive to a range of nutrients and other food components have been identified, and many are localized to intestinal chemosensory cells, eliciting hormonal and neuronal signaling to the brain and periphery. This review examines the role of G-protein-coupled receptors as signaling molecules in the gut, with a particular focus on pathways relevant to appetite and glucose homeostasis.
MeSH term(s)	Animals ; Cannabinoid Receptor Modulators/metabolism ; Chemoreceptor Cells/metabolism ; Humans ; Intestinal Mucosa/metabolism ; Neurotransmitter Agents/metabolism ; Receptors, G-Protein-Coupled/metabolism ; Signal Transduction
Chemical Substances	Cannabinoid Receptor Modulators ; Neurotransmitter Agents ; Receptors, G-Protein-Coupled
Language	English
Publishing date	2012-04-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2176834-1
ISSN	1932-7420 ; 1550-4131
ISSN (online)	1932-7420
ISSN	1550-4131
DOI	10.1016/j.cmet.2011.12.019
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells

Psichas, Arianna / Tolhurst, Gwen / Brighton, Cheryl A / Gribble, Fiona M / Reimann, Frank

Journal of visualized experiments. 2017 Apr. 20, , no. 122

2017

Abstract	The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.
Keywords	calcium ; cell lines ; epithelial cells ; epithelium ; genetically modified organisms ; hormone secretion ; image analysis ; mice ; models ; neuropeptides ; nutrients ; signal transduction ; small intestine
Language	English
Dates of publication	2017-0420
Size	p. e55687.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/55687
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells

Deborah A. Goldspink / Van B. Lu / Lawrence J. Billing / Pierre Larraufie / Gwen Tolhurst / Fiona M. Gribble / Frank Reimann

Molecular Metabolism, Vol 7, Iss , Pp 90-

2018 Volume 101

Abstract: Objectives: The aim of this study was to investigate the electrical properties of ileal Glucagon-like peptide 1 (GLP-1) secreting L-cells using murine organoid cultures and the electrophysiological and intracellular signaling pathways recruited following ...

Abstract	Objectives: The aim of this study was to investigate the electrical properties of ileal Glucagon-like peptide 1 (GLP-1) secreting L-cells using murine organoid cultures and the electrophysiological and intracellular signaling pathways recruited following activation of the Gαq-coupled free fatty acid receptors FFA1 and Gαs-coupled bile acid receptors GPBAR1. Methods: Experiments were performed using ileal organoids generated from mice transgenically expressing fluorescent reporters (Epac2-camps and GCaMP3) under control of the proglucagon promoter. Electrophysiology and single cell imaging were performed on identified L-cells in organoids, and GLP-1 secretion from cultured organoids was measured by immunoassay. Results: The FFA1 ligand TAK-875 triggered L-cell electrical activity, increased intracellular calcium, and activated a depolarizing current that was blocked by the TRPC3 inhibitor Pyr3. TAK-875 triggered GLP-1 secretion was Pyr3 sensitive, suggesting that the TRPC3 channel links FFA1 activation to calcium elevation and GLP-1 release in L-cells. GPBAR1 agonist triggered PKA-dependent L-type Ca2+ current activation and action potential firing in L-cells. The combination of TAK-875 and a GPBAR1 agonist triggered synergistic calcium elevation and GLP-1 secretory responses. Conclusions: FFA1 and GPBAR1 activation individually increased electrical activity in L-cells by recruiting pathways that include activation of TRPC3 and L-type voltage-gated Ca2+ channels. Synergy between the pathways activated downstream of these receptors was observed both at the level of Ca2+ elevation and GLP-1 secretion. Keywords: GLP-1, FFA1, GPBAR1, Organoid, Diabetes, Obesity
Keywords	Internal medicine ; RC31-1245
Subject code	571
Language	English
Publishing date	2018-01-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells.

Goldspink, Deborah A / Lu, Van B / Billing, Lawrence J / Larraufie, Pierre / Tolhurst, Gwen / Gribble, Fiona M / Reimann, Frank

Molecular metabolism

2017 Volume 7, Page(s) 90–101

Abstract	Objectives: The aim of this study was to investigate the electrical properties of ileal Glucagon-like peptide 1 (GLP-1) secreting L-cells using murine organoid cultures and the electrophysiological and intracellular signaling pathways recruited following activation of the G Methods: Experiments were performed using ileal organoids generated from mice transgenically expressing fluorescent reporters (Epac2-camps and GCaMP3) under control of the proglucagon promoter. Electrophysiology and single cell imaging were performed on identified L-cells in organoids, and GLP-1 secretion from cultured organoids was measured by immunoassay. Results: The FFA1 ligand TAK-875 triggered L-cell electrical activity, increased intracellular calcium, and activated a depolarizing current that was blocked by the TRPC3 inhibitor Pyr3. TAK-875 triggered GLP-1 secretion was Pyr3 sensitive, suggesting that the TRPC3 channel links FFA1 activation to calcium elevation and GLP-1 release in L-cells. GPBAR1 agonist triggered PKA-dependent L-type Ca Conclusions: FFA1 and GPBAR1 activation individually increased electrical activity in L-cells by recruiting pathways that include activation of TRPC3 and L-type voltage-gated Ca
MeSH term(s)	Animals ; Bile Acids and Salts/metabolism ; Calcium/metabolism ; Cells, Cultured ; Enteroendocrine Cells/metabolism ; Enteroendocrine Cells/physiology ; Fatty Acids/metabolism ; Glucagon-Like Peptide 1/metabolism ; Ileum/cytology ; Ileum/metabolism ; Membrane Potentials ; Mice ; Receptors, G-Protein-Coupled/metabolism ; TRPC Cation Channels/metabolism
Chemical Substances	Bile Acids and Salts ; Fatty Acids ; Ffar1 protein, mouse ; Gpbar1 protein, mouse ; Receptors, G-Protein-Coupled ; TRPC Cation Channels ; TRPC3 cation channel ; Glucagon-Like Peptide 1 (89750-14-1) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2017-11-11
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2708735-9
ISSN	2212-8778 ; 2212-8778
ISSN (online)	2212-8778
ISSN	2212-8778
DOI	10.1016/j.molmet.2017.11.005
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons.

Lu, Van B / Rievaj, Juraj / O'Flaherty, Elisabeth A / Smith, Christopher A / Pais, Ramona / Pattison, Luke A / Tolhurst, Gwen / Leiter, Andrew B / Bulmer, David C / Gribble, Fiona M / Reimann, Frank

Nature communications

2019 Volume 10, Issue 1, Page(s) 1029

Abstract: Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in ... ...

Abstract	Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y
MeSH term(s)	Adenosine Triphosphate/metabolism ; Afferent Pathways ; Animals ; Cell Line ; Eating ; Enterocytes/metabolism ; Enteroendocrine Cells/metabolism ; Female ; Ganglion Cysts/metabolism ; Ganglion Cysts/pathology ; Glucagon-Like Peptide 1/metabolism ; Incretins/metabolism ; Intestinal Mucosa/innervation ; Intestinal Mucosa/metabolism ; Intestinal Mucosa/pathology ; Intestines ; Male ; Mice ; Mice, Inbred C57BL ; Neurons/pathology ; Neurons, Afferent/metabolism ; Nodose Ganglion/metabolism ; Nodose Ganglion/pathology ; Peptide YY/metabolism ; Receptors, Purinergic P2X2/metabolism ; Receptors, Purinergic P2X3/metabolism ; Vagus Nerve/metabolism
Chemical Substances	Incretins ; Receptors, Purinergic P2X2 ; Receptors, Purinergic P2X3 ; Peptide YY (106388-42-5) ; Glucagon-Like Peptide 1 (89750-14-1) ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2019-03-04
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-019-09045-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons

Van B. Lu / Juraj Rievaj / Elisabeth A. O’Flaherty / Christopher A. Smith / Ramona Pais / Luke A. Pattison / Gwen Tolhurst / Andrew B. Leiter / David C. Bulmer / Fiona M. Gribble / Frank Reimann

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 13

Abstract: Glucagon-like peptide 1 (GLP-1) is released from intestinal L-cells following nutrient uptake and enhances insulin release as well as promotes satiety. Here, the authors demonstrate that GLP-1 secreting cells release ATP and that this stimulates nodose ... ...

Abstract	Glucagon-like peptide 1 (GLP-1) is released from intestinal L-cells following nutrient uptake and enhances insulin release as well as promotes satiety. Here, the authors demonstrate that GLP-1 secreting cells release ATP and that this stimulates nodose neurons and enterocytes in a paracrine manner in vitro.
Keywords	Science ; Q
Language	English
Publishing date	2019-03-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons

Van B. Lu / Juraj Rievaj / Elisabeth A. O’Flaherty / Christopher A. Smith / Ramona Pais / Luke A. Pattison / Gwen Tolhurst / Andrew B. Leiter / David C. Bulmer / Fiona M. Gribble / Frank Reimann

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 13

Abstract	Glucagon-like peptide 1 (GLP-1) is released from intestinal L-cells following nutrient uptake and enhances insulin release as well as promotes satiety. Here, the authors demonstrate that GLP-1 secreting cells release ATP and that this stimulates nodose neurons and enterocytes in a paracrine manner in vitro.
Keywords	Science ; Q
Language	English
Publishing date	2019-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Purification of native bone marrow megakaryocytes for studies of gene expression.

Tolhurst, Gwen / Carter, Richard N / Miller, Nigel / Mahaut-Smith, Martyn P

Methods in molecular biology (Clifton, N.J.)

2011 Volume 788, Page(s) 259–273

Abstract: Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of ... ...

Abstract	Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41. In the second method, individual megakaryocytes, identified by their distinct morphology, are extracted using glass suction pipettes. Despite the small numbers of cells that can be isolated via the latter technique, recent studies have demonstrated how this pure population can be used to detect mRNA transcripts encoding ion channels and other proteins in the native megakaryocyte.
MeSH term(s)	Animals ; Bone Marrow/metabolism ; Cell Separation/methods ; Gene Expression Profiling ; Megakaryocytes/cytology ; Megakaryocytes/metabolism ; Mice ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; RNA, Messenger/genetics ; RNA, Messenger/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction
Chemical Substances	RNA, Messenger
Language	English
Publishing date	2011-12-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-307-3_18
Database	MEDical Literature Analysis and Retrieval System OnLINE

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