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.

Publications

10 25 50

publication icon
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

publication icon
Ruas M (2015) Two-Pore Channels: Lessons from Mutant Mouse Models. in Messenger (Los Angeles, Calif. : Print)

 
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