Fluorescent monitoring of enteroendocrine cell development and function
Lead Research Organisation:
University of Cambridge
Department Name: Clinical Biochemistry
Abstract
The project will investigate how intestinal endocrine cells sense food intake, using fluorescence imaging techniques in live tissue. It will address the questions of how enteroendocrine cells are formed and how they detect changes in food intake and metabolism.
Enteroendocrine cells (EECs) are located in the intestinal epithelium and convert nutrient signals arriving in the gut lumen into chemical hormonal signals, which in turn regulate intestinal function, insulin release and food intake. Like the absorptive enterocytes, EECs are continuously formed from stem cells in intestinal crypt, and have a life span of about 5 days. The hormones they produce exhibit a longitudinal gradient along the gut, with some hormones exhibit preferential expression in the proximal gut, and others in the distal gut.
In recent years, we have generated a number of transgenic mouse models in which enteroendocrine cells are labelled by fluorescent markers, cre-recombinase, rtTA or fluorescent sensors of intracellular cAMP or calcium. These models enable the identification and purification of specific populations of EEC as well as dynamic monitoring of their intracellular signalling pathways in primary culture. In recent years we have exploited the combination of fluorescent imaging, electrophysiology and transcriptomic analysis to dissect how EECs detect a range of ingested nutrients. By assaying hormone release from mixed primary culture and intact tissue pieces mounted in Ussing chambers, we correlated cellular activity with hormone secretion. We identified a range of nutrient transporters, metabolic pathways and G-protein coupled receptors that variously result in the detection of digested carbohydrates, fat and protein. Transcriptomic analysis further revealed that several different EEC types, once thought to represent distinct populations, are actually highly related, and likely reflect a single cell type with a variable hormonal signature.
In the current project we will extend these techniques to the study of mouse and human EECs in organoid culture. Organoids will be grown from intestinal crypts from different regions of the mouse or human gut, and maintained in primary culture. Using this system, organoids can be maintained in culture indefinitely. Genes under specific hormonal promoters will be transduced into organoids using lentivirus or other methods, to introduce fluorescent markers and sensors specifically into the EEC population. Using the methodologies described above, we will address the following questions:
-What specifies the hormonal signature of an EEC? Is it determined by the position of the gastrointestinal tract from which the crypts originated? Can it be influenced by external factors such as nutrients, cytokines or other metabolic factors, and can it be changed during the lifetime of a single EEC (as monitored by time lapse imaging of fluorescent markers driven by hormonal promoters in organoids)?
-Do EECs in organoid culture exhibit similar secretory responsiveness to stimuli as EEC cell lines, L-cells in mixed primary culture, and L-cells in intact tissue pieces?
-Which nutrient detection machinery and intracellular signaling pathways are implicated in the detection by human EECs of nutrients, hormones and neurotransmitters, and are the pathways similar to those found in mice?
Enteroendocrine cells (EECs) are located in the intestinal epithelium and convert nutrient signals arriving in the gut lumen into chemical hormonal signals, which in turn regulate intestinal function, insulin release and food intake. Like the absorptive enterocytes, EECs are continuously formed from stem cells in intestinal crypt, and have a life span of about 5 days. The hormones they produce exhibit a longitudinal gradient along the gut, with some hormones exhibit preferential expression in the proximal gut, and others in the distal gut.
In recent years, we have generated a number of transgenic mouse models in which enteroendocrine cells are labelled by fluorescent markers, cre-recombinase, rtTA or fluorescent sensors of intracellular cAMP or calcium. These models enable the identification and purification of specific populations of EEC as well as dynamic monitoring of their intracellular signalling pathways in primary culture. In recent years we have exploited the combination of fluorescent imaging, electrophysiology and transcriptomic analysis to dissect how EECs detect a range of ingested nutrients. By assaying hormone release from mixed primary culture and intact tissue pieces mounted in Ussing chambers, we correlated cellular activity with hormone secretion. We identified a range of nutrient transporters, metabolic pathways and G-protein coupled receptors that variously result in the detection of digested carbohydrates, fat and protein. Transcriptomic analysis further revealed that several different EEC types, once thought to represent distinct populations, are actually highly related, and likely reflect a single cell type with a variable hormonal signature.
In the current project we will extend these techniques to the study of mouse and human EECs in organoid culture. Organoids will be grown from intestinal crypts from different regions of the mouse or human gut, and maintained in primary culture. Using this system, organoids can be maintained in culture indefinitely. Genes under specific hormonal promoters will be transduced into organoids using lentivirus or other methods, to introduce fluorescent markers and sensors specifically into the EEC population. Using the methodologies described above, we will address the following questions:
-What specifies the hormonal signature of an EEC? Is it determined by the position of the gastrointestinal tract from which the crypts originated? Can it be influenced by external factors such as nutrients, cytokines or other metabolic factors, and can it be changed during the lifetime of a single EEC (as monitored by time lapse imaging of fluorescent markers driven by hormonal promoters in organoids)?
-Do EECs in organoid culture exhibit similar secretory responsiveness to stimuli as EEC cell lines, L-cells in mixed primary culture, and L-cells in intact tissue pieces?
-Which nutrient detection machinery and intracellular signaling pathways are implicated in the detection by human EECs of nutrients, hormones and neurotransmitters, and are the pathways similar to those found in mice?
Organisations
People |
ORCID iD |
Fiona Gribble (Primary Supervisor) | |
Ming Yang (Student) |
Publications
Yang M
(2021)
Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells.
in Scientific reports
Yang M
(2021)
Chemosensing in enteroendocrine cells: mechanisms and therapeutic opportunities.
in Current opinion in endocrinology, diabetes, and obesity
Coll AP
(2020)
GDF15 mediates the effects of metformin on body weight and energy balance.
in Nature
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M011194/1 | 30/09/2015 | 31/03/2024 | |||
1645477 | Studentship | BB/M011194/1 | 30/09/2015 | 29/09/2019 | Ming Yang |
Description | In my research project, I investigate the effects of the anti-diabetes drug metformin in the gastrointestinal tract. One of the effects that makes metformin an attractive anti-diabetes agent is its efficacious blood glucose lowering effects, and previous studies have found that metformin greatly increases glucose consumption in the gut. In this part of the project, I investigate the cellular mechanisms that explain this phenomenon using intestinal cells grown in organoids. I found that metformin increases glucose uptake in intestinal cells, which is associated with increased glycolytic metabolism due to metformin inhibiting mitochondrial function. Another associated effect of metformin is its effects on weight loss, and metformin is currently used for the potential treatment of obesity. The stress marker GDF-15 is elevated in patients who had been orally administered metformin. In a multidisciplinary collaboration, we have found that the source of GDF-15 induction is the lower GI tract. I found that metformin stimulates GDF-15 secretion in intestinal cells, which is associated with mitochondrial dysfunction caused by metformin, which leads to the activation of the integrated stress response. |
Exploitation Route | Even though metformin is a well established glucose-lowering agent for the treatment of Type 2 Diabetes, the mechanisms involved are not well known. The gastrointestinal tract is believed to be an important site of action, and recent studies have shown that metformin increases intestinal glucose utilisation. The work from this project supports this hypothesis and may provide an explanation for the glucose-lowering and weight loss effects of metformin. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | MY Poster at Incretin Study Group 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Characterisation of Electrical Activity in STC-1 Cells and their relevance to study Voltage-gated Ion Channels in Primary K- and L-cells |
Year(s) Of Engagement Activity | 2017 |
Description | MY Poster at Incretin Study Group 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | MY poster at Incretin Study Group: "Amino acid sensing in K and L cells from mouse duodenal organoids" |
Year(s) Of Engagement Activity | 2019 |
Description | MY. Oral presentation at Diabetes UK Annual Professional Conference 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Metformin alters glucose uptake and metabolism in intestinal cells |
Year(s) Of Engagement Activity | 2019 |
Description | Science Festival 2016, 2017 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | Participated in running metabolic sciences stand of the Science Festival. Included events such as explaining the GI tract using knitted models, and the "glucose game" involving velcro and sand bags. |
Year(s) Of Engagement Activity | 2016,2017 |