Elucidation of the final stages in coupling of insulin signalling to GLUT4 translocation

Maintenance of blood glucose levels is critically dependent on insulin stimulated glucose transport in adipose tissue, heart and skeletal muscle. Insulin combines with its receptor on target tissues and this initiates a cascade of linked reactions that ultimately result in the fusion of vesicles containing the glucose transporter protein GLUT4 with the plasma membrane. This latter process increases the availability of transporter molecules and thereby increases glucose transport into the cell. Although many of the links in the cascade are well studied, many technical difficulties have prevented detailed study of the final steps in the sequence, namely the fusion process. The proposed project will utilise novel approaches to study the fusion reaction and the extent to which small G-proteins of the Rab family link with the fusion machinery. We have identified many similarities between the membrane fusion reactions that occur in the pancreatic beta cell (which lead to secretion of insulin) and in insulin target tissues (which are involved in GLUT4 vesicle fusion). These similarities will guide the research objectives and experimental plans. These plans will be directed at advancing knowledge of how specific and known components of the fusion mechanism are linked together by effector proteins that are downstream of Rab proteins. This work is important as the fundamental mechanism that links signalling to regulated membrane fusion underlies many processes in biology and is relevant to human health. Elucidation of common mechanisms for pancreatic insulin-vesicle traffic and insulin target cell GLUT4-vesicle traffic will allow a unification of our understanding of those key reactions that are critical for control of blood glucose. These may become dysfunctional by a common route in metabolic disease including obesity and type 2 diabetes.

We plan to study mechanisms involved in regulating the fusion of GLUT4 vesicles with the plasma membrane of insulin target cells. We have identified Rab3 as a target of insulin signalling and using a novel photolabelling procedure have found that the loading of this G-protein is increased by insulin action. This labelling approach has also led to the identification of Rap1 as another regulated G-protein target of insulin action. In the beta cell of the pancreas Rap1 is downstream of trimeric G-proteins that augment glucose-dependent insulin release. Furthermore, it is associated with Rab3 control of vesicle traffic. In addition Munc18 is known to be a critical component of the vesicle fusion in both systems. These similarities have led us to propose that there is a unifying mechanism underlying the membrane fusion roles of these components. We plan to examine whether known Rab3 effectors that link with Munc18 in the pancreas have similar counterparts involved in insulin regulation of GLUT4 vesicle fusion. Therefore, we plan to identify Rab3 effectors in insulin-target tissues and validate their involvement in the fusion reaction. We will then study the interaction of Rab3 and the Rab effectors with Munc18. We will also examine the extent to which Munc18 availability for fusion and interaction with SNARE proteins is dependent of its tyrosine phosphorylation. We will determine whether insulin receptor tyrosine kinase is responsible for this phosphorylation using intact receptor and intact plasma membrane as components in our cell-free fusion system. In addition, we will examine the extent to which Rab3 and its effectors are regulated downstream of trimeric G-proteins and through the intermediate Rap1, as occurs in pancreatic beta cells.

Economic impact
Potential Beneficiaries: Pharmaceutical companies (improved products and sales); NHS (improved therapies and interventions);
The Holman group has collaborated extensively with UK based Pharmaceutical companies including Glaxo-SmithKline and currently AstraZeneca that have helped translate our research into practice. One example outcome of these studies has been the testing of the potency of an insulin sensitizer that was developed into a highly successful anti-diabetes drug called Rosiglitazone. Information obtained in this collaboration was used to mechanistically validate the drug. Annual sales of this drug peaked at approx $2.5bn in 2006, but declined after reports of adverse effects. Our current work is partially supported AstraZeneca has been supported by two consecutive CASE studentships from 2006-2009 and currently 2009-2013. Both studentships have involved setting up high-throughput cell models for testing drug targets, including insulin sensitive Rab-GAPs. With these continuing links we will be able to translate new output on the proposed project into drug screening programs with an expected eventual impact on new drug sales. Diabetes treatment has economic consequences to the NHS (below).

Social and human health impact
Potential beneficiaries: General public; NHS; Charities; Policy Makers
Type 2 diabetes is a major global problem with more than 250 million people worldwide currently living with this condition and by 2025, this total is expected to increase to over 380 million people. Each year another 7 million people develop diabetes. 2.8m people in the UK are now diagnosed with diabetes and it is estimated that 1 million more people may have pre-diabetes. Diabetes can lead to complications including kidney failure, coronary heart disease, stroke, blindness and foot amputation. It is estimated that Diabetes treatment (mainly type 2) accounts for an estimated 5% of all NHS expenditure. A detailed understanding of the underlying biological system is key for developing effective diagnostic tools, causal intervention strategies and in supporting public health advice (through government departments and charities). Providing a full pathway from insulin receptor to glucose transporter will impact on strategies for the control of glycemia, energy homeostasis and bodyweight. Therefore there is potential to change behaviours with associated improvements in health outcomes.
The targeting of this fundamental research area will reduce animal use by generating more targeted drug discovery. In addition it will provide a focused and directed range of parameters to be monitored in human subjects undergoing clinical drug trials.

Skills and training impact
Potential beneficiaries: Next generation research students; Pharmaceutical companies; Health coaching
The proposed research on insulin action will involve postgraduate students who will benefit from training in the novel methods and approaches used by our group. We use a combination of cell physiology and biochemistry approaches. We also have chemical biology input with generation of new tools and reagents for analysis of aspects of insulin action. This wide range of skills will be passed on in training of new PhD students. We hope to continue to recruit students who can interact with pharmaceutical companies. Students trained in this way will be able to pass on skills to a range of employment sectors but specifically sectors concerned with treatments for obesity and type 2 diabetes. There are currently two PhD students in the Holman group. One of these is a CASE student with AstraZeneca. Holman has successfully trained 27 previous PhD students. Our interaction with Sports and Exercise Science PhD and MSc students allows us to share expertise in metabolism and signalling with those contemplating careers in human health related areas such as exercise and diet coaching.