The role of NAADP and the two-pore channel proteins in mediating insulin secretion in pancreatic beta cells
Lead Research Organisation:
University of Oxford
Department Name: Pharmacology
Abstract
Glucose is an important fuel for the cells of our bodies, allowing them to carry out their many functions. After a meal, glucose passes from our gut into the blood where it is carried to the pancreas. A set of specialized cells called pancreatic beta-cells respond to the increase of glucose levels in the blood by releasing the hormone insulin. Insulin is then carried by the blood to the organs of the body where it binds to a specific protein or receptor on the surface of cells and triggers a cascade of chemical events inside each cell to promote glucose transport into the cell for use as a fuel or for storage. How glucose stimulates insulin release is a key question, since a defect in this mechanism is responsible for type 2 diabetes. Diabetes is associated with decreased glucose uptake into cells, and chronically high levels of glucose in the blood, leading to multiple pathological events. One set of key proteins in the membranes surrounding beta-cells are called potassium channels. These close in response to uptake and metabolism of glucose, and also after treatment with some anti-diabetic drugs, leading to electrical changes resulting in an increase of calcium ions inside the beta-cell. Calcium ions act as a signal to promote release of insulin from storage granules from inside the cells into the blood. However, there is now evidence that glucose may also trigger insulin release by mechanisms independent of these potassium channels. Our preliminary work has shown that an important additional mechanism may involve a molecule called NAADP, which is generated inside the beta-cell in response to raised glucose levels and then releases calcium from internal stores within the cell to promote insulin release. Recently we made a major step forward by identifying the target receptor for NAADP in the cell as being the two-pore channel (TPC) proteins. We propose to study how production of NAADP is controlled in beta-cells and discover the precise way in which it acts upon the TPCs and other connected signalling proteins to stimulate release of insulin. Our work should thus uncover new molecular components involved in insulin release which may be impaired in diabetes and may represent new targets for drugs in the treatment of this disease.
Technical Summary
Changes in cytosolic calcium are a key signalling mechanism by which glucose triggers insulin secretion from the pancreatic beta-cell. Previous studies have emphasised the importance of KATP channels; however, our recent studies also implicate a central role for calcium mobilizing messenger NAADP. Recently we showed that the two-pore channel (TPC) proteins are integral components of the NAADP receptor. Here we will unravel the role of NAADP and TPCs in mediating insulin secretion by addressing a series of key questions:
A. Does glucose increase NAADP levels in primary pancreatic beta-cells? We will measure NAADP concentration changes following glucose and GLP-1 stimulation in beta-cells to assess their effects upon NAADP production.
B. How is NAADP synthesized in the beta-cell? We will investigate the role of ADP-ribosyl cyclase enzymes CD38 and CD157 as generators of NAADP in beta-cells using immuno-localisation and ARC knockout mice.
C. Which store is targeted by NAADP in beta-cells? NAADP-targeted stores are acidic organelles but their precise identity in beta-cells remains unresolved. We will investigate this using targeted indicators and immuno-based approaches.
D. Does NAADP-induced calcium mobilization generate glucose-induced calcium microdomains? Local calcium microdomains may be evoked beneath the plasma membrane of beta-cells in response to glucose but their origin is largely unknown. We will use TIRF and electrophysiology to study this issue in beta-cells from wild-type and TPC knockout mice.
E. What is the exact nature of NAADP-induced inward currents? NAADP-induced depolarizing currents may combine with closure of KATP channels in setting the excitability threshold by which glucose activates VDCCs. We will use a variety of approaches to characterize these currents.
F. What is the role of the NAADP receptor/TPCs in beta-cell physiology and insulin secretion?
We will study how NAADP signals mediated by TPCs act with KATP channels to trigger activation of VDCCs using TPC and KATP knockout/mutant mice.
G. What can TPC knockout mice tell us about beta-cell physiology and pathogenesis? TPC knockout animals can provide insights into the link between NAADP signalling and beta-cell physiology, but also highlight interactions with other signalling pathways. We will investigate this using DNA microarrays and proteomic approaches.
H. What relevance do NAADP and TPCs have for human beta-cell physiology and diabetes?
Our ultimate aim in this project will be to relate our findings to human beta-cell physiology and the understanding and treatment of diabetes. We will thus also investigate these questions in primary beta-cells obtained from donated human islets.
A. Does glucose increase NAADP levels in primary pancreatic beta-cells? We will measure NAADP concentration changes following glucose and GLP-1 stimulation in beta-cells to assess their effects upon NAADP production.
B. How is NAADP synthesized in the beta-cell? We will investigate the role of ADP-ribosyl cyclase enzymes CD38 and CD157 as generators of NAADP in beta-cells using immuno-localisation and ARC knockout mice.
C. Which store is targeted by NAADP in beta-cells? NAADP-targeted stores are acidic organelles but their precise identity in beta-cells remains unresolved. We will investigate this using targeted indicators and immuno-based approaches.
D. Does NAADP-induced calcium mobilization generate glucose-induced calcium microdomains? Local calcium microdomains may be evoked beneath the plasma membrane of beta-cells in response to glucose but their origin is largely unknown. We will use TIRF and electrophysiology to study this issue in beta-cells from wild-type and TPC knockout mice.
E. What is the exact nature of NAADP-induced inward currents? NAADP-induced depolarizing currents may combine with closure of KATP channels in setting the excitability threshold by which glucose activates VDCCs. We will use a variety of approaches to characterize these currents.
F. What is the role of the NAADP receptor/TPCs in beta-cell physiology and insulin secretion?
We will study how NAADP signals mediated by TPCs act with KATP channels to trigger activation of VDCCs using TPC and KATP knockout/mutant mice.
G. What can TPC knockout mice tell us about beta-cell physiology and pathogenesis? TPC knockout animals can provide insights into the link between NAADP signalling and beta-cell physiology, but also highlight interactions with other signalling pathways. We will investigate this using DNA microarrays and proteomic approaches.
H. What relevance do NAADP and TPCs have for human beta-cell physiology and diabetes?
Our ultimate aim in this project will be to relate our findings to human beta-cell physiology and the understanding and treatment of diabetes. We will thus also investigate these questions in primary beta-cells obtained from donated human islets.
Organisations
- University of Oxford, United Kingdom (Collaboration, Lead Research Organisation)
- Catholic University of Louvain, Belgium (Collaboration)
- University of Bristol, United Kingdom (Collaboration)
- Imperial College London, United Kingdom (Collaboration)
- Yale University (Collaboration)
- University of Geneva, Switzerland (Collaboration)
- University of Toronto (Collaboration)
- Open University of Israel (Collaboration)
- AstraZeneca plc (Collaboration)
- University of Milan, Italy (Collaboration)
- Ben-Gurion University of the Negev (Collaboration)
- University of Pisa, Italy (Collaboration)
- Cochin Institute (Collaboration)
- University of Alberta (Collaboration)
- Institute of Genetics, Molecular and Cellular Biology (IGBMC) (Collaboration)
Publications

Arredouani A
(2015)
Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic ß Cells.
in The Journal of biological chemistry

Cane MC
(2016)
The two pore channel TPC2 is dispensable in pancreatic ß-cells for normal Ca²? dynamics and insulin secretion.
in Cell calcium

Favia A
(2014)
VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.
in Proceedings of the National Academy of Sciences of the United States of America

Galione A
(2015)
A primer of NAADP-mediated Ca(2+) signalling: From sea urchin eggs to mammalian cells.
in Cell calcium

Gerasimenko JV
(2015)
Both RyRs and TPCs are required for NAADP-induced intracellular Ca²? release.
in Cell calcium

Hamilton A
(2018)
Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca Mobilization From Acidic Stores in Pancreatic a-Cells.
in Diabetes


Martinez-Sanchez A
(2016)
Disallowance of Acot7 in ß-Cells Is Required for Normal Glucose Tolerance and Insulin Secretion.
in Diabetes

Rorsman P
(2018)
Pancreatic ß-Cell Electrical Activity and Insulin Secretion: Of Mice and Men.
in Physiological reviews

Ruas M
(2015)
Expression of Ca²?-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.
in The EMBO journal
Description | Academy of Medical Sciences Sectional Committee Member |
Geographic Reach | National |
Policy Influence Type | Participation in advisory committee |
Description | Cellular mechanisms of Sleep (Galione) |
Amount | £137,000 (GBP) |
Funding ID | 102828/Z/13/A |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2016 |
End | 03/2018 |
Description | Senior Investigator Award |
Amount | £3,175,000 (GBP) |
Funding ID | 095531 |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2011 |
End | 09/2018 |
Description | Wellcome Trust Investigator award (Rutter) |
Amount | £2,000,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2013 |
End | 12/2020 |
Description | Wellcome Trust Senior Investigator Award (Galione) |
Amount | £1,500,000 (GBP) |
Funding ID | 102828/Z/13/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2014 |
End | 06/2019 |
Title | NAADP chemical tools |
Description | Syntesis of permeant NAADP antagonist of NAADP TPC antibodies |
Type Of Material | Technology assay or reagent |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | several research papers published using these |
URL | http://www.sciencedirect.com/science/article/pii/S0143416007001650 |
Title | TPC1/2 double knockout beta cells |
Description | mouse pancreatic beta cells lacking both isoforms of two-pore channels (TPCs) |
Type Of Material | Cell line |
Year Produced | 2011 |
Provided To Others? | Yes |
Impact | Elucidation of the role of TPCs in signalling pathways involving the messenger NAADP |
URL | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910865/ |
Title | conditional TPC2 KO mice |
Description | We have now successfully created a pancreatic beta cell specific TPC2 KO mouse |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Provided To Others? | No |
Impact | Ongoing studies analysing glucose-mediated calcium signals and insulin secretion |
URL | http://www.sciencedirect.com/science/article/pii/S014341601500175X |
Description | AMPK in central control of hepatic glucose production |
Organisation | University of Toronto |
Country | Canada |
Sector | Academic/University |
PI Contribution | Generation and use of adenoviral constructs |
Impact | Publication in Diabetes 2010 (x2) |
Start Year | 2008 |
Description | Conditional knockout mice |
Organisation | Cochin Institute |
Country | France |
Sector | Academic/University |
PI Contribution | Generation of tissue specifi KO mice |
Impact | Publication in Diabetologia, 2010 (Sun et al) |
Description | D Bosco (Université de Genève) |
Organisation | University of Geneva |
Department | Faculty of Diabetes |
Country | Switzerland |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | Sorcin links pancreatic ß cell lipotoxicity to ER Ca2+ stores - Marmugi A et al - PMID: 26822088 Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells. Gerber PA et al PMID: 24865615 Incretin-modulated beta cell energetics in intact islets of Langerhans. Hodson DJ et al - PMID: 24766140 ADCY5 couples glucose to insulin secretion in human islets. Hodson DJ et al PMID: 24740569 Lipotoxicity disrupts incretin-regulated human ß cell connectivity. Hodson DJ et al - PMID: 24018562 |
Start Year | 2013 |
Description | Denton |
Organisation | University of Bristol |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | manuscript (in press) Denton RM et al Biochem J 2016 |
Start Year | 2014 |
Description | Denton |
Organisation | University of Pisa |
Country | Italy |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | Sorcin links pancreatic ß cell lipotoxicity to ER Ca2+ stores - Marmugi A et al - PMID: 26822088 Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells. Gerber PA et al PMID: 24865615 Incretin-modulated beta cell energetics in intact islets of Langerhans. Hodson DJ et al - PMID: 24766140 ADCY5 couples glucose to insulin secretion in human islets. Hodson DJ et al PMID: 24740569 Lipotoxicity disrupts incretin-regulated human ß cell connectivity. Hodson DJ et al - PMID: 24018562 |
Start Year | 2014 |
Description | Generator of b-cell specfic knock-out mice for PASK |
Organisation | AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | Design of PASK flox'd mice |
Impact | None as yet |
Start Year | 2009 |
Description | Gerald Gradwohl (IGBMC) |
Organisation | Institute of Genetics and Molecular and Cellular Biology (IGBMC) |
Country | France |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | Piccard et al - http://www.ncbi.nlm.nih.gov/pubmed/25497096 |
Start Year | 2015 |
Description | James Shapiro (Alberta) |
Organisation | University of Alberta |
Country | Canada |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | ADCY5 couples glucose to insulin secretion in human islets. Hodson DJ et al PMID: 24740569 Sorcin links pancreatic ß cell lipotoxicity to ER Ca2+ stores - Marmugi A et al - PMID: 26822088 |
Start Year | 2014 |
Description | Lorenzo Piemouti (Milan) |
Organisation | University of Milan |
Country | Italy |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | ADCY5 couples glucose to insulin secretion in human islets. Hodson DJ et al - PMID: 24740569 Sorcin links pancreatic ß cell lipotoxicity to ER Ca2+ stores. Marmugi A et al - PMID: 26822088 |
Start Year | 2014 |
Description | Piero Marchetti (Pisa) |
Organisation | University of Pisa |
Country | Italy |
Sector | Academic/University |
PI Contribution | experiments for publications |
Collaborator Contribution | experiments for publications |
Impact | Sorcin links pancreatic ß cell lipotoxicity to ER Ca2+ stores - Marmugi A et al - PMID: 26822088 Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells. Gerber PA et al PMID: 24865615 Incretin-modulated beta cell energetics in intact islets of Langerhans. Hodson DJ et al - PMID: 24766140 ADCY5 couples glucose to insulin secretion in human islets. Hodson DJ et al PMID: 24740569 Lipotoxicity disrupts incretin-regulated human ß cell connectivity. Hodson DJ et al - PMID: 24018562 |
Start Year | 2014 |
Description | Role of AMPK in counter regulation |
Organisation | Yale University |
Country | United States |
Sector | Academic/University |
PI Contribution | Provision of adenoviral constructs |
Impact | None as yet |
Start Year | 2008 |
Description | Sekler |
Organisation | Ben-Gurion University of the Negev |
Country | Israel |
Sector | Academic/University |
PI Contribution | Experiments for research paper |
Collaborator Contribution | Experiments for research paper |
Impact | Publication Pancreatic beta-cell Na+ channels control global Ca2+ signaling and oxidative metabolism by inducing Na+ and Ca2+ responses that are propagated into mitochondria. DOI - 10.1096/fj.13-248161 |
Start Year | 2013 |
Description | TPCs in pancreatic islets |
Organisation | University of Oxford |
Department | Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Electrophysiology of beta cells |
Collaborator Contribution | cell biology of beta and alpha cells |
Impact | doi: 10.2337/db17-1102. doi: 10.1016/j.ceca.2015.12.004. doi: 10.1074/jbc.M115.671248. doi: 10.1172/JCI81975. |
Start Year | 2014 |
Description | TRPM4/5 channels |
Organisation | Catholic University of Louvain |
Department | Laboratory of Molecular Cell Biology |
Country | Belgium |
Sector | Academic/University |
PI Contribution | Use of TRPM4/5 knockout mice to generate pancreatic beta cells to study plasma membrane depolarizing currents |
Collaborator Contribution | Provision of the TRPM4/5 knockout mice |
Impact | Ongoing |
Start Year | 2013 |
Description | Two pore channels in beta cells |
Organisation | Imperial College London |
Department | Department of Medicine |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Co-PI on MRC Programme Grant |
Collaborator Contribution | Molecular biology of TPCs in beta cells |
Impact | Ongoing |
Start Year | 2010 |
Description | Two pore channels in beta cells |
Organisation | University of Oxford |
Department | Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Co-PI on MRC Programme Grant |
Collaborator Contribution | Molecular biology of TPCs in beta cells |
Impact | Ongoing |
Start Year | 2010 |
Description | Yuval Dor (Israel) |
Organisation | Open University of Israel |
Country | Israel |
Sector | Academic/University |
PI Contribution | Experiments for publications |
Collaborator Contribution | Experiments for publications |
Impact | LKB1 and AMPK differentially regulate pancreatic ß-cell identity - Kone M1 et al - FASEB J. 2014 Nov;28(11):4972-85. doi: 10.1096/fj.14-257667. Epub 2014 Jul 28. Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects. Swisa A1et al - J Biol Chem. 2015 Aug 21;290(34):20934-46. doi: 10.1074/jbc.M115.639237. Epub 2015 Jul 2. |
Start Year | 2014 |
Title | Role of TPCs in stimulus-secretion coupling in beta cells |
Description | TPC knockout or pharmacological antagonism inhibits stimulus-secretion coupling inn pancreatic beta cells |
IP Reference | WO2010030840 |
Protection | Patent application published |
Year Protection Granted | 2010 |
Licensed | No |
Impact | None |
Description | Seconded as science writer to The Times as a Media Fellowship |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | Yes |
Type Of Presentation | Paper Presentation |
Geographic Reach | National |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Published articles on science in the Times newspaper asked to provide other articles to other media outlets |
Year(s) Of Engagement Activity | 2012 |