The role of NAADP and the two-pore channel proteins in mediating insulin secretion in pancreatic beta cells

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.

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.